DEVELOPMENT OF MOLECULAR TOOLS FOR BREEDING IN LEGUME SPECIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0208928
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Plant Pathology

Non Technical Summary
Legumes provide an estimated 33% of protein in the human diet and compensate for the low lysine content of grain proteins. The majority of legume crops suffer significant yield loss from plant disease caused by viruses, fungi, bacteria and nematodes. This project will identify the molecular basis of disease resistance to important pathogens of grain legume species, including cowpea, peanut, chickpea and pigeonpea, and deploy this knowledge for purposes of crop improvement through plant breeding.

Our approach is to leverage knowledge gained from the study of well-characterized legume species, such as soybean and Medicago. Both of these species have fully sequenced genomes, and we have developed comparative data sets that permit us to transfer molecular genetic information from these well-characterized species to less characterized crop species. Using this approach, we can accelerate the characterization of disease resistance traits in less characterized species, and ultimately arrive at a detailed description of the genes that underlie disease resistance against specific pathogens.

Key outputs will be a better understanding of the genes that confer resistance to important plant pathogens of legumes. Such knowledge can lead to more precise and rapid breeding strategies to provide disease resistant varieties of key crop legumes.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1419	1040	30%
202	1419	1080	30%
206	1419	1060	20%
212	1419	1060	20%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 206 - Basic Plant Biology; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1419 - Leguminous vegetables, general/other;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1080 - Genetics;

Keywords

legumes

crop improvement

geomics

plant disease resistance

Goals / Objectives
To various extents, the majority of legume crops suffer from poorly developed infrastructure for genetic and genomic analysis. Lack of such infrastructure limits application of enabling biotechnologies for crop improvement. We will develop improved tools and access to information aimed at improving legume crops and will focus specifically on the large family of NBS-LRR disease resistance proteins.

We aim to a)increase the availability of high quality molecular genetic markers that can be used for purposes of marker assisted selection(MAS) in breeding programs, b)clone and characterize genes that underlie disease resistance traits in legumes, and c)improve the capacity for biotechnology by developing enabling physical and information resources related to disease resistance genes.
Objectives
1. Characterize genome regions that contain functional disease resistance genes.
2. Complete the map-based cloning and functional characterization of disease resistance genes for cowpea mosaic virus in cowpea and Fusarium oxysporum in pigeonpea.
3. Develop functional molecular markers for legume disease resistance traits.
Objective 1. We will combine a comparative genomics approach with traditional map-based cloning. Key elements of this approach are: (1) develop tightly linked genetic markers based on comparisons between the Medicago and soybean genomes. (2) Identify/construct BAC contigs containing the corresponding genetic interval. (3) Sequence the full extent of resistant and susceptible hapolotypes, followed by candidate gene selection.
Objective 2. Functional proof that a candidate gene is responsible for the disease resistance phenotype will require a combination of additional genetic analyses and complementation studies based transgenic approaches.
Genetic analyses will involve association genetics, using allele re-sequencing to determine patterns of linkage disequilibrium and haplotype-phenotype associations. We will pursue associations between specific candidate genes/gene regions and traits, rather than whole genome association studies. In parallel to association genetic studies, we will clone candidate genes into T-DNA expression vectors, under control of both native and strong exogenous promoters.
Objective 3. Characterization of susceptible and resistant haplotypes in pigeonpea and cowpea will provide the basis for discovery of tightly linked polymorphisms. We will convert genetic polymorphisms into genetic markers that are useful in breeding applications. Marker types/technology will vary according to the capacity of breeding partners, but are likely to include PCR-based markers as well as automated single nucleotide polymorphism protocols. In parallel, we intend to explore resistance-associated polymorphisms in core germplasm collections of each species, to predict the distribution of resistance haplotypes in breeding and pre-breeding materials.
Expected outputs
1. Cloned disease resistance determinants from cowpea and pigeonpea.
2. Better characterized germplasm resources in important food legume species.
3. Molecular markers to breed for disease resistance in cowpea and pigeonpea.

Project Methods
Genetic Analysis
Our approach to genetic analysis involves the standard practices of developing populations that segregate for disease resistance phenotypes, identification of genetic polymorphisms that are suitable as genetic markers, and genetic linkage analysis to place target phenotypes within the context of genetic linkage maps.

Our preferred polymorphisms are single nucleotide polymorphisms (SNP) within genes. SNP are mapped either by means of moderate throughput technologies such as allele re-sequencing or SNaPshot assays, or by high throughput methods such as Illumina GoldenGate genotyping.

Genome Sequencing
Once fine scale genetic maps are developed, BAC clones are isolated using a combination of linked molecular markers from the target species, and by compiling sets of genes that are inferred to reside in the corresponding genetic interval based on comparisons to Medicago and soybean. Once identified, the candidate BAC clones are subject to High Information Content Fingerprinting to develop local physical maps, which are then integrated with the available genetic data to delimit the candidate gene region.

