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

普通干豆(菜豆)是全球3亿多人的主食。加拿大豆类产量为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。白色背景的彩色豆子，如品托和小红莓豆，在储存过程中会变黑，导致市场价值下降，因为消费者会将与年龄相关的变黑与适口性下降和烹饪时间延长联系在一起。我们从Wit-rood中移出了一个不变黑的性状，Wit-rood是一种引入品豆和小红莓豆的种质中心植物。来自Witrood的不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。通过表型分析和SNP基因分型，我们从普通品系杂交的Witrood中创建了一个来自F5的重组自交系作图群体，我们确定了10号染色体上不变暗性状的一个主效QTL，并将其与先前发现的决定菜豆是否变暗的种皮颜色基因J相关联。对带有不变黑基因的豆子进行的生化分析表明，它们含有较低水平的原花青素，这是已知的与褐变反应有关的物质。一项RNAseq研究确定了在变暗和不变黑的豆类中差异表达的基因，并确定了其表达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候选基因的扩增测序，我们发现R2R3-MYB转录因子的非暗化等位基因中存在单核苷酸缺失。MYB基因非变暗的Witrood等位基因的缺失可能会导致翻译框架移位，从而扰乱R2R3-MYB C末端转录激活域的功能。这项拟议的研究将检测R2R3-MYB基因在豆类中的功能，以确认该基因在控制种子采后暗变反应中的中心地位，了解Witrood等位基因中的SNP如何扰乱其功能，并开发该性状的分子标记。特别是，我们将使用实时荧光聚合酶链式反应和瞬时表达研究来检测在种子发育过程中，在变暗和非变暗的豆类中，受MYB控制并与其相互作用的结构基因和转录因子的表达。此外，CRISPR将被用来影响Witrood种子早期突变的R2R3-MYB基因中SNP的同源修复，以明确地将突变与非暗化性状联系起来。拟议的研究将支持圭尔夫大学现有的豆类遗传学项目，该项目致力于在分子、全植物和作物水平上了解性状，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.