A second generation framework for the analysis of microsatellites in expressed sequence tags and the development of EST-SSR markers for a conifer, Cryptomeria japonica

Abstract

BackgroundMicrosatellites or simple sequence repeats (SSRs) in expressed sequence tags (ESTs) are useful resources for genome analysis because of their abundance, functionality and polymorphism. The advent of commercial second generation sequencing machines has lead to new strategies for developing EST-SSR markers, necessitating the development of bioinformatic framework that can keep pace with the increasing quality and quantity of sequence data produced. We describe an open scheme for analyzing ESTs and developing EST-SSR markers from reads collected by Sanger sequencing and pyrosequencing of sugi (Cryptomeria japonica).ResultsWe collected 141,097 sequence reads by Sanger sequencing and 1,333,444 by pyrosequencing. After trimming contaminant and low quality sequences, 118,319 Sanger and 1,201,150 pyrosequencing reads were passed to the MIRA assembler, generating 81,284 contigs that were analysed for SSRs. 4,059 SSRs were found in 3,694 (4.54%) contigs, giving an SSR frequency lower than that in seven other plant species with gene indices (5.4–21.9%). The average GC content of the SSR-containing contigs was 41.55%, compared to 40.23% for all contigs. Tri-SSRs were the most common SSRs; the most common motif was AT, which was found in 655 (46.3%) di-SSRs, followed by the AAG motif, found in 342 (25.9%) tri-SSRs. Most (72.8%) tri-SSRs were in coding regions, but 55.6% of the di-SSRs were in non-coding regions; the AT motif was most abundant in 3′ untranslated regions. Gene ontology (GO) annotations showed that six GO terms were significantly overrepresented within SSR-containing contigs. Forty–four EST-SSR markers were developed from 192 primer pairs using two pipelines: read2Marker and the newly-developed CMiB, which combines several open tools. Markers resulting from both pipelines showed no differences in PCR success rate and polymorphisms, but PCR success and polymorphism were significantly affected by the expected PCR product size and number of SSR repeats, respectively. EST-SSR markers exhibited less polymorphism than genomic SSRs.ConclusionsWe have created a new open pipeline for developing EST-SSR markers and applied it in a comprehensive analysis of EST-SSRs and EST-SSR markers in C. japonica. The results will be useful in genomic analyses of conifers and other non-model species.

Comparative in silico analysis of SSRs in coding regions of high confidence predicted genes in Norway spruce (Picea abies) and Loblolly pine (Pinus taeda)

“…Predominance of trimers in Picea abies [16] and Pinus taeda [17] was reported earlier only in two studies, likewise Yan et al [25] demonstrated higher frequency of trimers it in Pinus spp. Abundance of hexamers in gymnosperms is in accordance with earlier results in Picea [15, 16], Pinus [15], and Cryptomeria [36], as well as in comparative studies, which report hexamers to be more common among EST-SSRs in gymnosperms than angiosperms [14, 18]. The estimation of hexamer repeats was however under-estimated in earlier studies [14, 15], as a consequence of analysing only class I SSRs, whereas the current analysis reveals that there is very high abundance of hexamer repeats if class II SSRs are also taken into consideration (1100 and 971 per Mbp in spruce and pine, respectively).…”

“…Similar results were reported in Physcomitrella patens and Arabidopsis thaliana [18]. However, the GO term with the highest number of genes containing SSR loci in Cryptomeria [36] was cellular process instead of metabolic process as is the case in Pinus taeda and Picea abies . Therefore, we suggest that the GO distribution may be species specific rather than generalised for gymnosperms as such.…”

Development and Characterization of Genomic and Expressed SSRs in Citrus by Genome-Wide Analysis

“…A few higher repeat numbers were observed in di- and tri-nucleotide SSRs, but in tetra-, penta-, and hexa-nucleotide SSRs, no repeat number was found beyond 6 (Table 1). Similar trends have also been observed in other plant genomes [31], [32]. The tri-nucleotide were predominant in Clementine mandarin, in agreement with other SSR survey studies [33] supporting the relative distribution of motifs in these plant groups.…”

Identification of RAN1 orthologue associated with sex determination through whole genome sequencing analysis in fig (Ficus carica L.)

“…. Total 3,648 pairs of primers were designed using Primer340 according to Ueno et al 41. and 480 pairs were screened for the detectability of polymorphisms between the F1 parents by 10.0% PAGE (Poly-Acrylamide Gel Electrophoresis).…”