The candidate gene region is sequenced by shotgun sequencing of BAC clones, and the data is assembled using a combination of PHRED/PHRAP. Sequence is annotated by reference to other legume genomes, and using off-the-shelf tools such as FGenesH combined with BLASTn/BLASTx vs NCBI databases.

Association Mapping
For purposes of association mapping we use a combination of phenotyping and genotyping in selected individuals from large germplasm collections available for each species. Consideration is given to the potentially confounding effects of genetic structure. Preliminary studies indicate that linkage disequilibrium often decays of kb-sized intervals, increasing the power of such analyses.

Molecular Complementation
Complementation is conducted using transient assays that involve either leaf infiltration with A. tumefaciens or hairy root transformation with A. rhizogenes. In parallel to gain of function complementation studies, we are also poised to test gene function using RNAi knockdown experiments, using similar transformation methods.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Scientific colleagues reached through peer-reviewed publications; Advice to federal research programs through formal advisory relationships; Advice to non-governmental funding organizations through formal advisory relationships; International governmental and academic research programs through formal advisory relationships.Scientific colleagues reached through peer-reviewed publications; Advice to federal research programs through formal advisory relationships; Advice to non-governmental funding organizations through formal advisory relationships; International governmental and academic research programs through formal advisory relationships. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1.Training of UCDavis graduate students. 2.Training of postdoctoral research associates at UCDavis. 3.Training of graduate students at other US universities,in particular Florida International University and the University of Southern California.FIU is an Historically Hispanic Serving University 4.Training of graduate students atforeign institutions ,particularly inTurkey and Ethiopia. 5.Professional development for UC Davis technical and professional staff. How have the results been disseminated to communities of interest?1.Peer-reviewed publication of research results. 2.Invited research seminars. 3. New paper articles. 4. Web communications What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Completed a transcriptional profiling analysis of gene expression during establishment of nitrogen fixation symbiosis, published in the journal Plant Physiology. Made significant progress developing nested association mapping panels and advanced backcross introgression lines of chickpea for phenotyping and breeding. Characterize domestication-associated shifts in the genomes of symbiotic rhizobia and its chickpea host. In the bacterium, we have identified a wide range of microbial diversity that correlates with global cultivation patterns. This diversity is being assembled into a core set of bacterial strains to initiate field testing for improved nitrogen fixation. Analyzed population structure and local ecological and geological features of wild chickpea populations. Identified candidate genes for flowering time, seed coat condensed tannins and flower color, and green cotyledons. Each of these genes have uses in breeding. The condensed tannin/flower color gene was recently accepted for publication in New Phytologist. 7. Used comparative genomics between pigeonpea and soybean to identify a major gene for resistance against the Asian Soybean Rust pathogen. The corresponding manuscript was just accepted for publication in Nature Biotechnology. 8. Analyzed tolerance to salinity in natural population of Medicago truncatula from Portugal and Tunisia. Such salinity tolerance improves our understanding of natural adaptations from salinity tolerance and may underlie gene identification for use in agricultural situations. This work has been submitted for publication. 9. Collected and analyzed the wild progenitor species of cultivated chickpea, with the goal of re-domesticating cultivated chickpea for climate resilience. The collected material was deposited to the multi-lateral gene bank system.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Larrainzar E, Riely BK, Kim SC, Carrasquilla-Garcia, N, Yu HJ, Hwang HJ, Oh M, Kim GB, Surendrarao AK, Chasman D, Siahpirani AF, Penmetsa RV, Lee GS, Kim N, Roy S, Mun JH, Cook DR (2015) Deep sequencing of the Medicago truncatula transcriptome reveals a massive early response to Nod factor and ethylene signals. Plant Physiology 169:233-265.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Kawashima, CG, Guimaraes, GA, Nogueira, Sr, Rausher, G, MacLean, D, Steuernagel, B, Baek, J, Cook, DR, Bouyioukos, C, Wulff, BBH, Ward, E, Rairdan, GJ, Broglie, KE, van Esse, P, Jones, JDG and Brommonschenkel, SH (2016) A pigeonpea gene for resistance to Asian soybean rust functions in soybean. Nature Biotechnology, accepted.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: R. Varma Penmetsa, Noelia Carrasquilla-Garcia, Emily M Bergmann, Lisa Vance, Brenna Castro, Mulualem T. Kassa, Birinchi K. Sarma, Subhojit Datta, Anuja Dubey, Neha Gujaria, Jong-Min Baek1, Jimmy E Woodward, Andrew D Farmer, Clarice J Coyne, Eric J.B. von Wettberg, Rajeev K Varshney, Douglas R Cook (201x). Multiple post-domestication origins of kabuli chickpea through allelic variation in a diversification-associated transcription factor. New Phytologist, accepted.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Scientific colleagues reached through peer-reviewed publications; Advice to federal research programs through formal advisory relationships; Advice to non-governmental funding organizations through formal advisory relationships; International governmental and academic research programs through formal advisory relationships. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1. Training of UC Davis graduate students. 2. Training of postdoctoral research associates at UC Davis. 3. Training of graduate students at other US universities, in particular Florida International University and the University of Southern California. FIU is an Historically Hispanic Serving University. 4. Training of graduate students at foreign institutions, particularly in Turkey and Ethiopia. 5. Professional development for UC Davis technical and professional staff. How have the results been disseminated to communities of interest? 1. Peer-reviewed publication of research results. 2. Invited research seminars. 3. New paper articles. 4. Web communications. What do you plan to do during the next reporting period to accomplish the goals? 1. Continue research and chickpea, especially climate resilience and nitrogen fixation. 2. Disseminate research results through peer-reviewed publication, the media, and invited seminars. 3. Providing advice through formal advisory committee to national and international programs. 4. Train students and professional staff.

