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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

2017 Annual Report

1. Evaluate molecular markers and saturated genetic maps that can be used 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. The contamination of crops with aflatoxin is a threat to human health, global food safety and security. In our previous studies, isolates of Aspergillus (A.) flavus were found to exhibit different degrees of oxidative stress tolerance which appeared to correlate with their aflatoxin production capability suggesting that aflatoxin production may contribute to stress tolerance. In order to better understand the differences in isolate-specific responses to oxidative stress, and to further explore the potential role of aflatoxin production in stress alleviation in A. flavus, we examined the global transcriptional responses of several isolates of A. flavus to increasing oxidative stress. In this study, we report a detailed analysis of changes in the transcriptomes of different toxigenic A. flavus isolates with distinguished aflatoxin production capabilities to increasing oxidative stress in an aflatoxin conducive culture medium. Isolates which produced higher levels of aflatoxin tended to exhibit fewer differentially expressed genes than isolates with lower levels of production. Genes found to be differentially expressed in response to increasing oxidative stress included antioxidant enzymes, primary metabolism components, antibiosis-related genes, and secondary metabolite biosynthetic components specifically for aflatoxin, aflatrem, and kojic acid. Aflatoxin biosynthetic genes and antioxidant enzyme genes were also found to be co-expressed and highly correlated with fungal biomass under stress. This suggests that these secondary metabolites may be produced as part of coordinated oxidative stress responses in A. flavus along with antioxidant enzyme gene expression and developmental regulation. Aspergillus flavus is a facultative plant pathogen, which is capable of infecting corn and peanut, resulting in significant economic losses and a serious health issue to human and animal due to the production of carcinogenic aflatoxins. Outbreaks of aflatoxin contamination typically occur in regions prone to drought stress, which has been shown to stimulate the production of reactive oxygen species (ROS) in plant tissues. Recent studies have suggested that these ROS and their reactive byproducts may influence the production of aflatoxin by A. flavus. We observed that isolates, which produced higher levels of aflatoxin and possessed greater tolerance to oxidative stress, exhibited less differential gene expression compared to less tolerant, atoxigenic isolates. To examine the oxidative stress responses of A. flavus at the protein/enzymatic level, we examined the proteomic responses of select field isolates of A. flavus to oxidative stress. The selected isolates exhibited distinct responses to oxidative stress which provide insights into potential targets for enhancing host resistance and biological control. Greater numbers of differentially expressed proteins were detected in isolates with less oxidative stress tolerance. Correlative analysis between this proteome data and the transcriptome data showed a weak correlation (r = 0.1114) indicating a possible post-transcriptional regulation. Highly toxigenic isolates exhibited greater expression of lytic enzymes and sclerotial developmental proteins while less toxigenic isolates mainly displayed regulation of antioxidant and primary metabolic pathways. The environmental stress tolerance mechanisms employed by these isolates provide direction for the enhancement of host resistance through the manipulation of host antioxidant capacity and lytic enzyme inhibitor activity using biomarker selection in breeding programs and through novel biotechnologies such as genome editing in crops. The contamination of agricultural crops with aflatoxin and fumonisin is a major concern for global food security particularly in developing countries. Breeding for resistance is still considered to be one of the best strategies currently available to lower aflatoxin and fumonisin accumulation in maize. In these experiments, we screened 87 inbred lines for resistance to aflatoxin and fumonisin contamination using a field screening assay for two years. There were 53 inbred lines that had lower levels of aflatoxin accumulation than the resistant control, Mp717. The inbred lines TUN15, TUN61, TUN37, CY2 and TUN49 had the lowest aflatoxin accumulation, and CN1, GT601, TUN09, TUN61 and MP717 were found to have the lowest fumonisin accumulation. TUN61 exhibited the lowest accumulation of both mycotoxins, which has been used in breeding program. Also, this research indicated that high levels of aflatoxin could coexist with high levels of fumonisin in maize, and there was an overall positive correlation coefficient of 0.37. By identifying resistant maize germplasm to both aflatoxin and fumonisin contamination, it would be possible to enhance the resistance of corn hybrids in breeding programs to reduce the contamination of aflatoxin and fumonisin, and allow for further study of the specific mechanisms underlying resistance to both A. flavus and Fusarium spp. Peanut, also called groundnut, is the second most important legume oilseed in the world. Genetic improvement of yield and production is always the ultimate goal. Peanut is susceptible to many diseases and the cost of disease control can be high. Since molecular breeding has advantages over conventional breeding approach, it is advisable to identify linked markers and then use these markers in improving the target traits through molecular marker-assisted selection. Therefore, we have used a recombinant inbred line (RIL) population called T-population for phenotyping and genotyping followed by construction of an improved genetic map and identification of quantitative trait loci (QTLs) associated with three important diseases. This study included four years of field evaluations of disease severity for early leaf spot, late leaf spot and Tomato Spotted Wilt Virus (TSWV) from 2010 to 2013, and each year there were two planting dates (total eight field trials) in order to obtain better field rating (all naturally occurring). The genetic linkage map was improved to 418 marker loci. Major QTLs with larger contribution to the phenotypic variances were identified for all three diseases. Of the total 42 QTLs, 34 were mapped on the A sub-genome and eight mapped on the B sub-genome, suggesting that the A sub-genome chromosomes have more resistance genes than the B sub-genome. This genetic map was also compared with the diploid progenitor physical maps, and the co-linearity found was good. These QTLs will be further studied for fine mapping of linked markers and identification of potential candidate genes for validation in order to be used in marker-assisted breeding.

