| Wheat stripe(yellow)rust,caused by Puccinia striiformis Westend.f.sp.tritici Erikss.(Pst)is one of the most destructive fungal diseases of wheat worldwide.The major approaches for controlling stripe rust are growing resistant cultivars and applying fungicides.However,changes in virulence frequently occur in the pathogen population and fungicide applications generate additional costs and potentially are harmful to the environment.Therefore,it is important to develop wheat cultivars with durable resistance.Systemic research on the mechanisms between wheat-Pst has been extensively conducted in the fields of histology,cytology,molecular biology,and biochemistry.However,the stripe rust pathogen is a biotrophic obligate parasite in the basidiomycete group of fungi.The fungus has a heteroecious macrocyclic life cycle consisting of five spore stages,and the special features of hexaploidy with large and complex genome and difficulties for transformation.Studies of genes involved in the wheat-Pst interactions using genetic and molecular techniques were largely limited.The discovery of the complete life cycle and the sequencing of genome provide a convenient study for studying the genetic mechanism of wheat stripe rust.In the present study,a sexual population was developed through self-fertilizing Pst isolate 08-220 on barberry plants undercontrolled greenhouse conditions and the cluster population were collected include 372 isolate from different countries as the research object.Molecular techniques were used to study the role of of the virulence gene and mating type Gene on the evolution of wheat stripe rust.As a result,we obtained the following main innovative research results.(1)A segregating population of 119 F1 isolates was developed by self-crossing isolate 08-220(race PSTv-11)of P.striiformis f.sp.tritici,which can be used as a F2 generation for the genetic study of a trait controlled by heterokaryotic alleles in the parental isolate.Based on the virulence/avirulence patterns of the progeny isolates on the set of 18 Yr single-gene differentials,49 isolates were identified as race PSTv-11(virulent to Yr1,Yr6,Yr7,Yr8,Yr9,Yr17,Yr27,Yr43,Yr44,YrExp2,and Yr76;avirulent on Yr5,Yr10,Yr15,Yr24,Yr32,YrSP,and YrTrl),the same as the parental isolate;10 isolates as PSTv-71(virulent to Yr1,Yr6,Yr7,Yr9,Yr27,Yr43,Yr44,YrExp2,and Yr76;avirulent to Yr5,Yr8,Yr10,Yr15,Yr17,Yr24,Yr32,YrSP,and YrTrl),2 isolates as PSTv-76(virulence to Yr1,Yr6,Yr7,Yr8,Yr9,Yr17,Yr27,Yr43,Yr44,and YrExp2;avirulent to Yr5,Yr10,Yr15,Yr24,Yr32,YrSP,YrTrl,and Yr76);and 58 isolates as 27 new races.26 virulence loci were confirmed.(2)The population was segregated at 19 avirulence/virulence loci.The segregation ratios of avirulence/virlence showed that virulence could be controlled by one or two genes with different modes of inheritance and interactions.(3)Our study is the first to use the GBS technology to study genetics and mapping virulence genes in Pst.The GBS experiment generated 10163 SNP markers after filtering out low quality tags.Based on the genetic relationships among the progeny isolates,the 10,163 SNP markers were grouped in six clusters.The results indicated that Pst had six chromosomes.CHI consisted of 2483 markers,CH2 1396 markers,CH3 1393 markers,CH4 1793 markers,CH5 1253 markers,and CH6 1845 markers.Our study demonstrated that GBS was suitable for constructing genetic maps for Pst and other fungi.(4)To construct a base genetic map for Pst,1072 GBS-SNP markers were selected based on their P values(>0.1)of Chi-squared test for goodness of fit to the expected 3:1 ratio(presence:absence)for the self-fertilized progeny population.The 1072 markers,together with 30 SSR and 23 SP-SNP markers were used in linkage analysis.Of these 1104 markers,805 were assigned to linkage groups,whereas 299 were not assigned to any linkages.Using software Joinmap,six linkage groups were constructed with the 805 markers.These linkage groups spanned a total of 597.8 cM.The six linkage groups corresponded to the six clusters,presumed as six chromosomes.The individual chromosomes contained 52 to 167,on average of 137 marker loci.The genetic lengths of the linkage groups ranged from 65.8 cM to 148.9 cM with an average genetic distance of 0.73 cM between two loci.(5)When the virulence loci were integrated into the base map,17 of the 26 virulence loci were mapped whereas the other 10 were not mapped including the virulence loci to resistance genes Yr2 and Yr76.The mapped virulence loci mainly distributed on chromosomes 4 and 6.In chromosome 6,12 virulence loci(VYr17-1,VYrExp2-1.VYr35,VYr8,VYr25,VYr7,VYr9,VYr74,VYr6,VYr1,VYr27,and VYr28)were clustered to one end of the chromosome within a genetic distance of 35.3 cM without interruption by any molecular markers.The closest marker was GBS-SNP marker B64305,9.1 cM proximal from the VYr28 locus.The VYr31-1 locus mapped 26.7 cM away from VYr28 with nine markers in between,and it was tightly linked with GBS markers B6273(0.1 cM).In chromosome 6,VYr21-1 mapped 7.5 cM away from VYr31-1 with six markers in between,and the locus was flanked by GBS markers A212837(0.2 cM proximal)and B67965(0.4 cM distal).Using the expectation-maximization algorithm(EM)method,two QTL associated with the virulence phenotype to Yr43 mapped to Chromosome 4 using a cutoff value of LOD 3.48.Using the QTL mapping approach,one QTL(VYr43-1)conferring the Yr43 virulence phenotype was mapped in an interval of 4.2 cM flanked by GBS-SNP markers A264254 and A157785,and the other QTL(VYr43-2)for the same virulence phenotype was mapped in an interval of 1.7 cM flanked by GBS-SNP markers A417112 and A418952,which was about 13.5 cM from the first QTL.(6)We developed 28 mating type SNP markers and used these markers to study the population structure of Pst.from 372 isolates collected in the different countries.The 372 isolates were grouped into five genetic lineages.74 isolates from US were clustered into Clusterl.92 isolates from Uzbekistan,China,Russia,Tajikistan,Turkey,Nepal,Turkey,Pakistan,eight countries were clustered into Cluster2.63 isolates from Nepal,Mexico,Kyrgyzstan,Hungary,Chile,Spain,Algeria,Germany,Canada and Australia,ten countries were clustered into Cluster3.76 isolates from US and Ecuador were clustered into Cluster4.67 isolates from Italy,Ecuador and US were clustered into Cluster5.The genetic diversity of Nei’s was 0.39564,the Shannon information index(Ⅰ)was 0.5828,the allele frequency was 0.6832,the genetic diversity was 0.4007,the heterozygosity was 0.4555,and the gene flow was 0.3293.It was presumed that thegenetic diversity of mating type geneofwheat stripe rust was higher,and mating type gene diversity had nothing to do with the region.Mating type gene flow intensity was weak.Using the QTL mapping approach,MAT21 might be related to the virulence gene Yr1 and most of the SNP markers were not associated with virulence.(7)The gene expression of mating gene was studied by Q-PCR technique in different life cycle of wheat stripe rust.All genes were highly expressed in germinated spores,especially in pycnia spores and aecia spores,the results showed that mating genes played an important role in wheat stripe rust cycle.Fully characterized virulence genes and mating type gene should improve the understanding of the wheat-Pst interactions and the information can be used to select effective resistance genes for better combinations to be used in breeding wheat cultivars with high level and durable resistance to stripe rust. |
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