A STRUCUTRE analysis found that the optimal K was five . This is the same number of populations identified by Chacón-Sánchez and Martinez-Castillo . Rather than fitting neatly into the categories of MI, MII, AI, AII, and admixed, these samples were optimally divided into MII, AI, AII and two MI groups. The larger of the MI groups included a mixture of wild and domesticated lines while the smaller MI group consisted mainly of wild accessions collected in Mexico. Based on this analysis, 36 lines identified as belonging primarily to the two Andean gene pools were removed from the study. For future publication, a higher threshold of admixture may also be considered for removing some additional genotypes. With the Andean lines removed the remaining population showed significantly less population structure .A GWAS of volatile HCN production in the first 15 minutes of tissue rupture caused by thawing, identified several significant SNPs for flower tissue and one highly significant SNP for pod tissue . The most significant SNPs for flower tissue, on Chromosomes 2 and 4 are located near matches for the BLAST search of the white clover Li/li sequence. The SNP identified in pods is not near the significant alignment of the BLAST search against the Lima bean reference genome of the white clover sequence or the QTL identified in the biparental population. When considering cyanogenesis as a defense trait, the immediate release of HCN following tissue disruption deters an insect herbivore and therefore serves as a resistance trait . As such, it will be most successful against opportunistic, square plant pots generalist herbivores rather than specialist herbivores which would have experineced coevolution with the crop and had more opportunity to adapt to its defenses .

Additional study of these findings may yield great contributions to breeding efforst for L. hesperus resistance. Additional significant SNPs were found in the 15-30 minute exposure window . In flower tissue, SNPs on chromosomes 9, 5, and 7 were closely located to significant matches from the BLAST of the white clover Ac/ac gene sequence on the Lima bean reference genome. In pod tissue, a significant SNP on chromosome 6 was also closely located to a match for the Ac/ac sequence.Prior QTL analysis of HCN in floral buds, immature pods, and leaves of a RIL population identified significant loci for volatile HCN on chromosome 5 . This QTL is very close in position to one found by the GWAS analysis of HCN in flowers defrosting for 15-30 minutes, PL05_36471809. There is also a significant alignment with the white clover sequence for the Li/li gene in a nearby region of chromosome 5 . It is interesting to note that there is evidence of β-glucosidase activity being induced by the presence of insect herbivores . The greenhouse from which the samples in this study were collected had a stable infestation of thrips but was free of the larger herbivores typically found in field settings. It is therefore possible that if this study were repeated with field-collected samples, this locus would have a stronger effect.Cyanogenesis is a complex trait in Lima bean with multiple SNPs closely associated with the expression of cyanogenesis. Highly significant SNPs found in flowers during the first 15 minutes after tissue disruption are close matches for the white clover Li/li gene sequence. This could contribute to the effectiveness of cyanogenesis as a resistance trait that deters insect herbivores . Additional SNPS on chromosomes 9, 5, 7, and 6 found in the 15-30 minute exposure window may be associated with the biosynthesis of cyanogenic glucosides as they are close to matches of the white clover Ac/ac gene sequence.

Finally, a QTL on chromosome 5 was in close proximity to previously identified QTL for cyanogeneis in flowers, pods, and leaves as well as the white clover sequence for Li/li. Further analysis and research is needed to clarify the function and expression of genes located near the significant SNPs identified by this study and solidify understanding of the genetic architecture of cyanogenesis in Lima beans. Several additional steps will be take to advance this research prior to publicaiton. First, the STRUCTURE and GWAS analyses will be reexamined to consider higher thresholds of admixture. Next, confidence intervals and markers flanking the significant SNPS will be analyzed to increase certainty about the relationship between these findings and the BLAST search maches as well as previously identified QTL from the RIL population. A study of genome annotations and the expression atlas will also be undertaken to identify clues about the function of genes near these significant SNPs. The results from wild and domesticated accessions will also be compared to determine how the matches for Li/li and Ac/ac genes may have been affected by domestication. Lastly accessions with extreme phenotypes will be identified and their associated genotypes used for breeding, further mapping, and validation studies.Amplifying defense traits that protect plants from insect herbivores through plant breeding has the potential to increase yields while reducing pesticide use and associated concerns for human and environmental health . This is a particularly important strategy for organic systems in which conventional pesticides cannot be used. Lima beans are an important grain legume globally and the most economically important dry bean grown in California where their primary insect pest is the Western Tarnished Plant Bug . Lima beans are a model experimental organism for studying anti-herbivore defense traits . Within this body of literature, many studies have focused on the trait of cyanogenesis . Several experiments have been conducted in recent years to identify specific mechanisms that contribute to the tolerance or resistance traits that protect some Lima bean accessions from damage by L. hesperus .

