Author: naturehydro

A strawberry hydroponic gutter system is a method of growing strawberries using a hydroponic system that utilizes gutters as the growing medium. Hydroponics is a technique for cultivating plants without soil, where the plants receive all the necessary nutrients through a water-based solution.

In a hydroponic gutter system specifically designed for strawberries, a series of gutters are set up at an incline. The gutters typically have holes or channels where the strawberry plants are placed. The plants’ roots hang down into the flowing nutrient-rich water solution.

Here are the key components and considerations for a strawberry hydroponic gutter system:

1. Gutters: The gutters used in the system can be made of various materials such as PVC, metal, or plastic. They should be designed to hold the plants securely while allowing the roots to access the nutrient solution.
2. Nutrient Solution: The nutrient solution contains a balanced mix of essential nutrients that strawberries need for healthy growth. It typically includes macronutrients (such as nitrogen, phosphorus, and potassium) and micronutrients (such as iron, zinc, and calcium). The solution is regularly pumped or flowed through the gutters, ensuring the roots have access to the nutrients.
3. Substrate: A substrate or growing medium may be used in the gutter system to support the strawberry plants. Common options include coconut coir, perlite, vermiculite, or rockwool. The substrate holds moisture and provides stability for the plants.
4. Irrigation: An automated irrigation system is necessary to supply the nutrient solution to the gutters. This can be accomplished using drip irrigation, where small tubes or emitters deliver a regulated amount of nutrient solution to each plant or gutter.
5. Lighting: Depending on the location and available natural light, supplemental artificial lighting may be needed to ensure the strawberries receive sufficient light for photosynthesis. LED grow lights are commonly used in hydroponic systems to provide the specific light spectrum needed for plant growth.
6. Climate control: Controlling the temperature, humidity, and ventilation is crucial for optimal strawberry growth. Proper airflow and ventilation help prevent diseases and provide an environment suitable for plant development.
7. Planting and maintenance: Strawberry plants are typically propagated from runners or small transplants and placed into the gutter system. Regular monitoring of pH levels, nutrient concentrations, and overall plant health is necessary. Pruning, removing dead leaves, and ensuring pollination (if not using self-pollinating varieties) are also important for productive plants.

A hydroponic gutter system offers several advantages for growing strawberries. It allows for high-density planting, efficient use of space, and easier access to the plants for maintenance and harvesting. Additionally, since hydroponics avoids soil, the risk of soil-borne diseases and pests is minimized.

However, it’s important to note that designing and managing a hydroponic gutter system requires some expertise in hydroponic gardening. It may be beneficial to consult resources, attend workshops, or seek guidance from experienced growers to ensure the best results.

Low resolution phylogenetic trees made using the chloroplast regions mentioned above have been reported for other taxa, including Curcuma and Sisyrinchium . The inadequate resolution may be due to the lower substitution rates and lack of variation found in single plastid regions. Thus, we do not recommend single plastid regions as DNA barcodes for this the genus. Among the candidate barcode genes, the Consortium for the Barcode of Life Plant Working Group suggested that rbcL, matK, and the rbcL+matK combination should be sufficient for a plant barcode, and that this combination should be supplemented with additional markers as required . In addition, Kress et al. and Chase et al. proposed that trnH-psbA can be used in two-locus or three-locus barcode systems to improve resolution. For instance, two of the three combinations of the three chloroplast loci tested in this study, rbcL+trnH-psbA and rbcL+matK+trnH-psbA exhibited higher discriminatory performance than any single marker. Consequently, this highlights the need to use chloroplast multi-locus barcodes to improve the resolution of species identification in Pulsatilla. The nuclear ITS region provided the highest inter-and intraspecific divergences and had a higher success rate for the correct identification of species in TAXONDNA . However, as for the treebuilding method,large pot with drainge the discriminatory performance of ITS is not satisfactory, as its highest resolution is 39.02% .

As evidenced by previous studies, the multi-locus barcode is one of the combinations that demonstrated the highest species resolution rate, e.g., Aceraceae , Lysimachia , Oberonia , Rhodiola and Schisandraceae . However, in this study, addition of ITS to different kinds of combinations of chloroplast markers did not increase the resolution rate obviously . The resolution rate based on tree-building analyses was 51.22% for BI and 58.54% for PWG. In addition, we found no distinct barcoding gap. This phenomenon may be due to the one or more of several reasons. first, incomplete lineage sorting and non-homogeneous concerted evolution are likely to occur at the ITS locus . Second, the three chloroplast regions cannot compensate for the drawbacks of ITS because they are sourced from a different genome. Although the nuclear genome is inherited biparentally, the chloroplast genome is inherited uniparentally. Thus, the chloroplast genome experiences more complete lineage sorting than the ITS locus does. Third, hybridizations may cause conflicts between ITS and chloroplast loci, as well as problematic results in ITS phylogeny due to the possibility of homogenization to paternal copies in some lineages and maternal copies in others. A combination of DNA markers from different genomes— which have different modes of inheritance and conflicting phylogenies—can hinder our understanding of species delimitation and the evolutionary processes of speciation. Because of its myriad variable sites that can reliably distinguish species, resulting from a high mutation rate and rapid concerted evolution, we recommend ITS as a good single barcode for the genus Pulsatilla.Phylogenetic identification and species recognition are foundationally important for biology . The results of the phylogenetic analyses performed in this study may shed some light on the identification and taxonomy of the genus Pulsatilla . Here, we found that Pulsatilla formed a monophyletic group with high support. Moreover, the three recognized subgenera — i.e. subg. Pulsatilla, subg. Kostyczewianae, and subg.

Preonanthus— were resolved as distinct monophyletic groups, which is consistent with the recent phylogenetic result . Within subgenus Pulsatilla, our analyses found that P. camanella and P. ambigua were resolved as sister to one another with high support. These two species share many common morphological characters, such as almost fully expanded leaves at anthesis,black plastic planting pots and dense, long trichomes. The flowers of both species nod before anthesis . However, during anthesis, the sepals of P. camanella can easily be distinguished from those of P. ambigua by color . At the same time, the micro-morphological characters of the leaves are also different . Actinocytic and anomocytic stomata exist in both species, but most stomata in P. camanella are actinocytic, whereas most are anomocytic in P. ambigua. Thus, molecular data as well as micromorphological characters can distinguish between these two species relatively well. Both types of evidence may be helpful to accurately identify specimens that are damaged or lack sufficient diagnostic characters. In addition to its use in identifying specimens, DNA barcoding is also useful for resolving taxonomic uncertainty .They did not have distinct barcodes. The micro-morphological characters were also found to be the same, since both plants showed polygonal epidermal cells with striation, a dense distribution of stomata, and glabrous or sparsely short trichomes. In addition, the geographical distribution of these two species overlaps in Inner Mongolia. Taken together, these distinct lines of evidence collectively suggest that P. turczaninovii and P. tenuiloba are the same species. The discovery of hybridization, introgression, and/or incomplete lineage sorting among species is another useful application of DNA barcoding . The chloroplast region is inherited maternally, but the nuclear genome, including the ITS region, is inherited biparentally . Thus, if there are different results in different phylogenetic analyses from chloroplast and nuclear data, we speculate that these differences may be caused by hybridization and/or introgression among species, which could result in a non-monophyletic clade. In subg. Pulsatilla, we found several complex groups. The samples of P. chinensis and P. cernua in cluster III, were indistinguishable. In the Bayesian inference tree based on ITS sequences , the samples of P. cernua clustered in a clade along with sample P. chinensis. However, in the Bayesian inference tree based on the combination of chloroplast sequences , sample P. chinensis clustered in a clade with all other samples of P. chinensis. P. chinensisis a widespread species and has a geographical range that covers that of P. cernua; in addition, sample P. chinensis was collected near populations of P. cernua in Jilin Province, China. Hybridization or introgression might have occurred during the speciation of P. chinensisis and P. cernua. A similar situation was also found for sample P. ambigua108 and cluster I , suggesting hybridization may have occurred between P. ambigua and P. tenuiloba/P. turczaninovii.

In addition, as we strictly controlled cropload to a similar level each year by thinning flowers and young fruits, dry matter accumulation was not drastically different between the transgenic lines and the CK in our experiment.In response to a decreased sorbitol supply from source leaves, both the transcript level and the activity of SDH decreased in the transgenic fruit, which is consistent with previous findings in apple fruit cortex tissues fed with sorbitol and in shoot tips and fruit of the transgenic trees. As most of the fructose in apple fruit is converted from sorbitol by SDH, a significantly lower fructose level had been predicted in the transgenic fruit based on dramatically reduced import of sorbitol into the transgenic fruit and the associated lower SDH activity. However, the fructose level in the transgenic fruit was remarkably similar to that in the untransformed CK: no difference before 74 DAB and at harvest with only a slight difference detected at rapid fruit expansion between the transgenic fruit and the CK . This near homeostasis of fructose level in the transgenic fruit has clearly resulted from the response of the Sucrose cycle and the associated sugar transport system to increased availability of sucrose in the transgenic fruit, specifically, more sucrose is taken up into parenchyma cells in fruit after phloem unloading; more fructose is generated from sucrose breakdown by NINV and sucrose synthase and less fructose is phosphorylated by FK in the cytosol; and tonoplast sugar transporters are upregulated to take up more hexoses into the vacuole. In apple fruit, sucrose as well as sorbitol enters the parenchyma cells via the apoplastic pathway after being released from SE-CC complex. In many species that employ apoplastic unloading for sucrose in sink cells,draining plant pots sucrose is mainly converted to glucose and fructose by CWINV in the cell wall space and then transported into the parenchyma cells by hexose transporters. CWINV is typically considered as a sink-specific enzyme and its activity is usually very low in source leaves.

