Development of a panel of unigene-derived polymorphic EST-SSR markers in lentil using public database information

Dbjyot Sn Gupta,Pn Cn,Gaurav Sablok,Dl Tavaraja, Pusparaja Tavaraja,Clarc J.Coyn,SvKumar,Mcal Baum,Rbcca J.McG*

aDepartment of Plant Sciences，North Dakota State University，Fargo，ND 58108，USA

bICAR-ARS，Indian Institute of Pulses Research，Kanpur，208024，UP，India

cDivision of Plant Sciences，University of Missouri，Columbia，MO 65211-7140，USA

dDepartment of Biodiversity and Molecular Ecology，Research and Innovation Centre，Fondazione Edmund Mach，Via E.Mach 1，38010 San Michele all'Adige，Trento，Italy

eAgricultural and Environmental Sciences，Clemson University，Clemson，SC 29634，USA

fInternational College Beijing，China Agricultural University，Beijing，China

gUSDA-ARS，Western Regional Plant Introduction Station，Washington State University，Pullman，WA 99164，USA

hBIGM Program，International Center for Agricultural Research in the Dry Areas（ICARDA），Rabat-Instituts，Rabat，Morocco

iBIGM Program，International Center for Agricultural Research in the Dry Areas（ICARDA），Beirut，Lebanon

jUSDA-ARS，Grain Legume Genetics and Physiology Research Unit，Washington State University，Pullman，WA 99164，USA

Development of a panel of unigene-derived polymorphic EST-SSR markers in lentil using public database information

Debjyoti Sen Guptaa,b,1,Peng Chengc,1,Gaurav Sablokd,Dil Thavarajahe, Pushparajah Thavarajahf,Clarice J.Coyneg,ShivKumarh,Michael Baumi,Rebecca J.McGeej,*

aDepartment of Plant Sciences，North Dakota State University，Fargo，ND 58108，USA

bICAR-ARS，Indian Institute of Pulses Research，Kanpur，208024，UP，India

cDivision of Plant Sciences，University of Missouri，Columbia，MO 65211-7140，USA

dDepartment of Biodiversity and Molecular Ecology，Research and Innovation Centre，Fondazione Edmund Mach，Via E.Mach 1，38010 San Michele all'Adige，Trento，Italy

eAgricultural and Environmental Sciences，Clemson University，Clemson，SC 29634，USA

fInternational College Beijing，China Agricultural University，Beijing，China

gUSDA-ARS，Western Regional Plant Introduction Station，Washington State University，Pullman，WA 99164，USA

hBIGM Program，International Center for Agricultural Research in the Dry Areas（ICARDA），Rabat-Instituts，Rabat，Morocco

iBIGM Program，International Center for Agricultural Research in the Dry Areas（ICARDA），Beirut，Lebanon

jUSDA-ARS，Grain Legume Genetics and Physiology Research Unit，Washington State University，Pullman，WA 99164，USA

A R T I C L E I N F O

Article history：

29 June 2016

Accepted 11 July 2016

Available online 26 July 2016

Lens culinaris

EST-SSRs

Functional annotation

Unigene sequences

EST database

Genetic resources

Lentil（Lens culinarisMedik.），a diploid（2n=14）witha genomesizegreaterthan4000 Mbp，isan important cool season food legume grown worldwide.The availabilityof genomic resources is limited inthiscropspecies.Theobjectiveof thisstudy wastodeveloppolymorphicmarkersin lentil using publicly available curated expressed sequence tag information（ESTs）.In this study，9513 ESTs were downloaded from the National Center for Biotechnology Information（NCBI）database to develop unigene-based simple sequence repeat（SSR）markers.The ESTs were assembled into 4053 unigenes and then analyzed to identify 374 SSRs using the MISA microsatellite identification tool.Among the 374 SSRs，26 compound SSRs were observed. Primer pairs for these SSRs were designed using Primer3 version 1.14.To classify the functional annotation of ESTs and EST-SSRs，BLASTx searches（using E-value 1×10-5）against the public UniProt（http://www.uniprot.org/）and NCBI（http://www.ncbi.nlh.nih.gov/）databases were performed.Further functional annotation was performed using PLAZA（version 3.0）comparative genomics and GO annotation was summarized using the Plant GO slim category.Among the synthesized 312 primers，219 successfully amplified Lens DNA.A diverse panel of 24 Lens genotypes was used to identify polymorphic markers.A polymorphic set of 57 markers successfully discriminated the test genotypes.This set of polymorphic markers with functional annotation data could be used as molecular tools in lentil breeding.

