Identification of EST-SSRs and molecular diversity analysis in Mentha piperita

Birendr Kumr，Umesh Kumr，Hemnt Kumr Ydv，c，*

aCSIR-Central Institute of Medicinal and Aromatic Plant Sciences，P.O.CIMAP，Near Kukrail Picnic Spot，Lucknow-226 015，India

bCSIR-National Botanical Research Institute，Rana Pratap Marg，Lucknow-226 001，India

cAcademy of Scientific and Innovative Research（AcSIR），New Delhi，India

Identification of EST-SSRs and molecular diversity analysis in Mentha piperita

Birendra Kumara，Umesh Kumarb，Hemant Kumar Yadavb，c，*

aCSIR-Central Institute of Medicinal and Aromatic Plant Sciences，P.O.CIMAP，Near Kukrail Picnic Spot，Lucknow-226 015，India

bCSIR-National Botanical Research Institute，Rana Pratap Marg，Lucknow-226 001，India

cAcademy of Scientific and Innovative Research（AcSIR），New Delhi，India

A R T I C L E I N F O

Article history:

Received 8 July 2014

Received in revised form

2 February 2015

Accepted 16 February 2015

Available online 28 February 2015

EST-SSRs

Genetic diversity

Mentha piperita

Polymorphic information content Transferability

EST sequences of Mentha piperita available in the public domain（NCBI）were exploited to develop SSR markers.A total of 1316 ESTs were assembled into 155 contigs and 653 singletons and of these，110 sequences were found to contain 130 SSRs，with a frequency of 1 SSR/3.4 kb.Dinucleotide repeat SSRs were most frequent（72.3%）with the AG/CT（43.8%）repeat motif followed by AT/AT（16.2%）.Primers were successfully designed for 68 SSR-containing sequences（62.0%）.The 68 primers amplified 13 accessions of M.piperita and 54 produced clear amplicons of the expected size.Of these 54，33（61%）were found to be polymorphic among M.piperita accessions，showing from 2 to 4 alleles with an average of 2.33 alleles/SSR，and the polymorphic information content（PIC）value varied between 0.13 and 0.51（average 0.25）.All the amplified SSRs showed transferability among four different species of Mentha，with a highest in Mentha arvensis（87.0%）and minimum in Mentha citrata（37.0%）.The newly developed SSRs markers were found to be useful for diversity analysis，as they successfully differentiated among species and accessions of Mentha.

?2015 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.

1.Introduction

The genus Mentha（mint）is one of the most important taxa of the family Lamiaceae and comprises 25 to 30 species grown in different parts of the world.Only five of them，Japanese/menthol mint（Mentha arvensis L.var.piperascens forma Holmes），spearmint（Mentha spicata L.），peppermint（Mentha piperita L.），scotch spearmint（MenthacardiacaBaker.）andBergamotmint（Mentha citrata Ehrh.）are commercially grown in India and other countries［1，2］.These five species are the major natural source of aroma compounds of industrial importance；namely-menthol，menthofuran，carvone，linalool，and linalyl acetate.Because of their cooling，pleasant aroma and flavor，essential oils of mint are used in perfumery，cosmetics，confectionery，and the pharmaceutical industries.The oil of M.piperita，known as peppermint oil，is widely used for headache，nerve pain，toothache，oral inflammation，joint conditions，itchiness，allergic rash，repelling mosquitoes，rheumatism，muscular pains，etc.［3，4］.Menthol is the major constituent of the essential oil constituents of peppermint oil［5］.Peppermint oil of globally accepted quality contains high amounts of menthol，moderate amounts of menthone，and very low amounts of pulegone and menthofuran［6，7］.The presence and concentrations of certain chemical constituents of essential oils changeaccording to the season，soil，climate，and site of plant growth. Peppermint is cultivated in several parts of India and has great economic value and a strong export potential for its volatile oil extracts.

