title: Large-scale identification of polymorphic microsatellites using an in silico approach.
abstract: BACKGROUND
  Simple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.
  
  
  RESULTS
  We have developed PolySSR, a new pipeline to identify polymorphic SSRs rather than just SSRs. Sequence information is obtained from public EST databases derived from heterozygous individuals and/or at least two different genotypes. The pipeline includes PCR-primer design for the putatively polymorphic SSR markers, taking into account Single Nucleotide Polymorphisms (SNPs) in the flanking regions, thereby improving the success rate of the potential markers. A large number of polymorphic SSRs were identified using publicly available EST sequences of potato, tomato, rice, Arabidopsis, Brassica and chicken.The SSRs obtained were divided into long and short based on the number of times the motif was repeated. Surprisingly, the frequency of polymorphic SSRs was much higher in the short SSRs.
  
  
  CONCLUSIONS
  PolySSR is a very effective tool to identify polymorphic SSRs. Using PolySSR, several hundred putative markers were developed and stored in a searchable database. Validation experiments showed that almost all markers that were indicated as putatively polymorphic by polySSR were indeed polymorphic. This greatly improves the efficiency of marker development, especially in species where there are low levels of polymorphism, like tomato. When combined with the new sequencing technologies PolySSR will have a big impact on the development of polymorphic SSRs in any species.PolySSR and the polymorphic SSR marker database are available from http://www.bioinformatics.nl/tools/polyssr/.
authors.affiliation: Laboratory of Bioinformatics, Wageningen University, PO Box 8128, 6700 ET Wageningen, the Netherlands. jifeng.tang@gmail.com
authors.forename: Jifeng
authors.initials: J
authors.lastname: Tang
authors.forename: Samantha J
authors.initials: SJ
authors.lastname: Baldwin
authors.forename: Jeanne Me
authors.initials: JM
authors.lastname: Jacobs
authors.forename: C Gerard van der
authors.initials: CG
authors.lastname: Linden
authors.forename: Roeland E
authors.initials: RE
authors.lastname: Voorrips
authors.forename: Jack Am
authors.initials: JA
authors.lastname: Leunissen
authors.forename: Herman
authors.initials: H
authors.lastname: van Eck
authors.forename: Ben
authors.initials: B
authors.lastname: Vosman
mesh_terms: D000465:Algorithms; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D020224:Expressed Sequence Tags; D005819:Genetic Markers / Q000235:genetics*; D018895:Microsatellite Repeats / Q000235:genetics*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software
chemical_list: D005819:Genetic Markers
references.citation: Genome. 1996 Oct;39(5):866-73
references.pmid: 8890517
references.citation: Methods Mol Biol. 2000;132:365-86
references.pmid: 10547847
references.citation: Theor Appl Genet. 2004 Mar;108(5):881-90
references.pmid: 14647900
references.citation: Science. 2002 Apr 5;296(5565):92-100
references.pmid: 11935018
references.citation: Bioinformatics. 2004 May 1;20(7):1081-6
references.pmid: 14764542
references.citation: Plant Mol Biol. 2002 Mar-Apr;48(5-6):501-10
references.pmid: 11999831
references.citation: DNA Res. 2007 Feb 28;14(1):37-45
references.pmid: 17452422
references.citation: Trends Biotechnol. 2005 Jan;23(1):48-55
references.pmid: 15629858
references.citation: Trends Genet. 2006 May;22(5):253-9
references.pmid: 16567018
references.citation: In Silico Biol. 2005;5(5-6):439-63
references.pmid: 16268789
references.citation: Theor Appl Genet. 2003 Jan;106(2):363-73
references.pmid: 12582864
references.citation: Genome. 1999 Feb;42(1):139-49
references.pmid: 10208007
references.citation: Theor Appl Genet. 2002 Feb;104(2-3):399-407
references.pmid: 12582712
references.citation: Trends Genet. 2008 Mar;24(3):133-41
references.pmid: 18262675
references.citation: Genomics. 1997 Nov 15;46(1):24-36
references.pmid: 9403055
references.citation: Theor Appl Genet. 2004 Aug;109(4):783-91
references.pmid: 15205737
references.citation: Theor Appl Genet. 2005 Jul;111(2):291-312
references.pmid: 15926074
references.citation: Nucleic Acids Res. 2004 Jan 1;32(Database issue):D115-9
references.pmid: 14681372
references.citation: Genome Res. 1998 Mar;8(3):186-94
references.pmid: 9521922
references.citation: BMC Genomics. 2007 Feb 14;8:52
references.pmid: 17300727
references.citation: Plant Physiol. 2006 May;141(1):147-66
references.pmid: 16531485
references.citation: Nature. 2005 Sep 15;437(7057):376-80
references.pmid: 16056220
references.citation: In Silico Biol. 2006;6(6):607-20
references.pmid: 17518768
references.citation: Science. 2002 Apr 5;296(5565):79-92
references.pmid: 11935017
references.citation: Genome. 2004 Apr;47(2):389-98
references.pmid: 15060592
references.citation: Mol Genet Genomics. 2006 Nov;276(5):436-49
references.pmid: 16924545
references.citation: Theor Appl Genet. 2005 Aug;111(3):456-66
references.pmid: 15942755
references.citation: Theor Appl Genet. 2002 Sep;105(4):532-543
references.pmid: 12582502
references.citation: Genome Res. 1999 Sep;9(9):868-77
references.pmid: 10508846
references.citation: Mol Genet Genomics. 2007 May;277(5):469-80
references.pmid: 17237941
references.citation: Nat Rev Genet. 2004 Jun;5(6):435-45
references.pmid: 15153996
references.citation: Mol Genet Genomics. 2003 Dec;270(4):315-23
references.pmid: 14508680
references.citation: Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W656-9
references.pmid: 16845092