Genetic Linkage Map Construction And Identification Of Resistance Gene Associated With Marssonina Leaf Blotch In ‘Qinguan' Apple

China as the largest apple producer and consumer country occupies a pivotal position in the apple industry around the world.Marssonina leaf blotch is one of the most serious diseases in apple production area in China.At present,spraying fungicides is the main method to prevent the outbreak of Marssonina leaf blotch in China.However,with the appearance of fungicide-tolerant strains,Marssonina leaf blotch will become increasingly serious,leading to more complicated issues regarding environment protection and food safety.Therefore,the most economical and effective way to control Marssonina leaf blotch disease is to find out the resistance genes in Marssonina leaf blotch resistant cultivars and introgression these genes in the apple breeding programme.In this study,F₁hybrids derived fromNo.2'were used to construct a high-density SNP genetic linkage map of‘Qinguan'apple,further QTL mapping for Marssonina leaf blotch resistance was investigated according to the linkage map of‘Qinguan'apple,in the end candidate genes associated with Marssonina leaf blotch resistance were identified based on the published GDDH13 genome sequence.The aim of this study is to lay a foundation for identification of the key genes related to Marssonina leaf blotch resistance by using the modern molecular biology and bioinformatics technology.The main results of this dissertation are as follows:1. According to the results of disease resistance assessment of the apple F₁hybrids population in the field in 2018,each 17 progenies with extreme resistance and susceptibility were selected separately.After testing the polymorphism and repeatability of 216 SSRs,58SSRs which are both heterozygous in‘Qinguan'and evenly distributed on the apple genome were selected.Genomic scanning method?GSA?was performed to detect the genotype of the parents and the extreme phenotype progenies,chi-square test was used to determine if marker allele separation ratio would show bias towards the 17 resistant F₁hybrids.Two SSRs which might be associated with resistance genes were detected,C4065 on LG6 and CH01g12 on LG12,respectively.Further analysis for the surrounding SSRs of CH01g12 revealed that Hi02b07 and Hi02f12 might also be associated with resistance genes.Finally,it was predicted that the resistance locus of Marssonina leaf blotch might be located near C4065 on LG6 and within 29.5-43.0 c M on LG12 of‘Qinguan'.In addition,two SSRs that may be associated to lethal factors were found out,CH03c02 and NZ02b1,respectively.2. The 122 hybrids of No.2'were chosen as the mapping population.We used 20K SNP Illumina Infinium^?array to perform genotyping of 122 F₁hybrids and two parents in order to construct the high-density genetic linkage map of‘Qinguan'and‘Nagafu No.2'.The results showed that the‘Qinguan'map contains 17 linkage groups,with a total length of 1,112.3 c M and 1,100 SNPs.The average distance between adjacent markers is 1.59c M?858.18 kb?,covering 91.33%of the genome-wide physical map.The‘Nagafu No.2'map also contains 17 linkage groups,with a total length of 1,114.2 c M and 1,082 SNPs.The average distance between adjacent markers is 1.66 c M?848.33 kb?,covering 86.79%of the genome-wide physical map.3. In the two years of 2018 and 2019,the field disease resistance assessment was conducted and the resistance levels were artificially divided one each apple progenies.In2019,both spraying and quantitative suspension drop artificial inoculation on detached leaves of F₁population were carried out followed with disease resistance assessment and lesion sizes measure calsulation.Disease resistance QTL mapping was performed based on the newly constructed genetic map.5 QTLs on‘Qinguan'LG15 and LG17 were detected by Kruskal-Wallis test.Interval mapping verified a stable QTL on LG15,the marker SNP?FB?0269355 was stably associated with the LOD peak.The LOD values were 4.09 and5.07 in two years,respectively,which were able to explain 14.3%and 17.4%of disease resistance variation.The flanking markers were RB?CT?1872379 and SNP?FB?0271916.Multiple QTL-mapping limited the most significant QTL interval to 17.2-32.6 c M on LG15.4. According to the flanking marker of the QTL,its corresponding location on reference genome was determined to be Chr15:5.07-10.45 Mb.There are 676 genes in this region when comparing with the GDDH13 genome.In combination with the GO annotation and the functional description of homologous genes in other species,73 candidate genes were preliminarily identified,which are directly involved in the defense response of fungi and bacteria,disease resistance-related substances metabolism,hypersensitivity,cell wall modification,and scavenging of reactive oxygen species.Together with the previous transcriptome data of‘Qinguan'leaves inoculated with M.coronaria,it was identified that there were 14 candidate genes showing the different expression pattern between the control and treatment groups,which were thought to be the most likely genes related to the Marssonina leaf blotch resistance in‘Qinguan'.