Melampsora Genome Consortium - 2008 Summer Workshop
Microsatellites in the Mlp genome
Pascal Frey, Constance Xhaard, Axelle Andrieux,
Benoît Barrès, Fabien Halkett
INRA Nancy
Joint Research Unit « Tree – Microbe Interactions »
Champenoux, France
Reminder: what is a microsatellite?
• Microsatellites
= SSR (Simple Sequence Repeats)
= VNTR (Variable Number of Tandem
Repeats
• Motifs : di-, tri-, tetra-, penta-,
hexa-, or hepta-nucleotides
e.g. (TA)n, (TCA)n, (CAGT)n
• Highly variable genetic markers
Very useful for population genetics,
genetic mapping, diagnosis...
Why studying microsatellites in the Mlp genome?
1. To determine the number and the distribution of
microsatellite loci in the Mlp genome.
2. To build a genetic map of Mlp. Problems to obtain a large
offspring population.
3. To develop new microsatellite loci for population genetic
studies.
Microsatellite finding with Magellan (1)
Magellan software : Dee Carter, Univ. Sydney
www.medfac.usyd.edu.au/people/academics/profiles/dcarter.php
1st run (march 2007) on the 1st assembly of the Mlp genome (Jazz)
Genome size: 122 Mb; 2,682 scaffolds
7,530 microsatellite loci found (min 6 repeats for all motifs)
59% di, 34% tri, 3% tetra, 2% penta, 2% hexa-nucleotides
Culling settings : min 10 repeats for di-nucl., and 8 repeats for tri-nucl.
664 microsatellite loci found
17% di, 39% tri, 17% tetra, 11% penta, 14% hexa-nucleotides
Microsatellite finding with Magellan (2)
2nd run (July 2008) on the 2nd assembly of the Mlp genome (Arachne)
Genome size: 102 Mb; 462 scaffolds
7,293 microsatellite loci found (min 6 repeats for all motifs)
58% di, 35% tri, 3% tetra, 2% penta, 2% hexa-nucleotides
Culling settings : min 10 repeats for di-nucl., and 8 repeats for tri-nucl.
654 microsatellite loci found (652 in the main genome, 1 in the
mitochondrial DNA, 1 in Alt-haplotypes, none in Repetitive elements)
18% di, 39% tri, 16% tetra, 12% penta, 13% hexa-nucleotides
Microsatellite finding with Magellan (3)
Comparison of both runs (and of both assemblies)
Most of the microsatellite loci were found in both runs.
In some cases, 2 microsatellite loci were defined on 2 separate scaffolds
of the 1st assembly, and corresponded to only 1 locus in the 2nd
assembly.
-> both alleles for strain 98AG31
Validation of the microsatellites found (1)
• Design of 1-2 primer pairs for each of the 650 loci with
Primer3 software.
• In vitro PCR assays to validate the loci: 75% amplification
success
• Determination of the polymorphism of the loci on 2 sexual
populations of 40 Mlp isolates.
• Suppression of some loci not at Hardy-Weinberg equilibrium.
Validation of the microsatellites found (2)
Microsatellite loci resolved on a Beckman CEQ8000 DNA sequencer
homozygous loci
heterozygous locus
Validation of the microsatellites found (3)
40
35
30
Design of 2 multiplex panels
25
of 16 and 14 loci to be
amplified in a single tube
20
15
10
5
0
50
150
250
350
450
550
650
Dye4
Dye3
-5
Dye2
NA
-10
-15
Distribution of microsatellites on the 34 main scaffolds
4,5
4,0
Position (Mb)
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
0
5
10
15
20
25
30
Scaffold #
Of the 462 scaffolds, only 111 contain microsatellites.
Most of the smallest scaffolds contain no microsatellite.
Average density: 6.5 microsatellites/Mb
35