Month: February 2025

Previous research using mitochondrial and microsatellite DNA markers found some evidence of population structure, as well as evidence that the European invasion originated from a single population in central Chile . However, determination of higher-resolution population structure, migration events, divergence times, and population size can benefit from using a larger number of markers, such as what is produced from genome-wide sequencing studies . Additionally, few genetic studies have been conducted to understand how Tuta absoluta has performed so successfully as an agricultural pest beyond targeted examinations of known insecticide resistance alleles. One reason for this has been the lack of a highly contiguous genome with annotated genes. A short-read based assembly has been previously published for the purpose of developing molecular diagnostics ; however, it is highly fragmented and duplicated. In this study, we addressed these issues by using long-read sequencing technology to produce a highly contiguous genome assembly for Tuta absoluta. We then use short-read technology to sequence genomes of individuals collected across Latin America, as well as a Spanish population, blueberry pot to identify single nucleotide polymorphisms in an unbiased manner. We use these SNPs to detect population structure and estimate population history parameters to understand how and when Tuta absoluta spread across Latin America. Finally, we use genome scanning statistics to identify genes putatively under selection that may explain Tuta absoluta’s success as an agricultural pest. We expect the genome assembly and population data will be an asset toward developing new strategies to manage this pest.

For genome assembly, a single Tuta absoluta larva was collected from a colony originally sourced from the Institute of Agrifood Research and Technology , Cabrils, Spain and held in the Contained Research Facility in UC Davis and frozen on dry ice. The larva was pulverized in liquid nitrogen with a pestle in a 2 mL microcentrifuge tube using 740 mL of lysis buffer , 135 ug/mL Prot K. After a 37℃ overnight incubation step, 240 μL of 5M NaCl was added and gently mixed in by rocking before centrifuging at 10,000 RCF, 4℃, for 15 minutes. Supernatant was transferred using a wide-bore pipetteto a 2 mL DNA low-bind tube , precipitated with 1 mL of 100% ethanol, and centrifuged at 10,000 RCF, 4℃, for 5 minutes. The DNA pellet was washed with 500 μL of ice-cold 70% ethanol twice before air-drying for 5 minutes. Dry pellet was resuspended in DEPC-treated water and allowed to dissolve for 1 hour at room temperature before being stored at 4℃ for no more than 2 weeks. Absorbance ratios were measured with a Nanodrop Lite , DNA concentration was measured with a Qubit 4 Fluorometer using a dsDNA High-Sensitivity Assay , and DNA fragment size was measured with a Tapestation genomic DNA ScreenTape . Approximately 700 ng of DNA was sent to QB3-Berkeley for library preparation and PacBio HiFi sequencing with 1 SMRTcell.We analyzed whole-genome sequencing data from individuals previously collected from field and greenhouse sites across South America and Costa Rica, as well as a lab colony from Spain . Mapping rates to the new genome assembly ranged between 70%-90%, although sequencing depth per individual was low . One population from Argentina had extremely low mapping rates and read depth, so we excluded it from further analysis. Wherever possible, we used methods based on genotype likelihoods, rather than genotype calls, to account for uncertainty that results from the low read depth.

To investigate population structure in our samples, we used Principal Component Analysis and admixture estimation based on allele frequencies from over 900,000 SNPs. The first two PCs captured 18.7% and 16.3% of the total variance in the data, with the remaining PCs each capturing less than 5% of the total data variance . Samples primarily cluster together based on collection site but also formed three distinct regional groups . Samples from Chile, Peru, and Ecuador form an “Andes” cluster west of the Andes Mountains; samples from Brazil, Uruguay, Paraguay, and Argentina form a “Central” cluster, east of the Andes Mountains; and samples from Columbia and Costa Rica form a “North” cluster. Spanish samples grouped tightly with the Andes cluster, particularly the VA site. When three clusters were allowed in admixture estimation, samples group into the same three clusters as in PCA, while at four clusters, the Spanish samples become their own group, with VA samples sharing a large proportion of admixture. Compared to other Andes populations, the VA samples are more differentiated from Central and North sites as well, with little signal of admixture at all levels of k tested. The other Andes populations all had low admixture proportions from Central at k=2, although at k=3 we see that all RI samples exhibited admixture from the North populations. This suggests that the non-VA Andes populations are more closely related to Central populations than VA, and that VA could represent an admixture between the population that gave rise to the Spanish lineage and the other Andes populations. Additionally, we see that RI represents an intermediate population between the Andes and North, which makes sense given its geographic location between the two clusters. To further quantify population structure between these clusters, we calculated nucleotide diversity, Tajima’s D, and Fst using genotype likelihoods . For all clusters, nucleotide diversity was approximately 2%, which is fairly high compared to most Lepidopterans .

