Identification, development, and application of cross-species intron-spanning markers in lentil(Lens culinaris Medik.)

Dejyoti Sen Gupt*,Jitendr Kumr*,Sunnd GuptSonli DueyPriynk GuptNrendr Prtp Singh,Gurv Slokc

aDivision of Crop Improvement,ICAR-Institute of Pulses Research,Kanpur 208024,UP,India

bDivision of Plant Biotechnology,ICAR-Institute of Pulses Research,Kanpur 208024,UP,India

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

1.Introduction

Lentil(Lens culinaris Medik.)is one of the most important food legumes and is grown in many parts of the world including Canada,Australia,northwestern USA,Turkey,Syria,Nepal,India,and Bangladesh[1].The world annual production is nearly 5 Mt[1].Lentil originated in the Fertile Crescent and is reported to be one of the earliest domesticated food crops[2].Cultivation of lentil is affected by various biotic and abiotic stresses including foliar and root diseases,high temperature,drought,soil pH(＜5.4),and water logging.Most lentil-producing countries use conventional breeding approaches in their active breeding programs for developing high-yielding lentil cultivars with better grain quality.However,in recent years,molecular markers are being widely used for accelerating precise breeding in several crops including major pulse crops such as chickpea and pigeonpea[3].Further,the advent of next-generation sequencing technologies has allowed the rapid genome sequencing of pulses such as chickpea(Cicer arietinum)[4]and pigeonpea(Cajanus cajan)[5,6].Availability of the genome sequences in these species has permitted the development of genome-anchored maps,visualization of single nucleotide polymorphism SNPs,and identifying species-specific SNPs.In the past,limited efforts have been made toward the development of molecular marker systems such as simple sequence repeats(SSRs)and SNPs for enriching genomic resources in lentil[7–12].The plant genomes have genes with larger intronsand spliced alignment of transcripts to the genome has revealed a large diversity in intron size.Despite being of diverse lengths,introns have been a major resource for molecular-marker development in several crop species[13–16]and have been recently leveraged to develop marker resources for legumes through the development of intron-spanning markers(ISMs),which are codominant or dominant,reproducible markers that show multi-allelic patterns[13–16].Development of these markers is gaining importance in species for which a reference genome sequence is available.In lack of the reference genome cross-species reference genome mapping can be used to identify the intron-spanning markers.Briefly,development of intron-spanning markers canbedone by performing a spliced alignment of the gene transcripts to the reference genome and developing polymerase chain reaction(PCR)primers that are anchored in conserved exons that span target introns.Previously,intron-spanning markers were developed in legumes using CSGM Designer[17],which provides algorithm-or alignment-based identification of intron spanning markers and these can be validated on a set of diverse genotypes.Similarly,in lentil,transcript sequence databases available in the public domain can be used for de novo assembly and identification and design of primers for the amplification of ISM regions.The objectives of this study were to(1)develop polymorphic ISMs in lentil using expressed sequence tag(EST)sequences,and(2)validate polymorphic ISM markers in a diverse panel of Lens genotypes including wild lentil species.

2.Materials and methods

2.1.Development of intron-spanning markers,primer design,and functional annotation

A cross-species mapping-based approach was used for developing intron-spanning markers.In this approach,a well annotated and curated reference genome of Medicago truncatula was used because of its close phylogenetic positioning with L.culinaris.The L.culinaris putative unique transcripts(PUTs)from Plant GDB version 187[18]were mapped onto the M.truncatula genome.Prior to mapping,repeat masking of the genome was performed using Repeat Masker,available from http://www.repeatmasker.org/.The Rep Base libraries available from http://www.girinst.org/repbase/and the L.culinaris ESTs downloaded from Plant GDB version 187 were aligned to the genome using Gene Seqer[19],a spliced alignment tool available from http://brendelgroup.org/bioinformatics2go/GeneSeqer.php.Following alignment,intron-spanning coordinates were extracted and primers were designed for the respective coordinates using Primer3 version 1.1.4,available from http://primer3.sourceforge.net/releases.php[20].

