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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

2015 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 6048-21000-025-00D, "Developing Genomic Approaches to Improve Resistance to Diseases and Aflatoxin Contamination in Peanut and Corn," 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. Peanut is vulnerable to a range of diseases, such as leaf spots and Tomato spotted wilt virus (TSWV), which cause yield loss every year worldwide. The most promising solution for managing peanut diseases is using resistant cultivars. New breeding line NC94022 has been identified with the highest resistance to TSWV in the field. An improved genetic linkage map was developed for a population derived from SunOleic 97R × NC94022 with 248 markers. The quantitative trait locus (QTL) analysis using the improved genetic map and several years’ phenotype data generated from multiple locations and years (2009-2013) resulted in the identification of 76 QTLs with phenotypic variance (PV) ranging from 3.47 to 30.15%. Of the 76 QTLs, 43 QTLs were for resistance to early leaf spot (ELS), 20 for late leaf spot (LLS) and 13 for TSWV, which includes 11, 6 and 4 major QTLs (PV >10%), respectively. The consistent QTL that occurred in more than two environments for ELS, LLS and TSWV were also identified. These results also suggested that linkage groups of a01, a03 and b03 have “resistance gene rich” regions. The identified QTLs and associated markers upon validation could be used for improving disease resistance through molecular breeding in addition to further discovery of resistance genes and understanding the molecular basis of disease resistance mechanisms in peanut. Drought stress in the field has been shown to exacerbate aflatoxin contamination of maize and peanut. Drought and heat stress also produce reactive oxygen species (ROS) in plant tissues. Given the potential correlation between ROS and exacerbated aflatoxin production under drought and heat stress, the objectives of this study were to examine the effects of hydrogen peroxide (H2O2)-induced oxidative stress on the growth of different toxigenic (+) and atoxigenic (-) isolates of Aspergillus flavus and to test whether aflatoxin production affects the H2O2 concentrations that the isolates could survive. Ten isolates were tested: NRRL3357(+), A9(+), AF13(+), Tox4(+), A1(-), K49(-), K54A(-), AF36(-), and Aflaguard(-); and one A. parasiticus isolate, NRRL2999(+). These isolates were cultured under a H2O2 gradient ranging from 0 to 50 mM in two different media, aflatoxin-conducive yeast extract-sucrose (YES) and non-conducive yeast extract-peptone (YEP). Fungal growth was inhibited at a high H2O2 concentration, but specific isolates grew well at different H2O2 concentrations. Generally the toxigenic isolates tolerated higher concentrations than did atoxigenic isolates. Increasing H2O2 concentrations in the media resulted in elevated aflatoxin production in toxigenic isolates. In YEP media, the higher concentration of peptone (15%) partially inactivated the H2O2 in the media. In the 1% peptone media, YEP did not affect the H2O2 concentrations that the isolates could survive in comparison with YES media, without aflatoxin production. It is interesting to note that the commercial biocontrol isolates, AF36(-), and Aflaguard(-), survived at higher levels of stress than other atoxigenic isolates, suggesting that this testing method could potentially be of use in the selection of biocontrol isolates. Further studies will be needed to investigate the mechanisms behind the variability among isolates with regard to their degree of oxidative stress tolerance and the role of aflatoxin production. Drought stress decreases crop growth and 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 tolerance have been shown to possess both common and genotype-specific strategies to cope with drought conditions. In this study, the physiological and metabolic response patterns in the leaves of maize seedlings subjected to drought stress were investigated using six maize genotypes. During drought treatments, drought-sensitive maize seedlings displayed more severe symptoms such as chlorosis and wilting, exhibited significant decreases in photosynthetic parameters, and accumulated much 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 observed antioxidant enzyme activities were constitutively higher in the tolerant genotypes than in the sensitive genotypes, which showed a rapid induction following drought induction. These results suggest that drought stress causes differential responses to oxidative and nitrosative stress in maize genotypes with tolerant genotypes being better able to sequester ROS and RNS than sensitive ones. These differential patterns imply possible inter-connections in tolerant and sensitive line responses to drought stress which can be utilized as potential biological markers for use in marker assisted breeding. Cultivated peanut is grown worldwide as rich-source of oil and protein. A broad genetic base is needed for cultivar improvement. The objectives of this study were to develop highly informative simple sequence repeat (SSR) markers and to assess the genetic diversity and population structure of peanut cultivars and breeding lines from different breeding programs in China, India and the US. A total of 111 SSR markers were selected for this study, resulting in a total of 472 alleles. The mean values of gene diversity and polymorphic information content (PIC) were 0.480 and 0.429, respectively. Country-wise analysis revealed that alleles per locus in three countries were similar. The mean gene diversity in the US, China and India was 0.363, 0.489 and 0.47 with an average PIC of 0.323, 0.43 and 0.412, respectively. Genetic analysis using the STRUCTURE divided these peanut lines into two populations (P1, P2), which was consistent to the dendrogram based on genetic distance (G1, G2) and the clustering of principal component analysis. The groupings were related to peanut market types and the geographic origin with a few admixtures. The results could be used by breeding programs to assess the genetic diversity of breeding materials to broaden the genetic base and for molecular genetics studies.

