Progress 02/26/03 to 02/25/08
Outputs
Progress Report Objectives (from AD-416) Develop simple sequence repeat (SSR) DNA markers, appropriate for mapping in autotetraploid alfalfa. Develop four SSR-based maps of autotetraploid alfalfa and map markers to appropriate linkage groups, integrating them with previously mapped Amplified Fragment Length Polymorphism (AFLP) and Restriction Fragment Length Polymorphism (RFLP) markers. Identify loci that are genetically linked to quantitative and qualitative genes of agronomic importance through quantitative trait loci (QTL) mapping. Collect Medicago germplasm and evaluate diversity and phylogenic relationships using SSR markers and to develop SSR marker subsets for differentiating among selected alfalfa cultivars and populations. Approach (from AD-416) SSR markers from alfalfa genomic clones, and from ESTs of the closely related diploid M. truncatula, will be mapped in four populations using single dose analyses of F1 data : (1) 'ABI 408' purple flowered winter hardy parent X 'WisFal' yellow flowered parent; (2) 'Peruvian' non-winter hardy parent X a second 'Wisfal' parent; (3) embryogenesis-incompetent ineffective nitrogen fixing X embryogenesis-competent effective nitrogen fixing parents; and (4) Aphanomyces susceptible X Aphanomyces resistant parents. All parents exhibit the standard characteristics of cultivated alfalfa. These populations are the principal autotetraploid alfalfa mapping populations available at this time, and new markers can be used immediately for improving cultivated alfalfa. Single nucleotide polymorphisms may also be identified from EST sequencing data and will be integrated into the maps. New and existing markers will be used to identify quantitative trait loci (QTL) controlling important traits, in diversity and phylogeny studies in Medicago sp. germplasm, and for differentiating among selected alfalfa cultivars and populations. Significant Activities that Support Special Target Populations This project was redirected as of October 1, 2007. Work associated with the transition to the new set of objectives and milestones can be found in the report for project 1275-22000-254-00D. Accomplishments over the life of the project: Over 2000 potential DNA molecular markers [Simple Sequence Repeats (SSR) ] were identified from the model alfalfa-related species Medicago truncatula, and alfalfa (M. sativa), of which 300 were determined to be useful in fingerprinting alfalfa. With the identification of these DNA molecular markers of alfalfa, breeders will be able to utilize them to identify alfalfa cultivars and improve alfalfa by quickly identifying plants that have rapid regrowth after cutting for improving yield. A set of 23 SSR DNA molecular markers were developed, as well as Sequence Related Amplified Polymorphisms (SRAPs), which were effective in separating the major historic alfalfa germplasm sources varying in levels of fall dormancy. These accomplishments fall within Component 2 (Plant Resources) of ARS National Program 205 (Rangeland, Pasture, and Forages), specifically, Problem Areas 2.1 (Lack of Available Germplasm) and 2.2 (Plant Biology and Gene Discovery).
Impacts
(N/A)
Publications
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Develop simple sequence repeat (SSR) DNA markers, appropriate for mapping in autotetraploid alfalfa. Develop four SSR-based maps of autotetraploid alfalfa and map markers to appropriate linkage groups, integrating them with previously mapped Amplified Fragment Length Polymorphism (AFLP) and Restriction Fragment Length Polymorphism (RFLP) markers. Identify loci that are genetically linked to quantitative and qualitative genes of agronomic importance through quantitative trait loci (QTL) mapping. Collect Medicago germplasm and evaluate diversity and phylogenic relationships using SSR markers and to develop SSR marker subsets for differentiating among selected alfalfa cultivars and populations. Approach (from AD-416) SSR markers from alfalfa genomic clones, and from ESTs of the closely related diploid M. truncatula, will be mapped in four populations using single dose analyses of F1 data : (1) 'ABI 408' purple flowered winter hardy parent X 'WisFal' yellow flowered parent; (2) 'Peruvian' non-winter hardy parent X a second 'Wisfal' parent; (3) embryogenesis-incompetent ineffective nitrogen fixing X embryogenesis-competent effective nitrogen fixing parents; and (4) Aphanomyces susceptible X Aphanomyces resistant parents. All parents exhibit the standard characteristics of cultivated alfalfa. These populations are the principal autotetraploid alfalfa mapping populations available at this time, and new markers