Differentiation of the Historic Alfalfa Germplasm Sources Using Microsatillites

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_at_ba.ars.usda.gov

2
Objectives

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

3
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

4
Screening process based on DNA hybridization
(1) Primary screening
1-3 colonies/well
(2) Secondary screening
single colony/well
(3) Tertiary screening
5
PCR amplification of the DNA inserts(Colonies
showing positive signals)
480 bp
6
DNA sequencing
(CT)15
(AT)20
7
The profile of SSR development from an alfalfa
genomic library
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 Worka
ble 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 Worka
ble primer pairs 12 (100) Polymorphic SSR
loci 7 (58)
8
Germplasm of M. sativa and M. truncatula used for
screening SSR primers
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 171721 11 Arabian
Very Nondormant UC-1887 12 Arabian
Very Nondormant UC-1465 13 Varia Grimm
NSL 4162 14 M. sativa ssp. caerulea Wild
diploid alfalfa PI577548 15 M. sativa ssp.
falcata Wild diploid ssp. falcata PI258754
16 M. truncatula Jemalong PI442895
9
Distribution of historical alfalfa germplasm
MF
V
T
F
C
L
I
A
R
MF Falcata L Ladak V Varia F Flemish T
Turkistan C Chilean P Peruvian I Indian A
African R Arabian
P
C
10
Banding patterns generated by three SSR primer
pairs
Multiple alleles
2 alleles
2 alleles
11
Allelic variation for the 51 SSR loci with CT,
CAT and GAT repeats
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
12
Polymorphic primers
Including M. truncatula 70.6 Within M.
sativa 58.8
13
Genetic distance
0.7 0.6 0.5 0.4 0.3
0.2 0.1
Chilean (NSL 4144)
Ladak (NSL 4164)
Peruvian (NSL 4143)
African (NSL 4142)
Varia (NSL 4162)
Flemish (PI 206103)
Indian (PI 235736)
4x M. sativa
Turkistan (PI 171721)
Arabian (UC-1887)
Arabian (UC-1465)
African (NSL 4123)
Falcata (PI 214218)
Falcata (PI 560333)
2x Alfalfa (PI 577548)
2x M. sativa
2X Falcata (PI 258754)
2x M. truncatula
Truncatula (PI 442895)
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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

15
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.

16
AcknowledgementsSoybean Genomics Improvement
Lab.,Beltsville, MD

• Dr. Perry Cregan
• Dr. Qijian Song
• Edward Fickus
• Kristina Pilitt