Legume Genomics and Genetics (online), 2010, Vol. 1, No.6, 30-33
http://lgg.sophiapublisher.com
33
Mace et al., 2008). They should be valuable in genome
mapping and population studies. The EST-SSRs have
two major advantages over genomic SSRs. First, as
EST-SSRs are part of or adjacent to functional genes,
they can be used for the mapping and functional
analysis of candidate genes. Second, because ESTs are
more conserved than average genomic sequences,
EST-SSRs may be more stable and transferable across
species. The successful development of EST-SSR in
the cultivated peanut should encourage similar efforts
in other
Arachis
for which a large number of ESTs are
available.
3 Materials and Method
3.1 Identification of SSR-Containing ESTs
ESTs of the peanut were downloaded from the EST
database of NCBI GenBank (http://www.ncbi.nlm.
nih.gov/dbEST) in April 2009. And EST sequences of
cDNA libraries derived from leaf tissues of high-oil
acid accession E12 were obtained previously (data not
shown). All sequences were downloaded or trans-
formed to a text file in FASTA format.
3.2 ESTs sequences splicing
All EST sequences were spliced by CAP3 software
(http://genome.cs.mtu.edu/sas.html). These ESTs are
divided into singletons which are not spliced with
other ESTs and contigs which are related to one
another by overlap of their sequences.
3.3 EST-SSR screening
These singletons and contigs were screened for SSRs
using the MISA software (MicroSAtellite, http://pgrc.
ipk-gatersleben.de/misa/). For this study, the criteria
for SSRs were set as sequences having at least eight
repeats of dinucleotide and five repeats for all other
repeats (tri-, tetra-, penta-, and hexa-nucleotide).
3.4 Design Primers
All SSR-containing ESTs were individually inspected
for suitability for primer design. SSR-containing ESTs
that contain sufficient flanking sequences of good
quality (no unknown bases) were selected for primer
design. Primers were designed using the PRIMER 3
software(http://frodo.wi.mit.edu/cgi-bin/primer3/prim
er3_ www.cgi), with an optimal annealing tempe-
rature of 60
℃
and a fragment size between 100 bp and
300 bp. AGC clamp was added at the 3’ primer end when
possible.
Acknowledgements
This research was supported by Modern Agro-industry Technology
Research System (nycytx-19), National High-Tech Research and Develop-
ment Plan of China (2006AA10A114; 2007AA10Z189) and National Pro-
ject of Scientific and Technical Supporting Program (2008BAD97B04).
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