Genetic diversity and DNA fingerprinting in jute （Corchorus spp.）based on SSR markers

Liwu Zhang*，Rongrong Cai，Minhang Yuan，Aifen Tao，Jiantang Xu，Lihui Lin，Pingping Fang，Jianmin Qi*

College of Crop Science，F(xiàn)ujian Agriculture and Forestry University/Key Laboratory of Ministry of Education for Genetics，Breeding and Multiple Utilization of Crops/Fujian Provincial Key Laboratory of Crop Breeding by Design，F(xiàn)uzhou 350002，China

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Genetic diversity and DNA fingerprinting in jute （Corchorus spp.）based on SSR markers

Liwu Zhang*，Rongrong Cai，Minhang Yuan，Aifen Tao，Jiantang Xu，Lihui Lin，Pingping Fang，Jianmin Qi*

College of Crop Science，F(xiàn)ujian Agriculture and Forestry University/Key Laboratory of Ministry of Education for Genetics，Breeding and Multiple Utilization of Crops/Fujian Provincial Key Laboratory of Crop Breeding by Design，F(xiàn)uzhou 350002，China

A R T I C L E I N F O

Article history：

Received in revised form

17 May 2015

Accepted 23 June 2015

Available online 2 July 2015

Jute

Corchorus

Simple sequence repeats

DNA fingerprinting

Genetic diversity

A B S T R A C T

Genetic diversity analysis and DNA finger printing are very useful in breeding programs，seed conservation and management.Jute（Corchorus spp.）is the second most important natural fiber crop after cotton.DNA fingerprinting studies in jute using SSR markers are limited.In this study，58 jute accessions，including two control varieties（Huangma 179 and Kuanyechangguo）from the official variety registry in China were evaluated with 28 pairs of SSR primers.A total of 184 polymorphic loci were identified.Each primer detected 3 to 15 polymorphic loci，with an average of 6.6.The 58 jute accessions were DNA-fingerprinted with 67 SSR markers from the 28 primer pairs.These markers differentiated the 58 jute accessions from one another，with CoSSR305-120 and CoSSR174-195 differentiating Huangma 179 and Kuanyechangguo，respectively.NTSYS-pc2.10 software was used to analyze the genetic diversity in the 58 jute accessions.Their genetic similarity coefficients ranged from 0.520 to 0.910 with an average of 0.749，indicating relatively great genetic diversity among them.The 58 jute accessions were divided into four groups with the coefficient 0.710 used as a value for classification，consistent with their species and pedigrees.All these results may be useful both for protection of intellectual property rights of jute accessions and for jute improvement.

?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

Jute（Corchorus spp.）is an important natural fiber crop worldwide，comprising two cultivated species:white jute（Corchorus capsularis）and dark jute（Corchorus olitorius）.The two cultivated species differ markedly in growth habit and many agronomic traits［1］.Jute fiber is a biodegradable，renewable，and environment-friendly cellulose fiber and is called golden fiber. As the second most important natural fiber crop after cotton， jute is widely cultivated in subtropical and tropical regions in the world，mainly in India，Bangladesh，China，Uzbekistan，Nepal，Vietnam，Burma，Zimbabwe，Thailand，and Egypt［1，2］.

In recent years，a few elite parental lines have been used repeatedly in jute cross-breeding programs，resulting in relatively narrow genetic variation among new varieties［1，3］. Moreover，jute accessions derived from the same pedigree may have different names in different places owing to the exchange of germplasm across different countries or regions.Thus，it is difficult to distinguish jute accessions using only morphological traits.However，the conventional method of establishing the identity and purity of varieties is field evaluation，whichistime-consumingandaffectedbyenvironmental variation.

Table 1-List of 58 jute accessions and group assignment based on cluster analysis.