can be used immediately for improving cultivated alfalfa. Single nucleotide polymorphisms may also be identified from EST sequencing data and will be integrated into the maps. New and existing markers will be used to identify quantitative trait loci (QTL) controlling important traits, in diversity and phylogeny studies in Medicago sp. germplasm, and for differentiating among selected alfalfa cultivars and populations. Significant Activities that Support Special Target Populations An F1 population of alfalfa segregating for resistance to the potato leafhopper was developed. This population was created by crossing a purple flowered Medicago sativa ssp. sativa alfalfa plant with potato leafhopper resistance to a yellow flowered M. sativa ssp. falcata plant that is susceptible to the potato leafhopper. This population was produced to be used in the development of a molecular map of alfalfa to determine the genetic mechanisms of insect resistance. Accomplishments Simple Sequence Repeats (SSR) DNA Markers to �Fingerprint� Alfalfa: Because alfalfa (Medicago sativa) is a cross-fertilizing species with a complex polyploid genome, it is difficult to distinguish alfalfa varieties from one another. To solve this problem, a small set of 21 SSR DNA molecular markers were identified for fingerprinting the major historic alfalfa germplasm sources which vary in their levels of fall dormancy. These SSR markers will be very useful in diversity and phylogenetic studies in Medicago sp. germplasm, and for differentiating among selected alfalfa cultivars and populations. This accomplishment falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on Problem Statements 2.1, Lack of Available Germplasm and 2.2, Plant Biology and Gene Discovery.
Impacts
(N/A)
Publications
- Vandemark, G. J., J. J. Ariss, G. A. Bauchan, R. C. Larsen, and T. J. Hughes. 2006. Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with Sequence Related Amplified Polymorphisms. Euphytica 152:9-16.
- Jauhar, P.P. 2005. Cytogenetic architecture of cereal crops and their manipulation to fit human needs:opportunities and challenges. (book chapter in: germplasm resources, chromosome engineering, and crop improvement. CRC Press, Boca Raton, FL, pp. 1-25.
- Robins, J.G., G.R. Bauchan, and E.C. Brummer. 2007. Genetic mapping forage yield, plant height, and regrowth at multiple harvests in tetraploid alfalfa (Medicago sativa L.). Crop Sci. 47:11-18.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Alfalfa has lagged behind other crops in its improvement for yield and forage quality. One efficient method of improving crop plants is the development of molecular maps for the identification of genes important to agriculture. There are molecular maps of alfalfa available but not for cultivated tetraploid alfalfa. A more accurate and extensive map of alfalfa is needed such that markers closely linked to important agronomic traits can be identified. Simple sequence repeats (SSRs) have emerged as the marker system of choice. These markers can not only be used in the development of a molecular map of alfalfa but also be used as a powerful new tool for investigating germplasm diversity and for fingerprinting cultivars and populations. The project has four major goals: (1) develop SSR
DNA markers, appropriate for mapping in autotetraploid alfalfa, through screening alfalfa genomic clones and SSR primer pairs developed from M. truncatula expressed sequence tags (ESTs); (2) develop one or more SSR-based maps of autotetraploid alfalfa and map markers to appropriate linkage groups, integrating them with previously mapped Restriction Fragment Length Polymorphism (RFLP) markers; (3) identify loci that are genetically linked to quantitative and qualitative genes of agronomic importance through quantitative trait loci (QTL) mapping; and (4) collect Medicago germplasm and evaluate diversity and phylogenetic relationships using SSR markers and develop SSR marker subsets for differentiating among selected alfalfa cultivars and populations. This project falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on 2.1 (Lack of Available Germplasm), 2.2 (Plant Biology and Gene Discovery), and 2.3 (Overcoming Limitations to Plant Growth and
