Rapid isolation of Yr9 via MutIsoSeq and QTL analysis of durable stripe rust resistance in wheat cultivar Xingzi 9104

Abstract

The fungus Puccinia striiformis f. sp. tritici (Pst) is the causal agent of wheat stripe rust which constitutes a major limitation to wheat production. Cloning and applying disease-resistant genes are considered as an effective solution. Chinese wheat cultivar Xingzi 9104 (XZ9104) has exhibited durable resistance across multiple environments since its release. Through quantitative trait loci (QTL) analysis, eight QTL were found on chromosome arms 1BS, 1BL, 2AL, 2BL, 3BS, 4BL, 5BL and 7BL. YrXZ identified as 1RS.1BL translocation conferred race-specific all-stage resistance to Pst race CYR23. QYrxz.nwafu-1BL.6 and QYrxz.nwafu-3BS.7 were considered as the adult plant resistance genes Yr29 and Yr30, respectively. Notably, QYrxz.nwafu-2BL.5 accounted for 15.75-47.63% of the phenotypic variation across diverse environments and its pyramiding with Yr29 and Yr30 can confer high level of resistance. Other QTL were environment-dependent with minor effects. To clone the above resistance genes, we created a population of over 2,000 M₅ mutants in XZ9104 using ethylmethane sulfonate (EMS) mutagenesis and screened various types of susceptible mutants. Using the MutIsoseq approach with five mutant lines susceptible to race CYR23, we rapid isolated a candidate gene for YrXZ encoding coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) protein. Integrating cytological analysis, gene-based association analysis, transcriptomic profiling and virus-induced gene silencing (VIGS), we confirmed that the causal gene for YrXZ was indeed Yr9. This study demonstrated that multiple QTL with different effects contributed to the durable resistance in XZ9104. Understanding the molecular mechanisms and pathways involved in plant defense can inform future strategies for deploying resistance gene and engineering of genetic resistance against evolving diseases.

Keywords: Yr9; Durable resistance; QTL analysis; Stripe rust; Xingzi 9104.

Fig. 1

The overall response of XZ9104 (a) and AvS (b) to stripe rust at adult plant stage, and the typical stripe rust symptoms of wheat lines XZ9104 (c1-4) and AvS (c5-8) after inoculation with different Pst races at seedling stage. d Frequency distributions of mean infection type (IT) for 177 RILs from cross AvS × XZ9104 evaluated at Yangling, Jiangyou and Tianshui. e Frequency distributions of mean disease severities (DS) for 177 RILs from cross AvS × XZ9104 evaluated at Yangling, Jiangyou and Tianshui

Fig. 2

Deletion SNP analysis was performed on the whole genome and 1BS chromosome using a wheat 660 K SNP array (a, b). c The wheat disease-resistant material XZ9104 was identified by FISH. Oligo-pSc119.2 (green) and Oligo-pTa535 (red) were labeled as FISH probes. Scale bars, 10 μm (color figure online). d The molecular marker Yr9 (1B. 1R)-H20 was used to verify the materials involved in this study. AvS, MX169 and XY22 were Yr9 negative controls

Fig. 3

Graphical display of stripe rust QTL mapping regions on chromosome arms 1BL (a), 2BL (c), 3BS (d) in six environments in the field, and 1BS (b) in the seeding stage, respectively. The flanking markers colored with blue, and the red bar represents the target interval

Fig. 4

(a) Effects of different combinations of quantitative trait loci (QTL) on stripe rust reaction using infection type (IT) data for the AvS × XZ9104 RIL population from Yangling (YL), Tianshui (TS), and Jiangyou (JY). b Effects of different combinations of quantitative trait loci (QTL) on stripe rust reaction using disease severity (DS) data for the AvS × XZ9104 RIL population from Yangling (YL), Tianshui (TS), and Jiangyou (JY)

Fig. 5

(a) The response of the selected EMS susceptible mutants to the race CYR23 of wheat stripe rust. 1–10 were mutation lines, 11–12 were the susceptible parent AvS, 13–14 were the susceptible control MX169, 15–16 were the resistant control KN9204, and 17–18 were the wild type XZ9104. b Gene structure of The YrXZ (Yr9). c The predicted protein structure of YrXZ and the location of the mutation sites of each mutant (marked by blue inverted triangle). d Representative images showing the results of the VIGS experiment using barley stripe mosaic virus (BSMV). BSMV-γas1 and BSMV-γas2 indicate two silencing constructs targeting YrXZ, BSMV-γ indicates control, and BSMV-γPDS indicates silencing construct targeting the phytoene desaturase genes. Mock represents the results of wild-type inoculation without silencing any structure

Fig. 6

The graph of the frequency and distribution of Yr9. Using the developed Yr9-specific functional markers, several germplasms around the world were identified, and their frequency and distribution were counted. The orange area in the fan graph represents the presence of Yr9. The world map was downloaded at the website (http://bzdt.ch.mnr.gov.cn), and the map content approval number is GS(2016)1663