Progress 09/01/00 to 08/31/04
Outputs
The goal of this project is to develop microsatellite markers for cultivated peanut because of a few DNA markers available in peanut. We have completed all specific objectives for this project. Accomplishments and findings 1. We have established a cost-effective system for developing microsatellites in peanut. A collection of 5400 clones was sequenced. About 60% of the clones were found to containing microsatellite, most of which were (GA) motif (65%) and (GGC) motif (30%). A total of 373 peanut microsatellite sequences were submitted to GenBank. 2. From our newly developed microsatellite markers, 8 were identified as botanical variety-specific markers, which produced a specific band only presenting in certain botanical varieties. Such specific markers might be involved in shaping the evolution of cultivated peanut and are very useful in taxonomic identification. 3. We have also searched microsatellites from 1350 peanut expressed sequence tags (ESTs) in GenBank. Three
hundred fifty-three ESTs were found containing microsatellites. 4. Besides identifying and developing microsatellite markers from the peanut genome, searching transferable SSR markers from other legume is a useful way to increase available markers for peanut genomic studies. We have examined a total of 386 soybean SSR markers including 170 (AT)n, 181 (ATT)n, and 35 EST-SSR markers. Out of 386 markers, 145 (38%) were amplifiable in peanut. Among amplifiable markers, 33% detected polymorphism among 4 peanut cultivars. Because of a large number of SSR markers available in soybean, the high level of transferability of soybean SSR markers to peanut will easily increase to a large pool of available markers for peanut. A set of common markers between peanut and soybean will be useful for comparative mapping, which will allow us not only to better understand the evolution and speciation of legume crops, but also to predict the location of genes of interest from soybean which has a well
developed genetic map compared to peanut for which such information is scarce. Dissemination activities undertaken and resulting outcomes Three hundred seventy-three microsatellites were deposited at GenBank (accession number AY237736-237798, AY-310535-310564, AY526357-526456, AY731521-731698). Partnerships and collaborative ventures A research collaborative project Comparative mapping and genomic analysis of resistance genes between peanut and Medicago truncatula between Tuskegee University and University of California at Davis is initiated supported by an NSF supplement grant. Future initiatives A research collaborative project Mapping and genome organization of microsatellite sequences in cultivated peanut among Tuskegee University, UC Davis, USDA/ARS at Tifton, GA, and NCGR is being planned to pursue a funding from NSF.
Impacts
The developed microsatellite markers will provide useful tools to geneticists and breeders for peanut genetic research. The project has also enhanced the research capacity in peanut genomics at Tuskegee University. Several graduate and undergraduate students, and a high school student were trained in peanut genomic research under this project. Graduate students Ronghua Meng and Frieda Woullard, undergraduate students Ismaila Marong, and an Auburn high school student Jasmin Felton have worked on developing SSR markers including cloning fragments containing SSRs, sequencing clones, testing primers designed, identifying polymorphism among peanut genotypes. Also, a peanut research collaboration was established between Tuskegee University and USDA/ARS at Griffin and Tifton, GA. Drs. Roy Pittman and Melanie Newman at Griffin and Dr. Baozhu Guo at Tifton were involved in sequencing clones for this project.
Publications
- Meng, R.H. and G.H. He. 2003. Microsatellites in cultivated peanut. Tuskegee Horizons 2003: 21.
- He, G.H., R.H. Meng, Hui Gao, B.Z. Guo, G.Q. Gao, M. Newman, R.N. Pittman, and C. S. Prakash. 2004. Botanical variety-specific markers in cultivated peanut (Arachis hypogaea L.). Euphytica (submitted).
- He, G.H., F. E. Woullard, I. Marong, and B.Z.Guo. 2004. Transferability of soybean SSR markers to cultivated peanut. Peanut Sciences (submitted).
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Progress 10/01/02 to 09/30/03
Outputs
The objective of this study is to isolate microsatellite sequences from peanut genome. We have established a good system for developing microsatellites in peanut. Briefly, AFLP fragments were hybridized with SSR probes, the fragments containing microsatellite were then separated from those without microsatellite using magnetic bead method, and cloned into victors and transferred into bacteria. A collection of 1200 clones was sequenced. About 69% of the clones (800) were found containing microsatellite, most of which were (GA) motif (65%) and (GGC) motif (30%). Of 800 microsatellite sequences, 195 were unique and the rest were redundant. The 195 peanut microsatellite sequences were submitted to GenBank (accession number AY237736-237798, AY310535-310564, AY 526357-526457). We have designed primer pairs for these sequences and have tested 85 primers for dinucleotide repeats. Among 85 primer pairs, 29 could detect polymorphism among 24 genotypes of peanut. Up to 15
alleles were identified at one locus using primer pair PM50. Also, we have searched microsatellites from 1350 peanut ESTs in GenBank. Three hundred fifty-three ESTs were found containing microsatellites. Primers were designed for 44 ESTs and 9 of them detected polymorphism. We believe that microsatellites in peanut might be abundant like other legume species, such as soybean. From these newly developed microsatellite markers, 8 were identified as botanical variety-specific markers, which produced a specific band only presenting in certain botanical varieties. Such specific markers might be involved in shaping evolution of cultivated peanut and are very useful in taxonomic identification. All these primary results suggested that this system is a promising one for isolation and characterization of microsatellites in peanut.