Development). In developing this project, we took particular note of the conclusions in the NP205 Action Plan that new molecular biology technologies are needed for rapid identification of useful genes that can be manipulated to create new genetic combinations, and that a coordinated effort is needed to identify, characterize, and map genes that control agronomically important quantitative traits. Attaining these objectives will provide new basic knowledge on the alfalfa genome and on diversity and/or phylogenic relationships within Medicago sp. germplasm, cultivars and populations. A map with molecular markers linked to loci for important agronomic traits will assist in the production of alfalfa cultivars that will yield more forage of higher quality, persist longer, resist diseases, and survive in hostile environments, thus increasing profitability for the farmer. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2004) 1. Develop 50 SSR
markers. Year 2 (FY 2005) 1. Develop an additional 100 SSR markers. 2. Map with 150 markers completed. 3. Initial SSR subsets suitable for genetic diversity and phylogenic studies in selected Medicago sp. germplasm identified. Year 3 (FY 2006) 1. Develop an additional 100 SSR markers. 2. Maps with 250 markers completed. 3. Initial maps of selected qualitative traits completed. 4. Initial SSR subsets suitable for fingerprinting selected alfalfa cultivars identified. Year 4 (FY 2007) 1. Develop an additional 100 SSR markers. 2. Maps with 350 markers completed. 3. Final maps of qualitative traits and initial maps of selected quantitative traits completed. 4. Refine and expanded SSR subsets for genetic diversity, phylogeny, and fingerprinting studies available. Year 5 (FY 2008) 1. Develop an additional 100 additional SSR markers. 2. Consensus SSR map of alfalfa with 450 markers completed. 3. Final maps of quantitative traits completed with markers suitable for efficacious molecular marker
assisted selection. 4. Final SSR subsets for genetic diversity, phylogeny, and fingerprinting studies available. 4a List the single most significant research accomplishment during FY 2006. Identified a Set of DNA Molecular Markers for Fingerprinting the Fall Dormancy Classes of Alfalfa: This accomplishment falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on 2.1 (Lack of Available Germplasm) and 2.2 (Plant Biology and Gene Discovery. Our lab identified over 2000 potential DNA molecular markers [Simple Sequence Repeats (SSR]) from the model Medicago species, M. truncatula, of which 300 were determined to be useful in fingerprinting alfalfa. With the identification of these DNA molecular markers of alfalfa, breeders will be able to utilize these molecular markers to identify alfalfa cultivars and improve alfalfa by quickly identifying plants which have rapid regrowth of alfalfa after cutting for improving yield of alfalfa. 4b List other significant
research accomplishment(s), if any. Molecular Markers Identify Alfalfa Germplasm Sources: Developed a set of DNA molecular markers [Sequence Related Amplified Polymorphisms (SNAP)] which are effective in separating the major historic alfalfa germplasm sources varying in levels of fall dormancy. This accomplishment falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on 2.1 (Lack of Available Germplasm) and 2.2 (Plant Biology and Gene Discovery. Because alfalfa is a polyploid crop which is outcrossed it is difficult to identify alfalfa varieties from one another. DNA sequence-based genetic markers referred to as SNAP markers were developed to solve this problem. Thus, these SNAP markers will be very useful in diversity and phylogenetic studies in Medicago sp. germplasm, and for differentiating among selected alfalfa cultivars and populations. 5. Describe the major accomplishments to date and their predicted or actual impact. This project has been
able to develop a molecular map of alfalfa with a significant number of alfalfa genomic SSRs and SSRs derived from M. truncatula which were suitable for mapping in alfalfa, the discernment of the historically recognized alfalfa cultivars and fall dormancy check cultivars. This accomplishment falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on 2.1 (Lack of Available Germplasm) and 2.2 (Plant Biology and Gene Discovery. The customers of this research are alfalfa breeders and geneticists in the public and private sectors. As the molecular map of tetraploid alfalfa begins to be used by alfalfa breeders and geneticists the impact will be the more rapid development of improved cultivars for U.S. producers. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and
durability of the technology products? Data on the development of molecular markers to identify fall dormancy check cultivars were presented at the North American Alfalfa Improvement Conference. This technology will become available to scientists when data are published in the journal Crop Science in FY 2007. The major constraint to using these technologies could be the unpredictability of the use of molecular markers in cross pollinated autotetraploids such as alfalfa.
