Nature Agriculture

Our results also corroborated with this finding. Our results indicated that, apart from fruits, SS were mainly accumulated in the leaves at harvest, which accounted for about 90% of the total leaf NSC. Thus, the allocation of NSCs in different organs allowed the plants to persist when respiration rate was higher than photo assimilation in annual events, but also aided in responding to abiotic stresses such as drought . Our results indicated that plants that received 100% ETc had higher NSC content. Similarly, a previous study with potted grapevines reported increased starch and SS contents in the leaves from the grapevines with higher leaf area to fruit ratio that were well-watered . In shoots, sucrose and raffinose proportions were higher in 50 and 100% ETc treatments compared with 25% ETc. As a great part of the shoot biomass is vascular tissue, this may suggest an increase in NSC translocation in these treatments. Although sucrose is the main sugar for carbon translocation through the phloem into the sink tissues, recent research highlighted the roles of other sugars, such as raffinose, in carbon translocation and storage . On the other hand, previous research reported less NSC accumulation in grapevine canes under carbon starvation at a low leaf to fruit ratios, suggesting that sucrose may control starch accumulation through adjustment of the sink strength . Furthermore, Rossouw et al. also highlighted the role of raffinose toward root carbohydrate source functioning in grapevines with significantly lower leaf to fruit ratio due to defoliation from carbon starvation . When the photosynthetic supply of carbohydrates is limited,round flower buckets remobilization from perennial tissues can provide an alternative carbon source . Thus, previous research conducted on potted grapevines reported a concurrent starch remobilization from roots with a rapid berry sugar accumulation .

Conversely, under our experimental conditions , no effect of water deficits on NSC remobilization from roots to berries was observed despite the decreased leaf to fruit ratio. Likewise, Keller et al. did not observe higher amounts of sugars in berries from field-grown Cabernet Sauvignon subjected to 25% ETc compared with 70 or 100% ETc under field conditions. Under our experimental conditions, yield per plant was strongly related to shoot, leaf, and root BM. Similarly, Field et al. found that grapevines with the lowest shoot growth rate before veraison had significantly less fruit set than the other treatments, attributing these effects to the restoration of root carbohydrate reserves that occurred at the same time. Grapevines subjected to 25% ETc had reduced photo assimilates due to lower AN in both seasons resulting in less NSC in the source leaves available for new growth and exported to sinks. This resulted in a general lower plant BM . Contrarily, grapevines subjected to 100% ETc had higher photo assimilation rates throughout the course of the study that led to higher SS and starch content and, consequently, to the improvement of BM and, therefore, higher harvest index. Therefore, the reduced growth rate of both sink and source organs in response to water deficits indicated that the availability of carbon is a major growth constraint. The yield per plant of 50% ETc was lower than 100% ETc, but not as low as 25% ETc. However, canopy BM was greatly reduced in both 50% ETc and 25% ETc compared with 100% ETc. Accordingly, Field et al. reported that grapevine grown under warm soil conditions favored shoot and fruit development over carbohydrate reserve accumulation. In contrast, Candolfi-Vasconcelos et al. reported that a lower leaf area to fruit ratio increased the translocation of carbohydrates from permanent structures to reproductive organs to support grape ripening. The shoot to root ratio revealed a positive relationship with the total BM, leaf and root NSC, and N contents. Thus, the distribution of biomass relies on the C:N ratio as highlighted by the negative relationship between shoot to root and the sucrose:nitrogen ratios. Similarly, a linear relationship between NSC and root to shoot ratio in grapevines grown under stressful conditions was previously reported .

From a molecular point of view, the alterations of source:sink ratios led to transcriptional adjustments of genes involved in starch metabolism, including the upregulation of VvGPT1 and VvNTT for lower leaf area to fruit ratios . Furthermore, enhanced root biomass in 100% ETc likely resulted from higher sugar content in the roots as our data supported. It was recently reported that increases in root elongation and hexose contents were due to the VvSWEET4 over expression, a gene implied as a grapevine response to abiotic stress . Similarly, Medici et al. reported up- or down regulation of the genes encoding hexose transporters in grapevines subjected to water deficits corroborating this result. Therefore, although some genes may be expressed under water deficit, lack of carbon accumulation impaired the growth. The relationship between root to shoot ratio and plant nitrogen content was previously reported for grapevines, suggesting that dry matter partitioning is largely a function of the internal status of the plants . We found decreased N content in grapevines facing water deficits, which resulted in a decrease of total BM. Similarly, Romero et al. reported reductions in leaf nitrogen content when vines were subjected to water deficits. These authors suggested that nutrient uptake may be reduced due to deficits in soil water profile, and the slow root growth under these conditions consequently inhibited grapevine growth. In our study, N content was strongly related to photosynthetic pigments. Accordingly, previous studies reported lower leaf N and leaf chlorophyll in deficit-irrigated grapevines, suggesting quantitative losses in the photosynthetic apparatus and/or damage to the biochemical photosynthetic machinery, decreasing photosynthetic capacity as corroborated with the lower NSC leaf content with water deficits. Finally, molecular research over the last decades has suggested the important regulatory functions of sucrose and N metabolites in metabolism at the cellular and subcellular levels and/or in gene expression patterns, giving new insights into how plants may modulate over a longer period its growth and biomass allocation in response to fluctuating environmental conditions .

Optimism is a key facet of positive psychological well-being that is associated with reduced morbidity and mortality, independent of psychological distress . Despite accumulating evidence regarding the relationship between optimism and health, underlying mechanisms remain unclear. Prior work has linked optimism with higher levels of physical activity and better physiological adjustment to stress exposure . Potential beneficial effects of optimism on physical activity and stress response may be partly a function of its regulatory component. Optimism reflects having confidence in the future, which in turn results in a greater likelihood of employing strategies for achieving one’s goals, including effortful goal engagement, problem-focused coping with challenges, and goal adjustment when goals become unattainable . The ability to employ these strategies effectively and flexibly likely provides greater means with which to confront and manage life’s challenges and to adopt more health protective behaviors. Despite increasing evidence of optimism’s relationships with more physical activity and healthier physiological responses to stress, the vast majority of studies focusing on dispositional optimism and its associations with beneficial physical and physiological outcomes have been observational . As such, the direction of the effect is unclear,plastic flower buckets wholesale leaving open the question of whether optimism causally influences physical activity or physical activity increases optimism. Additionally, almost all previous studies have relied on self-reported optimism and positive affect which may be subject to social desirability and recall bias. Experimental research in which individuals are randomly assigned to an optimism intervention with targeted pathways related to improved health as outcomes – like physical activity and stress responses – is needed to establish the causal role of optimism in relation to these outcomes. While most research has focused on trait-based optimism, a growing number of studies have considered whether optimism can be encouraged, cultivated or modified, and if health-related effects of more deliberately cultivated optimism are similar . A number of studies have used experimental methods to manipulate optimism, typically through interventions involving writing tasks . These interventions successfully increase optimism and other self reported affective responses and decrease negative affect, depression, anxiety, and aggression, but a key outstanding question is whether these interventions can also sufficiently alter behavior and physiology in ways that explain downstream beneficial health effects. While some evidence suggests positive effects of these interventions, the generalizability of these effects to healthy community-dwelling adults is unclear. For example, many experimental studies to date have been conducted among patient populations with a recent disease diagnosis or acute event such individuals tend to be highly motivated to improve their health behaviors, willing to undergo relatively intensive intervention protocols, and also relatively unhealthy at the outset and therefore have substantial room to show improvement. Other studies testing short-term optimism manipulation strategies in non-patient populations also provide some evidence that writing task interventions could improve health and well-being; however, these have been conducted exclusively within student populations which may be more compliant than the general population .

Therefore, a key outstanding question is whether optimism levels can be altered to subsequently lead to changes in downstream health-relevant processes like physical activity and stress reactivity within the general population. The overall aim of this research was to examine the effects of an optimism intervention on physical activity and stress reactivity with community volunteers. We developed a short intervention designed to induce optimism in the short-term and randomized participants either to the optimism intervention or an active control condition. The same intervention was used in both studies, and we harmonized the self-reported dependent variables. We hypothesized that: 1) the intervention group relative to the control group would show increased self-reported optimism and positive affect, as well as decreased anxiety, depression, aggression, and negative affect; 2) in Study 1, the optimism intervention compared to the control group would show more engagement and persistence with physical activity tasks, as operationalized by a stepping task and a sit-stand task and self-reported exercise beliefs including perceived benefits and barriers of exercise as well as self-efficacy of exercising; 3) in Study 2, the optimism intervention compared to the control group would show healthier stress-related physiologic responses at rest and during an acute stressor; and 4) those who were rated as more optimistic would show more engagement and persistence in physical activity tasks and have better stress-related physiologic responses in the lab.Participants ages 22–60 years old were recruited through Craigslist, Student Employment Office websites at local universities, and flyers posted on community bulletin boards. Potential participants completed a telephone screening to determine their eligibility and willingness to adhere to the study protocol. Individuals with chronic diseases like heart disease, who were pregnant, had body mass index ≥30 kg/m2 , or had a current diagnosis of clinical depression or other major mental disorders were excluded. As a goal of Study 1 was to assess whether optimism has beneficial effects on willingness to engage in physical activity, further exclusions included not being able to engage in physical activity or self-reporting regularly spending more than 60-minutes per week doing strenuous or moderate exercise. Additionally, participants were asked to refrain from eating for a half-hour and exercising for 2 hours prior to lab visits. Eligible individuals completed secure online consenting procedures through Qualtrics. Written consent for the in-lab portion of the study was obtained from participants during their lab visits.The protocol for both studies included three online writing tasks and one phone interview prior to a lab visit. In the lab, participants completed another writing task and one in-person interview. All the writing tasks and the lab study were conducted over one to two weeks . The four writing tasks for the intervention group included a values assessment, remembering past achieved goals, writing a gratitude letter, and imagining one’s best possible self, all chosen based on prior literature suggesting such interventions may lead to greater optimism levels . The control condition provided comparable demands for attention and writing but focused on neutral and less future-oriented activities. Prompts for the intervention condition writing tasks were identical in both studies; however, the first three prompts in the control condition were slightly modified for Study 2 Table 1. After completing the first three writing tasks online, participants of both studies were invited to come to the lab to complete the final part of the study. At the lab, participants completed a brief final screening to confirm accuracy of previously self reported information, an imagery task, a fourth writing task, an in-person interview, and physical activity tasks or stress reactivity tasks . For Study 1, participants were asked to engage in a stepping task and a sit-to-stand task.

There is also the possibility of a loss of accessions occurring during the revival, either due to contamination or low recovery, especially for some specific Vitis varieties. Seed bank storage is also utilized for the ex situ conservation of trees, although the germinating progeny will show segregating genotypes. However, plants with recalcitrant and intermediate seeds cannot stand desiccation conditions and cold storage without losing viability; only orthodox seeds are highly suited to this procedure. Moreover, conservation via storing seeds is suitable only for wild grapevines, and not for cultivated ones. Therefore, field gene bank collections are important for such species. In field gene bank collections, tree species are maintained and multiplied indefinitely, using seeds or vegetative propagation; this emphasizes the importance of having a long-term plan for the sustainability of such collections. However, maintenance of such collections is costly , and extensive maintenance actions are required. Moreover, these collections are vulnerable to climate change, biotic and abiotic stresses, and sometimes natural disasters. The emergence of intense competition in the international wine market has encouraged the idea of recovering high-quality ancient and indigenous accessions. As a result, a large number of indigenous accessions have been collected from wild and old vineyards,procona system and maintained in the field collections of grapevine-growing countries, including Israel, the USA, Spain, Italy, and many other countries. For example, the United States Department of Agriculture—Agricultural Research Service collections of wild grapevines at the Plant Genetic Resource Unit and the National Clonal Germplasm Repository maintain around 5000 accessions as a field collection. The FEM grape germplasm collection at San Michele all’Adige, Italy, maintains 2273 accessions.

The IFAPA research center “Rancho de la Merced” hosts 930 accessions, and the “El Encín” Vine Varieties Collection, Spain, has 966 indigenous accessions. Finally, the Vitis International Variety Catalogue is a database of ~23,000 cultivars, breeding lines, and Vitis species, that provides a comprehensive overview of most international grapevine collections. The grapevine is one of the most sensitive cultivated plants; it is very responsive to its surrounding environment. Different varieties may have a wide range of responses to specific stressors; thus, maintaining a wide range of cultivars in a field collection using a uniform growth protocol is challenging. As mentioned above, grapevine field collections also require large spaces, are labor-intensive, expensive to maintain, and are exposed to natural disasters, climate change, and infection by pathogens. Therefore, the maintenance of field grapevine collections is challenging. In spite of these complications, the importance of maintaining grapevine field collections is growing in light of current and forecasted climate changes; the sustainability of current commercial grapevine varieties is under greater pressure as a result of global warming. New, resistant varieties will be greatly needed, and it is anticipated that the diversity found in germplasm collections will become increasingly valuable for future breeding programs . Genomics-assisted breeding will reduce the time and costs that are required to breed new cultivars that possess desirable traits. The methodical collection of data from a field collection can enhance our understanding of phenological variations, and of individual responses to various biotic and abiotic factors—for instance, in terms of cold hardiness, blooming time, drought stress , as well as important commercial traits such as wine characteristics. Due to the adaptability of such varieties in local microclimatic conditions, these varieties can provide root stocks that are resistant to the salt and drought stresses that are associated with climate change.

