E-microsatellite markers for Centella asiatica (Gotu Kola) genome: validation and cross-transferability in Apiaceae family for plant omics research and development.

Abstract Centella asiatica (Gotu Kola) is a plant that grows in tropical swampy regions of the world and has important medicinal and culinary use. It is often considered as part of Ayurvedic medicine, traditional African medicine, and traditional Chinese medicine. The unavailability of genomics resources is significantly impeding its genetic improvement. To date, no attempt has been made to develop Expressed Sequence Tags (ESTs) derived Simple Sequence Repeat (SSR) markers (eSSRs) from the Centella genome. Hence, the present study aimed to develop eSSRs and their further experimental validation and cross-transferability of these markers in different genera of the Apiaceae family to which Centella belongs. An in-house pipeline was developed for the entire analyses by combining bioinformatics tools and perl scripts. A total of 4443 C. asiatica EST sequences from dbEST were processed, which generated 2617 nonredundant high quality EST sequences consisting 441 contigs and 2176 singletons. Out of 1776.5 kb of examined sequences, 417 (15.9%) ESTs containing 686 SSRs were detected with a density of one SSR per 2.59 kb. The gene ontology study revealed 282 functional domains involved in various processes, components, and functions, out of which 64 ESTs were found to have both SSRs and functional domains. Out of 603 designed EST-SSR primers, 18 pairs of primers were selected for validation based on the optimum parameter value. Reproducible amplification was obtained for six primer pairs in C. asiatica that were further tested for cross-transferability in nine other important genera/species of the Apiaceae family. Cross-transferability of the EST-SSR markers among the species were examined and Centella javanica showed highest transferability (83.3%). The study revealed six highly polymorphic EST-SSR primers with an average PIC value of 0.95. In conclusion, these EST-SSR markers hold a big promise for the genomics analysis of Centella asiatica, to facilitate comparative map-based analyses across other related species within the Apiaceae family, and future marker-assisted breeding programs. To the best of our knowledge, this is the first report of development of EST-SSRs in Centella asiatica by in silico approaches, which offers a veritable potential in further use in plant omics research and development.

[1]  Jyoti Singh,et al.  De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural, functional and regulatory genes with special reference to secondary metabolism. , 2013, Gene.

[2]  Sarika Gupta,et al.  Development of eSSR-Markers in Setaria italica and Their Applicability in Studying Genetic Diversity, Cross-Transferability and Comparative Mapping in Millet and Non-Millet Species , 2013, PloS one.

[3]  A. Aydin,et al.  Cross-genera transferable e-microsatellite markers for 12 genera of the Lamiaceae family. , 2013, Journal of the science of food and agriculture.

[4]  J. Batley,et al.  Predicting polymorphic EST‐SSRs in silico , 2013, Molecular ecology resources.

[5]  S. W. Park,et al.  Development of EST-derived SSR markers in pea (Pisum sativum) and their potential utility for genetic mapping and transferability , 2012 .

[6]  K. Weising,et al.  In silico mining for simple sequence repeat loci in a pineapple expressed sequence tag database and cross-species amplification of EST-SSR markers across Bromeliaceae , 2011, Theoretical and Applied Genetics.

[7]  S. Tangphatsornruang,et al.  A genome scan for quantitative trait loci affecting cyanogenic potential of cassava root in an outbred population , 2011, BMC Genomics.

[8]  H. Okada,et al.  Phylogenetic relationships among subgenera, species, and varieties of Japanese Salvia L. (Lamiaceae) , 2011, Journal of Plant Research.

[9]  Shiv Kumar,et al.  Cross-genera amplification of informative microsatellite markers from common bean and lentil for the assessment of genetic diversity in pigeonpea , 2010, Physiology and Molecular Biology of Plants.

[10]  M. Modgil,et al.  Assessment of genetic fidelity of micropropagated apple rootstock plants, EMLA 111, using RAPD markers. , 2009, Indian journal of experimental biology.

[11]  S. Knapp,et al.  Ontology and diversity of transcript-associated microsatellites mined from a globe artichoke EST database , 2009, BMC Genomics.

[12]  Sarika Gupta,et al.  Development and characterization of genic SSR markers in Medicago truncatula and their transferability in leguminous and non-leguminous species. , 2009, Genome.

[13]  C. Schlötterer,et al.  Survey of microsatellite clustering in eight fully sequenced species sheds light on the origin of compound microsatellites , 2008, BMC Genomics.

[14]  Marta Matvienko,et al.  SSRs and INDELs mined from the sunflower EST database: abundance, polymorphisms, and cross-taxa utility , 2008, Theoretical and Applied Genetics.

[15]  Jiming Jiang,et al.  Major cytogenetic landmarks and karyotype analysis inDaucus carota and other Apiaceae. , 2008, American journal of botany.

[16]  H. Nagaraja,et al.  Protective antioxidant effect of Centella asiatica bioflavonoids on lead acetate induced neurotoxicity. , 2008, The Medical journal of Malaysia.