Production and hosting by Elsevier B.V.on behalf of Crop Science Society of China and

Institute of Crop Science，CAAS.This is an open access article under the CC BY-NC-ND

license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.

1.Introduction

Lentil（Lens culinaris L.Medik.）is a nutritious food legume crop grown throughout the world.Primary production regions include Canada，Australia，northwestern USA，Turkey，Syria，and the Indian subcontinent（Nepal，India，and Bangladesh）. World annual production is nearly five million tons［1］.Lentil is classified into several market classes，including extra small red，small red，large red，small green，medium green，large green，Spanish brown，zero tannin，and Puy.

Lentil originated in the Fertile Crescent（southwest Asia and Mediterranean region）and is believed to be one of the earliest domesticated food crops［2］.The cultivated lentil，L.culinaris，has two types，macrosperma and microsperma，based on seed and pod characteristics［3］.Like other food legumes，lentil has a narrow genetic base.Realization of potential yield is limited by various biotic and abiotic stresses such as foliar and root diseases，highorlowtemperature，soilpH（＜5.4），and waterlogging.Optimization of crop management is also important，as weed management，water availability，and soil fertility vary among growing environments.Breeding programs worldwide are working to breed high-yielding lentil cultivars with resistance to one or more of these stresses.Many breeding programs have implemented marker-assisted selection to speed up the selection process.

Availability of molecular markers and their ease of use in large breeding programs is a priority for many crop species. However，because of the unavailability of a full genome sequence as well as the complexity of the large（4063 Mbp）genome［4］，the number of available polymorphic markers is limited in lentil.Hamweigh et al.［5］developed 14microsatellite markers from a genomic library developed from the lentil cultivar“ILL5588”.The genetic diversity index calculated from the number of alleles amplified was high and markers could accurately discriminate cultivated from wild types.In another study，Kaur et al.［6］developed expressed sequence tag-simple sequence repeat（EST-SSR）markers by transcriptome sequencing of lentil and validated 79 polymorphic EST-SSRs among 13 lentil genotypes including one Lens nigricans accession. Verma et al.［7］developed EST-SSRs by transcriptome sequencing of the lentil genotype“Precoz”［8］and validated 54 polymorphic EST-SSRs among 22 lentil genotypes including one L.culinaris subsp.orientalis and two Lens lamottei genotypes.

The total number of lentil ESTs（9513）in the National Center for Biological Information （NCBI）database has remained constant.Development of genomic or transcriptome libraries is expensive and time-consuming.Researchers working in various crop species including rice［9］，wheat［9］，maize［10］，chickpea［10］，faba bean［11］，pea［12］，tea tree［13］，and Medicago［14］have developed polymorphic markers using sequence information available in public databases.

TheuseofgenicSSRmarkersorEST-SSRsisimportantfroma breeding point of view.Despite recent advances in molecular markers such as single-nucleotide polymorphisms（SNPs）or DNA array-based markers，SSRs hold promise as breederfriendly markers involving limited technical or operating difficulties.SSR markers are reproducible and PCR-based，resulting in easy application in breeding programs for marker-assisted selection or prediction of breeding values.Public databases such as NCBI NR（http://www.ncbi.nlm.nih.gov/），UniProt（http:// www.uniprot.org/），and TAIR（http://www.arabidopsis.org/）can be used to functionally annotate the ESTs or EST-SSRs.This algorithm-based or alignment-based prediction of gene functions can be verified in trait-specific cases.The synchronization between functional annotation and wet-lab validation depends largely on the standard of draft sequence available.Functional annotation of the SSRs provides an opportunity for expression analysis of specific genes.

