Mohammad Saleem Wani·Vikas Sharma,3·Raghbir Chand Gupta·Abid Hussain Munshi

Abstract Betula utilis D.Don.is an important species of alpine Himalaya and forms the major treeline component of western Himalaya.The different populations of B.utilis are declining and are under high risk.In the present study,novel expressed sequence tag-simple sequence repeat(EST-SSR) primers were developed from expressed sequence tag(EST)data of different Betula species.Of the 10,796 designed primers,the percentages of di-,tri-,tetra-,penta-,and hexa-repeats were 36% ,35% ,15% ,5.5% and 7.7% ,respectively.For validation,50 primers were synthesized randomly and were characterized in 20 different B.utilis accessions from north-western Himalaya.Of these,45 primers amplified fragments in a range of 1-6.The 24 polymorphic primers produced 111 fragments in aggregate with 4.6 fragments on average.Polymorphism information content(PIC)ranged from 0.288 in marker BUMS-24 to 0.497 in BUMS-3 and BUMS-7,with an average of 0.447 among polymorphic markers.Dendrogram based on Jaccard’s similarity coefficient and UPGMA method showed that newly developed SSR markers distinguished twenty accessions of B.utilis into two groups.As no SSR markers were available in this species,the newly developed markers will foster molecular genetics research and conservation efforts for this species.

Keywords Betula utilis·Simple sequence repeat(SSR)·Western Himalaya·Polymorphism information content(PIC)·Genetic diversity

Introduction

Betula utilisD.Don.,commonly known as Himalayan birch,belongs to the family Betulaceae.The species is a deciduous, tetraploid, perennial plant, forming treeline vegetation along the alpine Himalayan region.The bark is sparkling,ruddy white or white,with white,horizontal,smooth lenticels.The external bark consists of layers,peeling in expansive,even rolls.The leaves are ovateacuminate,elliptic,and sporadically serrate.The species is monoecious and wind pollinated(DeJong 1993;Jonsell 2000).Male catkins mature in summer,shedding pollen the following spring;a few days after female flowers have appeared.Their light dust grains(Johnsson 1974),are delivered in colossal amounts and passed on long distances by the wind(Hjelmroos 1991).Seeds develop as a result of cross-fertilization because of a biochemical self-incompatibility mechanism (Hagman 1971). The Himalayan Birch bloom with leaf flush in the spring,and seeds mature in October to November. Female catkins are smaller,shorter and more erect than the longer,pendulous,clustered male ones.The winged-seeds are dispersed by wind.The plant grows in light(sandy),medium(loamy)soils,but can develop in depleted or wet soils.The species inclines toward corrosive,nonpartisan and fundamental basic soils,and can develop in semi-shade(light forest)to full sun.Moreover,the survival of this birch species depends on adequate moisture and nutrients (Carlton and Bazzaz 1998).Ecologically,B.utilis plays a significant role in subalpine and alpine Himalaya,and is the only broadleaved,tree species found at these altitudes(Zobel and Singh 1997).B.utilis shows a high freezing tolerance(Sakai and Larcher 1987),which enables this species to form a treeline in the Himalayas.Over most of its range,it is profuse in a belt just below the treeline,and in a few sites,it may be mixed with Rhododendron,Abies and Pinus species,especially in gorges where the snow lies for long periods(Zobel and Singh 1997).B.utilis has an essential ecological role because of its easy regeneration and fast growth.It is a typical pioneer species,able to establish on bare,degraded lands.The ability of this species to grow at high altitudes also allows it to serve a vital role in watershed protection and soil stabilization.In addition,this species possesses several medicinal properties and is exploited for therapeutic purposes.Customarily,the bark of B.utilis has been utilized as an astringent lotion,an antibacterial compound, an anticonvulsant drug, an expectorant and as a tonic.The bark contains betulin,lupeol, oleanolic acid, acetyloleanolic acid, lupenone,sitosterol,methylebetulonate,methyl betulate,and another triterpenoid named Karachic acid.It is sweet-smelling and has disinfectant properties(Chauhan 1999;Selvam 2008).Therefore,it is a useful species of the alpine Himalayan region with numerous chemical attributes.However,the natural range of B.utilis is small and fragmented.Furthermore,habitat destruction,grazing and fuel wood cutting by local people are factors which have resulted in declining populations of B. utilis along the Western Himalaya and has pushed this species into an endangered species category(Samant et al.2007;Zaki et al.2011).The natural populations of B.utilis are decreasing due to various anthropogenic disturbances and it is a major concern.Therefore,conservation of the declining populations from the Himalayan region is required.In addition,estimation of the genetic diversity of the existing populations can provide useful information for developing conservation and management plans.Genetic diversity assumes an essential part in deciding a population’s viability(Allendorf and Ryman 2000)and within populations;it is frequently utilized as an indirect warning of the risk of extinction since it influences the evolutionary potential of a species(Soule and Simberloff1986;Holsinger and Gottlieb 1991).The level of genetic differentiation among populations can likewise give helpful information for designing operations such as reinforcement of existing populations through introduction of material by ex-situ culture or seeds(Schemske et al.1994;Gaudeul et al.2000).Genetic variations in a species also form the basis for its evolution and play vital roles in its survival and adaptability in a changing environment.

