Progress 08/01/06 to 07/31/11
Outputs
OUTPUTS: Massively parallel DNA sequencing technologies were used to develop short-read ESTs for A. duranensis and A. hypogaea, from leaf, seed, and root RNAs. The A. duranensis SR-ESTs (>2 mil total; SR-ESTs in SRA accessions SRX019980, SRX019981, SRX020011, SRX020013) were assembled with 35,291 long-read ESTs (PRJNA50587) and publicly released to NCBI. The diploid assembly was mined for single nucleotide polymorphisms which were used to generate an Illumina GoldenGate array for highly parallel SNP genotyping of an A-genome F2 population in order to produce a high-density, gene-based SNP map of A. duranensis containing 1054 SNP loci. For cultivated A. hypogaea, ~4.7 mil short-read ESTs (deposited at NCBI) of 19 genotypes, including the reference genotype Tifrunner, were assembled with 88,000 long-read ESTs to output ~212,000 contigs plus singletons. A first-generation assembly with a subset of these data resulted in 37,916 contigs (NCBI Project Accession PRJNA49471) plus 63,216 singletons from which 2,138 EST-SSRs were mined. These markers were transferrable across A- and B-genome species and enabled the construction of a B-genome map from A. batizocoi with 449 SSR loci. Furthermore A- and B-genome syntenic relationships were determined from shared SSR loci. A second generation A. hypogaea assembly was used for SNP discovery and production of a GoldenGate array for SNP genotyping in diploids and tetraploids. The feasibility of this highly parallel SNP genotyping platform was demonstrated for inbred lines of A. hypogaea. PARTICIPANTS: The PI (Steven J. Knapp) developed research plans, wrote several additional grant proposals, secured additional extramural funding to supplement funding from the USDA, developed thesis research plans, and supervised and mentored postdoctoral scientists, research technicians, and graduate and undergraduate students. The Co-PI (Peggy Ozias-Akins) developed research plans, supervised and mentored postdoctoral scientists and graduate students, and disseminated research results through presentations and publications. Several undergraduate students (Sarah Hafner, Rebecca Okashah, Jodie James) have assisted with greenhouse, field, and laboratory components of this research. This grant has supported the graduate research programs for two M.S. students (Sameer Khanal and Yan Li) and the research program for a postdoctoral scientist (Yufang Guo). Part of the research was completed by another postdoctoral scientist (Osman Radwan). Shunxue Tang (a senior research scientist) and Chris Taylor (a bioinformatician) played a crucial role in the development of the EST database and diploid mapping populations. Ed Johnson (a bioinformatician) assisted with the development of the peanut EST database. Four research technicians (Adam Heesacker, Jenny Wood, and Jason Prothro, and Nelly Khalilian) have assisted with greenhouse, field, and laboratory research activities. We are collaborating with scientists from several laboratories in the US and abroad. We initiated a collaboration in 2007 with David Hoisington, Rupakula Aruna, and Rajeev Varshney, the team at ICRISAT (Hyderabad, Andhra Pradesh, India) leading the Groundnut Genomics and Breeding Program, and with David Bertioli (EMBRAPA, Brazil). The ICRISAT team is leading an international program in peanut genomics funded by the Bill and Melinda Gates Foundation through the Generation Challenge Program. Greg May, Andrew Farmer, and others at the National Center for Genomic Resources (NCGR) are providing DNA sequencing and bioinformatics support for this research. We are collaborating with Doug Cook, Varma Penmetsa, and other scientists at UCD developing genomic resources for groundnut as part of an NSF Plant Genome Funded research program awarded to UCD. We have collaborative research activities underway in peanut with Corley Holbrook, Baozhu Guo, and Albert Culbreath at UGA and USDA-ARS, Tifton, Georgia and H. Thomas Stalker, Susanna Milla-Lewis, and Thomas Isleib, and Niels Nielsen at NCSU. TARGET AUDIENCES: Peanut breeders are a primary target audience. It is expected that as a consequence of the accomplishments of this project, breeders will have access to abundant