Israel is characterized by a dramatic climatic gradient, from the northern moderate temperature and humidity of the Golan Heights, to the southern hot and dry Negev. Here, we describe the advantages of maintaining a unique field indigenous collection in Ariel, Israel. This collection includes 96 accessions that were collected in Israel during the last decade, representing one of the most ancient grape populations in the world, and one that is suspected of having been domesticated from local Vitis sylvestris populations. Furthermore, individual grapevines in this collection were collected from very harsh ecosystems, including the Mediterranean Sea shore, the Negev desert, and dry mountainous areas; such grapevines represent a unique opportunity to study abiotic stress resistances. This collection was used to gain a better understanding of the genomic structure of the domesticated and wild grape populations in the south Levant. This study will argue the benefits of maintaining this unique in-field collection in order to study drought resistance, phenotypic and phenological variations, and grape aroma profiles of previously uncharacterized grape varieties. This study presents the physiological data retrieved from 33 varieties , measured at five time points during a drought stress experiment of 10 weeks. Using k-means analysis to classify the individual vines into groups [using the physiological information measured for each vine at all five time points—stem water potential , photosynthesis, and stomata conductance], we show that the vines, represented by dots, are divided into three groups that are based on their physiological behavior during the extended drought stress period . Next, we annotated every dot to the specific vine and variety, and left in the analysis only those varieties in which all four vines are classified into the same group. When looking into the physiological data for the vines represented in each group—the group of varieties marked by green dots—Nitzan 3 and Beer, which come from a group that is more separated from the other two groups, are varieties that are characterized by the most negative SWP and lowest stomata conductance at the later stages of the experiment . Meanwhile, the group of varieties marked by red dots—Shami and Batar Nitzamin—had improved parameters at the later stages. Finally, the intermediate group marked by the blue dots—Jandaly and Ramtania—showed moderate parameters at the late stages, and had some intermixing with the red group .The correlation between stomata conductance and photosynthetic efficiency of varieties under drought stress shows close to linear relations with high R 2 levels for most varieties.

Nevertheless, the slopes are higher for the stable varieties than for those drought-sensitive varieties that show a more extreme reaction , meaning that the red group transpires more water for the same photosynthetic efficiency. Next, we conducted a correlation between midday stem water potential and stomatal conductance for two representative varieties for each response strategy during all five measuring points . The correlation clearly shows the differences between the groups—the more stable varieties, Shami and Batar-Nitzanim, are only mildly affected by extended drought, reaching only circa -1MPa, and maintaining relatively high gs at the later stages of the experiment, while the sensitive varieties, Beer and Nitzan 3, are dramatically affected at the early stages, reaching more negative levels of SWP and with lower stomatal conductance. The Ramtania and Jandaly varieties represent the moderate group, dropping faster into a more negative SWP and lower gs than the stable group, but not as dramatically as the sensitive varieties.A comprehensive dating of the phenological stages for the accessions in the collection was conducted during the season of 2018. Determination of the phenological sequence is generally important for the characterization of each variety as an early, medium, or late blooming. When extended to the ripening period, it also enables planning for an extended harvest season for table grapes, and enables planning for early or late harvest for wine making purposes. Interestingly,procona valencia buckets when referring to the former division of the varieties by their drought stress reactions, we found that stable varieties show very late bud break and foliage development compared to sensitive varieties. By fruit set, most varieties show a similar development pattern.Studies of phenotypic variation using germplasm collections can improve our understanding of phenotypic plasticity in grapevine varieties, as they respond to changing climatic conditions. Therefore, it is essential to examine diverse germplasms to better understand variation in individual responses to changing conditions, and to identify germplasms that can withstand climatic variations for use in breeding programs. In this paper, we have shown that drought stress, applied uniformly to the whole range of the Israeli germplasm collection which contains six plants for each variety, enables the preliminary identification of drought-stable or drought-sensitive varieties. Stable varieties showed improved SWP and higher levels of stomata conductance under drought stress, while the sensitive varieties dropped in SWP rapidly, as well as in stomata conductance. This initial characterization should be followed by a deeper one, concentrating on the varieties that harbor the most pronounced phenotypes in larger dedicated plots and replications. Using a field germplasm collection for this initial screening has pros and cons. Advantageously, we can count the fact that one can conduct comprehensive surveys that encompass the entire range of varieties, to the trait in hand, and the ease of work, when all varieties are under the same conditions in one plot; this facilitates multiple analyses in a short time. In terms of disadvantages, a collection usually contains a very limited number of vines per variety, with no replicate vines spread in different parts of the vineyards, as we would in stationing a proper field experiment. Thus, when analyzing the plasticity of varieties to traits such as stress resistance, the data collected can serve only as preliminary observations to be verified by wider experiments.

Living germplasm collections can also be valuable resources for characterizing diversity in phenological sequences—bloom time, timing of bud break, and véraison, as well as fruit development, ripening processes, and cold hardiness, when responding to identical sets of environmental conditions on the same site across multiple years and conditions. As we have shown, our tracking of the early phenological sequence in some varieties in our collection is in concert with our findings during the drought stress experiment, and the separation of varieties into “stable” or “sensitive” to drought stress. It seems that the stable varieties are those which are late blooming, while those that are sensitive are early blooming . We suggest that the late bud breaking and foliar development of the “stable” varieties is possibly the mechanism that enables their mild reaction to the initiation of drought stress. The early varieties have around forty additional days to sustain their foliage, possibly causing rapid depletion of the soil water reservoir accumulated during winter. Thus, we suggest that at the point of drought stress initiation , their depleted soil water reservoir leads them into more profound stress, preventing them from keeping their stomata open; meanwhile, the late blooming varieties, which have much larger water reservoirs due to less time sustaining foliage, have improved water status. We will need to continue monitoring both phenological and physiological reactions of the stable and sensitive groups over an extended period of time, in order to understand and validate this suggestion; nevertheless, these surprising observations were possible only through maintaining an in-field collection, with meticulous data collection for different traits. Field germplasm collections can be utilized as reference materials for ampelographic studies. Recent research done at the indigenous grapevine collection at Ariel University revealed information about genetic diversity and single nucleotide polymorphisms among various accessions, which in combination with the ampelographic data collected in field, can enable the definition of varieties and clones. For example, we have previously shown that seed morphology analyses of three sets of SSR identical pairs of varieties—Shami and Tufahi, Karkashani and Zituni, Baluti and Bituni—found that the first two pairs are clearly distinguishable by seed morphology, while the third pair has very similar seed morphology, strengthening this pair’s definition assynonyms. The newly developed method for defining 3D seed morphology joins the more traditional straightforward traits of bunch and berry size and shape, berry color, and taste, which help distinguish synonyms from clones. Indeed, many such variations resulted in the development of popular cultivars such as ‘Pinot gris’ and ‘Pinot blanc’, which are lighter-colored variations of the grapevine cultivar, ‘Pinot noir’, which possesses 17 clones within the USDA grapevine germplasm collection. While such accessions may appear to be genotypic duplicates, especially when a limited number of genetic markers is used to evaluate their differences, they are actually valuable resources for studying plant developmental biology and gene function. Here, we deeply analyzed phenolic composition for some red varieties in the collection, and aroma profiles for the white ones. We show that most red indigenous varieties have relatively low anthocyanin and polyphenolics levels. Only four varieties, namely Marawani, Gilboa, Black Tzuriman, and Baluti, showed higher levels of anthocyanins and phenolic characteristics, which may indicate possible suitability for wine production.

Since these loci range in length from roughly 400 to 700 bp, this result would be consistent with an average recombination length of no more than a few kb, similar to the 2.6-kb average length observed by Nunney et al. in a comparison of two X. fastidiosa subsp. fastidiosa genomes . Similarly, the regions identified from the data of Parker et al. showed the same pattern, with a high proportion of recombination breakpoints identified within the sequenced loci . In this context, it is important to note that Rogers and Stenger have found a conjugative plasmid in X. fastidiosa. Furthermore, a high rate of transformation has been demonstrated in the lab , and it has been shown that this process can result in efficient recombination with only a few hundred bases of homologous sequence . Both conjugation and transformation may have been involved in the evolution of the recombinant group, since the data raise the possibility of both large-scale intersubspecific and smaller-scale intrasubspecific recombination . The results support the general conclusion that successful recombination is a rare but important event, a possibility emphasized by Wiedenbeck and Cohan in their review of bacterial adaptation to new environments. However, given the high rates of recombination observed experimentally in X. fastidiosa , this rarity is somewhat surprising, perhaps suggesting that in X. fastidiosa the majority of intersubspecific recombination events fail due to their negative fitness consequences. Fitness loss due to recombination is consistent with the high level of plant host speci- ficity observed among the genotypes of X. fastidiosa . On the other hand,blueberry in pot it is clear that recombination can create combinations that are beneficial to the species, enabling it to invade new plant hosts.

Specifically, the successful invasion of blueberry and blackberry appears to have resulted from large-scale recombination between two subspecies, a pattern that appears to be repeated in the invasion of mulberry . Furthermore, Nunney et al. suggested that introgression into X. fastidiosa subsp. pauca in South America from a donor may have enabled X. fastidiosa subsp. pauca to infect citrus, causing the economically devastating disease of citrus variegated chlorosis. This would help explain why CVC did not appear in Brazil until the 1980s , despite the presence of the native pathogen and vectors ever since citrus was introduced several hundred years ago. These observations raise an important concern: that mixing of genetically divergent forms of the same species can result in recombinant forms capable of invading new niches—in this case, new plant hosts. Thus, the presence of a pathogen in an area should not lead to the assumption that further introductions will cause no further harm; in fact, as a result of recombination, further introductions may result in a qualitative worsening of the problem.Agriculture is a key human activity in terms of food production, economic importance and impact on the global carbon cycle. As the human population heads toward 9 billion or beyond by 2050, there is an acute need to balance agricultural output with its impact on the environment, especially in terms of greenhouse gas production. An evolving set of tools, approaches and metrics are being employed under the term “climate smart agriculture” to help—from small and industrial scale growers to local and national policy setters—develop techniques at all levels and find solutions that strike that production-environment balance and promote various ecosystem services. California epitomizes the agriculture-climate challenge, as well as its opportunities. As the United States’ largest agricultural producing state agriculture also accounted for approximately 8% of California’s greenhouse gas emissions statewide for the period 2000–2013. At the same time, California is at the forefront of innovative approaches to CSA. Given the state’s Mediterranean climate, part of an integrated CSA strategy will likely include perennial crops, such as wine grapes, that have a high market value and store C long term in woody biomass.

Economically, wine production and retail represents an important contribution to California’s economy, generating $61.5 billion in annual economic impact. In terms of land use, 230,000 ha in California are managed for wine production, with 4.2 million tons of wine grapes harvested annually with an approximate $3.2 billion farm gate value. This high level of production has come with some environmental costs, however, with degradation of native habitats, impacts to wildlife, and over abstraction of water resources. Although many economic and environmental impacts of wine production systems are actively being quantified, and while there is increasing scientific interest in the carbon footprint of vineyard management activities, efforts to quantify C capture and storage in annual and perennial biomass remain less well-examined. Studies from Mediterranean climates have focused mostly on C cycle processes in annual agroecosystems or natural systems. Related studies have investigated sources of GHGs , on-site energy balance, water use and potential impacts of climate change on productivity and the distribution of grape production. The perennial nature and extent of vineyard agroecosystems have brought increasing interest from growers and the public sector to reduce the GHG footprint associated with wine production. The ongoing development of carbon accounting protocols within the international wine industry reflects the increased attention that industry and consumers are putting on GHG emissions and offsets. In principle, an easy-to-use, wine industry specific, GHG protocol would measure the carbon footprints of winery and vineyard operations of all sizes. However, such footprint assessment protocols remain poorly parameterized, especially those requiring time-consuming empirical methods. Data collected from the field, such as vine biomass, cover crop biomass, and soil carbon storage capacity are difficult to obtain and remain sparse, and thus limit the further development of carbon accounting in the wine sector .

