Development, cross-species/genera transferability of novel EST-SSR markers and their utility in revealing population structure and genetic diversity in sugarcane Ram K. Singh a, ⁎, Satya N. Jena b , Suhail Khan a, c , Sonia Yadav a , Nandita Banarjee a , Saurabh Raghuvanshi d , Vasudha Bhardwaj a , Sanjay K. Dattamajumder a , Raman Kapur a , Sushil Solomon a , M. Swapna a , Sangeeta Srivastava a , Akhilesh K. Tyagi d, e a Indian Institute of Sugarcane Research (ICAR), Rai Bareli Road, Lucknow-226002, U.P., India b Plant Molecular Biology Laboratory, National Botanical Research Institute, Lucknow-226001, India c Centre for Desert Agriculture, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia d Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India e National Institute of Plant Genome Research, Aruna Asaf Ali Marg, P.O. Box No. 10531, New Delhi-110 067, India abstract article info Article history: Accepted 27 March 2013 Available online 13 April 2013 Keywords: Expressed sequence tag (EST) Population structure Simple sequence repeat (SSR) Sugarcane Sugarcane (Saccharum spp. hybrid) with complex polyploid genome requires a large number of informative DNA markers for various applications in genetics and breeding. Despite the great advances in genomic tech- nology, it is observed in several crop species, especially in sugarcane, the availability of molecular tools such as microsatellite markers are limited. Now-a-days EST-SSR markers are preferred to genomic SSR (gSSR) as they represent only the functional part of the genome, which can be easily associated with desired trait. The present study was taken up with a new set of 351 EST-SSRs developed from the 4085 non redundant EST sequences of two Indian sugarcane cultivars. Among these EST-SSRs, TNR containing motifs were pre- dominant with a frequency of 51.6%. Thirty percent EST-SSRs showed homology with annotated protein. A high frequency of SSRs was found in the 5′UTR and in the ORF (about 27%) and a low frequency was observed in the 3′UTR (about 8%). Two hundred twenty-seven EST-SSRs were evaluated, in sugarcane, allied genera of sugarcane and cereals, and 134 of these have revealed polymorphism with a range of PIC value 0.12 to 0.99. The cross transferability rate ranged from 87.0% to 93.4% in Saccharum complex, 80.0% to 87.0% in allied gen- era, and 76.0% to 80.0% in cereals. Cloning and sequencing of EST-SSR size variant amplicons revealed that the variation in the number of repeat-units was the main source of EST-SSR fragment polymorphism. When 124 sugarcane accessions were analyzed for population structure using model-based approach, seven genetically distinct groups or admixtures thereof were observed in sugarcane. Results of principal coordinate analysis or UPGMA to evaluate genetic relationships delineated also the 124 accessions into seven groups. Thus, a high level of polymorphism adequate genetic diversity and population structure assayed with the EST-SSR markers not only suggested their utility in various applications in genetics and genomics in sugarcane but also enriched the microsatellite marker resources in sugarcane. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Modern sugarcane cultivars (Saccharum spp. hybrid) are the result of a limited cycle of crosses and backcrosses, predominantly involv- ing noble cane, Saccharum officinarum L. (2n = 80) and wild cane, Saccharum spontaneum (2n = 40 to 120). Another four closely related genera of Saccharum (Erianthus section Ripidum, Narenga, Miscanthus section Diandra, and Sclerostachya), have purportedly undergone inter- breeding, forming the ‘Saccharum complex’ (Daniels and Roach, 1987). However, the present day sugarcane cultivars are inter-specific poly- aneuploid hybrids, usually with 112–128 chromosomes assigned to eight homology groups (Aitken et al., 2005). Most of the traits in sug- arcane are quantitative, multigenic and/or multi-allelic, and continued Gene 524 (2013) 309–329 Abbreviations: EST-SSRs, expressed sequence tag-simple sequence repeats; UTR, untranslated region; gSSR, genomic simple sequence repeat; UPGMA, unweighted pair group method with arithmetic mean; PIC, polymorphism information content; SUCEST, sugarcane expressed sequence tag; DNR, dinucleotide repeat; TNR, trinucleotide repeat; TtNR, tetranucleotide repeat; PNR, pentanucleotide repeat; HNR, hexanucleotide repeat; IISR, Indian Institute of Sugarcane Research; Pop, population; ISMC, International Sugarcane Microsatellite Consortium; UGMS, unigene microsatellite markers. ⁎ Corresponding author at: Biotechnology Laboratory, Division of Crop Improvement, Indian Institute of Sugarcane Research (ICAR), Rai Bareli Road, Lucknow-226002, U.P., India. Tel.: +91 522 2480735, +91 522 2480726 Ext. 140; fax: +91 522 2480738. E-mail address: ikrps@yahoo.com (R.K. Singh). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.125 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene