Dissecting The Genetic Basis Of Stripe Rust Resistance In Wheat Cultivar Shumai126

Stripe rust,caused by Puccinia striiformis West f.sp.tritici（Pst）,is an important fungal disease affecting wheat production.Cultivation of stripe rust resistant cultivars is an economic and effective measure to control this disease.However,due to the rapid variation of stripe rust races and the emergence and prevalence of virulence physiological races,the stripe rust resistance of most wheat cultivars in production was quickly overcame and lost the value of production and utilization.Therefore,it is of great significance to exploit and apply new stripe rust resistance genes in wheat breeding that will facilitate to breed durable disease resistant cultivars and sustainable control of wheat stripe rust.Shumai126 is a new wheat cultivar bred by the Triticeae Research Institute of Sichuan Agricultural University.Shumai126 is a wheat cultivar with characteristics white grains,high yield,stable yield and high resistance to stripe rust.It was released as new wheat cultivar in Sichuan Province in 2016（no.2016004）.Shumai126 showed a high level of resistance in Sichuan Basin,an area with high and heavy incidence of stripe rust,since2012.In this study,we systematically analyzed the genetic basis of stripe rust resistance of Shumai126 by QTL mapping,histochemical observation,transcriptome analysis and virus induced gene silencing technology.The main results are as follows:（1）In order to identify the stripe rust resistance quantitative trait loci（QTL）in Shumai126,a recombinant inbred line（RIL）population was derived from the cross between cultivars Taichang29 and Shumai126.A genetic linkage map was constructed using Wheat55K single nucleotide polymorphism（SNP）array.The genetic map and stripe rust resistance phenotypes in different environments were further used to mapping the resistance QTL.Six resistance-QTL were mapped on chromosomes 1BL,2AS,2AL,6AS,6BS,and 7BL,respectively.The resistance alleles of all QTL were contributed by SM126except QYr.sicau-2AL.QYr.sicau-1BL and QYr.sicau-2AS are two major loci,explained27.00-39.91%and 11.89-17.11%of phenotypic variances,which may correspond to the reported resistance genes Yr29 and Yr69,respectively.QYr.sicau-2AL,QYr.sicau-6AS,and QYr.sicau-6BS had minor effects and may be novel.QYr.sicau-7BL was only detected at seedling stage,and QYr.sicau-2AS was stably detected at both seedling and adult-plant stages.SNP markers linked to QYr.sicau-1BL（AX-111056129 and AX-108839316）and QYr.sicau-2AS（AX-111557864 and AX-110433540）were converted to Kompetitive allele-specific PCR（KASP）markers,and that can be used for molecular marker assisted selection of these two QTL in wheat.（2）Histopathological techniques were used to reveal the histochemical characteristics of Shumai126 and 1BL line（QYr.sicau-1BL）and 2AS line（QYr.sicau-2AS）with single major QTL against CYR34.Shumai126（IT=3）and 2AS line（IT=2-3）were resistant to stripe rust at seedling stage,and the leaves of 14dpi showed obvious necrotic spots and sporadic spores can be seen on the leaves of Shumai 126;1BL line（IT=7-8）was susceptible to stripe rust,and the leaves showed a large number of uredinium.In addition,haustorial mother cells are established in both resistant and susceptible lines.The hypha growth in Shumai126 and 2AS line was significantly inhibited,which was manifested in shorter hypha length and the significant reduction of the number of hypha branches and haustoria.At the early stage of stripe rust infection,a large amount of H₂O₂was accumulated in the leaves of Shumai126 and 2AS line.（3）Shumai126 was inoculated with CYR34,a prevalent stripe rust race in China.The leaves at different time points after inoculation were sampled to perform RNA-Seq to identify the differently expressed gene（DEG）in response to stripe rust stress.In total,520,148 and 1439 DEGs were detected at 1,3,and 7 days post Pst infection（dpi）,respectively.Most of the DEGs expressed only at one or two sample time（s）,which showed transient expression patterns.GO and KEGG enrichment analysis revealed that many biological processes,such as photosynthesis,flavonoid biosynthesis,oxidative phosphorylation,MAPK signaling pathway,and phenylalanine metabolism were involved in Shumai126 in response to Pst.Some genes were involved in the plant-pathogen interaction pathway,including 202 typical resistance（R）genes and 16 transcription factors.These DEGs in response to Pst stress may be involved in the resistance of Shumai126.（4）One DEG encoding tyrosine-protein kinase was identified in plant-pathogen interaction pathway.This gene was temporarily named as TaTPK and differently expressed at 1dpi and 7dpi.The genomic DNA of TaTPK is 5666bp in length,and the coding sequence（CDS）is 1098bp.TaTPK encodes 365 amino acids with a tyrosine kinase catalytic domain.The CDS of TaTPK is highly conserved in Shumai126(TaTPK_-SM126)and Taichang29(TaTPK_-TC29).The expression level of TaTPK_-SM126was significantly higher than TaTPK_-TC29after Pst inoculation,which indicated that TaTPK may positively regulate the stripe rust resistance of Shumai126.TaTPK was localized on the cell membrane by tobacco transient expression system.Virus induced gene silencing experiment showed that transient silencing of TaTPK attenuates the resistance level of Shumai126 against stripe rust.The Pst growth was better and the accumulation of H₂O₂was less observed in TaTPK silenced leaves.The results showed that TaTPK may be involved in regulation of the accumulation of H₂O₂that lead to stripe rust resistance.