Following the identification of intron-spanning regions,primer pairs were designed using Primer3 with parameters defined as minimum amplicon size 100 bp and maximum amplicon size 300 bp,primer size 18–27 bp,primer Tm57–63 °C,primer GC content30%–70%,CG clamp0,maximum end stability 250,maximum Tmdifference2,maximum self-complementarity 6,maximum 3′end self-complementarity 3,maximum Ns accepted 0,and maximum poly-X5.The aligned PUTs to the Medicago genome have been annotated and then after annotation gene ontology has been defined based on the annotation.Functional annotation and gene ontology of the intron-spanning markers were performed using BLASTx searches(E-value,1 × 10?5) against GenBank (http://www.ncbi.nlm.nih.gov/),UniProt(http://www.uniprot.org/),and TAIR (https://www.arabidopsis.org/)databases.

2.2.Plant materials and DNA extraction

Thirty-two Lens genotypes were used for genotyping with 105 primers.A diverse panel of thirty-two Lens genotypes consisting of L.culinaris released cultivars,advanced breeding lines,parents of mapping populations,and genotypes of L.ervoides and L.culinaris subsp.orientalis was tested to identify polymorphic markers(Table 1).DNA samples were extracted from individual plant leaf tissue when seedlings were two weeks old using the cetyltrimethylammonium bromide(CTAB)procedure[21].The DNA concentrations of the extracted samples were recorded and were compared with after corresponding concentration with λDNA.The extracted DNA samples were diluted to a uniform concentration of 20 μg μL?1for PCR amplification.

2.3.PCR amplification

One hundred and five primer pairs(Table S1)were synthesized from Imperial Life Sciences(P)Limited,Gurugram,India and used in this study.PCR reactions(in 25-μL volumes)were conductedinaG-Strom(modelnumberGT-40319,UK)thermocycler.Each reaction contained 2.5 μL Taq buffer(Merck,Bangalore,India),1.5 μL MgCl2(25 mmol L?1)(Merck,Bangalore,India),0.20 mmol L?1of each dNTP(Merck,Bangalore India),0.50 mmol L?1of each primer[Imperial Life Sciences(P)Limited,Gurugram India],0.5 U of Taq polymerase(Merck,Bangalore,India),and 20 ng of template DNA.Primers amplifying Lens DNA were validated in a set of 32 diverse Lens genotypes using the following PCR conditions:94°C for 4 min,followed by 35 cycles of 94 °C for 1 min,58 °C for 1 min,and 72 °C for 1 min followed by a final elongation step of 72°C for 15 min.PCR products were resolved by 10%polyacrylamide vertical gel electrophoresis(Sigma-Aldrich,New Delhi,India)and visualized by silver staining.Fragments were scored visually after staining.

2.4.Molecular data scoring and statistical analysis

Polymorphism information content(PIC)values were calculated following Botstein et al.[22].The presence and absence of the band were scored as 1 and 0 and the binary data so obtained for all Lens genotypes for polymorphic markers were used to calculate a correlation matrix using Jaccard's similarity coefficient analysis[23].The similarity coefficient was used to construct a dendrogram based on the unweighted pair groupmethod with arithmetic average(UPGMA)using NTSYS pc-2.21q[24]software.The data were also subjected to principal coordinate analysis(PCA)using NTSYS.

Table 1–Details of plant materials used.

3.Results

3.1.Development and validation of ISM markers in lentil

A total of 1703 ISMs were developed in lentil using a cross-species mapping-based approach.For the identification of ISMs,16,279 EST sequences of L.culinaris from were mapped onto the M.truncatula genome(Table 2).RepBase libraries and L.culinaris ESTs were aligned which resulted in a total of 25,717 GeneSeqer alignments.These were further curated to identify 1703ISMs(TableS2).Among these,a set of 105 primer pairs were used for experimental validation,which resulted in successful amplification of 54 primer pairs(51%)on lentil genomic DNA.

Table 2–Summary statistics from bioinformatic analysis and wet-lab validation.