If we look at the weighted Fst, we see differentiation between clusters is high, particularly between North and all other clusters. The combination of high diversity levels and high Fst could mean these regions diverged from each other a long time ago, prior to the detection of Tuta absoluta by growers across Latin America in the 1960s to 1980s. If divergence had occurred recently, we might expect reduced diversity levels in invasive populations relative to the ancestral population.To detect potential migration events between populations, we used Treemix to build a maximum likelihood tree based on allele frequencies, as well as predict migration edges and calculate F3 statistics. As Treemix was designed to take allele count data per population, we called genotypes using PCAngsd using a 95% accuracy cutoff and counted alleles within each sampling location. After filtering out loci with missing data, 47,535 SNPs were available for use. In general, the tree topography aligns with results from PCA and admixture analyses. We see sampled sites cluster into the same three clusters, North, Andes, and Central, with the Spanish samples sister to the VA site . In agreement with Fst estimates, North populations have experienced more genetic drift from the Andes and Central populations, compared to the Andes and Central populations with each other. Interestingly, the RI site does not form a clade with other Central populations but descends from the common ancestor of the Central/Andes group. Based on admixture analysis that showed low levels of admixture in RI from the North, the position of RI in the tree could be further evidence that Ecuador represents an intermediate mixing zone between populations north and south of it. To investigate further, we re-ran Treemix allowing between one to five migration events and calculated the F3 statistic between all combinations of three populations to see if admixture was supported. At m=2,4 and 5, Treemix reported a strong migration from the Spain or Spain/VA branch to RI, while at m=3 and 5, Treemix reported a weak migration event from the North to RI. F3 statistics F3 and F3 were significantly negative , indicating that a simple bifurcating tree does not explain RI’s relationship with CH, CR, and SP. While Treemix infers a migration from the Spanish branch to the RI branch, it is important to remember that this migration is inferred to have occurred somewhere along the branch between the current day Spanish population and the most recent common ancestor of Spain and VA . This migration could have occurred early in the branch, nursery pots when the population was still in Chile, or late in the branch, when the population moved to Spain. Based on fresh tomato trade between the two countries, in 2006 Chile shipped over 29,000 kg of fresh tomatoes to Spain while importing none back . This makes admixture from Spain back to RI unlikely and suggests that RI contains admixture from North and Chilean populations. In addition to admixture in RI, Treemix and F3 statistics also detected admixture in AR from the Spanish population. At m=1,2,3, and 4, Treemix detected a migration edge from the Spanish branch to AR with migration weight varying between 9% to 17%. F3 statistics of AR and Spain with any population from North or Central resulted in a significantly negative value, providing strong evidence of a migration event from a Spanish ancestor like the signal detected with RI.

This suggests that the admixture signal we see in AR is from the same Chilean population that gave rise to the Spanish invasion and RI admixture.Based on the high levels of nucleotide diversity and Fst between the Andes, Central, and North clusters, we hypothesized that the three regions may have diverged many generations ago, before the appearance and detection of Tuta absoluta in agricultural crops throughout South America in the mid- 20th century. This would suggest a model in which Tuta absoluta may have adapted from local, wild host plants to nearby tomato fields independently, rather than a single population that became adapted to tomatoes and was spread through human activity. To investigate this, we calculated the folded two dimension site frequency spectrum between populations and estimated parameter values under various population models using maximum likelihood coalescent methods . We excluded the VA samples from the Andes cluster to avoid potential modeling issues due to VA appearing to originate from a distinct ancestor than other Andes populations. The simplest model allows for two population splits with constant population sizes, while the exponential growth model adds an exponential growth rate to each population. As exponential growth may not be appropriate if divergence times are long, we also tested a model with a simple resizing event for each population at some point in time. We used a post-hoc comparison of simulated linkage disequilibrium decay rates between models to test model fit. We found that while all three models simulated decay rates within the 95% confidence interval of the Andes population data, none simulated decay rates that overlapped with Central and North decay rate estimates, although the resizing population model was closest. Under the resizing population model, divergence of the North occurred 252,383 generations ago , followed by a Central population divergence 187,034 generations ago . Reports of Tuta absoluta generation times can be as high as 6 to 12 or more generations per year , dating these divergence events to tens of thousands of years ago. This suggests that Tutaabsoluta was already present across Latin America prior to the 1960s, and as tomato agriculture surged, adapted locally to the new host plant.The Population Branch Statistic is an Fst-based statistic that uses Fst data between 3 populations to calculate the population-specific allele frequency changes. Regions of the genome with abnormally high PBS may be under strong selective forces, causing the loci allele frequencies to change faster than expected by drift. We calculated PBS across the genome for all three populations and found several peaks in contigs 2, 9, 15, and 22 that were exceptionally high and broad, particularly in the North cluster . The peak in contig 9 contained the gene paralytic , a neuronal sodium channel protein that is the active target of pyrethroid insecticides . While PBS peaks in the North population between 13.1 and 13.2Mb on contig009, we note that the allelic diversity was low in the Andes and Central clusters relative to the North . We calculated allele frequencies of known resistance-inducing mutations in each cluster , and found one mutation, an alanine to leucine substitution at position 1014, was fixed in the Central and Andes, while at 41% frequency in the North . In addition, we found low to intermediate frequencies of other resistance alleles, including M918T, T929I, V1016G, L925M, and I254T. A similar selective sweep signal was also seen in the PBS hotspot on contig 2 , with high PBS and diversity levels in the North, and a large region of low allelic diversity in the Andes and Central .