One mechanism that has been considered is the production of various polygalacturonase inhibiting proteins in the cell walls of Lima bean that bind to L. hesperus salivary enzymes and mitigate attempted digestion of the cell wall . This trait was found to be strongly influenced by environmental variables such as pest pressure and insecticide treatments but the study design did not permit differentiation of these results as the primary goal was QTL mapping . Cyanogenesis is a trait of particular interest since it is known to be an effective anti-herbivore defense trait in wild Lima beans that has been selected against during domestication . Several QTL have been identified for cyanogenesis in flowers, immature pods, and leaves . However, these studies have not yet determined if cyanogenesis is an effective trait in the defense of Lima beans against L. hesperus specifically. L. hesperus predominantly feed on the flowers and immature pod tissue of Lima bean and if cyanide is an effective deterrent or toxin for L. hesperus then increased expression of cyanogenesis in these tissue types could be amplified through breeding without risk to the human consumers of mature seeds which are known to have low cyanogenic capacity . The final part of this study aims to determine how cyanogenesis affects L. hesperus survival and development as well as test if cyanogenic capacity can be induced by the presence of L. hesperus.Many economically important crops with high protein content and great importance for indigenous food systems are members of the legume family . Several of these legume crops are cyanogenic . It appears that this trait has evolved independently several times in the legume family. Within the legume genus Phaseolus, there are five domesticated crop species but only one, Lima bean, is cyanogenic . In addition to Lima bean, five other cross-compatible, Phaseolus species within the Polystachios group of section Paniculati are also cyanogenic . This and other evidence indicate that despite being a widespread trait, cyanogenesis evolved independently multiple times through the recruitment of similar genes .In an extensive screening of wild, weedy, garden pots square and cultivated forms of Lima bean, all were found to be cyanogenic. However, there is variability within and between populations . Domesticated forms typically have much less cyanogenic potential , the amount of stored cyanogenic glucosides, and cyanogenic capacity , the amount of cyanide released when damage occurs . Cyanogenic potential is determined by the biosynthesis and accumulation of cyanogenic glucosides . Cyanogenic capacity is primarily determined by genetic factors but there is also a significant influence of plant age and other environmental factors . Cyanogenesis is typically considered a constitutive trait with strong genetic control by two Mendelian genes . However, there is great variation in the trait within populations and even within an individual plant . Previous studies have found cyanogenic potential and capacity to vary based on the age and tissue type being measured . For example, in Lima bean, young leaves have higher cyanogenic potential than mature leaves .

Wild Lima bean seeds by contrast have very high cyanogenic potential but low cyanogenic capacity, likely due to the low moisture content inhibiting β-glucosidase activity . Additionally, there is evidence that cyanogenic capacity may be locally induced by the presence of insect herbivores even if cyanogenic potential is constitutive . Temperature, humidity, seasonal dynamics, water-stress, and nutrient availability may also affect cyanogenesis .Cyanogenesis is very nitrogen intensive with a one-to-one ratio of nitrogen and carbon in each molecule of hydrogen cyanide . The availability of nitrogen can be a limiting factor for plant growth . Therefore, it has been hypothesized that cyanogenic glucosides evolved first as an intermediate nitrogen storage compound and only later evolved into a defense compound . In the case of Lima beans, evidence supports the hypothesis that cyanogenic glucosides primarily serve as an anti-herbivore defense more so than a nitrogen storage mechanism . Lima bean plants with high cyanogenic glucoside content in leaves had lower above ground biomass than low cyanogenic glucoside content plants when no herbivores are present, but this difference was less in the presence of herbivores . This could indicate that there is a high cost to producing cyanogenic glucosides. Alternatively, these plants may be investing in a strong defense of their vegetative tissue so that a smaller above ground biomass can produce higher yield. Additionally, seeds of Lima bean with high cyanogenic glucoside content had lower germination rates but produce seedlings that had high cyanogenic glucoside content and supported lower growth rates of the generalist herbivore Spodoptera littoralis . In addition to having tradeoffs with growth and vigor, plants with high cyanogenic glucosides have lower investment in other defense mechanisms . In Lima bean, a negative correlation was found between cyanogenic glucosides and volatile organic compound emissions . This evidence indicates that in Lima bean, cyanogenicglucosides serve primarily as an antiherbivore defense compound rather than a nitrogen storage mechanism.Cyanogenesis is an anti-herbivore defense trait found in many plant families . It is especially common in crop plants. While an estimated 11% of all plant species are cyanogenic, the trait is present in approximately 21% of the major world food crops . Given that humans have long known of several effective methods of detoxifying cyanogenic foods, including leaching, cooking, and fermenting, it is possible that crops with this trait were specifically selected by early farmers for their superior defense against insect herbivores . Despite its value as an anti-herbivore defense trait, cyanogenesis has been selected against during the process of domestication. With the notable exception of sorghum, most crops have lower levels of pre-cyanogenic compounds than their wild relatives . This may be because, though satisfactory, our methods of detoxifying foods do not fully eliminate cyanide. Specifically, in the example of Lima bean, the enzyme linamarase rapidly hydrolyzes cyanogenic glucosides during cooking but becomes denatured at 141 °C . If cyanogenic glucosides remain unhydrolyzed when that cooking temperature is reached, their cyanide will be released within the consumers digestive track . This can be tolerated at low levels, but chronic cyanide intoxication can cause severe symptoms including degenerative neuropathy, paralysis, blindness, and premature death . Given the severe consequence of chronic or acute cyanide intoxication as well as the bitter taste, it is understandable that cyanogenesis was selected against in crop plants . The toxicity of HCN comes from asphyxiation when it binds to cytochrome oxidase, a key enzyme in the mitochondrial respiratory pathway . This chemistry makes it toxic to both animal and plant cells. It is therefore necessary for plants to store pre-cyanogenic compounds, typically cyanogenic glycosides, separately from enzymes that cleave the compound and form HCN . The result of this arrangement is a possible difference in the HCNp and the HCNc of a plant.