Impacts
What was accomplished under these goals? Our main accomplishments during the 2014 reporting year were: 1. Completed a transcriptional profiling analysis of gene expression during establishment of nitrogen fixation symbiosis, wrote manuscript and submitted for publication (in review). 2. Initiated development of nested association mapping panels and advanced backcross introgression lines of chickpea. 3. Continued to characterize domestication-associated shifts in the genomes of symbiotic rhizobia and its chickpea host, with the long term goal of understanding and improving symbiotic efficiency in crop legumes. 4. Analyzed population structure and local ecological and geological features of wild chickpea populations. 5. Continued analysis of flowering time, nitrogen and nitrogen fixation-related traits in chickpea.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Warschefsky E, Penmetsa RV, Cook DR, von Wettberg EJ (2014). Back to the wilds: tapping evolutionary adaptations for resilient crops through systematic hybridization with crop wild relatives. Am J. of Botany. 101:1791-1800.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Friesen ML, von Wettberg EJB, Badri M, Moriuchi KS, Barhoumi F, Cuellar-Ortiz C, Chang PL, Cordeiro MA, Vu WT, Arraouadi S, Djebali N, Zribi K, Badri Y, Porter SS, Aouani MA, Cook DR, Strauss SY, Nuzhdin SV. (2014). The ecological and genomics basis of salinity adaptation in Tunisian Medicago truncatula. BMC Genomics, 15:1160.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Leal-Bertioli, SCM, Santos, SP, Dantas, KM, Inglis, P, Nielen, S, Ara�jo, ACG, Joseane, JP, Cavalcante, U, Guimar�es, PM, Brasileiro, AM, Carrasquilla-Garcia, N, Penmetsa, RV, Cook, D, Morezsohn, MC, Bertioli, DJ (2014) Arachis batizocoi: a study of its relationship to cultivated peanut (A. hypogaea L.) and its potential for introgression of wild genes into the peanut crop using induced allotetraploids. Annals of Botany. doi10.1093/aob/mcu237.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Gujaria-Verma, N, Vail, SL, Carrasquilla-Garcia, N, Penmetsa, RV, Cook, DR, Farmer, AD, Vandenberg, A, Bett, KE (2014) Genetic mapping of legume orthologs reveals high conservation of synteny among wild and cultivated lentil species and the sequenced genomes of Medicago and chickpea. Frontiers in Plant Science. 5:676. doi:10.3389/fpls.2014.00676.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Cordeiro, MA, Moriuchi, KS, Fotinos, T, Miller, KE, Badri, M, Friesen, ML, Nuzhdin, SV, Strauss, SY, von Wettberg, E, Cook, DR (2014) Population differentiation for germination and early seedling root growth traits under saline conditions in the annual legume Medicago truncatula (Fabaceae). American Journal of Botany 101(3):488-498.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Laporte, P., Lepage, A., Fournier, J., Catrice, O., Moreau, S., Jardinaud, M-F., Mun, J-H., Larrainzar, E., Cook, D.R., Gamas, P. and Niebel A. (2014) The CCAAT box-binding transcription factor MtNF-YA1 controls rhizobial infection. Journal of Experimental Botany 65:481-94.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Scientific colleagues reached through peer-reviewed publications; Advice to federal research programs through formal advisory relationships; Advice to non-governmental funding organizations through formal advisory relationships; International governmental and academic research programs through formal advisory relationships. Changes/Problems: No major change, but a more defined focus on the issue of chickpea domestication and incorporating traits for climate resilience into cultivated germplasm. What opportunities for training and professional development has the project provided? 1. Training of UC Davis graduate students. 2. Training of graduate students at other US universities, in particular Florida International University, which is an Historically Hispanic Serving University, through close research collaboration. 3. Training of graduate students at foreign institutions, particularly in Turkey and Ethiopia. 4. Professional development for UC Davis technical and professional staff. How have the results been disseminated to communities of interest? 1. Peer-reviewed publication of research results. 2. Invited research seminars. What do you plan to do during the next reporting period to accomplish the goals? 1. Continue research and chickpea, especially climate resilience and nitrogen fixation. 2. Disseminate research results through peer-reviewed publication, the media, and invited seminars. 3. Providing advice through formal advisory committee to national and international programs. 4. Train students and professional staff.