Dynamics of pp highlighted the typical inversion phenomenon of the diel curve in olive leaves from trees under deficit irrigation . In NB, DI-66 and DI-33 leaves exhibited the half-inverted state , whereas DI-0 leaves showed a total inversion of the curve . In MN trees, a clear shift from state I to II was not observed, despite the slight tendency to enter state II at 219 and 221 DOY, with no apparent differences among irrigation levels . This suggests that MN leaves can maintain high cell turgor, probably by reduced cell wall elasticity or osmotic adjustments, as found in other olive genotypes . The highest RRfruit always occurred early in the night as fruit quickly rehydrated their tissues . As expected, the most negative RRfruit rate always occurred in the warmest hours of the day. RRfruit dynamics were also affected by deficit irrigation in NB, as the diel RANGE was greater in DI-0 and DI-33 than in DI-66 and FI fruit . A completely different behavior was observed in MN fruit, which instead had the widest diel RANGE in FI trees . In addition, the overall diel RANGE of RRfruit in MN was almost double than in NB, implying larger water in- and out-flows per unit of fruit volume in the former, determined by high fruit sink power for water. A general positive peak of RRleaf was exhibited early in the morning , representing a quick leaf turgor loss after pre-dawn highest turgor in the 24-hour timeframe. Even in this case, the two cultivars responded differently to water deficit, growing blueberries in pots with NB DI-0 trees exhibiting minimal diel fluctuations while MN DI-0 trees showing the largest RANGE. This suggested that the oscillations of RRleaf might be linked to those of RRfruit. Another 5-day interval was considered at stage III of fruit development .

Differently from stage II, FD responses were characterized by an evident diameter increase across the 5 days and within the 24-hour period in both cultivars, as in stage III fruit are in full cell enlargement phase . Daily curves of pp did not show pronounced inversion phenomena, as this week was characterized by high rainfall and general higher midday 9 stem . Only NB DI-0 trees showed a partially inverted pp curve. Diel RANGE of RRfruit was found to be greatly reduced at stage III compared to stage II . In the former, low VPD and good soil water availability determined by abundant precipitations led to an increase of water content in fruit and lower fruit water exchanges. For similar reasons, the diel RANGE of RRleaf was reduced in stage III , although NB and MN maintained the same differences in response to deficit irrigation levels observed at stage II . Considering the interesting findings from RRfruit and RRleaf dynamics, these two indices were further related to each other regressing their diel data at 15-minute intervals in a day at stage II and stage III . Scatter plots in Figure 10 show anti-clockwise hysteretic relationships between RRfruit and RRleaf, both for NB and MN . Hysteresis are common when relating outputs from different sensors of plant water status mounted on different organs , as there is generally a lag in tissue water de- and re-hydration, and in our case, also a likely different pattern of the RRleaf to RRfruit relationship between day and night. An overall decrease of the hysteretic loop area occurred from stage II to stage III in both cultivars . This is probably driven by the different fruit growth pattern at stages II and III which induced a reduction of the RRfruit diel range . In both DOY 223 and 287, the hysteretic loops in NB progressively flattened along the RRfruit axis with increasing water deficit due to the change in the ratio between RRfruit and RRleaf.

In other words, on one hand, at increasing water deficit and in a diel interval, it seems that NB leaves significantly reduce water exchanges, as the values of RRleaf stay around 0 Pa kPa−1 min−1 . On the other hand, increasing water deficit caused MN loops to flatten along the RRleaf axis , with MN leaves keeping high water exchanges at low 9 stem, while fruit water exchanges were significantly reduced, as RRfruit did not change much from 0 µm mm−1 min−1 .This opposite trend suggests a completely different mechanism of leaf and fruit water exchanges in response to increasing water deficit in the two cultivars, which might be driven by different osmotic adjustments, cell-wall elasticity and tissue water content. The statistical diel, nocturnal and diurnal parameters of RRfruit were associated to the corresponding RRleaf parameters to assess fruit and leaf responses to water deficit. Subsequently, data were analyzed by MANOVA to determine whether the combined response of parameters was affected by cultivars, irrigation levels, and cultivar × irrigation interaction. The cultivar did not influence significantly diel, diurnal and nocturnal RRfruit/RRleaf when statistical parameters were considered together . Diel and diurnal RRfruit/RRleaf parameters changed significantly in response to irrigation levels, but the cultivar × irrigation interaction had the strongest effect , indicating that RRfruit/RRleaf responses to deficit irrigation differ between the two genotypes under study. Specifically, the highest F was found in the MANOVA that tested diurnal RRfruit/RRleaf responses to cultivar × irrigation. These results suggest that, under increasing water deficit, the differences in genotype-specific fruit and leaf sink power to water are predominant in day hours.Human persistence depends on many natural processes, termed ecosystem services, which are usually not accounted for in market valuations. Global degradation of such services can undermine the ability of agriculture to meet the demands of the growing, increasingly affluent, human population . Pollination of crop flowers provided by wild insects is one such vulnerable ecosystem service , as their abundance and diversity are declining in many agricultural landscapes . Globally, yields of insect-pollinated crops are often managed for greater pollination through the addition of honey bees as an agricultural input . Therefore, the potential impact of declines in wild pollinators on crop yields is largely unknown, as is whether increasing application of honey bees compensates for losses of wild pollinators, or even promotes these losses. Wild insects may increase the proportion of flowers that develop into mature fruits or seeds , and therefore crop yield , by contributing to pollinator abundance, species number , and equity in relative species abundance . Increased pollinator abundance, and therefore visitation rate to crop flowers, should augment fruit set at a decelerating rate until additional individuals do not further increase , or even decrease fruit set . Richness of pollinator species should increase the mean, and reduce the variance, of fruit set , because of complementary pollination among species , facilitation , or “sampling effects” , among other mechanisms . Pollinator evenness may enhance fruit set via complementarity, or diminish it if a dominant species is the most effective pollinator . To date, drainage gutter the few studies on the importance of pollinator richness for crop pollination have revealed mixed results , the effects of evenness on pollination services remain largely unknown, and the impact of wild insect loss on fruit set has not been evaluated globally for animal pollinated crops.

