Chaudhary et al. Hereditas (2016) 153:16 DOI 10.1186/s41065-016-0019-8 RESEARCH Open Access PineElm_SSRdb: a microsatellite marker database identified from genomic, chloroplast, mitochondrial and EST sequences of pineapple (Ananas comosus (L.) Merrill) Sakshi Chaudhary†, Bharat Kumar Mishra, Thiruvettai Vivek, Santoshkumar Magadum and Jeshima Khan Yasin*† Abstract Background: Simple Sequence Repeats or microsatellites are resourceful molecular genetic markers. There are only few reports of SSR identification and development in pineapple. Complete genome sequence of pineapple available in the public domain can be used to develop numerous novel SSRs. Therefore, an attempt was made to identify SSRs from genomic, chloroplast, mitochondrial and EST sequences of pineapple which will help in deciphering genetic makeup of its germplasm resources. Results: A total of 359511 SSRs were identified in pineapple (356385 from genome sequence, 45 from chloroplast sequence, 249 in mitochondrial sequence and 2832 from EST sequences). The list of EST-SSR markers and their details are available in the database. Conclusions: PineElm_SSRdb is an open source database available for non-commercial academic purpose at http://app.bioelm.com/ with a mapping tool which can develop circular maps of selected marker set. This database will be of immense use to breeders, researchers and graduates working on Ananas spp. and to others working on cross-species transferability of markers, investigating diversity, mapping and DNA fingerprinting. Keywords: Ananas, Genome wide marker analysis, Organelle, Pineapple, Simple sequence repeats Background industrial crop during 20th century [2,3]. In addition to The extremely surprising flavour and fragrance of fresh fruit consumption, pineapple is used for canned pineapple (Ananas comosus L.) delighted mankind at that slices, juice and juice concentrate, extraction of bromelain time of its discovery by Christopher Columbus and (a meat-tenderizing enzyme), high-quality fibre, animal even today. Pineapple, a perennial monocot plant feed and medicines [2]. At present, gross production value belongs to Bromeliales order, Bromelioideae subfamily of pineapple is approaching $9 billion due to its cultiva- and Bromeliaceae family. Pineapple is a tropical plant tion on 1.02 million hectares of land in over 80 countries native to South America, domesticated more than and annual production of 24.8 million metric tonnes of 6000 years ago [1]. At the end of the sixteenth fruit [4]. Wild varieties of pineapple are self-compatible, century, pineapple had become pantropical and is the whereas cultivated pineapple, A. comosus (L.) Merr., is third most economically important tropical fruit crop self-incompatible [5], which provides an opportunity to after banana and mango. Pineapple has become scrutinize the molecular basis of self-incompatibility in monocots. Over the last few decades, a wide range of molecular * Correspondence: [email protected]; [email protected] †Equal contributors markers have been developed and used in crop improve- Division of Genomic Resources, ICAR- National Bureau of Plant Genomic ment as molecular markers are helpful in assessing Resources, PUSA campus, 110012 New Delhi, India © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chaudhary et al. Hereditas (2016) 153:16 Page 2 of 6 Fig. 1 Homepage of the web app germplasm diversity, testing of hybridity, trait mapping, consumption, expensive process for generation of gen- marker assisted selection etc. [6]. Among all the markers omic libraries and sequencing of large number of clones till date, Simple Sequence Repeats (SSRs) are the most later to find the SSR-containing DNA regions [20] and ideal, powerful and reliable markers for molecular plant labour-intensive. To expedite this task, the traditional breeding applications because of their high abundance, methods of SSR markers generation from genomic co-dominant inheritance and multiple alleles [7]. In libraries [21] have been recouped briskly by in-silico addition, BES-SSR markers serve a useful resource for mining of SSRs from DNA sequences available in bio- integrating genetic and physical maps [8,9]. logical databases [22,23] and from expressed sequence SSRs consists of 2–7 base pair tandem repeat motifs of tags (ESTs) that represent only the coding region of the mono-, di-, tri-, tetra and penta-nucleotides (A, T, AT, genome [24–26]. GA, AGG, AAAG etc.) with different lengths of repeat motifs. These repeats are extensively distributed Methods throughout plants and animal genomes. A high level of Retrieval of genome sequences genetic variation is observed between and within species The complete genome sequence of pineapple (Ananas due to differences in the number of tandem repeating comosus (L.) Merrill) was retrieved from the CoGe units at a locus which produces a highly polymorphic Genome (Genome ID- 25734) page (https://genomevolu banding pattern [10] and is detected by the Polymerase tion.org/coge/GenomeInfo.pl?gid=25734) in FASTA for- Chain Reaction (PCR) using locus specific flanking mat. The chloroplast genome (Genome ID- 25280) and primers [11]. Molecular markers are widely recognized mitochondrial genome (Genome ID- 25281) of pineapple as a tool in generating linkage maps [12] as they define were also downloaded from CoGe Genome info respectively specific locations in the genome unambiguously [13,14]. (https://genomevolution.org/coge/GenomeInfo.pl?gid=2528 There are few valuable software and tools available for 0&81) in FASTA format. Total 5978 EST sequences of pine- SSRs identification and in-silico marker development. apple were downloaded from NCBI http://www.ncbi.nlm. Important sources for SSR identification are with bene- nih.gov/nucest/?term=ananas+comosus in FASTA format. fits from the advanced next generation sequencing tech- nology such as TROLL [15], MISA [16], SciRoko [17], SSR Locator [18] and GMATo [19]. MISA is the most SSRs identification common tool used for SSR identification. Generation of MISA tool allows the identification and localization of SSR markers have been exhaustive due to the time- perfect microsatellite as well as compound microsatellite Fig. 2 Main page of the database Chaudhary et al. Hereditas (2016) 153:16 Page 3 of 6 Fig. 3 EST-SSRs list which are interrupted by a certain number of bases. Results and discussion MISA uses Perl script for SSRs analysis. It requires a The home page of the database provides the complete set of sequences in FASTA format and a parameter access of the database (Figs. 1 and 2). Genomic sequence file that defines unit size and minimum repeat num- of Ananas comosus (L.) Merrill is available as 3133 scaf- ber of each SSR. MISA is available at http://pgrc.ipk- folds of 381,905,120 bp length. Of these scaffolds only gatersleben.de/misa/. MISA tool provides two result 2726 contain SSRs. From genomic sequence 356385 files; misa file and statistical file. MISA file provides SSRs were identified. Two thousand four hundred the information about SSR repeat types like simple, eighty-six sequences contain more than one SSR and interrupted or compound, size of SSR and SSR pos- 19086 compound SSRs are also exists. From NCBI 5978 ition in genome sequence. Statistical file contains the EST sequences of 4,294,909 bp were downloaded of statistical information like the frequency chart of SSR which 1886 sequences yielded 2832 SSRs (Fig. 3). Of motif and distribution of SSR to differently repeat these 1886 sequences 638 were found to contain more type classes. Classification of SSRs was done manually than one SSR region and 83 with compound repeat on the basis of their presence in coding region and regions. The access to the database can be obtained non-coding region of the genome sequences. from http://app.bioelm.com/ by creating user account. This will allow the users to generate images and search results. Genomic markers can be viewed as listed in Database development Fig. 4 and as a circular map of the selected markers An open, non-commercial database PineElm_SSRdb is using incorporated tools (Additional file 1). designed for educational purpose. PineElm_SSRdb is There is only one chloroplast genome sequence of available at http://app.bioelm.com/. 159,600 bp available for Ananas comosus (L.) Merrill in Fig. 4 Genomic SSR marker list Chaudhary et al. Hereditas (2016) 153:16 Page 4 of 6 which there are 45 SSRs exists and only one complex of cultivars may be required to protect the breeders repeat (Additional file 2). There are 13 sequences of right. Genome wide identification of markers can serve 881,399 bp Ananas comosus (L.) Merrill mitochondrial this purpose as SSR markers have been handy for inte- genome yielded 249 SSRs of which 13 sequences contain gration the physical, genetic and sequence-based phys- more than one SSR and 8 SSRs were found in com- ical maps in plant species, and concurrently equipped pound formation (Fig. 5 and Additional file 3). The EST- breeders and geneticists with an effective tool to bridge SSR statistics were represented in Additional file 4. SSRs phenotypic and genotypic variation. SSR markers have identified from chloroplast and mitochondrial sequences been handy for integration the physical, genetic and are highly specific and unique to pineapple. These SSRs sequence-based physical maps in plant species, and con- are not present in any other NCBI database and also not currently equipped breeders and geneticists with an effect- present in the nuclei genome of pineapple. ive tool to bridge phenotypic and genotypic variation [33].