Simple yet accurate methods are needed to allow vineyard managers to measure C stocks in situ and thereby better parameterize carbon accounting protocols. Not only would removing this data bottleneck encourage broader participation in such activities, it would also provide a reliable means to reward climate smart agriculture.Building on research that has used empirical data to compare soil and above ground C stocks in vineyards and adjacent oak woodlands in California, this study sought to estimate the C composition of a vine, including the relative contributions of its component parts . By identifying the allometric relationships among trunk diameter, plant height, and other vine dimensions, growers could utilize a reliable mechanism for translating vine architecture and biomass into C estimates. In both natural and agricultural ecosystems, several studies have been performed using allometric equations in order to estimate above ground biomass to assess potential for C sequestration. For example, functional relationships between the ground-measured Lorey’s height and above ground biomass were derived from allometric equations in forests throughout the tropics. Similarly, functional relationships have been found in tropical agriculture for above ground, below ground, and field margin biomass and C. In the vineyard setting, however, horticultural intervention and annual pruning constrain the size and shape of vines making existing allometric relationships less meaningful, though it is likely that simple physical measurements could readily estimate above ground biomass. To date, most studies on C sequestration in vineyards have been focused on soil C as sinks and some attempts to quantify biomass C stocks have been carried out in both agricultural and natural systems. In vineyards, studies in California in the late 1990s have reported net primary productivity or total biomass values between 550 g C m−2 and 1100 g C m−2 . In terms of spatial distribution, some data of standing biomass collected by Kroodsma et al. from companies that remove trees and vines in California yielded values of 1.0–1.3 Mg C ha−1 year−1 woody C for nuts and stone fruit species, and 0.2–0.4 Mg C ha−1 year−1 for vineyards. It has been reported that mature California orchard crops allocate, on average,plastic planters wholesale one third of their NPP to the harvested portion and mature vines 35–50% of the current year’s production to grape clusters. Pruning weight has also been quantified by two direct measurements which estimated 2.5 Mg of pruned biomass per ha for both almonds and vineyards. The incorporation of trees or shrubs in agroforestry systems can increase the amount of carbon sequestered compared to a monoculture field of crop plants or pasture. Additional forest planting would be needed to offset current net annual loss of above ground C, representing an opportunity for viticulture to incorporate the surrounding woodlands into the system. A study assessing C storage in California vineyards found that on average, surrounding forested wild lands had 12 times more above ground woody C than vineyards and even the largest vines had only about one-fourth of the woody biomass per ha of the adjacent wooded wild lands.The objectives of this study were to: measure standing vine biomass and calculate C stocks in Cabernet Sauvignon vines by field sampling the major biomass fractions ; calculate C fractions in berry clusters to assess C mass that could be returned to the vineyard from the winery in the form of rachis and pomace; determine proportion of perennially sequestered and annually produced C stocks using easy to measure physical vine properties ; and develop allometric relationships to provide growers and land managers with a method to rapidly assess vineyard C stocks.

Lastly, we validate block level estimates of C with volumetric measurements of vine biomass generated during vineyard removal.The study site is located in southern Sacramento County, California, USA , and the vineyard is part of a property annexed into a seasonal floodplain restoration program, which has since removed the levee preventing seasonal flooding. The ensuing vineyard removal allowed destructive sampling for biomass measurements and subsequent C quantification. The vineyard is considered part of the Cosumnes River appellation within the Lodi American Viticultural Area, a region characterized by its Mediterranean climate— cool wet winters and warm dry summers—and by nearby Sacramento-San Joaquin Delta breezes that moderate peak summer temperatures compared to areas north and south of this location. The study site is characterized by a mean summer maximum air temperature of 32 °C, has an annual average precipitation of 90 mm, typically all received as rain from November to April. During summer time, the daily high air temperatures average 24 °C, and daily lows average 10 °C. Winter temperatures range from an average low 5 °C to average high 15 °C. Total heating degree days for the site are approximately 3420 and the frost-free season is approximately 360 days annually. Similar to other vineyards in the Lodi region, the site is situated on an extensive alluvial terrace landform formed by Sierra Nevada out wash with a San Joaquin Series soil . This soil-landform relationship is extensive, covering approximately 160,000 ha across the eastern Central Valley and it is used extensively for wine grape production. The dominant soil texture is clay loam with some sandy clay loam sectors; mean soil C content, based on three characteristic grab samples processed by the UC Davis Analytical Lab, in the upper 8 cm was 1.35% and in the lower 8–15 cm was 1.1% . The vineyard plot consisted of 7.5 ha of Cabernet Sauvignon vines, planted in 1996 at a density of 1631 plants ha−1 with flood irrigation during spring and summer seasons. The vines were trained using a quadrilateral trellis system with two parallel cordons and a modified Double Geneva Curtain structure attached to T-posts . Atypically, these vines were not grafted to root stock, which is used often in the region to modify vigor or limit disease .In Sept.–Oct. of 2011, above ground biomass was measured from 72 vines. The vineyard was divided equally in twelve randomly assigned blocks, and six individual vines from each block were processed into major biomass categories of leaf, fruit, cane and trunk plus cordon . Grape berry clusters were collected in buckets, with fruit separated and weighed fresh in the field. Leaves and canes were collected separately in burlap sacks, and the trunks and cordons were tagged. Biomass was transported off site to partially air dry on wire racks and then fully dried in large ventilated ovens. Plant tissues were dried at 60 °C for 48 h and then ground to pass through a 250 μm mesh sieve using a Thomas Wiley® Mini-Mill . Total C in plant tissues was analyzed using a PDZ Europa ANCA-GSL elemental analyzer at the UC Davis Stable Isotope Facility. For cluster and berry C estimations, grape clusters were randomly selected from all repetitions. Berries were removed from cluster rachis. While the berries were frozen, the seeds and skins were separated from the fruit flesh or “pulp”, and combined with the juice .

Soil acidity can be adjusted to optimize plant growth, but adjusting acidity is not always the best course of action. Instead, it may be best to grow plants adapted to the native soil. For gardeners who decide to adjust the acidity of soil, it is important to understand when and how to do so. Acidity, measured on a logarithmic scale called pH, depends on the concentration of hydrogen in a solution. In the case of soils, acidity depends on how much hydrogen is dissolved in the liquid that soil particles hold. Possible pH values range from 0 to 14. A soil with a pH of 7.0 is considered neutral— that is, it is neither acidic nor alkaline. A pH value below 7 is acidic while a value above 7 is alkaline. Distilled vinegar used in cooking is very acidic and has a pH of about 2.4. Household chlorine bleach is very alkaline, with a pH of about 11. Each whole-number change in pH value represents a 10-fold change in acidity. For example, a solution with a pH of 6 is 10 times more acidic than a solution with a pH of 7.As noted, most crops perform best when the soil is neither too acidic nor too alkaline. For many crops, a soil pH somewhere between 5.5 and 7.5 works well. In this range, most plant nutrients are chemically available to plant roots , though some plants require a soil acidity outside this range. Soils in California generally range in pH from 5 to 8.5, but most are higher than pH 7.While it is possible to add amendments to soil to adjust the pH, doing so is not always the best course of action. Ideally, gardeners should select plants that are adapted to the native soil. If a gardener wishes to grow a plant that is not tolerant of local soil conditions,plastic planters bulk the best long-term solution may be to grow it in a container or raised bed filled with a suitable soil mix.

All soils have a natural pH that depends on the minerals in the soil and conditions that arise over very long time periods, during soil development. In a garden, fighting a soil’s natural pH can be challenging. When gardeners attempt to adjust soil acidity, the results are often temporary. Moreover, products that raise or lower pH are slow to react in the soil. If gardeners decide to adjust the pH in a garden, they should do so based on careful evaluation of the condition of their plants. A soil may not need pH adjustment, even if it is technically outside the ideal range for a garden, unless plants show symptoms of nutrient deficiencies or toxicities. The pH of the soil can influence whether a nutrient is chemically available to plants. An essential plant nutrient such as iron may be present in the soil but unavailable to the plant due to the soil pH. Plants unable to obtain essential nutrients, either due to soil pH or scarcity in the soil, show deficiency symptoms. When plants show symptoms associated with excessively alkaline or acidic soil conditions, such as yellowing between veins—known as interveinal chlorosis—gardeners should attempt to determine whether the pH of the soil is the cause. Urban and suburban soils can be significantly modified during housing construction, so their acidity may differ significantly from values available online. Other activities that disturb the soil, such as gardening, can affect soil pH. Therefore, a measurement of acidity in a soil sample taken directly from a garden is more accurate than online soil survey data. Though the most accurate way to determine soil pH is through a soil test conducted by a laboratory, gardeners can purchase equipment such as a chemical test kit, paper pH test strips, or a digital pH meter to evaluate soil pH. All of these products should come with instructions for use. With most soil chemical test kits, drops or tablets of specially selected chemicals are added to a solution extracted from soil, causing a color change or change of appearance. Chartsor cards included with the kit translate the results into numerical values. Some kits rely on paper test strips to measure soil pH. Paper test strips are easier to use than pH meters because they do not require setup or calibration, but they are less accurate.

Furthermore, they must cover the correct range of pH values expected in the soil; most test strips only cover a narrow measurement range. Digital meters can be purchased online or at tool supply stores for less than $20. Many devices are marketed as pH meters, but not all measure pH accurately. For the most reliable results, look for a device with a probe consisting of a glass orb and a small piece of metal at the end, similar to the item shown in figure 2. A new meter may be calibrated incorrectly, so follow the unit’s calibration instructions to improve accuracy before use. The calibration process may require gardeners to purchase an inexpensive buffer solution. The recommended technique for home gardeners to prepare a soil sample for a pH test is to collect a dry soil sample and add to it an equal amount of distilled water. Stir the mixture until it resembles a slurry and then let it set for 1 hour. Stir the mixture again and take the pH measurement .In many areas of California, particularly the drier regions, it may be necessary to increase the acidity of the soil—to lower the pH—to grow some popular garden crops. This is usually accomplished in gardens by adding elemental sulfur to the soil. Bagged sulfur products for acidifying soil can be purchased at garden centers and nurseries.Some plants, such as blueberries, are especially sensitive to high pH. A common symptom of plants grown in alkaline soil is yellowing of new growth, caused by a lack of available iron . This is called iron chlorosis. Correcting the soil pH usually solves the problem. In most cases, however, growing acid-loving plants in containers filled with a suitable soil mix is a better long-term solution than amending native, alkaline soil in a garden bed. See table 1 for suggested pH values for common plants.Table 2 shows how much elemental sulfur is needed to lower the soil pH to a desired level. Adding too much sulfur in one application can harm plants. Apply no more than 15 pounds per 1,000 square feet of soil in a single application. Wait 6 months, retest, and apply more sulfur if needed. Adjusting soil acidity for acid-loving plants is best performed prior to planting, when adjustments can be made quickly and plant damage is not as great a concern. Compost is sometimes recommended as an acidifying product for garden soils.

Compost can improve many aspects of soil health, including nutrient availability, but it probably will have little effect on soil pH when typical amounts are applied. Mature compost can range in pH from 6 to 8, so it is unlikely to lower soil pH into the range preferred by acid-loving plants. For an extensive overview of techniques for acidifying alkaline soils, see Locke et al. .While naturally acidic soils are rare in California, some fertilizers and amendments can, over time, cause soils to become acidic. But when naturally acidic soils do occur, as in some areas of California, it may be necessary to raise the pH of the soil to make it more alkaline. When soil pH needs to be increased in the home garden, the usual method is to amend the soil with pulverized limestone or dolomite. These materials react to neutralize the acid in the soil, much as an antacid works to relieve heartburn. Limestone and dolomite move into and react with soil very slowly. If these amendments are used to raise soil pH,collection pot the process should be begun before planting to allow time for the reaction to occur. The correct amount of limestone or dolomite to apply to a soil is difficult to determine without a laboratory soil test that characterizes the composition of the soil and identifies ions dissolved in the soil that can affect pH. The appropriate quantity of product to apply can be influenced by the amount of calcium in the soil, the soil texture , and other factors that are not easy to test at home. If a pH test indicates a need to raise the pH and a laboratory soil test cannot be obtained to determine the appropriate amount of limestone or dolomite, a good starting point is to attempt to raise the pH approximately 1 point by incorporating limestone or dolomite into soil as follows: for sand, incorporate 20 pounds per 1,000 square feet; for loam, 45 pounds per 1,000 square feet; for clay loam, 90 pounds per 1,000 square feet . Retest the pH value in 1 year to evaluate the results. As suggested above, these recommendations are less accurate than recommendations given by a laboratory because the amount of lime needed to decrease acidity depends on many factors beyond the pH value. The choice between using dolomite or limestone depends on the soil’s need for magnesium, an essential plant nutrient. California’s soils vary greatly in their magnesium content. If the decision is made to raise the soil pH, a product should be selected that matches plants’ nutrient needs. Dolomite should be used in soils low in magnesium. Limestone only provides calcium and is suitable for soils with ample magnesium. Without a laboratory soiltest, the only way to know if soil is deficient in magnesium or other elements is to identify plants’ nutrient deficiency symptoms. Gardeners are often tempted to apply wood ashes, hydrated lime, or other products to soil to alter the pH. While these amendments can be effective, they are easy to apply incorrectly and therefore should be avoided. A careful application of limestone or dolomite is less likely to cause unwanted effects in the garden.The growth of the “critical zone” paradigm has added impetus to closer investigation of soil-plant atmosphere interactions in ecohydrology . This follows from work emphasizing the importance of vegetation in regulating the global terrestrial hydrological cycle, with transpiration being the dominant “green water” flux to the atmosphere compared to evaporation from soils and canopy interception in most environments .