DNA-based molecular markers have been shown to be an efficient tool to assist conventional plant breeding in various ways，such as by assessing the gene pool for diverse parental lines，hybridity testing，QTL mapping，gene tagging，and marker-assisted selection.Despite the importance of peppermint as an aromatic and medicinal plant，no comprehensive molecular marker systems are available.A few studies have assessed genetic diversity in species of Mentha based on RAPD［8-10］and AFLP fingerprinting［11］.There is a complete lack of Mentha-specific molecular markers for use in genetic studies and genetic improvement programs.

Simple sequence repeats（SSRs）also called microsatellites，are 1-6-base tandem repeats of DNA sequences，abundant in both prokaryotic and eukaryotic organisms in coding and noncoding regions［12］.SSRs are a preferred marker system owing to their codominant inheritance，multiallelic nature，abundance in the genome，high reproducibility，hyperpolymorphism，and high rate of transferability across genera and species［13-15］.Expressed sequence tags（ESTs）available in the public domain are the easiest and cheapest source for SSR development.EST-SSRs offer various advantages including ease of access，presence in gene-rich regions，and high transferability across species and genera［16］，which enable them to serve as anchor markers for comparative mapping and evolutionary studies［15］.Given that no SSR markers are reported in Mentha，the present study was undertaken to exploitthe ESTdatabase of M.piperita to（1）analyze the frequency and distribution of SSRs in ESTs，（2）develop and characterize EST-SSRs（3）test their transferability in related species and（4）detect polymorphism/diversity among accessions and species of Mentha.

2.Materials and methods

2.1.Plant materials and DNA isolation

The plant material included 13 accessions of M.piperita，5 of M.arvensis，4 of M.spicata，and 1 each of Mentha longifolia and M.citrata.These accessions were previously evaluated for essential oil content and other components.The details of these plant materials are given in Table 1.M.piperita is characterized by moderate oil content and high menthofuran. M.arvensis，also known as menthol mint，contains comparatively high oil content rich in menthol.The accessions of M.spicata and M.longifolia contain carvone-rich essential oils and M.citrata is rich in linalool and linalyl acetate.These accessions were grown during the 2012-2013 crop season in the experimental field of the Central Institute of Medicinal and Aromatic Plants（CIMAP）Lucknow，Uttar Pradesh，India. Fresh leaves combined from five randomly chosen plants of each accession were used to isolate total genomic DNA with a Qiagen DNeasy Plant Mini Kit（Qiagen，Valencia，CA，USA）following the manufacturer's instructions.The quality of DNA was assessed on 0.8%agarose gel and quantity was checked with a NanoDrop spectrophotometer ND 1000（NanoDrop Products，USA）.Finally，the DNA was normalized to 10 ng μL-1for PCR amplification.

2.2.Data mining and EST-SSR identification

A total of1316 raw EST sequences of M.piperita were downloaded from the National Center for Biotechnology Information（NCBI；http://www.ncbi.nlm.nih.gov/dbest/）on January 14，2013.The 5′or 3′end poly A or poly T stretches were removed from the raw EST sequences using EST-Trimmer software（http://pgrc.ipk-gatersleben.de/misa/download/est_trimmer.pl）.EST sequences were then assembled using the CAP3 assembler［17］with criteria of 40 bp overlap and 90%identity.The assembled EST sequences were subjected to SSR search using MISA（http://pgrc.ipk-gatersleben.de/misa/）with criteria of minimum number of repeats of 5 for dinucleotide（DNR）and trinucleotide（TNR）and of 4 for tetra-，penta-，and hexanucleotide SSRs.

Table 1-Details of cultivars and species of Mentha used in the study.

