Identification of Novel Candidate Genes Associated With Scab Disease Resistance in Common Bean (Phaseolus Vulgaris)

Abstract

Scab is a fungal disease of common beans caused by the pathogen Elsinoë phaseoli. The disease results in major economic losses on common bean, and there are efforts to develop integrated pest management strategies to control the disease. In this study, modern computational biology and bioinformatics tools were deployed to identify resistance genes for scab disease in common bean. A diverse set of 182 common beans accessions were evaluated for phenotypic variation to scab disease in two sites in Western Kenya. The phenotypic variations observed was a pre-requisite for genomic analysis to identify the resistant genes associated with scab disease resistance. The diverse accessions were analyzed for genetic association with scab disease resistance using a Genome-Wide Association Study (GWAS) design of infected plants and non-infected plants (controls). A fixed and random model circulating probability unification (FarmCPU) model of these two covariates that considers a minor allele frequency threshold value of 0.03 and population structure analysis guided by a 7 components principal component analysis were deployed during the analysis. Annotation of genes proteins with significant association values was conducted using a machine learning algorithm of support vector machine on prPred using python3 on Linux Ubuntu 18.04 computing platform with an accuracy of 0.935. Subsequently, molecular markers associated with resistance to scab disease were identified. Common bean accessions tested showed varying phenotypes of susceptibility to scab disease. There were significant differences within the various genotypes at p=5.551e-15 for the treatments. A total of 16 and 163 accessions were observed to be resistant and susceptible, respectively, to scab disease caused by Elsinoë phaseoli. The dataset generated was further preprocessed and tested for normality using the Shapiro-Wilk’s normality test, and no significant difference from normal distribution was observed (W = 0.98901, P = 0.1812). On genomic analysis, a significant association was detected on chromosome one SNP position 6571566 and within the same locus a SNP on position 6231746 was also identified. The protein-coding sequence on position 6571566 had a resistant possibility of 55% and annotated to the Enhancer of Poly-comp like (EPL1) protein while position 6231746 with a resistant possibility of 64% was annotated to Adenosine triphosphate Binding Cassette (ABC) transporter protein. Nine primer pairs were designed for validation targeting the EPL1, ABC transporter, and the PHD finger genes in the common bean. Differences were observed for the EPL1 on the second primer pair of reverse outer and forward inner primer targeting the alternate SNP. The Third primer pair with forward-outer and reverse-inner primer was able to distinguish the resistant accessions. The significant difference in the phenotypic variability for scab disease indicates wide genetic variability among the common bean accessions. The resistant gene associated with scab disease was successfully identified by GWAS analysis and confirmed by designed EPL1 primers which showed amplification only in the resistant common bean accessions. The identified common bean accessions resistant to scab disease can be adopted into breeding programs as sources of resistance. The primer can be used in marker assisted selection targeting the identified scab resistant genes.