More locally, the role vegetation plays in partitioning precipitation into such “green water” fluxes and alternative “blue water” fluxes to groundwater and stream flow has increased interest in the feedbacks between vegetation growth and soil development in different geographical environments . The emerging consequences of climatic warming to changes in vegetation characteristics and the implications of land use alterations add further momentum to the need to understand where plants get their water from, and how water is partitioned and recycled in soil-plant systems . Stable isotopes in soil water and plant stem water have been invaluable tools in elucidating ecohydrological interactions over the past decade . Earlier work by Ehleringer and Dawson explained the isotope content of xylem water in trees in terms of potential plant water sources. Building on that, Brooks et al. showed that the isotope characteristics of xylem water did not always correspond to bulk soil water sources as plant xylem water was fractionated and offset relative to the global meteoric water line compared to mobile soil water, groundwater and stream flow signatures. This led to the “Two Water Worlds” hypothesis which speculated that plant water was drawn from a “pool” of water that was “ecohydrologically separated” from the sources of groundwater recharge and stream flow . Research at some sites has found similar patterns of ecohydrologic separation and suggested it may be a ubiquitous characteristic of plant-water systems . Others have found that differences between plant water and mobile water may be limited only to drier periods , or may be less evident in some soil-vegetation systems . Direct hypothesis testing of potential processes that may explain the difference between the isotopic composition of xylem water and that of potential water sources has been advanced by detailed experiments in controlled environments, often involving the use of Bayesian mixing models which assume all potential plant water sources have been sampled . However, as field data become increasingly available from critical zone studies, more exploratory, inferential approaches can be insightful in terms of quantifying the degree to which xylem water isotopes can or cannot be attributed to measured soil water sources .

“Sweet” or low acid pomegranates typically have been reported to have citric acid concentration less than 0.50% , but standards have not been established for low acid cultivars. Standards have been established for titratable acidity and total soluble solids of ‘Wonderful’ pomegranate. Generally, citric acid is the most abundant organic acid in pomegranate juice, so TA is expressed in citric acid equivalents. Minimum maturity of ‘Wonderful’ pomegranate fruit is 1.85% TA , so fruit are picked when fruit measure below that threshold. Maturity index is a measure of maturity in many fruit crops, including pomegranate, and is the ratio of °Brix to TA . The optimum MI for ‘Wonderful’ has been calculated to be greater than 8.1, at which point the fruit are ready for harvest. Other cultivars may have significantly different quantities of organic sugars and acids, so MI may be different for pomegranates harvested for different taste preferences and use which require fruit to be insipid, sweet, sweet-tart, tart, sour, bitter, etc. Fruit quality is not only related to sugar content, titratable acidity and spoilage, but also to phenolic compounds that lend to the fruit’s flavors, antioxidant activity, and colors . Cultivar is more influential in determining fruit juice composition than site of cultivation, year of harvest, or length of storage , so it is important to study differences among cultivars. In addition, it allows for a greater understanding of biodiversity of pomegranate germplasm. Knowing levels of phenolicsand the antioxidant activity of a cultivar’s juice is important to the beverage industry and consumers, because printed and televised commercials use antioxidant activity as the main selling point of their product. If any cultivar were to demonstrate a greater antioxidant activity than Wonderful,blueberry containers it would possibly be competitive in the pomegranate market or could be utilized by a breeder for increasing antioxidant activity. Having lower antioxidant activity than ‘Wonderful’ would make for an undesirable candidate for commercial production.

As previously mentioned, pomegranate germplasm in the United States is highly diverse and is comprised of hundreds of pomegranate cultivars sourced from domestic and international origins . Most cultivars are in the public domain with the potential to be developed into commercial cultivars. Stover and Mercure indicated that cultivars with softer seeds or lower acidity could increase consumer demand for the fruit. Wonderful, the industry standard in several countries, has relatively tart, bitter, acidic, moderately hard-seeded fruit and astringent juice compared to other cultivars previously analyzed from the USDA-ARS NCGR collection . Pomegranate cultivation in the United States is predominantly a monoculture of ‘Wonderful.’ The 14 cultivars included in the research presented herein have been described to have one or more of the following traits: unique color, soft seeds, low acidity, and/or unique flavor. Most of the cultivars in USDA germplasm have yet to be phenotyped for use by commercial growers, the food and beverage industries, and breeders. Recent developments in the California pomegranate industry have led to beverage companies predominantly desiring ‘Wonderful’ juice from growers for their products . This preference for ‘Wonderful’ juice is likely due to the clinical studies associated with this cultivar, such as the positive effect of ‘Wonderful’ juice used in conjunction with radiotherapy or surgery on men suffering from prostate cancer . Several methods exist to analyze the components and quality of fruit juices, including gas chromatography and liquid chromatography coupled with mass spectrometry, Fourier transform infrared spectroscopy , acid titration, refractometry , and spectrophotometry . All of these methods are useful, but they can be quite different in terms of sensitivity, selectivity and specificity and have disadvantages including sample destruction, incomplete sample separation in a chromatography column, and inability to profile specific quantities or classes of metabolites.

The ability of nuclear magnetic resonance to universally and quickly detect organic compounds makes it an efficacious method for the identification of differences in metabolic profile among fruit juices from different cultivars without prior separation of compounds. Additionally, NMR is advantageous because it requires relatively easy sample preparation, and allows compound identification and quantification without authentic standards . The NMR methodology has proven useful to the food and beverage industries for juice beverage quality control, including such applications as the detection of adulterants, quality control of fruit juice products during mixture analysis, and determination of juice authenticity . The pomegranate juice industry in the United States has experienced high profile lawsuits and federal investigations involving both false advertising of commercial juice composition and human health benefits from its consumption . Therefore, it is important to be able to detect beverage adulterants, such as sucrose and high fructose corn syrup, and distinguish between the juices of different fruits, vegetables and cultivars for economic, quality evaluation and public health reasons. Knowledge of a particular cultivar’s metabolic and physicochemical juice profile allows for a better understanding of pomegranate fruit juice quality, diversity of flavors and fruit maturity. For example, profiles of organic acids are known to affect flavor stability, nutrition and consumer acceptability . In regards to climate, heat has been shown to influence pomegranate fruit juice acidity. Groves planted in climates that experience hot summers typically result in fruit with reduced acidity and groves planted in climates with mild summers typically result in fruit with higher acidity . The amino acids glutamine and glutamate not only play very important physiological roles in plants, which include biochemical pathways related to amino acid synthesis, nitrogen metabolism, and ammonium detoxification, they are also important components of the human diet, with glutamine consumption becoming essential during major trauma, major surgery, sepsis, and radio- and chemotherapies .

Metabolic juice profiles might also be useful in determining fruit maturation and targeted harvest windows of pomegranate cultivars, which may be variable depending on climate and other agroecological factors. Additionally, pomegranate cultivars have differences in harvest dates, and this information is important for growers: 1) it allows them to pick fruit during harvest windows which afford the highest fruit quality for the fresh fruit, food and beverage industries; and 2) it allows growers to choose cultivars that have different maturity periods than Wonderful so they are not competing with Wonderful in domestic and international markets. Additionally, if growers start growing earlier cultivars, it could diversify the market and increase the length of the season during which high quality pomegranate fruit are available to both the food industry and consumer. The primary research objective of this dissertation was to evaluate a diverse set of preselected USDA-ARS pomegranate cultivars to determine which would meet and expand grower, consumer, and industry demands for the crop. This objective was met by: 1) conducting experiments to determine the capacity of cultivars to be propagated using conventional,best indoor plant pots inexpensive propagation methods; 2) evaluating the germplasm in field trials in hot inland and cool coastal climates to test for early orchard establishment rates, tree precocity and yield to determine commercial suitability and to aid in regional cultivar selection for growers; and 3) evaluating fruit and juice quality of the germplasm to compare important quality and nutritional traits to the industry standard ‘Wonderful.’ The pomegranate cultivars used in the following studies were: Ambrosia, Desertnyi, Eversweet, Golden Globe, Green Globe, Haku Botan, Ki Zakuro, Loffani, Nochi Shibori, Parfianka, Phoenicia, and Wonderful. All cultivars used in this experiment were located at the USDA NCGR, but the cultivars conserved in this national germplasm collection were originally sourced from different countries. ‘Ambrosia,’ ‘Eversweet,’ ‘Golden Globe,’ ‘Green Globe,’ ‘Loffani,’ ‘Phoenicia,’ and ‘Wonderful’ originated in the United States. ‘Desertnyi’ and ‘Parfianka’ originated in Turkmenistan. Haku Botan, Ki Zakuro, and Nochi Shibori originated in Japan, with Haku Botan a fruiting edible ornamental cultivar with no red pigment and Ki Zakuro and Nochi Shibori sterile cultivars, producing no fruit. All three Japanese cultivars have “double flowers” with up to hundreds of petals per flower and an upright growth habit.

Eversweet and Desertnyi have dwarf-like growth characteristics in the field, with trees typically being approximately 20% to 30% shorter or less vigorous than Wonderful as measured in the University of California, Riverside, pomegranate cultivar trials. Parfianka has a canopy with a bushy growth habit and is thornier than many other cultivars. There were no differences in bark characteristics. The first experiment included all above mentioned cultivars, and the second experiment consisted of the following cultivars: Ambrosia, Green Globe and Wonderful. Cuttings were sourced from the USDA NCGR in Winters, CA. Dormant stems were collected from basal suckers in February of two successive years for Experiments 1 and 2, respectively, placed in a plastic bag with a wet paper towel to keep them moist, driven to Davis, CA, boxed, and shipped via private delivery service from Davis, CA, to Riverside, CA, for second-day delivery. Upon arrival, the plant material was unwrapped and rew rapped with new moistened paper towels and stored at 5 to 6 °C in the dark for approximately 2 months before preparation of cuttings. All cuttings were sourced from one-year-old growth and mean stem diameter ranged from 3.8 to 5.8 mm among cultivars. Cuttings were 10.5 ± 1.0 cm long with a minimum of two nodes , treated with IBA or control, and inserted 3 to 5 cm deep in 2.5 cm x 2.5 cm plastic pots containing #4 Sunshine potting mix and perlite . Treatments were separated by block in plastic flats. Both experiments were conducted in a greenhouse at the University of California, Riverside , with mean temperatures of 24.1 °C and 26.3 °C for Experiment 1 and 2, respectively, and natural photoperiod. Cuttings were hand-watered with deionized water every day for 5 weeks and then watered as needed . Starting at week 10, the water source was switched to municipal water and all plants were treated with PlantexTM water soluble fertilizer, which contained N-P-K plus micronutrients, at a rate of 0.2 g·L-1 nitrogen. This experiment was conducted to determine if differences in rooting percentages and vegetative growth attributes exist among cultivars. The experiment ran from 11 March until 09 August of year 1 and consisted of a randomized complete block design with eight blocks. Each block included four hardwood stem cuttings of each cultivar, totaling 32 cuttings per cultivar. All cuttings were treated for 5 s to a depth of 2 to 4 cm in a gelatinous formulation of 3 g×L -1 IBA before insertion in the rooting medium. This experiment was conducted to test effects of IBA concentration on rooting percentages and vegetative growth attributes of Wonderful , Green Globe, and Ambrosia . Experiment 2 ran from 02 April until 28 December 2014 and used a RCBD with four blocks, each with four cuttings per cultivarIBA combination, totaling 48 cuttings for each of the three cultivars. IBA was applied in the same manner as in experiment 1, except that cuttings were dipped in either DI water or IBA at 1.5 g×L -1 or 3 g×L -1 IBA before insertion in potting mix. Attributes measured in both experiments included initial stem diameter, rooting percentage, plant height, number of shoots, and total shoot growth . Rooting percentages were based on cuttings that successfully rooted and developed into plants. Plant height, number of shoots, and total shoot growth were assessed at the end of each experiment: day 154 for Experiment 1 and day 270 for Experiment 2. Experiment 2 was conducted over a longer time to allow for assessment of growth after a full growing season. Plant height was measured using the top of the potting container as the reference point to determine if there were differences among cultivar phenotypes for upright versus drooping growth habit. Wonderful pomegranate grows in willowy tree form, with a tall, often thin, spreading and bending branching pattern but there are cultivars with upright growth habit, so this growth habit trait was assessed by measuring both apical shoot growth and plant height. Number of primary shoots represented a count of shoots growing from above ground, lateral buds on the cutting. Attributes measured at the end of Experiment 1 were apical shoot growth , branching , and relative chlorophyll content . SPAD values were used to assess plant health by quantifying leaf relative chlorophyll content using a SPAD-502 chlorophyll meter . Attributes measured at the end of Experiment 2 were total leaf area and root dry mass. Total leaf area was measured with a LI-COR Model 3100 Area Meter . Dry root mass was determined by removing the roots from the plant, drying the roots in a forced air oven to constant weight, and weighing the dried roots.

