Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species

Abstract

Genic microsatellites or EST-SSRs derived from expressed sequence tags (ESTs) are desired because these are inexpensive to develop, represent transcribed genes, and often a putative function can be assigned to them. In this study we investigated 2,553 coffee ESTs (461 from the public domain and 2,092 in-house generated ESTs) for identification and development of genic microsatellite markers. Of these, 2,458 ESTs (all >100 bp in size) were searched for SSRs using MISA--search module followed by stackPACK clustering that revealed a total of 425 microsatellites in 331 (13.5%) non-redundant ESTs/consensus sequences suggesting an approximate frequency of 1 SSR/2.16 kb of the analysed coffee transcriptome. Identified microsatellites mainly comprised of di-/tri-nucleotide repeats, of which repeat motifs AG and AAG were the most abundant. A total of 224 primer pairs could be designed from the non-redundant SSR-positive ESTs (excluding those with only mononucleotide repeats) for possible use as potential genic markers. Of this set, a total of 24 (10%) primer pairs were tested and 18 could be validated as usable markers. Sixteen of these markers revealed moderate to high polymorphism information content (PIC) across 23 genotypes of C. arabica and C. canephora, while 2 markers were found to be monomorphic. All the markers also showed robust cross-species amplifications across 14 Coffea and 4 Psilanthus species. The apparent broad cross-species/genera transferability was further confirmed by cloning and sequencing of the amplified alleles. Thus, the study provides an insight about the frequency and distribution of SSRs in coffee transcriptome, and also demonstrates the successful development of genic-SSRs. It is expected that the potential markers described here would add to the repertoire of DNA markers needed for genetic studies in cultivated coffee and also related taxa that constitute the important secondary genepool for coffee improvement.

Polymorphic information content of SSR markers for Coffea spp.

“…These results demonstrate the potential of these SSR markers for genetic studies in related species of Coffea. Cross-species transferability primers in Coffea has been observed previously for EST-SSR markers (Bhat et al 2005, Aggarwal et al 2007). …”

“…Previous studies using different molecular markers showed low levels of polymorphism among C. arabica accessions (Poncet et al 2006, Aggarwal et al 2007, Missio et al 2009a, hindering further genetic research in this species. Simple Sequence Repeats (SSRs) or microsatellite markers are potentially useful in this situation, especially for exploring highly variable regions of the genome among individuals or populations of the same species.…”

“…Notwithstanding all the advantages of SSR molecular markers for genetic studies, few SSR primers have been developed for coffee (Combes et al 2000, Baruah et al 2003, Moncada and McCouch 2004, Bhat et al 2005, Poncet et al 2006, Aggarwal et al 2007, Hendre et al 2008, Missio et al 2009b), compared to other crops (http:// www.gramene.org/). In this study, the potential of 33 SSR markers, developed from C. arabica were used for assessed the Polymorphism Information Content (PIC) in 24 accessions of the Coffea genus, including C. arabica.…”

Bioinformatics tools for development of fast and cost effective simple sequence repeat (SSR), and single nucleotide polymorphisms (SNP) markers from expressed sequence tags (ESTs)

“…The tool is written in Perl and is therefore platform independent, but it requires as installation of Primer3 for primer search (Thiel et al, 2003). MISA has been applied for SSR identification in coffee (Aggarwal et al, 2007), barley (Thiel et al, 2003;Kota et al, 2001), wheat (Yu et al, 2004), rye (Khlestkina et al, 2004) and peanut (Liang et al, 2009). Another SSR search tool called as 'Repeat Finder' (Volfovsky et al, 2001) (http://www.cbcb.umd.edu/software/RepeatFinder/) finds SSRs in four steps.…”

Analysis of Genetic Diversity and SSR Allelic Variation in Rubber Tree (Hevea brasilensis)

“…Simple sequence repeat (SSR) marker has been used as an ideal molecular marker to investigate the genetic diversity because of its multi-allelic nature, reproducibility, codominant inheritance, high abundance and extensive genome coverage (Gupta & Varshney, 2000) in many crops, such as rubber tree (Lekawipat et al, 2003;Saha et al, 2005;Gouvêa et al, 2010), wheat (Liu et al, 2007;Hao et al, 2006), bean (Choi et al, 1999), barley (Brantestam et al, 2007), cole (Hasan et al, 2006), jowar (Marco et al, 2007), triticale (Tams et al, 2004), rice (Song et al, 2003) and coffee (Aggarwal et al, 2007).…”