3.2.Application of ISMs in assessing genetic diversity in Lens species

Fifty-four ISM primer pairs were tested for identification of polymorphic markers in a diverse panel of 32 Lens genotypes consisting of released cultivars,advanced breeding lines,parents of mapping populations(L.culinaris)and genotypes of L.ervoides and L.culinaris ssp.orientalis.Thus,a total of 40 ISMs were found polymorphic(Table 3,Fig.1),so that these ISMs showed high polymorphism(74%).The number of alleles ranged from 2 to 11 with an average of 3.7 alleles for each primer pair,while the PIC ranged between 0.10 and 0.50 with an average of 0.31.The sizes of alleles varied from 52 to 390 bp.

In the present study,polymorphic ISMs were used to assess the genetic diversity among the 32 genotypes and to establish the genetic relationships among them.Ten accessions belonging to different Lens wild species amplified 667 alleles with an average of 17 alleles per marker,while 22 accessions belonging to cultivated species amplified 1404 alleles with an average of 35 alleles per marker.These results showed high allelic diversity among the lentil genotypes for ISMs,indicating that these markers can be used further in lentil breeding programs for several purposes.Cluster analysis based on genotyping data of 40 polymorphic ISMs clustered the 32 Lens genotypes into two groups(Fig.2).Group I contained 19 genotypes which belonged mostly to cultivated and wild species of lentil(L.culinaris subsp.culinaris,L.culinaris subsp.orientalis,and L.ervoides genotypes).Genotypes of L.ervoides were clustered within group I(Fig.2).Another 13 accessions were clustered in group II and belonged only to L.culinaris subsp.culinaris.The first three components of PCA accounted for 13%,9%,and 8%of total observed variation reflected as a measure of the polymorphism,respectively.In total,three PCA components accounted for 30% of total variation for the 32 genotypes.

Table 3–Tm,allelesize,and polymorphism information content(PIC)of each polymorphic intron-spanning marker.

Fig.1–PCR amplification profiles of 32 lentil genotypes for the primer PUT 11770_1.1,IG-72632;2,IG-136668;3,IG-136655;4,IG-72678;5,IG-136620;6,IG-136615;7,IG-72860;8,IG-72861;9,IG-72636;10,IG-116039;11,DPL-62;12,IPL-220;13,DPL-58;14,IPL-526;15,IPL-81;16,IPL-221;17,JL-01;18,ILL-7663;19,IPL-316;20,IPL-325;21,IPL-219;22,IG3973;23,IG3364;24,IG3575;25,IG3568;26,DPL15;27,IG2507;28.IG4258;29,FLIP2009-55L;30,IG3327;31,IG3330;32,IG3546.Lane M contains a molecular ladder.

We also studied the functional annotation of ISMs.Protein databases available in the public domain were searched for the designed ISMs.Annotations of the entire set of 1703 ISMs showed sequence similarity with legumes encoding genes,as presented in Table S3 and summarized in Fig.S1.

4.Discussion

Various molecular markers have been developed in lentil[7–12,25].PCR-based markers such as SSRs have been the choice of plant breeders.Because the full genome sequence of lentil is not available in the public domain[11],development of polymorphic markers in lentil is slower than in other sequenced food legumes such as chickpea and pigeonpea.However,it is possible to develop intron-spanning markers(ISMs)by exploiting exon conservation to develop highly polymorphic,highly transferable,and codominant markers[26,27].Earlier,using a cross-species mapping based approach,91 ISMs markers were developed from pairs of highly similar genes of M.truncatula,a model legume,and Glycine max and validated on six different legume species including M.truncatula,Pisum sativum,Lotus japonicus,L.filicaulis,Vigna radiata,and Phaseolus vulgaris[28].In this study,genomic synteny between M.truncatula and Lens was used for developing ISMs in lentil.Because ISMs can contribute toward the development of informative markers by saturating the chromosomal region of interest[26,27].In our study,51%of markers could be amplified on Lens DNA.However,in grass species,intron-spanning markers showed high(79%–95%)amplification rates on bulk DNA samples made from six forage species(Lolium perenne,L.multiflorum,Festuca pratensis,F.arundinacea,Phleum pretense,and Dactylis glomerata)[29].ISMs developed in the present study showed high polymorphism,74%(40 of 54 ISMs)with an average PIC of 0.31.Similarly,ISMs were highly polymorphic in other crop species such as mustard[30]and rice[13,31].Also,in another food legume species,pigeonpea,55%of ISR(intron-spanning region)markers were polymorphic with an average PIC value of 0.16 and alleles amplified ranged from one to three[32].More recently,119,169 and 110,491 ISMs were developed from introns of desi and kabuli chickpea genes,respectively[33]and a set of 2405 ILP markers showed high polymorphism(86.2%)in 32 accessions of chickpea[33].