However, we found that, except for MdCWINV3 in 40- DAB fruit, the expression of MdCWINVs was much lower in the fruit than in the shoot tips where sucrose unloading is symplastic . In yeast cells expressing apple SOTs, sorbitol uptake is competitively inhibited by glucose and fructose but not by sucrose. So we postulate that most sucrose is directly transported into the parenchyma cells by plasma membrane-bound SUCs in apple fruit to avoid inhibition of sorbitol uptake by sucrose-derived glucose and fructose. Increased sucrose import into transgenic fruit did not alter the activity of CWINV but significantly elevated the transcript levels of both MdSUC1 and MdSUC4 , indicating that more sucrose is taken up into the parenchyma cells in the transgenic fruit. There are two pathways for sucrose breakdown in the cytosol of fruit parenchyma cells: conversion to fructose and glucose by NINV or to fructose and UDP-glucose by SUSY. The upregulation of transcript levels of MdNINV1, MdNINV3, and MdSUSY1-3 and activities of NINV and SUSY in the transgenic fruit , which is indicative of higher availability of sucrose in the cytosol, generates more fructose. This, combined with a lower FK2 transcript level and a lower FK activity , makes enough fructose available in the cytosol for accumulation in the vacuole of the transgenic fruit to largely compensate for the reduced level of sorbitolderived fructose. The higher NINV activity is also expected to elevate the glucose level in the cytosol, which may have led to higher transcript levels of MdHKs and a higher HK activity through glucose signaling and a higher dark respiration rate in the transgenic fruit . The higher HK activity detected in the transgenic fruit is similar to that of rice leaves in response to glucose manipulation. However, we found that increases in both HK activity and the glucose concentration did not enhance, but rather diminished, the accumulation of G6P in the transgenic fruit. This is likely due to a decrease in F6P flux from phosphorylation of fructose along with an increase in dark respiration such that more G6P was reversibly converted to F6P. Our result is consistent with the finding that glucose derived from sucrose contributes to the hexose phosphate pool more than fructose derived from sorbitol or sucrose in the apple fruit.

Despite a higher glucose flux going through dark respiration in the transgenic fruit, more glucose is still available for transport into vacuole for accumulation as indicated by the 3–6-fold increase in glucose concentration in the transgenic fruit at harvest. Higher fruit glucose levels have also been reported for these antisense plants by Teo et al.but to a lesser degree. It is interesting that greater import of sucrose did not significantly increase its concentration in transgenic fruit except at 74 DAB. We think that two factors may have contributed to this near homeostasis of sucrose in the transgenic fruit. first, upregulation of sucrose breakdown described above uses more sucrose. Second, downregulation of MdSPS3 and MdSPS6 transcript levels and SPS activity in the transgenic fruit makes less sucrose re-synthesized from F6P and UDP-glucose. While upregulation of SUSY in response to increased sucrose supply was observed in both fruit and shoot tips of the transgenic plants, NINV responded in the transgenic fruit but not in the shoot tips. The exact reason for this difference is not known, but differences in sucrose concentration and/or presence of different isoforms of NINV between fruit parenchyma cells and shoot tips might exist. Our findings on activities of SUSY, NINV, FK, HK, and SPS are not in agreement with those reported by Teo et al.. We believe that the discrepancy might be related to the difference in the way fruit samples were taken. In our study, it took about 2 min to cut and freeze fruit samples on site in the orchard, but in Teo et al.all harvested fruits were placed on ice before being transported to the laboratory and it was only after several quality indices were measured that the cortical tissues were frozen for further analysis. Because import of sorbitol and sucrose into fruit stops upon detachment from the tree, both enzyme activity and gene expression may be altered if they are not frozen in liquid nitrogen in a very short period of time. In addition, strict cropload CK in our study as reflected in much larger fruit might have made the difference between the transgenic fruit and the CK easier to be detected. Most of the hexoses and sucrose in fruit parenchyma cells are stored in the central vacuole that occupies >80% of the cell volume.

The uptake of these sugars into the vacuole is carried out by sugar transporters located on the tonoplast. Transcript levels of MdvGT1 and MdvGT2, both of which are vacuolar glucose transporters encoded by two Malus orthologs of AtvGT, were higher in the transgenic fruit than in the CK , suggesting that more glucose is transported into the vacuole of the transgenic fruit. This is consistent with the glucose concentration measured on bulk fruit samples . In addition, tonoplast monosaccharide transporters can transport both glucose and fructose into the vacuoles,drainage gutter and Arabidopsis TMT1 activity for fructose is approximately 30% of that for glucose. In the five Malus orthologs of TMT, it is possible that proteins encoded by MdTMT1 and/or MdTMT2 have high ability to transport fructose, and the enhanced expression by MdTMT1 in the transgenic fruit might indicate a regulatory response to the reduced flux of fructose derived from sorbitol. Alternatively, as fructose-specific TMTs have not been identified in fructose-accumulating fleshy fruits, the upregulation of MdTMT1 could be triggered by higher levels of glucose derived from sucrose in the transgenic fruit. In addition to hexoses, the vacuoles in ripening apple fruit accumulate a high concentration of sucrose. So far, no SUC has been identified to have proton-coupled antiport activity for loading sucrose into the vacuole, but AtTMT1/2 probably represents a proton-coupled antiporter capable of transporting both glucose and sucrose into the vacuole. A recent report on TMTs in sugar beet indicates that one of the two TMT2 proteins has developed specific affinity to sucrose and is responsible for sucrose accumulation in the taproots. The expression patterns of both MdTMT1 and MdTMT2 are in general agreement with that of sucrose accumulation in our apple fruit. It has been demonstrated that interruption of carbohydrate import into fruit by girdling or adjustment of cropload did not alter the fructose level in apple fruit. Contrasting light exposure did not appear to affect peel fructose level either. The data obtained from the transgenic fruit in this study provides further evidence for supporting the idea that the Sucrose cycle and the associated transport system operates to maintain the homeostasis of fructose in the apple fruit. From an evolutionary perspective, having fructose homeostasis in the apple fruit may help seed dispersal for this species because fructose is the sweetest among all the soluble sugars present in fleshy fruits. In conclusion, when sorbitol synthesis is decreased by antisense suppression of A6PR in the source leaves of apple trees, less sorbitol but more sucrose is transported from the leaves to the fruit. In response to the lower sorbitol/higher sucrose supply, sorbitol metabolism is down regulated, whereas breakdown of sucrose is upregulated in the transgenic fruit to compensate for the decreased flux of fructose derived from sorbitol.

This altered sugar metabolism, together with corresponding changes in the sugar transport system, leads to near homeostasis of fructose and sucrose and much higher levels of glucose and galactose in the transgenic fruit. This study clearly demonstrates the metabolic flexibility and the advantages of having two transport carbohydrates in sorbitol-synthesizing Rosaceae tree fruit species and the central role of the Sucrose cycle and the sugar transport system in determining sugar metabolism and accumulation in fleshy fruits.Five-year-old trees of the untransformed CK and transgenic lines of “Greensleeves” apple with antisense suppression of A6PR expression were used. A6PR activity in mature leaves of A27 and A04 was decreased to about 30% and 15% of that of CK, respectively. All trees were grafted onto M.26 root stocks and grown outdoors at Ithaca, NY, USA, under natural conditions, in 55-L plastic pots containing a sand:MetroMix 360 medium . There were five replicates for each genotype with three trees each arranged in a completely randomized design. The trees were trained as a spindle system at a density of 1.5 × 3.5 m2 . They were moved into a screen house for the entire bloom period to prevent pollen escape and hand-pollinated using mixed pollen of crab apple and several commercial varieties. The cropload of these trees was adjusted by hand-thinning to four fruits per cm2 trunk cross-sectional area at 10-mm king fruit size. During the growing season, the trees were supplied twice weekly with 15 mM N using Plantex® NPK with micro-nutrients .At 40 DAB , 74 DAB , 108 DAB , and 134 DAB , fruits were sampled from the south side of the tree canopy between 12 noon and 2:00 P.M. under full sun exposure. On each sampling date, five replicates per genotype with at least six fruits each from three trees were harvested. The fruits were immediately weighed, cut into small pieces after removing the core, and frozen in liquid nitrogen on-site. The entire process took approximately 2 min. To estimate the levels of transport carbohydrates, five replicates of leaf petioles and fruit pedicels with four each were covered with aluminum foil for 7 days prior to sampling on 75 DAB and were frozen in liquid nitrogen along with mature leaves. All samples were stored at −80 °C.Net CO2 assimilation rates of bourse shoot leaves were measured using a CIRAS-1 portable photosynthesis system with a broad leaf chamber at 5 tree developmental stages from 30 DAB to fruit harvest. On each sampling date, two bourse shoot leaves per replicate were measured at mid-day under full sun exposure and ambient temperature and relative humidity conditions. Fruit respiration was measured with the CIRAS-1 gas exchange system connected to a custom-made chamber, which accommodated the entire fruit for each sample. In the early afternoon on each of those four sampling dates, fruits under full sun exposure were detached from the trees and dark-adapted at 25 °C for 30 min before taking respiration measurements, and one fruit per replicate was measured.Although a large literature describes how recessions affect non-agricultural labor markets, few studies examine the effects of recessions in the seasonal agricultural labor market.

The extreme remodeling of pods by St and To eliminates pod shattering, but makes pods extremely difficult to thresh , and the alleles are therefore impractical for dry bean market classes. Since arid conditions are predicted to increase in coming decades , shattering-resistance alleles will be of increasing value for plant breeders. Despite this, little information exists on the degree of pod shattering in major market classes, the pattern of dominance and epistasis between resistance alleles, or the diversity available at each of these loci. Crucially, breeders also still lack genetic assays to evaluate the trait in segregating populations. Addressing these barriers will be critical to improve the productivity of a major source of nutrition globally. Three populations were evaluated in this study: a biparental population and two diversity panels, which represent each of the two domestication events of common bean. The biparental population was developed to study two shattering-related QTLs, their patterns of dominance and their interactions. Cultivars ‘Maylower’ and ‘Bill Z’ showed total resistance to pod shattering when field grown in Davis in 2017 . These varieties were among the most distantly related accessions in the MDP, with neither showing any evidence of admixturebetween ecogeographic races . May lower is a navy bean type , which possesses a SNP allele on Pv08 that is weakly associated with resistance to pod shattering in race Mesoamerica. Bill Z is a pinto bean type and has a SNP variant on Pv03 associated with strong PvPdh1-mediated shattering resistance common in race Durango. The population can therefore be used to determine if a reduction in pod shattering was independently selected in each of these ecogeographic races. An F3 population of 138 individuals was developed by hybridization between these cultivars. Each F3 individual was descended from a distinct F2 plant,best vertical garden system and all of the F2 s were the progeny of a single F1 developed by cross-pollinating Maylower and Bill Z. This 138-member Maylower x Bill Z population was used to validate the possible alleles on Pv03 and Pv08 and test any patterns of dominance and epistasis between the loci.

