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

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

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

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

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

(CT) 15 (AT) 20 DNA sequencing

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

Number Germplasm Source Cultivar Name Accession 1African African NSL African Moapa NSL Chilean Arizona Common NSL Falcata WISFAL PI Falcata Wild tetraploid ssp.falcataPI Flemish DuPuit PI Indian Sirsa Type 9 PI Ladak Ladak NSL Peruvian Hairy Pervian NSL Turkistan Kaysari PI Arabian Very Nondormant UC Arabian Very NondormantUC Varia Grimm NSL M. sativa ssp. caerulea Wild diploid alfalfaPI M. sativa ssp. falcata Wild diploid ssp. falcataPI M. truncatula JemalongPI Germplasm of M. sativa and M. truncatula used for screening SSR primers

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

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

No. allelesNo. SSR loci% Allelic variation for the 51 SSR loci with CT, CAT and GAT repeats

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

Truncatula (PI ) 2X Falcata (PI ) 2x Alfalfa (PI ) Falcata (PI ) Falcata (PI ) African (NSL 4123) Arabian (UC-1465) Arabian (UC-1887) Turkistan (PI ) Varia (NSL 4162) African (NSL 4142) Peruvian (NSL 4143) Chilean (NSL 4144) Ladak (NSL 4164) Flemish (PI ) Indian (PI ) Genetic distance 4x M. sativa 2x M. sativa 2x M. truncatula

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;

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.

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