| Wheat is an important crop and wheat stripe rust is a common and destructive wheat disease caused by Puccinia striiformis f.sp.Tritici(Pst).Analysis of genetic structure,virulence variation,origin and evolution,Migration rule and co-evolution with wheat of this fungus are all hotspots.SNP is an important molecular marker to study these scientific problems,but it has not been popularized yet.Triazolone,as an important Fungicide of plant pathogenic fungi,reports about its resistance are endless.And the mechanism of its resistance has been already confirmed in many crop pathogenic fungi.However,the research on triazolone resistance of wheat stripe rust is lagging behind for a long time.Previous triazolone resistance surveillance experiments of Pst in our lab showed that Pst in China had evolved resistance.However,due to the cumbersome steps and harsh conditions,it was difficult to popularize the experiment on a large scale.At the same time,the mechanism of triazolone resistance of Pst needs to be further clarified and confirmed.In this study,the genetic diversity and triazolone resistance KASP-SNP molecular markers of Pst were developed.The practicability of the two markers was preliminarily tested and evaluated,and the resistance of triadimefon in Pst in China was preliminarily investigated.The main results are as follows:1)The genome sequence of the epidemic race CYR32 in China was used as a reference in this study,and compared with race PST78 in US and 38S102 in India.462 pairs of KASP-SNP primers were designed according to the 29929 SNP sites,polymorphism at each SNP locus was determined using 64 Chinese Pst isolates,based on which 43 pairs of polymorphic SNP primers were obtained.For comparison purpose,13 pairs of polymorphic SSR markers were also used to analyze this Pst population.The average PIC(polymorphism information content)index of the SNP markers was 0.346 and the gene diversity index was0.420,both indexes were higher than those of the SSR primers,which were 0.238,0.265,respectively.Structure software(edition 2.3)was used to analyze the population genetic structure revealed from these two molecular marker systems.The results were very similar,for example,the optimum K value were both 4,Yunnan Pst population had the simplest genetic structure and Hubei population had the most complex structure.The two marker systems supported similar patterns of population structures,while the genetic division of individual isolates was quite different.The KASP-SNP molecular marker was more polymorphic than SSR molecular markers.Compared with regular gel detection,KASP-SNP has the advantages of high efficient,high throughput,high specificity and high sensitivity.2)The CYP51 gene sequence of 70 wheat stripe rust samples was amplified,and one SNP loci was mapped,which is located on the codon of the 134 th amino acid encoding the CYP51 gene and lead to a difference between tyrosine and phenylalanine in translation.Association analysis display that the SNP loci was significantly correlated with the results of bioassays for triadimefon resistance in 47 samples.Based on the above results,four pairs of KASP-SNP primers were developed,and the best one was selected as the candidate.This pair of primers were used to simultaneously perform the triazolone resistance bioassay and KASP test.The experiment was repeated three times and the two results were exactly the same.Which proved that the primers are of high precision.The KASP test of triazolone resistance on samples collected from nine provinces in China showed that all the other eight provinces had developed different degrees of resistance,except Tibet.It is worth noting that the resistant Pst in Gansu and Qinghai provinces accounted for the highest proportion,reaching 83% and 73% respectively,while no resistant Pst were found in Tibet. |
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