The two diversity panels were grown to evaluate the degree of pod shattering across diverse accessions of common bean. In 2016, 98 members of major market classes in the Andean Diversity Panel were ffield-grown in Davis, California, to evaluate each variety’s susceptibility to pod shattering. In 2017, 278 varieties of the BeanCAP Middle American Diversity Panel were similarly ffield-grown in Davis to evaluate pod shattering. At maturity, a sample of pods was harvested from each variety. Mature pods of all phenotyped varieties were harvested and then exposed to seven days of desiccation at 65 °C and a further seven days of re-equilibration to room temperature. The desiccation conditions for all varieties were identical, and desiccation was conducted using the same drying chamber. The proportion of pods dehiscing in this treatment was recorded, along with the market class of each variety. For evaluation of pod twists in the MxB population, all non-shattering pods were fractured by hand, and then, all pods were subjected to the desiccation treatment and re-equilibration again. The number of twists was counted for ten pods of each genotype, with “1” indicating a complete 360° rotation of the valve.DNA was extracted from young trifoliate leaves of the greenhouse-grown biparental MxB F3 generation, using a modiied CTAB protocol . DNA was quantiied with a NanoDrop spectrophotometer and genotyped using the BARCBean6K_3 BeadChip , yielding 5398 initial SNPs. SNPs that were missing or heterozygous in either parent or identical between the parents, were filtered from further analysis. The remaining SNPs were arranged into a linkage map using the ASMap R package . SNPs that did not map to one of the 11 major linkage groups were removed, leaving 1794 SNPs for QTL mapping. QTL mapping was conducted using the expectation maximization method in R/qtl . Phenotypes for QTL mapping were generated by harvesting all the pods from each greenhouse-grown F3 plant , then subjecting them to seven days at 65 °C and seven further days of re-equilibration to room temperature. Pods that had fractured to the tip of the beak due to this treatment were counted as shattered, while those with no opening or only issuring along the sutures were considered non-shattering.The maximum LOD score of 1000 randomized analyses of the data was used as a significance threshold. To test dominance, F3 individuals were subset by genotype at highly significant SNPs, and comparisons were made between groups by t-test.Next, the 43 SNPs within 100 kb of PvPdh1 in the MDP data set were analyzed to identify patterns of selection and diversity around the gene.

To simplify and visualize the data, principal component analysis was performed on the SNPs using R. Sequence variation was converted to integer values and the imputePCA function of the missMDA package was used to impute missing data . The genotype data were also sorted to identify unique haplotypes within the populations. The degree of similarity between the PCA and haplotype diversity was then compared. Individuals with missing data for SNPs distinguishing the haplotypes or haplotype clusters were not shown in plots and not numbered in plots as they could not be unambiguously placed within any haplotype group.The PvPdh1 putative causal polymorphism was used to develop a Cleaved Ampliied Polymorphic Sequence marker for efficient screening of breeding populations. The sequence surrounding the SNP was extracted using Phytozome 12 . Restriction enzymes that would differentially cut the alternative alleles were identified using RestrictionMapper version 3.0 . PCR primers were developed for the locus based on the sequence of accession G19833 , using the NCBI primer BLAST tool, and were then checked against the genome sequence of BAT93 to ensure that the sequences were identical and would successfully amplify membersof both major gene pools. The sequence surrounding the SNP was ampliied using the primers PDH1-TAQII-2F and PDH1-TAQII-2R . PCR was conducted with Takara ExTaq and included an initial elongation at 95 °C for three minutes, 44 cycles with denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, elongation at 72 °C for 60 s, and a final elongation of 72° for five minutes. PCR products were cleaved with ChimerX TaqII during a 65 °C degree incubation for seven hours, and run on a 2.5% agarose gel. The SNPs tightly linked to PvPdh1 in the MDP data set were then screened for other positions that could be useful for conversion to additional CAPS markers. The SNP closest to PvPdh1 in this data set, at Pv03 position 49,132,438 , is distinguishable by EcoRI and is highly correlated with pod shattering. Unlike the TaqII-based CAPS marker, the allele cleaved by EcoRI is the shattering-resistant variant, reducing the risk of falsely identifying a susceptible individual as resistant due to technical errors in digestion. The SNP distinguished by EcoRI is separated from the PvPdh1 causal polymorphism by less than 7 kb. The sequence surrounding this SNP was ampliied using the primers PDH1-ECORI-1F and PDH1-ECORI-1R . Marker development used the same methods as the TaqII-distinguishable marker, and the same PCR conditions successfully amplified both fragments.

The amplicons were then digested by Promega EcoRI at 37 °C for 15 min, and the PCR products were resolved on a 2.5% agarose gel. Major discrepancies in pod shattering exist between the major market classes of common bean . In the Andean gene pool, pod shattering is highest in the cranberry market class,vertical farming equipment with a mean value of 41% of pods shattering after desiccation. The purple speck/mottled market class has the greatest degree of shattering resistance among Andean beans, with only 3% of pods shattering after the same treatment. In Middle American beans, pod shattering is highest in the black and navy/small white market classes of race Mesoamerica, and lowest in the pinto , great northern and pink classes of race Durango. PvPdh1-mediated resistance to pod shattering is found almost exclusively in pinto, great northern, and pink market classes and is therefore associated with levels of pod shattering which may be the lowest of those of any major economic groups of common beans grown for grain.Three major SNP haplotype clusters were identified in the sequence surrounding the PvPdh1 gene . The most distinct of these included six individuals, several of which are of known Andean ancestry. The first principal component of the genetic data explained 64% of the variation and separated this group from the two other major clusters. The second principal component explained 25% of the variation and separated varieties belonging to race Mesoamerica from race Durango. Five individuals with missing data for the ten SNPs that distinguish these ecogeographic races were filtered from subsequent analyses. Additionally, cv. ‘Tepary 22′ and cv. ‘Jackpot’ exhibited highly unique haplotype patterns. Jackpot shows recombination in the region between the predominant haplotype of the Andean gene pool and race Durango, while P. acutifolius is a separate but closely related species . Races Durango and Mesoamerica differed in haplotype diversity around the PvPdh1 locus . The race Mesoamerica haplotype cluster includes six unique haplotypes. The most common of these includes 137 of the 148 varieties that can be clustered into a group unambiguously , without missing data in the SNP positions distinguishing the sub-groups. The race Mesoamerica haplotypes displayed 18% shattering on average. In contrast, race Durango varieties display only three haplotypes. The most common of these haplotypes includes 178 of 182 unambiguous varieties , with an average proportion shattering of 0.6% in this group. The two low-frequency race Durango haplotypes showed no shattering when field-grown in 2017 .The TaqII-based CAPS marker of the PvPdh1 causal polymorphism leads to cleavage of susceptible alleles, while resistant alleles are not cut. The total pre-digestion amplicon length in G19833 was 578 bp, comparable to the 580 bp amplicon of BAT93. After digestion, susceptible alleles were cleaved into fragments of 449 and 129 bp in G19833. While digestion was seen in all shattering-susceptible samples after digestion with TaqII, this enzyme led to only partial digestion in a minority of cases. The EcoRI-digestible CAPS marker was extremely robust, and never led to partial digestion. After digestion, this marker led to resistant alleles that were cut into fragments of approximately 332 bp and 310 bp, while susceptible alleles remained uncleaved at 642 bp in BAT 93 . Andean varieties showed comparable fragment sizes, such as 639 bp in G19833. The EcoRI-digestible CAPS marker never experienced partial digestion or ambiguity. The SNP used for this marker has a strongly significant correlation with pod shattering , and is one of the 10 SNPs contributing to the haplogroup differentiation between race Durango and race Mesoamerica . The median proportion of pod shattering among 97 varieties with the shattering susceptible allele was 0.14, equal to the maximum level of shattering seen in any of the 160 varieties carrying the resistant allele . Themedian proportion shattering of those varieties with the resistant allele was 0.00%. Testing for multiple origins of Middle American shattering resistance and allelic effects Only one major locus was identified with an effect on pod shattering in the Middle American domesticated gene pool of common bean. These results highlight the important role of the Pv03 PvPdh1 locus in this population. The lack of a major QTL on Pv08 in the MxB population suggests that Maylower has no shattering resistance allele on that chromosome which is not also found in the distantly related Bill Z. Because the Pv08 SNP identified through GWAS did not reach significance after a Bonferroni correction and only 11 of 280 members of the MDP possessed this SNP, our results indicate that this chromosome does not have a major, widespread role in regulating pod shattering in the Middle American domesticated gene pool. This does not preclude the possibility that the QTL has a role in shattering of Andean beans, the latter of which has been demonstrated with much greater conidence . The locus may also have a role in regulating pod shattering in a very small proportion of Middle American bean varieties, possibly through de novo mutation in race Mesoamerica or introgression from the Andean gene pool.

