SSR and RAPD Profile Based Grouping of Selected

J. Plant Biochemistry & Biotechnology Vol. 17(1), 29-35, January 2008

SSR and RAPD Profile Based Grouping of Selected Jute Germplasm with Respect to Fibre Fineness Trait Javid Iqbal Mir1, Pran Gobinda Karmakar2, Swapan Chattopadhyay2, Subrata Kumar Chaudhury2 Subrata Kumar Ghosh1 and Anirban Roy1* 1 Plant Virus Laboratory and Biotechnology Unit, Division of Crop Protection, and 2Division of Crop Improvement, Central Research Institute for Jute and Allied Fibres, Barrackpore, Kolkata 700 120, India

With an aim to develop mapping population on fibre fineness trait, grouping of 16 selected jute accessions, eight each from Corchorus olitorius and Corchorus capsularis which showed promising agronomic characteristics, was carried out using fibre fineness data and DNA fingerprinting using SSR and RAPD primers. Based on fibre fineness trait two subgroups depicting the fine and coarse fibre yielding accessions were obtained in each species. A total of 26 RAPD primers and 22 pairs of SSR primers yielded 277 and 41 scorable bands, respectively. High level of polymorphism was detected between fine and coarse fibre yielding jute accessions. Dendrogram showed that all the accessions formed two main clusters between C. olitorius and C. capsularis and each main cluster subdivided in two clusters containing fine and coarse fibre jute accessions. RAPD and SSR marker data-sets showed high levels of positive correlation (Mantel test, r = 0.97). Grouping of jute accessions based on morphological and molecular data was highly correlated. This study will be useful in future jute breeding programs. Key words: Corchorus capsularis, Corchorus olitorius, fibre fineness, genetic diversity, parent selection , random amplified polymorphic DNA, simple sequence repeat.

Jute (Corchorus capsularis L and Corchorus olitorius L; 2n = 14), is the most important natural, renewable, and biodegradable fibre after cotton. Traditionally jute is being used as raw material in the packaging industries for more than 160 years. The fibre of commerce is obtained from the bast tissue of the stem of cultivated varieties of these two species. Plants of C. capsularis produce finer but weaker quality white colour fibre than those of C. olitorius, which produce stronger but coarser tossa commercial fibre. A combination of the useful characters of the two species in a single genotype is desirable. Traditional plant breeding program evoked limitations as neither of the released varieties of the two species have the desired quality parameters and moreover there is lack of genetic diversity among these cultivated jute varieties though variation exist within the germplasm in both the species (1- 3). Problem becomes more intricate as there is a sexual incompatibility between the two jute species and between wild Corchorus species (4, 5). In recent years, molecular approaches are being considered as alternatives for crop improvement through precise selection. The first step in implementation of such approach essentially demands the use of divergent *Corresponding author. E-mail: [email protected]

germplasm lines within a species for development of a mapping population followed by identification of molecular markers linked to the fibre fineness quantitative trait loci (QTLs). Very limited efforts have been reported so far on the use of molecular approaches in jute improvement. Genetic diversity analysis of jute with random amplified polymorphic DNA (RAPD) markers has been reported using indigenous varieties, germplasm lines and wild relatives (6, 7). Inter simple sequence repeat (ISSR) markers were used to classify wild species of jute (8). Use of RAPD and amplified fragment length polymorphic (AFLP) markers for grouping of cold tolerant germplasm lines has also been reported by Hossain et al (9). In India, diversity analysis of different exotic germplasm accessions has been carried out using eight chloroplast genome specific microsatellite primers developed from Nicotiana tabacum and 10 pairs of AFLP primers (2). Recently, few microsatellite markers have been developed and a comparative analysis was done using different markers (RAPD, ISSR and STMS) to evaluate molecular diversity among Indian varieties of both the species and exotic germplasm along with wild relatives of jute (3). A perusal of literature indicated that no work has so far been carried out regarding QTL identification of fibre fineness

30 J Plant Biochem Biotech

trait. With a goal to develop a mapping population for fibre fineness trait, we report here the identification and grouping of promising jute accessions with respect to fibre fineness characteristics using SSR and RAPD profiling.