Impacts
What was accomplished under these goals? Our main accomplishments during the 2013 reporting year were: 1. Analyzed tolerance to salinity in natural population of Medicago truncatula from Portugal and Tunisia. Such salinity tolerance improves our understanding of natural adaptations from salinity tolerance and may underlie gene identification for use in agricultural situations. This work has been submitted for publication. 2. Collected and initiated archiving and analysis of the wild progenitor species of cultivated chickpea, with the goal of re-domesticating cultivated chickpea for climate resilience. 3. Identified domestication-associated shifts in the genomes of symbiotic rhizobia and its chickpea host, with the long term goal of understanding and improving symbiotic efficiency in crop legumes.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Choi, HK, Iandolino, A, Goes da Silva, F, Cook, DR (2013) Water deficit modulates the response of Vitis vinifera to the Pierce's disease pathogen Xylella fastidiosa. MPMI 26:643-657.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Garz�n, LN, Oliveros, OA, Rosen, B, Ligarreto, GA, Cook, DR, and Blair, MW (2013) Isolation and Characterization of Nucleotide-Binding Site Resistance Gene Homologues in Common Bean (Phaseolus vulgaris). Phytopathology 103:156-168.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Varshney, R.K. et al., & Cook, D.R. (2013) Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nature Biotechnology 31:240-246.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Major outputs during 2012 include:

1. Development of genetic maps and molecular markers in peanut, cowpea, common bean, lentil and chickpea.

2. Sequencing of the chickpea genome.

3. Molecular and phylogenetic analysis of legume NB-LRR disease resistance genes.

4. Molecular validation of the cowpea mosaic virus disease resistance gene.

5. Delimited genome regions containing disease resistance genes in chickpea against Fusarium oxysporum fsp ciceri.

PARTICIPANTS: This project has contributed to the training of graduate students and professional researchers in the United States, and also to the training of visiting students and sabbatical faculty from a range of international educational and research institutions. TARGET AUDIENCES: Target audiences include (1) commodity group organizations focused on legumes, (2) USDA-ARS scientists working on pulse legumes, and (3) international centers with mandates that include the legume species under investigation on this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Molecular markers developed for legume species are now being used by legume breeders in India, Africa and Canada.

2. Sequencing of the chickpea genome identified regions of the genome under selection during domestication and breeding, including candidate genome intervals for disease resistance. Moreover, re-sequencing of 90 cultivated accessions provides a wealth of genetic polymorphisms for chickpea improvement.

3. Based on the chickpea genome sequence and related high-density genetic maps we have delimited the flowering time control genes. These genes have important for the development of early maturing chickpea varieties, which have been a primary mechanism used by breeders to avoid terminal drought stress. The advent of molecular markers for these genes will permit more precise molecular breeding approaches in chickpea.

4. Genome regions containing Fusarium wilt resistance genes in chickpea have been identified, and this advance open the possibility of directly validating genes for resistance against the major agronomic races of this pathogen.

