Research On Important Biological Characteristics And Transcriptome Of All Spore Stages Of Puccinia Striiformis F.sp.Tritici

Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici（Pst）,is an important fungal disease.It happens all over the world,threatening the safety of wheat production.Sexual reproduction is an important way of virulence variation of wheat rust.Inheritance of avirulence/virulence and heterozygosity of Pst can be determined by genetic analysis of a sexual population.Based on genetic analysis of sexual population,correlation analysis between whole genome sequencing data and phenotype provides us with an effective way to rapidly map the avirulence genes of Pst,which provides a basis for further understanding the interaction mechanism between avirulence genes in stripe rust and the resistant genes in wheat.The use of fungicide for decades lead to the emergence of resistant isolates of pathogen.Analyses on sequence variation of CYP51 gene and sensitivity to triazolone of progeny isolates acquired by sexual reproduction make us understand the triazolone resistance mechanism of Pst,which provides theoretical basis for rational use of fungicides.The five spore types in the life cycle of Pst play distinctive roles in terms of fungal infection and survival.Mechanisms underlying these functional differences,however,are unclear.Therefore,this study focused on the population acquired by selfing isolates of Pst,combining with traditional genetics,genomics,transcriptomics and other means,to study the virulence variation of Pst,avirulence gene,mechanism of triazolone resistance and different functions of five spore stages in the life cycle.The main results are as follows:1)One hundred and twenty progeny isolates were obtained by selfing an isolate GS-2013 collected in Gansu Province.These progeny isolates were divided into 51pathotypes using 25 lines of wheat containing Yr genes.All these progeny isolates were avirulent on lines with Yr5,Yr10,Yr15,Yr24 and Yr26 and virulent on lines with Yr17,Yr25and Yr A.Segregation was found for wheat lines with Yr1,Yr2,Yr4,Yr6,Yr7,Yr8,Yr9,Yr27,Yr28,Yr32,Yr43,Yr44,Yr Exp2,Yr Sp,Yr Tr1,Yr Tye and Yr V23.The avirulence/virulence of the parental isolate was controlled by one or two genes.These 120 progeny isolates were divided into 55 genotypes by 11 SSR（Simple Sequence Repeats）molecular markers.In this study,four avirulence genes and 11 SSR molecular markers were used to construct a genetic linkage map.The linkage relationship indicated that CAvr7,CAvr8 and CAvr Exp2 may be clustered at a specific location.CAvr SP was far away from these three loci.2)An isolate collected from Gansu province named Pinglan17-7 and 52 progeny isolates acquired by selfing this isolate were sequenced.BSA（Bulked Segregant Analysis）and correlation analysis were used to map the avirulence gene Avr6.Three hundred and forty-one SNPs were obtained by BSA method.Seventy-three SNPs were located in 18genes.Among these genes,six genes（PSTCY32＿10201,PSTCY32＿10203,PSTCY32＿10707,PSTCY32＿17500,PSTCY32＿17501 and PSTCY32＿17717）encoded secreted proteins.A total of 78 significantly correlated SNPs were obtained by association analysis.Among these SNPs,27 were located within nine genes,and PSTCY32＿08746 encoded secreted protein.Among these seven genes encoding secreted protein,PSTCY32＿08746,PSTCY32＿10203and PSTCY32＿10707 were predicted to be effector genes by Effector P v2.0.According to the results of Pfam v31.0,PSTCY32＿17501 contained protein domain with unknown function,and PSTCY32＿17717 contained extension factor Tu GTP binding domain.Finally,in addition to PSTCY32＿17717,the other six genes were identified as candidate genes for avirulence gene Avr6.3)An isolate collected in Gansu province named Pinglan17-7 and some of progeny isolates acquired by selfing were examined for their sensitivity to triadimefon by detached leaf method.According to the resistance criteria used in this study,the parental isolate showed resistance to triazolone,with the EC50 value of 1.88mg/L.A wide range of EC50were detected among the progeny isolates ranging from 0.06 mg/L to 7.89 mg/L.A mutation at codon 134（Y134F）was identified in resistant isolates.Isolates with mutation presented higher EC50 than wild type isolates.Significant difference was found between log EC50 of mutant isolates and wild type isolates(P=2.2e^-16).However,difference between log EC50 of isolates with homozygous and heterozygous point mutations was not obviously discrepant（P=0.21）.Separation ratio of progeny isolates with homozygous point mutation,heterozygous point mutation and wild type was in accord with 1:2:1（P>0.05）.There was a negative correlation between triadimefon resistance and lesion growth rate（r=-0.53;P=0.01）.No significant difference on lesion growth was found between isolates with homozygous point mutation and isolates with heterozygous point mutation（P=0.83）.In the cases of latent period and sensitivity to triadimefon,no correlation was found（P>0.05）.4)High-depth transcriptome sequencing was performed on five spore stages.Of the29591 transcripts identified,4817 transcripts were expressed at all spore stages,and these transcripts were significantly enriched in the GO term related to biosynthesis,transport and metabolism.Nine hundred and fifty-one（10.5%）transcripts in basidiospores were specifically expressed,follow by 920（8.5%）,761（10.2%）,266（2.7%）and 110（1.3%）in teliospores,pycniospores,aecidiospores and urediniospores respectively.Principal component analysis and hierarchical clustering showed that the expression patterns of urediniospores,teliospores,and aecidiospores were similar,while the expression patterns of pycniospores and basidiospores were different from other spore stages.Genes with specific functions at specific spore stages were also identified in this study.