2.3.Primer design and PCR amplification

The SSR-containing sequences were used to design flanking primers with PRIMER3 software（http://frodo.wi.mit.edu/primer3）with major primer design parameters as follows:product length 100-300 bp，primer size 18-25 bp，and melting temperature 57-63°C（optimum 60°C）.In some cases，where primers could not be designed，the criteria were relaxed. The primers were synthesized with an additional 18-base（5′-TGTAAAACGACGGCCAGT-3′）tag at the 5′end to all the forward primers［18］.Additionally，four 18-base primer named as“M13 tag”was also synthesized labeled with either FAM，VIC，PET，or NED fluorescent dye.PCR amplification of genomic DNA was performed in a 10 μL reaction volume in an Veriti Thermal Cycler PCR（Applied Biosystems，F(xiàn)oster City，CA，USA）containing 10 ng genomic DNA，1×PCR Master Mix（AmpliTaq Gold 360），5 pmol forward primer（tailed with M 13 tag），15 pmol reverse primer，and 15 pmol“M13 tag”.The PCR programs employed initial denaturation for 5 min at 95°C followed by 35 cycles of denaturation for 1 min at 94°C，annealing for 45 s at 48-52°C（primer-specific）and extension for 1 min at 72°C.These were followed by 10 cycles of denaturation for 30 s at 95°C，annealing for 45 s at 53°C，and extension for 45 s at 72°C followed by a final extension for 12 min at 72°C.The PCR products were separated by capillary electrophoresis using the ABI 3730xl DNA Analyzer（Applied Biosystems，F(xiàn)oster City，CA，USA）.After PCR amplification confirmation on 1.5%agarose gel，post-PCR multiplex sets were constructed based on fluorescence-labeled primer dyes.For post-PCR multiplexing，1 μL of FAM and 2 μL each of VIC，NED，and PET-labeled PCR products representing different SSRs were mixed with 60 μL water.The mixed product（2 μL）was then added to 8 μL of Hi-Di formamide containing 0.20 μL GeneScan 600 LIZas internalsize standard，denatured for 5 min at 95°C，quick-chilled on ice for 5 min，and loaded on the ABI3730xl DNA Analyzer for electrophoresis.SSR amplicon size was determined with GeneMapper 4.0 software（Applied Biosystems，USA）.

2.4.Data acquisition and statistical analyses

The PCR-amplified SSR markers were scored by allele size as well as presence（1）or absence（0）.Statistical analysis for the calculation of observed heterozygosity（Ho），gene diversity or expected heterozygosity（He），major allele frequency，and polymorphic information content（PIC）of EST-SSR markers was performed with Power Marker 3.25［19］.PIC was calculated following Botstein et al.［20］:where Piand Pjare the frequencies of the i th and j th alleles. A pairwise similarity matrix among the accessions was calculated with the 0-1 data matrix using Jaccard's coefficient.This matrix was used to construct a dendrogram using the unweighted pair-group method with arithmetic mean（UPGMA）with NTSYS-pc 2.2［21］.

3.Results

3.1.Frequency and distribution of EST-SSRs

A total of 1316 raw EST sequences varying in length from 105 to 1147 bases（average 525）were downloaded，cleaned，and assembled（Table 2）.The assembly resulted in 155 contigs and 653 singletons.The length of contigs varied between 409 and 2118 bp with an average of 743 bp.Of the 808 assembled sequences，110 were found to contain 130 SSRs，with a frequency of 1 SSR/3.4 kb of the available ESTs.Of the 110 SSR-containing ESTs，14（13%）contained more than one SSR，and 15 SSRs were found in compound form.Among the types of SSRs，the highest proportion was represented by dinucleotide repeat（DNR）（72.3%），followed by trinucleotide（TNR）（21.5），tetranucleotide（3.8%），and hexanucleotide（2.3%）repeats（Table 3）.No pentanucleotide SSR was identified under the criteria used for the SSR search.The majority of the SSR motifs were of smaller repeat length and only 8 were found to contain 10 or more repeats（Table 3）.The most common type of SSR motif was AG/CT（43.8%），followed by AT/AT（16.2%），AC/GT（12.3%），AAG/CTT（10.8%），and AAT/ATT（3.8%）（Table 3）.

