Genetic Structure Of Phytophthora Infestans Population In Southwestern China And Dual Transcriptome Analysis On The Interactions Of Phytophthora Infestans And Potatoes

Potato is known as“the food of the future”and is the fourth largest crop after rice,corn and wheat in China.Potato plays an active role in ensuring national food security,restructuring the planting industry,and fighting poverty.Potato also plays a decisive role in southwestern China,and is the main driving force and important industry for fighting poverty and rural revitalization in southwestern China.Due to the unique geographical conditions,climatic conditions,and potato planting structure in southwestern China,potato late blight,caused by Phytophthora infestans（Mont.）de Bary,has always been a very devastating disease in potato production in southwestern China.In this study,the genetic structure of P.infestans collected and isolated from different potato production areas and different years in southwestern China was analyzed.The transcriptome analysis of the interaction between P.infestans and potato were performed to analyze the expression patterns of genes of P.infestans and excavation of important effector protein gene resources,with a view to providing a theoretical and practical basis for molecular genotype study of P.infestans and comprehensive management of potato late blight in China.After research,the following research results have been obtained.1.The phenotype of P.infestans population in southwestern ChinaThe phenotype of P.infestans population in southwestern China was complex,with sexual reproduction,resistance to metalaxyl,and high physiological race diversity.（1）The mating types of P.infestans from 5 regions in southwestern China such as Sichuan,Chongqing,Guizhou,Yunnan and Hubei were determined by culturing with standard strains and microscopic examination.The results showed that there were A1,A2 and self-fertile types in the 679 strains of P.infestans tested,and the distribution of mating types in the populations of P.infestans was very different between different regions.113 A1 mating types were detected with the frequency of 16.64%of the tested strains,53 A2 mating types with the frequency of 7.81%and 513 self-fertile strains with the frequency of 75.55%.At the same time,there are oospores in potato leaves in the field.（2）The sensitivity of metalaxyl to 299 strains of P.infestans in southwestern China was determined.The results showed that the sensitivity of metalaxyl in different mating types of P.infestans was significantly different,but most of the strains were resistance to metalaxyl.There were nine strains showed sensitive to metalaxyl with the frequency of 3.01%,52medium-resistant strains with the frequency of 17.39%and 238 highly resistant strains with the frequency of 79.60%.（3）The results of physiological races of 365 strains of P.infestans in southwestern China showed that there were a large number of races that can overcome all known vertical resistance genes.92 physiological race types were found,of which the dominant physiological race was 1.2.3.4.5.6.7.8.9.10.11 with the frequency of 26.58%of the tested strains,13.70%of the physiological races of P.infestans appeared only once,and the Shannon diversity index of different physiological race groups was 0.55.More than 99%of P.infestans can overcome more than 5 R genes,and each strain can overcome 9.04 resistance genes on average,and the virulence frequency was extremely significantly different between different sampling years.2.The genotypes of P.infestans population in southwestern ChinaThe P.infestans population in southwestern China was a"new"population with high genotype diversity,containing the Avr3a^EMcoding sequence.（1）Three mt DNA haplotypes existed in the 362 strains of the“new”population of P.infestans tested,among which type Ia was prevalent in southwestern China,with the frequency of 78.73%,and types IIa and IIb were 4.70%and 16.57%,respectively.Three mt DNA haplotypes were detected only in Sichuan.A1,A2 and self-fertile mating type of P.infestans existed in each mt DNA haplotype.The self-fertile strains were dominant in the Ia haplotype,and the A1 mating type was dominant in the IIb haplotype.（2）A total of 118 genotypes were identified in 241 strains of P.infestans using 14 SSR markers,of which the genotype SW-48 was dominant with the frequency of 8.30%and distributed in Sichuan,Chongqing,Yunnan and Hubei.The secondary genotype was SW-40,with the frequency of 7.88%,which was widely distributed in Sichuan,Chongqing,Guizhou,Yunnan and Hubei.The standard Shannon diversity index（Hs）of the genotypes of the P.infestans population was 0.78,reflecting a higher genetic diversity within the population.The genetic variation and significant genetic drift existed in P.infestans population in southwestern China.Through further cluster analysis,it revealed that the genetic distance between the populations of P.infestans in southwestern China was close,and 79.25%of the strains were closer to the highly pathogenic genotype Blue＿13.（3）The nucleotide sequences of 110 sequenced P.infestans were 99.77%identical to avirulence gene Avr3a,mainly differences in amino acid sequences at positions 19,80 and103.The tested P.infestans all contain the gene sequence encoding Avr3a^EM,which can infect the potato varieties containing R3a.The Avr3a sequence of the populations of P.infestans showed high haplotype diversity and low nucleotide diversity,which can be divided into 17haplotypes.The haplotype diversity（Hd）and nucleotide diversity（Pi）of the P.infestans populations were 0.743±0.030 and 0.0036,respectively.The neighbor-Joining tree and haplotype network of Avr3a of different P.infestans populations showed that 27.27%of the tested strains were relatively close to the gene avr3a＿9479695,and the Avr3a haplotype of P.infestans was complicated.3.Dual transcriptome analysis on the interactions of P.infestans and potatoesDual transcriptome analysis of the pathogen-host interactions of potato late blight revealed response-related genes,laying a foundation for the subsequent research on response-related genes.For in-depth analysis of the interaction mechanism between P.infestans and potato,the late blight resistant variety Ziyun No.1（incompatible interaction）and the susceptible variety Favorita（compatible interaction）were used as hosts to inoculate the dominant strain of P.infestans in southwestern China.The strain and the leaves were taken at 0 h before inoculation,24 h and 48 h after inoculation to perform Dual RNA-Seq sequencing.A large number of host genes specifically expressed and significantly differentially expressed under the mode of incompatible interaction and compatible interaction were detected,which were significantly enriched in metabolic pathways such as biological stress responses and defense response,phenylpropane-like metabolism.In this study,559 RXLR effector protein genes were expressed in samples,and a large number of RXLR genes were up-regulated at 48 hpi.Combined with the results of published gene microarray,it was found that 39 RXLR effector protein genes were up-regulated by more than 2 times after infection of the host,and 10 of which had no functional report.The expression of most of the 92 potato NBS-LRR genes was low,but a few showed up-regulated or specific expression patterns.After 48 h of infection,114 and 177 kinase genes showed a significant up-regulation trend in the resistant host and susceptible host,respectively.Several NAC,MYB,WRKY,b ZIP,AP2/ERF transcription factor genes were induced by P.infestans,and this induction had nothing to do with whether the host is resistant to disease.The genes encoding RXLR effector,NBS-LRR protein and kinase that exhibited significant expression differences and the genes encoding WRKY,MYB,AP2/ERF,b ZIP,and NAC transcription factors were identified in this study provide a large number of candidate genes for the subsequent analysis of RXLR effector function and potato disease resistance mechanism.