联合全基因组关联和转录组分析筛选玉米拟轮枝镰孢穗腐病的抗性候选基因

Screening candidate resistance genes to ear rot caused by Fusarium verticillioides in maize by combined GWAS and transcriptome analysis

玉米是我国第一大粮食作物,实现玉米的高产稳产对于我国粮食安全、农业稳定有重要意义。穗腐病是一种严重危害全球玉米的真菌性病害,会造成玉米大幅减产和品质劣变。本研究选用我国玉米穗腐病的优势致病菌拟轮枝镰孢(Fusariumverticillioides),对241份来源广泛的玉米自交系进行2年田间人工接种抗性鉴定,同时利用20,586个高质量SNP标记通过全基因组关联分析(genome-wideassociationstudy,GWAS),鉴定拟轮枝镰孢穗腐病抗性显著关联的SNP位点;在此基础上选取对拟轮枝镰孢穗腐病表现高抗和高感的玉米自交系各1份,其籽粒在室内接种拟轮枝镰孢,通过对3个不同侵染时间的籽粒进行转录组测序(RNA-seq),分析抗感材料差异表达基因(differentiallyexpressedgenes,DEGs)及其富集情况;结合GWAS和RNA-seq结果,共同定位筛选抗病候选基因。主要研究结果如下:(1)综合2年田间抗性鉴定结果,筛选到4份抗拟轮枝镰孢穗腐病的玉米自交系,其中含有热带血缘的玉米种质对拟轮枝镰孢穗腐病表现出更好的抗性。(2)2年GWAS分析共检测到26个与拟轮枝镰孢穗腐病抗性显著关联的SNP位点,其中有18个位点位于前人定位到的QTL范围内。(3) RNA-seq结果表明,抗感材料对病原菌的响应基因不同。与感病材料相比,抗病材料均显示出更多的DEGs,且都有更多的上调基因;在抗感材料特异性DEGs共同富集的GO条目和KEGG通路中,抗病材料中富集到的DEGs占比显著多于感病材料;一些与植物防御病原菌相关的条目和通路也仅在抗病材料中被特异富集。(4)在GWAS检测到的显著关联位点上下游100 kb范围内筛选与转录组DEGs共同定位到的候选基因,结果 16个基因被GWAS和RNA-seq同时检测到;根据这些基因的蛋白功能及相关文献报道,从中预测到6个与拟轮枝镰孢穗腐病抗性相关的候选基因。综上所述,本研究筛选出4份抗拟轮枝镰孢穗腐病的玉米自交系,来自热带、亚热带的玉米种质可以作为抗逆性品种选育的研究重点;对抗感玉米自交系响应拟轮枝镰孢侵染的DEGs及其相关抗病机制进行了初步解析,联合GWAS和RNA-seq共定位到6个与拟轮枝镰孢穗腐病抗性相关的候选基因,研究结果为玉米穗腐病抗性基因的克隆验证与抗性品种的培育提供了一定的理论依据。

Maize,as the largest grain crop in China,holds great significance for food security and agricultural stability,making it crucial to achieve high and stable yields.Ear rot,a fungal disease,poses a serious threat to global corn production,resulting in significant yield reduction and quality deterioration.This study focused on the dominant pathogen Fusarium verticillioides and aimed to identify resistance in 241 maize inbred lines from diverse sources through two years of artificial inoculation in the field.Additionally,a genome-wide association study(GWAS)was conducted using 20,586 high-quality SNP markers to identify SNP loci significantly associated with ear rot resistance.Based on this research,one maize inbred line with high resistance and one with high susceptibility to F.verticillioides-induced ear rot were selected.The young kernels of these lines were subjected to RNA transcriptome sequencing(RNA-seq)at three different infection stages,and differentially expressied genes(DEGs)and enrich-ment analysis were conducted.The results of GWAS and RNA-seq were combined to identify candidate resistant genes.The main findings were as followed:(1)Four inbred lines resistant to F.verticillioides-induced ear rot were selected based on two years of field resistance identification.Maize germplasm with tropical consanguinity exhibited higher resistance.(2)A total of 26 SNP loci associated with ear rot resistance were detected through two years of GWAS analysis,with 18 loci located within previously iden-tified QTL regions.(3)RNA-seq analysis revealed differential response genes between resistant and susceptible lines upon F.verticillioides infection.The resistant line exhibited a greater number of DEGs and up-regulated genes.In the co-enriched GO entries and KEGG pathways specific to the DEGs of resistant and susceptible lines,the proportion of DEGs enriched in the resis-tant line was significantly higher.Moreover,some entries and pathways related to plant defense against pathogens were specifi-cally enriched in the resistant line.(4)Sixteen genes,detected by both GWAS and RNA-seq,were identified as candidate genes by screening those co-located with transcriptome DEGs within a range of 100 kb upstream and downstream of significant association loci.Based on protein function and relevant literature reports,six candidate genes related to ear rot resistance were predicted.In summary,this study identified four maize lines resistant to F.verticillioides-induced ear rot.Maize germplasm from tropical and subtropical regions holds promise for stress-tolerant variety breeding.The preliminary analysis of DEGs and related resistance mechanisms of maize in response to F.verticillioides infection was conducted.Furthermore,six candidate genes associated with ear rot resistance were identified through the integration of GWAS and RNA-seq.These findings provide a theoretical basis for the cloning and validation of maize ear rot resistance genes,as well as the breeding of resistant varieties.