3.2.Polymorphism analysis and cross-species transferability

All of the 110 SSR-containing sequences were used to design primers flanking the SSR motif.We successfully designed primers for 68（62%）SSR-containing sequences（Table S1）.The remaining 42（38%）SSR containing sequences were not found suitable for designing primers，owing either to marginal SSRs or inappropriate flanking sequences.All the 68 primer pairs were synthesized and characterized for various marker attributes with 13 accessions of M.piperita and also used to assess cross-species transferability in four species of Mentha. Of the 68，54 primer pairs produced clear amplicons of the expected sizes.The 54 EST-SSRs amplified with M.piperita accessions resulted in 33 polymorphic SSRs with a total of 77alleles and 21 monomorphic SSRs（Table 4，F(xiàn)ig.S1）.The number of alleles varied from 2 to 4 with an average of 2.33 alleles/SSR.The PIC values of polymorphic SSRs ranged between 0.13 and 0.51 with an average of 0.325±0.09.The primer pair EMM_003 showed the highest PIC（0.51），followed by EMM_049（0.48），EMM_055（0.36），and EMM_41（0.36）.The expected heterozygosity（He）varied from 0 to 0.69（EMM_026）with an average of 0.21（Table 4）.The potential cross-species transferability of the 54 EST-SSRs was assessed in four species of Mentha including five accessions of M.arvensis，four of M.spicata，and one each of M.citrata and M.longifolia.Among these，M.arvensis showed the highest EST-SSR transferability（87.0%）followed by M.spicata（83.0%），M.longifolia（55.0%），and M.citrata（37.0%）.

Table 2-Details of ESTs and SSRs identified in M.piperatai.

Table 3-SSR frequencies by repeat type and motif in M.piperita.

3.3.Genetic diversity analysis

Jaccard's similarity coefficients for 24 mint accessions were calculated based on the genotypic data for 54 ESTSSRs and used to prepare a dendrogram（Fig.1）.The genetic similarity coefficient varied from 0.32 to 0.87 with an average of 0.57±0.12.The maximum similarity was found between CIM-Indus and CIMAP-Patra（0.87）followed by Kosi and MAS-10-11-45（0.84），MPS-16 and MPS-20（0.83），and MPS-21 and MPS-20（0.81）.The minimum similarity or maximum dissimilarity was found between M.citrata and MPS-16（0.32）and between M.longifolia and MPS-20（0.32）. UPGMA clustering classified all the accessions into three major clusters（Fig.1）.Cluster I was the largest，containing all the accessions of M.piperita and M.arvensis.This cluster could be further subdivided into two subclusters，one containing all the accessions of M.piperita and other all the accessions of M.arvensis.The main cluster II contained all four accessions of M.spicata.The species M.citrata and M. longifolia grouped together as an outgroup（cluster III）in the dendrogram.