Impacts
(N/A)
Publications
- Bauchan, G.R., Xia, Z., Van Berkum, P.B. 2006. Molecular dna markers utilized to discern alfalfa fall dormancy check cultivars. North American Alfalfa Improvement Conference. July 16-19, 2006, Minneapolis, MN. p.6.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Alfalfa has lagged behind other crops in it's improvement for yield and forage quality. One efficient method of improving crop plants is the development of molecular maps for the identification of genes important to agriculture. There are molecular maps of alfalfa available but not for cultivated tetraploid alfalfa. A more accurate and extensive map of alfalfa is needed such that markers closely linked to important agronomic traits can be identified. Simple sequence repeats (SSRs) have emerged as the marker system of choice. These markers can not only be used in the development of a molecular map of alfalfa but also be used as a powerful new tool for investigating germplasm diversity and for fingerprinting cultivars and populations. The project has four major goals: (1) develop SSR DNA markers,
appropriate for mapping in autotetraploid alfalfa, through screening alfalfa genomic clones and SSR primer pairs developed from M. truncatula expressed sequence tags (ESTs); (2) develop one or more SSR-based maps of autotetraploid alfalfa and map markers to appropriate linkage groups, integrating them with previously mapped Restriction Fragment Length Polymorphism (RFLP) markers; (3) identify loci that are genetically linked to quantitative and qualitative genes of agronomic importance through quantitative trait loci (QTL) mapping; and (4) collect Medicago germplasm and evaluate diversity and phylogenetic relationships using SSR markers and develop SSR marker subsets for differentiating among selected alfalfa cultivars and populations. This project falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on 2.1 (Lack of Available Germplasm), 2.2 (Plant Biology and Gene Discovery), and 2.3 (Overcoming Limitations to Plant Growth and Development). In
developing this project, we took particular note of the conclusions in the NP205 Action Plan that new molecular biology technologies are needed for rapid identification of useful genes that can be manipulated to create new genetic combinations, and that a coordinated effort is needed to identify, characterize, and map genes that control agronomically important quantitative traits. Attaining these objectives will provide new basic knowledge on the alfalfa genome and on diversity and/or phylogenic relationships within Medicago sp. germplasm, cultivars and populations. A map with molecular markers linked to loci for important agronomic traits will assist in the production of alfalfa cultivars that will yield more forage of higher quality, persist longer, resist diseases, and survive in hostile environments, thus increasing profitability for the farmer. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) 1. Develop 50 SSR markers. Year 2 (FY 2005) 1.
Develop an additional 100 SSR markers. 2. Map with 150 markers completed. 3. Initial SSR subsets suitable for genetic diversity and phylogenic studies in selected Medicago sp. germplasm identified. Year 3 (FY 2006) 1. Develop an additional 100 SSR markers. 2. Maps with 250 markers completed. 3. Initial maps of selected qualitative traits completed. 4. Initial SSR subsets suitable for fingerprinting selected alfalfa cultivars identified. Year 4 (FY 2007) 1. Develop an additional 100 SSR markers. 2. Maps with 350 markers completed. 3. Final maps of qualitative traits and initial maps of selected quantitative traits completed. 4. Refine and expanded SSR subsets for genetic diversity, phylogeny, and fingerprinting studies available. Year 5 (FY 2008) 1. Develop an additional 100 additional SSR markers. 2. Consensus SSR map of alfalfa with 450 markers completed. 3. Final maps of quantitative traits completed with markers suitable for efficacious molecular marker assisted selection. 4. Final
SSR subsets for genetic diversity, phylogeny, and fingerprinting studies available. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Develope an additional 100 SSR markers. Milestone Fully Met 2. Map with 150 markers completed. Milestone Fully Met 3. Initial SSR subsets suitable for genetic diversity and phylogenic studies in selected Medicago sp. germplasm identified. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? A molecular map of alfalfa will be available which will be saturated with SSR markers which can be used by breeders to identify genes for improved yield, fall dormancy, forage quality, pest resistance and abiotic stress resistance. SSR molecular markers will be available to assess
genetic diversity, phylogeny and fingerprint alfalfa cultivars for the identification of specific cultivars and utilization in the management of germplasm collections. 4a What was the single most significant accomplishment this past year? Developed a molecular map of tetraploid alfalfa. This map identified several locations for the genes involved in bio-mass production, rapid regrowth after cutting which is related to fall dormancy, and erect-tall growth habit. Until this study was conducted we had no idea where the genes were located which were involved in the inheritance of these traits. Our lab identified over 1500 potential simple sequence repeats (SSR) from the model Medicago species, M. truncatula, of which 210 were determined to be useful for mapping in alfalfa. We discovered that 27 of the markers were closely linked to bio-mass production, fall dormancy and erect upright growth habit which are important to the improvement of alfalfa. This map was constructed with the
collaborative efforts of colleagues at the University of Iowa. With the development of a molecular map of alfalfa, breeders will be able to utilize these molecular markers to improve alfalfa by quickly identifying plants which have these traits to combine them and develop alfalfa with improved yield, winter hardiness and lodging resistance. 4b List other significant accomplishments, if any. Identified 81 simple sequence repeats (SSR) from a genomic library of alfalfa which are effective in separating the major historic alfalfa germplasm sources varying in levels of fall dormancy. Because alfalfa is a polyploid crop which is outcrossed it is difficult to distinguish alfalfa varieties from one another. These SSR markers were identified via screening of the historic alfalfa germplasm sources with a large set of SSR markers available in our laboratory. The 81 SSR markers will be very useful to alfalfa geneticists and breeders in diversity and phylogenetic studies in Medicago sp.