tools for marker assisted selection (MAS). MAS already is having an impact on development of nematode resistant cultivars, and its application on a broader scale and for additional traits will be enabled by the tools developed in this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The genomic resources developed through this research are currently being used in several collaborative research activities, in addition to enabling our own research, and have greatly expanded the foundation for applying genomics approaches in peanut breeding and genetics research. The information we have generated has been applied to the discovery of candidate genes for nematode resistance and is currently being applied in genetic analyses focusing on resistance to tomato spotted wilt virus, early and late leaf spot, and rust, seed morphology and quality traits, and other agronomic traits. Molecular markers developed through this research have been implemented in a molecular breeding program for nematode resistant cultivar development. Subsequent to securing funding from NRI, we reached agreements with two groups developing ESTs to collaborate on the development of an EST database. NRI funding also enabled us to leverage funding from several public sources (the Georgia Seed Development Commission, the Peanut Foundation, the National Peanut Board, the Georgia Peanut Commission, and the Generation Challenge Program). This funding has enabled us to broaden the scale and scope of our research and significantly increase the effort directed towards DNA marker development and genetic mapping in cultivated peanut. Our NRI research primarily focused on the development of diploid models for tetraploid peanut, but with collaborations established with UC-Davis and ICRISAT, a critical mass of DNA markers for genetic mapping in intraspecific tetraploid populations also was developed. A significant emphasis was placed on SNP discovery, validation, and mapping (using highly parallel arrays), although the SSR markers have had a more immediate impact on the peanut research community because of their transportability, hypervariability, versatility, and broad applicability in tetraploids. Nevertheless, the feasibility of highly parallel SNP arrays for genotyping of tetraploid peanut was demonstrated. While low levels of polymorphism in cultivated peanut continue to constrain intraspecific genetic mapping, the abundant molecular polymorphism found in the wild species along with alleles for traits of interest will provide additional genetic tools for peanut crop improvement through interspecific hybrids.
Publications
- 9. Nagy, E., Y. Guo, S. Khanal, C. Taylor, S. Knapp, P. Ozias-Akins, H.T. Stalker, and N. Nielsen. 2010. Developing a High-Density Molecular Map of the A-Genome Species A. duranensis. p. 25, APRES Proceedings, Clearwater Beach, FL, 12-15 July. http://www.apresinc.com/pdf/Proceedings/Volume%2042,%20Proceedings_20 10.pdf
- 10. Chu, Y., C. C. Holbrook, P. Faustinelli, X.Y. Deng, A. Bhattacharya, E.D. Nagy, S. Knapp, and P. Ozias-Akins. 2010. Application of biotechnology in peanut cultivar improvement. http://www.intl-pag.org/18/abstracts/W49_PAGXVIII_366.html
- 11. Nagy, E.D., Y. Chu, Y. Li, W.B. Dong, P. Timper, P. Ozias-Akins, C.C. Holbrook, B. Rosen, D. Cook, S.J. Knapp. Discovery of NBS-LRR encoding genes linked to a dominant nematode resistance gene (Rma) in groundnut lines carrying a genomic segment introgressed from a wild diploid donor . Plant and Animal Genome Meeting, San Diego, California, January, 2009.
- 5. Varshney, R.K., D.J. Bertioli, M.C. Moretzsohn, K. Ravi, V. Vadez, L. Krishnamurthy, R. Arunal, S.N. Nigam, B.J. Moss, K. Seetha, G. He, S.J. Knapp, and D.A. Hoisington. 2009. The first SSR-based genetic linkage map for cultivated groundnut (Arachis hypogaea L.). Theor. Appl. Genet. 118: 729-739.
- 12. Taylor, C.A., S. Tang, E. Bachlava, A. Farmer, S. Ayyampalayam, J. Huntley, G.D. May, and S.J. Knapp. SNP Discovery by massively parallel transcriptome resequencing in sunflower and development of a bioinformatic pipeline and database for mining and displaying SNPs in next-generation sequence assemblies. Plant and Animal Genome Meeting, San Diego, California, January, 2009.
