Differentiation of the Historic Alfalfa Germplasm Sources

Using MicrosatillitesC. 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

Objectives

To develop SSR from alfalfa genomic

library

To determine the genetic relationships

among

ten historically recognized alfalfa

germplasm

sources

Materials and Methods

Genomic library

DNA from pest resistant germplasm

“W10”

Size-selected fragments: 400-900 bp

Vector: pUC19

Transformation and colony lift

Colony hybridization and signal

development

DNA sequencing

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): 41Useful DNA sequences: 53

Redundant DNA sequences: 11Unique DNA sequence: 42

PCR primers designed: 42Workable primer pairs: 39 (93%)

Polymorphic SSR loci: 29 (69%)

Trinucleotide (CAT, GAT)

Positive signals: 24Unuseful DNA sequences(no SSR, terminal SSR, unreadable): 11Useful DNA sequences: 13

Redundant DNA sequences: 1Unique DNA sequence: 12

PCR primers designed: 12Workable 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

1 African African NSL 4142 2 African Moapa NSL 4123 3 Chilean Arizona Common

NSL 4144 4 Falcata WISFAL PI 560333 5 Falcata Wild tetraploid ssp.falcata PI 214218 6 Flemish DuPuit PI 206103 7 Indian Sirsa Type 9 PI 235736 8 Ladak Ladak NSL 4164 9 Peruvian Hairy Pervian NSL 4143 10 Turkistan Kaysari PI 17172111 Arabian Very Nondormant

UC-1887 12 Arabian Very Nondormant UC-146513 Varia Grimm NSL 4162 14 M. sativa ssp. caerulea Wild diploid alfalfaPI577548 15 M. sativa ssp. falcata Wild diploid ssp. falcata

PI258754 16 M. truncatula JemalongPI442895

Germplasm of M. sativa and M. truncatula used for screening

SSR primers

MFV

T

IL

F

A

C

CP

R

MF = FalcataL = LadakV = VariaF = FlemishT = TurkistanC = ChileanP = PeruvianI = IndianA = AfricanR = Arabian

Distribution of historical alfalfa germplasm

Banding patterns generated bythree SSR primer pairs

2 alleles

2 alleles

Multiple alleles

No. alleles No. SSR loci%

1 15 29.4 2 9 17.6 3 5 9.8 4 10 19.6 5 3 5.9 6 6 11.8 7 2 3.9 10 1 2.0

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 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 0.1Genetic distance

4x M. sativa

2x M. sativa

2x M. truncatula

Summary

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;

About 70% of the 51 SSRs were

polymorphic among the germplasm and

produced 154 different alleles;

Summary (continue)

The number of alleles generated from the

36

polymorphic SSR primer pairs ranged from

1-10;

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