germplasm, and for differentiating among selected alfalfa cultivars and populations. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project is only 2 years into the 5 year planned research and already we have been able to develop a molecular map of alfalfa with a significant number of alfalfa genomic SSRs and SSRs derived from M. truncatula which were suitable for mapping in alfalfa and discernment of the historically recognized alfalfa cultivars. The customers of this research are alfalfa breeders and geneticists in the public and private sectors. As the molecular map of tetraploid alfalfa begins to be used by alfalfa breeders and geneticists the impact will be the more rapid development of improved cultivars for U.S. producers. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other
scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Data on the SSR map and the location of quantitative trait loci for bio- mass production, regrowth after fall cutting and erect-tall growth habit discovery, as well as on using SSR marker polymorphisms to separate genotypes, were presented at the International Symposium on Molecular Breeding for the Genetic Improvement of Forages Crops and Turf. This technology will become available to the scientist when data are published in the journals Crop Science and Genetics in FY 2006. The major constraint to using these technologies could be the unpredictability of marker assisted selection in cross pollinated autotetraploids such as alfalfa.
Impacts
(N/A)
Publications
- He, C.N., Xia, Z., Campbell, T.A., Bauchan, G.R. 2003. Isolation and characterization of SSR markers in alfalfa [abstract]. Plant and Animal Genome Conference Proceedings. P242.
- He, C.N., Xia, Z., Campbell, T.A., Bauchan, G.R. 2003. Development and characterization of simple sequence repeat (SSR) markers in medicago sativa [abstract]. Agronomy Abstracts. P1027.
- Campbell, T.A., Brummer, E.C., Luth, D., Bauchan, G.R., Xia, Z., He, C.N. 2003. Screening autotetraploid alfalfa and medicago truncatula SSR makers for inclusion in a cultivated alfalfa linkage map [abstract]. International Symposium on Molecular Breeding of Forage Crops Proceedings. P927.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? In the past 20 years, molecular marker maps of diploid and/or tetraploid alfalfa have been constructed based on Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), and Amplified Fragment Length Polymorphism (AFLP) DNA markers. Unfortunately, many of these maps were based on F2 mapping populations where inbreeding can severely distort map distances. In addition, a number of the maps were based on diploid alfalfa to avoid the complexity inherent in evaluating segregation in autotetraploid cultivated alfalfa. The utility of diploid maps is still under investigation, but their ability to capture the complexity of tetraploid relationships is questionable. The lack of an accurate genetic map that is populated with highly informative DNA markers is hindering progress
in alfalfa gene discovery and genetic improvement. Thus, a more accurate and extensive map of cultivated alfalfa is needed such that markers closely linked to important agronomic traits can be identified. Simple Sequence Repeats (SSRs) have emerged as the marker system of choice because they are abundant and informative, PCR-based, co-dominant, multi-allelic, highly polymorphic and reproducible. Genetic diversity of alfalfa and related species is seriously eroding, particularly in areas of China and the Middle East. While numerous forage germplasm expeditions have been conducted in these areas, especially the People's Republic of China, there is still a need for continuing germplasm exploration and evaluation. It is important that germplasm be collected and characterized to serve as a source of genes for future genetic improvements. If such germplasm is not collected it is possible that future genetic improvement will be restricted. Molecular techniques are a powerful new tool
for investigating germplasm diversity and phylogeny, and for fingerprinting cultivars and populations. The ability to fingerprint and classify germplasm allows plant geneticists to identify the most promising sources of new and useful genetic variants. Thus, the lack of a well developed and efficient genetic fingerprinting system hinders the effective use of alfalfa germplasm. The project has four major goals: (1) develop SSR DNA markers, appropriate for mapping in autotetraploid alfalfa, through screening alfalfa genomic clones and SSR primer pairs developed from M. truncatula Expressed Sequence Tags (ESTs); (2) develop one or more SSR-based maps of autotetraploid alfalfa and map markers to appropriate linkage groups, integrating them with previously mapped RFLP markers; (3) identify loci that are genetically linked to quantitative and qualitative genes of agronomic importance through Quantitative Trait Locus (QTL) mapping; and (4) collect Medicago germplasm and evaluate diversity