Publications

• Hiremath PJ, Kumar A, Penmetsa RV, Farmer A, Schlueter JA, Chamarthi SK, Whaley AM, Carrasquilla-Garcia N, Gaur PM, Upadhyaya HD, Kavi Kishor PB, Shah TM, Cook DR, Varshney RK (2012) Large-scale development of cost-effective SNP marker assays for diversity assessment and genetic mapping in chickpea and comparative mapping in legumes. Plant Biotechnol J. 10:716-732.
• Bohra A, Saxena RK, Gnanesh BN, Saxena K, Byregowda M, Rathore A, Kavikishor PB, Cook DR, Varshney RK (2012) An intra-specific consensus genetic map of pigeonpea [Cajanus cajan (L.) Millspaugh] derived from six mapping populations. Theor Appl Genet DOI 10.1007/s00122-012-1916-5.
• Kassa, M.T., Penmetsa, R.V., Carrasquilla-Garcia, N., Sarma, B.K., Datta, S., Upadhyaya, H.D., Varshney, R.K., von Wettberg, E.J.B., Cook, D.R. (2012) Genetic patterns of domestication in pigeonpea (Cajanus cajan (L.) Millsp.) and wild Cajanus relatives. PLoS ONE 7(6):e39563.
• Rachit K Saxena, R Varma Penmetsa, Hari D Upadhyaya, Ashish Kumar, Noelia Carrasquilla-Garcia, Jessica A. Schlueter, Andrew Farmer, Adam M. Whaley, Birinchi K. Sarma2, Gregory D May, Douglas R Cook and Rajeev K Varshney (2012) Large-scale development of cost-effective SNP marker assays for genetic mapping in pigeonpea and comparative mapping in legumes. DNA Research, PMID 23103470 Oct 26. [Epub ahead of print]
• Varshney, R.K. et al., & Cook, D.R. (2013) Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nature Biotechnology doi:10.1038/nbt.2491.
• Blair, MW, Cortes, AJ, Penmetsa, RV, Farmer, A, Carrasquilla-Garcia, N, Cook, DR (2012) A high-throughput SNP marker system for parental polymorphism screening, and diversity analysis in common bean (Phaseolus vulgaris L.) Theoretical and Applied Genetics DOI 10.1007/s00122-012-1999-z.
• Garzon, LN, Oliveros, OA, Rosen, B, Ligarreto, GA, Cook, DR, and Blair, MW (2013) Isolation and Characterization of Nucleotide-Binding Site Resistance Gene Homologues in Common Bean (Phaseolus vulgaris)doi.org/10.1094/PHYTO-07-12-0180-R.
• Nagy, ED, Guo, Y, Tang, S, Bowers, JE, Okashah, RA, Taylor, CA, Zhang, D, Khanal, S, Heesaker, AF, Khalilian, N, Farmer, AD, Carrasquilla-Garcia, N, Penmetsa, RV, Cook, D, Stalker, HT, Ozias-Akins, P, and Knapp, SJ (2012) A high-density map of Arachis duranensis, a diploid ancestor of cultivated peanut. BMC Genomics 13:469-479.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: All results are disseminated through peer review publications. Sequence data is deposited to the National Center for Biotechnology Information databases. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During the past year we have extended the description of genome evolution in the Papilionoid legumes. In particular, two new data types have been added, including genetic data for Lentil (a cool season legume species) and the explicit inclusion of ~500 duplicated genes within the comparative framework. This later data type has helped define a series of ancestral duplications that in total define a whole genome duplication event at that base of Papilionoid evolution that has been predicted, but not previously described. Within this polyploid, ancestral species, we identify clusters of disease resistance genes whose genome locations have been conserved in modern day legume species. These results demonstrate that, despite that fast and chaotic process of disease resistance gene (NB-LRR genes) evolution, their genome locations have been largely conserved over the past ~60 million years. Working with soybean, we have deduced a framework genome structure of its diploid progenitor species, and we predict the subgenome structure corresponding to the distinct diploid parents of allopolyploid soybean. From the analysis we make the surprising conclusion that the majority of NB-LRR genes in modern day soybean derive from only one of the two subgenomes, and from this we speculate that genome attrition has occurred predominantly in only one of the two subgenomes. We have also contributed to the genome sequencing of pigeonpea, Cajanus cajan. We have annotated the genome of C. cajan for NB-LRR proteins and compared these annotations to our previous NB-LRR cloning efforts in this species. We have developed a single nucleotide genotyping assay that will increase the frequency with which NB-LRR genome regions are accurately assigned to the pigeonpea genetic map.

Publications

• Varshney, R, et al. (2011) Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers. Nature Biotechnology 30(1):83-9.
• Wang et al., (2011) Development and characterization of BAC-end sequence derived SSRs, and their incorporation into a new higher density genetic map for cultivated peanut (Arachis hypogaea L.). BMC Plant Biology, in press.
• Pandey MK, Monyo E, Ozias-Akins P, Liang X, Guimaraes P, Nigam SN, Upadhyaya HD, Janila P, Zhang X, Guo B, Cook DR, Bertioli DJ, Michelmore R, Varshney RK. (2011) Advances in Arachis genomics for peanut improvement. Biotechnol Adv. PMID: 22094114
• Dutta, S, Kumawat, G, Singh, BP, Gupta DK, Singh, S, Dogra, V, Gaikwad, K, Sharma, TR, Raje, RS, Bandhopadhya, T, Datta, S, Singh, MN, Bashasab, F, Kulwal, P, Wanjari, KB, Varshney, RK, Cook, DR, Singh, NK (2011) Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus Cajan (L.) Millspaugh]. BMC Plant Biology 11:17.
• Thudi M, Bohra A, Nayak SN, Varghese N, Shah TM, Penmetsa RV, Thirunavukkarasu N, Gudipati S, Gaur PM, Kulwal PL, Upadhyaya HD, Kavikishor PB, Winter P, Kahl G, Town CD, Kilian A, Cook DR, Varshney RK. (2011) Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.). PLoS One 6:e27275
• Jixian Zhai, Dong-Hoon Jeong, Emanuele De Paoli, Sunhee Park, Benjamin D. Rosen, Yupeng Li, Alvaro J. Gonzalez, Zhe Yan, Sherry L. Kitto, Michael A. Grusak, Scott A. Jackson, Gary Stacey, Douglas R. Cook, Pamela J. Green, D. Janine Sherrier, and Blake C. Meyers (2011) microRNAs as Master Regulators of the Plant NB-LRR Defense Gene Family via the Production of Phased, Trans-acting siRNAs. Genes and Development 25:2540-53.
• Young, N.D et al. (2011) The Genome Sequence of Medicago truncatula and the Evolution of Nodulation. Nature 480:520-4.
• Choi HK and Cook DR (2011) Bridging Comparative Genomics and DNA Marker-aided Molecular Breeding. Kor. J. Breed. Sci. 43: 103-120.
• Bohra A, Dubey A, Sazena RK, Penmetsa RV, Poornima KN, Kumar N, Farmer AD, Srivani G, Upadhyaya HD, Gothalwal R, Ramesh R, Singh D, Saxena KB, Kavikishor PB, Cook DR and Varshney RV (2011) Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea (Cajanus spp.). BMC Plant Biology 11:56.
• Gujaria N, Kumar A, Dauthal P, Dubey A, Hiremath P, Prakash AB, Farmer, A, Bhide M, Shah T, Gaur PM, Upadhyaya HD, Bhatia S, Cook DR, May GD, Varshney RK (2011) Development and use of genic molecular markers (GMMs) for construction of a transcript map of chickpea (Cicer arietinum L.) Theor Appl Genet 122:1577-1589.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: During the past year we have finalized a description of genome evolution within the Papilionoid legumes, allowing ancestral genome segments to be tracked across essentially all modern legume crop species. The comparative framework includes 1,100 low copy unique genes.