The limits of genetic characterization using morphological traits via field evaluation may be overcome by use of DNA markers.Among several marker types，simple sequence repeats（SSR）or microsatellite markers are considered a desirable tool for DNA fingerprinting and genetic diversitystudies，owing to their many advantages including codominant Mendelian inheritance，high polymorphism，and rapid and convenient detection［4—8］.For this reason，SSR markers have been used to characterize genetic diversity in jute［4—6］. Banerjee et al.［4］analyzed the genetic diversity in 292 jute genotypes using 172 SSRs and found that most of the accessions of the two cultivated species could be clearly delineated into separate groups，a finding similar to that of Ghosh et al.［5］using 6 polymorphic SSRs and amplified fragment length polymorphism（AFLP）markers with 63 jute genotypes.However，few studies have used SSR markers for DNA fingerprinting in jute.Aside from 13 DNA fingerprints of jute varieties with SSR markers by Wu et al.［9］，other DNA fingerprints have been constructed with sequence-related amplified polymorphism （SRAP）［9，10］and inter-simple sequence repeat（ISSR）markers［9］.

Since the International Plant Variety Protection Act（http:// en.wikipedia.org/wiki/Plant_Variety_Protection_Act_of_1970）was signed，the protection of intellectual property rights of varieties has received attention worldwide.Since the 1940s，several elite jute varieties have been released and registered in main fiber production regions or countries［1］.To support the protection of these varieties，it is desirable first to acquire their DNA fingerprints.The aim of the present study was DNA fingerprinting and assessment of genetic diversity in a panel of 58 jute accessions，including two control varieties （Huangma 179 and Kuanyechangguo）from the official variety register of China，using 28 pairs of SSR primers developed in our laboratory.

2.Materials and methods

2.1.Plant materials and DNA isolation

A set of 58 jute accessions from 8 countries，provided by the Natural Fiber Crop Genetics and Breeding Laboratory of Fujian Agriculture and Forestry University，China，having different genetic backgrounds，were used as test materials（Table 1）. Among them，12 were dark jute and 46 white jute accessions.All of the accessions were planted on the experimental farm of Fujian Agriculture and Forestry University，China on May 1，2013.

Genomic DNA of these jute accessions was extracted from 30-day-old seedlings using a modified cetyltrimethyl ammonium bromide method［11］and diluted to 50 ng μL?1with double distilled H2O.

2.2.SSR primers

For screening SSR with sharp DNA fragments，all SSR primers prefixedwithCcSSRandCoSSRdevelopedbyourlaboratorywere tested in a set of six diverse jute accessions［3］.Finally，28 SSR primers were selected to amplify the set of 58 jute accessions on the basis of clear polymorphic bands（Table 2）.

2.3.PCR analysis

PCRamplificationswereperformedina10-μLvolume containing 50 ng μL?1DNA 2.0 μL，10 μmol μL?1left primer 0.5 μL，10 μmol μL?1rightprimer0.5 μL，0.5U μL?1Taq polymerase 0.1 μL，10 mmol L?1dNTPs 0.2 μL，10×PCR buffer 1 μL，50 mmol L?1Mg2+0.8 μL，and dd H2O 4.9 μL.The PCR procedure，electrophoresis，and silver staining were as described by Zhang et al.［3］.

Table 2-SSR primers used in this study and their amplification results.

2.4.Data analysis

Amplified DNA fragments were scored as 1（present）or 0 （absent）.Polymorphism information content（PIC）was estimated with PowerMarker 3.51［12］.Genetic similarity coefficients（GSC）comparing all pairs of the 58 jute accessions were calculated by the unweighted pair group of arithmetic means （UPGMA）method with NTsys pc2［13］.SSR markers used in DNA fingerprinting were named according to the primer pairs and the estimated molecular weight of the fragment.For example，CcSSR001-260 is a specific SSR marker of length 260 bp amplified from CcSSR001.

3.Results

3.1.SSR primer statistics

A total of 28 pairs of SSR primers were used to screen for polymorphisms among 12 dark and 46 white jute accessions. In total，184 bands were obtained，of which 134 bands were polymorphic（Table 2）.The reason for the high level ofpolymorphism（72.8%）was possibly that the tested materials belonged to two cultivated jute species that are crossincompatible and quite different.The number of fragment samplified by each primer varied from 3 to 15 with an average of 6.6.The sizes of amplified fragments ranged from 80 to 900 bp.The average PIC，which is a measure of heterozygosity，was as low as 0.1436（CoSSR053）and as high as 0.7319 （CoSSR232），with an average of 0.4962.

