Identification of causal SNPs in promoter sequence of Co 2 and development of functional markers for anthracnose resistance in common bean Phaseolus vulgaris L

Abstract

newline Anthracnose caused by the fungus Colletotrichumlindemuthianumis one of the most devastating diseases of common beans causing yield loss up to 100 per cent. The study was conducted to identify single nucleotide polymorphism sequences (SNPs) in the promoter region of the Co-2 gene, to identify causal SNPs linked to anthracnose disease resistance using phenotypic and SNP data and to develop functional marker to differentiate the alleles of causal SNPs and validation for anthracnose resistance. In the present study, the Colletotrichumisolates collected from Doda and Rajouri districts of Jammu and Kashmir were identified based on micromorphology and cultural characteristics. Multigene sequence data (ITS, GAPDH, CHS-1, HIS3, ACT and TUB2) and phylogenetic analyses (ML) of the isolates authenticated them as Colletotrichumlindemuthianum. Results showed that all the tested isolates of C. lindemuthianumwere pathogenic to Phaseolus vulgaris cv. Bhaderwah-Rajmash (local name). Besides this, 87 collected diverse genotypes of common bean were screened morphologically against this isolate under epiphytotic conditions. Most of the genotypes showed high resistance and moderate resistance. Identified resistant genotypes were further confirmed by screening the common bean genotypes for Co-2 gene using SCAR marker SCAreoli1300/1000. Out of these genotypes, 10 common bean genotypes vizEC-500250,IC-274530,S1,S3,EC-398527,EC-398565,BR36,EC-398591, KB12, EC-121013 genotypes indicated the presence ofresistanceallele. Using NCBI data, promoter region of Co-2 gene was identified and marker was developed using Primer 3 software. Three anthracnose resistant and susceptible genotypes were amplified, cloned and sequenced for identification of SNPs associated to disease resistance. Twenty-three SNPs were identified at the regulatorymotifsregionsusingNsite(www.softberry.com)atpositions30, 32, 38, 39, 188, 223, 228, 326, 329, 475, 477, 481, 499, 503, 1017, 1019, 1024, 1369, 1373, 1469, 1471, 1472 and 1476. Fourteen SNPs were found