4.Discussion

Among various molecular markers，SSR markers have been widely exploited for several plant genetics and breeding studies and applications，including evaluation of genetic relationship between individuals，tagging useful genes/alleles，linkage/QTL mapping，and phylogenetic analyses. Their features including hypervariability，multiallelic nature，wide genome coverage，relative abundance，and amenability to automation and high-throughput genotyping make SSRs preferential markers［22］.In the present investigation，we exploited the publically available EST database of M.piperita to develop EST-SSRs for various genetic studies.A total of 110（8.4%） of the M.piperita EST sequences contained microsatellites，yielding 130 SSRs.This was a relatively high abundance of SSRs as compared to those reported earlier for maize（1.4%），barley（3.4%），sorghum （3.6%），and rice（4.7%）［23］.However，it was lower than those reported for tea（15.5%）［24］，castor bean（28.4%）［25］，and opium poppy（18.8%）［26］. The relative frequency of EST-SSRs was found to be 1/3.4 kb，comparable to those reported for pepper（1/3.8 kb）［27］，and tea（1/3.5 kb）［28］and much higher than those reported for lotus（1/13.0 kb）［29］，wheat（1/15.6 kb）［23］，tomato（1/11.1 kb）［30］，and lily（1/15.9 kb）［31］.However，comparing the frequency and abundance of SSRs reported in different plant species may not give conclusive information，as these values are dependent on SSR search criteria，size of data set，database mining tools，and EST sequence redundancy［15］.DNR and TNR have been reported to be the predominant types of repeat motif in EST-SSRs in several plants，but the most abundant motif varied with species［15］.In M.piperita EST-SSRs，DNR was the most dominant motif（72.3%）followed by TNR（21.5%），with both accounting for 93.8%of EST-SSRs.A high proportion of DNR was also reported in EST-SSRs in coffee［32］，lotus［29］，cassava［33］，and Jatropha［34］.However，TNR was reported as the most common repeat motif in EST-SSRs in citrus［35］，Hawaiian mint［36］，peanut［37］，and lily［31］.The EST dataset used in the present investigation was small，and accordingly various features of EST-SSRs such as frequency，abundance，and motif type might vary if a larger dataset were used.AG/CT（43.8%）and AAG/CTT（10.8%）were the most common DNR and TNR respectively.Similar findings have also been reported earlier by Cardle et al.［30］，Kantety et al.［23］，Raji et al.［33］，Pan et al.［29］，Zhang et al.［38］，Akash and Myers［39］.In the present study no GC/GC repeat motif SSRs were identified. The GC/GC motif was also not reported in EST-SSRs of Medicago truncatula［40］，cassava［33］，coffee［32］，peanut［37］，sesame［37］，or alfalfa［41］.

The 68 SSR flanking primers developed in the present study showed a higher rate of successful primer design（62.0%）than reported for alfalfa（14.0%）［41］，sesame（49%）［38］，or faba bean（29.0%）［39］，but lower than reported for opium poppy（86.4%）［26］.Of 54 primers，33 amplified across 13 accessions of M.piperita，showing 61.0%of polymorphism. The percent polymorphism in the present investigation wasfound to be higher than that observed across 24 accessions of sesame（11.6%）［38］but lower than that reported among 28 alfalfa accessions（97.0%）［41］and among 37 accessions of opium poppy［26］.The polymorphism found in the EST-SSRs of M.piperita suggested the potential of these markers for use in future genetic studies.However，most of the SSRs showedlow PIC values，with an average of 0.25±0.09.The low average PIC value may reflect the small number of accessions surveyed or low allelic polymorphism.The utility of the SSRs could be expanded by studying their transferability to closely associated genera/species.Transferability of EST-SSRs has been reported in many crop plants，including sugarcane［42］，pear［43］，Prunus［44］，lettuce［45］，alfalfa［41］，faba bean［39］，and opium poppy［26］.M.arvensis and M spicata showed the highest transferability，indicating the closeness of their relationship to M.piperita.This close relationship has also been previously established and reported by Khanuja et al.［8］，Gobert et al.［11］，and Shiran et al.［10］based on RAPD and AFLP analysis.Reports of the interspecific hybrids M.arvensis×M.spicata［2］and M.arvensis×M.piperita［46］also support these findings.The EST-SSRs developed in the present study were also used to evaluate genetic relatedness among accessions and species of Mentha.The EST-SSRs were able to distinguish different accessions within and among Mentha species.All 13 accessions of M.piperita were grouped in one cluster and accessions of M.arvensis clustered together. The adjacent clustering of M.arvensis with M.piperita further supports their close relatedness.Likewise，4 accessions of M.spicata clustered together and M.citrata and M.longifolia did not group with any cluster.This result indicates that the EST-SSRs reported here have potential for various genetic studies in Mentha.The dendrogram clustering of M.citrata and M.longifolia close to M.spicata and M.arvensis indicates some common ancestry.Gobert et al.［11］showed high similarity between M.spicata and M.longifolia based on AFLP analysis.