[17]  J. Slate,et al.  Simple sequence repeats in zebra finch (Taeniopygia guttata) expressed sequence tags: a new resource for evolutionary genetic studies of passerines , 2007, BMC Genomics.

[18]  T. Kocher,et al.  Genetic and developmental basis of cichlid trophic diversity , 2006, Heredity.

[19]  V. Poncet,et al.  SSR mining in coffee tree EST databases: potential use of EST–SSRs as markers for the Coffea genus , 2006, Molecular Genetics and Genomics.

[20]  L. Sun,et al.  Genetic analyses and mapping of a new thermo-sensitive genic male sterile gene in maize , 2006, Theoretical and Applied Genetics.

[21]  Young A. Choi,et al.  Mining and characterizing microsatellites from citrus ESTs , 2006, Theoretical and Applied Genetics.

[22]  Snehasis Mukhopadhyay,et al.  Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species. , 2005, Genome.

[23]  M. Crowe,et al.  PineappleDB: An online pineapple bioinformatics resource , 2005, BMC Plant Biology.

[24]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[25]  C. Feuillet,et al.  High transferability of bread wheat EST-derived SSRs to other cereals , 2005, Theoretical and Applied Genetics.

[26]  A. A. Garcia,et al.  Survey in the sugarcane expressed sequence tag database (SUCEST) for simple sequence repeats. , 2004, Genome.

[27]  Liangjiang Wang,et al.  Tall fescue EST-SSR markers with transferability across several grass species , 2004, Theoretical and Applied Genetics.

[28]  L. Fraser,et al.  EST-derived microsatellites from Actinidia species and their potential for mapping , 2004, Theoretical and Applied Genetics.

[29]  S. Decroocq,et al.  Development and transferability of apricot and grape EST microsatellite markers across taxa , 2003, Theoretical and Applied Genetics.

[30]  R. Varshney,et al.  Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.) , 2003, Theoretical and Applied Genetics.

[31]  Mark L. Blaxter,et al.  Making sense of EST sequences by CLOBBing them , 2002, BMC Bioinformatics.

[32]  M. Sorrells,et al.  Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat , 2002, Plant Molecular Biology.

[33]  M. Morgante,et al.  Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes , 2002, Nature Genetics.

[34]  L. Zane,et al.  Strategies for microsatellite isolation: a review , 2002, Molecular ecology.

[35]  M. Morgante,et al.  A simple sequence repeat-based linkage map of barley. , 2000, Genetics.

[36]  R. J. Henry,et al.  Analysis of SSRs derived from grape ESTs , 2000, Theoretical and Applied Genetics.

[37]  X. Huang,et al.  CAP3: A DNA sequence assembly program. , 1999, Genome research.

[38]  N. Freimer,et al.  Compound microsatellite repeats: practical and theoretical features. , 1999, Genome research.

[39]  D. Struss,et al.  The use of microsatellite markers for detection of genetic diversity in barley populations , 1998, Theoretical and Applied Genetics.

[40]  P. Sivakumar,et al.  Effects of Centella asiatica extract on dermal wound healing in rats. , 1996, Indian journal of experimental biology.

[41]  M. Maroof,et al.  Development of simple sequence repeat DNA markers and their integration into a barley linkage map , 1996, Theoretical and Applied Genetics.

[42]  M. Boguski,et al.  dbEST — database for “expressed sequence tags” , 1993, Nature Genetics.

[43]  G. Churchill,et al.  Optimizing parental selection for genetic linkage maps. , 1993, Genome.

[44]  Archana Sharma,et al.  Chromosome studies and estimation of nuclear DNA in different varieties of Cuminum cyminum L. and Carum copticum Benth. and Hook. , 1990 .

[45]  T. Chuang,et al.  Chromosome Numbers of the Vascular Plants of Taiwan I , 1962 .

[46]  A. Sharma,et al.  Cytogenetics of some of the Indian umbellifers , 1955, Genetica.

[47]  Wei Li,et al.  Development, characterization and cross-species/genera transferability of EST-SSR markers for rubber tree (Hevea brasiliensis) , 2008, Molecular Breeding.

[48]  L. Singh,et al.  Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species , 2006, Theoretical and Applied Genetics.

[49]  Zhenlin Ju,et al.  An in silico Mining for Simple Sequence Repeats from Expressed Sequence Tags of Zebrafish, Medaka, Fundulus, and Xiphophorus , 2005, Silico Biol..

[50]  Andreas Graner,et al.  Genic microsatellite markers in plants: features and applications. , 2005, Trends in biotechnology.

[51]  Thomas Thiel,et al.  In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species. , 2002, Cellular & molecular biology letters.

[52]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[53]  D. Metzgar,et al.  Selection against frameshift mutations limits microsatellite expansion in coding DNA. , 2000, Genome research.

[54]  A. McClung,et al.  Microsatellites and a single-nucleotide polymorphism differentiate apparentamylose classes in an extended pedigree of US rice germ plasm , 1997, Theoretical and Applied Genetics.