Materials and Methods Plant material — A total of 16 jute (Corchorus sp) accessions (eight from each cultivated species) collected from Crop Improvement Division of our Institute were used in the study (Table 1). These accessions were selected based on their initial screening of the desirable traits as mentioned in the catalogues on germplasm characterization (morphological) and evaluation published by our Institute. Among these accessions, three commercial varieties of C. olitorius (JRO 524, JRO 878 and JRO 7835) and two commercial varieties of C. capsularis (Padma and UPC 94) were included along with promising germplasm lines of C. olitorius (OIJ 249, OIJ 255, OIN 572, OIN 574 and OIN 576) and C. capsularis species (CIJ 031, CIN 117, CIN 146, CIN 149, CIN 206 and CIN 312). The classification of genotypes into fine fibre category and coarse fibre category in the two species is based on different values of tex (unit of measurement for fibre fineness) (10). Hence in the present study, genotypes having fibre fineness upto or less than 2.60 tex were considered as fine fibre C. olitorius, whereas genotypes Table 1. List of jute accessions with respective fineness of fibre Species

Fibre group

Genotype

C. olitorius

Fine fibre

JRO 878

2.60 ± 0.054

OIN 572

2.18 ± 0.050

OIN 574

2.07 ± 0.071

Coarse fibre

C. capsularis

Fine fibre

Coarse fibre

Fineness (tex)*

OIN 576

2.38 ± 0.098

JRO 524

3.40 ± 0.088

JRO 7835

3.50 ± 0.064

OIJ 249

3.81 ± 0.091

OIJ 255

3.84 ± 0.112

CIJ 031

1.47 ± 0.054

CIN 206

1.25 ± 0.067

CIN 312

1.30 ± 0.046

UPC 94

1.60 ± 0.052

CIN 117

2.32 ± 0.072

CIN 146

2.28 ± 0.067

CIN 149

2.26 ± 0.064

Padma

2.58 ± 0.052

* Mean value of 15 replications of each accession ± SEM

having fineness upto or less than 1.60 tex were considered as fine fibre C. capsularis. Evaluation on fibre fineness — The fibre fineness was measured by Air-flow method (11). The working principle of the instrument is based on the measurement of rate of flow of air through a fibre plug of specified mass and volume placed in a cylindrical cell and subjected to air current at a known pressure. As the fibre fineness is proportional to the rate of flow of air, the flow-meter is calibrated in terms of fineness (in tex). Thus by measuring the rate of flow of air through the fibre plug we determined the fibre fineness. DNA extraction — DNA was extracted from young jute leaves using the cetyl trimethyl ammonium bromide (CTAB) method (12) and purified by RNase treatment. The mucilage and polyphenolic compounds were removed by passing DNA through purification column. The DNA was quantified on 0.8% agarose gel stained with ethidium bromide in the presence of different concentrations of undigested λ-DNA and a final concentration of 25 ng μl-1 was used for PCR. RAPD and SSR analyses — DNA from an individual plant of each jute accession was screened with 26 random primers (Operon Technologies Inc). The PCR reaction (25 μl) contained the following: 1x reaction buffer (20 mM TrisCl pH 8.4, 50 mM KCl), 0.2 mM dNTPs, 2 mM MgCl2, 10 pM primer, 1.0 Unit of Taq DNA polymerase, and 25 - 50 ng genomic DNA. The DNA was amplified in a thermal cycler (Thermo Px2 Thermal Cycler) that was programmed as o follows: initial DNA denaturation for 5 min at 94 C; 45 cycles o o of 60 sec at 94 C (denaturation), 60 sec at 37 C (annealing), o and 120 sec at 72 C (extension); and a final extension at o 72 C for 7 min. In addition 22 pairs of SSR primers designed for polymorphism survey from the jute sequences deposited in the GenBank were used (Table 2). For standardization of annealing temperatures of SSR primers, gradient PCR was carried out in a gradient thermal cycler (T-gradient 96, BIOMETRA, Germany). For SSR, initial o denaturation at 94 C for 5 min was followed by 35 cycles o o o at 94 C for 1 min, 50 – 53 C for 1 min and 72 C for 2 min. o The final extension was carried out at 72 C for 7 min. The RAPD amplified-DNA was analyzed by electrophoresis on 2% agarose gel in a 0.5x TBE buffer. The SSR amplified fragments were resolved on 3% metaphore agarose gel in a 0.5x TBE buffer. The gels were stained with ethidium bromide (0.5 μg ml-1) and visualized under UV light.