Fig.2–Dendrogram based on Jaccard's similarity coefficient using UPGMA clustering.The black dotted line denotes the reference line.Group I consisted of 19 genotypes(IG72632,IG72636,IG136668,IG116309,DPL62,IPL220,IPL221,JL01,ILL7663,IPL81,IPL316,DPL58,IPL526,IG136655,IG72678,IG136620,IG136615,IG72860,IG72861)and Group II consisted of 13 genotypes(IPL325,IG2507,IG4258,FLIP2009-55L,IG3327,IPL219,IG3973,IG3364,IG3575,IG3568,IG7663,IG3330,IG3546).Name of the respective Lens species of each genotype were given beside their names.

Our results also showed high allelic diversity among the lentil genotypes for ISMs,indicating that these markers can be useful in lentil breeding programs for several purposes.The cluster analysis based on genotyping data of 40 polymorphic ISMs clustered the 32 Lens genotypes into two groups.The first group showed subclustering of wild and cultivated accessions separately and further subclustering of the lentil cultivars along with a few other genotypes from ICARDA(International Center for Agricultural Research in the Dry Areas)might be due to the use of ICARDA materials in lentil breeding programs of India.The second group,comprised mostly lentil germplasm or landraces of exotic origin,indicating that they have different genetic constitution compared to cultivated accessions.Thus,the present ISMs clearly differentiated the 32 Lens genotypes.These ISMs will enrich current genomic resources of lentil.Further,ISMs can be used for gene expression studies for a set of candidate genes in lentil.

5.Conclusions

A set of 1703 ISMs were designed using spliced alignment of lentil EST sequences against the Medicago genome.A panel of 57 ISM primer pairs(polymorphic as well as monomorphic ISMs)was validated in a group of cultivated and wild lentils.These markers have been developed from gene sequences of lentil that encode several functional proteins.Therefore,these ISMs will be useful as functional or genic markers in lentil genetics and breeding.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2017.09.004.

Acknowledgments

The authors thank the Indian Council of Agricultural Research,New Delhi,India for research support.This work was partially funded by the Department of Biotechnology,Government of India,New Delhi,India through a grant support(BT/PR10921/AG11/106/943/2014).Gaurav Sablok acknowledges bioinformatics server from Fondazione Edmund Mach,Italy for performing the bioinformatics analysis and development of the intron-spanning markers.

[1]FAOSTAT Database,http://www.fao.org/faostat/en/#data/QC,Accessed date:10 January 2017.

[2]S.S.Yadav,A.Z.Rizvi,M.Manohar,A.K.Verma,R.Shrestha,C.Chen,G.Bejiga,W.Chen,M.Yadav,P.N.Bahl,Lentil growers and production systems around the world,in:S.S.Yadav,L.McNeil,P.C.Stevenson(Eds.),Lentil:An Ancient Crop of Modern Times,Springer,Dordrecht,The Netherlands,2007.

[3]R.K.Varshney,Exciting journey of 10 years from genomes to fields and markets:some success stories of genomics-assisted breeding in chickpea,pigeonpea and groundnut,Plant Sci.242(2016)98–107.

[4]R.K.Varshney,C.Song,R.K.Saxena,S.Azam,S.Yu,A.G.Sharpe,S.Cannon,J.Baek,B.D.Rosen,B.Taran,T.Millan,Draft genome sequence of chickpea(Cicer arietinum)provides a resource for trait improvement,Nat.Biotechnol.31(2013)240–246.

