1. Differentiation of the Historic Alfalfa Germplasm Sources Using Microsatillites C. He, Z.L. Xia, T.A. Campbell, and G. Bauchan, Soybean Genomics and Improvement Lab USDA-ARS, Plant Sciences Institute Beltsville, MD 20705 hec@ba.ars.usda.gov

2. Objectives n To develop SSR from alfalfa genomic library n To determine the genetic relationships among ten historically recognized alfalfa germplasm sources

3. Materials and Methods n Genomic library  DNA from pest resistant germplasm “W10”  Size-selected fragments: 400-900 bp  Vector: pUC19 n Transformation and colony lift n Colony hybridization and signal development n DNA sequencing n Primer design and germplasm screening

4. (1) Primary screening (2) Secondary screening (3) Tertiary screening single colony/well 1-3 colonies/well Screening process based on DNA hybridization

5. PCR amplification of the DNA inserts (Colonies showing positive signals) 480 bp

6. (CT) 15 (AT) 20 DNA sequencing

7. Dinucleotide (CT) Positive signals: 94 Unuseful DNA sequences (no SSR, terminal SSR, unreadable): 41 Useful DNA sequences: 53 Redundant DNA sequences: 11 Unique DNA sequence:42 PCR primers designed:42 Workable primer pairs:39 (93%) Polymorphic SSR loci: 29 (69%) Trinucleotide (CAT, GAT) Positive signals: 24 Unuseful DNA sequences (no SSR, terminal SSR, unreadable): 11 Useful DNA sequences: 13 Redundant DNA sequences: 1 Unique DNA sequence:12 PCR primers designed:12 Workable primer pairs:12 (100%) Polymorphic SSR loci: 7 (58%) The profile of SSR development from an alfalfa genomic library

8. Number Germplasm Source Cultivar Name Accession 1African African NSL 4142 2African Moapa NSL 4123 3Chilean Arizona Common NSL 4144 4Falcata WISFAL PI 560333 5 Falcata Wild tetraploid ssp.falcataPI 214218 6Flemish DuPuit PI 206103 7Indian Sirsa Type 9 PI 235736 8Ladak Ladak NSL 4164 9Peruvian Hairy Pervian NSL 4143 10Turkistan Kaysari PI 171721 11 Arabian Very Nondormant UC-1887 12 Arabian Very NondormantUC-1465 13Varia Grimm NSL 4162 14M. sativa ssp. caerulea Wild diploid alfalfaPI577548 15M. sativa ssp. falcata Wild diploid ssp. falcataPI258754 16M. truncatula JemalongPI442895 Germplasm of M. sativa and M. truncatula used for screening SSR primers

9. MF V T I L F A C C P R MF = Falcata L = Ladak V = Varia F = Flemish T = Turkistan C = Chilean P = Peruvian I = Indian A = African R = Arabian Distribution of historical alfalfa germplasm

10. Banding patterns generated by three SSR primer pairs 2 alleles Multiple alleles

11. No. allelesNo. SSR loci% 1 1529.4 2 9 17.6 3 5 9.8 4 1019.6 5 3 5.9 6 611.8 7 2 3.9 10 1 2.0 Allelic variation for the 51 SSR loci with CT, CAT and GAT repeats

12. Polymorphic primers Including M. truncatula: 70.6% Within M. sativa:58.8%

13. Truncatula (PI 442895) 2X Falcata (PI 258754) 2x Alfalfa (PI 577548) Falcata (PI 560333) Falcata (PI 214218) African (NSL 4123) Arabian (UC-1465) Arabian (UC-1887) Turkistan (PI 171721) Varia (NSL 4162) African (NSL 4142) Peruvian (NSL 4143) Chilean (NSL 4144) Ladak (NSL 4164) Flemish (PI 206103) Indian (PI 235736) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Genetic distance 4x M. sativa 2x M. sativa 2x M. truncatula

14. Summary n In developing SSRs, 118 colonies with positive signals were identified and sequenced for the repeats of CT, CAT and GAT, 54 primer pairs were obtained; n About 70% of the 51 SSRs were polymorphic among the germplasm and produced 154 different alleles;

15. Summary (continue) n The number of alleles generated from the 36 polymorphic SSR primer pairs ranged from 1-10; n The dendrogram largely represents the true relationship among the 24 germplasm.

16. Acknowledgements Soybean Genomics & Improvement Lab., Beltsville, MD Dr. Perry Cregan Dr. Qijian Song Edward Fickus Kristina Pilitt