Microsatellite Marker Development in Switchgrass

1
Microsatellite Marker Development in Switchgrass

• Environmental Sciences Division
• Oak Ridge National Laboratory
• BFDP Subcontractors Workshop
• November 7, 2001

2
Objective

• To develop a robust set of genetic markers that
  are PCR-based, portable across labs and genotypes
  that can be applied to population analysis,
  marker-assisted selection, mapping and QTL
  analysis

3
Overall Objectives

• Develop genetic markers
• Create mapping pedigrees
• Construct a framework genetic map for switchgrass

4
What Are Microsatellites?

• Genetic markers based on variation of unique DNA
  sequences
• 1-6 nucleotide core element tandemly repeated,
  e.g.
• atatatatatatatatatat (at)10
• Allele size based on repeat number of core
  elements

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How Can Microsatellites Be Used?

• Identity-unique individual
• Relationship-relatedness among genotypes
• Structure-partitioning of variation among and
  within populations
• Location-mapping

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Mining Microsatellites

• Cloning and sequencing
• Sequence analysis and primer design
• Primer testing and marker analysis

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Cloning and Sequencing
1. Extract switchgrass genomic DNA
2. Digest, clone into plasmid
3. Amplify,sequence DNA
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Sequence Analysis
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Primer Design
CGNATCCATATGCTGCAAGTGCTTGCCACTTGCAGTTTATGCTCTGAAAC
ATGGAGCAGCAAGATGGCCCAACGAGCATA TGTAAAGAAAGCATCAGGG
ACAGAGACCAGAACGATCTGTTGACCACAACTTGACTCGTCTAATCCTCG
CAACAAAAAAC TTGACTCATCTAAGACAACAACTAGCTATGTATGTTCC
ACATCATCAGCGCACAACATGCCACTGATCAGGGAGCGAGCT CATCAGA
TTTGGCAAAGACGTGCAGGCCTCGCACCTGAATTGGGAAGGCCAAATCTG
CCTGGCCGGGAGAAGCCAGGGC GCTGGGTCCGGACCTAAACCCTGCCCG
GCCGGCAACTCCTCCTCCTCCTTGGCCGGNCAAGCTCAGCTCTCACAGTC
TCA CACCCCACCGACNAAAAGCTAACGCCGCCTTGGGGCCCAACCCAAC
GGAGAGCACAACCCACTANCCTTTCANAGCAAGC   OLIGO
3' seq LEFT PRIMER ACATCATCAGCGCACAACA
T RIGHT PRIMER TGGGGTGTGAGACTGTGAGA   PRODUCT
SIZE 209
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Primer Testing
a
c
b
d
1. Extract DNA
2. PCR amplification
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Marker Analysis
Single Locus
10 Alleles
1
2
3
4
5
6
7
8
9
10
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Microsatellite Analysis

• 1824 sequences
• 960 clones
• 664 total microsatellites

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Dinucleotide Microsatellites
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Microsatellite Polymorphism
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Marker Survey VK15, VS16
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Marker Survey P13, VS16
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Marker Survey P13, P19
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Future Directions

• Continue microsatellite RD (ORNL)
• Microsatellite map construction (with UGA)
• Framework map construction (with UGA)
• Comparative mapping (with UGA)

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Acknowledgments

• Zamin Yang-ORNL
• Shreni Keniya-Knox College
• Ken Vogel-USDA-ARS, University of Nebraska
• Joe Bouton, Ali Missaoui-University of Georgia
• DOE/ OFD/ BFDP