and phylogenetic relationships using SSR markers and develop SSR marker subsets for differentiating among selected alfalfa cultivars and populations. This project falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on 2.1 (Lack of Available Germplasm), 2.2 (Plant Biology and Gene Discovery), and 2.3 (Overcoming Limitations to Plant Growth and Development). In developing this project, we took particular note of the conclusions in the NP205 Action Plan that new molecular biology technologies are needed for rapid identification of useful genes that can be manipulated to create new genetic combinations, and that a coordinated effort is needed to identify, characterize, and map genes that control agronomically important quantitative traits. Attaining these objectives will provide new basic knowledge on the alfalfa genome and on diversity and/or phylogenic relationships within Medicago sp. germplasm, cultivars and populations. A map with molecular markers
linked to loci for important agronomic traits will assist alfalfa geneticists and breeders in the development of alfalfa cultivars that will yield more forage of higher quality, persist longer, resist diseases, and survive in hostile environments, thus increasing profitability for the farmer. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Develop 50 SSR markers. Year 2 (FY 2005) Develop an additional 100 SSR markers. Map with 150 markers completed. Initial SSR subsets suitable for genetic diversity and phylogenic studies in selected Medicago sp. germplasm identified. Year 3 (FY 2006) Develop an additional 100 SSR markers. Maps with 250 markers completed. Initial maps of selected qualitative traits completed. Initial SSR subsets suitable for fingerprinting selected alfalfa cultivars identified. Year 4 (FY 2007) Develop an additional 100 SSR markers. Maps with 350 markers completed. Final maps of qualitative traits and initial maps of selected
quantitative traits completed. Refined and expanded SSR subsets for genetic diversity, phylogeny, and fingerprinting studies available. Year 5 (FY 2008) Develop an additional 100 additional SSR markers. Consensus SSR map of alfalfa with 450 markers completed. Final maps of quantitative traits completed with markers suitable for efficacious molecular marker assisted selection. Final SSR subsets for genetic diversity, phylogeny, and fingerprinting studies available. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. All milestones were fully met. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2005 Develop an additional 100 SSR
markers. Map with 150 markers completed. Markers will be identified and mapped. Initial SSR subsets suitable for genetic diversity and phylogenic studies in selected Medicago sp. germplasm identified. FY 2006 Develop an additional 100 SSR markers. Maps with 250 markers completed. Markers will be identified and mapped. Initial maps of selected qualitative traits completed. Initial SSR subsets suitable for fingerprinting selected alfalfa cultivars identified. FY 2007 Develop an additional 100 SSR markers. Maps with 350 markers completed. Markers will be identified and mapped. Final maps of qualitative traits and initial maps of selected quantitative traits completed. Refine and expanded SSR subsets for genetic diversity, phylogeny, and fingerprinting studies available. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004 (one per Research Project): Medicago truncatula EST and BAC library sequence databases were
mined for SSR-containing sequences and 116 new SSR markers were developed, tested, and genetically mapped in a mapping population developed from a cross of [WISFAL-6 (M. sativa ssp. falcata) x ABI-408 (M. sativa ssp. sativa)]. This accomplishment greatly expands the number of useful SSR markers that are available to alfalfa breeders and geneticists. As result of the genetic mapping which was done in collaboration with Iowa State Univ. eight consensus linkage groups were identified and the map spanned approximately 673 cM with 3.9 cM between marker loci. Mapping of the SSR markers allowed a large phenotypic database to be analyzed and new QTLs for biomass production, forage quality, and winter hardiness to be identified. B. Other significant accomplishments A genomic DNA library of tetraploid alfalfa was developed and 78 clones containing simple sequence repeat (SSR) sequences were identified from the library. These SSRs were effective in identifying a majority of the historic
alfalfa germplasm sources varying in levels of fall dormancy. Genomic SSRs were screened to determine if they could identify 16 different accessions of the historically recognized germplasm sources of modern alfalfa cultivars. Of the 718 SSRs screened, 61 were proven effective in discerning 8 out of the 16 accessions. SSRs will be very useful in diversity and phylogenetic studies in Medicago species germplasm, and for differentiating among selected alfalfa cultivars and populations. C. Significant activities that support special target populations. None. D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project is only 15 months into the 5 year planned research and already we have
been able to develop a significant number of genomic and EST derived SSRs which have been suitable for mapping in alfalfa and discernment of the historically recognized alfalfa cultivars.