In parallel, we have annotated the genomes of Medicago and soybean for the large and diverse family of NB-LRR disease resistance genes. When viewed within the context of the comparative framework, we determined that disease resistance genes within these crop species derive from a set of conserved ancestral locations, even if the genes themselves lack simple 1:1 orthology.

We have previously cloned ~3,500 NB-LRR genes from six additional legume species. During the past year, we have identified thousands of single nucleotide polymorphisms associated with this important class of candidate genes and we are preparing to conduct genetic analysis to place these genes also in the comparative framework.

In parallel studies, we have begun efforts to clone and functionally characterize NB-LRR genes that confer resistance to cowpea mosaic virus in cowpea and Fusarium wilt in pigeonpea. We have delimited the genomic/genetic interval that contains these genes and sequenced BAC clones that span these regions. In each case we have identified a cluster of NB-LRR genes that are now being tested for function. Knowledge of the genome regions that contain these disease resistance genes can used for trait breeding and introgression, as well as more basic studies of gene function. PARTICIPANTS: This project has contributed to the training of graduate students and professional researchers in the United States, and also to the training of visiting students and sabbatical faculty from a range of international educational and research institutions TARGET AUDIENCES: Target audiences include (1) commodity group organizations focused on legumes, (2) USDA-ARS scientists working on pulse legumes, and (3) international centers with mandates that include the legume species under investigation on this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Detailed characterization of disease resistance gene regions in cowpea and pigeonpea.

2. Molecular markers for >1,000 of NB-LRR disease resistance genes in pigeonpea, chickpea and cowpea.

Publications

• Dutta S, Kumawat G, Singh BP, Gupta DK, Singh S, Dogra V, Gaikwad K, Sharma TR, Raje RS, Bandhopadhya TK, Datta S, Singh MN, Bashasab F, Kulwal P, Wanjari K, K Varshney R, Cook DR, Singh NK. (2011) Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh]. BMC Plant Biol. 11:17.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: We have completed the identification of single nucleotide polymorphisms (SNP) in 6 different legume crops: lentil, chickpea, common bean, cowpea, pigeonpea and peanut. In total we have identified ~25K polymorphisms in ~1400 genes across all species.

The data was used to construct Illumina GoldenGate SNP mapping assays and develop gene-based genetic maps for chickpea, common bean, cowpea, pigeonpea and peanut, with genetic mapping on going in lentil. Because a common set of genes was mapped in each species, we were able to assemble a comparative genetic frame work that links each species to all other species, and also to the fully sequenced genomes of Medicago truncatula and soybean. In total, the comparative framework includes ~1000 genes.

The primary outcome of this work are:
(1) We can deduce the genomic structure of the last common ancestor of this set of species, and define the history of major genome rearrangements that define all modern crop legumes. The common ancestor existed ~55 million years ago.

(2) This comparative genetic framework permits us to infer gene content in crop legumes based on reference to the sequenced legume genomes. We have used this information to rapidly identify a small genome interval that contains candidate genes for resistance to the agronomically important viral pathogen of cowpea, cowpea mosaic virus. Efforts are underway to functionally validate the disease resistance gene and to transfer the corresponding molecular information into breeding programs for marker assisted selection.

We have completed the cloning of NBS-LRR disease resistance genes in chickpea, common bean, cowpea, pigeonpea, peanut and Cercis, and we have annotated the genomes of Medicago and soybean for the corresponding gene homologs. In total we have identified ~4200 NBS LRR proteins.

These NBS-LRR genes were used to probe Bacterial Artificial Chromosome (BAC) libraries for each species, and thus identify BAC clones that carry these disease resistance gene homologs. The corresponding BAC clones were BAC end sequenced to generate linked sequence information and fingerprinted by High Information Content Fingerprinting to produce physical contigs at each resistance gene locus in each genome.

