Genome-Wide Association Study Select The Resistance Genes Of Aspergillus Flavus Infection In Maize Kernels

Aflatoxin is a highly toxic substance and designated as a class I carcinogens by the World Health Organization（WHO）.Statistics from the United Nations Food and Agriculture Organization showed that about 25%of the food crops were contaminated by mycotoxins and inedible each year in the world,caused direct economic loss of hundreds of billions of dollars.Maize is the world’s largest food crops and its seed is the most susceptible to aflatoxin contamination in the three major grains,screening of kernel Aspergillus flavus resistance gene of maize and cultivating kernel Aspergillus flavus resistant varieties of maize is an effective way to solve this problem,and it is also very important for the national economy and for the food and feed safety.In this dissertation,a natural populationcontains 110 maize inbred lines were selected as experimental material,the artificial infection experiment were carried out to screen the Aspergillus flavus highly resistance germplasm resources and then the Genome-Wide Association Study（GWAS）was applied to dissect the candidate resistance genes related to the Aspergillus flavus infection in maize kernels.The results provided germplasm resources for maize breeding of kernel resisitance to Aspergillus flavus infection and also provided excellent gene resources for maize molecular breeding of kernel resistance to Aspergillus flavus infection.The main findings are as follows:1.A natural population contains 110 maize inbred lines were used as experimental material and a Randomized complete block design with one to two replications was used for field trials in three environments,including Nanchong in 2014,Ya’an and Chongzhou in 2016.The resistance of the harvested seedsto the Aspergillus flavus infection was identified by artificial infection and then cultivated the seeds on wet filter paper for two weeks.A total of 12 germplasms were identified as Aspergillus flavus infection resistance germplasms in the three environments,which provided germplasm resources for kernel Aspergillus flavus infection resistance in maize breeding in future.2.The GWAS was performed using 50118 high-quality SNPs covering the maize genome,considering the population structure and the genetic relationship and in combination with phenotypic identification data,then using the association analysis software TASSEL4.3 and selecting the MLM model for GWAS.A total of 43 SNPs（P<1.99E-05）were significantly associated with the kernel resistance to Aspergillus flavus infection.Among them,24 SNPs were obtained in Nanchong environment,25 SNPs were obtained in Chongzhou environment and 23 SNPs were obtained in Ya’an environment.In addition,13 same SNPs were obtained in Nanchong and Chongzhou environments which distributed on the chromosomes 1,2,3,4,5,7,8 and 10.11 same SNPs were obtained in Nanchong and Ya’an environments which distributed on the chromosomes 2,4,5,7,8,9,10.And 13 same SNPs were obtained in Chongzhou and Ya’an environments which distributed on the chromosomes 2,3,4,5,6,7,8,10,And 8 same SNPs were obtained in Nanchong,Chongzhou and Ya’an environments which distributed on the chromosomes 2,4,5,7,8,10.3.The results of GWAS yielded 8 candidate genes that were significantly associated with the kernel resistance to Aspergillus flavus infection.They are GRMZM2G140116,GRMZM2G084035,GRMZM2G057000,GRMZM2G700188,GRMZM2G305280,GRMZM2G148783,GRMZM5G880300,GRMZM2G414915.Among these,The S₁97231098 marker locus on the chromosome 2 is located in the second exon of the GRMZM2G140116 gene coding region;the S₁57630797 marker locus on chromosome 4 is located at the second exon of the GRMZM2G084035 gene coding region;the S₁08388330 marker locus on the chromosome 7 is located in the first exon of the GRMZM2G700188 gene coding region;the S₁59727632 marker locus on the chromosome 7 is located in the second exon of the GRMZM2G100741 gene coding region;the S₁65988462 marker locus on chromosome 8 is located in the first exon of the GRMZM2G148783 gene coding region;the S₁36294379 marker locus on the chromosome 10 is located in the first exon of the GRMZM2G414915 gene coding region.Among them,the GRMZM2G140116 gene that located on the chromosome 2 was annotated as ribosomal protein L14;the GRMZM2G084035 gene that on the chromosome 4 was annotated as F-box/RNI-like superfamily protein;the GRMZM2G057000 gene that on the chromosome 5 was annotated as（CBB1,DIM,DIM1,DWF1,EVE1）cell elongation protein/DWARF1/DIMINUTO（DIM）and FAD-linked oxidoreductase protein;the GRMZM2G414915 gene that on the chromosome 10 had the greatest contribution to resistance to Aspergillus fiavus infection and was annotated as Sub45-Putative Subtilisin,recently,it has been reported that the Subtilisin can inhibit the growth of Aspergillus flavus.Therefore,the GRMZM2G414915 gene located on chromosome 10 may play an important role in the resisitance of Aspergillus flavus infection in maize kernel.