Impacts
(N/A)
Publications
- Campbell, T.A., Brummer, E.C., Luth, D., Bauchan, G.R., Xia, Z., He, C.N. 2003. Screening autotetraploid alfalfa and medicago truncatula SSR makers for inclusion in a cultivated alfalfa linkage map [abstract]. International Symposium on Molecular Breeding of Forage Crops Proceedings. P927.
- He, C.N., Xia, Z., Campbell, T.A., Bauchan, G.R. 2003. Development and characterization of simple sequence repeat (SSR) markers in medicago sativa [abstract]. Agronomy Abstracts. P1027.
- He, C.N., Xia, Z., Campbell, T.A., Bauchan, G.R. 2003. Isolation and characterization of SSR markers in alfalfa [abstract]. Plant and Animal Genome Conference Proceedings. P242.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? In the past 20 years, molecular marker maps of diploid and/or tetraploid alfalfa have been constructed using RFLP, RAPD, and AFLP DNA marker systems. Unfortunately, many of these maps were based on F2 (inbred) mapping populations where inbreeding can severely distort map distances. Also, the ability of diploid maps to capture the complexity of tetraploid relationships is questionable. An additional problem we are addressing is the serious erosion of genetic diversity, particularly in areas of China and the Middle East. While numerous forage germplasm expeditions have been conducted in these areas, especially China, there is still a need for continuing germplasm exploration and evaluation. Simple sequence repeats (SSRs) have emerged as the marker system of choice because they are highly abundant and informative. We plan to develop a tetraploid SSR linkage map based on F1 (outcrossed)
rather than inbred progeny, as well as add SSR markers to existing F1 linkage maps. It is anticipated that the resulting maps will more accurately define genetic distances in the alfalfa genome and will also assist in an understanding of the complexities of inheritance in an autotetraploid. These maps will also be used to identify markers that are closely linked to important agronomic traits (quantitative trait loci or QTL), investigate germplasm diversity and phylogeny, and fingerprint cultivars and populations. An important objective of our work is the collection of germplasm of alfalfa and related species from China and the Middle East and the characterization of this germplasm using subset of SSR markers from the tetraploid alfalfa maps. 2. How serious is the problem? Why does it matter? Alfalfa is the most important forage legume, and the third most widely cultivated agronomic crop, in the United States. Between 10 and 11 million hectares of alfalfa are grown throughout the
50 states. Alfalfa can be harvested as hay, used directly as pasture, or ensiled, making it an attractive crop for most livestock and dairy systems. Alfalfa cannot approach its full potential, however, unless several serious problems are solved. Among these are a yield barrier, sensitivity to acid soil, bloat, and loss of protein in the rumen. Considerable conventional research effort has been expended on these problems with little success and new technologies must be developed and employed. One very powerful new technology for solving intractable breeding problems is QTL discovery coupled with marker assisted selection (MAS). We plan to use the new SSR maps to locate QTL that will help break the yield barrier, adapt alfalfa to acid soil, reduce bloat, and enhance protein utilization. Subsequently, new cultivars will be developed using MAS techniques. Other problems associated with alfalfa production are the need to develop robust sets of genetic markers for plant variety
protection and for assessing genetic diversity accurately during selection programs and germplasm evaluations. SSRs are the marker system of choice for fingerprinting cultivars and for estimating genetic distances among potential parents and germplasms, as well as for assessing phylogenetic relationships. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This project falls within Component 2 (Plant Resources) of NP 205. The project focuses primarily on 2.1 (Lack of Available Germplasm), 2.2 (Plant Biology and Gene Discovery), and 2.3 (Overcoming Limitations to Plant Growth and Development). In developing this project, we took particular note of the conclusions in the NP205 Action Plan that "new molecular biology technologies are needed for rapid identification of useful genes that can be manipulated to create new genetic combinations, and that a coordinated effort is needed to identify, characterize, and map genes that