PCR primers have been designed against 1-2K BAC ends in each species, and are being used to develop SNP data sets that target the disease resistance gene repertoire of each species. Already this approach has helped us clone the cowpea mosaic virus disease resistance genes, and we anticipate that further development of this resource will enable similar approaches for a large number of agronomically important disease resistance genes. PARTICIPANTS: This project is part of the INDO-US Agricultural Knowledge Initiative. Partner organizations include USDA FAS in the United States, and ICRISAT and ICAR institutes in India.

This project has contributed to the training of graduate students and professional researchers in the United States, and also to the training of visiting Indian students and sabbatical faculty from a range of Indian educational and research institutions. TARGET AUDIENCES: Target audiences include (1) commodity group organizations focused on legumes, (2) USDA-ARS scientists working on pulse legumes, and (3) international centers with mandates that include the legume species under investigation on this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Genetic maps produced for 5 legume crop species, with a 6th almost complete.

2. Detailed analysis of genome synteny, connecting 6 crop species and 3 model species to their common ancestor, ~55 million years ago.

3. Synteny framework permits cloning of agronomically important genes, at a faster rate and with less expense than previously possible. We have demonstrated this using the cowpea mosaic virus disease resistance locus as an example, and we are in the process of transferring molecular marker information to breeders.

Publications

• Nyak, SN, Zhy, H, Varghese, N., Datta, S., Choi, HK, Horres, R, Jungling, R, Singh, J, Kavi Kishor, PB, Sivaramakrishnan, S, Hoisington, DA, Kahl, G, Winter, P, Cook, D and Varshney, RK (2010) Integration of novel SSR and gene-based SNP marker loci in the chickpea genetic map and establishment of new anchor points with Medicago truncatula genome. Theoretical and Applied Genetics DOI 10.1007/s00122-010-1265-1
• Douglas R Cook and Rajeev K Varshney (2010) From genome studies to agricultural biotechnology: closing the gap between basic plant science and applied agriculture. Current Opinion in Plant Biology, in press.
• R. K. Varshney, R. V. Penmetsa, S. Dutta, P. L. Kulwal, R. K. Saxena, S. Datta, T. R. Sharma, B. Rosen, N. Carrasquilla-Garcia, A. D. Farmer, A. Dubey, K. B. Saxena, J. Gao, B. Fakrudin, M. N. Singh, B. P. Singh, K. B. Wanjari, M. Yuan, R. K. Srivastava, A. Kilian, H. D. Upadhyaya, N. Mallikarjuna, C. D. Town, G. E. Bruening, G. He, G. D. May, R. McCombie, S. A. Jackson, N. K. Singh, D. R. Cook (2009) Pigeonpea genomics initiative (PGI): an international effort to improve crop productivity of pigeonpea (Cajanus cajan L.) Molecular Breeding DOI 10.1007/s11032-009-9327-2.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: We have completed allele re-sequencing of 1440 orthologous genes in seven legume species: lentil, chickpea, cowpea, common bean, pigeonpea, Arachis duarnensis, and peanut. We have established agreements to sequence this gene set in mungbean and red clover. In all cases, we have sequenced alleles from at least 2 genotypes that represent the parents of reference mapping populations.

A bioinformatics pipeline was developed to assemble individual sequence reads within a genotype and then construct cross-genotype alignments for automated identification of single nucleotide and multiple neighbor nucleotide polymorphisms. Polymorphisms have been validated by means of manual curation step, yielding >forty thousand polymorphisms.

Polymorphisms are being converted into ADT files for production of an Illumina GoldenGate mapping platform containing 768 loci in each of the target genomes.

We have completed the first phase of characterizing the NBS-LRR disease resistance gene homologs of lentil, chickpea, common bean, cowpea, pigeonpea and peanut, as well as from the basal Cesalpinoid legume Cercis occidentalis. In total we have identified >3,000 disease resistance gene homologs.

We constructed bacterial artificial chromosome (BAC) libraries for peanut, chickpea, cowpea and pigeonpea, and we have obtained BAC libraries from collaborators working on common bean and lupin. High density membrane filters were prepared for all species (except lupin) and interrogated by hybridization with a representative set of NBS domains. In this manner, we have identified what we believe to be a comprehensive set of BAC clones that encompass the vast majority of all disease resistance genes (NBS-LRR genes) in each species. By way of example, the peanut (a tetraploid species) resistance gene BACs include ~4200 clones, while the cowpea resistance gene BAC set includes ~2500 clones.

We have initiated BAC clone fingerprinting and physical map assembly in each species. This work is complete in peanut, and scheduled for completion in the other species during spring of '09.

We have sequenced BAC ends for 25K BAC clones, representing ~35 million base pairs of data, in chickpea, cowpea and Arachis duranensis. For pigeopea, we completed BAC end sequencing for 50K clones, representing ~60 million base pairs of data; this work was a shared project with Rajeev Varshney's group at ICRISAT in India.

BAC end sequences have been generated for the resistance gene-containing BAC clones, yielding 1 to 2 million base pairs of sequence tags linked to disease resistance genes in each species.

