2009 Annual Report
1a.Objectives (from AD-416)
The three objectives of the research are firstly, to define linkage disequilibrium and recombination rates across the soybean genome to facilitate efficient discovery of quantitative trait loci (QTL) through Association Analysis and efficient introgression of exotic germplasm, secondly, to define genome regions in cultivated soybean that are associated with domestication for the discovery of genetic variation lost through the domestication bottleneck that can be used to improve soybean and thirdly, to discover QTL and genes controlling biotic and abiotic stress resistance and quality traits in soybean and wheat, and develop DNA markers that define haplotype variation across these and previously identified regions.
1b.Approach (from AD-416)
Single nucleotide polymorphism (SNP) DNA markers will be discovered using high throughput genome sequence analysis in combination with the newly developed whole genome soybean sequence from the Department of Energy, Joint Genome Institute. A set of 50,000 SNPs, selected from across the genome, will be identified and genetically mapped in cultivated soybean as well as in a newly created cultivated x wild soybean population. The same SNPs will be used to characterize 16,795 soybean landraces as well as a set of 96 elite soybean cultivars and 1,116 wild soybean genotypes. This will allow an assessment of linkage disequilibrium and population structure across the genomes of the landraces, elite cultivars and wild soybeans. Association Analysis will be assessed as a new approach to detect genes/QTL underlying the important trait of seed protein concentration. The high resolution genetic maps in both cultivated x cultivated and cultivated x wild soybean populations combined with QTL analysis of traits related to soybean domestication will facilitate the identification of regions in cultivated soybean which, in comparison to wild soybean, have little or no genetic variation as a result of “selective sweeps” that occurred during soybean domestication. A universal set of 1536 soybean SNPs with high rates of polymorphism and even distribution across the genome will be developed and used to discover QTL underlying a number of disease resistance and quality traits in soybean. In addition, DNA marker development in hexaploid wheat will be continued and these markers and other SSR markers previously developed in our laboratory will be used in QTL analysis for a number of important traits in hexaploid wheat.
Progress was made in discovery of SNPs for the genetic analysis of soybean germplasm with 37,000 SNP markers. Reduced representation DNA libraries were created from genomic DNA of the soybean cultivars Evans, Essex, Archer, Minsoy, Peking, Noir 1 as well as of the wild soybean PI 468916. In addition, similar libraries were created with a mix of DNA of the genotypes Minsoy, Noir 1, Archer, Evans, and Peking. The genomic DNA libraries consisted of restriction digested fragments that were “size selected” to only include DNA fragments in the 100-150 basepair size range. Following numerous runs on the Solexa/Illumina Genome Analyzer more than 7 billion bases of DNA sequence was obtained from these various libraries. These were aligned with the whole genome sequence of soybean to facilitate the discovery of more than 60,000 putative SNPs.
Progress was made in the anchoring of the whole soybean genome sequence produced by the Department of Energy, Joint Genome Institute to the 20 chromosomes that make-up the soybean genome. The Consensus Soybean Linkage Map 4.0 contains more than 4,800 DNA sequence-based markers including 3792 SNP-containing sequence tagged sites and 1009 simple sequence repeat markers. An additional 1,240 SNP markers were developed specifically to anchor segments of the genome that were not anchored by markers in Consensus Soybean Linkage Map 4.0. These markers were genetically mapped in a high resolution mapping population of 470 recombinant inbred lines developed from a cross of Williams 82 with the wild soybean PI 468916. As result of these efforts a total of 97% of the whole soybean genome sequence was anchored to the 20 soybean chromosomes.
Progress was made in the discovery of wheat SNP markers using next generation Solexa/Illumina DNA sequencing. Genomic DNA libraries were constructed by restriction enzyme digestion and size selection of DNA fragments in the 100-150 basepair size range of the parents of the ITMI (International Triticeae Mapping Initiative) genetic mapping parents Opata 85 and W7984. These DNA libraries were sequenced on the Solexa/Illumina Genome Analyzer. Homologous fragments were aligned and compared for the identification of defining DNA sequence differences or SNPs. To date a total of 1742 putative SNPs have been identified.
Development and application of the Universal 1536 Soy Linkage Panel 1.0 (USLP 1.0). The analysis of hundreds of DNA markers on hundreds of soybean lines in populations developed by geneticists for the discovery of genetic factors that underlie traits of importance for soybean genetic improvement is laborious and time consuming. Single nucleotide polymorphisms (SNPs) are the DNA markers of choice for many researchers due to their abundance and the rapid high-throughput methods available for the characterization of many markers in one analysis. ARS scientists in the Soybean Genomics and Improvement Laboratory in Beltsville, MD with collaborators at the Univ. of Nebraska, the Univ. of Maryland, and with ARS scientists at Ames, IA and Raleigh, NC developed a selected set of 1536 SNP DNA markers that can be analyzed on 192 soybean DNA samples in a three day period using the Illumina GoldenGate assay. The set of 1536 markers is referred to as the Universal Soy Linkage Panel 1.0 (USLP 1.0) and was selected from more than 3000 markers, each of which was genetically mapped to identify markers with equidistant spacing across each of the 20 soybean consensus linkage groups that define the 20 soybean chromosome pairs. In addition, the 3000 markers were used to analyze 96 elite soybean varieties and 96 diverse accessions from the USDA Soybean Germplasm Collection in order to identify those markers that were maximally informative. The 1536 SNP markers in the USLP 1.0 will permit rapid genetic analysis of soybean populations that are segregating for important genetically controlled traits affecting disease and insect resistance, environmental stress such a drought, seed quality traits including soybean oil quality and protein concentration as well as seed yield and to discover the genome positions of genes controlling these traits.
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Gaitan-Solis, E., Choi, I., Quigley, C.V., Cregan, P.B., Tohme, J. 2008. Single nucleotide polymorphisms in common bean: their discovery and genotyping using a multiplex detection system. The Plant Genome. 1:125-134.
Hyten, D.L., Smith, J.R., Frederick, R.D., Tucker, M.L., Song, Q., Cregan, P.B. 2009. Bulk Segregate Analysis using the GoldenGate Assay to Locate the Rpp3 Locus that Confers Resistance to Phakopsora pachyrhizi (Soybean Rust) in Soybean. Crop Science. 49:265-271.
Wu, X., Blake, S., Sleper, D.A., Shannon, J.G., Cregan, P.B., Nguyen, H.T. 2008. QTL and Additive and Epistatic Effects for SCN Resistance in PI 437654. Theoretical and Applied Genetics. 118:1093-1105.
Malkus, A., Song, Q., Cregan, P.B., Arseniuk, E., Ueng, P.P. 2009. Genetic linkage map of Phaeosphaeria nodorum, the causal agent of stagonospora nodorum blotch disease of wheat. European Journal of Plant Pathology. 124:681-690.
Chakroaborty, N., Curley, J., Frederick, R.D., Hyten, D.L., Nelson, R.L. Hartman, G.L., Diers, B.W. 2009. Mapping and Confirmation of a New Allele at Rpp1 from Soybean PI 504538A Conferring RB Lesion Type Resistance to Soybean Rust. Crop Science. 49:783-790.