Table 4-Details of 54 EST-SSRs of M.piperita and their cross-species transferability among four other species of Mentha.

Fig.1-UPGMAdendrogramof Mentha species/accessions.Genetic distance was based on Jaccard's similarity coefficientcalculated from EST-SSR data.

In the present study，we have developed EST-SSRs using the M.piperita EST database.The EST-SSRs showed a high level of polymorphism and were transferable across several species of Mentha.Genetic relatedness among different accessions and species of Mentha was established using the EST-SSRs.The present study demonstrates the potential of EST-SSRs for genetic and phylogenetic studies in Mentha.

Supplementary material

Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.cj.2015.0x.00x.

［1］N.K.Patra，H.Tanveer，N.K.Tyagi，S.Kumar，Cyto-taxonomical status and genetical and breeding perspectives of Mentha，J.Med.Arom.Plant Sci.22（2000）419-430.

［2］N.K.Patra，H.Tanveer，S.P.S.Khanuja，A.K.Shasany，H.P.Singh，V.R.Singh，S.Kumar，A unique interspecific hybrid spearmint clone with growth properties of Mentha arvensis L.and oil qualities of Mentha spicata L，Theor.Appl.Genet.102（2001）471-476.

［3］S.Behnam，M.Farzaneh，M.Ahmadzadeh，A.S.Tehrani，Composition and antifungal activity of essential oils of Mentha piperita and Lavendula angustifolia on post-harvest phytopathogens，Commun.Agric.Appl.Biol.Sci.71（2006）1321-1326.

［4］S.Kizil，N.Hasimi，V.Tolan，E.Kilinc，U.Yuksel，Mineral content，essential oil components and biological activity of two mentha species（M.piperita L.，M.spicata L.），Turk.J.Field Crops.15（2010）148-153.

［5］W.A.Court，R.C.Roy，R.Pocs，Effect of harvest date on the yield and quality of the essential oil of peppermint，Can.J. Plant Sci.73（1993）815-824.

［6］S.S.Mahmoud，R.B.Croteau，Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase，Proc.Natl.Acad.Sci.U.S.A.100（2003）14481-14486.

［7］R.B.Croteau，E.M.Davis，K.L.Ringer，M.R.Wildung，Menthol biosynthesis and molecular genetics，Naturwissenschaften 92（2005）562-577.

［8］S.P.S.Khanuja，A.K.Shasany，A.Srivastava，S.Kumar，Assessment of genetic relationships in Mentha species，Euphytica 111（2000）121-125.

［9］A.L.Fenwick，S.M.Ward，Use ofrandomamplified polymorphic DNA markers for cultivar identification in mint，Hort.Sci.36（2001）761-764.

［10］B.Shiran，M.Soheila，R.Khorshid，Assessment of genetic diversity among Iranian mints using RAPD markers，in:J. Vollmann，H.Grausgruber，P.Ruckenbauer（Eds.），Genetic Variation for Plant Breeding.Proceedings of the 17th EUCARPIA General Congress，Tulln，Austria，8-11 September，2004，2004，pp.121-125.

［11］V.Gobert，S.Moja，M.Colson，P.Taberlet，Hybridization in the section Mentha（Lamiaceae）inferred from AFLP markers，Am. J.Bot.89（2002）2017-2023.

［12］D.Field，C.Wills，Abundant microsatellite polymorphism in Saccharomyces cerevisiae，and the different distributions of microsatellites in eight prokaryotes and S.cerevisiae，result from strong mutation pressures and a variety of selective forces，Proc.Natl.Acad.Sci.U.S.A.95（1998）1647-1652.

［13］W.Powell，G.Machray，J.Provan，Polymorphism revealed by simple sequence repeats，Trends Plant Sci.1（1996）215-222.［14］P.K.Gupta，S.Rustgi，S.Sharma，R.Singh，N.Kumar，H.S. Balyan，Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat，Mol. Gen.Genomics.270（2003）315-323.