Several of the cache pits were made entirely of flat slabs of quartz diorite that evidently had fallen from the ceiling of the shelter. In each case, these tabular rocks were carefully overlapped to make as tight a construction as possible .Other slab-lined cache pits differed in that the rocks were placed in a mosaic fashion rather than overlapping. No small chinking rocks were noted in any of these features. One of these was constructed globular, as opposed to tabular, form. In some cases, smaller rocks were used to chink spaces between the larger rocks . One cache pit , constructed of irregularly shaped rocks, lacked any small rocks or predominantly of tabular rocks and was placed such that two massive boulders were part of the overall construction. Incorporated into its construction were two metate fragments, both of granitic material. The fill of the feature consisted of miscellaneous rocks and soil, a mano and two mano fragments, and one complete unifacial block metate. Some ofthe milling implements or other rocks from the fill may have been used in its construction. The majority of the cache pits excavated at Indian Hill Rock shelter were constructed of medium- to large-sized rocks of overall chinking, and there were large spaces between the rocks. Some rocks may have been taken from this feature and reused in the construction of the overlapping cache pit or for another purpose. One of the rocks used in the feature is a bifacial block metate; also present was a rock with a patch of red hematite paint. Feature 19 was a well-constructed rock-Hned basin. It was fairly shallow and apparently represented the floor of a cache pit. Construction material consisted of about 40 rocks,hydroponic vertical garden including one core-hammerstone, nine block metate fragments of granitic material , and one unifacial mano.

This feature was unique among the cache pits in that it contained so many large metate fragments. The situation at Indian Hill Rock shelter, then, is one in which rock-lined cache pits were built in sandy matrix using either flat slabs or other rocks, including metate fragments. Care was taken in some of the cache pits to overlap the slabs used in their construction, and in others to chink interstices between rocks. This suggests attempts to make the structures rodent-proof. We therefore conclude that bags or baskets of foodstuffs, rather than equipment, most likely were stored in these cache pits. We are inclined to classify these cache pits as group specific, permanent furnishings at Indian Hill Rock shelter. Each cache pit may have been used many times, each time carefully roofed over, perhaps with bunch grass to keep the sand out, and then rocks, and concealed beneath the sand that formed the floor of the shelter. Concealment may have been considered important in the event other groups used the shelter. We thus interpret the cache pits as more or less “permanent” site furnishings of whatever group used Indian HiU Rock shelter, but not publicly available site furniture. In all cases the contents of the cache pits had been removed in antiquity. In most cases, only the rock-paved floors of the cache pits remained intact, and most of the rocks that formed the walls were lacking. In some cases large rocks all but filled the cache pits, apparently having been discarded there upon abandonment of the cache. The roofing rocks and wall rocks, which may have extended to near the former ground surface, must have been pulled up out of the site matrix and used elsewhere for the same or another purpose. The ethnographic description of “pulling up” the site furniture , or “site furnishings,” in this case using it elsewhere at the site for the same or for another purpose, appears to describe the situation at Indian Hill Rock shelter. The levels from which cache pits are believed to have been dug have few or no ceramic sherds, but ceramics are common in overlying levels.

This distribution leads to the conclusion that ceramic ollas may have replaced rock-lined cache pits after the inception of ceramic technology in the region. Ollas may have been buried in the same sheltered sites that formerly contained rock lined cache pits. Rock-lined cache pits are reported from two sites, Chapman Rock shelter No. 1 and Resurrection Shelter . Four rock lined cache pits were excavated at Chapman Rock shelter No. 1. These cache pits were lined with basalt slabs, and metates were used in the construction of two of them. They ranged from ca. 75 to 95 cm. in diameter and from 45 to 60 cm. in depth. Two of these cache pits contained historic artifacts ; the third was built from a comparable level below the surface . Linings in these cache pits included such material as bunch grass; buckwheat plant parts, Joshua tree fiber, tule matting, and twined basketry. The fourth cache pit possibly may be older, as it was built far below the others, at about 1.2 m. below the present surface. This cache pit was not lined, but contained debitage, a small biface, a slate pendant fragment, a basalt mano, a bone bead, pinyon hulls, and twined basketry. A large, rock-lined cache pit was partially exposed during test excavations at Resurrection Shelter on the eastern slope of the Coso Range near Darwin Wash. The maximum diameter of this feature is estimated to be about 2 m., the depth about 40 cm. It was constructed of large, blocky rocks. None of these appeared to be metates, nor was any chinking with smaller rocks noted. The pit was lined with alternate layers of bunch grass plants and Joshua treebark. A covering of rocks was not discernible, but a large pile of rocks just inside the entrance of the shelter and next to this cache pit once may have covered it. Artifacts in and around the cache pit include basket fragments, a remnant of a fiber brush, fiber cordage, pinyon nut shells, fiber cordage wrapped with strips of skin, and painted reed fragments . The shelter is very small, and it may have been used only for storage. Some or all of the artifacts could have found their way into the cache when opened. Since pinyon trees grow several kilometers away at higher elevations, the nut shells apparently did not get into the cache pit accidentally. The available evidence suggests the cache served for storage of pine nuts rather than equipment. A detailed report by the investigators is in progress, and radiocarbon analyses are planned for selected materials from this cache pit .

Insufficient information is available to make a determination of the organizational strategy of the people that built the cache pits in the Coso Range. Ethnographically, the region was occupied by Panamint Shoshoni, who shifted from a dispersed forager strategy during the spring and summer to a clustered collector strategy during the fall and winter. Crown gall of grapevine reduces the grape yield by 20% to 40% and trunk diameter approximately by 9%, compared to uninfected healthy grapevines . The causal agent of the grapevine crown gall, Agrobacterium vitis, is a different biovar from other Agrobacterium spp. because it can induce a grapevine specific crown gall . A. vitis has host specificity-related genes, such as pehA, which encodes polygalacturonase that is hypothesized to facilitate A. vitis attachment and systemic colonization on susceptible grapevine tissues . Another host specificity-related gene function is tartrate utilization, and the host specificity is explained by the use of tartaric acid from specific organic acids in grapevines . Crown gall disease is one of the difficult diseases to eradicate by chemicals in vineyards. As a strategy to prevent crown gall disease without chemical sprays, vertical vegetable tower the use of resistance plant cultivars and antagonistic microbial species have been developed. Some grapevine species, mostly Vitis vinifera, are susceptible to crown gall disease, and thus, root stocks from resistant species or crossed seedlings are used by many vineyards to prevent crown gall disease on the scions of the susceptible grapevine cultivars . Another prevention strategy is the use of a non-tumorigenic A. vitis strain as host-specific biological control of grapevine crown gall, in which its pretreatment inhibits transformation by tumorigenic A. vitis strains on the grapevine, leading to the inhibition of crown gall induction . Recently, various platforms have been used to attempt to identify disease resistance-related metabolites in grapevine, including nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry . However, when using gas chromatography mass spectrometry , extracted metabolite-profiling data can annotate information from the metabolite libraries . Derivatization techniques are required to process GC-MS metabolite profiling . Silylation is the most suitable derivatization method for non-volatile metabolites, such as hydroxyl and amino compounds . One silylation agent, Nmethyl-N–trifluoro-acetamide , has been used to reveal the whole range of metabolites in plants . However, the highest advantages of GCMS analysis in metabolomics are noted as well-organized stable protocols from sampling to data analysis, covering relatively a broad range of compound classes .

Principal component analysis is an unsupervised method that attempts to create a model of the data without a priori information, which is able to provide an overview of the whole metabolite profile and find the differential metabolite profiling within the group using a qualitative analysis . Projections of latent structures analysis is a supervised analytical method that enhances the separation between groups, combining variable importance for projection scores to obtain a proper cutoff value and increase its performance . Orthogonal projections of latent structures analysis add a single component to the PLS analysis as a predictor of the model, and the other components describe the variation orthogonal to the first predictive component . OPLS discriminant analysis provides useful information about putative biomarkers using an S-plot that combines the covariation and correlation from the model in a scatter plot . The VIP score in PLS-DA and correlation value in OPLS-DA provide cutoff values for finding significant metabolites. This experimental method using GC-MS and multivariate data analysis can identify new aspects of pathogenicity-related metabolites. Genetic, physiological and morphological changes of the host plant to the infection of Agrobacterium species and in the crown gall development have been well documented in relation to plant responses to the tumorigenic bacterium before T-DNA transfer and during gall development . However, relatively little has been studied on those changes occurring in the non-tumorigenic plant tissues that have been influenced by the disease development , which may broaden the holistic understandings of plant responses to the crown gall pathogen infection and disease development. This may enhance their practical applicability in the disease control; e.g., use of crown gall-resistant root stocks. Therefore, in the present study, we examined metabolite profiling in the intermodal tissues of grapevine species in relation to plant responses to the crown gall development in grapevines induced by the infection of A. vitis using GC-MS and statistical multivariate data analysis. Grapevines used in our study were obtained from the National Clonal Germplasm Repository . For Vitis spp., 11 to 22 green shoot cuttings were collected from NCGR fields in June 2015 and placed in a perlite bed for rooting at 25o C with mist for one month. The green shoot cuttings with root systems were transferred to a pot with bed soil for pathogen inoculation test. The pathogen, kindly provided by Dr. Burr, Cornell University, was A. vitis. K305. The bacterium was cultured on potato-dextrose agar  at 25o C for three days. The bacterial cultures grown on PDA were collected by the agar surface scratching in sterile distilled water , and the population density was examined by dilution plate method , which was diluted with SDW to make a bacterial suspension with a concentration of 109 colony-forming units ml–1. For inoculation, a wound was made in the internode of the green shoot using a 2-mm-day drill and 10 µl of the bacterial suspension was injected into the wound using a micropipette. After inoculation, the wound site was immediately wrapped with sealing film and the green shoot cuttings were grown at 25o C in a greenhouse. Symptom development was examined every other day after inoculation throughout the experimental period. The GC-MS system consisting of a gas chromatograph and mass detector was used for GC-MS analysis of the metabolites extracted from the tissue samples.

