Identication of Candidate Resistance Genes against Rhynchosporium in Barley
Rhynchosporium caused by the fungus Rhynchosporium commune is one of the most economically important and damaging diseases of barley worldwide. Research was carried out to identify candidate genes for resistance against rhynchosporium in barley, using genetics and next generation sequencing approaches, with the aim of improving our understanding of the molecular mechanisms behind host resistance to rhynchosporium and developing tightly linked molecular markers that can be used by breeders. QTL mapping of a Steptoe x Morex (SxM) Doubled Haploid (DH) population using R. commune isolates 271 and UK7 identified a significant QTL on the short arm of chromosome 6H, close to the Rrs13 locus. Fine mapping using select SxM BC1 lines, R. commune isolate L73a and KASP markers designed from Steptoe exome capture variant calling data was carried out to fine map the QTL to a 660 kb interval, between 10.92 and 11.58 Mb on 6H. The Morex reference genome sequence showed that flanking markers for the QTL mapped closer to the telomere than the flanking markers for Rrs13, to a rhynchosporium resistance locus identified previously in a CIho 3515 x Alexis (CxA) DH population that had been named Rrs18. Rrs18 resistance was shown to be dominant, providing partial resistance, causing a reduction but not an eradication of symptoms. A preliminary field trial showed Rrs18 in the CxA population to be effective in the field. A total of 17 genes were identified within the Rrs18 interval using the Morex genome annotation with only six of these genes expressed in leaves of SxM BC1 lines, CIho 3515 and Alexis used for RNA-seq. Of these a protein kinase was identified as the most likely candidate for Rrs18. A comparison of SNPs present in RNA-seq datasets identified six candidate SNPs for the causative variant of Rrs18 resistance. Rrs18 appears to be present in a number of Syrian and Jordanian landraces, along with the relatively well characterised rhynchosporium resistance gene Rrs1. Rrs1 has previously been mapped to the centromeric region of chromosome 3H using a genome wide association study, identifying three SNPs in a wall associated kinase (HORVU3Hr1G064130) highly associated with resistance. A close study of exome capture variant calls and read mappings from Rrs1 carrying lines revealed that a set of reads had mismapped to HORVU3Hr1G064130 from a closely linked paralog. PCR validation and Sanger sequencing, combined with further next generation sequencing enabled part of the gene sequence and structure of the Rrs1 candidate to be deciphered. qRT-PCR revealed the Rrs1 candidate to be expressed at a low level, with no evidence of signicant changes in expression after inoculation with R. commune. Virus induced gene silencing (VIGS) was carried out in an attempt to validate the Rrs1 candidate. Tightly linked molecular markers for Rrs1 and Rrs18 will provide a useful tool for breeders aiding marker assisted selection.
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