1. Identification of peanut resistance markers for TSWV and leaf spot diseases. Peanut is vulnerable to a range of diseases, such as leaf spots and Tomato spotted wilt, which cause yield loss and increase chemical control cost every year. The most promising solution for managing peanut diseases is using resistant cultivars. ARS researchers in Tifton, Georgia, in collaboration with researchers at the University of Georgia, Tuskegee University, and the University of Florida have developed a genetic map and identified molecular markers linked to resistance to the diseases of Tomato spotted wilt, early leaf spot and late leaf spot of peanut. Molecular markers will shorten the breeding cycle in half. Peanut growers have been supporting research to develop molecular markers for resistance and molecular breeding, which will give farmers better and more effective management tools for diseases.

2. Identification of bio-markers for drought tolerant corn and peanut. Drought stress decreases crop growth and yield, and can further exacerbate aflatoxin contamination in corn and peanut. Tolerance and adaptation to drought stress is an important trait of agricultural crops. ARS researchers in Tifton, Georgia, in collaboration with researchers at the University of Georgia, the University of Florida, and Florida A&M University have studied the physiological and metabolic response patterns in corn, and discovered the accumulation of reactive oxygen species, reactive nitrogen species and the observed antioxidant enzyme activities. The differential patterns can be utilized as potential biological markers for use in marker assisted breeding and screening for drought stress tolerant corn and peanut cultivars. The bio-markers have been published for public use.

Review Publications
Yang, L., Jiang, T., Fountain, J.C., Scully, B.T., Lee, R.D., Kemerait, R.C., Chen, S., Guo, B. 2014. Protein profiles reveal diverse responsive signaling pathways in kernels of two maize inbred lines with contrasting drought sensitivity. International Journal of Molecular Sciences. 15:18892-18918. doi: 10.3390/ijms151018892.
Wang, M.L., Khera, P., Pandey, M.K., Wang, H., Qiao, L., Feng, S., Tonnis, B.D., Barkley, N.L., Pinnow, D.L., Holbrook Jr, C.C., Culbreath, A.K., Varshney, R.K., Guo, B. 2015. Genetic mapping of QTLs controlling fatty acids provided insights into the genetic control of fatty acid synthesis pathway in peanut (Arachis hypogaea L.). PLoS One. 10(4):e0119454. doi: 10.1371/journal.pone.0119454.
Pandey, M.K., Upadhyaya, H.D., Rathore, A., Vadez, V., Sheshshayee, M.S., Sriswathi, M., Govil, M., Kumar, A., Gowda, M.V., Sharma, S., Hamidou, F., Kumar, V.A., Khera, P., Bhat, R.S., Khan, A.W., Singh, S., Li, H., Monyo, E., Nadaf, H.L., Mukri, G., Jackson, S.A., Guo, B., Liang, X., Varshney, R.K. 2014. Genomewide association studies for 50 agronomic traits in peanut using the 'reference set' comprising 300 genotypes from 48 countries of the semi-arid tropics of the world. PLoS One. 9(8):e105228. doi: 10.1371/journal.pone.0105228.
Zhang, H., Scharfenstein, L.L., Zhang, D., Chang, P., Montalbano, B.G., Guo, B., Meng, X., Yu, J. 2014. Peanut resistant gene expression in response to Aspergillus flavus infection during seed germination. Journal of Phytopathology. 163(3):212-221.
Fountain, J.C., Khear, P., Yang, L., Nayak, S., Scully, B.T., Lee, R.D., Chen, Z., Kemerait, R.C., Varshney, R.K., Guo, B. 2015. Resistance to Aspergillus flavus in maize and peanut: Molecular biology, breeding, environmental stress and future perspectives. The Crop Journal. 3(2015):229-237. doi: org/10.1016/j.cj.2015.02.003.