- 13. Knapp, S.J. SNP discovery, validation, and mapping in groundnut. Generation Challenge Program Annual Meeting, September, 2008, Bangkok, Thailand (Invited Presentation).
- 14. Nagy E, Chu Y, Li Y, Dong WB, Timper P, Ozias-Akins P, Holbrook CC, Radwan O, Rosen B, Cook D, Knapp SJ. Discovery and genetic mapping of NBS-LRR encoding resistance gene candidates linked to a root knot nematode resistance gene (Rma) introgressed from a wild diploid donor in groundnut. Generation Challenge Program Annual Meeting, September, 2008, Bangkok, Thailand.
- 15. Khanal S, Tang S, Nagy E, Guo Y, Li Y, Beilinson V, San Miguel P, Guo B, Nielsen N, Stalker T, Cordonnier-Pratt MM, Pratt LH, Johnson VE, Taylor CA, Wiley GB, Macmil SL, Roe B, Ravi K, Naidu G, Hoisington D, Varshney R, Knapp SJ. EST-SSR marker resources for groundnut. Generation Challenge Program Annual Meeting, September, 2008, Bangkok, Thailand.
- 1. Qin, H., S. Feng, C. Chen, Y. Guo, S. Knapp, A. Culbreath, G. He, M. Wang, X. Zhang, C. C. Holbrook, P. Ozias-Akins, and B. Guo. 2011. An integrated genetic linkage map of cultivated peanut (Arachis hypogaea L.) constructed from two RIL populations. Theor. Appl. Genet. (in press).
- 2. Gautami, B., M.K. Pandey, V. Vadez, S.N. Nigam, P. Ratnakumar, L. Krishnamurthy, T. Radhakrishnan, M.V.C. Gowda, M.L. Narasu, D.A. Hoisington, S.J. Knapp, and R.K. Varshney. 2011. Quantitative trait locus analysis and construction of consensus genetic map for drought tolerance traits based on three recombinant inbred line populations in cultivated groundnut (Arachis hypogaea l.). Mol. Breed. (in press).
- 3. Pandey, M.K., E. Monyo, P. Ozias-Akins, X. Liang , P. Guimaraes, S.N. Nigam, H.D. Upadhyaya, P. Janila , X. Zhang, B. Guo, D.R. Cook, D.J. Bertioli , R. Michelmore, R.K. Varshney. 2011. Advances in Arachis genomics for peanut improvement. J. Biotech. Adv. (doi:10.1016/j.biotechadv.2011.11.001)
- 4. Sujay, V., M.V.C. Gowda, M.K. Pandey, R.S. Bhat, Y.P. Khedikar, H.L. Nadaf, B. Gautami, C. Sarvamangala, S. Lingaraju, T. Radhakrishnan, S.J. Knapp, and R.K. Varshney. 2011. Quantitative trait locus analysis and construction of consensus genetic map for foliar disease resistance based on two recombinant inbred line populations in cultivated groundnut (Arachis hypogaea l.). Mol. Breed. (in press).
- 6. Nagy, E.D., Y. Chu, Y. Guo, S. Khanal, S. Tang, Y. Li, W.B. Dong, P. Timper, C. Taylor, P. Ozias-Akins, C.C. Holbrook, V. Beilinson, N.C. Nielsen, H.T. Stalker, and S.J. Knapp. 2010. Recombination is suppressed in an alien introgression on chromosome 9 of peanut harboring Rma, a dominant root-knot nematode resistance gene. Mol. Breed. 26:357-370.
- 7. Chu, Y., C. Wu, P. Ozias-Akins, and C.C. Holbrook. 2011. Marker-assisted breeding for wild species-derived traits in Arachis. APRES Proceedings, San Antonio, TX, 11-14 July.
