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ARS Home » Southeast Area » Tifton, Georgia » Crop Protection and Management Research » Research » Research Project #425281

Research Project: Developing Genomic Approaches to Improve Resistance to Diseases and Aflatoxin Contamination in Peanut and Corn

Location: Crop Protection and Management Research

2016 Annual Report

1. Develop molecular markers and saturated genetic maps to identify quantitative trait loci (QTLs) associated with important agronomic traits of peanut and develop effective marker-assisted selection methods for peanut breeders. 1.A. Construction and use of saturated genetic map for identification of quantitative trait loci (QTLs) associated with disease resistance and oil quality. 1.B. Application of marker-assisted selection method for breeding to combine two traits into one genotype. 2. Evaluate corn germplasm for drought tolerance, understand the underlying molecular mechanisms, and develop molecular markers for identifying drought tolerant corn germplasm. 2.A. Identification and re-sequencing of genes in response to drought stress and development of polymorphic markers associated with drought tolerance in corn. 2.B. Corn germplasm and breeding lines re-evaluation for preharvest aflatoxin resistance and drought tolerance and genotyping with the polymorphic markers for association study.

1. Genotype and phenotype data for a genetic segregation population can be associated with QTLs and markers for trait of study and a genetic linkage map could be constructed. Peanut germplasm accessions have variable levels of disease resistance. The mapping population(s) will be genotyped using primarily SSRs, and ultimately the sequence-based markers will be added into this collection when Peanut Genome Sequence Project will be completed soon, in which the four parental line, Tifrunner, GT-C20, SunOleic 97R and NC94022, and their RILs will be sequenced. Field phenotyping for TSWV, leaf spots and other agronomic traits will be conducted for at least two years and at least two different locations with at least three replications. 2. Marker-assisted breeding will be employed as an example to combine two different traits with known linked marker(s) for faster and accurate transfer of trait from donor to elite lines through a back cross program or pedigree selection. Two traits can be combined into one productive peanut cultivar. Available markers for nematode, rust, and high oleic traits and new markers identified for TSWV and leaf spots will be compiled. The outcome of these efforts will enable more precise and effective molecular breeding for peanut improvement. 3. Drought stress during the late kernel development enhances aflatoxin contamination before harvest. The differences in drought-tolerance or -sensitivity of different corn accessions will display different profiles of expressed genes in developing kernels in response to A. flavus infection and the drought stress. It is possible to identify different genes responding to drought stress, and characterize the genes that may be associated with drought tolerance in different corn lines. Genes/markers associated with drought tolerance will be identified as “candidate” genes for association studies of resistance to Aspergillus flavus and preharvest aflatoxin contamination (PAC) and used in germplasm screening for drought tolerance. 4. Drought tolerance is a characteristic that has the potential to serve as an indirect selection tool for resistance to preharvest aflatoxin contamination (PAC). The outcome of these efforts will enable effective method to screen germplasm for drought tolerance and resistance to PAC in breeding program using marker-assisted selection.

Progress Report
The primary focus of this project is to develop and employ genomic tools and resources to identify genetically diverse corn and peanut germplasm that harbor resistance genes/markers and to elucidate resistant mechanisms. Corn and peanut are staple food crops throughout the world and their contamination with carcinogenic aflatoxins poses a global threat to food safety and human health. Aflatoxin production by Aspergillus (A.) flavus is exacerbated by drought stress in the field and by oxidative stress in vitro. Over the last decade, we have learned much about the biological pathway for aflatoxin production and are beginning to characterize the factors controlling its production. We examined differences in genes expressed by six isolates of Aspergillus flavus, three that do not produce aflatoxin and three that do. Isolates were grown in media known to be either conducive or non-conducive to aflatoxin production, both of which were amended with hydrogen peroxide at multiple levels to generate oxidative stress. We found that toxigenic and atoxigenic isolates employ distinct mechanisms to remediate oxidative damage, and that carbon source affected the isolates’ expression profiles. Iron metabolism, monooxygenases, and secondary metabolism appeared to participate in isolate oxidative responses. The results suggest that aflatoxin and aflatrem biosynthesis may remediate oxidative stress by consuming excess oxygen and that kojic acid production may limit iron-mediated, non-enzymatic generation of reactive oxygen species. Together, secondary metabolite production might enhance A. flavus stress tolerance in nature and during pathogenesis. Drought stress decreases crop growth, yield, and can further exacerbate pre-harvest aflatoxin contamination. Tolerance and adaptation to drought stress is an important trait of agricultural crops like maize. However, maize genotypes with contrasting drought tolerances have been shown to possess both common and genotype-specific adaptations to cope with drought stress. In this research, the physiological and metabolic response patterns in the leaves of maize seedlings subjected to drought stress were investigated using six maize genotypes including: A638, B73, Grace-E5, Lo964, Lo1016, and Va35. During drought treatments, drought-sensitive maize seedlings displayed more severe symptoms such as chlorosis and wilting, exhibited significant decreases in photosynthetic parameters, and accumulated significantly more reactive oxygen species (ROS) and reactive nitrogen species (RNS) than tolerant genotypes. Sensitive genotypes also showed rapid increases in enzyme activities involved in ROS and RNS metabolism. However, the measured antioxidant enzyme activities were higher in the tolerant genotypes than in the sensitive genotypes in which increased rapidly following drought stress. The results suggest that drought stress causes differential responses to oxidative and nitrosative stress in maize genotypes with tolerant genotypes with slower reaction and less ROS and RNS production than sensitive ones. These differential patterns may be utilized as potential biological markers for use in marker assisted breeding. Peanut is vulnerable to a range of diseases, such as Tomato spotted wilt virus (TSWV) and leaf spots which will cause significant yield loss. The most sustainable, economical and eco-friendly solution for managing peanut diseases is development of improved cultivars with high level of resistance. We developed a recombinant inbred line population from the cross between SunOleic 97R and NC94022, named as the S-population. An improved genetic linkage map was developed for the S-population with 248 marker loci and a marker density of 5.7 cM/loci. This genetic map was also compared with the physical map of diploid progenitors of tetraploid peanut, resulting in an overall co-linearity of about 60% with the average co-linearity of 68% for the A-genome and 47% for the B-genome. The analysis using the improved genetic map and multi-season (2010-2013) phenotypic data resulted in the identification of 48 quantitative trait loci (QTLs) with phenotypic variance explained (PVE) from 3.88 to 29.14%. Of the 48 QTLs, six QTLs were identified for resistance to TSWV, 22 QTLs for early leaf spot (ELS) and 20 QTLs for late leaf spot (LLS), which included four, six, and six major QTLs (PVE larger than 10%) for each disease, respectively. A total of six major genomic regions (MGR) were found to have QTLs controlling more than one disease resistance. The identified QTLs and resistance gene-rich MGRs will facilitate further discovery of resistance genes and development of molecular markers for these important diseases.