The Avalon Green Alley project in south Los Angeles is designed to allow water to percolate into the soil and recharge the water table . Beneath the permeable pavement of the green alley, catch basins store storm water temporarily where it can be bio-remediated before flowing into surrounding soil. Scanlon et al. describe three categories of methods: physical techniques such as measuring flows , tracer techniques which include the use of chemical, isotopic, or historical tracers , and numerical modeling techniques . Challenges still exist such as those posed by spatial and temporal variability , but there are efforts to address them. For example, Tubau et al. developed a temporally explicit model for quantifying groundwater recharge. The value of groundwater recharge can be calculated using various methods; the most appropriate is dependent on the end-use of water. For example, in 2010, California withdrew 12,700 million gallons of groundwater per day of which approximately 22.2% was used for domestic purposes . The value of groundwater recharge can therefore be associated with the price of water to consumers. Replacement costs can also be used to assign a value to groundwater recharge. Artificial groundwater recharge is the spread of water on land to increase infiltration or the injection of water directly into the aquifer . These actions have associated costs and can be used to estimate a value for the same service performed by NTS.As storm water runoff flows over urban surfaces, it can acquire contaminants such as suspended solids, heavy metals, nutrients, and pathogens . Because southern California receives little precipitation, it allows for the build-up of contaminants and results in the “first flush” phenomenon: elevated levels of storm water pollution at the beginning of storm events . These contaminants can then enter larger bodies of water and degrade the local environment . Many highly urban areas,vertical hydroponics such as Los Angeles County, are coastal which can exacerbate this issue of water quality because storm water runoff can drain directly into the ocean with little opportunity for treatment.

There is a range of common storm water runoff pollutants. Industrial processes and traffic produce copper, lead, and zinc . Heavy metals can persist in the environment and accumulate in sediment, plants, and animals, leading to the degradation of environmental and human health . Major contributors to nutrient pollution are fertilizers, sewage, and erosion . The input of excess nutrients into streams, lakes, and the ocean can cause degradation of habitat and changes in community structure. Eutrophication can lead to harmful algal blooms, hypoxia, and anoxia, which can have cascading effects on the local ecosystem . Harmful organics from herbicides, pharmaceuticals, and industrial processes can be found in urban storm water runoff and be detrimental to biology . Pathogens also pose a risk to human health through exposure . Lim et al. found that captured storm water can be used for toilet-flushing with acceptable risk, but it does not meet required standards for showering and food-crop irrigation. Beach closures and advisories are often the result of bacteria levels exceeding water quality standards . In addition to the threat to human health, the resulting closures have associated economic costs, e.g. less use of parking lots, restaurants, and shopping . NTS remove contaminants through several pathways. Physical filtration removes debris and suspended solids . While this process may lead to clogging, and subsequent deterioration of bio-remediation functions, informed NTS design, such as plant selection, can help maintain infiltration capacity .A significant portion of bio-remediation in these systems is performed by soil microbial communities that can be stimulated by moisture . NTS can prevent contaminants from traveling further to pollute local bodies of water, but they can also concentrate pollutants in plants and filter media . These concentrated contaminants can leach into surrounding soils, e.g. due to lower oxygen levels that increase metal solubility. As a result, some maintenance is likely required in order to prevent build up and transport of contaminants into the environment. Contaminant removal may be the most well-studied service provided by NTS. Figure 5.3 shows examples of systems installed to remove debris and contaminants from runoff. The Grand Boulevard tree wells consists of seven water filtration systems that capture urban runoff from 6.8 acres of residential and commercial area before the water reaches the Santa Monica Bay .

The Filterra™ system utilized on Grand Boulevard has been shown to effectively remove suspended solids, heavy metals, and nutrients, at efficiency ratios ranging from 83-88%, 33-77%, and 9-70% respectively, by comparing pollutant concentrations between inflow and outflow . Removal rates of heavy metals by bio-retention systems can be quite high . Sediment and nutrient removal by NTS have also been found to be relatively high in several laboratory experiments . While these laboratory experiments provide a deep understanding of how NTS may function under specified conditions , in situ studies are also needed to put those laboratory experiments into context. How NTS operate over time is still an open question, both over long and short time scales, as well as how the timing of water quality measurements can affect results . There are many studies that estimate the value of improved water quality using a variety of methods. Contingent valuation, including discrete choice experiments, asks a sample of respondents about their willingness-to-pay for a spectrum of water quality . A production function approach can value improved water quality by comparing the cost of alternative methods for contaminant removal, such as a storm water treatment plant. The appropriate method is dependent on the fate of the storm water and the final ecosystem service it provides, e.g. clean water for drinking or clean water that hosts higher biodiversity.There are efforts to better quantify and value the water services discussed above , but NTS can also provide a range of non-targeted ecosystem services, linked to their utilization of natural structures and functions, and other co-benefits. Filter media and plant communities, which increase infiltration and remove contaminants, host biodiversity which contributes to ecological processes that can result in beneficial ecosystem services . These ecosystem services and other co-benefits are not generally considered during the design and assessment of NTS. One unique feature of NTS is that they are human-made, so they can be designed to provide specific benefits, which are discussed here to further expand design options that can enhance variety and value of services that can be provided by NTS.Vegetated NTS act as man-made ecosystems that contain a diversity of plants, animals, and microbes from which ecosystem services can be generated . They provide patches of habitat within an urban landscape, and potentially act as corridors through which organisms can move. This can be important for population connectivity and resilience in a changing environment due to removal of natural habitat, habitat fragmentation, and anthropogenic climate change .

Biodiversity is here loosely defined to encompass plant, animal, and microbial species richness, abundance, and distribution. While NTS biodiversity can be measured using a range of methods , how biodiversity translates into ecosystem services is more complex and will need targeted studies. Plants act as ecosystem engineers in bio-retention systems, influencing both hydrological and ecological features. Vegetation captures precipitation , undergoes evapotranspiration , maintains media porosity with roots, and assimilates pollutants . Plants determine photosynthesis and respiration rates, organic matter in soil, and ultimately, carbon sequestration and storage in NTS . Additionally, plant communities influence microbial and in faunal communities which subsequently impact ecosystem function. Microbial communities are most often assessed in terms of harmful taxa present or as functional groups . Fauna, except in wetland settings, are usually not considered in NTS design,hydroponic vertical farming systems despite their role in facilitating targeted functions and services. Kazemi et al. and Mehring et al. identify common bio-filter taxa, such as Megadrilacea , Enchytraeidae , and Collembola . Earthworms are known to increase water infiltration via burrows , while spring tails can impact plant growth and nutrient cycling . As a result, these soil invertebrates can be considered ecosystem engineers that move and aerate soil, shaping the microbial, floral, and faunal communities from which more ecosystem services can stem. Because urban NTS can receive more water than native ecosystems, they can host elevated biodiversity of local soil invertebrates relative to natural habitats . Higher biodiversity can be beneficial if they enhance service provision, or detrimental if they involve invasive species that disrupt function. The Ballona Freshwater Marsh is reported to have provided breeding and foraging grounds for 217 bird species in 2012 . Wetland bird species are used internationally to designate areas for conservation and, in addition to contributing to wetland biodiversity, provide recreational services . Increases in biodiversity have also been shown to increase human well-being in urban settings . Studies exist on the economic value of biodiversity in urban and engineered settings . These often employ contingent valuation, travel cost analysis, and hedonic property pricing methods to estimate society’s WTP for biodiversity and its conservation. Travel cost analysis equates the value of an amenity with the amount of resources used to enjoy that amenity. Hedonic pricing considers goods as a bundle of attributes that can then be manipulated in order to determine how people value those attributes. In urban areas, Dupras et al. estimated that urban forests in the Greater Montreal area in Canada host biodiversity that creates a value of $2623 2013 CAD per hectare annually. Brander et al. conducted a meta-analysis of wetland biodiversity that resulted in an average value of $17,000 1995 USD per hectare of wetland annually. Biodiversity associated with other human-made ecosystems, such as agricultural land, may also be relevant to constructed NTS .

Carbon dioxide emissions are the largest contributor to anthropogenic climate change , and as a result, climate-regulating services related to carbon , have become increasingly important especially in urban areas that contribute disproportionately to global emissions . NTS plants have the potential to contribute to this effort by converting atmospheric carbon dioxide into biomass through photosynthesis. How long this carbon is subsequently stored is dependent on several factors. While some carbon is quickly rereleased during respiration, some is stored as plant biomass and soil detritic compounds. Turnover rates vary with types of biomass. For example, woody biomass has slower turnover rates than biomass that contains chlorophyll . Turnover rates can also vary with soil moisture , soil oxygenation, soil organic matter , and microbial communities. Some bioretention systems contain saturated or submerged zones, designed to create anaerobic conditions for denitrification , but may also help prevent microbial breakdown of organic matter. Quantifying carbon sequestration and storage would necessitate measurements that include net carbon fluxes , soil and plant carbon density , and biomass turnover rates. This has been done in urban green spaces but not specifically in NTS. Nowak et al. estimated annual carbon sequestration in U.S. urban forests to be 25.6 million tonnes which, at $36 2015 USD per tonne of carbon , has a value of over $900 million 2015 USD annually. On smaller scales, green roofs have also been shown to sequester 375 g per m2 annually, additionally decreasing carbon emissions due to lowered electricity usage for cooling . Researchers at the University of California, San Diego, are currently working on evaluating net greenhouse gas fluxes over different urban landscapes, including NTS, that can potentially be associated with an economic value. In addition to physically storing carbon, increased green space can reduce air and surface temperatures, reducing electricity use and emission of greenhouse gases . The urban heat island effect occurs due to increased air temperature in urban settings, relative to undeveloped areas, as a result of replacing vegetation with pavement . Pavements, such as asphalt and cement, have lower surface albedo than natural vegetation and therefore absorb more heat. Evapotranspiration also contributes to plant regulation of micro-climates by increasing the amount of water in the air for a cooling effect. Additionally, vegetation can provide shade. Even small green spaces can have a significant impact on micro-climate . Factors that affect microclimate regulating ecosystem services include UV intensity, wind, and size of the green space. The value of micro-climate regulation by urban green space can, in many instances, be calculated using avoided costs methods. For example, if a green space makes an area cooler, people may not run their air conditioning as long or intensively.

