Mining and characterization of SSRs from pomegranate (Punica granatum L.) by pyrosequencing K UNDAPURA V. R AVISHANKAR 1,3 ,K ANUPRIYA C HATURVEDI 1 ,N ISCHITA P UTTARAJU 1 ,S ANTHOSHKUMAR G UPTA 1 and S AMPATHKUMAR P AMU 2 1 Division of Biotechnology, Indian Institute of Horticultural Research, Hessarghatta Lake Post, Bengaluru, 560089, India; 2 Division of Fruit Crops, Indian Institute of Horticultural Research, Hessarghatta Lake Post, Bengaluru, 560089, India; 3 Corresponding author, E-mail: kv_ravishankar@yahoo.co.in, kvravi@iihr.ernet.in With 1 figures and 4 tables Received November 14, 2013/Accepted October 18, 2014 Communicated by H. Flachowsky Abstract Pomegranate (Punica granatum L.) is an important fruit-bearing decidu- ous shrub which has been under cultivation since ancient times. Genomic resources like microsatellite markers are limited in pomegranate; there- fore, this study was undertaken with the objective to develop and charac- terize microsatellite markers using Roche 454 GS FLX Titanium pyrosequencing technology. The total length of non-redundant sequences obtained was 19.7 Mbp, consisting of 59 603 reads assembled into 7361 contigs, of which, 567 (7.70%) contained microsatellite repeats. The average G+C content was 41.3%. Functional annotation to Arabidopsis thaliana sequences yielded 734 unigenes from 7361 contigs which were classified based on GO terms. Primer pairs were designed for total of 171 loci, out of which 167 produced reproducible polymorphic bands in 12 genotypes of pomegranate. A total of 951 alleles were identified, ranging from 1 to 14 per locus (average of 5.56). The polymorphism information content (PIC) value ranged between 0.00 and 0.910 (mean of 0.5427). The markers developed here will be useful for genetic diver- sity studies, gene mapping and genotyping in pomegranate. Key words: microsatellites — genomics — genetic diversity — next-generation sequencing Pomegranate (Punica granatum L.; 2n = 2x = 18) is a diploid, perennial, woody plant and belongs to the monogeneric family Lythraceae which possesses two species viz, Punica granatum L. and Punica protopunica Balf. f. The genome size of pome- granate has been ascertained to be 704 Mbp, about six times the size of Arabidopsis thaliana (Bennett and Leitch 2010). This fruit is believed to have originated in Iran (Levin 1994) from where it diversified to other regions like Mediterranean coun- tries, India, China, Afghanistan, through ancient trade routes. It is one of the oldest known edible fruit (Damania 2005) and highly prized for its nutritional and medicinal properties (Noda et al. 2002). In the recent years, demand for pomegranate has increased due to the presence of high amount of antioxidants and other phytochemicals in fruit and peel. Although some effort has been made for the development of superior cultivars by con- ventional breeding of this crop, the genomic resources like expressed sequence tags (ESTs), sequenced genes or high- density genetic maps are lacking (Ophir et al. 2014). Evaluations of genetic diversity of pomegranate have been conducted in the past using dominant markers like randomly amplified polymor- phic DNA (RAPD) by Dorgac et al. (2008), Sarkhosh et al. (2006), Talebi et al. (2003), Zarei et al. (2009), inter simple sequence repeats (ISSR) by Talebi et al. (2005), amplified frag- ment length polymorphism (AFLP) by Jbir et al. (2008), La Malfa (2010), Yuan et al. (2007), Rahimi et al. (2006), sequence related amplified polymorphism (SRAP) by Soleimani et al. (2012) and directed amplification of minisatellite DNA (DAMD) by Narzary et al. (2009). In 2007, Koohi et al. published the first report describing the isolation of 15 microsatellites in pome- granate. Since then 178 SSR markers have been isolated by dif- ferent workers in this crop, and all of them have been developed using the enrichment method and with hybridization of probes (Curr o et al. 2010, Ebrahimi et al. 2010, Hasnaoui et al. 2010, Pirseyedi et al. 2010, Soriano et al. 2011). This method is costly, labour intensive and produces many redundant clones. The recent introduction of next-generation sequencing technolo- gies (NGS), such as pyrosequencing (454 Life Sciences, Bran- ford, USA) is revolutionizing the field of molecular biology. One of the important applications of NGS is the rapid and cost- effective discovery of simple sequence repeat (SSR) or microsat- ellite loci (Seo et al. 2012). Currently, Roche/454, Solexa/Illu- mina and AB SOLiD are the technologies that are predominantly being used in sequencing for crop genetics and breeding applica- tions. Roche’s 454 pyrosequencing method uses fragmented (300–800 bp) nucleic acid template outfitted with two different adaptor sequences at each end, which are later used as priming sites for emulsion PCR (ePCR) and sequencing reactions. At present, 454 technology offers better reads, around 400 bp on the GS FLX Titanium platform. Therefore, this platform was used in this study to examine microsatellite sequences in pome- granate genomic data and to evaluate genetic relationships among a set of pomegranate cultivars. We also performed func- tional annotation to identify genes in assembled contigs. Materials and Methods Plant material and DNA isolation: Twelve genotypes of pomegranate, namely ‘Amlidana’ (bearing internal number IIHR 223474), ‘Bhagwa’ (IIHR 223475), ‘Ganesh’ (IIHR 223476), ‘Jodhpur Red’ (IIHR 223477), ‘Daru’ (IIHR 223478), ‘Daya’ (IIHR 223479), ‘Jallore Seedless’ (IIHR 223480), ‘Kabul Yellow’ (IIHR 223481), ‘Muscot’(IIHR 223482), ‘Nana’(IIHR 223483), ‘Naina’(IIHR 223484) and ‘Ruby’(IIHR 223485), were used in this study. Young leaf samples were collected from the pomegranate germplasm collection maintained at Indian Institute of Horticultural Research, Bengaluru. The leaf samples were washed three times in sterile distilled water, frozen in liquid nitrogen and kept at 80°C until used. Total genomic DNA was extracted from the leaf material using the modified CTAB method (Ravishankar et al. 2000). The concentration of DNA was determined by spectrophotometer (Gene Quanta, Amersham Biosciences, New Jersey, USA) at 260 nm. The integrity was determined by agarose gel electrophoresis (0.8%). A total of 10 lg of ‘Ganesh’ genomic DNA was used to perform shotgun sequencing using 454 GS FLX Titanium pyrosequencing platform (454 Life Sciences, Branford, USA). Plant Breeding doi:10.1111/pbr.12238 © 2015 Blackwell Verlag GmbH