1. Aflatoxin production a response to stress? Aflatoxin production by Aspergillus (A.) flavus is increased by drought stress in the field and by oxidative stress in vitro. ARS researchers at Tifton, Georgia, examined gene expression differences in six Aspergillus flavus isolates, three each that do or do not produce aflatoxin. Isolates were grown in media known to be either conducive or non-conducive to aflatoxin production, both of which were amended with hydrogen peroxide to generate a range of oxidative stress. Results indicate that distinctly different mechanisms are employed by the aflatoxin-producing strains to remediate oxidative-stress damage, and that carbon source affects gene-expression. Iron metabolism, monooxygenases, and secondary metabolites appear to participate in isolate oxidative responses. Results suggest that aflatoxin and aflatrem biosynthesis may remediate oxidative stress by consuming excess reactive oxygen and that kojic acid production may limit iron-mediated, non-enzymatic generation of reactive oxygen species. These results are the first to suggest that aflatoxin production may be as a secondary metabolite to enhance A. flavus stress tolerance and ednurance.

Research experience is one of the most effective avenues for attracting students to and retaining them in science and engineering, and for preparing them for careers in these fields. In collaboration with Florida A&M University, students participated in the REU (research experiences for undergraduates) by conducting field experiments in peanut drought stress and proteomic study and discussed aflatoxin contamination and food safety issues.

Review Publications
Yang, L., Fountain, J., Wang, H., Ni, X., Ji, P., Lee, R.D., Kemerait, R.C., Scully, B.T., Guo, B. 2015. Stress sensitivity is associated with differential accumulation of reactive oxygen and nitrogen species in maize genotypes with contrasting levels of drought tolerance. International Journal of Molecular Sciences. 16:24791-24819. doi: 10.3390/ijms161024791.
Wang, H., Khera, P., Huang, B., Yuan, M., Katam, R., Zhuang, W., Harris-Shultz, K.R., Moore, K.M., Culbreath, A.K., Zhang, X., Varshney, R.K., Xie, L., Guo, B. 2016. Analysis of genetic diversity and population structure of peanut cultivars and breeding lines from China, India and the US using SSR markers. Journal of Integrative Plant Biology. 58:452-465. doi: 10.1111/jipb.12380.
Fountain, J.C., Scully, B.T., Chen, Z., Gold, S.E., Glenn, A.E., Abbas, H.K., Lee, R.D., Kemerait, R.C., Guo, B. 2015. Effects of hydrogen peroxide on different toxigenic and atoxigenic isolates of Aspergillus flavus. Toxins. 7:2985-2999.
Bertioli, D., Cannon, S.B., Froenicke, L., Huang, G., Farmer, A.D., Cannon, E., Dash, S., Liu, X., Barkley, N.L., Guo, B., Scheffler, B.E., et al. 2016. The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut. Nature Genetics. 48:438-446. doi: 10.1038/ng.3517.
Guo, B., Khera, P., Wang, H., Peng, Z., Sudini, H., Wang, X., Osiru, M., Chen, J., Vades, V., Yuan, M., Want, C.T., Zhang, X., Waliyar, F., Wang, J., Varshney, R.K. 2016. Annotation of trait loci on integrated genetic maps of Arachis species. In: Stalker, H.T., Wilson, R.F., editors. Peanuts: Genetics, Processing, and Utilization. Academic Press and the American Oil Chemists' Society (AOCS) Press. p. 163-207.