This would not only require an investment in seed multiplication and breeding, but also a reorientation of farmer attitudes to see groundnuts as a cash crop rather than something grown mainly for household subsistence. Since 1985, communal and resettlement farmers have produced less than 4 500 tons per year compared with an annual total of more than 100 000 tons for commercial growers; in 1999/2000 LSC farmers produced a record harvest equal to nearly 140 000 metric tons. Most soybeans are grown in the high-potential areas of Natural Region II where approximately one third of the area planted on LSC farms is under irrigation. Zimbabwe is self-sufficient in soybean meal and regularly exports cake to South Africa, Zambia and other regional markets. Domestic market outlets for soybeans include use as a high protein cake for stockfeed; a high energy feed for livestock; a source of protein in corn-soy blends; a meat extender; and a direct human food. Large refining companies process more than 90% of Zimbabwe’s soybean harvest using solvent extraction methods that yield about 18% oil. Zimbabwe is not self-sufficient in edible oil, however, and regularly imports about one third of its annual crude oil requirement for domestic refining.13 Although price controls are not imposed on soybeans, it is sometimes difficult to obtain export permits for trade with higher-value international markets. Soybeans are typically grown by most LSC farmers on very large plots in rotation with irrigated winter wheat. This rotation is an important part of many farm systems, not only in terms of the revenue generated,equipment for vertical farming but also because soybeans help maintain soil fertility and offer a good way to share limited irrigation equipment. On the other hand, both soybeans and wheat grow best on heavier soils than those suited to tobacco and many farmers also prefer to cultivate these crops in different sections of their farm depending on soil type.

Most LSC farmers achieve a yield between 2.2 and 2.5 tons per hectare; smallholder yields are around 1.0 tons per hectare when fertiliser is used. The financial indicators for dryland and irrigated soybeans are summarised in Table 21. Compared with all other LSC enterprises, these data show that soybeans are highly unprofitable except for the fact that they are relatively inexpensive to produce and usually grown in large quantities throughout the year. It must also be kept in mind that soybeans are normally grown in rotation with irrigated wheat, which is highly profitable and can easily offset the net losses shown below. With high input management, the gross profits from dryland and irrigated soybeans are reasonably attractive, but still only sufficient to finance 6% to 8% of the preharvest cash costs for a single hectare of dryland flue-cured tobacco respectively. In terms of labour requirements, LSC soybeans are the least labour intensive crop analysed and rarely demand additional casual workers. Although relatively few smallholder farmers grow soybeans, a quantitative analysis was carried out to assess the overall viability of this enterprise. Efforts were made a few years ago to promote soybeans as a cash crop among smallholder farmers through a seed distribution program. However, most of the harvest was retained for home consumption and there has been little work to support smallholder soybeans since. The smallholder soybeans results are summarised in Table 22.These data show that soybeans are one of the least expensive crop options available to smallholder farmers. On the other hand, the estimated gross and net profits are also very low and all other enterprises offer a potential for greater farmer income. In interpreting these results, however, it should be noted that that the daily returns to family labour are higher than for any other crop that does not require additional hired workers.

The returns would be even greater if measured by the crop’s imputed food security value, suggesting that soybeans may be an especially good food security choice for households with a shortage of active workers. As a cash enterprise, however, smallholder farmers do not have the capacity to grow soybeans in large quantities and so are unable to achieve the same economies of scale that help to justify LSC production. LSC farmers grow virtually all the wheat produced in Zimbabwe as an irrigated winter crop. Total production is normally between 225 000 and 250 000 metric tons from an area of about 50 000 hectares. Zimbabwe is a net importer of wheat, but still exports several thousand tons annually to Zambia, Malawi and other regional buyers. Very little wheat is traded with South Africa, however, since farmers in that country benefit from production and export subsidies with which Zimbabwe cannot compete. In this respect, there has been some recent debate over the efficiency of growing wheat in Zimbabwe since high irrigation costs make domestic production expensive compared with other world producers. Once transportation costs are taken into account, however, it is generally acknowledged the costs of local production are lower than import parity. Zimbabwe also enjoys a competitive edge in other regional markets like Zambia where production costs are even higher. Total domestic demand is around 400 000 metric tons per year. Wheat has been an important catalyst for the development irrigation in Zimbabwe and is a central part of most LSC farm systems. The crop was mainly introduced during the 1970s as part of the strategy to cope with international sanctions imposed on what was then Rhodesia. A revolving fund was established specifically to help LSC farmers invest in the irrigation equipment needed for wheat. Although farmers were obliged to grow wheat as part of this scheme, they quickly started to use the new equipment for other crops as well including irrigated tobacco groundnuts, soybeans and maize.

Wheat grows best on relatively heavy soils and is typically planted in rotation with rain fed soybeans. Although not ideal complements in terms of soil type, a typical rotation for many LSC farmers is to follow flue-cured tobacco by irrigated winter wheat and then smaller plots of soybeans, groundnuts and maize in the next rainy season. This section considers the costs and returns for three non-traditional crops: coffee, paprika and marigold.14 Horticultural exports including supermarket vegetables and roses are considered separately in the next section. As a group, the crops covered here all have the potential to grow well in tobacco areas and are indicative of some of the diversification opportunities available in Zimbabwe. The costs of production for these crops are generally higher than for most traditional field crops,vertical farming systems but lower than for tobacco and horticultural exports. In most cases, tobacco still provides more income on a per hectare basis, but the returns from nontraditional crops are attractive in their own right and could perhaps even substitute for tobacco as the foundation of a highly profitable farm system. Specific production and marketing issues for each crop are discussed below. 114. Beyond the crops covered here, many other niche products also offer diversification potential for smallholder and LSC farmers including mushrooms, flower seeds, game ranching, medicinal plants and spices. These enterprises are all being pursued on a limited basis in Zimbabwe and can provide an important source of farm income and improved cash flow. Compared with tobacco, however, the market outlets for these products are more limited and each farmer must find the right mix of enterprises that works best for them. A good area for further analysis would be to calculate production budgets and farm models based on these and other diversification options.Citrus crops including oranges, grapefruit and lemons, for example, are an especially important diversification option with more than 88 000 hectares of permanent orchards planted on LSC farms as of 1999. Unfortunately, reliable data on the costs of production for these crops were not available and it would be misleading to comment on their costs and profitability. Anecdotal evidence, however, suggests that the returns may not be very high and one farmer with a fifty-hectare orchard complained of serious marketing problems and large net losses. One of the problems this grower identified with citrus is that these crops are perishable and so cannot be stored for a long time to take advantage of seasonal price variations. On the other hand, this grower also pointed out that citrus can be sold for foreign exchange and so can be justified as part of the farm system even at a net loss. Citrus trees take about three to four years to produce their first fly crop and only reach full maturity after ten years.

Game ranching on LSC farms is another popular diversification activity. In tobacco areas, wildlife ranching began in the mid-1970s mainly as a hobby on unused land not suited for intensive cropping. Since then, the wildlife sector has developed into a fully integrated part of many farm systems with production for hunting and photographic safaris; game meat; and sale of live animals to other ranches or private tourist parks. Although wildlife could never substitute for intensive cropping in high potential areas, game management has become an important source of supplemental income and can easily generate an income of several thousand USD annually. Establishment costs include game fencing, animal stock, watering points plus road construction and guest cottages for safari visitors. As an industry that caters primarily to overseas tourists, the wildlife sector has been especially hard hit by recent instability in Zimbabwe. Large-scale commercial farmers. A summary of key production costs for the three non-traditional LSC crops covered here is given in Table 24. Compared with tobacco, each non-traditional crop costs about the same or less than tobacco except for long-season paprika. Marigold costs about the same as many traditional field crops whereas coffee and paprika are among the most expensive enterprises analysed because of high irrigation requirements and intensive use of fertilisers and agrochemicals. These 3 crops all demand less labour than tobacco, but still have a high overall wage bill and generate anywhere from 50 to 250 days casual employment per hectare plus additional jobs in downstream processing. Because coffee and paprika are grown to fairly uniform standards only two management levels have been analysed for these crops; marigold is relatively new to Zimbabwe and just one management level is considered in this case. Importantly, the results above clearly show that gross and net profits from other crops can rival and even surpass tobacco on LSC farms. Although the returns from coffee are rather low with current prices, farmer profits improve significantly at values closer to the long-term average. The returns from paprika are also very attractive, especially when grown as a long-season crop with high input management. Although limited world demand means that paprika prices are highly sensitive to production increases, inclusion of this crop as part of a mixed farm system has the potential to reduce growers’ dependence on tobacco. Estimated profits for marigold are considerably lower than for coffee and paprika, but still rival the income that can be earned from most traditional crops including wheat, cotton, groundnuts and maize consumed on-farm. 120. Smallholder farmers. Due to various barriers including high production costs, lack of extension support, uncertain returns and special infrastructure requirements, smallholder farmers have fewer opportunities to grow and market non-traditional cash crops than LSC farmers. Previous efforts to promote coffee and paprika among smallholder growers have been uneven at best and certainly not on a scale needed to attract broad segments of the tobacco growing population away from this crop. Smallholder coffee is perhaps the best developed, but production has been based entirely in the Eastern Highlands and there has been no work to develop the type of communal irrigation and pulping facilities needed to support farmers in flue-cured tobacco areas. Even as an alternative to burley tobacco grown in the Eastern Highlands, much work is still needed to develop appropriate management skills and infrastructure. As yet, marigold has not been promoted to any great extent as a smallholder crop but is likely to do well on communal lands since it is not fastidious to soil type or moisture.Key production costs for non-traditional smallholder crops are summarised in Table 26.Although paprika looks expensive in its own right, and certainly costs more than coffee, total variable costs are still only about 57% of those for flue-cured tobacco at each corresponding management level.

