Pooled DNA sequencing to identify SNPs associated with a major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.)

We used pooled DNA sequencing to characterize a major QTL for bacterial wilt resistance of Italian ryegrass and to develop inexpensive sequence-based markers to efficiently target resistance alleles for marker-assisted recurrent selection. From the mapping population segregating for the QTL, DNA of 44 of the most resistant and 44 of the most susceptible F₁ individuals were pooled and sequenced using the Illumina HiSeq2000 platform. Allele frequencies of 18 x 10⁶ single nucleotide polymorphisms (SNP) were determined in the resistant and susceptible pool. A total of 271 SNPs on 140 scaffold sequences of the reference parental genome showed significantly different allele frequencies in both pools. We converted 44 selected SNPs to KASP markers, genetically mapped these proximal to the major QTL and thus validated their association with bacterial wilt resistance.