［15］R.K.Varshney，A.Graner，M.E.Sorrells，Genic microsatellite markers:features and applications，Trends Biotechnol.23（2005）48-55.

［16］T.Thiel，W.Michalek，R.K.Varshney，A.Graner，Exploiting EST database for the development and characterization of gen-derived SSR-markers in barley（Hordeum vulgare L.），Theor.Appl.Genet.10（2003）6411-6422.

［17］X.Huang，A.Madan，CAP3:a DNA sequence assembly program，Genome Res.9（1999）868-877.

［18］M.Schuelke，An economic method for the fluorescent labeling of PCR fragments，Nat.Biotechnol.18（2000）233-234.［19］K.Liu，S.V.Muse，Power marker:integrated analysis environment for genetic marker data，Bioinformatics 21（2005）2128-2129.

［20］D.Botstein，R.L.White，M.Skolnick，R.W.Davis，Construction of genetic linkage map in man using restriction fragment length polymorphisms，Am.J.Hum.Genet.32（1980）314-331.［21］F.J.Rohlf，NTSYS-pc Version 2.2:Numerical Taxonomy and Multivariate Analysis System，Exeter Software，New York，2000.

［22］R.K.Kalia，M.K.Rai，S.Kalia，R.Singh，A.K.Dhawan，Microsatellite markers:an overview of the recent progress in plants，Euphytica 177（2011）309-334.

［23］R.V.Kantety，M.L.Rota，D.E.Mathews，M.E.Sorrells，Data mining for simple sequence repeats in expressed sequence tags from barely，maize，rice，sorghum and wheat，Plant Mol. Biol.48（2002）501-510.

［24］J.Q.Ma，Y.H.Zhou，C.L.Ma，M.Z.Yao，J.Q.Jin，Identification and characterization of 74 novel polymorphic EST-SSR markers in the tea plant，Camellia sinensis（Theaceae），Am.J. Bot.97（2010）153-156.

［25］L.Qui，C.Yang，B.Tian，J.B.Yang，A.Liu，Exploiting EST databases for the development and characterization of EST-SSR markers in castor bean（Ricinus communis L.），BMC Plant Biol.10（2010）278.

［26］H.Selale，I.Celik，V.Gultekin，J.Allmer，S.Doganlar，A. Frary，Development of EST-SSR markers for diversity and breeding studies in opium poppy，Plant Breed.132（2013）344-351.

［27］G.Yi，J.M.Lee，S.Lee，D.Choi，B.D.Kim，Exploitation of pepper EST-SSRs and an SSR-based linkage map，Theor.Appl.Genet. 114（2006）113-130.

［28］R.K.Sharma，P.Bhardwaj，R.Negi，T.Mohapatra，P.S.Ahuja，Identification，characterization and utilization of unigene derived microsatellite markers in tea（Camellia sinensis L.），BMC Plant Biol.9（2009）53.

［29］L.Pan，Q.Xia，Z.Quan，H.Liu，W.Ke，Y.Ding，Development of novel EST-SSRs from sacred lotus（Nelumbo nucifera Gaertn）and their utilization for the genetic diversity analysis of N.nucifera，J.Hered.101（2010）71-82.

［30］L.Cardle，L.Ramsay，D.Milbourne，M.Macaulay，D.Marshall，R.Waugh，Computational and experimental characterization of physically clustered simple sequence repeats in plants，Genetics 156（2000）847-854.

［31］S.Yuan，G.Liang，C.Liu，J.Ming，The development of EST-SSR markers in Lilium regale and their cross-amplification in related species，Euphytica 189（2013）393-419.

［32］R.K.Aggarwal，P.S.Hendre，R.K.Varshney，P.K.Bhat，V. Krishnakumar，L.Singh，Identification，characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species，Theor.Appl. Genet.114（2007）359-372.

