No abstract

The powdery mildew resistance gene Pm21 was physically and comparatively mapped by newly developed markers. Seven candidate genes were verified to be required for Pm21 -mediated resistance to wheat powdery mildew. Pm21, a gene derived from wheat wild relative Dasypyrum villosum, has been transferred into common wheat and widely utilized in wheat resistance breeding for powdery mildew. Previously, Pm21 has been located to the bin FL0.45-0.58 of 6VS by using deletion stocks. However, its fine mapping is still a hard work. In the present study, 30 gene-derived 6VS-specific markers were obtained based on the collinearity among genomes of Brachypodium distachyon, Oryza and Triticeae, and then physically and comparatively mapped in the bin FL0.45-0.58 and its nearby chromosome region. According to the maps, the bin FL0.45-0.58 carrying Pm21 was closely flanked by the markers 6VS-03 and 6VS-23, which further narrowed the orthologous regions to 1.06 Mb in Brachypodium and 1.38 Mb in rice, respectively. Among the conserved genes shared by Brachypodium and rice, four serine/threonine protein kinase genes (DvMPK1, DvMLPK, DvUPK and DvPSYR1), one protein phosphatase gene (DvPP2C) and two transcription factor genes (DvGATA and DvWHY) were confirmed to be required for Pm21-mediated resistance to wheat powdery mildew by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) and transcriptional pattern analyses. In summary, this study gives new insights into the genetic basis of the Pm21 locus and the disease resistance pathways mediated by Pm21.

Pm21, originating from wheat wild relative Dasypyrum villosum, confers immunity to all known races of Blumeria graminis f. sp. tritici (Bgt) and has been widely utilized in wheat breeding. However, little is known on the genetic basis of the Pm21 locus. In the present study, four seedling-susceptible D. villosum lines (DvSus-1 ∼ DvSus-4) were identified from different natural populations. Based on the collinearity among genomes of Brachypodium distachyon, Oryza, and Triticeae, a set of 25 gene-derived markers were developed declaring the polymorphisms between DvRes-1 carrying Pm21 and DvSus-1. Fine genetic mapping of Pm21 was conducted by using an extremely large F2 segregation population derived from the cross DvSus-1/DvRes-1. Then Pm21 was narrowed to a 0.01-cM genetic interval defined by the markers 6VS-08.4b and 6VS-10b. Three DNA markers, including a resistance gene analog marker, were confirmed to co-segregate with Pm21. Moreover, based on the susceptible deletion line Y18-S6 induced by ethyl methanesulfonate treatment conducted on Yangmai 18, Pm21 was physically mapped into a similar interval. Comparative analysis revealed that the orthologous regions of the interval carrying Pm21 were narrowed to a 112.5 kb genomic region harboring 18 genes in Brachypodium, and a 23.2 kb region harboring two genes in rice, respectively. This study provides a high-density integrated map of the Pm21 locus, which will contribute to map-based cloning of Pm21.

Pm21, derived from wheat wild relative Dasypyrum villosum, is one of the most effective powdery mildew resistance genes and has been widely applied in wheat breeding in China. Mapping and cloning Pm21 are of importance for understanding its resistance mechanism. In the present study, physical mapping was performed using different genetic stocks involving in structural variations of chromosome 6VS carrying Pm21. The data showed that 6VS could be divided into eight distinguishable chromosomal bins, and Pm21 was mapped to the bin FLb4–b5/b6 closely flanked by the markers 6VS-08.6 and 6VS-10.2. Comparative genomic mapping indicated that the orthologous regions of FLb4–b5/b6 carrying Pm21 were narrowed to a 117.7 kb genomic region harboring 19 genes in Brachypodium and a 37.7 kb region harboring 5 genes in rice, respectively. The result was consistent with that given by recent genetic mapping in diploid D. villosum. In conclusion, this study demonstrated that physical mapping based on chromosomal structural variations is an efficient method for locating alien genes in wheat background.

Abstract:Common wheat (Triticum aestivum L.) is one of the most important cereal crops. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a continuing threat to wheat production. The Pm21 gene, originating from Dasypyrum villosum, confers high resistance to all known Bgt races and has been widely applied in wheat breeding in China. In this research, we identify Pm21 as a typical coiled-coil, nucleotide-binding site, leucine-rich repeat gene by an integrated strategy of resistance gene analog (RGA)-based cloning via comparative genomics, physical and genetic mapping, BSMV-induced gene silencing (BSMV-VIGS), large-scale mutagenesis and genetic transformation.not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/177857 doi: bioRxiv preprint first posted online Common wheat (Triticum aestivum L.) is the most widely grown cereal crop occupying ~17% of all cultivated land worldwide and providing ~20% of the calories consumed by humankind (Fu et al. 2009). However, wheat production is challenged constantly by powdery mildew, which is caused by Blumeria graminis f. sp. tritici (Bgt). Utilization of powdery mildew resistance (Pm) genes is an effective and economical way to reduce yield losses caused by Bgt. Up to now, more than 100 Pm genes in wheat and its relatives have been documented (McIntosh et al. 2017). Among them, Pm21 that originates from Dasypyrum villosum confers a high level of resistance to all known Bgt races (Chen et al. 1995;Cao et al. 2011). It is important to clarify the genetic basis and functional mechanism of Pm21.Previously, several candidate genes, including Stpk-V and DvUPK located in chromosome 6VS bin FL0.45-0.58 carrying Pm21, were reported to be required by Pm21 resistance (Cao et al. 2011; He et al. 2016); however, due to lack of a fine map, the relationships of these candidate genes and Pm21 are unclear.In the present study, four D. villosum lines (DvSus-1 ~ DvSus-4) susceptible to Bgt isolate YZ01 at the seedling stage were identified from a total of 110 accessions ( Fig. 1E and Table S1). Fine genetic mapping of Pm21 was conducted on an F 2 population derived from a cross between resistant line DvRes-1 carrying Pm21 and susceptible line DvSus-1. Among the total 10,536 F 2 plants, 64 recombinants between markers 6VS-00.1 and Xcfe164 (Qi et al. 2010) on 6VS were identified. Pm21 was then mapped to a 0.01-cM interval flanked by the markers 6VS-08.4b and 6VS-10b ( Fig. 1A and Fig. S1), in which, genes DvEXO70 (6VS-08.8b; encoding a putative exocyst complex component EXO70A1-like protein) and DvPP2C (6VS-09b) co-segregated with Pm21 (Fig. S2), whereas candidate genes reported previously, such as Stpk-V (Cao et al. 2011) , were not.A conserved coiled-coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR)-encoding resistance gene analog (RGA) locus was found between wheat and Brachypodium by comparative mapping (Fig. 1B, 1C and 1D). Subse...