BAC end sequences have been annotated by means of a bioinformatics pipeline, including the identification of simple sequence repeat motifs. ~7000 primer pairs are being tested for amplification and polymorphism in cowpea, chickpea, pigeonpea and peanut. PARTICIPANTS: This project is part of the INDO-US Agricultural Knowledge Initiative. Partner organizations include USDA FAS in the United States, and ICRISAT and ICAR institutes in India.

This project has contributed to the training of graduate students and professional researchers in the United States, and also to the training of visiting Indian students and sabbatical faculty from a range of Indian educational and research institutions. TARGET AUDIENCES: Target audiences include (1) commodity group organizations focused on legumes, (2) USDA-ARS scientists working on pulse legumes, and (3) international centers with mandates that include the legume species under investigation on this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. SSR markers identified in random BAC end sequences or from BAC clones that contain disease resistance genes have been shared with the research communities working on the respective species. These molecular markers have substantially increased the volume of molecular makers available for breeding.

2. All BAC end sequence data has been deposited to the National Center for Biotechnology Information Genome Survey Sequence database. Data on single nucleotide polymorphisms has been incorporated into a relational database, developed specifically for this project. Once data are curated, the database will be made available for public access.

3. We have developed a database for legume disease resistance genes. This database contains a curated set of hundreds of NBS domains in each of ten different legume species. Once curation of this data set is complete, we will enable public access to the data, which will facilitate both basic and applied studies of this important gene set.

4. We are building linkages with plant breeders in each of our target crop legume species, with the goal of applying our molecular marker resource to breeding for disease resistance. One aspect of this focus is a recent agreement with the Peanut Foundation to characterize resistance gene diversity in cultivated germplasm collections.

Publications

• Varshney, R.K., Close, T.J., Singh, N.K., Hoisington, D.A., and Cook, DR (2009) Orphan legume crops enter the genomics era! Current Opinion in Plant Biology 11:1-9.

Progress 01/01/07 to 12/31/07

Outputs
During the project period, the Cook laboratory has been involved in the development of enabling data sets for breeding in a range of legume species. We have focused on several species of important, to varying degrees, in California, the US and in India and Africa. Species of primary interest include chickpea, cowpea, common bean, pigeonpea, lupin, peanut and redbud (an evolutionarily basal species). Activities included generation of a suite of gene-based single nucleotide polymorphism genetic markers, cloning and analysis of NBS-LRR disease resistance genes, and development of a set of simple sequence repeat genetic markers linked to physical maps.

In cowpea and chickpea, we sequenced a combined total of ~75 million base pairs of genomic DNA from the ends of bacterial artificial chromosome (BAC) clones. As a result, we have discovered ~4,500 genes in each species and we have designed and tested ~1,100 simple sequence repeat (SSR) genetic markers in each species. These SSR markers have been used for analysis of genetic polymorphisms in bi-parental populations of interest to plant breeders, and also to assess and understand genetic diversity in the World's germplasm collections for these species. In peanut, lupin and pigeonpea, we have either completed BAC libraries (peanut), obtained BAC libraries from collaborators (lupin), or are currently constructing BAC libraries.

We have developed a PCR-based approach to discover single nucleotide polymorphisms (SNPs) within the intron regions of genes. We have focused our efforts on a set of ~1400 genes, representing orthologous genes in each of our target species. We have either completed, or are currently completing, analysis of polymorphism in the parents of selected mapping populations for each target species, representing tens of thousands of sequenced amplicons and thousands of SNPs in each species. In the case of cowpea and peanut a subset of these markers have been used to explore genetic diversity and population structure in germplasm collections. Our current near term goal is to complete SNP discovery in common bean, pigeonpea, cowpea and chickpea, so that we can develop and Illumina OPA assay for genetic mapping and diversity analysis in each species.

As our data sets are curated, they are deposited to the National Center for Biotechnology Information (NCBI), for open access by other research. All BAC end data is at NCBI and we anticipate depositing genetic marker data towards the end of this calendar year.

I have hosted three conferences this year, including legume genomics meetings in San Diego and Washington State, and a grape genomics meeting in San Diego. My laboratory collaborates closely with a national and international network of scientists, including scientists at USDA ARS, at other UC campuses and US universities, at international centers of the CGIAR system, and at national programs of the Indian Council for Agricultural Research and at the National Institute for Plant Genome Research.

Impacts
The activities described above represent represent a new direction in the Cook laboratory. As such, we are mainly involved in building enabling data sets and the results have yet to find application in breeding programs. However, the initial results from the project have informed us about the structure and evolution of legume genomes, and they have helped organize germplasm collections into pseudo-populations, and thus provide a rationale basis for further germplasm exploration.

Perhaps the most important impact of this project during the past year is that it has acted to nucleate discussion and interaction among legume researchers (breeders, germplasm curators and genome specialists) to make decisions regarding resource development, collaborative research projects, and new grant proposals.

Publications

• No publications reported this period