control agronomically important quantitative traits". 4. What were the most significant accomplishments this past year? A. This research was undertaken to develop simple sequence repeat (SSR) DNA markers, and to integrate these markers into an autotetraploid AFLP/RFLP-based alfalfa linkage map that can be efficiently used to identify markers associated with agronomically important genes, and to evaluate diversity and phylogenic relationships in cultivated alfalfa and Medicago sp. germplasm. To date, 501 SSRs, discovered in Medicago truncatula (a closely related species) expressed sequence tags (ESTs) and 118 SSRs identified in an alfalfa genomic library have been screened by the Soybean Genomics and Improvement Laboratory (T. A. Campbell, G.R. Bauchan, Z.L. Xia, and C. He) in collaboration with the Department of Agronomy at Iowa State University (E. C. Brummer, D. Luth, and J. Robins). Marker patterns for 188 EST-derived SSRs and 36 genomic library SSRs distinguished the parents of
our mapping population and are being integrated into the AFLP/RFLP linkage map and screened for their ability to separate test sets of Medicago sp. clones varying in genetic distance as well as10 alfalfa germplasms differing in fall dormancy characteristics. These markers will constitute the SSR framework of an alfalfa genomic map, assist in the discovery of important quantitative trait loci, and will enhance the efficacy with which genetic diversity can be studied and phylogenetic relationships can be established. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Developing and utilizing a SSR-based genomic map of autotetraploid alfalfa is a new project, however, the current project builds upon the former CRIS project, which, in part, was focused upon the development of molecular techniques for studying genetic diversity and phylogenetic relationships. Methods for assessing genetic diversity and/or establishing
phylogenetic relationships, require continual refinement, and in the previous CRIS we exploited variation in ribosomal and organelle DNA as well as in RAPD, anchored microsatellite, and RFLP markers. The SSR technologies employed in the current CRIS should be more efficacious than any of the earlier technologies we utilized. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2004: Develop 100 additional SSR markers and complete an alfalfa genome map consisting of 150 markers. Complete selection of initial SSR subsets suitable for genetic diversity and phylogenetic studies. FY 2005: Develop 100 additional SSR markers and complete an alfalfa genome map consisting of 250 markers. Develop initial maps of selected QTL and identify initial SSR subsets suitable for fingerprinting selected alfalfa cultivars. FY 2006: Develop 100 additional SSR markers and complete the mapping of a total of 350 new SSR markers. Complete the final mapping of selected QTLs. Refine
and expand SSR subsets for genetic diversity, phylogeny, and fingerprinting studies. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A map with 150 SSRs, 200 RFLPs, and 100 AFLPs should be available in 2004. Quantitative trait loci for traits such as biomass production should be available soon thereafter. The usefulness of this technology to the scientist will be partially contingent upon the efficacy of marker assisted selection in an outcrossing species. The usefulness and durability of the QTL discovery, marker assisted selection, and genotype identification technologies still require significant amounts of evaluation over a number of years.
Impacts
(N/A)
Publications
- Campbell, T.A., Young N., Huguet, T., Brummer, E.C., Bauchan, G.R., Xia, Z.L., He, C. Screening Medicago truncatula microsatellite markers for inclusion in a cultivated alfalfa microsatellite map. Proceedings of the North American Alfalfa Improvement Conference. 2002. p. 30.
- Campbell, T.A. Investigation of variations in NBS motifs in alfalfa (Medicago sativa), M. edgeworthii, and M ruthenica. Canadian Journal of Plant Science. 2003. v. 83. p. 371-376.
- Campbell, T.A., Bauchan, G. R. Organelle based molecular analyses of the genetic relatedness of cultivated alfalfa (Medicago sativa L) to Medicago edgeworthii Sirjaev, and Medicago ruthenica (L.) Ledebour. Euphytica. 2002. v. 125. p. 51-58.
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