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

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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.