The objectives of this study were to（1）develop polymorphic SSR markers in lentil using EST sequences，（2）validate polymorphic EST-SSR markers in a diverse panel of Lens genotypes including wild lentil species，and（3）functionally annotate the EST-SSRs using public protein databases.

Table 1-Summary of data mining of unigene sequences of Lens culinaris.

2.Materials and methods

2.1.EST sequence assembly,SSR detection,and functional annotation

Microsatellite or SSRs were developed from 9513 expressed sequence tags（ESTs）downloaded from the NCBI database using the queries“Lens culinaris”［Organism］OR Lens culinaris［All Fields］AND “Lens culinaris”［porgn］（Table 1）.ESTs representing（“Lens culinaris/Colletotrichum truncatum mixed EST library”［porgn:__txid880151］）were excluded and only L.culinaris-specific ESTs were used for the analysis.The downloaded ESTs were cleaned of contamination using UniVec（http://www.ncbi.nlm.nih.gov/tools/vecscreen/univec）.Cleaned ESTs were assembled using the Overlap-Layout-Consensus assembler MIRA（parameters:job=denovo，est，accurate，454 using the-notraceinfo option）［15］.Following the MIRA assembly，unigenes were created using CAP3［16］with parameters -p 95，-o 49，and-t 10，000 as previously described［17-19］.In addition to the parameters described in Zheng et al.［18］，the parameter-t was assigned a value of 10，000，a choice that can substantially improve the quality of the assembly using the maximumavailablememory.Thisdecisionavoidedthe misassembly of the ESTs and formation of counterfeit long assemblies，as previously suggested［18，19］.The assembled unigenes were searched for SSRs using MISA［20］（http://pgrc. ipk-gatersleben.de/misa/）.SSRs were defined by a minimum repeat sequence of 10 nucleotides as mono-，a sequence of six consecutive repeat units as di-，and a sequence of five repeat units for tri-，tetra-，penta-and hexanucleotide sequences.To identify and classify compound repeats，theminimum distancebetween two repetitive units was kept at≤100 bp as suggested in MISA［20］.Open reading frames were extracted from the assembled unigenes using the getorf function of the EMBOSS package（http://emboss.sourceforge.net/）.

2.2.Database mining

2.2.1.Development of EST-SSRs and primer design

Following the identification of SSRs，primer pairs were designed using Primer3 version 1.1.4（http://primer3.sourceforge.net/）withminimumandmaximumampliconsize100-300 bp，primer size（minimum，optimum，and maximum）18-27 bp，primer Tm（minimum，optimum，and maximum）57-63°C，primer GC content 30-70%，CG clamp 0，maximum end stability 250，maximum Tmdifference 2，maximum self-complementarity 6，maximum 39 self-complementarity 3，maximum Ns accepted 0，and maximum poly-X 5.

2.2.2.Functional annotation of unigenes and EST-SSRs

Functional annotation and gene ontology of the ESTs and EST-SSRs were performed using BLASTx searches（E-value，1×10-3）againstGenBank （http://www.ncbi.nlm.nih.gov/），UniProt（http://www.uniprot.org/），and TAIR（https://www. arabidopsis.org/）databases.Additional functional annotation and gene ontology were obtained using FastAnnotator［21］.GO annotations obtained were further analyzed using GO-SLIM（Plant）（http://www.geneontology.org/ontology/subsets/goslim_ plant.obo）and functional GO-SLIM categories were defined.

Table 2-Details of plant materials used.