DNA markers are routinely used for the estimation of genetic diversity in plants.Among the different DNA markers,SSR markers are preferred over others.This is due to their many advantages such as multi-allelic nature,abundance,locus specificity,co-dominant,high polymorphic,genome-wide coverage and easy reproducible nature.They are also easily and automatically scored(Powell et al.1996),and are considered ideal genetic markers for estimating plant genetic diversity and relationships between species,populations and individuals(Kostova et al.2006;Tu et al.2007).Moreover,they are easily cross-transferable between different species and genera;this property of SSR markers has been exploited to study genetic diversity and relationships of various species from related species and genera(Sharma et al.2008,2015a,2015b;Rana et al.2015,2016;Gupta et al.2016;Koul et al.2017).Therefore,in this study we developed and characterized the first set of EST-SSR markers in B.utilis which should be useful for genetic studies in B.utilis and other related species.

Materials and methods

Expressed sequence tag(EST)data of Betula species was examined at the National Centre for Biotechnology Information(NCBI,USA,https://www.ncbi.nlm.nih.gov/) and a total of 133,030 EST sequences were found as of June 20,2015.These EST sequences were downloaded,checked and processed for SSR primer designing.They were processed using EGassembler(Masoudi-Nejad et al.2006),to remove the redundancy of sequences.Microsatellite search and primer designing was performed according to Sharma et al.(2009).The resulting unigenes/contigs were then searched for the presence of SSRs. Simple sequence repeats having a minimum repeat length of 10 bp were searched using SSRIT(Temnykh et al.2001).SSR primers were designed using PRIMER 3 software (Rozen and Skaletsky 2000)with default settings.These EST-SSRs were named Betula utilis MicroSatellite(BUMS).Of the total primers designed,50 were synthesized and characterized in 20 accessions of B.utilis collected from different regions of north-western Himalaya.The accessions were-Sinthontop-1, Sinthontop-2, Sinthontop-3, Sinthontop-4,Sinthontop-6, Sinthontop-7, Sinthontop-8, Sinthontop-9,Sinthontop-10,Sinthontop-11,Sinthontop-12,Sinthontop-13, Sinthontop-14, Sinthontop-16, Sinthontop-18,Sinthontop-19, Sinthontop-20, Razdan-1, Razdan-2 and Razdan-3.The young leaf samples of these accessions were collected,kept in coarse silica gel in small polybags until DNA isolation,and then total genomic DNA was extractedusing the CTAB method(Doyle and Doyle 1990).The quality and concentration of the extracted DNA was estimated on 0.8% agarose gel by comparing with diluted uncut λ DNA as a standard(Fermentas,Lithuania).PCR reactions were performed in a 10-μL reaction volume containing 25 ng of template DNA,15 ng of each primer,200 μM of each dNTP,10 mM Tris-HCL(pH 8.3),50 mM KCL,1.5 mM MgCl2,0.01% gelatin and 0.5 U of Taq DNA polymerase(High Media Pvt.Ltd.,India).The PCR conditions were:one cycle of 5 min at 94°C,35 cycles of one minute at 94°C,1 min at the respective annealing temperature for each primer(Table 1),1 min at 72°C,and final extension for 7 min at 72°C.All the PCR reactions were conducted in a 96 well thermal cycler (Applied Biosystems,USA).Amplification products were initially checked on 3.5% agarose gel and then separated on 6% denaturing polyacrylamide gel in 1×TBE buffer.Fragments were visualized by silver staining and sized by 50 bp DNA ladder(Fermentas,Lithuania).Only unambiguously amplified fragments were scored for further analysis.As the species is tetraploid,scoring was done for dominant markers,i.e.,1 for presence of a fragment and 0 for absence.The scored data was converted into a binary matrix and analyzed by different software.Distance-based cluster analysis was performed by generating a dendrogram based on Jaccard’s similarity coefficient and the unweighted pair group method with arithmetic mean(UPGMA)method using DARwin(Perrier and Jacquemoud-Collet 2006).Polymorphism Information Content(PIC)values were calculated using the formula of Roldan-Ruiz et al.(2000).

