Development of Molecular Markers Linked to Powdery Mildew Resistance Gene Pm4b by Combining SNP Discovery from Transcriptome Sequencing Data with Bulked Segregant Analysis (BSR-Seq) in Wheat.

Peipei Wu,Zhongjun Zhang,Yang Zhou,Miaomiao Li,Hongjun Zhang,Hongjie Li,Hongwei Liu,Li Yang,Jingting Li,Zhiyong Liu,Dan Qiu,Jinghuang Hu,Jingzhong Xie

doi:10.3389/fpls.2018.00095

Abstract

Powdery mildew resistance gene Pm4b, originating from Triticum persicum, is effective against the prevalent Blumeria graminis f. sp. tritici (Bgt) isolates from certain regions of wheat production in China. The lack of tightly linked molecular markers with the target gene prevents the precise identification of Pm4b during the application of molecular marker-assisted selection (MAS). The strategy that combines the RNA-Seq technique and the bulked segregant analysis (BSR-Seq) was applied in an F2:3 mapping population (237 families) derived from a pair of isogenic lines VPM1/7∗Bainong 3217 F4 (carrying Pm4b) and Bainong 3217 to develop more closely linked molecular markers. RNA-Seq analysis of the two phenotypically contrasting RNA bulks prepared from the representative F2:3 families generated 20,745,939 and 25,867,480 high-quality read pairs, and 82.8 and 80.2% of them were uniquely mapped to the wheat whole genome draft assembly for the resistant and susceptible RNA bulks, respectively. Variant calling identified 283,866 raw single nucleotide polymorphisms (SNPs) and InDels between the two bulks. The SNPs that were closely associated with the powdery mildew resistance were concentrated on chromosome 2AL. Among the 84 variants that were potentially associated with the disease resistance trait, 46 variants were enriched in an about 25 Mb region at the distal end of chromosome arm 2AL. Four Pm4b-linked SNP markers were developed from these variants. Based on the sequences of Chinese Spring where these polymorphic SNPs were located, 98 SSR primer pairs were designed to develop distal markers flanking the Pm4b gene. Three SSR markers, Xics13, Xics43, and Xics76, were incorporated in the new genetic linkage map, which located Pm4b in a 3.0 cM genetic interval spanning a 6.7 Mb physical genomic region. This region had a collinear relationship with Brachypodium distachyon chromosome 5, rice chromosome 4, and sorghum chromosome 6. Seven genes associated with disease resistance were predicted in this collinear genomic region, which included C2 domain protein, peroxidase activity protein, protein kinases of PKc_like super family, Mlo family protein, and catalytic domain of the serine/threonine kinases (STKc_IRAK like super family). The markers developed in the present study facilitate identification of Pm4b during its MAS practice.

Highlights

In wheat (Triticum aestivum L.), powdery mildew is caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) (Green et al, 2014)
A powdery mildew resistance gene in VPM1 was localized on a T. persicum chromosomal segment that was translocated onto the long arm of wheat chromosome 2A, and proved to be an allele in locus Pm4, designated Pm4b (Bariana and McIntosh, 1994)
Line VPM1/7∗Bainong 3217 F4 was resistant to 72.7% of isolates that were collected from Hebei province, and it was effective against half of the isolates from Henan, and Shandong provinces

Summary

Introduction

In wheat (Triticum aestivum L.), powdery mildew is caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) (Green et al, 2014). A powdery mildew resistance gene in VPM1 was localized on a T. persicum chromosomal segment that was translocated onto the long arm of wheat chromosome 2A, and proved to be an allele in locus Pm4, designated Pm4b (Bariana and McIntosh, 1994). Even though it was identified over 30 years ago, Pm4b is still effective in certain regions of China and the United States (Wang et al, 2005; Parks et al, 2008; Zeng et al, 2014). It was used to enhance powdery mildew resistance in triticale (× Triticosecale Wittmack) (Kowalczyk et al, 2011)

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