3.2.DNA fingerprinting

A total of 134 polymorphic bands obtained from the 28 pairs of SSR primers were used as markers for DNA fingerprinting.The amplified fragments of specific SSRs were encoded as a string of 0 sand1 s.Thesestringswerearrangedinadigitalfingerprintof the 58 tested jute accessions.Table 3 shows that the digital DNA fingerprintswere formedby 67SSR markers and thatallthese58 jute accessions could be uniquely identified.The names of the accessions and the corresponding specific SSR primers are presented in Table S1.Accessions of Zijinhuangma and SM/034 were identified uniquely by as many as three pairs of SSRs，followed by accessions of Minhuang 3，Xinxuan 1，Zipimaixinpi，Maliyesheng，Bachang 4，and Yindumolvzi by two primer pairs，whereas each of the other fifty accessions was identified by one specific pair of SSR primers.The amplified fragments of primer CoSSR184 for Lubinyuanguo are shown in Fig.S1 as an example. Among the 28 pairs of SSR primers，21 identified either one or two accessions（Table S2）.CcSSR045 identified 14 cultivars，followed by CoSSR146 and CoSSR305 with six cultivars each. CoSSR122 identified five cultivars，CoSSR362 and CoSSR434 four，and CoSSR192 three.CoSSR305-120 and CoSSR174-195 differentiated Huangma 179 and Kuanyechangguo，respectively.These two control varieties are listed in the official jute variety register in China.

3.3.Genetic diversity

To characterize the genetic diversity among these 58 jute accessions，cluster analysis based on SSR markers was performed（Fig.1）.The genetic similarity coefficient（GSC）ranged from 0.520 to 0.910，with an average of 0.749.When a GSC of 0.613 was selected as a value for classification，the jute accessions fell into two main groups:dark jute and white jute，designated as Gc and Go respectively.The Gc group contained 46 white accessions and the Go group 12 dark accessions.This result suggested the presence of high genetic variation between the two cultivated species of jute.When a GSC of 0.710 was used，the Gc and Go groups were further classified into Gc1 and Gc2，and Go1 and Go2 subgroups，respectively. The Gc1 subgroup contained 29 accessions.Of these，20 were from China and the remaining 9 were from other Asian countries.The Gc2 subgroup contained 17 accessions，16 of which were from China and the remaining one from India. The Go1 subgroup contained 11 accessions，3 of which were from China and the remaining 8 from Mali，Pakistan，India，Nepal，and Kenya.The Go2 subgroup contained one accession （Maliyeshengchangguo），whichisnoteworthybecauseit displayed the lowest average GSC（0.594）.The locations of different accessions within different subgroups suggest that cluster analysis among these jute accessions was not influenced by accessions from a particular location.However，most of these jute accessions were consistent with pedigrees.For example，Bachang 4 and Kuanyechangguo had the highest GSC（0.97），indicating a close interrelationship between them. According to the pedigree analysis，Kuanyechangguo is a pure cultivar selected from a cross between Bachang 4 and Guangfengchangguo.To increase the genetic variation in jute cross-breeding programs，parental lines should be selected from different subgroups instead of from different geographical or collection regions.

4.Discussion

4.1.SSR reliability for DNA fingerprinting construction

In the last two decades，a variety of DNA markers have been widely used in major crops for DNA fingerprinting，germplasm evaluation，genetic mapping，and genetic diversity analysis［4—10，12，14］.As suggested by Guidelines for Molecular Marker Selection and Database Construction［15］，SSRs and single nucleotide polymorphisms（SNP）are favored as molecular markers for DNA fingerprinting.The development of high-throughout sequencing has allowed calling SNPs based on the genomic sequence of varieties，greatly improving the precision of variety differentiation［16］.However，it has not been widely used in DNA fingerprinting，owing to its cost. Compared with SNP，SSRs are considered an alternative choice for DNA fingerprinting and genetic diversity studies with the advantages of low cost，reliability，and multiplicity of alleles.SSR markers have been found useful in studies of differentiation of cotton［17］and identification of different soybean species［18］.In the present study，28 pairs of SSR primers differentiated 58 jute accessions from one another，indicating that SSRs are an effective molecular marker type for DNA fingerprinting.