2.3.Plant materials and DNA extraction

Four Lens genotypes were used for initial screening of 312 primers，including three L.culinaris（Red Chief，ILL669，and WA8649041）and one L.nigricans（PI 572340）genotype.A diverse panel of 22 Lens genotypes，consisting of L.culinaris advanced breeding lines，parents of mapping populations，wild taxa，and genotypes of L.nigricans，L.culinaris ssp.orientalis，and L.lamottei was used to identify polymorphic markers among primers amplifying Lens DNA（Table 2）.DNA samples were extracted from individual plant leaf tissue（100 mg）when seedlings were twoweeks oldusing a DNeasy Plant MiniKit（QIAGEN，Valencia，CA，USA）.The DNA concentrations of the extracted samples were recorded using a Nanodrop 2000c spectrophotometer（Nanodrop，Wilmington，DE USA）.The extracted DNA samples were diluted to a uniform concentration of 20 μg μL-1for successful PCR amplification.

Table 3 - Tm， allele size， and polymorphism information content （PIC） of expressed sequenced tagged-simple sequence repeat （EST-SSRs） polymorphic primers； putative functions were assigned based on BLASTx search against the nr protein database （NCBI）.

（continued on next page）

Table 3 （continued）

2.4.PCR amplification

Three hundred and twelve primer pairs were synthesized by Eurofins MWG Operon（Louisville，KY，USA）and used in this study.PCR reactions（25 μL volume）were conducted in an ABI 7500（Applied Biosystems，Foster，CA，USA）thermocycler，with each reaction containing 2.5 μL of Taq buffer，1.5 μL of MgCl2（25 mmol L-1），0.20 mmol L-1of each dNTP（all from Promega，Madison，WI，USA），0.50 mmol L-1of each primer（Eurofins MWG Operon），0.25 μL of Hot Start Taq polymerase（Promega）and 20 ng of template DNA.For initial screening of primers，touchdown PCRs were performed using DNA from four lentil genotypes and the following program:94°C for 3 min，followed by 18 cycles of 94°C for 50 s，65-55°C for 50 s，and 72°C for 50 s，followed by 20 cycles of 94°C for 50 s，55°C for 50 s，and 72°C for 50 s，and a final elongation of 72°C for 7 min.The PCR products were resolved in 2%agarose gels（molecular biology grade，Sigma，USA）and bands were scored using a gel documentation system.Primers amplifying Lens DNA were validated on a set of 22 diverse Lens genotypes in an ABI 7500 thermocycler using the following program:94°C for 5 min，followed by 42 cycles of 94°C for 1 min，50°C for 1 min，and 72°C for 1 min，followed by a final elongation step of 72°C for 5 min.Forward primers were tagged with M13 sequence（5′-CACGACGTTGTAAAACGAC-3′）at the 5′end. Four dyes were used to set up the multiplex PCR reactions. PCR products were separated using an ABI 3730xl（Applied Biosystems，Foster，CA，USA）according to the manufacturer's instructions with the addition of an ABI GeneScan LIZ500 size standard，and amplification product sizes were determined with GeneMapper v3.7 software（Applied Biosystems）.

3.Results

3.1.Assembly and SSR detection

A set of 9513 ESTs were downloaded in FASTA file format from NCBI［22］and clustered by an identity of 0.95 into 4106 unigene sequences.Unigenes shorter than 100 bp were removed and the homopolymer ends of unigenes longer than 100 bp were trimmed.MIRA assembly of the 9513 EST sequences ultimately generated 4053 unigene sequences.The total length of the analyzed sequences was 2，574，487 bases. MIRA detected 374 SSR-bearing EST sequences among these unigenes.Of these EST-SSRs there were 36 sequences with more than one SSR.Also，26 compound SSRs were observed. For further analysis，348 EST-SSRs were chosen.Using Primer3，primer pairs were designed for these 348 EST-SSRs（Table S1）.In addition，279 primer pairs（Table S2）were designed based on the plant GDB assembly（version 187a）of L.culinaris（http://www.plantgdb.org/download/download. php？dir=/Sequence/ESTcontig/Lens_culinaris/current_version）. These were designed based on the detected EST-SSRs，and were further e-validated using iPCRessipcress software（http://www.ebi.ac.uk/about/vertebrate-genomics/software/ ipcress-manual）.Primers co-amplifying products are listed in Table S2.In the validation experiment，312 primers were used: 48 primers from the e-validated list and 264 primers from Table S1.E-validated primers are coded with prefix“PUT”and other primers with“UN”（Table 3）.