1. Research on the drought stress induced aflatoxin production offers insight on breeding targets for corn and peanut. ARS researchers at Tifton, Georgia, examined Aspergillus flavus responses at the protein level in aflatoxin conducive medium amended with varying levels of peroxide. Greater numbers of differentially expressed proteins were detected in isolates with less oxidative stress tolerance. Highly toxigenic isolates exhibited greater expression of lytic enzymes and sclerotial developmental proteins while less toxigenic isolates mainly displayed regulation of antioxidant and primary metabolic pathways. The environmental stress tolerance mechanisms employed by these isolates provide a direction for the enhancement of host resistance through the manipulation of host antioxidant capacity and lytic enzyme inhibition activity using biomarker selection in breeding programs and through novel approaches such as genome editing in crops.

Review Publications
Fountain, J.C., Bajaj, P., Pandey, M., Nayak, S.N., Yang, L., Kumar, V., Jayale, A.S., Chitikineni, A., Zhuang, W., Scully, B.T., Lee, R.D., Kemerait, R.C., Varshney, R.K., Guo, B. 2016. Oxidative stress and carbon metabolism influence Aspergillus flavus transcriptome composition and secondary metabolite production. Scientific Reports. 6:38747. doi: 101038/srep38747.
Guo, B., Ji, X., Ni, X., Fountain, J.C., Li, H., Abbas, H.K., Lee, D., Scully, B.T. 2017. Evaluation of maize inbred lines for resistance to pre-harvest aflatoxin and fumonisin contamination in the field. The Crop Journal. 5:259-264. doi:10.1016/j.cj.2016.10.005.
Katama, R., Sakata, K., Suravajhala, P., Pechand, T., Kambiranda, D., Naik, K., Guo, B., Basha, S. 2016. Comparative leaf proteomics of drought-tolerant and -susceptible peanut in response to water stress. Journal of Proteomics. 143:209-226. doi:10.1016/j.jprot.2016.05.031.
Fountain, J.C., Bajaj, P., Nayak, S.N., Yang, L., Pandey, M.K., Kumar, V., Jayale, A.S., Chitikineni, A., Lee, R.D., Kemerait, R.C., Varshney, R.K., Guo, B. 2016. Responses of Aspergillus flavus to oxidative stress are related to fungal development regulator, antioxidant enzyme, and secondary metabolite biosynthetic gene expression. Frontiers in Microbiology. 7:2048 doi: 10.3389/fmicb.2016.02048.
Khera, P., Pandey, M.K., Wang, H., Feng, S., Qiao, L., Culbreath, A.K., Kale, S., Wang, J., Holbrook Jr, C.C., Zhuang, W., Varshney, R.K., Guo, B. 2016. Mapping quantitative trait loci of resistance to Tomato spotted wilt virus and leaf spots in a recombinant inbred line population of peanut (Arachis hypogae L.) from SunOleic 97R and NC94022. PLoS One. 11(7):e0158452. doi:10.1371/journal.pone.0158452.
Pandey, M.K., Agarwal, G., Kale, S.M., Clevenger, J., Nayak, S.N., Sriswathi, M., Chitikineni, A., Chavarro, C., Chen, X., Bertioli, D.J., Guo, B., Jackson, S.A., Ozias-Akins, P., Varshney, R.K. 2017. Development and evaluation of a high density genotyping 'Axiom_Arachis' array with 58K SNPs for accelerating genetics and breeding in groundnut. Scientific Reports. 7:40577 doi: 10.1038/srep40577.
Pandey, M.K., Khan, A.W., Singh, V.K., Vishwakarma, M.K., Shasidhar, Y., Kumar, V., Garg, V., Bhat, R.S., Chitikineni, A., Janila, P., Guo, B., Varshney, R.K. 2017. QTL-seq approach identified genomic regions and diagnostic markers for rust and late leaf spot resistance in groundnut (Arachis hypogaea L.). Plant Biotechnology Journal. 2017:1-15. Available:
Pandey, M.K., Wang, H., Khera, P., Vishwakarma, M.K., Kale, S.M., Culbreath, A.K., Holbrook Jr, C.C., Varshney, R.K., Guo, B. 2017. Genetic dissection of novel QTLs for resistance to leaf spots and Tomato spotted wilt virus in peanut (Arachis hypogaea L.). Frontiers in Plant Science. 8:25. doi: 10.3389/fpls.2017.00025.
Luo, H., Ren, X., Li, Z., Xu, Z., Li, X., Huang, L., Zhou, X., Chen, Y., Chen, W., Lei, Y., Liao, B., Pandey, M.K., Varshney, R.K., Guo, B., Jiang, X., Liu, F., Jiang, H. 2017. Co-localization of major quantitative trait loci for pod size and weight to a 3.7 cM interval on chromosome A05 in cultivated peanut (Arachis hypogaea L.). BMC Genomics. 18:58. doi:10.1186/s12864-016-3456-x.
Song, H., Wang, P., Li, C., Han, S., Zhao, C., Xia, H., Bi, Y., Zhang, X., Guo, B., Wang, X. 2017. Comparative analysis of NBS-LRR genes and their response to Aspergillus flavus in Arachis. PLoS One. 12(2):e0171181. doi: 10.1371/journal.pone.0171181.