Computational and Experimental Analysis of Microsatellites in Rice (Oryza sativa L.): Frequency, Length Variation, Transposon Associations, and Genetic Marker Potential

Abstract

A total of 57.8 Mb of publicly available rice (Oryza sativa L.) DNA sequence was searched to determine the frequency and distribution of different simple sequence repeats (SSRs) in the genome. SSR loci were categorized into two groups based on the length of the repeat motif. Class I, or hypervariable markers, consisted of SSRs Ն20 bp, and Class II, or potentially variable markers, consisted of SSRs Ն12 bp <20 bp. The occurrence of Class I SSRs in end-sequences of EcoRI-and HindIII-digested BAC clones was one SSR per 40 Kb, whereas in continuous genomic sequence (represented by 27 fully sequenced BAC and PAC clones), the frequency was one SSR every 16 kb. Class II SSRs were estimated to occur every 3.7 kb in BAC ends and every 1.9 kb in fully sequenced BAC and PAC clones. GC-rich trinucleotide repeats (TNRs) were most abundant in protein-coding portions of ESTs and in fully sequenced BACs and PACs, whereas AT-rich TNRs showed no such preference, and di-and tetranucleotide repeats were most frequently found in noncoding, intergenic regions of the rice genome. Microsatellites with poly(AT)n repeats represented the most abundant and polymorphic class of SSRs but were frequently associated with the Micropon family of miniature inverted-repeat transposable elements (MITEs) and were difficult to amplify. A set of 200 Class I SSR markers was developed and integrated into the existing microsatellite map of rice, providing immediate links between the genetic, physical, and sequence-based maps. This contribution brings the number of microsatellite markers that have been rigorously evaluated for amplification, map position, and allelic diversity in Oryza spp. to a total of 500.[Clone sequences for 199 markers (RM1-RM88, RM200-RM345) developed in this lab are available as GenBank accessions AF343840-AF343869 and AF344003-AF344169.]Microsatellites are tandemly arranged repeats of short DNA motifs (1-6 bp in length) that frequently exhibit variation in the number of repeats at a locus. Because of their abundance and inherent potential for variation, these simple sequence repeats (SSRs) have become a valuable source of genetic markers. Previous studies in rice have contributed to the development of several hundred microsatellite markers and a genetic map consisting of 320

Isolation and characterization of SSR and EST‐SSR loci in Chamaecyparis formosensis (Cupressaceae)

“…The contigs ranging from 80 to 530 bp in length were merged, and a total of 10,487,858 contigs were assembled. These contigs were screened using the Simple Sequence Repeat Identification Tool (SSRIT; Temnykh et al., 2001) and at least five di‐, tri‐, tetra‐, penta‐, and hexanucleotide repeats were selected, resulting in a total of 305,556 SSR‐containing sequences.…”

A Helitron-Like Transposon Superfamily from Lepidoptera Disrupts (GAAA)n Microsatellites and is Responsible for Flanking Sequence Similarity within a Microsatellite Family

“…Additional investigation of O. sativa (TA) n microsatellites indicated that nearly 45% were flanked by sequence that Fig. 2 Putative Ostrinia nubilalis microsatellite-associated interspersed nuclear element (OnMINE-1) secondary structure elements had homology to Micron superfamily members (Temnykh et al 2001). Primate Alu elements also are located near multiple microsatellite repeat motifs (Arcot et al 1995), with the most ancient AluJ class associated with (GAAA) n repeats (Yandava et al 1997).…”

“…Genome associations between TEs and microsatellite loci are also observed when integrations occur preferentially into tandem repeats (Akagi et al 2001;Temnykh et al 2001) or when tandem repeats hitchhike within mobile elements (Wilder and Hollocher 2001;López-Giráldez et al 2006;Coates et al 2009). Modification of microsatellite loci by TE insertion has been documented.…”

“…Subfamilies of human Alu elements are associated with microsatellite repeats (Zuliani and Hobbs 1990;Jurka and Pethiyagoda 1995), wherein AluJ subfamily members are preferentially located near (GAAA) n repeats (Yandava et al 1997). Also, the wheat genome is composed of microsatellite loci with flanking nucleotide sequences that contain TEs (Ramsey et al 1999), whereas rice (TA) n microsatellites are a known target for the insertion of the Micron family of MITEs (Akagi et al 2001;Temnykh et al 2001). TE associations with microsatellite loci often result in molecular markers that are non-Mendelian, show PCR co-amplification of [1 locus, or weak PCR amplification due to oligonucleotide primer competition (Economou et al 1990;Zhang 2004;Meglécz et al 2004; Van't Hof et al 2007), and are problematic during the development of genetic and genomic marker loci.…”

Characterization of simple sequence repeats (SSRs) from Phlebotomus papatasi (Diptera: Psychodidae) expressed sequence tags (ESTs)

“…The dataset was divided into small files, each containing 100 FASTA formatted sequences. SSR-containing sequences were identified using SSRIT web based SSR identification tool [46] available at http://www.gramene.org/db/markers/ssrtool. Any sequence was considered as an SSR where a repeat motif of one to six nucleotides in length was repeated at least five times for dinucleotide, trinucleotide, tetranucleotide and pentanucleotide SSRs.…”