4.2.Jute DNA fingerprinting using SSR markers

In previous studies，the locations of jute genetic accessions in cluster analyses were not in accord with their geographical origins［10，21，22］.This phenomenon maybe accounted for by germplasm exchange across borders.Such exchange may result in jute accessions derived from the same pedigree having different names in different places.For this reason，it is useful to construct DNA fingerprints，also called molecular identities，for a panel of jute accessions using molecular markers.Wu et al. ［9］constructed DNA fingerprints of jute varieties with SRAP，ISSR，and SSR markers，as an example for DNA fingerprinting in jute.But only 13 DNA fingerprints were constructed using SSR markers.Since SSRs are considered an alternative choice for DNA fingerprinting in comparison with SRAP and ISSR，it is desirable to perform DNA fingerprinting using SSR markers.The main finding in the present study is that all 58 jute accessions could be differentiated from one another using 28 pairs of SSR primers.ThesepairsofprimersconsistingofCcSSR045，CoSSR146，CoSSR305，CoSSR122，CoSSR362，CoSSR434，and CoSSR192 were found to be effective for jute accession discrimination.Each of the other 50 accessions was identified by one pairofSSRs.In fact，the probabilityofDNAfingerprintidentityof any two accessions depends on the type of molecular markersandthenumberoftestedmaterials.Thus，thenumberofspecific SSR primers decreased as the number of tested materials increased.To construct DNA fingerprints in a large panel of jute germplasm，it is necessary to increase the number of SSR markers or call SNPs using high-throughput sequencing.

Table 3-DNA fingerprinting of 58 jute accessions by SSR markers.

4.3.Genetic diversity of jute based on SSR markers

Another achievement in the present study is assessing the genetic diversity of 58 jute accessions.The jute accessions were divided into two distinct groups（Go and Gc），indicatingthat high genetic variation was present in white and dark jutes. This classification is in accord with the genetic diversity reported in previous studies［18—21］.As reported by Xiong［1］，the two cultivated species in jute may have originated in differentgeographicallocations；darkjute possiblyoriginatedin Africa and white jute in India and southern China.The reason for the differencebetweenwhite anddarkjute could be that the two species had different modes of evolution.The sexual incompatibility barrier between white and dark jute supports this speculation［1，22］.This study also showed that the genetic diversity of jute accessions is relatively large，with genetic similarity coefficients ranging from 0.520 to 0.910.To broaden thegeneticvariationinjute，theparentallinesincross-breeding programs should be selected from different subgroups instead of different geographical or collection regions.Moreover，other biotechnologies，such as somatic hybridization and genetic transformation，could be used to overcome the sexual incompatibility barrier between dark and white jute.

Fig.1-Genetic diversity of 58 jute accessions based on SSR data.Dendrogram constructed from cluster analysis by UPGMA. The 58 jute accessions are classified into major groups of dark jute（Go）and white jute（Gc），and further into subgroups（Go1，Go2，Gc1，and Gc2）.

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Acknowledgments

This project was sponsored by funds from the Doctoral Program of Higher Education of China（20133515120002）；Introduction Breeding and Varieties Demonstration of Featured Crops between China and Benin（2015I0001）；Distinguished Young Research Fund of Fujian Agriculture and Forestry University（xjq201401），China；China Agriculture Research System（nycytx-19-E06）；and Construction of Germplasm Resources Platform for Bast Fiber Crops in Fujian，China（2010N2002）.

Supplementary material

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cj.2015.05.005.

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*Corresponding authors.Tel.:+86 591 87644898.

E-mail addresses:lwzhang@fafu.edu.cn，zhang_liwu@hotmail.com（L.Zhang），qijm863@163.com（J.Qi）.

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

http://dx.doi.org/10.1016/j.cj.2015.05.005

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/）.

9 February 2015