2009 Annual Report 1a.Objectives (from AD-416) Objective 1, the improvement of yield potential, will deal with QTL analysis of breeding populations derived from exotic germplasm and with a method to identify appropriate exotic germplasm for study. Objective 2, the improvement of seed composition, will focus on raising oleic acid content of soy oil, reducing phytate in the protein meal, and in determining the impact of altered seed composition on vulnerability to disease. Objective 3, the improvement of drought tolerance, will identify QTLs for drought tolerance derived from exotic germplasm and test a rapid screening method that may streamline breeding for drought tolerance. 1b.Approach (from AD-416) The research will be accomplished by combining conventional breeding technology with Quantitative Trait Loci (QTL) analysis and near isogenic line development. Drought tolerance is treated as a topic distinct from yield, because it is the greatest agronomic limitation to soybean production in the USA. Using molecular marker technology in combination with field and lab studies, we will assess the genetic potential of accessions in the USDA soybean germplasm collection for improving soybean yield potential, seed composition, disease resistance, and drought tolerance. We will identify the genomic location of alleles which govern these traits. 3.Progress Report Soybean is among the least diverse crops in the USA. Such narrow genetic diversity renders a crop vulnerable to changing pests and environmental extremes, also limits the ability of breeders to improve the crop. New genetics are needed to address this diversity problem. This project will i) identify exotic germplasm in the soybean collection which carry key economic traits, ii) determine the genomic location of the genes governing these traits using DNA markers, and iii) use DNA markers and diverse breeding lines derived from exotic germplasm in practical crop improvement. The primary traits of interest, corresponding to the three objectives of this project, are improved seed yield, improved seed composition, and improved drought tolerance. Approximately 15,000 experimental yield plots were grown in pursuit of project objectives. Research was carried out at 5 diverse sites in North Carolina. To improve yield using exotic germplasm, Quantitative Trait loci (QTL) populations and related advanced breeding lines carrying exotic pedigree were grown in more than 7000 yield plots. From this work, 27 advanced breeding lines derived from exotic Asian germplasm were submitted to testing in regional trials. In addition, the use of F2 bulk yield as a criterion for selection of superior breeding populations derived from exotic germplasm was evaluated. The F2 generations of 6 different bi-parental combinations were tested in field experiments along with a set of random F6 lines derived from each population. To improve seed composition using exotic germplasm, 144 high protein lines, 7 high oil lines, and 14 low phytate lines, and 48 oil quality lines were tested for seed composition and agronomic quality in field experiments. Quantitative-RT PCR assays were developed to monitor fungal pathogen growth in soybean tissues following inoculation. Together with the q-RT PCR assays developed in 2008 for measuring the induction profiles of 13 soybean pathogen defense genes these assays are being used to characterize the defense gene activation of soybean leaves and seeds in response to attack by the fungal pathogens Cercospora kikuchii, causal agent of leaf blight and purple seed stain, and Diaporthe phaseolorum var. meridionales, causal agent of southern stem canker and seed decay. To improve drought tolerance, more than 5000 yield plots were evaluated at a drought-prone field site. From this work, 13 advanced breeding lines derived from exotic Asian germplasm were submitted to testing in regional trials. Two genetically diverse plant populations have been developed, one for DNA analysis, and the other for integrating plant canopy temperature, prior to the occurrence of drought stress, as a possible early indicator of drought tolerance. 4.Accomplishments 1. Drought-tolerant germplasm developed. A new generation of breeding lines was developed and tested for drought response. In regional and local testing, two of them, N04-9646 and N01-11771, N05-7432, N05-7462 were slow wilting and showed a substantial yield benefit when grown under dry conditions. They also yielded reasonably well in good environments which had minimal stress conditions. These genetic materials have been made available to commercial breeders as parental stock. These new lines are very likely the most drought tolerant soybean materials in the world. They are being requested and used by industry. The impact on the soybean production will be realized as commercial breeding programs release new cultivars derived from this USDA stock. 