High-resolution bulked segregant analysis enables candidate gene identification for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.).

Bacterial wilt, caused by Xanthomonas translucens pv. graminis (Xtg), is a serious disease of economically important forage grasses, including Italian ryegrass (Lolium multiflorum Lam.). A major QTL for resistance to Xtg was previously identified, but the precise location as well as the genetic factors underlying the resistance are yet to be determined. To this end, we applied a bulked segregant analysis (BSA) approach, using whole-genome deep sequencing of pools of the most resistant and most susceptible individuals of a large (n = 7484) biparental F2 population segregating for resistance to Xtg. Using chromosome-level genome assemblies as references, we were able to define a ~300 kb region highly associated with resistance on pseudo-chromosome 4. Further investigation of this region revealed multiple genes with a known role in disease resistance, including genes encoding for Pik2-like disease resistance proteins, cysteine-rich kinases, and RGA4- and RGA5-like disease resistance proteins. Investigation of allele frequencies in the pools and comparative genome analysis in the grandparents of the F2 population revealed that some of these genes contain variants with allele frequencies that correspond to the expected heterozygosity in the resistant grandparent. This study emphasizes the efficacy of combining BSA studies in very large populations with whole genome deep sequencing and high-quality genome assemblies to pinpoint regions associated with a binary trait of interest and accurately define a small set of candidate genes. Furthermore, markers identified in this region hold significant potential for marker-assisted breeding strategies to breed resistance to Xtg in Italian ryegrass cultivars more efficiently.

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

KEY MESSAGE: SNPs and candidate genes associated with bacterial wilt resistance in Italian ryegrass were identified by sequencing the parental plants and pooled F1 progeny of a segregating population. Italian ryegrass (Lolium multiflorum Lam.) is one of the most important forage grass species in temperate regions. Its yield, quality and persistency can significantly be reduced by bacterial wilt, a serious disease caused by Xanthomonas translucens pv. graminis. Although a major QTL for bacterial wilt resistance has previously been reported, detailed knowledge on underlying genes and DNA markers to allow for efficient resistance breeding strategies is currently not available. 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 was pooled and sequenced using the Illumina HiSeq 2000 platform. Allele frequencies of 18 × 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. This study highlights the power of pooled DNA sequencing to efficiently target binary traits in biparental mapping populations. It delivers genome sequence data, SNP markers and potential candidate genes which will allow to implement marker-assisted strategies to fix bacterial wilt resistance in outcrossing breeding populations of Italian ryegrass.