High Resolution Mapping of Rph MBR1012 Conferring Resistance to Puccinia hordei in Barley (Hordeum vulgare L.).

Leila Fazlikhani,Holger Deising,Jens Keilwagen,Dragan Perovic,Frank Ordon,Doris Kopahnke

doi:10.3389/fpls.2019.00640

Abstract

Isolation of disease resistance genes in barley was hampered by the large genome size, but has become easy due to the availability of the reference genome sequence. During the last years, many genomic resources, e.g., the Illumina 9K iSelect, the 50K Infinium arrays, the Barley Genome Zipper, POPSEQ, and genotyping by sequencing (GBS), were developed that enable enhanced gene isolation in combination with the barley genome sequence. In the present study, we developed a fine map of the barley leaf rust resistance gene RphMBR1012. 537 segmental homozygous recombinant inbred lines (RILs) derived from 4775 F2-plants were used to construct a high-resolution mapping population (HRMP). The Barley Genome Zipper, the 9K iSelect chip, the 50K Infinium chip and GBS were used to develop 56 molecular markers located in the target interval of 8 cM. This interval was narrowed down to about 0.07 cM corresponding to 0.44 Mb of the barley reference genome. Eleven low-confidence and 18 high-confidence genes were identified in this interval. Five of these are putative disease resistance genes and were subjected to allele-specific sequencing. In addition, comparison of the genetic map and the reference genome revealed an inversion of 1.34 Mb located distally to the resistance locus. In conclusion, the barley reference sequence and the respective gene annotation delivered detailed information about the physical size of the target interval, the genes located in the target interval and facilitated the efficient development of molecular markers for marker-assisted selection for RphMBR1012.

Highlights

Leaf rust of barley is a serious disease caused by the biotrophic fungus Puccinia hordei Otth., which, under favorable conditions, may cause yield losses of up to 62% (Park et al, 2015), while in general loses are about 15–25% (Whelan et al, 1997)
Disease resistance provided by major Rph genes is often overcome due to the emergence of new P. hordei pathotypes (Niks, 1982; Steffenson et al, 1993; Park, 2003) indicating the need for introducing new sources of resistance into barley breeding as well as the need for isolating known ones toward deciphering the structure and function offering the possibility of developing functional markers for breeding and create new alleles by e.g., CRISPR/Cas9 (Kumar et al, 2018)
In this study we have shown the efficient use of the barley reference sequence in physical mapping and especially in marker saturation

Summary

Introduction

Leaf rust of barley is a serious disease caused by the biotrophic fungus Puccinia hordei Otth., which, under favorable conditions, may cause yield losses of up to 62% (Park et al, 2015), while in general loses are about 15–25% (Whelan et al, 1997). To combat leaf rust epidemics caused by newly occurring/generated virulent races and to achieve a sustainable disease control, the employment of new resistance genes using functional molecular markers in breeding schemes as well as the isolation of known ones in order to get detailed information on the structure and function is of prime importance. Since the first genetic study on leaf rust resistance (Waterhouse, 1927), 25 Rph (Resistance to P. hordei) genes have been mapped in barley (Kavanagh et al, 2017). Among all known Rph genes, only Rph has been isolated recently, using the newly developed cloning approach called Mutant Chromosome Sequencing (MutChromSeq) (Steuernagel et al, 2016) in combination with genetic mapping (Dracatos et al, 2018)

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Discussion

Conclusion