[5]R.K.Varshney,W.Chen,Y.Li,A.K.Bharti,R.K.Saxena,J.A.Schlueter,M.T.Donoghue,S.Azam,G.Fan,A.M.Whaley,A.D.Farmer,2012:draft genome sequence of pigeonpea(Cajanus cajan),an orphan legume crop of resource-poor farmers,Nat.Biotechnol.30(2012)83–89.

[6]N.K.Singh,D.K.Gupta,P.K.Jayaswal,A.K.Mahato,S.Dutta,S.Singh,S.Bhutani,V.Dogra,B.P.Singh,G.Kumawat,J.K.Pal,The first draft of the pigeonpea genome sequence,J.Plant Biochem.Biotechnol.21(2012)98–112.

[7]A.Hamwieh,S.M.Udupa,A.Sarkar,C.Jung,M.Baum,Development of new microsatellite markers and their application in the analysis of genetic diversity in lentils,Breed.Sci.59(2009)77–86.

[8]S.Kaur,N.O.Cogan,L.W.Pembleton,M.Shinozuka,K.W.Savin,M.Materne,J.W.Forster,Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery,BMC Genomics 12(2011)1.

[9]S.Kaur,N.O.Cogan,A.Stephens,D.Noy,M.Butsch,J.W.Forster,M.Materne,EST-SNP discovery and dense genetic mapping in lentil(Lens culinaris Medik.)enable candidate gene selection for boron tolerance,Theor.Appl.Genet.127(2014)703–713.

[10]P.Verma,N.Shah,S.Bhatia,Development of an expressed gene catalogue and molecular markers from the de novo assembly of short sequence reads of the lentil(Lens culinaris Medik.)transcriptome,Plant Biotechnol.J.11(2013)894–905.

[11]K.Bett,L.Ramsay,C.Crystal,A.G.Sharpe,D.R.Cook,P.R.Varma,P.Chang,C.J.Coyne,R.McGee,D.Main,A.Vandenberg,LenGen:The International Lentil Genome Sequencing Project,Plant and Animal Genome XXIII Conference,January10–14,2015(San Diego,California USA).

[12]D.Sen Gupta,P.Cheng,G.Sablok,P.Thavarajah,C.J.Coyne,S.Kumar,M.Baum,R.J.McGee,Development of a panel of unigene-derived polymorphic EST-SSR markers in lentil using public database information,Crop J.4(2016)425–433.

[13]S.Badoni,S.Das,Y.K.Sayal,S.Gopalakrishnan,A.K.Singh,A.R.Rao,P.Agarwal,S.K.Parida,A.K.Tyagi,Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice,Sci.Rep.6(2016)23765.

[14]P.J.Hiremath,A.Farmer,S.B.Cannon,J.Woodward,H.Kudapa,R.Tuteja,A.Kumar,A.Bhanuprakash,B.Mulaosmanovic,N.Gujaria,L.Krishnamurthy,Large-scale transcriptome analysis in chickpea(Cicer arietinum L.),an orphan legume crop of the semi-arid tropics of Asia and Africa,Plant Biotechnol.J.9(2011)922–931.

[15]S.Choudhary,R.Gaur,S.Gupta,S.Bhatia,EST-derived genic molecular markers:development and utilization for generating an advanced transcript map of chickpea,Theor.Appl.Genet.124(2012)1449–1462.

[16]N.Gujaria,A.Kumar,P.Dauthal,A.Dubey,P.Hiremath,A.B.Prakash,A.Farmer,M.Bhide,T.Shah,P.M.Gaur,H.D.Upadhyaya,Development and use of genic molecular markers(GMMs)for construction of a transcript map of chickpea(Cicer arietinum L.),Theor.Appl.Genet.122(2011)1577–1589.

[17]J.H.Kim,C.Lee,D.Hyung,Y.J.Jo,J.S.Park,D.R.Cook,H.K.Choi,CSGM Designer:a platform for designing cross-species intron-spanning genic markers linked with genome information of legumes,Plant Methods 11(2015)30.

[18]J.Duvick,A.Fu,U.Muppirala,M.Sabharwal,M.D.Wilkerson,C.J.Lawrence,B.Volker,PlantGDB:a resource for comparative plant genomics,Nucleic Acids Res.36(2008)D959–D965.

