关键词: 宽皮柑橘, 褐斑病, 分子标记, 全基因组关联分析, 群体遗传分化指数分析

Abstract: 【Objective】The study objective was to explore candidate genes associated with resistance to Alternaria brown spot disease in citrus mandarin and provide the basis for developing suitable molecular markers for further citrus resistance breeding work. 【Method】In summer and autumn of 2022, the young leaves of 136 citrus mandarin accessions were picked and inoculated with the fungus mycelium of Alternaria alternata in the laboratory. The combined results of two experiments were used to obtain the reliable results of 121 citrus mandarins responding to Alternaria brown spot. The phenotypes of 121 citrus mandarins were selected to verify the effectiveness of the CAPS marker developed in a previous study. Then, the phenotypes of 121 citrus mandarins and corresponding SNP genotype data from GBS sequencing were analyzed respectively with PCA, GWAS, and F_st methods for obtaining the candidate SNPs related to Alternaria brown spot resistance. Candidate genes had selected from the flanking region of 25 kb sequences surrounding the candidate SNPs site, and then they were screened out according to phytozome annotations. The expression of candidate genes analyzed after inoculating with the pathogen fungus on leave of Chengtuo hongju and Clementina (Algeria) for 24, 48, and 72 h. 【Result】Among 121 citrus mandarins, 67 varieties such as Clementine and Satsuma were resistant to Alternaria brown spot disease, whereas 54 varieties such as Hong Ju and Ponkan were susceptible. Some varieties resistance to Alternaria brown spot disease could discriminated by CAPS, but its accuracy only accounts for 76.86% in the study. GWAS analysis identified six significant SNPs highly associated with disease resistance, among which SNP1 located in Ciclev10021676 at 24 838 146 bp of chromosome 3 could be used to predicate the resistance of varieties, which genotype showed a strong correlation with phenotype. Eight significant SNPs highly associated with disease resistance selected by F_st analysis. Finally, five genes of Ciclev10018604, Ciclev10023485, Ciclev10023486, Ciclev10024586 and Ciclev10019874 were screened out. The expression levels of these five genes in Chengtuo Hongju were up-regulated extensively after inoculating leaves with the pathogen fungus, and their expression levels reached the highest 48 hours later. 【Conclusion】Through GWAS, the SNP at 24 838 146 bp on chromosome 3 was found to be the most significant one with high resistance correlation to Alternaria brown spot disease, and the genotype at this location could be effectively use to distinguish the resistant varieties. The candidate genes responding to Alternaria brown spot disease in mandarin, i.e., Ciclev10019874, Ciclev10018604, Ciclev10023485, Ciclev10024586, and Ciclev10023486 were discussed.

Key words: citrus mandarin, Alternaria brown spot, molecular marker, genome-wide association study, fixation index