Impacts
The results will lead to more productive genetic studies, which will benefit U.S. peanut improvement and production.
Publications
- He, G. H., Meng, R. H., Newman, M., Gao, G. Q., Pittman, R. N., and Prakash, C. S. 2003. Microsatellites as DNA markers in cultivated peanut (Arachis hypogaea L.). BMC Plant Biology, 3:3.
- Luo, M., Dang, P., He, G. H., Guo, B. Z., Holbrook, C. C., Lee, R. D., Bausher, M., and Lynch, R. E. 2004. Generation of expressed sequence tags (EST) for gene discovery and marker development in cultivated peanut (Arachis hypogaea L.). Crop Science (accepted).
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Progress 10/01/01 to 09/30/02
Outputs
The goal of this project is to develop microsatellite markers in cultivated peanut. First, we have tried to use the procedure reported by Akkaya et al. (2000) to identify microsatellites, because of no library involved. However, we have found that it was very difficult to find positive bands on the gel after the amplification of AFLP fragments that were picked up by SSR probes. Then, we gave up this procedure and have constructed a SSR-enriched library following the techniques described in our proposal with some modification. Our recent results showed that it is a good system to develop microsatellite in peanut. Two hundred and eight (60%) cloned fragments were found containing microsatellites from 349 clones randomly picked up from the library. The result was consistent with those from other reports using SSR-enriched library, such as 50%-70% of the clones contained at least one microsatellite in several plants (Edwards et al., 1996), 60% positive clones showed in
onion and shallot (Fischer and Bachmann, 1998). However, the rate of redundancy was very high in peanut, 61% of 208 fragments containing SSR were redundant. Only 81 fragments were unique, 62 of which could be designed for primer pairs, the rest fragments were too short to have primers. One third of these novel primers have detected DNA polymorphism based on 28 genotypes. Using one of these primer pairs, 10 alleles were found within these genotypes. It is the first time such high number of alleles identified in cultivated peanut. The 62 novel primers will be soon submitted to GeneBank for patent. The project is ongoing to sequencing as many clones as we can to ensure developing more than 100 microsatellites in peanut.
Impacts
The developed microsatellite markers would be very useful for peanut genetic research because of lack of genetic markers in this important crop.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
The primary goal of this project is to develop microsatellite markers in cultivated peanut. Microsatellite have become ideal genetic markers in plant genomic research due to its highly abundant and evenly distributed, highly polymorphic and codominant. However, detailed information on the abundant of microsatellite in peanut still remains blank. The procedure we used to develop microsatellite was reported by Akkaya et al. (2000), because of no library involved. Briefly, cultivated peanut genomic DNAs were used to generate AFLP segments by standard methods. AFLP fragments containing microsatellite were hybridized with biotinylated single sequence repeat (SSR) probes. The hybridized fragments were immobilized onto magnetic beads coated with streptavidin. After several washing steps with low and high stringency solutions, they were eluted by water, re-amplified by PCR, and separated on polyacrymide gel. The positive bands were cut from gel, purified and then sequenced.
For the sequences containing microsatellite and the reliable flanking region, PCR primers to the flanking region were designed using Primer3 software. The primer pairs were tested for their ability to detect polymorphic loci among a set of 48 cultivated peanut accessions with diversified origins. Nine biotinyated di-, tri- and tetranucleotide SSR oilgos reported to be rich in other plant species were included to screen AFLP fragments. In this way, we have found nine di- or tri-nucleotide microsatellites and one minisatellite containing 4 repeats of 9 base motif. From the SSR-containing sequences, we were able to design 9 primer pairs and 3 of them produced different level of polymorphisms. The results showed that the GA/CT repeat was the most frequently dispersed microsatellite in peanut followed by GGC/CCG motif, while the tetra-nucleotide repeats were not found in this study. The microsatellite DNA markers are more likely to produce DNA polymorphism in cultivated peanut than other
DNA markers. This approach of SSR isolation, however, has a problem of non-specific binding between magnetic beads and AFLP fragments. We are trying to solve this problem, so that more microsatellite markers will be developed rapidly and efficiently.
Impacts
Development of microsatellite markers will be very useful in peanut genomic research. More DNA markers will be added to saturate our peanut genetic map.
Publications
- Gao, G., G. He & C.S. Prakash, 2002. Development of microsatellite markers in cultivated peanut. Abstract for Plant, Animal & Microbe Genomes X.
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