The Population Genetic And Genomic Study On Chinese V26 Lineage Of Puccinia Striiformis F.sp. Tritici

Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most important fungal diseases of wheat and poses a severe threat to wheat production worldwide.Due to virulence variation frequently,resistances of wheat cultivars were often overcome by new virulent races of Pst,resulting in control of the disease becoming a long-term problem in the world.Because the occurrence frequency of Chinese V26 lineage was increasing since the first dectection of V26 isolates in 2008,many wheat cultivars carrying Yr26 were overcome,leading to subsequent epidemics.While the origin and evolution of V26 lineage remain unclear.Pst can generate genetic variation through mutation,heterokaryosis,somatic recombination and sexual recombination.Previous studies demonstrated that sexual reproduction is an important way for causing virulence variation of Pst.However,how sexual reproduction affects the evolution of the pathogen,especially the emergence of the new virulent races under natural conditions,remains largely unknown.In addition,the absence of a complete V26 genome seriously restricts the accurate identification of genetic variation.Therefore,combining with traditional biology,genomics,population genetics and bioinformatics,this study analysed detailedly the population structure,linkage disequilibrium and selective pressure of Pst,identified candidates for avirulence genes,assemblied the genome of a V26 isolate and built a pan-genome of Pst.The major results were as follows:(1)Sexual recombination plays an important role in the evolution of Chinese V26 lineage of Pst.We sequenced whole genomes of 42 Pst isolates from China and other countries.Virulence profiles of Yr26-virulent isolates(called V26)were significantly broader than that of the Yr26-avirulent isolates(called Av26)according to the phenotypes on the set of wheat Yr(yellow rust)single-gene differential lines.Using population structure analysis based on genome-wide genetic variation,all isolates grouped into four genetic lineages(termed GL1,GL2,GL3,and GL4).All GL3 isolates were collected from China and avirulent to Yr26.GL4 only included all Chinese V26 isolates,and had very close relationships with GL3.Meanwhile,based on genome heterozygosity estimation,we found the heterozygosity of all GL3 and GL4 isolates was significantly lower than that of isolates from other countries,in which most countries are mainly or even completely dominated by asexual reproduction.Using the phylogenetic neighbor-net network and linkage disequilibrium simulations analysis for testing genetic recombination of Pst,we confirmed the frequent recombination events between and within GL3 and GL4,indicating that sexual reproduction has greatly impacted on the evolution of Pst virulent races in China,specifically for V26 lineage.Furthermore,combining the analyses of population structure and genetic differentiation,Chinese V26 lineage differentiated from Chinese Av26 lineage,and the V26 lineage had significantly lower nucleotide diversity than that of the Av26 lineage,suggesting Chinese V26 lineage have been exerted selective pressure.Selective sweep analysis revealed 94 genes located in candidate selected regions.(2)Six genes were identified as Avr Yr26 candidates.Through association analysis,total719 genes were significantly associated with Avr Yr26 locus,in which six genes encoding secreted proteins located in candidate selected regions.These six genes,including PSTG＿11420,PSTG＿12323,PSTG＿13164,PSTG＿13955,PSTG＿13957 and PSTG＿13967,were identified as Avr Yr26 candidates.Moreover,the expression of four candidates,including PSTG＿11420,PSTG＿13955,PSTG＿13957 and PSTG＿13967,were up-regulated either in haustoria or infected leaves relative to germinated urediniospores.We analysed the basic function of the gene PSTG＿13957,which was the most associated with Avr Yr26 locus among the six candidates.The functional signal peptide of PSTG＿13957 was confirmed by the yeast secretion system,and PSTG＿13957 was identified to suppress cell death caused by mouse BAX(BCL2-associated X)protein in Nicotiana benthamiana via PVX virus(potato virus X)mediated by Agrobacterium tumefaciens.Furthermore,PSTG＿13957 was highly induced at 48 hpi(hour post inoculation)by q RT-PCR(quantitative real-time PCR).These results indicate that PSTG＿13957 probably has the pathogenicity during the infection process of Pst.(3)We assembled a chromosome-level genome of a Pst isolate GS-15 of Chinese V26 lineage.A 76.61-Mb genome assembly was obtained,with 73.60 Mb anchored into 18 pseudochromosomes and a scaffold N50 of 4.01 Mb.Total 17,062 protein-coding genes were predicted,including 2,689 predicted secreted-protein genes and 1,511 predicted effector genes.In addition,we obtained a complete and circular mitogenome of GS-15 with 101,838 bp in length,containing 14 protein-coding genes and 24 t RNA genes.Compared with previous published Pst genomes,the quality and integrity of this assembly were better.By comparing 8 de novo assemblies of representative Pst with GS-15 assembly,total 45.49 Mb non-redundant novel sequences absent from GS-15 genome were identified.The final novel sequences and GS-15 genome were merged as the Pst pan-genome,with the mapping rates of re-sequencing data increasing 0.90～2.21%.In summary,this study demonstrated that sexual reproduction plays an important role in the evolution of Pst V26 lineage in China,and revealed that V26 lineage has been exerted selective pressure.Association analysis identified six genes encoding secreted proteins as Avr Yr26 candidates.This study also assembled a chromosome-level genome of Chinese V26 lineage,and built a pan-genome of Pst,which would contribute to a more comprehensive and accurate analysis of genetic variation,and avirulence genes mining.The results laid the foundation for in-depth analysis of virulence variation pathway and population evolution of Pst,as well as provided a theoretical basis for continuous and effective control of wheat stripe rust.