We tested four predictions arising from the assumption that wild insects effectively pollinate a broad range of crops, and that their role can be replaced by increasing the abundance of honey bees in agricultural fields: for most crops, wild-insect and honey bee visitation enhances pollen deposition on stigmas of flowers; consequently, for most crops, wild insect and honey bee visitation improves fruit set; visitation by wild insects promotes fruit set only when honey bees visit infrequently ; and pollinator assemblages with more species benefit fruit set only when honey bees visit infrequently . To test these predictions we collected data at 600 fields on all continents, except Antarctica, for 41 crop systems . Crops included a wide array of animal-pollinated, annual and perennial fruit, seed, nut, and stimulant crops; predominately wind-pollinated crops were not considered . Sampled fields were subject to a diversity of agricultural practices, ranging from extensive monocultures to small and diversified systems , fields stocked with low to high densities of honey bees , and fields with low to high abundance and diversity of wild insects . For each field, we measured flower visitation per unit of time for each insect species, from which we estimated species richness and evenness . We quantified pollen deposition for 14 systems as the number of pollen grains per stigma, and fruit set for 32 systems as the percentage of flowers setting mature fruits or seeds. Spatial or temporal variation of pollen deposition and fruit set were measured as the coefficient of variation over sample points or days within each field . The multilevel data provided by fields within systems were analyzed with general linear mixed-effects models that included crop system as a random effect, and wild-insect visitation, honey bee visitation, evenness, richness, and all their interactions as fixed effects. Best-fitting models were selected based on Akaike’s Information Criterion . In agreement with the first prediction, crops in fields with more flower visits received more pollen on stigmas, with an overall 74% stronger influence of visitation by honey bees than by wild insects . Honey bee visitation significantly increased pollen deposition in seven of ten crop systems, and wild insects in ten of 13 systems . Correspondingly, increased wild insect and honey bee visitation reduced variation in pollen deposition among samples . Contrary to the second prediction, fruit set increased significantly with wild-insect visitation in all crop systems, but with honey bee visitation in only 14% of systems . In addition, fruit set increased twice as strongly with visitation by wild insects than by honey bees . These partial regression coefficients did not differ simply because of unequal abundance, or disparate variation in visitation between wild insects and honey bees. In crop systems visited by both honey bees and wild insects, honey bees accounted for half of the visits to crop flowers , and among-field CVs for visitation by honey bees and by wild insects were equivalent. Furthermore, wild-insect visitation had stronger effects than honey bee visitation, regardless of whether honey bees were managed or feral and, comparing across systems, even where only wild insects or honey bees occurred . Moreover, wild-insect visitation alone predicted fruit set better than honey bee visitation alone . Correspondingly, the CV of fruit set decreased with wild-insect visitation, but varied independently of honey bee visitation . This contrast likely arose from pollen excess, filtering of pollen tubes by post pollination processes, and seed abortion , and so reflects pollination quality, in part. Intriguingly, the difference in coefficients between pollen deposition and fruit set for honey bees greatly exceeds that for wild insects , indicating that wild insects provide better quality pollination, such as greater cross-pollination . These results occurred regardless of which crop systems were selected , sample size , the relative frequency of honey bees in the pollinator assemblage among systems, the pollinator dependence of crops, or whether the crop species were herbaceous or woody, or native or exotic . Poor-quality pollination could arise if insect foraging behavior, based on focal resources typical of honey bees , causes pollen transfer between flowers of the same plant individual or the same cultivar within a field, thereby limiting cross pollination and increasing the incidence of self-pollen interference and inbreeding depression . The smaller difference in coefficients between pollen deposition and fruit set for wild insects, and the stronger effect on fruit set of wild-insect visitation, suggest that management to promote diverse wild insects has great potential to improve global yield of animal-pollinated crops. The third prediction was also not supported, as fruit set increased consistently with visitation by wild insects, even where honey bees visited frequently . In particular, the best-fitting model for fruit set included additive effects of both visitation by wild insects and honey bees , suggesting that managed honey bees supplement the pollination service of wild insects, but cannot replace it. Overall, visitation by wild insects and honey bees were not correlated among fields , providing no evidence for either competition for the resources obtained from crop flowers , or density compensation between wild insects and honey bees at the field scale.

