Genetic control of postharvest darkening in bean (Phaseolus vulgaris), creating understanding and developing applications

普通的干豆（Phaseolus vulgaris）是全世界3亿多人的主食。加拿大大豆产量为30万吨/年，价值超过2.5亿美元。豆类因其营养和植物化学成分而对人类健康产生有益影响，并通过其固定大气氮的能力对环境产生积极影响。具有白色背景的彩色豆，如平托豆和蔓越莓豆，在储存期间变暗，导致市场价值降低，因为消费者将与年龄相关的变暗与适口性降低和烹饪时间增加相关联。 我们从Wit-rood（一种种质中心植物）中移走了一个非深色性状，将其引入了平托和蔓越莓豆中。来自Witrood的非变黑性状是一种简单遗传的隐性性状，在保持豆类的新鲜外观方面非常有效。通过表型和SNP基因分型的F5衍生的重组自交系作图群体，我们创建了，从一个Witrood的常规平托豆杂交，我们确定了一个主要的QTL的nondarkening性状的染色体10号，并将其与以前确定的种皮颜色基因称为J，决定是否豆将变暗。对带有非变黑基因的豆类进行的生化分析表明，它们含有低水平的原花青素，而原花青素是已知的与布朗宁反应有关的物质。一项RNAseq研究确定了在变暗和非变暗豆类之间差异表达的基因，并确定了表达与原花青素积累高度相关的基因。使用扩增子测序的候选基因的nondarkening QTL的10号染色体上，我们发现一个单核苷酸缺失的nondarkening等位基因的R2 R3-MYB转录因子。MYB基因的非变暗Witrood等位基因中的缺失可能引起翻译移码，其破坏R2 R3-MYB的C末端中的转录激活结构域的功能。这项研究将检查R2 R3-MYB基因在豆类中的功能，以确认该基因在控制采后储存期间种子变暗反应中的中心地位，了解Witrood等位基因中的SNP如何破坏其功能，并开发该性状的分子标记。特别是，我们将研究的结构基因和转录因子的表达，控制和相互作用的MYBs在种子发育过程中，在变暗和nondarkening，豆使用真实的时间PCR和瞬时表达的研究。此外，CRISPR将用于影响早期Witrood种子中突变R2 R3-MYB基因中SNP的同源修复，以明确地将突变与非变暗性状联系起来。拟议中的研究将支持圭尔夫大学现有的豆类遗传学项目，该项目致力于了解分子、整株植物和作物水平的性状，并以创新的方式将这些见解应用于豆类育种。

The common dry bean (Phaseolus vulgaris) is the staple food for more than 300 million people worldwide. Canadian bean production of 300,000 tons/ year is worth more than $250 M. Beans have beneficial effects on human health because of their nutritional and phytochemical compositions and positive effects on the environment through their ability to fix atmospheric nitrogen. Coloured beans with white backgrounds, like pinto and cranberry beans, darken during storage, resulting in a reduced market value because consumers associate age related darkening with decreases in palatability and increased cooking time. We moved a nondarkening trait from Wit-rood, a germplasm centre plant introduction into pinto and cranberry beans. The nondarkening character from Witrood is a simply inherited, recessive trait, that is very effective in retaining the fresh appearance of beans. By phenotyping and SNP genotyping a F5-derived recombinant inbred line mapping population we created, from a Witrood by regular pinto bean cross, we identified a major QTL for the nondarkening trait on chromosome 10 and associated it with a previously identified seed coat colour gene called J, that determines whether a bean will darken. A biochemical analysis of beans with the nondarkening gene showed that they have low levels of proanthocyanidins, which are known to be involved in browning reactions. A RNAseq study identified genes that were differentially expressed between darkening and nondarkening beans and identified genes whose expression was highly correlated with proanthocyanidin accumulation. Using amplicon sequencing of candidate genes underlying the nondarkening QTL on chromosome 10, we found a single nucleotide deletion in the nondarkening allele of a R2R3-MYB transcription factor. The deletion in the non-darkening Witrood allele of the MYB gene likely causes a translational frame shift that disrupts the function of a transcriptional activation domain in the C-terminus of the R2R3-MYB. The proposed research will examine the function of the R2R3-MYB gene in beans to confirm the centrality this gene in controlling the seed darkening reaction during postharvest storage, understand how the SNP in the Witrood allele disrupts its function and develop molecular markers for the trait. In particular, we will examine the expression of the structural genes and the transcription factors that are controlled and interact with MYBs during seed development, in darkening and nondarkening, beans using real time PCR and transient expression studies. In addition, CRISPR will be used to affect homologous repair of the SNP in the mutant R2R3-MYB gene in early stage Witrood seeds to unambiguously link the mutation with the nondarkening trait. The proposed research will support an existing bean genetics program at the University of Guelph that strives to understand traits at molecular, whole plant and crop levels and apply those insights in bean breeding in innovative ways.