- 8. Holbrook C. C., Y. Chu, P. Ozias-Akins, E. D. Nagy, S. J. Knapp, and B. Z. Guo. Use of marker-assisted selection to develop disease resistant cultivars with high O/L ratio. Proc. 4th Intern. Conf. on Advances in Arachis through Genomics and Biotechnology. Bamako, Mali, 19-22 Oct. 2010.
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Progress 08/01/06 to 07/31/07
Outputs
OUTPUTS: We have processed, annotated, and assembled 88,000 ESTs developed from RNAs isolated from normalized and non-normalized developing seed and leaf cDNA libraries; developed a relational EST database with a suite of bioinformatic pipelines for BLAST analyses, contig display, and DNA polymorphism discovery; produced 330,000 short-read ESTs (using 454-FLX) from normalized developing seed cDNAs of two genotypes and 1.3 Gbp of short-read ESTs (using Solexa) from non-normalized developing seed cDNA of two genotypes; initiated bioinformatic analyses for SNP discovery in Sanger-454-Solexa assemblies; developed non-normalized and normalized cDNA libraries from the diploid progenitors of peanut (A. duranensis and A. ipaensis) for transcriptome sequencing and resequencing; developed non-normalized and normalized cDNA libraries from multiple tissues of a reference genotype (Tifrunner) of cultivated peanut (A. hypogaea); completed the development of two A- and two B-genome diploid F2
mapping populations; completed the development and screening of 200 SSR markers from methylation-filtered and unfiltered genomic DNA sequences and 400 SSCP markers for NBS-LRR class resistance gene candidates; initiated the development and screening of 2,000 EST-SSR markers; completed a survey of EST-SSR polymorphisms among early and modern cultivars and progenitors of modern cultivars; and screened 900 SSR markers for polymorphisms among the parents of several diploid and tetraploid mapping populations. These genomic resources have been described at four scientific meetings and are currently being shared and used in several collaborative research activities in the US and abroad.
PARTICIPANTS: The PI (Steven J. Knapp) developed research plans, wrote several additional grant proposals, secured additional extramural funding to supplement funding from the USDA, developed thesis research plans, and supervised and mentored postdoctoral scientists, research technicians, and graduate and undergraduate students. Several undergraduate students (Sarah Hafner, Rebecca Okashah, Jodie James) have assisted with greenhouse, field, and laboratory components of this research. This grant has supported the graduate research programs for two M.S. students (Sameer Khanal and Yan Li) and the research program for a postdoctoral scientist. Part of the research was completed by another postdoctoral scientist (Osman Radwan). Shunxue Tang (a senior research scientist) and Chris Taylor (a bioinformatician) played a crucial role in the development of the EST database and diploid mapping populations. Ed Johnson (a bioinformatician) assisted with the development of the peanut EST database.
Four research technicians (Adam Heesacker, Jenny Wood, and Jason Prothro, and Nelly Khalilian) have assisted with greenhouse, field, and laboratory research activities. We are collaborating with scientists from several laboratories in the US and abroad. We initiated a collaboration in 2007 with David Hoisington, Rupakula Aruna, and Rajeev Varshney, the team at ICRISAT (Hyderabad, Andhra Pradesh, India) leading the Groundnut Genomics and Breeding Program, and with David Bertioli (EMBRAPA, Brazil). The ICRISAT team is leading an international program in peanut genomics funded by the Bill and Melinda Gates Foundation through the Generation Challenge Program. Greg May, Andrew Farmer, and others at the National Center for Genomic Resources (NCGR) are providing DNA sequencing and bioinformatics support for this research. We are collaborating with Doug Cook, Varma Penmetsa, and other scientists at UCD developing genomic resources for groundnut as part of an NSF Plant Genome Funded research
program awarded to UCD. We have collaborative research activities underway in peanut with Corley Holbrook, Peggy Ozias-Akins, Baozhu Guo, and Albert Culbreath at UGA and USDA-ARS, Tifton, Georgia and H. Thomas Stalker, Susanna Milla-Lewis, and Thomas Isleib, and Niels Nielsen at NCSU.