Molecular Profiling and Grouping of Jute Germplasm

31

Table 2. SSR markers developed and used in the present study Primer-ID

Sequence ID

Motif

Reverse primer

Forward primer

Product size (bp)

JSSR-07

DQ108589

AAG

CAAAGCTTTAAGAGAGAGGAAA

AATGGCTATAACGTTTAGTTGG

195

JSSR-08

DQ108589

TCCTT

CAAAGCTTTAAGAGAGAGGAAA

TTAACCTCAAGCAAGTAGAATG

336

JSSR-09

DQ108428

TG

GATTAAGGTTTATCGGTTTCTG

TATCTCAAAGCTATAGGTCGGT

282

JSSR-10

AY562124

CAA

AATCACACACACACTAGTCAGG

CAATCTCTCCATAGGATCAACT

296

JSSR-11

AY562123

TC

GAGTGGTACAAATTCAAGAGGT

AGAGACCAAGTGATACTCGAAC

204

JSSR-13

DQ108592

TTTA

CCAATTTATTTGATCCAATTCT

CAAAGAAGAAGAAGAAGGAAAG

354

JSSR-14

DQ108581

TTTTC

GGCTCAACAAGATACATCAGTA

CCTAGCCCATAAGAAATAAGAA

390

JSSR-15

DQ108576

CT

TACCTTATCCTTTATGGGACAG

TCTTTAGGTATATCGGGTATGG

321

JSSR-16

DQ108575

CT

TACCTTATCCTTTATGGGACAG

TCTTTAGGTATATCGGGTATGG

321

JSSR-17

DQ108574

AT

TTACCTCTTTCAGGTTTCAATC

ATACAAGGAGGGTACAAGAATG

320

JSSR-18

DQ108572

ACC

TTAACCAATGAAGTTCAACAAA

TTGAGGCTCTCTAATTTCATCT

114

JSSR-19

DQ108520

TC

GAGTGGTACAAATTCAAGAGGT

AGAGACCAAGTGATACTCGAAC

204

JSSR-20

DQ108452

CAA

AATCACACACACACTAGTCAGG

CAATCTCTCCATAGGATCAACT

296

JSSR-42

DQ108581

TTA

GGCTCAACAAGATACATCAGTA

CCTAGCCCATAAGAAATAAGAA

390

JSSR-43

DQ108581

TTTTC

TCTTCTTTCTTCTTCGTTATGG

TTTGGAATTGATTGTAGTGAAA

369

JSSR-44

DQ108580

CT

GGAAGGAAGGATACAACTAACA

TAAGAATTCCTCACAATGAACA

375

JSSR-45

DQ108577

CTT

AAGGTGTGGTCTTTATGTGAAT

TAGTCGATCTAGAGAGAGGAGC

363

JSSR-46

DQ108576

GTTT

ACTCAAATTCACTCGATCTCTC

CTTGTTACAACACAAATTGACC

384

JSSR-47

DQ108575

GTTT

ACTCAAATTCACTCGATCTCTC

CTTGTTACAACACAAATTGACC

384

JSSR-48

DQ108574

TTTC

TTACCTCTTTCAGGTTTCAATC

ATACAAGGAGGGTACAAGAATG

320

JSSR-49

DQ108574

CTT

TTACCTCTTTCAGGTTTCAATC

ATACAAGGAGGGTACAAGAATG

320

JSSR-50

DQ108573

CTT

AAGGTGTGGTCTTTATGTGAAT

TAGTCGATCTAGAGAGAGGAGC

363

The prominent DNA bands that were amplified by a given primer were scored as present (1) or absent (0) for all of the samples that were studied. The total number of bands, polymorphic band and percent polymorphism across the species, between and within the sub-groups from each species were calculated using MS Excel (Table 3). The Polymorphism Information Content (PIC) value of individual primers were calculated based on the formula PIC = 2 × F (1- F) (13). The Jaccard’s similarity index was calculated using NTSYS-pc version 2.02e (Applied BioStatistics, Inc., Setauket, NY, USA) package to compute pairwise Jaccard’s similarity coefficients (14) and this similarity matrix was used in cluster analysis using an unweighted pair-group method with arithmetic averages (UPGMA) and sequential, agglomerative, hierarchical and nested (SAHN) clustering algorithm to obtain a dendrogram. The correlation of matrices obtained from SSR and RAPD profiles was judged by two-way Mantel test (15) using MxComp Module of NTSYS-pc version 2.02e package. To judge the confidence of the group revealed in the

dendogram, bootstrap analysis was performed using the WINBOOT program (16) with 1000 replications.

Results Grouping of germplasm accessions based on fibre fineness trait — The fibre fineness of the jute accessions under study is presented in Table 1. The values for the fibre fineness trait ranged between 2.07 to 3.84 in case of C. olitorius and 1.25 to 2.58 in case of C. capsularis. With this diversity both C. olitorius and C. capsularis accessions were divided into two groups, one containing the accessions with fine fibre and the other group having the accessions yielding coarse fibre. This indicated that fine fibre jute accessions and coarse fibre jute accessions in both the cultivated species were distinct with respect to fibre fineness trait. But this distinction is not sufficient enough to identify the most divergent parental lines in each species. Furthermore as the fineness trait is influenced by several environmental factors, use of molecular markers for

0.27 3.77 1.85 10.23 Polymorphic band per primer

1.45

2.65

0.05

2.27

0

4.23

0.09

2.5

0.05

0.14

1 6.23 5 10.65 Total band per primer

1.86

5.62

0.95

5.35

0.95

6.73

1

5.23

0.95

0.91

6 98 48 32 266 Polymorphic band

69

1

59

0

110

2

65

1

3

22 162 130 41 Total scorable band

277

146

21

139

21

175

22

136

21

20

3 21 15 1 19 2 19 0 16 1 16 18 26 Total polymorphic primer

RAPD SSR RAPD SSR RAPD Attribute

1

SSR RAPD SSR RAPD SSR

RAPD

SSR

RAPD

SSR

Within coarse C. olitorius Between fine and coarse C. capsularis Within coarse C. capsularis Within fine C. capsularis

Within C. capsularis group Between

C. olitorius and C. capsularis

Table 3. Total and polymorphic DNA markers obtained from RAPD and SSR analyses of jute accessions

Within fine C. olitorius

Within C. olitorius group

Between fine and coarse C. olitorius

32 J Plant Biochem Biotech

assessing the divergence pattern of the parents appeared to be more suitable and reliable. Development and use of SSR markers — Taking advantage of the availability of genomic sequences of C. olitorius (cultivar O-4) from the GenBank database, a set of 22 pairs of SSR primers was designed and used in the present study (Table 2). In these available sequences, seven dinucleotide, eight trinucleotide, four tetranucleotide and three pentanucleotide repeats were observed. Sequences DQ108589, DQ108574 and DQ108581 yielded two repeat motifs each. These repeats could be grouped into 13 distinct classes namely, AAG, TCCTT, TG, CAA, TC, TTA, TTTTC, CT, AT, ACC, CTT, GTTT and TTTC. On the basis of cross transferability and polymorphism, these SSR markers could be grouped into four classes, (a) Cross species transferable but non-polymorphic SSR : five SSR markers (JSSR 10, JSSR 11, JSSR 18, JSSR 49 and JSSR 50); (b) Cross species transferable polymorphic SSR: seven SSR markers (JSSR 8, JSSR 9, JSSR 13, JSSR 15, JSSR 17, JSSR 19 and JSSR 43); (c) C. olitorius specific polymorphic SSR : two SSR markers (JSSR 46 and JSSR 47) are specific to C. olitorius and showed intraspecific polymorphism; and (d) C. olitorius specific non-polymorphic SSR : eight SSR markers (JSSR 7, JSSR 14, JSSR 16, JSSR 20, JSSR 42, JSSR 44, JSSR 45, and JSSR 48). Out of 22, 18 primers gave an average polymorphism of 78% from a total of 41 bands between C. olitorius and C. capsularis with an average of 1.86 bands including average 1.45 polymorphic bands per primer (Table 3). In case of C. capsularis, two primers gave an average polymorphism (9%) between fine and coarse fibre accessions from a total of 22 bands. The corresponding results between fine and coarse C. olitorius accessions revealed that three primers gave an average 27% polymorphism from a total 22 bands. While JSSR 13 and JSSR 15 showed polymorphism within fine fibre yielding accessions of C. capsularis and C. olitorius respectively, none of the SSR primers used could detect any polymorphism within coarse fibre accessions of any of these two species. Allele number per locus varied from one (JSSR 07) to four (JSSR 13). RAPD analysis — All of the jute accessions were tested for identification of diversity using the RAPD technique. The data on RAPD profile are presented in Table 3. All the 26 RAPD primers used gave an average polymorphism of 96.03% from a total of 277 bands with an average of 10.65 bands per primer including 10.23 polymorphic bands per

Molecular Profiling and Grouping of Jute Germplasm

primer. In case of C. capsularis, 19 primers (73.08%) showed 62.66% polymorphism between fine fibre and coarse accessions from a total of 175 bands with average number of 6.73 bands per primer. Results between fine and coarse C. olitorius accessions revealed that 21 primers gave 60.49% polymorphism from a total of 162 bands with 6.23 bands per primer. When the polymorphism profile was analyzed within fine and coarse accessions of each species, it was observed that fine and coarse fibre accessions of C. capsularis gave higher number of total bands (146 and 139, respectively) than fine (136 bands) and coarse (130 bands) of C. olitorius. While fine fibre accessions of C. olitorius gave higher average polymorphism (47.8%) with an average of 2.50 polymorphic bands per primer than fine fibre accessions of C. capsularis (42.45%) with an average of 2.65 polymorphic bands per primer. However, less polymorphism (36.9%) was noticed within coarse fibre accessions of C. olitorius with 1.85 polymorphic bands per primer than its counterpart in C. capsularis (42.45%) with 2.27 polymorphic bands per primer. Primers OPI06, OPL10 and OPM20 with PIC values 0.5 appeared more informative between C. capsularis and C. olitorius species. The primers OPA09, OPO14 and OPP09 were more informative with PIC values >0.3 between fine and coarse accessions of C. capsularis,

33

primers OPA09, OPJ01, OPK01 and OPP03 with PIC values >0.37 were more informative within fine accessions of C. capsularis, primers OPA09, OPP03 and OPP09 with PIC valve >0.3 were more informative within coarse accessions of C. capsularis, primers OPK01, OPP09, OPV08 and OPW10 with PIC > 0.3 were more informative between fine and coarse accessions of C. olitorius, primers OPK01, OPP03 and OPP09 with PIC >0.4 were more informative within fine accessions of C. olitorius and primers OPP09, OPV08 and OPW10 with PIC >0.33 were more informative within coarse accessions of C. olitorius jute. Two way Mantel test was done between the SSR and RAPD data matrices. The correlation coefficient was estimated to be 0.97 between matrices generated by SSR and RAPD markers using the Mantel test (t = 10.05, P = 1.00). Genetic relationship among jute accessions — In order to find out the genetic relationship between different jute accessions, SSR and RAPD data sets were combined together and used for analysis using NTSYS-pc version 2.02e. The Jaccard’s similarity coefficient ranged from 0.120.18 with an average of 0.14 between C. capsularis and C. olitorius species (Fig. 1). In C. capsularis, the average similarity between fine and coarse fibre yielding accessions was 69%, whereas within the fine fibre yielding accessions it was 75% and within coarse fibre yielding accessions it was 78%. In C. olitorius, the average similarity

Fig. 1. UPGMA dendrogram showing clustering pattern of C. capsularis and C. olitorius accessions. The bootstrap values are given on the nodes.

34 J Plant Biochem Biotech

between the fine and coarse fibre yielding accessions was 70% whereas within the fine fibre yielding accessions it was 75% and within coarse fibre yielding accessions it was 83%. The genetic relationship between the accessions was clearly depicted in the dendrogram which was constructed from the DNA profile and the confidence of the cluster was further confirmed by bootstrap analysis. The dendrogram showed that all the accessions formed two main clusters between C. olitorius and C. capsularis and each main cluster formed two sub clusters of fine and coarse fibre yielding accessions of jute species (Fig. 1). The grouping obtained with the SSR and RAPD analysis showed correspondence with the morphological grouping.

