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Legume Genomics and Genetics (online), 2010, Vol. 1, No.6, 30-33
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of which are come from the intergenic regions with no
gene function. Secondly, the procedures for developing
those markers are difficult, complex, high-cost. At the
same time, large-scale sequencing projects have pro-
duced a large amount of single-pass sequences of
complementary DNAs (cDNAs), more and more EST
sequences have been developed from different plant
species http://www.ncbi.nlm.nih.gov/). ESTs contain
SSR sequences in both coding and noncoding regions
(Temnykh et al., 2001), and SSRs have been success-
fully developed from ESTs in many species (Thiel et al.,
2003; Gao et al., 2004; Nicot et al., 2004; Sera-pion et
al., 2004; Perez et al., 2005; Wang et al., 2005).
31
Recently, more than 80 000 ESTs are now available
for peanut in GenBank of NCBI, and the number of
these ESTs is increasing year by year. But develop-
ment of EST-SSR markers are lagged behind com-
pared of other species. In this research, totally 86 132
peanut ESTs from NCBI and 12 501 ESTs in cDNA
library derived from E12 which comes from a Chinese
landrace contains high oleic acid are analyzed. Here
we report the successful development and character-
rization of EST-SSR markers in peanuts. These markers
can enrich the molecular biological resources in peanut.
1 Results
1.1 EST sequences screening
86 132 ESTs of the EST database in GenBank were
found and 12 501 ESTs from cDNA library of E12
were obtained. All the 98 633 EST sequences from
NCBI and cDNA library were used for searching
singletons and contigs (Table 1). In the Genbank of
NCBI, there were 14 141 singletons and 9 892 contigs.
And in cDNA library derived from E12, the number
of singletons and contigs was 3,910 and 80 res-
pectively. After screening EST-SSRs using MISA
software in all singletons and contigs from NCBI and
cDNA library, 2 463 (9.52%) EST-SSRs in NCBI and
641 (16.4%) EST-SSRs in cDNA library were found
(Table 1). In NCBI database 2,443 ESTs contained
one EST-SSR, and 20 ESTs had more than two
EST-SSRs. In cDNA library, 641 ESTs contained one
SSR loci, 82 ESTs have two EST-SSRs and four ESTs
had three EST-SSRs (Table 1).
1.2 Distribution and frequency of EST-SSRs
All these EST-SSRs could be divided into six kind
motifs, such as di-nucleotide, thi-nucleotide, tetranu-
Table 1 EST and EST-SSR distribution in NCBI and cDNA
library
NCBI
cDNA library
singleton
14 141
3 910
contig
9 892
80
SSR
2 463
641
ESTs with one SSR loci
2 287
555
ESTs with two SSR loci
156
82
ESTs with three SSR loci
17
4
ESTs with four SSR loci
2
0
ESTs with five SSR loci
1
0
cleotide, penta-nucleotide, hexa-nucleotide and multi-
nucleotide etc., The tri-nucleotide motif was the most
frequent motif in both NCBI and cDNA library. And
the frequency of tri-nucleotide was 43.0% and 56.8%
respectively. The number of di- and pentanucleotide
motifs was second and third in all motifs. And the hexa-
nucleotide was the least motif in NCBI and cDNA library
(Figure 1). The number of multinucleotide motif was 329
and 53, accounting for 13.4% and 8.3%, respectively.
Figure 1 Comparison of frequency distribution of different
types of SSR motif between NCBI and cDNA library
In terms of nucleotide repeat motifs, the AG/TC was
the most motifs in all repeat types nucleotide in NCBI
and cDNA library, and accounted for 8.65% and
13.42%, respectively. The following di-nucleotide repeat
motif was AT/TC and CT/GA motifs. Among the tri-
nucleotide repeats, CTT/GAA was the most frequent
motif, accounting for 6.7% and 13.42%, respectively.
And this type motif repeat is almost six and four times
than the other tri-nucleotide repeats in NCBI and cDNA
library respectively. The number of tetra-, penta- and
hexa-nucleotide motifs is less than 2% in both NCBI
and cDNA library. The AAAT/TTTA motif accounted
for 1.26% in NCBI but there was no this type motif in