3.2.Structural and functional annotation of ESTs and EST-SSRs

Contig length ranged between 199 and 2599 bp（Fig.S1）.The most prevalent contig length was 600-799 bp，followed by 400-599 and 800-999.Functional GO-SLIM analysis showed that the distribution of unigenes among GO，Domain，and Enzyme categories was 55.57%，39.91%，and 4.52%，respectively（Fig.S2）.This distribution was consistent with the categorization of the EST-SSRs into functional GO，Domain，and Enzyme categories，which accounted for 54.25%，42.38%，and 3.70%，respectively（Fig.S3）.In the GO category Biological Process，the first four processes were oxidation-reduction process，ribosome biogenesis，translation and regulation of transcription，and DNA dependent（Fig.S4）.A similar trend was observed using GO Biological Process analysis of the ESTSSRs，in which the ranking of processes was as follows: oxidation-reduction process，regulation of transcription，DNA dependent，ribosome biogenesis，and translation（Fig.S5）.The first four functions of the unigenes in the GO category Molecular Function were DNA binding，nutrient reservoir activity，structural constituent of ribosome，and zinc ion binding（Fig.S6）.However，in the GO category Molecular Function，for the EST-SSRs，the first four functions were ATP binding，DNA binding，structural constituent of ribosome，and zinc ion binding（Fig.S7）.In the GO category Cellular Process，the first four functions of the unigenes were nucleus，cytosol，plasma membrane and chloroplast（Fig.S8）and for the ESTSSRs the first four functions were cytosol，plasma membrane，nucleus，and integral to membrane（Fig.S9）.

3.3.Frequency and distribution of EST-SSRs

The frequency of SSRs was 6.89 per kb of sequence analyzed（374 SSRs/2575 kb of sequence）.There were 21 SSR repeat patterns（Fig.S10）.The most prevalent were trinucleotide，followed by mono-，tetra-，andpentanucleotide repeatpatterns. The most frequently observed repeat was AG/CT，followed by AAG/CTT，AAC/GTT，ATC/ATG，and AT/AT（Fig.S10）.

3.4.Validation of EST-SSRs

Amongthesynthesized312primers，219successfully amplified Lens DNA.A diverse panel of 22 Lens genotypes，consisting of L.culinaris advanced breeding lines，parents of mapping populations，wild taxa，and genotypes of L.nigricans，L.culinaris ssp.orientalis，and L.lamottei was tested to identify polymorphic markers.A total of 57 polymorphic primers were found.The number of alleles amplified ranged from 2 to 17 for each primer and the polymorphic information content（PIC）ranged between 0.10 and 0.91.The average number of alleles produced per primer was seven.