Discussion In order to group the selected accessions of both the cultivated species of jute with respect to fibre fineness trait and to find out the variability within and between these groups, the present investigation was conducted using SSR and RAPD generated DNA profile. When the morphological relationship with respect to fibre fineness was examined, the accessions of both the species could be divided into two categories with clear difference in their fibre fineness characteristics. Morphological characterization is an effective discriminating tool for jute varieties (1). However, this approach appears to be less informative as the trait is under polygenic control and requires more time and cost in developing and identifying the superior line from a breeding population. Recently, a number of studies have demonstrated the capacity of molecular markers in discriminating between varieties of wide range of species, including tomato (17), oilseed rape (18, 19), maize (20) and evergreen azaleas (21). Our immediate objective was to determine whether polymorphism was sufficient to distinguish the jute accessions yielding fine and coarse fibre and to identify the most divergent parental combination in both the species, which could be utilized in the development of trait specific mapping population. In the present study, 22 C. olitorius SSR primers were custom designed. These primers based on the genomic sequences of C. olitorius were not fully transferable to the C. capsularis as higher degree of polymorphism was shown in C. olitorius than in C. capsularis species. This suggested considerable divergence in the DNA sequence between the two cultivated species.

RAPD primers showed more polymorphism in discriminating fine and coarse fibre yielding accessions of C. olitorius than the corresponding C. capsularis accessions. The fine fibre yielding accessions of C. olitorius and C. capsularis were found more divergent than their respective counterparts. Earlier studies with commercial varieties and exotic germplasm revealed lower diversity within C. capsularis species (3), but in this study similar range of similarity index in C. capsularis indicated the existence of similar pattern of variability in the selected accessions of this species and that could be explained by the fact that diversity depends upon the choice of accessions taken for the study. The two cultivated species remained in two different major clusters with a very high degree of divergence. This suggested that they have a polyphyletic origin that supports the earlier proposition by Kundu (22), based on morphological and crossability relationships. The diversity analysis with jute germplasm done by earlier workers (2, 3, 6) also revealed a similar situation. The fine fibre yielding accessions (as revealed in morphological analysis) in both the cultivated species were clearly differentiated from the respective coarse fibre accessions indicated high degree of correlation between the morphological descriptors with the molecular data. However, the relationship between the individual accessions within fine or coarse fibre group did not show much correlation with the fineness data and that could be due to the influence of other morphological parameters which were not taken in account in this study. The Jaccard’s similarity coefficient values indicated the sufficient diversity exists between the fine and coarse fibre yielding accessions in both the species. The parent selection for development of mapping population with respect to fibre fineness trait can be done by selecting parents in any combination between the two clusters representing fine and coarse fibre yielding accessions in both the species. SSR and RAPD marker generated data-sets showed high level of positive correlation as revealed by higher value of r (r = 0.97) using the Mantel test (t = 10.05, P = 1.00). Thus, in the present study fibre fineness pattern of the selected accessions of jute was evaluated and the results were correlated with molecular profile data. This study is the first effort to group jute accessions according to a commercially desired trait, and suggested parents for developing mapping population to initiate marker assisted breeding in jute.

Molecular Profiling and Grouping of Jute Germplasm

Acknowledgement The authors are grateful to Dr H S Sen, Director, CRIJAF, Barrackpore, Kolkata, India for providing necessary help in the study.

9

Hossain MB, Awal A, Rahman MA, Haque S & Khan H, J Biochem Mol Biol, 36 (2003) 427.

10

NIRJAFT, In Data book on jute (BC Mitra, Editor), National Institute of Research on Jute and Allied Fibre Technology, Kolkata, India (1999) p 112.

11

Sinha NG & Bandyopadhyay SB, J Text Inst, 59 (1968) 148.

12

Murray MG & Thomson WF, Nucl Acids Res, 8 (1980) 4321.

13

Anderson JA, Churchill GA, Autrique JE, Tanksley SD & Sorrells ME, Genome, 36 (1993) 181.

14

Jaccard P, Bull Soc Vaud Sci Nat, 44 (1908) 223.

15

Mantel N, Cancer Res, 27 (1967) 209.

16

Yap IP & Nelson RJ, IRRI Discuss Paper Ser. 14, IRRI, Manila, Philippines, (1996).

17

Noli E, Salvi S & Tuberosa R, Genome, 40 (1997) 607. Lee D, Reeves JC & Cooke RJ, Electrophoresis, 17 (1996) 261.

Received 29 May, 2007; accepted 24 September, 2007.

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