An international LCA conference is held every two to three years that focuses on sustainable transport infrastructure, in particular pavement and hardscape. The most recent of these conferences was held in Champaign, Illinois in 2017 . This symposium was a continuation of earlier conferences and workshops beginning with the Pavement LCA workshop held in Davis, California in 2010 ; the RILEM Symposium on LCA for Construction Materials held in Nantes, France in 2012 ; and the Pavement LCA Symposium held in Davis, California in 2014 . These conferences act as a platform to bring together international construction material industries, contractors, consultants, researchers, academia and the transport administrations . One of the major aims of these conferences is to implement life cycle thinking for transport infrastructures and promulgating the use of quantitative environmental assessment methods that help in decision support, such as the LCA. The most recent conference focused on data collection, inventory development, and data quality. Data essential for the UM-LCA approach is rapidly being improved through the work of various collaborators in industry and government, many of whom come together semi-annually through the Federal Highway Administration Sustainable Pavement Task Group . A framework has been developed for pavement LCA which can easily be extended to all urban hardscapes . Data availability for urban hardscape materials is becoming more widely available through several efforts being organized by consortia of government, industry and academia, such as the Federal LCA Commons ,vertical tower planter and the other efforts .Climate change is a pressing problem that cities will have to deal with and adapt to, but they have often not started making changes in infrastructure engineering and management.

Reasons for this delay include short term priorities driving design and planning, and inefficient use or omission of climate change data in infrastructure planning processes . A group of academics and transportation practitioners who are involved in the Infrastructure and Climate network in the northeastern region of the United States has addressed the systematic incorporation of climate change in infrastructure engineering and has worked to connect climate experts to front-line transportation infrastructure managers and designers. This approach provides a model for further improvement of communication between climate change modelers and infrastructure managers, particularly with regard to converting climate change predictions into actionable data for engineers. Permeable pavement is one approach to using hardscape for the multiple functions of transportation, storm water quality management, groundwater recharge and flood control, but it has been used in a small fraction of the potential places where it might provide multifunctional benefits. In early 2017, the University of California Pavement Research Center and the National Center for Sustainable Transportation , working with the Interlocking Concrete Pavement Institute , identified gaps in knowledge and other barriers to wider implementation that were perceived to be holding back the full potential for deployment of pavements that can simultaneously solve transportation, storm water quality, and flood control problems. A workshop was organized in November 2017 based on those discussions with the goal of identifying knowledge, information, and communication barriers to adoption of permeable pavement of all types, and creation of a road map to address and overcome them. The workshop brought together a diverse group of stakeholders from the planning, storm water quality, flood control, and pavement communities to listen to presentations, exchange and discuss unanswered questions identified by the group, and then to discuss a proposed road map to fill the gaps in knowledge, processes, and guidance. Some of the major findings in the final report from the workshop regarding the institutional and informational obstacles to making use of permeable pavement more acceptable to designers echo the principles identified by Chester et al. for improving the resiliency of flood control infrastructure.

These include recognition of the benefits of designing decentralized, autonomous infrastructure systems, such as making major portions of urban paved areas permeable instead of, or in addition to, building centralized storm water conveyance and storage systems; and, encouraging communication and collaboration that transcend disciplinary barriers rather than involving multiple, but distinct disciplinary perspectives. In this case, difficulties in identifying roles and relationships between urban transportation departments, storm water regulatory agencies, and flood control agencies in determining responsibility for maintaining the multi-functionality of permeable pavements emerged as an obstacle. Technical issues were also identified by the workshop, as well as the need for better cost and performance data. A presentation at a workshop by Haselbach discussed how the risk of flood damage increases in downstream cities built along rivers in long flood plains, such as those along rivers in Texas, when upstream cities expand their urban hardscape and flood conveyance to rapidly shed water. Thus, there is increased risk of flooding in each city as the area covered by hardscape increases, and that risk cascades and is multiplied significantly for downstream cities as upstream systems release water to protect the integrity of their own infrastructure and local urban areas. In addition to the review of the published literature, meetings were held with researchers at UC Los Angeles , UC Davis and the University of Southern California to review the concept for the UM-LCA framework presented in this white paper and potential data sources. The consulted experts included Stephanie Pincetl and Eric Fournier at UCLA, Jay Lund and Jon Herman at UCD and George Ban-Weiss at USC. As an example of data collection, Pincetl and Fournier pointed to the LCA framework and tool called City Road Network LCA developed for the roadway system in Los Angeles and used by Fraser and Chester and other studies. The data tracks the location of streets and highways over time and was developed in a GIS environment.

To complement this source of information for hardscape, potential data sources for soil permeability information were identified to assess where permeable hardscape could successfully be used to infiltrate water into the soil versus areas where permeable hardscape can only store water for later discharge. The SSURGO and STATSGO spatial data layers from the National Resources Conservation Service of the U.S.Information from the Permeable Pavement Road Map Workshop indicated that the scale of these maps is not sufficiently fine to capture the permeability for project design purposes, but should still provide adequate data on the scale of urban metabolism studies. Pincetl and Fournier pointed to use of fly-over maps to identify hardscape paved areas that can augment the estimates of surface areas of highways, streets and roads available from state and local government sources. Data regarding material flows can be obtained down to a sub-urban area level from the IMPLAN database and tool. They indicated that data regarding building ages can be obtained from the California Economic Development Division by business data centers. This is the data approach used by Reyna and Chester to also indicate the rate of demolition of buildings. The rate of demolition of publicly owned roads and streets would have to be estimated from maintenance and rehabilitation data from local and state government sources, inferred for private sources, and compared with estimates of civil infrastructure demolition hauling ton-miles.An UM-LCA framework was developed that can quantify water and hardscape material metabolism for a defined area/boundary as shown in Figure 7. It is based on the literature survey and discussions with experts in water management systems,lettuce vertical farming storm water runoff quality, freight systems and materials across number of institutions and agencies. For initial use in California, Caltrans adjusted urban area boundaries derived from the 2010 census and approved by FHWA are being considered as the system boundaries for the framework. The framework consists of horizontal and vertical flows. Horizontal flows are hardscape material flows which include asphalt products, concrete products, aggregates, crushed concrete demolition materials from building and other structures, additives, admixtures and recyclable materials. The vertical flow is the hydrologic cycle which is considered in the framework. The goal for development and use of the framework is to reduce environmental impacts of urban hardscape through analysis and rethinking of hardscape management, including design, construction, maintenance, rehabilitation and end-of-life to produce and compare alternatives to current practices. In addition to the typical environmental impact-indicators in the FHWA pavement LCA framework , it is proposed that later development should consider inclusion of the social LCA indicators developed in a separate recent report . Initial indicators have been developed for thermal comfort, focused on outdoor or pavement related thermal comfort rather than inside-building comfort, aural comfort and glare comfort . Only some of these references look at development of life cycle impact indicators; most are focused on comfort indices that could serve as mid-point indicators rather than indicators of effects on human health over the life cycle and considering all stages of the life cycle. Effects of pavement on the overall urban heat island for the entire urban area and the resultant impacts on thermal comfort, summertime cooling, wintertime heating and energy use from heating and cooling can also be considered.

Consideration of overall urban heat island effects requires climate modeling, and consideration of the energy sources for heating and cooling. A recent study in California found that the energy and greenhouse gas emission impacts of changing pavement surfaces to increase albedo and decrease urban area heat island were primarily dependent on the changes in pavement materials, and the effects of resultant changes in building energy use were much smaller. This result will change depending on the climate region and sources of electrical energy for air conditioning and building heating.There is no single source for data for hardscape material flows. Some of the common data sources include databases, LCA literature, public documents, surveys, etc. A comprehensive list of data sources for different materials were compiled in previous research conducted for development of an LCA framework for aviation pavements as well as the FHWA pavement LCA framework report as shown in Table 1, and which can be translated for use in the UMLCA framework. In LCA, the terms foreground and background data are often used to differentiate between data that defines or represent the study application and the datasets used to describe the background systems on which they rely datasets derived from databases. Table 1 include references to both background and foreground data sources, but mostly background data sources.For California, the UCPRC has previously developed LCI datasets for construction materials tailored to California conditions, which are particularly suitable for projects in California. UCPRC updated LCA models with new inventories, processes, and models recently to expand the capacities of LCA for improving the sustainability of pavement operations in California . The updates included new inventories for pavement materials such as asphalt, concrete, terminal blend rubberized asphalt concrete, warm-mix asphalt, open-graded friction courses, pre-cast concrete slabs, reinforced concrete, aggregate bases, cement treated bases, asphalt treated bases, maintenance treatments such as chip seals, slurry seals, and reflective coatings, and the different types of full- and partial-depth recycling strategies that are typically used by Caltrans. Furthermore, the UCPRC LCIs have been critically reviewed by three LCA experts for data assumptions, collection, processes and quality. These LCIs and impact calculations are included in a new LCA tool called eLCAP . Similar activities could be undertaken at any state or city level to generate geographically appropriate LCAs and eLCAP or other similar tools could include updated regionally adjusted inventories for locations outside California. Quantification of flows of materials that are transported in and out of the urban boundary are needed to be able to apply the unit process data. The Commodity Flow Survey is conducted under the partnership of the Bureau of Transportation Statistics and the U.S. Census Bureau every few years. CFS data covers movement of forty-three goods that are transported in and out of the boundaries of the U.S. and U.S. cities by road transport. The Freight Analysis Framework Data Tabulation Tool  is another data source that can be used to collect data for a specific city or region in the U.S. that can then be used in the UM-LCA framework. The FAF4 tool is divided into four flow types; total flows, domestic flows, import flows and export flows. Total flows include domestic and foreign shipments between domestic origins and destinations whereas domestic flows do not include foreign trade flows. Import and export flows includes goods movement between domestic and foreign origins.