However, we also observed upregulation of biosynthetic genes involved in production of trichothecenes , which indicates that F. acuminatum also relies on other toxins during infection of tomato fruit concordant with the classification of F. acuminatum as strong toxin producer . Additionally, the AA6 family that appears during RR infections of F. acuminatum and R. stolonifer may be involved in metabolism of host defense compounds. These enzymes are 1,4-benzoquinone reductases, which have been shown to function in fungal protection against destructive host-produced quinones . Another physiological factor which may influence the success of infection is the pH of the pathogen-host interface. As the tomato fruit ripens, the apoplast becomes more acidic . Furthermore, B. cinerea has been shown to acidify the host environment through the production and secretion of oxalic acid . A key enzyme in oxalic acid biosynthesis is BcOAH1 , which encodes oxaloacetate hydrolase . This gene is not upregulated during interaction with tomato fruit in any of the treatments. However, there is significant downregulation of this gene in RR fruit compared to MG fruit. This suggeststhat, if B. cinerea utilizes oxalic acid to acidify tomato fruit, it does so to a much lesser extent in RR fruit where the pH is already comparatively acidic. In contrast, during infection of Arabidopsis roots, F. oxysporum relies on alkalinization via peptides known as rapid alkalinizing factors . However, a BLAST search of RALF sequences, as was performed to identify fungal RALFs in Thynne et al. , square pot revealed no clear RALF genes in our transcriptome of F. acuminatum. The importance of fruit ripening for the success of fungal infections was confirmed by comparing fungal growth and disease development in fruit from wild-type and a non-ripening mutant after fungal inoculation.

Growth and morphology of B. cinerea, F. acuminatum and R. stolonifer on nor MG and RR-like tomato fruit was comparable to that on wild-type MG fruit. This result is in agreement with our previous report that nor tomato fruit is resistant to B. cinerea infections . The inability to infect non-ripening tomato fruit highlights the dependency of these fungi on the activation and progression of ripening events that transform the host tissues into a favorable environment for disease development. Altogether, our results confirm that infection success of the three pathogens B. cinerea, F. acuminatum and R. stolonifer largely depends on fruit ripening stage. This is due to all three pathogens sharing similar lifestyles and necrotrophic infection strategies. However, the capacity to infect different plant tissues differs between the three fungi. B. cinerea shows distinct strategies in both ripening stages likely due to its ability to induce susceptibility in the host , whereas R. stolonifer is active almost exclusively in RR fruit. The ability of F. acuminatum to infect both MG and RR fruit may be reflective of its especially wide host range, which includes insects in addition to fruit . A summary of infection strategies utilized by the three pathogens during infection of MG and RR tomato fruit is shown in Table 2. Further research on which processes identified are required for successful infection would lead to a greater understanding of fruit-pathogen interactions and, ultimately, strategies for their management.The tomato is a functional genomics model for fleshy-fruited species and is one of the most popular and economically important crops globally . However, storage at temperatures below 12.5°C followed by rewarming to room temperature, compromises fruit quality, hampering the post harvest handling of this commodity .

This cold-induced damage to the fruit called post harvest chilling injury may only be detectable as a loss of flavor, or in severe cases, as fruit spoilage, the extent of which depends on the storage temperature, length of exposure, genotype and fruit developmental stage . The progression of PCI in fruit tissues is complex. It is marked by a loss of selective membrane permeability, increased solute leakage, reactive oxygen species accumulation and metabolic dysfunction . After the fruit is transferred to room temperature for rewarming or reconditioning, higher respiration ensues within days , and within a week, secondary symptoms such as uneven color formation, surface pitting,water soaking and decay are visible . Symptoms are more intense in green compared to riper fruit, since maturation processes are disrupted by chilling . Because of the negative effect on tomato quality and shelf-life, our goal is to better understand PCI development and regulation in this species. First, we investigated the spatial and temporal evolution of PCI in the whole tomato fruit using MRI. Most studies of tomato PCI have focused on the pericarp, ignoring the internal tissues, which can account for 30% and 70% of the fresh mass of round and cherry tomatoes, respectively. Tao et al. , investigated changes in chilled ‘Micro-Tom’ fruit using non-invasive MRI. They showed that the columella and locular region differed from the pericarp in their response to cold, which has implication for understanding the underlying causes of PCI. The fruit in that study were subjected to a severe cold stress , since this genotype is not as sensitive to chilling temperatures as many commercial varieties . Further, only one developmental stage was chosen . It is not known if their findings are applicable to other cultivars, storage conditions or maturation stages. Second, we investigated if 5-azacytidine could alter PCI. This chemical inhibits DNA and RNA methylation , epigenetic modifications that regulate gene expression, in response to developmental and environmental stimuli in a tissue-specific manner .