A recent comprehensive review paper on nutritional management of CKD by Kalantar-Zadeh and Fouque has suggested an intake of 4.7 g/day in the early stages of CKD without risk of hyperkalaemia, but a dietary potassium restriction of less than 3 g per day in CKD patients who tend to develop hyperkalaemia . A low potassium diet is defined as a dietary intake of 2–3 g/day as shown in Table 1.The majority of the regulation of potassium balance occurs at the renal level. Following a dietary potassium load, renal excretion increases after a few minutes reaching maximum levels after 2 h, thus preventing hyperkalaemia. This occurs by means of increased aldosterone production. Potassium secretion may also be facilitated by a recently postulated enteric sensor that reduces sodium reabsorption in the proximal tubule and facilitates potassium secretion by increased delivery of sodium to the distal tubule. Additional renal responses to potassium loading include reduced sodium reabsorption and increased potassium-channel conductance. In patients with advanced CKD the kidneys’ ability to adapt to increased potassium intake diminishes and in ESRD the renalmechanisms of potassium excretion often become negligible, making these patients extremely prone to hyperkalaemia. Dietary potassium is absorbed mostly in the duodenum and jejunum and the net intestinal potassium absorption is approximately 90%. Under physiologic circumstance faecal excretion is quite constant at about 10 mmol/day,blueberries in pots with a maximum level of 15–20 mmol/day. The capacity of the colon/rectum to secrete potassium is inversely related to residual kidney function and becomes the main route of potassium excretion in patients with ESRD. Potassium concentration in the faeces is very high , so that diarrhoea may lead to profound hypokalemia. Hence, it is conceivable that slow faecal transit time along the intestinal tract favours potassium absorption, whereas faster intestinal transit time reduces potassium absorption.

This suggests that constipation, instead of potassium dietary load, is the main determinant of hyperkalaemia in CKD and ESRD patients. Potassium is present in a large variety of foods, both from animal and plant sources. Potassium is found mainly intracellularly in animals, where it has a crucial role in determining the electric potential of cell membranes and then the excitability of nervous system and muscle cells. Hence it is not surprising that food rich in cells, such as meat or fish, are relevant sources of potassium. Indeed a recent study showed that high protein intake in maintenance dialysis patients has direct correlation with hyperkalaemia. In this study higher dietary potassium intake was associated with increased death risk in long-term haemodialysis patients, even after adjustments for serum potassium level; dietary protein; energy and phosphorus intake; and nutritional and inflammatory marker levels. The potential role of dietary potassium in the high mortality rate of dialysis patients warrants clinical trials. Notwithstanding the importance of protein intake, fruits and vegetables are supplying the majority of dietary potassium in most diets. Potassium is crucial for many processes in a plant’s life cycle, with its importance considered second only to nitrogen for plant growth and composition. Plants require potassium ions for protein synthesis, enzyme activation and maintenance of cation/anion balance in the cytosol. Potassium is also involved in the opening and closing of stomata regulating proton pumps; it plays an important role in photosynthesis and in photo-protection and it takes part in protein synthesis and in downward solute transport from the leaves. Potassium is important for crop yield as well as for the quality of edible parts of crops; its deficiency has a strong impact on plant metabolism. Plant responses to low potassium involve changes in the concentrations of many metabolites as well as alteration in the transcriptional levels of many genes and in the activity of many enzymes.

Potassium levels in plants are also associated with disease resistance through its effects on decreased cell permeability and decreased susceptibility to tissue penetration. When adequate levels of potassium are present a greater amount of silica is incorporated into the cell walls, strengthening the epidermal layer which represent a physical barrier to pathogens. Moreover, potassium seems to directly contribute to an adequate thickening of cell walls. Purely plant-based LPD may or may not lead to a more consistent potassium load than animal-based LPD. Therefore, in the case of a need for a protein restricted diet in advanced CKD, animal-based LPD could be favoured over vegetarian LPD, combined with educational strategies to reduce the effective potassium load and close monitoring of serum potassium level. However, such a strategy entails a therapeutic compromise, as it would abandon the additional cardiovascular benefits of a plant-based diet. Plant-based foods have favourable effects on systemic hypertension, on glomerular hemodynamics and perm-selectivity, leading to reduction of proteinuria. They supplylessbio-available phosphorus with favourable effect on the CKD mineral and bone disease and they are associated with effective renal protection probably by means of the reduced acid load. However, plant-based foods also supply a high content of fibres and alkali as well as anti-oxidant vitamins and trace elements. Of consequence, a lower net acid load and favourable effects on intestinal motility and microbiota are expected. Prevention or correction of metabolic acidosis and of constipation represent mechanisms that counteract the hyperkalaemia-inducing effects of high potassium intake, and may explain why vegetarian diets, more or less associated with a reduction in protein intake did not induce increase of serum potassium or overt hyperkalaemia in CKD patients. Similarly, during high-fruit intake, no changes in serum potassium levels were reported. Fibre intake has a major role in the modulation of intestinal microbiota, with high-fibre diets promoting the growth of bacteria with saccharolytic metabolism and lowering proteolytic-derived uremic toxins and also leading to faster bowel transit time.

Conversely, reduced bowel motility and constipation can induce dysbiosis of the intestinal microbiota, contributing to uremic intoxication and increase net absorption of potassium, leading to hyperkalaemia. Therefore, a high fibre content in the diet should be preserved even when the potassium intake is to be lowered. In clinical practice, a common dilemma in the management of advanced CKD patients with chronic hyperkalaemia is the patients’ deprivation of the beneficial effects of RAAS inhibitors or of the favourable effects of vegetarian diets in order to control hyperkalaemia. A solution may be the use of intestinal potassium binders. However, the capacity of intestinal potassium -binders to remove potassium is limited and they could be expensive in the long run. Therefore, a careful control of the dietary potassium load is in an important aspect of the management of CKD and heart failure patients with, or at risk of hyperkalaemia. In patients with stage 4–5 NDD CKD and ESRD, dietary potassium management also has to be synchronized with additional nutritional goals, namely the amount of protein intake , high fibre intake, reduced net fixed acid production and cardiovascular effects and the favouring of a heart-healthy diet. Another method of food selection may be based on the potassium content normalized for unit of fibre, namely the reporting of the potassium content of vegetables and fruits also as “mg per 1 g fibre”. Foods with low potassium to fibre ratio may be allowed whereas foods with very high potassium to fibre ratio should be avoided . Similarly, since protein intake must be increased in haemodialysis and peritoneal dialysis patients,square plant pots food selection should be addressed to reduce potassium intake without reducing dietary protein intake. Hence, reporting potassium intake per unit of protein may be another method that can make it easier to limit the intake of foods that supply more potassium for the same protein intake in ESRD patients. Additional aspects useful to limit effective potassium intake is education about the use of cooking procedures in order to obtain food demineralization: boiling is able to remove up to 60–80% of the potassium content of several raw foods Jones analyzed the mineral content of a wide variety of foods after different processing procedures. Food samples were subjected to aqueous mineral extraction after a pre-treatment which was different depending on the food group . They were then exposed to different water temperatures and time depending on cell type and the initial state : for example, vegetables were placed in 2 liters of hot tap water , stirred vigorously for 15–20 s and allowed to stand for a predetermined time period. Ham and hot dogs were placed in boiling water bath, stirred and allowed to boil for 3 min. Avocado and banana from the fruit group were placed in cold tap water, stirred gently and allowed to stand for the predetermined time period.

The reduction range for potassium was 59% ± 40% for vegetables, 78.5% ± 20.5% for legumes, 57% ± 41% for meats, 94% ± 3% for flours, 99% for cheddar cheese and 43% ± 16% for fruits.Burrows and Ramer confirmed these results, finding that soaking was not effective in the leaching of significant amounts of potassium from tuberous root vegetables while the double cooking method leached more potassium than did the normal cooking method. Similar results were found also by Aiimwe et al. who showed that soaking did not change potassium content in a particular type of banana while boiling at 200 ◦C reduced potassium concentration from 1.4 ppm to 1 ppm after 60 min. Poor results with soaking were found also by Picq et al. with various types of foods. Preparation of food seems to be important: boiling potatoes after cubing or shredding results in a much greater loss of potassium. These procedures are generally considered “negatives” as they can affect food nutritional properties, taste and appearance but giving patients appropriate instructions on how to process food after boiling this obstacle can be overcome with the advantage that many restricted foods become permissible.Finally, attention should be paid to hidden sources of potassium, such as salt substitutes and certain food additives. The former contain potassium instead of sodium and are usually recommended in hypertensive patients to reduce sodium intake and to increase potassium intake. However, in the case of treatment with RAASi and/or in patients with reduced renal function the risk of hyperkalaemia from these agents may be considerable. Two categories of salt substitutes are available on the market: low-sodium salts and sodium-free salts. In the low-sodium salt the sodium chloride content must not exceed 35% and cannot be less than 20% with a potassium: sodium ratio of at least 1.5:1. In the sodium-free salts potassium content ranges between 20% and 30% and sodium content is fixed at a maximum value of 0.12%. Hence, for instance, 5–6 g of a current salt substitute can supply from 1000–1200 mg to 1500–1800 mg of extra-potassium. This represents a significant potassium load on top of the potassium derived from food intake, which is of concern in patients with reduced capacity of potassium excretion. Special attention should be paid to the use of these salt substitutes because people believe that they are safer than regular salt and they tend to use greater amounts of them. The effect of potassium-based additives on potassium burden is not widely recognized, with limited literature. Sherman and Mehta found that potassium content in foods with additives varied widely and that uncooked, enhanced meat and poultry products had potassium levels up to threefold greater than similar unenhanced food products. The use of additives in packaged poultry, fish or meat foods can increase the effective dietary potassium load and of special concerns in patients with CKD, are sodium-reduced products. For instance, additive-free products had an average potassium content <387 mg/100 g, whereas five of the 25 products with additives analyzed in that investigation contained at least 692 mg/100 g with a maximum of 930 mg/100 g. Table 5 reports the most frequently used potassium-based additives and their acceptable daily intake. A temporary ADI of 3 mg/kg is currently established, while an ADI of 25 mg/kg for potassium sorbate is under evaluation. Paying attention to the food labels may be useful although the quantity of potassium added as an additive is not generally available.Nutritional therapy in CKD is very complex, as it has to consider concomitantly the intake of protein, energy, sodium, phosphorus and potassium. Individualized nutritional education programs and regular counselling are all important aspects of clinical management, which also look to improve patients’ lifestyle.

Some of the pitfalls of this assumption are highlighted in Figure 5.2a. Consider a scenario where we want to compare samples 1-3. An analysis schema that does not account for the chemical relationships among the molecules in these samples , will assume that the sugars in samples 2 and 3 are as chemically related to the lipids in sample 1 as they are to each other. This would lead to the naive conclusion that samples 1 and 2, and samples 2 and 3 are equally distinct, yet they are not from a chemical perspective. On the other hand, if we account for the fact that sugar molecules are more chemically related to one another than they are to lipids, we can obtain a chemically-informed sample-to-sample comparison. Sedio and coworkers developed the chemical structural compositional similarity metric to account for relationships between molecules based on the similarity of their fragmentation spectra. While CSCS compares samples based on modified cosine scores obtained from molecular networking, we calculate chemical relationships based on structurally-informed molecular fingerprints. We express these relationships in the form of a hierarchy which enables the use of other tree-based tools for downstream data analyses. For example, in Figure 5.2a, we show that by using a tree of structural relationships between molecules in samples 1-3, we can apply UniFrac , a tree-informed distance metric and demonstrate that the composition of sample 1 is distinct from samples 2 and 3.The importance of comparing samples by accounting for their molecular relatedness is highlighted when we contrast the results from ignoring the tree structure to those which integrate it . With the structural context provided by Qemistree,square planter pots the differences between replicates across batches are comparable to the within-batch differences .

The retention time shift in this dataset leads to a strong technical signal that obscures the biological relationships among the samples pseudo-F=120.75, p=0.001 vs. tree informed pseudo-F=18.2239, p=0.001. We observed and remediated a similar pattern originating from plate-to-plate variation in a recently published study investigating the metabolome and microbiome of captive cheetahs . In this study, placing the molecules in a tree using Qemistree reduced the observed technical variation , and highlighted the dietary effect that was expected . These results show how systematic and spurious molecular differences can be mitigated in an unsupervised manner using chemically-informed distance measures based on a tree structure. As a case study, we used Qemistree to explore chemical diversity in a set of food samples collected as a part of the Global FoodOmics initiative . We selected a diverse range of food ingredients to represent animal, plant, and fungal groupings. We first performed feature-based molecular networking using MZmine to obtain spectral library matches for a subset of the chemical features . Understanding the chemical relationships between different foods is challenging because most molecules within foods are unannotated. Using Qemistree, we collated GNPS spectral library matches and in silico predictions from CSI:FingerID to annotate ~91% of the chemical features with molecular structures. Using ClassyFire , we assigned a chemical taxonomy to 60% of these structures; the remaining 40% returned no ClassyFire taxonomy. Labeling annotations allowed us to retrieve subtrees of distinct chemical classes such as flavonoids, alkaloids, phospholipids, acyl-carnitines, and O-glycosyl compounds in food products. We propagated ClassyFire annotations of chemical features to each internal node of the tree and labeled the nodes by pie charts depicting the distribution in chemical superclasses and classes of its tips. The molecular fingerprint-based hierarchy of chemical features agreed well with ClassyFire taxonomy assignment, further demonstrating that molecular fingerprints can meaningfully capture structural relationships among molecules in a hierarchical manner.

