Development of Genome-Wide Unique Indel Markers for a Heat-Sensitive Genotype in Wheat (Triticum aestivum L.)

A chromosome segment substituted line (CSSL) represents an ideal resource for studying quantitative traits like thermotolerance. To develop wheat inter-varietal CSSLs with E6015-3S (a heat-sensitive genotype) being the recipient parent, genome-wide unique DNA markers are urgently needed for marker-assisted selection. In this study, 11,016 primer pairs targeting 5036 indel sites were successfully designed for E6015-3S, with an average density of 0.36 indels per Mbp. These primer pairs are believed to be unique and polymorphic in the wheat genome; as gathered from the evidence, (i) 76.18 to 99.34% of the 11,016 primer pairs yielded a single hit during sequence alignment with 18 sequenced genomes, (ii) 83.59 to 90.98% of 1042 synthesized primer pairs amplified a single band in 16 wheat accessions, and (iii) 59.69 to 99.81% of the tested 1042 primer pairs were polymorphic between E6015-3S and 15 individual wheat accessions. These primer pairs are also anticipated with excellent resolvability on agarose or polyacrylamide gels, since most of them have indel sizes from 15 to 46 bp, amplicon sizes from 141 to 250 bp, and polymorphism ratios from 6.0 to 25.0%. Collectively, these primer pairs are ideal DNA markers for inter-varietal CSSL development and more broad applications, like germplasm classification, seed purity testing, genetic linkage mapping, and marker-assisted breeding in wheat, owing to their uniqueness, polymorphism, and easy-to-use characteristics.