DNA methylation is a key regulatory process for tomato fruit ripening ; injecting AZA in round tomato fruit accelerated ripening . It was shown that chilling-induced reductions in red fruit volatiles correlated with methylation of key ripening genes. Co-regulation of the ripening and cold response regulatory networks in fruit undergoing chilling stress seems likely . Since differential methylation is essential to both processes, we wanted to determine if AZA could influence PCI symptoms in tomato fruit. In this study, two questions were asked: 1) is it possible to detect spatio-temporal differences in chilled tomato fruit differing in maturation stage, and temperature × time of storage by low-resolution MRI?, and 2) would AZA influence PCI response? For the former, we used commercial cherry tomatoes and mild to moderate chilling stress. For the latter, fruit were injected with AZA weekly in order to detect changes in PCI by methylation , specifically on respiratory activity. Fruit from a commercial cherry cultivar and the functional genomics model ‘Micro-Tom’ were used in this study.At this developmental stage in ‘Sweet 100’, the pericarp, columella and locular tissue showed a differentiated pattern in terms of their D-values after 7 days of chilling . Values were highest in the pericarp followed by the locular tissue and columella. Similar patterns were seen in freshly-harvested breaker fruit . These three tissues have heterogeneous transcriptional and metabolic profiles due to their distinct origin and functionality . This likely contributed to the distinct D-values observed. When D-values for each region were compared as over each chilling period, no changes were observed except for the columella in fruit held at 5°C. Unchanged D-values may be due to cold-induced reductions in free water movement within tissues, and pectin solubilization . Fruit exposed to warmer temperatures, i.e., after storage at the control temperature for 7 days, or after transfer from the cold to 20°C, showed more dynamism in D-values. The different tissue fractions, which had distinct D-values during chilling, changed and became more similar when exposed to warmer temperatures . These non-chilling temperatures may have allowed ripening and other physiological eventsto take place, leading to these changes.Figure 3A shows the D-values of ‘Sunsugar’ ripened fruit. These data, gathered from breaker, pink, square plastic planter and red fruit immediately after harvest, suggest that as ripening progresses, the D-values of the columella and locular region become more similar . Ripening increases the proportion of free water and metabolites within tissues, due to liquefaction of the locules and breakdown of the structural components of the cell . These changes may have underscored the increased Dvalues seen here, and in other studies . A similar occurrence was seen when red fruit was stored at 2.5°C for 5 days . When D-values for each region were compared over time, there was no significant difference. Tissue liquefactionin red fruit was so extended as a consequence of ripening, that cold did not generate any detectable increase by the MRI, or did not increase membrane leakage since it was already fluid. The observations of pink fruit stored in the cold and then rewarmed are less clear. Both chilling-induced damage during low-temperature storage, and ripening-related tissue deconstruction during rewarming would lead to increased membrane permeability and Dvalues , thus making it difficult to attribute higher Dvalues to one or the other biological phenomenon.

There are some points to emphasize with respect to the data when analyzed across cultivars and conditions. First, pericarp D-values did not vary as much as those in the columella and locular regions . Second, there was a weak correlation between MRIderived values for the pericarp and the physical changes caused by cold, visible on the pericarp e.g., poor color development, pitting and decay as reported by the CII data . In contrast, there was more synchrony for the columella and CII which is similar to the data published by Tao et al. . Surprisingly, the locular fraction showed a similar r-value to the pericarp when CII was considered. Therefore, other mechanisms besides the increased water mobility we were able to detect under the experimental conditions used, may have a higher contribution to the development of chilling induced external symptomatology. Third, different D-values were recorded in the three tissues as ripening progressed: they decreased in the pericarp, increased in the columella and were unchanged in the locular tissue , exemplifying the unique response of each tissue-type. Fourth, MRI could only detect changes after transfer of chilled fruit to room temperature. Loss of membrane selective permeability due to a cold-induced membrane phase transition was not sufficiently advanced to produce detectable increases in free water mobility during cold storage. This supports the view that, increased membrane permeability is unlikely to be one of the earliest events in PCI response, but occurs at a significant rate during rewarming .Fruit undergoing PCI normally exhibit a transitory burst of CO2 when transferred from chilling to room temperature, which acts as a reliable marker for the early stages of cold injured tissue . If AZA-treated fruit show differences in respiratory activity after cold stress compared to the water-treated fruit, this could be indicative of an effect of methylation on PCI. Different responses were observed across varying cold stress in ‘Micro-Tom’ and ‘Sun Cherry’ and are described in turn.MRI was useful for detecting fruit ripening, its attenuation by cold, and fruit tissue specificity in the cold response. MRI non-invasively differentiated among tomato pericarp, columella and locular fractions. However, when fruit were scanned after reconditioning, or when at an advanced stage of ripening, these distinctions were lost. Chilling-induced damage was detected by the MRI in the columella but not in the pericarp and locular tissue. MRI scans in the columella throughout the experiment better reflected the CII. Cold stress likely repressed the mechanisms leading to fruit free water production or increased water mobility. AZA was used to determine if demethylation could modulate the effect of PCI in chilled tomato fruit. The effect of AZA on PCI was determined by many multilayered factors, e.g., genotype, severity of stress and how it influences the underlying ripening pathways. This complexity is probably a consequence of the ubiquity of epigenetic methylation on the genome and transcriptome and the multitude of factors that influence its status. Even though in some conditions, the effect of AZA was not detected, it does not mean that methylation is not important to PCI, since the phenotypes assessed probably do not reflect all methylated regulation of fruit gene expression.Strawberry is an important soft fruit crop that is grown worldwide on more than 370 000 hectares and, for the United States alone, the total value of the annual strawberry production exceeds US$2.3 billion . Strawberries are beneficial to the human diet as a source of macro- and micronutrients, vitamins and health promoting antioxidants . Strawberry is a perennial herbaceous plant with short stems and densely spaced leaves. Strawberry produces complex accessory and aggregate fruit composed of achenes and a receptacle .