Furthermore, Qemistree coupled the chemical tree to sample metadata, revealing distinct chemical classes expected for each sample type. In contrast, honey, although categorized as an animal product, shared most of its chemical space with plant products, reflective of the plant nectar and pollen-based diet of honey bees. We observed a clade of flavonoids in both plant products and honey , but no other animal-based foods.While it is expected that a complex food such as blueberry kefir contains molecules from both blueberries and dairy, we can now visualize how individual ingredients and food preparation contribute to the chemical composition of complex foods. We noted that metabolite signatures that stem directly from particular ingredients, such as phosphoethanolamine from eggs, are present in egg scramble , but not in the other two foods highlighted . We can also observe the addition of ingredients in foods that were not listed as present in the initial set of ingredients. We were able to retrieve that there is black pepper in the egg scramble with chorizo and orange chicken, but that this signal is absent from the blueberry kefir . We show that our tree-based approach coherently captures chemical ontologies and relationships among molecules and samples in various publicly available datasets. Qemistree depends on representing chemical features as molecular fingerprints, and shares limitations with the underlying fingerprint prediction tool CSI:FingerID. For example, fingerprint prediction depends on the quality and coverage of MS/MS spectral databases available for training the predictive models, and these will improve as databases are enriched with more compound classes. Qemistree is also applicable in negative ionization mode; however, less molecular fingerprints can be confidently predicted due to less publicly available reference spectra, resulting in less extensive trees. In summary, we introduce a new tree-based approach for computing and representing chemical features detected in untargeted metabolomics studies. A hierarchy enables us to leverage existing tree-based tools, and can be augmented with structural and environmental annotations, greatly facilitating analysis and interpretation.

We anticipate that Qemistree, as a data organization strategy, will be broadly applicable across fields that perform global chemical analysis, from medicine to environmental microbiology to food science, and well beyond the examples shown here.We use SIRIUS , ZODIAC and CSI:FingerID to predict molecular substructures within mass spectrometry features in the MGF files imported as Mass Spectrometry Features. SIRIUS computes fragmentation trees for each molecular formula candidate of a feature and ranks these by score. SIRIUS uses MS1 spectrum in the MGF file to determine the candidate ion adduct to be used for the fragmentation tree computation of each feature. ZODIAC takes the top SIRIUS candidates as input and re-ranks molecular formula candidates considering reciprocal compound similarities in the dataset to increase correct molecular formula assignments. Subsequently, CSI:FingerID predicts molecular fingerprints for each feature based on the molecular formula with the highest ZODIAC score. Note that all spectra provided to the Qemistree pipeline do not necessarily produce a fingerprint. Indeed, SIRIUS does not compute fragmentation trees for multiply charged compounds and CSI:FingerID does not predict molecular fingerprints from spectra with less than 3 explained peaks. To ensure that high confidence molecular formulas are used in Qemistree, we only consider compounds with a ZODIAC score above 0.98 .Samples were analyzed using ultra high performance liquid chromatography coupled to a quadrupole-Orbitrap mass spectrometer . The quadrupole Orbitrap mass spectrometer was fitted with an electrospray source operating in positive ionisation mode. The source used the following parameters: spray voltage, +3500 V; heater temperature, 437.5°C; capillary temperature, 268.75°C; S-lens RF, 50 ; sheath gas flow rate, 52.5 ; and auxiliary gas flow rate, 13.75 . The samples were acquired in non-targeted MS2 acquisition mode,growing blackberries in containers with up to four MS2 scans of the most abundant ions per MS1 scan. The spectra were recorded from 0.48 to 17 min. The following parameters were used for full MS scan: resolution , Automatic Gain Control target , maximum injection time , scan range . For the datadependent in MS2 , the following parameters were used: resolution , AGC target , maximum injection time , loop count , isolation window fixed first mass . and up to four MS/MS scans of the most abundant ions per duty cycle. Higher energy collision induced dissociation was performed with a normalized collision energy of 30 . The data-dependent settings were set as follows: minimum AGC , apex trigger 3 to 15 s, charge exclusion 3-8 and > 8, exclude isotopes , dynamic exclusion .

Samples were collected, extracted, and MS data were acquired as a part of the Global FoodOmics project according to the sampling and data acquisition protocols described in Gauglitz et al., 2020 Food Chemistry. Briefly, 126 food samples were selected from the Global FoodOmics dataset. 119 simple food samples were selected to cover a broad spectrum of fruits, vegetables, meat and fungi. Each food was represented in at least triplicate in the data subset. Additionally 7 complex samples were selected that contained simple foods from the simple food subset in their ingredient lists. The complex foods were from two separate meals of orange chicken, a cooked cucumber and the sauce from a meal , sour cream, blueberry kefir, and egg scramble with chorizo. Sample metadata describes the food samples based on a food hierarchy beginning with plant vs. animal vs. fungus and increasing in detail down to persian cucumber vs. cherry tomato etc. . Briefly, samples were extracted in 95% LC-MS grade Ethanol; 5% LC-MS grade water. Samples were analyzed using the same LC-MS/MS setup and software as described above for the maXis II QTOF mass spectrometer , using a Phenomenex Kinetex C18 1.7 µm 100 x 2.1 column equipped with a guard cartridge . The instrument tuning and internal calibrant remained the same as described above. MS spectra were acquired in a positive ion mode in the range m/z 50–1,500. The mobile phases consisted of A and B , and the flow rate was set to 0.5 µL/min throughout the experiment, and the column maintained at 40℃.AT conceived the concept and managed the project. AT and YVB developed the algorithm and wrote the code for Qemistree. AT and YVB contributed equally to the work. LFN, RK, PCD supervised method implementation. KD, MW, JJJvdH, ME, DM, and AG tested and provided suggestions on how to improve the method. MW managed the deployment of Qemistree on GNPS. AT and MW developed the GNPS-Qemistree Dashboard. DA and AT wrote the documentation for the GNPS-Qemistree workflow. YVB, QZ, and AT developed Qemistree-iTOL visualization. LFN and MNE performed the mass-spectrometry for the evaluation dataset. AT, YVB, and LFN analyzed and interpreted the evaluation data. JMG performed mass spectrometry of the Global Foodomics samples. AT, JMG analyzed and interpreted the Global Foodomics data. KD, MF, ML, and SB supported the integration of SIRIUS, Zodiac, and CSI:FingerID. AT, YVB, PCD, and RK wrote the manuscript. LFN, JMG, MNE, JJJvdH, ME, KD, QZ, DM, AG, JH, MF, ML, SB, and RK improved the manuscript. The co-authors listed above supervised or provided support for the research and have given permission for the inclusion of the work in this dissertation.Flavor chemicals in electronic cigarette fluids , which may negatively impact human health, have been studied in a limited number of countries/locations. To gain an understanding of how the composition and concentrations of flavor chemicals in ECs are influenced by product sale location, we evaluated refill fluids manufactured by one company and purchased worldwide. Flavor chemicals were identified and quantified using gas chromatography-mass spectrometry . We then screened the fluids for their effects on cytotoxicity and proliferation and tested authentic standards of specific flavor chemicals to identify those that were cytotoxic at concentrations found in refill fluids. One hundred twenty-six flavor chemicals were detected in 103 bottles of refill fluid, and their number per/bottle ranged from 1 – 50 based on our target list. Two products had none of the flavor chemicals on our target list, nor did they have any non-targeted flavor chemicals. Twenty-eight flavor chemicals were present at concentrations ≥ 1 mg/mL in at least one product, and 6 of these were present at concentrations ≥ 10 mg/mL. The total flavor chemical concentration was ≥ 1 mg/mL in 70% of the refill fluids and ≥ 10 mg/mL in 26%. For sub-brand duplicate bottles purchased in different countries, flavor chemical concentrations were similar and induced similar responses in the in vitro assays . The levels of furaneol, benzyl alcohol, ethyl maltol, ethyl vanillin, corylone, and vanillin were significantly correlated with cytotoxicity. The margin of exposure calculations showed that pulegone and estragole levels were high enough in some products to present a non-trivial calculated risk for cancer.

Much of our knowledge of the human microbiome comes from association studies that use either a cross-sectional or case–control design. Well-designed case–control studies are critical to demonstrate a potential relationship between microbes and a disease of interest. However, these studies cannot establish causality, and are often subject to confounding variables such as differences in diet or medication between cases and controls. Most studies are conducted at a single time point in a population with the disease, and no long-term follow-up is performed. Consequently, these studies can only identify microbes that differentiate individuals with the disease and the control population. Although these microbes identified might have been causative agents, it is nearly impossible to separate this association from secondary effects associated with the condition. For example, medication plays a major partin shaping the microbiome; a study of patients with type II diabetes mellitus found that treatment with metformin had a larger effect on the microbiome than the disease . Similarly, we hypothesize the physiology of the disease might also contribute to changes in community structure. Association studies are also often confounded by the selection of poor controls. The microbiome is dynamic , and cumulative exposures over an individual’s life, shaped by their diet , lifestyle , medical history , genetics and other factors create a unique community. Thus, if cases and controls are not correctly selected,blueberry container association studies might detect differences due to confounding factors. Matching cases and controls based on age and sex is often not sufficient. In cases in which this matching to control for confounding variables is not possible, it is critically important to collect information about potential confounding factors.

Comparisons across current cross-sectional studies are also challenging due to large effects caused by inter-study differences in technical parameters, including sample collection, storage, primer selection and analysis techniques . Differences across studies increase the challenge of meta-analysis and make identifying causative clades more difficult . Some of these problems can be ameliorated by using consistent methodology between . Efforts like the Microbiome Quality Control Project are exploring sources of technical variation , while analysis platforms like Qiita provide a database of consistently annotated studies for comparison.Twin studies provide a potential antidote to some of the problems with association studies. Twin pairs are naturally controlled for age and some early life exposures. Monozygotic twin pairs also share the same genetic background, further limiting potential confounders . Twin studies can be leveraged in two ways. First, identifying differences between discordant and concordant twin pairs represent more powerful association studies, due to the partial internal control. Although these studies are particularly useful in young children due to shared environment, the approach can also be used with adults . Second, twin studies are critical to examine genetic control of the microbiome. A study published in 2016 of the UK Twins cohort suggested strong association of the microbiome and genes, including those associated with dietary preference and serum lipids . Twin studies provide a unique opportunity to assess if the familial risk factors are either genetic or environmental in nature. These studies have been applied to study heritability for studying hepatic steatosis and fibrosis now that advanced magnetic resonance imaging based assessment can be used to phenotype individuals . However, the sample size requirements for microbiome assessment in twins is large compared to the sample sizes heeded to study heritability may making recruitment for such a study challenging .

Twin studies may not be appropriate for other rare causes of liver diseases e.g. alpha-1 antitrypsin deficiency, cirrhosis, primary biliary cholangitis, and for such low prevalence diseases a trio family design would be more appropriate and would provide the highest power with the most efficient study design to detect association of a trait such as the role of microbiome on the risk of liver diseases .As the cost of microbiome analysis decreases, longitudinal studies are becoming more common. Understanding temporal fluctuation in the microbiome, and the role of microbes in contributing to disease etiology, will rely on studies over time. Work suggests that community instability might, in and of itself, be a characteristic of an unhealthy ecosystem . Prospective studies, such as an investigation examining death from HCC in individuals with NAFLD, have helped identify the role of exposures and etiological factors in contributing to disease outcomes . Currently, the appropriate sampling frequency for understanding the microbiome in prospective studies is unknown, in part due to an overall lack of long term follow up with microbiome studies. Initially, sample collection during standard clinic visits may provide information about the population-scale changes in the population. However, incorporating microbiome samples into these long-term studies will help examine the role of microbial communities—either at a single time point or the community dynamics—as a contributing factor to complex conditions .Model animals also have an important role in shaping our understanding of the microbiome in disease . Although rodent microbial communities are distinct from the human microbiota, there are some shared physiological and microbial traits . Both rodent and human communities are dominated by the same set of bacterial phyla, although a smaller percentage of genera are shared .