Impacts
The genomic resources developed through this research are currently being used in several collaborative research activities, in addition to enabling our own research, and have greatly expanded the foundation for applying genomics approaches in peanut breeding and genetics research. The information we have generated has been applied to the discovery of candidate genes for nematode resistance and are currently being applied in genetic analyses focusing on resistance to tomato spotted wilt virus, early and late leaf spot, and rust, seed morphology and quality traits, and other agronomic traits. Subsequent to securing funding from NRI, we reached agreements with two groups developing ESTs to collaborate on the development of the aforementioned EST database. This supplied us with the transcript assembly we needed for SNP discovery by massively parallel transcriptome resequencing. NRI funding enabled us to leverage funding from several public sources (the Georgia Seed
Development Commission, the Peanut Foundation, the National Peanut Board, the Georgia Peanut Commission, and the Generation Challenge Program). This funding has enabled us to broaden the scale and scope of our research and significantly increase the effort directed towards DNA marker development and genetic mapping in cultivated peanut. Our NRI research primarily focuses on the development of diploid models for tetraploid peanut. This is still the primary focus and critical to our collaborations with UC-Davis and ICRISAT; however, we now have sufficient resources to develop a critical mass of high-throughput DNA markers for genetic mapping in intraspecific tetraploid populations, are currently developing 2,000 EST-SSR markers for peanut, and are in the process of identifying SNPs among elite lines and developing an Illumina GoldenGate array for SNP validation and mapping. While we are placing a significant emphasis on SNP discovery, validation, and mapping (using highly parallel
arrays) and envision SNPs playing an important role in peanut, SSR markers will have a more immediate impact on the peanut research community than SNPs because of their transportability, hypervariability, versatility, and broad applicability in tetraploids. Even though SNPs can be genotyped much more rapidly and inexpensively using highly parallel array approaches (which is something we are doing), the development of individual SNP assays from array-mapped SNPs will require a significant and costly long-term effort by the peanut research community. When coupled with previously developed SSR markers, we should have 2,800 SSR markers for peanut by early spring (2008), which is six-fold more than the number available in the public domain at the start of this research. The newly developed SSR markers will be supplied to ICRISAT for genetic mapping in tetraploid populations and will be used for genetic mapping in diploid and tetraploid populations developed in our laboratory and others.
Medium-density genetic mapping in tetraploid peanut should be completed in 2008 and is the most important unanticipated outgrowth of the NRI funded work.
Publications
- Khanal, S. Tang, S., Beilinson, V, SanMiguel, P., Guo, B., Nielsen, N., Stalker, H.T., Cordonnier-Pratt, M.M., Pratt, L.H., Johnson, E.V., Taylor, C.A., Knapp, S.J. (2008) ESTs are a rich source of polymorphic SSRs for genomics and molecular breeding applications in peanut. Plant and Animal Genome Meeting, San Diego.
- Ma, W., Yan, L., Guo, B., Culbreath, A.K., Milla-Lewis, S.R., Shyamalrau, T.P., Holbrook, C.C., Isleib, T., Stalker, H.K., Tang S., Knapp, S.J. (2007) Simple sequence repeat polymorphisms in peanut. Plant and Animal Genome Meeting, San Diego, CA.
- Knapp, S.J. (2007) Peanut genomics progress report. American Peanut Council Meeting, Washington, D.C.
- Knapp, S.J. (2007) Translational and comparative genomics in sunflower and peanut. Department of Horticulture and Crop Science Seminar Series, Ohio State University, Wooster.
- Knapp, S.J. (2007) Peanut genomics: past, present, and future. International Peanut Genomics Initiative Meeting, Atlanta, Georgia.
- Knapp, S.J. (2008) Peanut genomics progress report. American Peanut Council Meeting, Washington, D.C.
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