Map-based Cloning And Functional Analysis Of Extreme Resistance Gene Ry_chc To Potato Virus Y

Potato is an important economic crop in China.Virus diseases,however,have seriously reduced potato yield and quality.Potato virus Y（PVY）is one of the ten most important viruses that seriously affects the development of China’s potato industry.Previously,the diploid potato wild species S.chacoense 40-3 with extreme resistance to PVY was crossed with the susceptible wild species S.berthaultii 143-6 to construct a segregating population,and the gene locus was located in the range of 141.7kb between M2526-2 and M4950-1.On this basis,the PVY extreme resistance gene Ry_chc was further finely mapped and cloned through map-based cloning,and was stably transformed into the diploid susceptible cultivar AC142 for functional identification.Finally,its resistance mechanism was preliminarily elucidated by transcriptome profiling analysis.The main results are as follows:1.The genomic DNA of 2 parents and 2 pools of resistant and susceptible offspring was sequenced by Ren Seq,respectively.The consistent specific SNPs obtained from resistant parents and resistant pool were located in or nearby the location interval identified previously,which confirmed that the early mapping result was correct.Inspired by this technique,the PCR amplification and sequencing were performed,using the resistant and susceptible materials as templates,directly with the primers of the pre-developed gene markers without polymorphism to search for the above type of polymorphic SNP loci.Finally,one closely linked SNP marker was developed,and the gene was located in the range of 113.4kb between M2526-2 and SNP48.For further fine mapping,an expanded F1 population with 2512 progeny was constructed using the PVY extremely resistant diploid wild species S.chacoense 40-3 as male parent and the susceptible diploid wild species S.berthaultii 143-6 as female parent.The flanking markers M28 and M50 were used to identify the genotypes of these 2512 plants.Five recombinant plants were found.Based on the phenotype and genotype of five recombinants,the gene interval was reduced to 107.7kb.2.A bacterial artificial chromosome（BAC）library covering about 10 times of potato genome was constructed using the resistant parent S.chacoense 40-3.8 positive clones were screened from the BAC library using the Ry_chc gene left flanking markers M2526 and M28 and right flanking markers SNP48 and M50 developed earlier in this study,and the three clones,71-24H,74-24B and 100-8O,which could cover the finely mapping region to the greatest extent were sequenced.Using the sequence of 71-24H as a template for gene marker development,a new positive clone 64E14 was screened and sequenced using three pairs of newly developed linkage markers M71-20,M71-21-1 and M71-28-3.But it failed to completely cover the entire fine mapping interval.Primers were designed using the susceptible BAC clone 100-8O as the template,and the sequence of missing part of the fine mapping interval was obtained by TA cloning technique.The total length of the obtained sequences was297.7kb,and the assembly sequence of the fine mapping region between M28 and SNP48 was174.1kb,which was 66.4kb more than that of the reference genome.3.A total of 9 pairs of tightly linked DNA markers were developed based on the fully assembled S.chacoense 40-3 fine mapping segment sequence,which eventually shortened the region to 26.1kb between M71-28-3 and M64-17.Sequence analysis showed that there were 4candidate genes in this region,among which C2 encoded oxidoreductase,C3 and C4 were TIR-NBS-LRR resistance genes,and C5 did not predict to have conserved domains.4.In order to determine the functional gene Ry_chc,candidate genes C2,C3 and C4 were introduced into the PVY-sensitive diploid potato cultivar AC142 through Agrobacterium-mediated stable transformation.And transgenic plants were inoculated with PVY^O-FL for phenotypic identification.The results showed that only the overexpression lines of C4 show resistant to PVY^O-FL,which proved that C4 was the target gene Ry_chc.The transgenic plants of Ry_chc were further inoculated with three viruses,PVY^N,PVY^NTN and PVA,and the results showed that these transgenic plants of Ry_chc were resistant to all three viruses,proving that Ry_chcwas an extreme resistance gene.5.Analysis of Ry_chc transcripts revealed two types of Ry_chc spliceosomes,with variable splice types as intron retention,and both spliceosomes had intact TIR-NBS-LRR domain.The stable Nicotiana benthamiana transformed lines of Ry_chc did not show any disease resistance,suggesting that Ry_chc may require the involvement of potato-specific endogenous genes to exercise its function.To study the regulatory network of Ry_chc-mediated extreme resistance,transcriptome sequencing was performed on the transgenic lines of Ry_chc and wild type AC142at 0 h,0.5 h,12 h and 24h after virus inoculation.The enrichment analysis of differentially expressed genes showed that there were a large number of differentially expressed genes overrepresented in cell wall organization and synthesis at all 4 time points after virus induction,suggesting that Ry_chc may play a role in disease resistance by regulating the structure of cell wall.Genes related to the synthesis of（-）-jasmonoyl-isoleucine,jasmonoyl-valine and jasmonoyl-leucine in Ry_chc transgenic lines were significantly up-regulated at 12 h and 1 d after virus induction compared with AC142,suggesting that Ry_chc may trigger a series of immune defense responses by synthesizing these signaling molecules.6.Left flanking DNA marker M71-20,functional marker MG64-17 and right flanking DNA marker M64-17 were used to detect with 12 wild species of S.chacoense line and 22susceptible resources.The genotype detection result of MG64-17 was 100%consistent with the phenotype so this functional marker can be used for later molecular marker-assisted selection breeding with S.chacoense as breeding resource.