As such, experimental findings implicating individual organisms or genera in rodents should be taken with caution until they are validated in humans. Instead, rodent models can show phenotypic consequences of microbiome manipulation. This aspect makes rodent models a useful model system to investigate causality, explore interactions and test early interventions. Both antibiotics and probiotics have been used to study the effect of changing the conventional mouse microbiome on a phenotypic outcome. Broad spectrum antibiotics decrease the total bacterial load, as well as causing major perturbations in the microbial communities . In some cases, such as in liver disease models, this approach can demonstrate the role of bacterial products like LPS in modulating inflammation . In other cases, like a reported addiction model, it can be used to demonstrate the importance of an intact microbiome in regulating behavior . Probiotics can also be used to investigate the effect of a specific bacteria or bacterial cocktail within a controlled environment. A study of alcoholic fatty liver disease demonstrated an attenuation of the microbiome-mediated inflammation when a probiotic was used . Gnotobiotic or germ-free mice can be used in multiple contexts. Comparisons of specific pathogen-free laboratory mice and germ-free mice can be used to examine the role of the microbiome in modulating an expressed or induced phenotype . More importantly, gnotobiotic mice can be humanized with a donor’s stool. This approach creates a system in which an individual’s microbiota can be tested, either for its ability to modulate a disease phenotype or as a target for intervention . For instance, in a small study, mice received their microbiome from a donor with either severe alcoholic hepatitis or no liver disease. Following alcohol treatment, the mice with the microbiome from the patient with alcoholic hepatitis showed greater liver damage than mice that received stool from the healthy donor . Well-designed mouse models that combine our current understanding of liver disease with humanized microbiomes offer some of the greatest potential for preclinical interventions. Avatar, or sometimes called patient-derived xenograft mice, are widely used in the cancer community to test the efficacy of chemotherapeutics for individual tumors, including HCC . This model better re-capitulates the complexity of a tumor than cell culture. Avatar mice can be further personalized by introducing a human immune system into an immuno compromised mouse, along with the tumor . Generating this model in germ-free mice with a humanized microbiome and immune system expands our capacity to understand the role of the microbiome in modulating cancer. For example,growing blueberries in containers this model could be used to study whether the microbiome of a patient with ALD leads to more tumor growth than the microbiome from a healthy individual as control. An accumulating body of research suggests that the disparate observations in liver-disease related studies can be unified and explained by the microbiome. It is now widely accepted that liver damage can result from extensive interplay between gut microbiota via specialized molecules and host-immune system via Kupffer-cell-mediated liver inflammation. However, a comprehensive understanding of the interactions between the microbiome and the liver still evades us. Animal models, particularly rodents, have been instrumental in elucidating many important mechanistic pathways in liver disease etiology. The introduction of the microbiome into these models will provide a more complete view of the cancer ecosystem. Because microbiome research is sensitive to technical variability that often masks underlying biological signals, there is a need for consistency in technical platforms and standardized protocols, so that findings from different laboratories can be replicated and validated. Additionally, it is critical to use an animal model that mimics human disease as closely as possible in all its physiological and metabolic manifestations. We are slowly advancing from observation-based studies in humans as research establishes grounds for microbiome-based therapeutic modalities such as FMT and probiotic interventions. However, effectively translating and applying findings accrued through animal models to humans requires well-designed, large-scale clinical trials spanning multiple disease etiologies and patient characteristics.

As the role of microbiota in liver disease development, prognosis and treatment is increasingly recognized, we emphasize the need for focused, microbiome-aware efforts to efficiently tackle the socioeconomic burden of this spectrum of liver diseases. The authors would like to thank D. McDonald, T. Kościółek, Z. Xu and A. Plymoth for their helpful discussions. M.K. is supported by the NIH grants R01 AI043477 and R01 CA118165. R.L. is supported in part by the grant R01-DK106419-03. Research reported in this publication was supported in part by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number P42ES010337. B.S. is supported by NIH grants R01 AA020703, U01 AA021856, U01AA24726, and by Award Number I01BX002213 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development. J.D. is supported by the Robert Wood Johnson Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Primary biliary cholangitis is characterized by inflammation-mediated damage to the small bile ducts inside the liver, which gradually progresses to liver fibrosis and cirrhosis . Previously considered a typical autoimmune disorder, the modified etiological understanding of PBC considers proinflammatory changes in the gut-microbiota, intestinal bile acid disruptions and gut-barrier dysfunction . Consequently, microbe-associated molecular patterns ascend the biliary duct, perpetuating infection. An immune attack against the biliary epithelial cells is mediated by antibodies that recognize E2 subunit of pyruvate dehydrogenase complex due to cross-reactivity with conserved proteins in Escherichia coli, Lactobacillus delbrueckiiand Novosphingobium aromaticivoransIn fact, genetically susceptible mouse strains developed liver lesions mimicking PBC when infected with Novosphingobium aromaticivoransm, which further implicates a role for the microbiome in this disease . Ursodeoxycholic acid, a tertiary bile acid produced by Ruminococcus, has been approved for PBC treatment . Thus, microbiome-based treatment modalities hold promise for managing PBC and should be studied further. Primary sclerosing cholangitis is also an immune-mediated disease of the bile ducts . However, unlike PBC, PSC can affect bile ducts, both inside and outside of the liver. Gut dysbiosis-mediated bile dysregulation, intestinal permeability and translocation of proinflammatory molecules in the portal vein characterizes PSC . The immune reaction in PSC is mediated by autoantibodies, including perinuclear antineutrophil cytoplasmic antibody, that recognize the ubiquitously expressed bacterial antigen FtsZ . Furthermore, increase in microbe-associated Toll-like receptor expression and T helper type 17 cells has been reported in PSC, which strongly suggests microbiome involvement in disease pathogenesis . PSC is closely associated with IBD , in particular ulcerative colitis and shares some of its characteristic features . Thus, a common disease mechanism might be at play, and novel treatment avenues by targeting microbe associated immune pathways can be explored.Limited data exist concerning the diagnostic accuracy of gut-microbiome-derived signatures for detecting NAFLD-cirrhosis. Here we report 16S gut-microbiome compositions of 203 uniquely well-characterized participants from a prospective twin and family cohort, including 98 probands encompassing the entire spectrum of NAFLD and 105 of their first-degree relatives , assessed by advanced magnetic-resonanceimaging . We show strong familial correlation of gut-microbiome profiles, driven by shared housing.

Table 3.1 summarized the diet composition. During 4 weeks of feeding, fresh food was provided to hamsters weekly. Their body weight and food intake were recorded weekly. Food efficacy, known as the weight gain per gram of diet consumption was calculated. At the end of the 4th week, hamsters were fasted for 14 h, anesthetized using isoflurane , and sacrificed based on the procedures in the previous study . 5 mL blood was drained by cardiac puncture into EDTA rinsed syringes, then centrifuged at 2000 g, 4 °C for 15 mins to collect plasma and stored at -80 ⁰C. The liver, kidney, and epididymal adipose were excised, weighed, and frozen in liquid nitrogen then stored at -80⁰C. Feces were collected during the last day of feeding and stored at -20 ⁰C. Plasma lipoproteins, including very-low-density lipoprotein -, low-density lipoprotein -, and high-density lipoprotein – cholesterol, were determined using size-exclusion chromatography as described . Specifically, A cholesterol reagent was delivered using Hewlett-Packard HPLC pump 79851-A at a flow rate of 0.2 mL/min. Lipoproteins were separated by injecting 15 μL of plasma onto the Superose 6HR HPLC column in Agilent 1100 HPLC chromatography system. Then the lipoproteins were eluted with a solution containing 0.15 M NaCl and 0.02% sodium azide at a flow rate of 0.5 mL/min. Base signals of the peak areas were calibrated by applying bovine cholesterol lipoprotein standards . Total cholesterols in plasma were counted as the sum of the VLDL-, LDL-, and HDL-cholesterol concentrations, interpolating from the standard curve. Plasma triglyceride was quantified using an enzyme colorimetric assay kit based on the absorbance at 505 nm . The liver and feces samples were ground in mortar and pestle, snap clamps for greenhouse then the lipid was extracted with hexane/isopropanol at 60 °C using Dionex ASE 350 accelerated solvent extractor . The lipid extracts were evaporated to dryness at 37 °C under nitrogen.

Total hepatic and fecal lipid contents were calculated based on the weighed extracted lipid over liver weight. Hepatic lipids were re-dissolved in hexane/isopropanol solvent and mixed with Triton X-100. After drying and diluting the mixture in DI water, total cholesterol and free cholesterol were determined with enzymatic kits , then esterified cholesterol was calculated based on the difference between TC and FC. Liver TG was measured using the same method for plasma TG.The body weight gains over 4-weeks of feeding were shown in Figure 3.1. All the diets elevated the total weight gain ranging from 18.14 to 33.82g, in order of HP, HF, HE, LP, and LE . The corresponding feed efficacy ratio was from 0.11 to 0.18 g/g. In both total weight gain and feed efficacy ratio, the HP diet was significantly lower while the LE diet was significantly higher compared to HF and HP diets . Possible reasons could be that the high fiber content in peel promoted earlier satiety , reduced the digestion and absorption of lipid content. At the same time, liquid extract in LE and HE diets tended to dissolve more reducing sugars from the pomegranate peel. Therefore, it promoted absorption efficiency and further weight gain. Sangüesa et al investigated the different adverse effects of glucose and fructose on the metabolism of female rats and observed that fructose had a greater impact on metabolic dysfunction . The carbohydrate composition of the extract and peel could be examined in the future for a better understanding of the mechanisms. As unusual LDL-elevating effects in plasma after PPP and PPE ingestion were observed, real-time qPCR was applied to investigate the potential mechanisms of diets modulating the lipid and cholesterol metabolism. As shown in Figure 3.4, the expression level of CYP7A1 , a gene that controls bile acid synthesis rate from cholesterol, was increased by 2.1-folds in the HP diet, while that was reduced to 0.86-, 0.91- and 0.90- fold in LP, LE, and HE diets. HMG-CoAR and Cyp51 are two important genes in cholesterol biosynthesis. Compared with the control diet, LP, HP, LE, HE demonstrated the same up-regulating pattern in these two genes — 1.03-, 1.51-, 1.11- and 1.18-folds for HMG-CoAR , as well as 1.18-, 1.72-, 1.25- and 1.22-folds for Cyp51 , respectively. LDL receptor facilitates the hepatic LDL uptake from circulation.

In the present study, LP and HP diets up-regulated LDLR expression by 1.18- and 1.38-folds , while that was slightly down-regulated by 0.95-fold in both LE and HE diets. These findings were in line with hepatic LDL cholesterol — all the reformulated diets prompted hepatic cholesterol synthesis. HP diet increased bile acid synthesis while other diets decreased it. Peel-formulated diets elevated LDL uptake and extract-formulated ones alleviated it. For peel-formulated diets, bile acid synthesis was dominating in lowering hepatic cholesterol, whereas for extract formulated diets it lowered LDL uptake. PPARα was an essential transcription factor regulating fatty acid β-oxidation. It was up-regulated in LP and HP diet-fed hamsters by 1.04- and 1.61- folds. In contrast, a lower expression of 0.90- and 0.93-fold of PPARα was observed in LE and HE diets . SREBP-1c is a gene encoding transcription factor for fatty acid synthesis . It targets SCD-1 to catalyze the synthesis of monounsaturated fatty acids, which is a substrate for TG synthesis and storage. LP diet slightly up-regulated the expression of SREBP-1c by 1.07-folds, while HP, LE, and HE diet down regulated that by 0.74-, 0.85- and 0.92-fold . All the diets significantly reduced SCD-1 expression level by 0.42-, 0.24-, 0.73- and 0.61-fold . These results indicated that all the formulated diets induced lower uptake of fatty acids. Peel-enhanced diet slightly promoted fatty acid β-oxidation in a dose-dependent manner, while extract-enhanced diet mitigated that, which was aligned with liver TG levels and hepatic lipid contents. To identify the microbiota-changing effects of different diets on lipid metabolism, relative abundance was assessed at the phylum level . Human gut microbiota is generally dominated by the bacterial phyla Bacteroidetes and Firmicutes . Besides these, Proteobacteria is considered correlated for the variation of the functionality of gut microbiota . Compared to the control HF diet, HE and HP diets reduced the relative abundance of Firmicutes by 9.33% and 18.3% while increasing the RA of Bacteroidetes by 43.1% and 41.9% and nearly triplicated the RA of Proteobacteria.

The corresponding ratio of Firmicutes/ Bacteroidetes dropped by 39.4% and 42.4%, with an increase of Proteobacteria/ Bacteroidetes ratio by 89.6% and 105.1%, indicating that a shift of fecal microbiota towards leaner phenotypes. Verrucomicrobia was boosted to 4.0% and 8.5% for HP and HE diets, which was non-detectable for the HF diet. Similar observations of Verrucomicrobia increase were found in formulated diets with PPE , cranberry , and black raspberry , which could be attributed to the abundant polyphenol content. At the same time, Cyanobacteria was elevated from 0.3% in the HF diet to 2.3% in HE and shown of associated with improved gut health . To the best of our knowledge, no other research reported the change of Verrucomicrobia and Cyanobacteria after PPP incorporation in hypolipidemic diets. Further metagenomics studies are required to understand how Verrucomicrobia and Cyanobacteria modulate the lipid metabolism pathways. As shown in Figure 3.6, the expression of hepatic HMG-CoAR was significantly correlated with obesity-related indices, with a positive correlation with plasma concentrations of the total- and LDL-cholesterol , and a negative correlation with liver , adipose and body weight . The expression of LDLR also exhibited a significant positive correlation with total cholesterol. This was consistent with previous research from Teh et al. , who concluded that HMG-GoAR and LDLR were the two major regulating factors in meditating hypo cholesterol effects of hamsters fed with fruit and vegetable seed meals. As for types of bacteria, total plasma cholesterol exhibited a significant positive correlation with the phylum Bacteroidetes,greenhouse snap on clamp and a significant negative correlation with the F/B ratio in response, suggesting that the increase of Bacteroidetes attributed to the decrease of F/B ratio and played a role in elevating total cholesterol level. Pomegranate peel, a commonly underutilized by-product with high phenolic and fiber content, was incorporated into the hypolipidemic diet in the form of powder and extract to investigate its hypoalipidemic potential. PPP and PPE containing a rich mixture of phytonutrients demonstrated sufficient effects to suppress weight gain, hepatic lipid profile and ameliorate the symptoms of metabolic syndrome in Golden Lakeview Golden Syrian hamsters with HF diet-induced obesity. These observations can be at least partially explained by hepatic metabolism changes and changes in gut microbiota composition. In this study, PPP and PPE lowered Firmicutes and boosted Bacteroidetes, Verrucomicrobia, and Cyanobacteria to lower the F/B ratio, as well as increased microbiota diversity. These indices were significantly correlated with obesity-related indices, indicating that microbiota might play an important role in the hypolipidemic effects of PPP and PPE. 2 hepatic genes were closely related to modulating the plasma and lipid profile, suggesting the ingested cholesterol and LDL uptake level were crucial metabolic changes. However, adverse plasma LDL-elevating effects were observed in a higher dose of PPP and PPE intake, which required further study on the potential toxicity.

