Identification And Association Analysis Of Shell Resistance To Infection Of Aspergillus Flavus In Peanut (Arachis Hypogaea L.)

Peanut(Arachis hypogaea L.)is an important source of vegetable oil and protein worldwide.During the past three decades,China has been the largest country in terms of peanut production and domestic consumption and the crop has played a unique role in assuring food security.However,aflatoxin contamination caused by Aspergillus flavus and other Aspergillus fungi has been a serious factor affecting the food safety and industry development of peanut.Hence,genetic improvement for resistance has been regarded as a high priority in peanut research for reducing aflatoxin risks.The pod shell is the preliminary physical protection mechanism of peanut seeds against infection of microorganisms,but very limited research effort has been made on the shell resistance,which has largely impeded enhancement and utilization of this resistance component.In this research,a reliable method was developed for evaluating the shell resistance to infection of Aspergillus flavus by comparing reaction to pod inoculation of different peanut varieties under laboratory conditions.The peanut mini-core collections from China,ICRISAT and USA were evaluated for shell resistance using the techniques developed.In addition,SSR and SNP markers were used for association studies of the Chinese peanut mini-mini core.The results of this research were as follows.1.In this research,the degree of infection of peanut shell by Aspergillus flavus was divided into 1to 9 scales,and the calculation method of infection index of peanut shell was established.Four peanut varieties including J11,Xuhua 13,Zhonghua 6 and Zhonghua 12 were selected for artificial inoculation with Aspergillus flavus suspensions in laboratory.Several combinations of inoculum concentration and incubation period after inoculation were compared for distinguishing the infection severity,from which,the optimal combination of an inoculum concentration as 2×10~6 spores per milliliter and incubation for7 days was determined.Through verifying trials by testing three peanut mini core collections from China,ICRISAT and USA,the method developed could effectively distinguish the resistance among different peanut varieties,and thus provide a useful technique for research on the shell resistance to A.flavus infection in peanut.2.By using the above novel method for shell resistance identification,the Chinese peanut mini-mini core collection consisting of 99 accessions was systematically evaluated for resistance to A.flavus infection during 2016 to 2018.The results showed that five peanut accessions,Zh.h1837,Zh.h2339,Zh.h2359,Zh.h4712 and Zh.h6180,were repeatedly found as relatively resistant in all three trials with their average infection index less than 0.5,while those of the highly susceptible lines were stably around 1.0.From the ICRISAT and USA peanut mini-core collections,totally 8 lines including PI196635,PI494034,PI476432,PI494034,PI337406,PI268586,PI259851 and PI296550 were repeatedly identified as moderately resistant with infection index less than 0.5.This is the first systematic evaluation of peanut shell resistance to A.flavus infection in the world,and the 13 identified germplasm lines are valuable for genetic improvement of resistance to A.flavus and study on shell resistance mechanism in peanut.3.Through group structure analysis,the 99 Chinese peanut mini-mini core lines were divided into2 subgroups by SNP.Based on this,37,152 SNP markers were used for association analysis,and a total of 9 SNP loci were detected to be related with shell resistance with the explanatory rate of phenotypic variation ranging from 8.60%to 31.95%.Interestingly,7 of the 9 SNP loci also located on the chromosome A03,concentrated in a length of chromosome segment 216 bp indicating that this region has a significant effect on phenotypic differentiation of shell resistance.In addition,one locus was detected on chromosome A04 and another one on B02 with phenotypic variation rates as 23.71%and22.51%respectively.The results of association analysis showed that the significantly-associated loci were accurate and reliable,and could be used as key sites for candidate gene discovery.Meanwhile,554SSR markers were used for association analysis of the Chinese mini-mini core lines with their reaction to shell infection.A total of 3 marker sites were found to be significantly associated with the shell resistance,among them,SSR markers AGGS1483 and AI124P23 had higher explanatory rates of phenotypic variation,which were 6.54%and 6.82%respectively,and all located on chromosome A03.Both the SNP and SSR markers identified would be of great significance for promoting molecular marker-assisted breeding for shell resistance in peanut.