84个苹果栽培品种对斑点落叶病的抗性评价和全基因组关联分析

Resistant Evaluation of 84 Apple Cultivars to Alternaria alternata f.sp.mali and Genome-Wide Association Analysis

【目的】斑点落叶病是中国苹果产区发生的主要病害之一,严重影响苹果的产量和品质。本研究旨在发掘具有高抗病性的苹果栽培品种和探寻调控斑点落叶病抗性的关键基因,为苹果品种改良提供科学依据。【方法】利用苹果斑点落叶病菌(Alternaria alternata f.sp.mali)对84份苹果栽培品种进行离体叶片接种鉴定,从病斑面积和病斑面积增长率两方面进行聚类分析,评价苹果栽培品种对斑点落叶病的抗性。用接种后叶片的病斑面积作为表型性状,以全基因组深度重测序获得的1243071个高质量SNP位点为遗传标记,采用EMMAX方法进行全基因组关联分析。【结果】84份苹果栽培品种接种后统计病斑面积发现,不同的苹果栽培品种在对斑点落叶病的抗病性方面表现出显著的多样性,其中感病和中抗的品种占绝大多数,而高抗和高感的品种占比较少;苹果斑点落叶病抗病性具有正态分布特征,呈现数量性状遗传特征。全基因组关联性状分析最终获得6个SNP位点呈现显著水平P≤0.0000001(-LgP≥7),深入分析将其关联到7个关键候选基因,包括整合素连接蛋白激酶、FMN连锁氧化还原酶、B-box型锌指蛋白、GATA型转录因子等,并验证了整合素连接蛋白激酶在苹果抗病中的作用。【结论】经过两年数据的综合分析,最终从84个苹果栽培品种里,鉴定到稳定抗性品种7份,稳定易感品种2份。通过全基因组关联性状分析鉴定到与苹果斑点落叶病抗病性显著相关的6个SNP位点,关联到7个关键候选基因,并验证了其中一个基因的功能。

【Objective】Alternaria alternata f.sp.mali is a main disease occurring in the apple orchards in China,which seriously threatens the yield and quality of apples.The aim of this research was to identify apple cultivars and genes responsible for high resistance to Alternaria alternata f.sp.mali,thus providing the basis for disease resistance breeding of apples.【Method】In this study,the isolated Alternaria alternata f.sp.mali strain was used to inoculate leaves of 84 apple cultivars.To evaluate the resistance of apple cultivars to Alternaria alternata f.sp.mali,the cluster analysis was carried out from two aspects including lesion area and lesion area growth rate.The genome-wide association study was performed based on the lesion area.The disease spot area of the inoculated leaves was used as a phenotypic trait,and the 1243071 high-quality SNP loci obtained by whole genome deep resequencing were used as genetic markers.Genome wide association analysis was performed by EMMAX method.【Result】The statistical analysis of lesion area showed significant diversity of 84 apple cultivars in resistance to Alternaria alternata f.sp.mali.Most apple cultivars were susceptible or resistant to Alternaria alternata f.sp.mali,while only a few cultivars were highly resistant and highly susceptible.The disease resistance of Alternaria alternata f.sp.mali has the characteristics of normal distribution,showing the genetic characteristics of quantitative characters.The genome-wide association trait analysis finally obtained 6 SNP loci with a significant level of P≤0.0000001(-LgP≥7),and in-depth analysis linked them to 7 key candidate genes,including Integrin-linked protein kinase,FMN-linked oxidoreductase superfamily protein,B-box zinc finger family protein,GATA type transcription factor,etc.The biological function of Integrin-linked protein kinase in disease resistance was further verified.【Conclusion】After two years of comprehensive analysis of data,7 stable resistant varieties and 2 stable susceptible varieties were identified from 84 apple cultivars.Through genome-wide association trait analysis,six SNP loci and seven genes significantly related to the disease resistance of apple leaf spot were identified,and function of one gene was verified.