The evolutionary and genetic aspects of the process of domestication have been studied intensively in temperate cereal crops and pulses and are fairly well understood for those crops . In contrast, we have a much less comprehensive knowledge of domestication in perennial crops such as tree fruits and nuts , even though these species make up an important component of human nutrition throughout the world . It has been suggested that the study of tree crops may reveal fundamental differences in the process of domestication and its genetic consequences . The fact that tree crops have long lifespans and that they are often asexually propagated limits the number of generations that have passed since their first cultivation by humans and the degree of evolution that has occurred under human selection in these taxa . Clement described five stages in the domestication process: 1) wild , 2) incidentally co-evolved , 3) incipiently domesticated , 4) semi-domesticated , and 5) domesticated . In species that are still in the early stages of domestication , cultivated and wild-type individuals will coexist in close proximity in the region of origin, where we can study how traits have responded to anthropogenic selection in the face of continued gene flow between wild and improved types. Some of our best-known examples of plants in the early stages of domestication come from Mesoamerica, where native crop species are frequently cultivated in traditional agroecosystems such as home gardens . We expect fruit and seed characteristics of tree crops to change in predictable ways under anthropogenic selection during the domestication process, 25 liter pot leading to a type of domestication syndrome for edible fruits .

Compared to their wild relatives, edible fruits of domesticated taxa tend to be larger and sweeter or higher in oil content . The ratio of edible product to waste, or “economic ratio” , goes up. We also expect to see a reduction in toxic compounds that confer defense against natural enemies at the cost of palatability . In addition to changing mean trait values, anthropogenic selection could have contradictory effects on the variance of traits. Due to selection bottlenecks, we expect neutral variation to decline with cultivation in domesticated species . For particular genes under strong directional selection, the reduction in genetic variation may be even more dramatic . However, if distinct cultivated varieties are selected for contrasting phenotypes, then phenotypic variance will be higher in cultivated plants . Some even have used changes in the variance and shape of the distribution of traits as an indicator of the degree of domestication shown by populations of indigenous fruit trees . Chrysophyllum cainito L. , commonly known as caimito or star apple, is highly desired throughout the tropics for its value as anornamental tree and its production of large, edible fruits. Caimito is presently cultivated throughout the Caribbean, Central America, and parts of South America as well as in Southeast Asia . Various hypotheses have been suggested for its geographic and taxonomic origin, as well as its center of domestication . Historical accounts made by plant explorers in the 16th and 17th centuries mention C. cainito as occurring in the islands of the Lesser and Greater Antilles as well as on the mainland and islands of the Bay of Panama . Patrick Brown mentions C. cainito as being cultivated all over the island of Jamaica and that it “thrives with very little care” . Seemann reports the presence of both wild and cultivated caimito in the Isthmus of Panama. Our preliminary molecular genetic results show much greater overall diversity of the species in Panama compared to northern Central America, Mexico, and the Caribbean, and support the hypothesis that C. cainito was domesticated in Panama.

In contemporary Panama, C. cainito is widely distributed as a natural part of tropical lowland forest. In fact, this species is promoted as a native tree for environmentally sensitive reforestation efforts, for example, by the Proyecto de Reforestación con Especies Nativas . The forest plot network that bisects the Panamanian Isthmus documents the presence of C. cainito at many sites on the Pacific side of central Panama . In the wild, C. cainito is found at low density, does not produce fruit until the tree reaches the canopy of the forest, maintains few branches below 10 m, and produces ripe fruits over only a short time interval; taken together, these aspects complicate the study of this species in wild populations. Cultivated caimito is a common component of Panamanian home gardens and small ranchos. Usually only one or two individuals are planted per property, as fruits are not sold on a commercial scale. While an owner will occasionally report that a tree “arrived on its own,” presumably dispersed by a bird or mammal, usually trees in gardens have been planted from seeds selected from a fruit, usually a fruit provided by a friend or neighbor . Sometimes plants have been purchased as seedlings from a nursery. During the Canal Zone era , C. cainito was planted horticulturally as a street tree in some communities within and surrounding Panama City. Thus the cultivation of C. cainito trees in Panama has included a combination of individual selection via fruits and seeds, with some relatively small-scale central production and distribution of plants. In addition, human migration between the Antilles and Panama may have resulted in the importation of caimito seeds from the Antilles. Our goal was to test the hypothesis that cultivated individuals of C. cainito show signs of a domestication syndrome: Distinctive fruit and seed characteristics that are typically associated with domestication relative to wild phenotypes growing in the same region. As a putative region of domestication for this species and an area with extensive populations in protected forests, Panama is the ideal place for such a study. We compared fresh collections from wild and cultivated trees in central Panama for a range of morphological and fruit quality traits to infer how human selection has influenced these traits. We then used Discriminant Analysis to investigate the relative importance of various traits for distinguishing cultivated fruits from wild fruits. In this paper, we also present information on the variance in traits, correlations among traits, and variation in trait values from year to year.We chose to focus our collections in central Panama, where caimito is cultivated in both rural and urban communities set within the context of wild populations in protected forests. Central Panama includes extensive areas of semi-deciduous, tropical forest, primarily associated with the Panama Canal watershed. There is a natural rainfall gradient of 1,750–3,000 mm per year from the Pacific to the Caribbean side of the isthmus, with a pronounced dry season .