[19]V.Brendel,L.Xing,W.Zhu,Gene structure prediction from consensus spliced alignment of multiple ESTs matching the same genomic locus,Bioinformatics 20(2004)1157–1169.

[20]A.Untergasser,I.Cutcutache,T.Koressaar,J.Ye,B.C.Faircloth,M.Remm,S.G.Rozen,Primer3–new capabilitiesand interfaces,Nucleic Acids Res.40(2012)e115.

[21]J.J.Doyle,J.L.Doyle,A rapid DNA isolation procedure for small quantities of fresh leaf tissue,Phytochem.Bull.19(1987)11–15.

[22]B.Botstein,R.L.White,M.Skolnick,R.W.Davis,Molecular markers in plant genome analysis,Am.J.Hum.Genet.32(1980)314–331.

[23]P.Jaccard,Nouvellesrecherchessur la distribution florale,Bull.Soc.Vaud.Sci.Nat.44(1908)223–270(in French).

[24]F.J.Rohlf,NTSYS-pc Numerical Taxonomy and Multivariate Analysis System,Version 2.21q,Exeter Publ,New York,USA,2009.

[25]E.E.Andeden,F.S.Baloch,E.?ak?r,F.Toklu,H.?zkan,Development,characterization and mapping of microsatellite markers for lentil(Lens culinaris Medik.),Plant Breed.134(2015)589–598.

[26]U.M.Quraishi,F.Murat,M.Abrouk,C.Pont,C.Confolent,F.X.Oury,J.Ward,D.Boros,K.Gebruers,J.A.Delcour,C.M.Courtin,Z.Bedo,L.Saulnier,F.Guillon,S.Balzergue,P.R.Shewry,C.Feuillet,G.Charmet,J.Salse,Combined meta-genomics analyses unravel candidate genes for the grain dietary fiber content in bread wheat(Triticum aestivum L.),Funct.Integr.Genomics 11(2011)71–83.

[27]U.M.Quraishi,M.Abrouk,F.Murat,C.Pont,S.Foucrier,G.Desmaizieres,C.Confolent,N.Riviere,G.Charmet,E.Paux,A.Murigneux,Cross-genome map based dissection of a nitrogen use efficiency ortho-metaQTL in bread wheat unravels concerted cereal genome evolution,Plant J.65(2011)745–756.

[28]H.K.Choi,M.A.Luckow,J.Doyle,D.R.Cook,Development of nuclear gene-derived molecular markers linked to legume genetic maps,Mol.Genet.Genomics 276(2006)56–70.

[29]K.I.Tamura,T.Kiyoshi,J.I.Yonemaru,The development of highly transferable intron-spanning markers for temperate forage grasses,Mol.Breed.30(2012)1–8.

[30]P.Panjabi,A.Jagannath,N.C.Bisht,K.L.Padmaja,S.Sharma,V.Gupta,A.K.Pradhan,D.Pental,2008:comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism(IP)markers:homoeologous relationships,diversification and evolution of the A,B and C Brassica genomes,BMC Genomics 9(2008)113.

[31]X.M.Xu,K.J.Liang,S.G.Zhang,W.Shang,Y.Y.Zhang,X.Y.Wei,B.Ke,Analysis of Indica-Japonica differentiation in rice parents and derived lines using ILP markers,Agric.Sci.China 8(2009)1409–1418.

[32]H.Kudapa,A.K.Bharti,S.B.Cannon,A.D.Farmer,B.Mulaosmanovic,R.Kramer,A.Bohra,N.T.Weeks,J.A.Crow,R.Tuteja,T.Shah,A comprehensive transcriptome assembly of pigeonpea(Cajanus cajan L.)using Sanger and secondgeneration sequencing platforms,Mol.Plant 5(2012)1020–1028.

[33]R.Srivastava,D.Bajaj,Y.K.Sayal,P.K.Meher,H.D.Upadhyaya,R.Kumar,S.Tripathi,C.Bharadwaj,A.R.Rao,S.K.Parida,Genome-wide development and deployment of informative intron-spanning and intron-length polymorphism markers for genomics-assisted breeding applications in chickpea,Plant Sci.252(2016)374–387.