Table 1 Diversity characteristics of SSR primers characterized in the present study

Table 1 continued

Table 1 continued

Results and discussion

Of the downloaded sequences,only 13,200(10% )contained SSR sequences with different types of repeat motifs in them.Out of these 13,200 sequences,we designed 10,796 SSR primer pairs.Of these,36% were di-repeats,35% tri-repeats,15% tetra-repeats,5.5% penta-repeats and 7.7% were hexa-repeats.A detailed account of these primers is given in the supplementary materials.From these 10,796 newly designed primers,50 were synthesized and validated in 20 B.utilis accessions from different locations in north-western Himalaya.Of the validated primers,five(BUMS-17,BUMS-35,BUMS-44,BUMS-45 and BUMS-49)failed to produce the reliable amplifications,while 45 amplified a minimum of one fragment across the different accessions.A representative screening amplification profile of eight primers(BUMS-1,BUMS-2,BUMS-6,BUMS-7,BUMS-8,BUMS-10,BUMS-13 and BUMS-14)using six accessions is shown in Fig.1.Of the successfully amplified primers (45), 24 were polymorphic while 21 were monomorphic. The polymorphic primers produced 111 fragments in aggregate with an average of 4.62 fragments per primer.Seven SSR primers(BUMS-10,BUMS-11,BUMS-19,BUMS-23,BUMS-25,BUMS-31 and BUMS-42)produced the maximum of six fragments while two SSR(BUMS-24 and BUMS-30)produced a minimum of two fragments among the 24 polymorphic primers.The size of amplified fragments ranged from 100 to 320 bp.Polymorphism Information Content (PIC) of the polymorphic primers ranged from 0.288 in primer BUMS-24 to 0.497 in primers BUMS-3 and BUMS-7,with an average of 0.447.Primer-wise diversity estimates,such as number of fragments amplified and PIC values, are given in Table 1.The marker data produced by newly developed SSR primers largely distinguished the different accessions of B.utilis on the basis of their geographic origin and grouped twenty accessions into two groups(Fig.2).

Genetic diversity estimates of B.utilis are not available till date.This is attributed to a lack of genomic resources,difficulty in sampling,and the endemic nature of this species.However,the lack of genomic resources may be the main reason,and without which we cannot proceed for genetic characterization of a species at a molecular level.The availability of EST sequence data in the public domain has proven very useful in generating genomic resources in many important but neglected plant species(Sharma et al.2014,2017;Kaur et al.2016).B.uitilis is also one species which requires immediate attention for its characterization and conservation in western Himalaya.The SSR markers developed in this study show high potential for detecting polymorphism and diversity in this species.The number of fragments generated and PIC values of polymorphic SSR markers show high genetic variability in the characterized accessions.As there are no markers data is available for this species,the newly developed SSR markers can be valuable in detecting genetic diversity and in other such genetic studies in B.utilis.

Fig.1 A representative picture of the screening of 8 primers(BUMS-1,BUMS-2,BUMS-6,BUMS-7,BUMS-8,BUMS-10,BUMS-13 and BUMS-14)from newly synthesized SSR primers in 6 accessions of B.utilis L=100 bp DNA ladder,bp=base pair

Fig.2 Dendrogram of 20 B.utilis accessions based on 24 polymorphic SSR markers data showing two major clusters

Information on the genetic diversity of a species is required to initiate successful conservation and management programs.The SSR markers developed and characterized in this study will be helpful in executing genetic diversity studies in B.utilis. The different accessions characterized have shown high polymorphic potential of the SSR primers developed.Thus,this first set of SSR markers will serve as a tool for the analysis of genetic diversity,hybrid detection and for revealing population structure of B.utilis and other Betula species.

AcknowledgementsWe are grateful to the Department of Biotechnology(DBT),Government of India for providing facilities and financial support under DBT-IPLS programme at Punjabi University,Patiala.The first author is thankful to UGC for providing a Fellowship under the UGC-BSR scheme.The corresponding author is thankful UGC for providing support under DSK PDF scheme and SERB for N-PDF scheme.