Wild individuals of Chrysophyllum cainito are distributed at low density throughout the forestsof the Canal Zone, particularly on the Pacific side of the continental divide, where they are found in old-growth as well as second-growth forests . For the purpose of our sample, we defined trees as wild if they were in natural forest habitat and if, to the best of our knowledge, people did not plant them. We used surveys and the knowledge of local indigenous people to locate over 150 adult wild trees, but only a percentage of these produced fruit in any given year. We included in our study every wild tree from which we could obtain enough fruits, including trees from Parque Nacional Soberanía, Old Gamboa Road, Venta de Cruces, Parque Nacional Camino de Cruces, and Parque Natural Metropolitano . Sample sizes differ among variables and years and are provided below; our sample sizes increased from 2006 to 2008 as we found new individuals and added new sites. We defined trees as cultivated if they were obviously planted either alongside rural dwellings or as horticultural plantings in more urbanized areas. Whenever possible, we interviewed owners to obtain direct information about who planted the tree; we did not use trees whose origin was ambiguous. Chrysophyllum cainito is primarily propagated by seed, apparently exclusively so in Panama. We roughly matched the sample sizes and locations of our cultivated sample to the wild tree sample, 25 liter plant pot including trees from the communities of Gamboa, Chilibre, Chilibrillo, Paraíso, Ciudad del Saber, Los Rios, Burunga, and Balboa . From among the available trees, we chose our cultivated sample based primarily on access, as it was not always possible to get permission from owners to collect fruits.We collected five to thirty fruits per tree, and of these, five or three were randomly selected for analysis. We completed measurements on fruits almost always within several hours of collection, and always within three days. For each fruit, we took a photograph, noted color, measured fresh weight, length and diameter, and toughness using a penetrometer . We then cut the fruit open and measured the thickness of the exocarp, or rind . We extracted fluid from the fruit pulp by passing it through a fine filter and assessed sugar content with a hand-held refractometer . We counted the number of seeds and measured mean seed mass . One wild tree produced no healthy mature seeds, decreasing the sample size for seed mass to 28. Fruit shape is expressed as diameter/length, and proportion allocation to exocarp as /diameter.To compare wild and cultivated classes, we used nested ANOVA with trees nested within class , and samples nested within tree. Nested ANOVA also allowed us to test for significant variation among parent trees within the classes. We used Levene’s test to test for a difference between cultivated and wild trees in the variance of each trait. For any trait that showed heterogeneity of variance, P-values from the ANOVAwere suspect. Therefore, for the subset of traits that tested significant for Levene’s test, we performed Welch’s t-test assuming unequal variances on the comparison between wild and cultivated fruits—but this test could not be used to test for significant variance among trees within classes. Chemistry data from 2006 were analyzed separately from the 2008 data. While the geographical logistics of this system did not allow for the sampling of truly independent but comparable regions, our sample in 2008 was large enough to support separating the data into Northern and Southern sub-regions. When the analyses were done separately on these sub-regions, we obtained essentially the same patterns of difference between wild and cultivated classes for the various traits. Therefore, only the combined data are presented here. Pairwise correlation coefficients were calculated among traits in the 2008 sample and among chemical traits in the 2006 sample . Correlations between fruit and leaf phenolics could not be estimated because they came from different sets of trees. We used Discriminant Analysis to test whether the classes of wild and cultivated trees could be distinguished from each other, and what traits contribute to this discrimination. From the 2008 dataset, we included the following variables: Fruit mass, fruit diameter/length, exocarp proportion, toughness, seed number, seed mass, and sugar concentration . We left out the most redundant variables: Fruit length, diameter, and absolute exocarp width. We used forward stepwise variable selection to choose variables that significantly contributed to the model , then ran the model. For all trees in the sample that produced fruits both in 2007 and in 2008 , we calculated correlation coefficients between the mean trait value in 2007 and the mean trait value in 2008.We found many traits that differ significantly between wild and cultivated fruits of C. cainito. Fruits from cultivated genotypes were larger and had more pulp and less exocarp, resulting in a higher “economic ratio” sensu Clement . Fruit pulp had significantly higher concentrations of sugar, was less acidic, and had lower concentrations of phenolics. Seeds were bigger and more numerous and were less defended with phenolics in cultivated than in wild fruits. Historical accounts mention caimito already being cultivated in Panama in the mid-1500 s . Although these data are scant, we can infer that caimito has been utilized as a fruit crop for at least 450 years and possibly much longer. Caimito trees may come into bearing within about 15 years, allowing us to estimate that a minimum of 30 generations or possible rounds of human-mediated selection have occurred to date. Interestingly, early historical accounts mention the presence of large and small fruited types , suggesting that substantial selection may have already occurred by that time. The sugar concentration in C. cainito is extremely high. In 2008 we measured a mean °Brix of 14.9, with a maximum value of 28.