Over the following half-century, most physicians and researchers among the Pima Indians avoided the conclusion that a more sedentary lifestyle was the decisive factor in the degradation of health on communities. Instead, many drew a correlation with the increase in processed starches and high sugar beverages among reservation Pima communities, in contrast to those who lived away from those regions that came to rely on federally-sanctioned processed foods. The health of these Native American communities, then, was impacted by new federal food and land policies; just as earlier colonial interventions impacted their nutritional lives in problematic ways.Among other resources for such a conclusion and/or postscript to their course, students could use Annual Reports of the Commissioner of Indians Affairs. The reports provide evidence from Indian Territory between the 1820s and the 1930s, describing nutritional changes, diseases, perceived health problems, and ecological and agricultural developments. Students could also assess Indian and Pioneer Papers, which detail thousands of interviews of residents of Oklahoma in the 1930s. Many of those residents were elderly and recalled nutritional, agricultural, and health developments that took place over the previous century. Students – and future researchers – might even consult advertisements from “Indian Territory” newspapers. Their proliferation of cures, tonics, and “snake-oil” medicines for digestive problems after the Civil War era might provide clues to problematic dietary changes among Native Americans; changes that resulted in part from disruptive state and federal policies that were contiguous with challenging interventions that took place during the earlier colonial eras.Into the most recent era, indeed, reservation communities have been more susceptible to certain problems that are associated with the modern western diet,vertical farm as broadly defined during the second half of the twentieth century – particularly its reliance on refined carbohydrates in packaged and processed goods.

From the 1960s to the 1970s, for example, medical researchers began to define a clear correlation between the appearance of obesity and Type II diabetes in Native American communities and their adoption of processed foods in federal welfare programs that targeted newly formed reservations. Reports of Type II diabetes among Oklahoma communities increased after their move into federally-mandated reservations through the 1940s. A review of literature from 1832 to 1939 by the epidemiologist Kelly West found no reports of diabetes among the Kiowa, Comanche or Apache communities living in the region .Similarly, diabetes seems to have been much less prevalent among Pima Indian populations who lived away from reservations in the American Southwest before 1940. By the turn of the twenty-first century, however, around half of all Pima Indians have been reported to suffer from diabetes.Following their analysis of early contact history, students might consider these more recent phenomena in light of modern scientific literature on metabolic syndrome. According to federal statistics from the present day, moreover, Native American communities continue to suffer from diabetes, cirrhosis, influenza, pneumonia, and perinatal and early infancy diseases at greater rates than the general American population.Yet as the USDA nutritional guidelines for the Food Distribution Program on Indian Reservations program demonstrate, Native American populations will continue to receive food welfare in the form of packaged and processed starches, seed oils, and low fat animal sources – foods that have potentially contributed to increasing metabolic syndrome in the US population more generally.As they try to understand possible correlations between the two scholarly fields of history and nutritional science, therefore, students and researchers might begin to think about problematic nutritional interventions and paradigms outside the Native American community, among the American public more generally.

The study of early-American history and the problematic European intervention in Native American nutritional life should inform their future careers in fields such as public health, scientific research, nutritional science, and medicine. Approximately five to seven million years ago, nectar spurs arose in the ancestor of Aquilegia , which then diversified into ∼ 70 species in two major clades: one distributed across Eurasia, and the other primarily distributed across North America. Spur morphology varies substantially across the genus, ranging in length from ∼ 1-16 cm, and varying in other morphological characteristics such as the degree of curvature and color. These petal characteristics contribute to pollinator specificity, which plays an important role in reproductive isolation between taxa . For example, species that occur in both Eurasia and North America have the ancestral morphology of the genus — shorter, curved spurs, usually ranging in color from blue to purple — and are typically pollinated by bees. In North America, convergent evolution has lead to several lineages with red and yellow flowers, and straight, medium-length nectar spurs that are primarily pollinated by hummingbirds. Multiple North American lineages that generally lack floral anthocyanins and are yellow or white have evolved long nectar spurs and are pollinated by hawk moths. In addition to these three pollination syndromes, one species native to montane regions of central China, A. ecalcarata, has secondarily lost nectar spurs and is primarily pollinated by syrphid flies. Given the role that nectar spurs have played in the diversification of Aquilegia, understanding the genetic and developmental basis of how they form is key to understanding both the initial evolution of this three dimensional structure as well as the generation of subsequent modifications that serve as adaptations to different pollinators.Phase I broadly comprises the mitotic phase of petal development in which localized cell-divisions establish the spur cup. Initially, cell divisions are dispersed throughout the petal primordium, but they cease in a wave that begins at the margins of the petal, moving basipetally and causing divisions to become concentrated in the nascent spur.

Mitotic activity is maintained in the developing spur until the spur reaches 5-9 mm in length, but it is progressively restricted toward the spur tip, where the nectary develops. As cells begin to differentiate in Phase II, anisotropic cell expansion becomes a major contributor to spur shape,nft vertical farming particularly length. Differences in spur length across Aquilegia species have been primarily attributed to differences in cell length, rather than cell number, although the basis of spur curvature and other aspects of shape have yet to be studied in detail. In an effort to understand the genetic basis of nectar spur development in Aquilegia, a previous study examined gene expression differences between the petal blade and spur at several early developmental stages in the horticultural variety A. coerulea ‘Origami’. This study ruled out a role for type I KNOX genes, which maintain cellular indeterminacy in meristematic tissue, indicating that the prolonged mitotic activity in the spur is not meristematic in nature. However, a number of other genes whose homologs play a role in regulating the transition between cell proliferation and differentiation in the petals of Arabidopsis thaliana were highly differentially expressed between the petal blade and spur. For example, the TEOSINTE BRANCHED/CYCLOIDEA/PCFgene, AqTCP4, whose homolog in A. thaliana, TCP4, controls petal size by repressing cell-division, was more highly expressed in petal blades where mitotic activity first ceases. In contrast, a homolog of an A. thaliana GRF-INTERACTING FACTORgene that controls petal size by promoting cell-division, AqGIF1/AN3, was up-regulated in the Aquilegia spur where mitotic activity is maintained. The most highly differentially expressed gene identified in this study is a member of the STYLISHgene family, whose homologs in A. thaliana are best known for their functions in carpel, rather than petal, development. Subsequent analyses of this gene, AqSTY1, and two additional Aquilegia homologs in the STY gene family, AqSTY2 and Aq LATERAL ROOT PRIMORDIUM , revealed that in addition to a conserved role in carpel development, these genes are critical to nectary development. Although the AqSTY-like genes do not appear to function in the earliest phases of nectar spur development, these results highlight the utility of gene expression studies to identify novel candidates involved in unique roles of Aquilegia petal development. The previous gene expression and functional analyses were conducted on a single Aquilegia cultivar, and did not address questions regarding the conservation and divergence of gene expression patterns during early petal development across the diverse morphologies in the genus Aquilegia. Here we conduct transcriptomic analyses of Phase I petals at five different developmental stages from four different Aquilegia species, A. ecalcarata, A. sibirica, A. formosa, and A. chrysantha, representing a diverse set of pollination morphologies and spanning the Aquilegia phylogeny . This broad sampling allowed us to take a comparative approach, combining differential expression and weighted gene correlation network analyses to explore both commonalities in the genetic basis of petal development across the genus Aquilegia as well as taxon-specific differences.

To that end, a set of genes commonly DE between the three spurred species in our study, A. sibirica, A. formosa, and A. chrysantha, and the spurless species, A. ecalcarata was compared to the set of DE genes between the petal blade and spur previously conducted in A. coerulea ‘Origami’. This comparison revealed only 35 genes that are either more highly expressed in both A. ecalcarata and the petal blade or in the spurred taxa and the petal spur. In addition to these unbiased analyses, we explored the expression of Aquilegia homologs of genes known to regulate the transition between cell division and differentiation in A. thaliana petals in order determine the potential for broad functional conservation as well as any potential role in Aquilegia spur development.Petals were dissected from floral buds spanning Phase I of development from each of four species of Aquilegia – A. ecalcarata, A. sibirica, A. formosa, and A. chrysantha. Although it can be challenging to determine homologous developmental stages across taxa, a combination of petal morphology and stamen development was used to group samples across the four species into five developmental stages , DS1-DS5, with petals from at least three flowers collected for each species and stage. Representative samples of each species at each of the five developmental stages are presented in Fig. 2. The first petal stage assessed was collected from buds approximately equivalent to floral stage 8. At this point, the morphology of petals from all four species examined was quite similar. Petals were approximately 0.5 mm wide and, although they exhibited slight concave adaxial curvature, the spur had yet to initiate. Notably, this is the first time that a pre-initiation stage petal has been sampled for RNA analysis in Aquilegia. The second petal stage examined came from floral buds at approximately stage 9. At this stage, the spur had begun to form in the spurred taxa and was approximately 0.5 mm long while petals were approximately 1 mm wide in the blade region. Even at this early stage of nectar spur development, morphological differences between the taxa were already detectable, with the developing spur of A. formosa being wider than those of A. sibirica and A. chrysantha . These differences in the spurred taxa continued through the third petal stage sampled where developing spurs continued to elongate but largely maintained their shape as established in DS2. By the fourth petal stage sampled , additional morphological differences became apparent. For example, although spurs were only between 1 and 2 mm long at this stage, curvature in the A. sibirica petal was already being established. Some of the spurs at this stage began to appear more bulbous at the tip of the spur, indicating the incipient development of the nectary. Although A. ecalcarata does not develop a spur or a nectary, at DS4 a small pocket is visible below the petal attachment point. In addition to spur differences, several subtle differences in petal blade morphology across the taxa were apparent, including the shape of the blade apex and its relative length compared to the spur. The developmental gap between the fourth and fifth petal stages collected was larger than the gaps between the other stages. At the fifth time point collected , the spurs of the spurred taxa ranged in length from 4-7 mm and morphological differences were accentuated. A. sibirica spurs exhibited more extreme curvature, A. formosa spurs were broad through most of the length of the spur, narrowing just before the nectary, and A. chrysantha spurs were long and narrow. In general, this stage should represent the transition out of phase I into phase II, where cell elongation is the dominant factor affecting spur length.We started by comparing changes in the transcriptional profiles of petals between serial developmental stages for each species. For A. ecalcarata, A. sibirica, and A. chrysantha, the differences in gene transcription were minimal between each of the first four stages, whereas there was a larger jump between DS4 and DS5 .