Delineating the elusive BaMMV resistance gene rym15 in barley by medium-resolution mapping

Abstract

Barley mild mosaic virus (BaMMV), transmitted by the soil-borne protist Polymyxa graminis, has a serious impact on winter barley production. Previously, the BaMMV resistance gene rym15 was mapped on chromosome 6HS, but the order of flanking markers was non-collinear between different maps. To resolve the position of the flanking markers and to enable map-based cloning of rym15, two medium-resolution mapping populations Igri (susceptible) × Chikurin Ibaraki 1 (resistant) (I × C) and Chikurin Ibaraki 1 × Uschi (susceptible) (C × U), consisting of 342 and 180 F2 plants, respectively, were developed. Efficiency of the mechanical inoculation of susceptible standards varied from 87.5 to 100% and in F2 populations from 90.56 to 93.23%. Phenotyping of F2 plants and corresponding F3 families revealed segregation ratios of 250 s:92r (I × C, χ2 = 0.659) and 140 s:40r (C × U, χ2 = 0.741), suggesting the presence of a single recessive resistance gene. After screening the parents with the 50 K Infinium chip and anchoring corresponding SNPs to the barley reference genome, 8 KASP assays were developed and used to remap the gene. Newly constructed maps revealed a collinear order of markers, thereby allowing the identification of high throughput flanking markers. This study demonstrates how construction of medium-resolution mapping populations in combination with robust phenotyping can efficiently resolve conflicting marker ordering and reduce the size of the target interval. In the reference genome era and genome-wide genotyping era, medium-resolution mapping will help accelerate candidate gene identification for traits where phenotyping is difficult.

High-Resolution Mapping of Barley mild mosaic virus Resistance Gene rym15

“…(A) An initial genetic map of rym15 based on a set of 217 DH lines derived from the cross of Chikurin Ibaraki 1 × Plaisant ( Le Gouis et al, 2004 ). (B) Medium-resolution map of rym15 based on a set of 180 F 2 lines derived from the population Igri×Chikurin Ibaraki 1 ( Wang et al, 2021 ). (C) High-resolution map of rym15 based on a set of 139 F 4 segmental RILs in the population Igri ×Chikurin Ibaraki 1.…”

“…DNA of F 2 plants was extracted at the two-leaf stage using the efficient 96-sample multiplex DNA extraction protocol described by Milner et al (2019) . All F 2 plants were analyzed using the co-dominant flanking markers rym15 _1 and rym15 _8 which we identified in a previous study ( Wang et al, 2021 ). Those F 2 plants carrying a recombination event within the target interval were self-pollinated and selfed seeds were harvested.…”

“…Based on the physical position of the previous flanking markers rym15 _1 and rym15 _8, a set of 28 SNPs derived from the 50 K Illumina Infinium iSelect SNP chip (8 SNPs), GBS (8 SNPs) and assembly data (12 SNPs) located in the target interval was converted to KASP markers using BatchPrimer3 and PolyMarker ( You et al, 2008 ; Ramirez-Gonzalez et al, 2015 ) algorithms. Furthermore, another two KASP markers located between markers rym15 _1 and rym15 _8 were selected from a previous study ( Wang et al, 2021 ; Supplementary Table S1 ).…”

“…The first genetic mapping of the Chikurin Ibaraki 1 derived BaMMV resistance locus rym15 revealed that it is inherited recessively and located on chromosome 6HS ( Le Gouis et al, 2004 ). In a previous publication ( Wang et al, 2021 ), two medium-resolution maps were constructed by using a set of 180 (I × C) and 342 (C × U) F 2 plants. In this publication mapping was done by the use of six SSR markers and eight KASP markers ( rym15 _1 to rym15 _17) that were developed based on a 50 K Illumina Infinium iSelect screen of three parental lines and phenotyping of corresponding F 2 -F 3 families, the gene was fixed between KASP markers rym15 _1 and rym15 _8 in an interval around 137 Mb according to the barley reference assembly Morex v2 ( Wang et al, 2021 ).…”

“…In a previous publication ( Wang et al, 2021 ), two medium-resolution maps were constructed by using a set of 180 (I × C) and 342 (C × U) F 2 plants. In this publication mapping was done by the use of six SSR markers and eight KASP markers ( rym15 _1 to rym15 _17) that were developed based on a 50 K Illumina Infinium iSelect screen of three parental lines and phenotyping of corresponding F 2 -F 3 families, the gene was fixed between KASP markers rym15 _1 and rym15 _8 in an interval around 137 Mb according to the barley reference assembly Morex v2 ( Wang et al, 2021 ). Based on this information, in a current study, two high-resolution mapping populations comprising 2,218 (I × C) and 5,870 (C × U) F 2 plants were developed and corresponding F 4 segmental RILs were phenotyped using the BaMMV-ASL isolate, the present study aimed to (1) construct a high-resolution mapping population of rym15 , (2) narrow down the target region, and (3) predict potential candidate genes for BaMMV resistance gene rym15 .…”