2010 Annual Report
1a.Objectives (from AD-416)
(1) Develop peanut genomic tools and strategies to elucidate the molecular mechanisms that define crop defense pathways and regulation of resistance to diseases such as tomato spotted wilt virus (TSWV), leaf spots and aflatoxin contamination in peanut. (2) Evaluate corn germplasm that harbors resistance genes that reduce aflatoxin contamination and understand the responding genes and pathways in corn.
1b.Approach (from AD-416)
(1) Replicated laboratory and field screening and evaluation of peanut accessions for disease resistance will be conducted in order to identify the “resistant” germplasm for further genomic studies. The resistant germplasm will be utilized in molecular marker development for marker-assisted breeding. (2) ESTs (expressed sequence tags) will be generated from cDNA libraries constructed from seed and leaf tissues of two genotypes, Tifrunner and GT-C20. EST-derived SSR markers will be developed and peanut oligo-microarray will be produced for gene expression study. (3) Genetic mapping populations (RILs, recombinant inbred lines) will be produced from crosses of Tifrunner and GT-C20, and SunOleic 97R x NC94022. QTL mapping will be conducted for resistance to tomato spotted-wilt virus (TSWV) and leaf spots, and aflatoxin contamination. (4) Microarray experiments will be used to identify candidate genes in corn-Aspergillus flavus and drought stress interactions that are turned on or off during corn kernel development. The candidate genes identified from microarray will be verified or confirmed through real time PCR or other well established methods. Another goal is to develop a macroarray tool (membranes) using these candidate genes from microarray to assess resistance or drought tolerance in corn germplasm for their stability of expression in native crops under environmental conditions (e.g., drought) known to be conducive to aflatoxin contamination. The genes identified in corn kernels also will be applied in searching possible ‘orthologs’ in peanut genome and peanut germplasm.
Applications of MAS (marker-assisted selection) in plant breeding have been shown to increase significantly the rate of genetic gain when compared to conventional breeding. The cost of genotyping and throughput are still a concern in marker-assisted selection in peanut breeding. We have developed a simple, low-cost, and high-throughput protocol for genotyping in peanuts. The developed system was based on polyacrylamide gel to separate PCR amplified DNA fragments and silver stain to visualize the bands. In this system, one electrophoresis unit can hold two vertical 52-sample slab gels. The electrophoresis runs about 1 hr and 40 min at 180 V for a 9% polyacrylamide gel or 1 hr and 20 min at 160 V for a 6% polyacrylamide gel. The silver stain takes 30 min. After stained, the gels can be placed on the light-box for genotyping score and the gel image can be photographed using digital camera. The cost per gel is estimated at $0.54 and the cost for silver stain is estimated at $0.37. Therefore, the total cost could be as low as $0.018 per data point, excluding PCR reaction and DNA extraction cost. This system has been successfully used in our peanut genetic mapping, and could generate over 1,200 data points by one person a day.
A genetic linkage map is critical for identifying the QTL (quantitative trait loci) underling targeted traits. A high resolution linkage map with sufficient markers will increase the chances of QTL identification. Two intra-specific F2:7-RIL (recombinant inbred line) populations of 248 and 352 lines derived by single seed descent from crosses between ‘Tifrunner’ × GT-C20 and ‘SunOleic 97R’ × NC94022’ have been developed. The primary phenotype evaluation conducted in 2009 has demonstrated that a significant divergence among RILs of both populations was obvious. The populations are suitable for linkage map construction and QTL analysis. We have screened 4,574 SSR markers for polymorphisms in the parents. Of these SSRs, 269 and 173 markers were polymorphic in these two populations, respectively, and used for the genetic map constructions. In 2009, we conducted field evaluation of F2:5 lines for disease resistance to TSWV with two replications. From our preliminary result, one QTL for TSWV resistance has been identified. The identified QTL may explain 37% phenotypic variation. Furthermore, an integrated map will be constructed from these two populations with more markers to better cover the peanut genome.
Drought tolerance is a complex agronomic trait and root characteristics logically play an important role in determining the response of plants to drought stress. Studies were conducted to investigate genotypic variations in morphological and physiological responses of roots to drought stress in corn. Two inbred lines, Lo964 and Lo1016, were planted in the field, greenhouse, and laboratory growth chamber for examination of the morphological and physiological alteration of root traits under drought stress versus no stress (well-water) conditions. The results revealed that Lo964 had a strong lateral root system, a high root/shoot ratio, and high production of ABA in comparison with Lo1016 under drought stressed conditions.