2. Release of high-yielding, high seed protein soybean germplasm N6202. Such a breeding line has been thought impossible to produce. However, the project developed a new USDA breeding line with this desirable combination. This new diverse release has 50% exotic germplasm. All high protein soybean releases in the South suffer from the problem of low yield. Using exotic genetic diversity, this new line overcame this problem and will likely be a major basis for development of higher protein soybean cultivars in the future. The release of this new germplasm adds diversity to the narrow genetic range of soybean breeding materials in the Southern USA. This germplasm is serving as a parent for a QTL study which seeks document this uncoupling of high protein with low yield. 3. New breeding method - hybrid vigor used to predict desirable soybean parents for inbred line development. When two high yielding varieties are cross-pollinated, the progeny in the F2 (second) generation may show hybrid vigor when compared with the two parents. If so, hybrid vigor of the F2 generation might be a criterion for deciding which parental combinations are the most likely to generate high yielding inbred soybean varieties. The F2 generations from six cross combinations were field tested along with their parents and 37 inbred lines derived from that mating. The one cross, which exhibited hybrid vigor in the F2 generation, was also the one cross with an inbred line that was significantly higher yielding than either parent. This is evidence that F2 generation performance can be used to pick parental combinations with good combining ability. This will aid in the elimination of expensive tests of breeding lines that are unlikely to be productive varieties. 4. Screening method developed to identify genetic resistance to diseases caused by Cercospora and Diaporthe. Expression of defense genes in pathogen-inoculated, detached green seeds harvested 35 days after flowering, was monitored over a 48 h post-inoculation period during which both pathogens grew in seed tissue around the site of inoculation. Gene expression analysis showed that five of seven genes were upregulated in seeds by both C. kikuchii and Diaporthe phaseolorum with four of the seven, PR1, MMP2, CHS and PAL strongly upregulated. PR2 was weakly upregulated by C. kikuchii, and LOX7 was weakly upregulated by D. phaseolorum. Defense gene induction was generally higher in inoculated seeds compared to leaves. There were similarities in pattern but differences in magnitude of induction of defense genes in soybean seeds for Cercospora and Diaporthe. In seeds, but not leaves, both pathogens elicited a remarkably high expression (55 to 161 fold over the control) of the MMP2 by 48 h post inoculation. Results show that defense gene induction occurs and can readily be measured in detached green soybean seeds, thus this approach appears feasible for identifying and characterizing genotypes with seed resistance to these pathogens. 6.Technology Transfer None Review Publications Lee, J., Smothers, S.L., Dunn, D., Villagarcia, M.R., Shumway, C.R.,Carter Jr, T.E., Shannon, G. Evaluation of a Simple Method to Screen Soybean Genotypes for Salt Tolerance. Crop Sci. 2008 48: 2194-2200. Carter Jr, T.E., Burton, J.W., Fountain, M.O., Rzewnicki, P.E., Villagarcia, M.R., Bowman, D.T. 2008. Registration of soybean cultivar ‘N8001’. Journal of Plant Registrations, 2: 22-23. Burkey, K.O., Carter Jr, T.E. 2009. Foliar Resistance to Ozone Injury in the Genetic Base of U.S. and Canadian Soybean and Prediction of Resistance in Descendent Cultivars Using Coefficent of Parentage. Field Crops Research. 111:207-217. Charlson, D.V., Bhatnagar, S., King, C.A., Ray, J.D., Sneller, C.H., Carter Jr, T.E., Purcell, L.C. 2009. Polygenic Inheritance of Canopy Wilting in Soybean [Glycine max (L.) Merr.]. Theoretical and Applied Genetics. DOI 10.1007/s11033-009-9496-4 Carter Jr, T.E., Burton, J.W., Rzewnicki, P.E., Villagarcia, M.R., Fountain, M.O., Taliercio, E.W., Bowman, D. 2009. Registration of ‘N8101’ small-seeded soybean. Journal of Plant Registrations, 3:22-27. Bachlava, E., Burton, J.W., Brownie, C., Wang, S., Auclair, J., Cardinal, A. 2008. Heritability of Oleic Acid Seed Content in Soybean Oil and its Genetic Correlation with Fatty Acid and Agronomic Traits. Crop Sci. Vol. 48:1764-1772.