Since the 21st century, society has an increasing awareness of health and is switching to healthier lifestyles and eating habits . Several sectors for product development are responsible for the change, such as food industries, researchers, health professionals, and regulatory authorities . In this context, functional foods have great potential. Functional foods represent the portion of the human diet that could provide health benefits and reduce the risk of chronic diseases beyond nutrition. Polyphenol-containing products are a common type of functional food with proven health benefits, such as protecting against certain cancers, cardiovascular diseases, type 2 diabetes, osteoporosis, pancreatitis, gastrointestinal problems, lung damage, and neurodegenerative diseases . According to Scalbert & Williamson , 1 g of daily consumption of polyphenols in long term is suggested to fulfill all the aforementioned health benefits of polyphenols. U.S. dietary guidelines recommended daily food intake to satisfy certain nutrient needs. However, polyphenols are not included and only 552 mg of polyphenol is satisfied through the recommended diet based on our calculation . Yogurt is a popular fermented dairy product known for its high nutritional value, especially the significant content of proteins and essential minerals, such as calcium. Greek Style Yogurt is a type of nutrient-dense yogurt with increasing popularity among consumers. According to Statista , from 2015 to 2020, the consumption of GSY in the U.S. significantly improved 50%, worth $3.7B and accounting for 52% of the U.S. yogurt market share. Compared to regular yogurts, GSY contains a higher solids content and is often perceived as being less acidic. The nutritional information commonly claims “twice the amount of protein as in regular yogurt” . However, they are never considered a source of functional foods. Therefore, it is of great interest to investigate the GSY fortified with polyphenol ingredients for improved polyphenol intake and increased consumption of dairy foods . El-Said et al., evaluated the antioxidant activities and physical properties of stirred yogurt fortified with the extracts of oven-dried and solar-dried pomegranate peel. Results showed that increasing the percentage of the added pomegranate peel extract statistically increased antioxidant activities and reduced the viscosity without significantly affecting the sensory attributes. However, since stirred yogurt has lower protein content and viscosity than GSY, a modified manufacturing process is needed for GSY. Kharchoufi et al. investigated quality changes of GSY fortified with pomegranate juice and arils during 18 days of storage. Researchers directly incorporated PJ and PA into GSY and quantified the total phenolic content, antioxidant activity, and sensory 1, 6, 12, and 18 days. This research demonstrated that the addition of PJ and PA in fermented GSY could significantly increase the antioxidant content and activity without negatively altering the sensory quality. However, incorporation of PPE in GSY prior to fermentation was not discussed, which could provide unique product characteristics and health benefits, as research has shown that bacteria could transform polyphenolic compounds into smaller units for increased extractability and stability . According to Ozcan , several main processing parameters influence the yogurt texture, including . fortification level and materials used, . stabilizers type and usage levels, . fat content and homogenization conditions, . milk heat treatment conditions, . starter culture , . incubation temperature , . pH at breaking, . cooling conditions and . handling of product post manufacture . Based on these considerations, this study aimed to investigate the effects of different contents of protein and PPE on the sensory, nutritional, and functional attributes of GSY.

Breeders increasingly are focused on meeting the needs of consumers, but genetic improvement of flavor is challenging as a consequence of the chemical and genetic complexities of the flavor phenotype . These challenges are accentuated in heterozygous, polyploid species. For example, fewer significant single nucleotide polymorphisms were detected in genome-wide association study of tetraploid blueberry when diploid models were applied ; in octoploid strawberry, structural variation underlying a locus affecting volatile production was difficult to resolve using a single reference genome . Recent advances have been made via chemical–sensory studies to identified specific volatiles associated with consumer preference . Although important volatile compounds in fruit crops are being identified, too little is known about the metabolomic and genetic diversity within species and breeding populations. Some volatiles have been lost during domestication and breeding as a combined result of negative selection and linkage drag in tomato and watermelon . Likewise, gain and loss of terpene compounds during strawberry domestication and its genetic causes have been investigated . Recent advances in sequencing technology and analytical approaches have opened new opportunities to understand the chemistry and genetics of fruit flavor. Meanwhile, genomes-wide expression quantitative trait loci studies have the capability to bridge the gaps between GWAS signals and their underlying causative genes. Integration of GWAS and eQTL studies has led to discovery of a master metabolite regulator in tomato and a flesh-color-determining gene in melon .

Long-read sequencing now allows assembly of genomes with high contiguity, and when coupled with parental short-read data ,large pot with drainage the two haplotypes of a heterozygous individual can be fully resolved. Phased assemblies have improved variant discovery, especially for large structural variants . The extent, diversity and impact of SVs increasingly are being studied in horticultural crops and have been shown to alter fruit flavor, fruit shape and sex determination . Great opportunity exists to coherently integrate these multi-omics resources for the discovery of flavor genes. Garden strawberry is an allo-octoploid species with highly palatable non-climacteric fruit . It increasingly has been utilized as a model for Rosaceae fruit crops genomics and flavor research as a result of its short generation time, wide cultivation and high value. Through exploration of spatiotemporal changes in gene expression and homolog search, several flavor genes have been cloned and validated, including an alcohol dehydrogenase and several alcohol acyltransferases for esters, a nerolidol synthase 1 for terpenes and a quinone oxidoreductase for furaneol. Recently, QTL studies and transcriptome data analyses for strawberry volatiles using biparental crosses have detected QTL and causative genes for mesifurane and gamma-decalactone . Nevertheless, low mapping resolution and a lack of subgenome-specific markers have hampered further characterization of causal genes underlying other QTL. This problem recently was addressed by the development of 50K Fana SNP array using probe DNA sequences physically anchored to the octoploid ‘Camarosa’ genome . High heterozygosity combined with an allopolyploid genome presents difficulties for resolving causative genes and their haplotypes. To further the goal of discovering causative genes affecting flavor in strawberry, association studies with larger sample sizes and additional genetic resources such as eQTL and additional genomes are required. Furthermore, these resources must span the breadth of natural variation in breeding germplasm.

Here we present multi-omics resources consisting of an eQTL study representing the genetic diversity of strawberry breeding programs in the US, phased genome assemblies of a highly- flavored University of Florida breeding selection, a structural variation map in octoploid strawberry and a volatile GWAS of 305 individuals. These are combined to leverage the extensive metabolomic, genomic and regulatory complexity in strawberry for the discovery of natural variation in genes affecting flavor. Ultimately, the functional alleles identified will be selected in breeding to achieve superior flavor.The eQTL population consisted of 196 genotypes including 133 newly sequenced accessions . The University of Florida genotypes were grown at GCREC and collected in the spring of 2020 and 2021. The University of California-Davis collection of diverse selections from multiple breeding programs were grown at either Santa Maria CA or Oxnard CA, for day-neutral and short-day accessions, respectively, and collected in the spring of 2021. Four UC genotypes were collected at both sites to ensure sequencing and SNP quality. Total RNA was extracted from a bulk of three fully ripe fruits using a Spectrum™ Plant Total RNA Kit , after flash freezing in liquid nitrogen. Illumina 150-bp pair-end sequencing was performed on the Illumina NovoSeq platform by Novogene Co. . On average, 6.9 Gb of sequence data were obtained for each sample. Raw RNA-Seq data of 63 samples from previous published studies were retrieved from the NCBI SRA database . In order to quantify gene expression, short reads were trimmed for adapter sequences and low-quality reads with TRIMMOMATIC v.0.39 and aligned against the reference genome using STAR v.2.7.6a in the two-pass mode . Only unique aligned reads were scored by HTSEQ v.0.11.2 in the union mode with the ‘–nonunique none’ flag supplied with the latest Fragaria_ananassa_v1.0.a2 annotation . All count files were compiled in R and normalized with the DESEQ package . To generate the marker dataset for eQTL mapping, SNPs and InDels were called using the mpileup and call commands. Markers were further hard-filtered using BCFTOOLS with the following steps: individual calls with lower than sequencing depth of three were set to missing using + setGT plugin; marker sites with quality < 30, missing rate > 0.3, heterozygous call rate > 0.98, minor allele frequency < 0.05, or number of alternative alleles > 1 were purged; the filtered markers were imported and analyzed in R, and only markers showing more than three matched calls in four duplicated sample pairs were retained.

A total of 491 896 markers passed the three stages of filtering. The missing calls were imputed, and all calls were phased using BEAGLE v.5.2 using the default settings . The eQTL mapping was performed for 62 181 fruit expressed genes using the filtered markers. Linear mixed models implemented in GEMMA were used for association analysis . The relationship matrix was computed in GEMMA and supplied to explain relationship within populations, and the top five principal components with a total of 25.0% variance explained were imported as covariates to reduce effects from population stratification to signify the genetic variance underlying the target traits. The Bonferroni corrected 5% significance threshold was used, determined the by number of LD-pruned markers . The approach to define an eQTL was similar to that used in previous studies . Briefly, we first clustered all significant markers with distance < 100 kb and purged clusters with fewer than three markers. The lead marker with lowest P-value was used to identify the eQTL, and boundaries of eQTL were defined as the furthest flanking significant markers. Clusters in LD were merged and boundaries were updated. The longest distance between cis-eQTL boundaries and eGene boundaries was limitedto 500 kb. Trans-eQTL hotspots were searched using the density function in R .In this study we leveraged eQTL, GWAS and haplotype-resolved genome assemblies of a heterozygous octoploid to identify allelic variation in flavor genes and their regulatory elements. Fine tuning of metabolomic traits such as amylose content in rice and sugar content in wild strawberry recently were made possible via CRISPR-Cas9 gene-editing technology. Similar approaches can be taken in cultivated strawberry for flavor improvement, but not before the biosynthetic genes responsible for metabolites production and their regulatory elements are identified. Our pipeline has proven to be effective in identification of novel causal mutations for flavor genes responsible for natural variation in volatile content and can be further applied to various metabolomic and morphological aspects of strawberry fruit such as anthocyanin biosynthesis , sugar content and fruit firmness. These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Strawberry also shares common volatiles with a variety of fruit crops. Specific esters are shared with apple ,drainage collection pot certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and subgenome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication .

Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, but rather is concentrated at a few hotspots controlled by putative master regulators . The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species . In cotton, physical interactions among chromatins from different subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes and limited length of Illumina reads, false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals. Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits. Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.The gastrointestinal tract, especially the large intestine, houses the most abundant and complex microbiota in humans. Most of intestinal bacteria belong to the phylum Firmicutesand Bacteroidetes , which make up more than 90% of known phylogenetic categories and dominate the distal gut microbiota. Other lower abundance bacteria include Actinobacteria, Fusobacteria, Proteobacteria, and Verrucomicrobia. Diet is one of the important factors contributing to the gut microbial composition that ultimately affects human health. Obesity and associated metabolic diseases, including type 2 diabetes, are intimately linked to diet . A number of recent in vitro, in vivo, and human studies showed that polyphenols or polyphenol-rich dietary sources, particularly tea, wine, cocoa, fruits, and fruit juices, influence the relative abundance of different bacterial groups within the gut microbiota byreducing the numbers of potential pathogens and certain gramnegative Bacteroides spp. and enhance beneficial bifidobacteria and lactobacilli . Spices are derived from bark, fruit, seeds, or leaves of plants and often contain spice-specific phytochemicals. Spices have been used not only for seasoning of foods but also for medicinal purposes, and have a number of demonstrated disease preventive functions such as antimicrobial, antiinflammatory, antimutagenic activities, and are known to reduce the risk of cancer, heart disease, and diabetes . They are best known for their strong antioxidant properties that exceed most foods. It was reported that of the 50 food products highest in antioxidant concentrations among 1113 U.S. food samples, 13 were spices. Among them, oregano, ginger, cinnamon, and turmeric ranked #2, 3, 4, and 5, respectively .