Development of a low-cost and high-throughput genotyping method for peanut. Traditionally, cultivar development has been dominated by conventional breeding methods, which have and will continue to play an important role in the genetic improvement of various crops. Applications of marker-assisted selection (MAS) in plant breeding have been shown to increase significantly the rate of genetic gain when compared to that of conventional breeding. In the past, given the cost involved in PCR amplification and gel electrophoresis of the large numbers of markers required in genotyping studies, laboratories that possess a small budget and/or limited or no access to expensive equipments are severely hindered to conduct this type of research. In this study, we introduce and describe a low-cost, relatively high-throughput PAGE system utilizing silver staining for genotyping applications successfully in peanut genotyping and linkage map construction with SSR (simple sequence repeat) markers. The cost could be as low as $0.018 per data point, excluding PCR reaction and DNA preparation cost. A scientist has the potential to generate over 1,200 data points per day. This system has been successfully utilized for peanut genotyping and linkage map construction, and could be utilized for other commodities.
Peanut multi-gene family encoding germin-like proteins: Peanut is one of the major economically-important legumes. On a global basis, peanut is a major source of protein and vegetable oil for human nutrition. However, peanut producers are facing many challenges such as a high cost of production due to chemical control of diseases in the US, and food safety concerns because of aflatoxin contamination. Peanut GLP (germin-like protein) genes have not yet been reported to be associated with an enzymatic activity that can be directly or indirectly related to peanut resistance to diseases. In this report, peanut AhGLP proteins associated with SOD (superoxide dismutase) activity may protect the E. coli cells from oxygen free radical mediated oxidative damage caused by the herbicide paraquat, resulting a better resistance to free radicals toxicity. Further studies are needed to elucidate the roles of the peanut GLP family in plant cell growth and stress biology. In summary, this study provides information of the diverse nature of the peanut GLP family and suggests that some of AhGLPs might be involved in peanut disease resistance.
Registration of Maize Inbred Line GT603. Infection of corn with Aspergillus flavus and consequent contamination with aflatoxin, a by-product of fungal metabolism and the most potent naturally-occurring carcinogen, are a serious threat to agricultural production and to human and animal safety. There is an increasing need to seek new resistant germplasm for the prevention of preharvest aflatoxin contamination. GT603 is a yellow dent corn inbred line developed and released by the USDA-ARS Crop Protection and Management Research Unit in cooperation with the University of Georgia Coastal Plain Experiment Station. GT603 was developed through seven generations of self-pollination from a corn population GT-MAS:gk which was released as a source of resistance to Aspergillus flavus. GT603 was initially selected from early self-pollinated lines under the experimental name GT-P50. Laboratory and field studies demonstrated that GT603 had aflatoxin levels similar to or lower than the related inbred lines GT601 and GT602 and the controls Mp313E and Mp715, but the maturity was earlier than Mp313E and Mp715. In hybrid performance tests in 2005 and 2009, GT603 exhibited better combining ability and heterosis with the Stiff Stalk Synthetic (SSS) inbred (B73) than the non-Stiff Stalk Synthetic inbred (Mo17) for aflatoxin levels, yields and biomass. GT603 is being registered and released to the public as a source of resistance, and could be used in other breeding programs to develop Aspergillus/aflatoxin-resistant corn for corn growers.
5.Significant Activities that Support Special Target Populations
Conducted collaborative research with Tuskegee University and Florida A&M University and had two female Young Scholars this summer and one Hispanic high school student as summer intern.
Luo, M., Liu, J., Lee, R.D., Guo, B.Z. 2008. Characterization of gene expression profiles in developing kernels of maize (Zea mays) inbred Tex6. Plant Breeding. 127:569-578.
Guo, B., Butron, A., Scully, B.T. 2010. Maize silk antibiotic polyphenol compounds and molecular genetic improvement of resistance to corn earworm (Helicoverpa zea Boddie) in sh2 sweet corn. International Journal of Plant Biology. 1(e3):13-18.
Jiang, H.F., Ren, X.P., Liao, B.S., Huang, J.Q., Lei, Y., Chen, B.Y., Guo, B., Holbrook, C.C., Upadhyaya, H.D. 2008. Peanut core collection established in China and compared with ICRISAT mini core collection. Acta Agronomica Sinica. 34(1):25-30.
Dang, P.M., Scully, B.T., Lamb, M.C., Guo, B. 2010. Analysis and RT-PCR identification of viral sequences in peanut (Arachis hypogaea L.) expressed sequence tags from different peanut tissues. Plant Pathology. 9(1):14-22.2010.
Hong, Y., Chen, X., Liang, X., Liu, H., Zhou, G., Li, S., Wen, S., Holbrook, C., Guo, B. 2010. A SSR-based composite genetic linkage map for the cultivated peanut (Arachis hypogaea L.) genome. Biomed Central (BMC) Plant Biology. 10:17.