［33］A.A.Raji，J.V.Anderson，O.A.Kolade，C.D.Ugwu，A.G. Dixon，I.L.Ingelbrecht，Gene-based microsatellites for cassava（Manihot esculenta Crantz）:prevalence，polymorphisms，and cross-taxa utility，BMC Plant Biol.9（2009）118.

［34］H.K.Yadav，A.Ranjan，M.H.Asif，S.Mantri，S.V.Sawant，R. Tuli，EST-derived SSR markers in Jatropha curcas development，characterization，polymorphism，and transferability across the species/genera，Tree Genet. Genomes 7（2011）207-219.

［35］C.Chen，P.Zhou，Y.Choi，S.Huang，F(xiàn).Gmitter，Mining and characterizing microsatellites from citrus ESTs，Theor.Appl. Genet.112（2006）1248-1257.

［36］C.Lindqvist，A.C.Scheen，M.J.Yoo，P.Grey，D.G. Oppenheimer，J.H.L.Mack，D.E.Soltis，P.S.Soltis，V.A.Albert，An expressed sequence tag（EST）library from developing fruits of an Hawaiian endemic mint（Stenogyne rugosa，Lamiaceae）:characterization and microsatellite markers，BMC Plant Biol.6（2006）16.

［37］X.Liang，X.Chen，Y.Hong，H.Liu，G.Zhou，S.Li，B.Gu，Utility of EST-derived SSR in cultivated peanut（Arachis hypogaea L.）and Arachis wild species，BMC Plant Biol.9（2009）35.

［38］H.Zhang，W.Libin，M.Hongmei，Z.Tide，W.Cuiying，Development and validation of genic-SSR markers in sesame by RNA-seq，BMC Genomics 13（2012）316.

［39］M.W.Akash，G.O.Myers，The development of faba bean expressed sequence tag-simple sequence repeats（EST-SSRs）and their validity in diversity analysis，Plant Breed.131（2012）522-530.

［40］I.M.Eujayl，K.Sledge，L.Wang，G.D.May，K.Chekhovskiy，Medicago truncatula EST-SSRs reveal cross-species genetic markers for Medicago spp，Theor.Appl.Genet.108（2004）414-422.

［41］Z.Wang，Y.Hongwei，F(xiàn).Xinnian，L.Xuehui，G.Hongwen，Development of simple sequence repeat markers and diversity analysis in alfalfa（Medicago sativa L.），Mol.Biol.Rep. 40（2013）3291-3298.

［42］G.M.Cordeiro，R.Casu，C.L.McIntyre，J.M.Manners，R.J.Henry，Microsatellite markers from sugarcane（Saccharum spp.）ESTs cross transferable to Erianthus and Sorghum，Plant Sci.160（2001）1115-1123.

［43］L.Fan，M.Y.Zhang，Q.Z.Liu，T.L.Li，Y.Song，L.F.Wang，S.L. Zhang，J.Wu，Transferability of newly developed pear ssr markers to other rosaceae species，Plant Mol.Biol.Report.31（2013）1271-1282.

［44］M.Mnejja，M.J.Garcia，J.M.Audergon，P.Arús，Prunus microsatellite marker transferability across rosaceous crops，Tree Genet.Genomes 6（2010）689-700.

［45］I.Simko，Development of EST-SSR markers for the study of population structure in lettuce（Lactuca sativa L.），J.Hered.100（2009）256-262.

［46］B.Kumar，A.K.Shukla，A.Samad，Development and characterization of menthofuran-rich inter-specific hybrid peppermint variety CIMAP-Patra，Mol.Breed.34（2014）717-724.

*Corresponding author at:CSIR-National Botanical Research Institute，Rana Pratap Marg，Lucknow-226 001，India.Tel.:+91 522 2297938.

E-mail address:h.yadav@nbri.res.in（H.K.Yadav）.

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science，CAAS.

http://dx.doi.org/10.1016/j.cj.2015.02.002

2214-5141/?2015 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.