4.Discussion

MIRA assembly is very flexible.Short reads，such as ESTs，can easily be assembled into contigs and specific trimming further improves the quality of the sequences（http://mira-assembler. sourceforge.net/docs/DefinitiveGuideToMIRA.html）［23］.Thenumber of SSR-containing sequences detected was very high compared to those in other studies；however，they yielded fewer polymorphic markers.These polymorphic markers successfully discriminated the test genotypes and grouped genetically more closely related individuals.Simple sequencebased markers are the most robust and easy to use in marker systems.Moreover，sequencing-based gel separation technologies can now detect differences of a very few bases among alleles.It can be seen from the results of the present experiment that each polymorphic marker generated multiple alleles and that for several，up to 17 alleles were observed. Similar results were obtained with other crops for which EST databases were used to develop robust genic SSR markers［10-14，29］.Recently，Kumar et al.［25］reviewed the recent development of genic SSR markers in lentil［6，7，26，27］and Andeden et al.［28］developed 78 polymorphic SSR markers in lentil.However，the number of polymorphic genic SSR markers is still limited.Kaur et al.［6］validated a subset of de novo discovered 192 EST-SSR markers across a panel of 12 cultivated lentil genotypes，observing 47.5%polymorphism using a set of 2393 EST-SSR markers.Kaur et al.［26］found 40 polymorphic markers after testing 516 EST-SSRs.Andeden et al.［28］developed（CA）n，（GA）n，（AAC）n，and（ATG）n repeatenriched libraries and by sequencing these libraries found 78 polymorphic SSR markers using a set of 15 Turkish lentil genotypes.They observed 21.6%polymorphism （of the 360 primers validated，78 were polymorphic）.In the present study，a test of 219 markers on 22 cultivated and wild lentil genotypes yielded 26%polymorphism.Verma et al.［7］reported 42.59%polymorphism in 54 markers tested on 22 lentil，Medicago，Glycine，and Vigna genotypes.The inclusion of additional genera contributed to the high percentage of polymorphism that they reported.The use of SSR markers for diversity analysis or grouping of genotypes based on genetic relatedness in lentil or other closely related food legumes has been reported by several workers［29-32］.Kaur et al.［6］and Verma et al.［7］also found comparable grouping ability of the test polymorphic markers.Verma et al.［7］and Andeden et al.［28］reported a lower number of alleles amplified per locus（2.3 and 5.1，respectively）than found in our study（7 alleles）.Wong et al.［33］classified four gene pools in lentil using genotyping by sequencing（GBS）of 60 genotypes.These were primary，secondary，tertiary，and quaternary gene pools，formed by L.culinaris/Lens orientalis/ Lens tomentosus，L.lamottei/Lens odemensis，Lens ervoides，and L. nigricans，respectively.

Thedistributionsoffunctionalannotationcategories were dissimilar between the total ESTs and EST-SSRs.It is noteworthy that reducing the detail of the GO categories improved the authenticity of the annotation data.It was observed that most functional annotations remain the same between the unigenes and EST-SSRs.Functional annotations of the EST-SSR flanking regions indicated the involvement of the translated portion of the genome.This finding is important for the development of functional markers in lentil. The lentil genome sequencing project is under way.The most recent draft（version 0.7）has approximately 150×coverage，producing scaffolds covering about half of the genome. The initial assembly resulted in useful SNPs suitable for marker-assisted selection［34］.

5.Conclusions

A polymorphic set of 57 markers was developed in lentil.Of these，14 amplified the same SSRs reported by Kaur et al.［6］. The markers were further validated among diverse lentil genotypes.They could be used by the lentil research community for molecular breeding.Development of dense genetic maps is a prerequisite for adoption of genomic tools in lentil［35］and recently EST-SSR and SNPs were mapped in lentil［26，36］.Lists of unigene sequences for the polymorphic markers are available in the Cool Season Food Legume database（https://www.coolseasonfoodlegume.org/）.

Supplementary data for this article can be found online at http://dx.doi.org/10.1016/j.cj.2016.06.012.

Acknowledgments

Financial assistance from ICARDA，Morocco，in the form of a brief project，and grant support from the Northern Pulse Growers Association and the USA Dry Pea and Lentil Council are gratefully acknowledged.Debjyoti Sen Gupta thanks the Indian Council of Agricultural Research，New Delhi，India for the ICAR International Fellowship for Doctoral Study.

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7 April 2016

in revised form

.

E-mail address:rjmcgee@wsu.edu（R.J.McGee）.

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

1Debjyoti Sen Gupta and Peng Cheng contributed equally to this work.

http://dx.doi.org/10.1016/j.cj.2016.06.012

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