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

普通的干豆（Phaseolus vulgaris）是全球3亿多人的主食。加拿大豆类产量为每年30万吨，价值超过2.5亿美元。豆类因其营养和植物化学成分对人类健康有益，并通过其固定大气氮的能力对环境产生积极影响。白色背景的有色豆，如平托豆和蔓越莓豆，在储存过程中会变暗，导致市场价值下降，因为消费者将年龄导致的变暗与适口性下降和烹饪时间增加联系在一起。我们从种质中心引种植物Wit-rood中移植了一个不变黑的性状到斑豆和蔓越莓豆中。Witrood的不变黑特性是一种简单的遗传隐性性状，它非常有效地保持了豆子的新鲜外观。通过表型分型和SNP基因分型，我们创建了一个f5衍生的重组自交系定位群体，从一个普通平豆杂交的Witrood中，我们确定了10号染色体上不变黑性状的主要QTL，并将其与先前确定的一个名为J的种皮颜色基因相关联，该基因决定了豆类是否会变黑。对带有不变黑基因的豆子进行的生化分析表明，它们的原花青素含量较低，而原花青素被认为与褐变反应有关。一项RNAseq研究发现了变黑和非变黑豆子之间表达差异的基因，以及与原花青素积累高度相关的基因。通过对10号染色体上非暗化QTL的候选基因进行扩增子测序，我们发现R2R3-MYB转录因子的非暗化等位基因存在单核苷酸缺失。MYB基因非暗Witrood等位基因的缺失可能导致翻译框移位，从而破坏R2R3-MYB c端转录激活域的功能。该研究将检测R2R3-MYB基因在豆类中的功能，以确认该基因在采后贮藏过程中控制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，我们将使用实时PCR和瞬时表达研究来检测在变暗和非变暗大豆种子发育过程中与MYBs相互作用的结构基因和转录因子的表达。此外，CRISPR将用于影响早期Witrood种子突变体R2R3-MYB基因SNP的同源修复，以明确将突变与非变黑性状联系起来。拟议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.