ORIGINAL ARTICLE

Vol 5 Issue 1 Feb 2015 ISSN No : 2230-7850

International MultidisciplinaryResearch Journal

Indian Streams

Research Journal

Executive Editor Editor-in-Chief

Ashok Yakkaldevi H.N.Jagtap

Welcome to ISRJ RNI MAHMUL/2011/38595 ISSN No.2230-7850

Indian Streams Research Journal is a multidisciplinary research journal, published monthly in English, Hindi & Marathi Language. All research papers submitted to the journal will be double - blind peer reviewed referred by members of the editorial board.Readers will include investigator in universities, research institutes government and industry with research interest in the general subjects.

International Advisory Board

Flávio de São Pedro FilhoFederal University of Rondonia, Brazil

Kamani PereraRegional Center For Strategic Studies, Sri Lanka

Janaki SinnasamyLibrarian, University of Malaya

Romona MihailaSpiru Haret University, Romania

Delia SerbescuSpiru Haret University, Bucharest, Romania

Anurag MisraDBS College, Kanpur

Titus PopPhD, Partium Christian University, Oradea,Romania

Pratap Vyamktrao NaikwadeASP College Devrukh,Ratnagiri,MS India

R. R. PatilHead Geology Department Solapur University,Solapur

Rama BhosalePrin. and Jt. Director Higher Education, Panvel

Salve R. N.Department of Sociology, Shivaji University,Kolhapur

Govind P. ShindeBharati Vidyapeeth School of Distance Education Center, Navi Mumbai

Chakane Sanjay DnyaneshwarArts, Science & Commerce College, Indapur, Pune

Awadhesh Kumar ShirotriyaSecretary,Play India Play,Meerut(U.P.)

Mohammad HailatDept. of Mathematical Sciences, University of South Carolina Aiken

Abdullah SabbaghEngineering Studies, Sydney

Ecaterina PatrascuSpiru Haret University, Bucharest

Loredana BoscaSpiru Haret University, Romania

Fabricio Moraes de AlmeidaFederal University of Rondonia, Brazil

George - Calin SERITAN Faculty of Philosophy and Socio-Political Sciences Al. I. Cuza University, Iasi

Editorial Board

Iresh Swami Ex - VC. Solapur University, Solapur

N.S. DhaygudeEx. Prin. Dayanand College, Solapur

Narendra Kadu Jt. Director Higher Education, Pune

K. M. BhandarkarPraful Patel College of Education, Gondia

Sonal Singh Vikram University, Ujjain

G. P. Patankar S. D. M. Degree College, Honavar, Karnataka

Maj. S. Bakhtiar ChoudharyDirector,Hyderabad AP India.

S.Parvathi DeviPh.D.-University of Allahabad

Sonal Singh,Vikram University, Ujjain

Hasan BaktirEnglish Language and Literature Department, Kayseri

Ghayoor Abbas ChotanaDept of Chemistry, Lahore University of Management Sciences[PK]

Anna Maria ConstantinoviciAL. I. Cuza University, Romania

Ilie Pintea,Spiru Haret University, Romania

Xiaohua YangPhD, USA

......More

Rajendra ShendgeDirector, B.C.U.D. Solapur University, Solapur

R. R. Yalikar Director Managment Institute, Solapur

Umesh Rajderkar Head Humanities & Social Science YCMOU,Nashik

S. R. PandyaHead Education Dept. Mumbai University, Mumbai

Alka Darshan Shrivastava Shaskiya Snatkottar Mahavidyalaya, Dhar

Rahul Shriram Sudke Devi Ahilya Vishwavidyalaya, Indore

S.KANNAN Annamalai University,TN

Satish Kumar Kalhotra Maulana Azad National Urdu University

Address:-Ashok Yakkaldevi 258/34, Raviwar Peth, Solapur - 413 005 Maharashtra, India Cell : 9595 359 435, Ph No: 02172372010 Email: ayisrj@yahoo.in Website: www.isrj.org

Indian Streams Research Journal ISSN 2230-7850 Impact Factor : 3.1560(UIF)

Volume-5 | Issue-1 | Feb-2015 Available online at www.isrj.org

MOLECULAR FINGERPRINTING AND PHYLOGENETIC RELATIONSHIPS AMONG

THREE EGYPTIAN MOULOKHYIA GENOTYPES (CORCHORUS OLITORIUS L.) USING RAPD, ISSR AND SRAP

MARKERS

1 2Hadia A. Heikal , Reham M. Abd El-Azeem and 3Hossam E. El-Wakil

1Molecular Biology Department, Genetic Engineering & Biotechnology Research Institute (GEBRI),Sadat City University, Sadat City, Egypt

2Environment Biotechnology Departments, Genetic Engineering & Biotechnology Research Institute (GEBRI),Sadat City University, Sadat City, Egypt

3Agricultural Botany Dept., Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria, Egypt

Abstract:-The purpose of this study was to document three Egyptian Moulokhya genotypes (namely; Fallahy, Saedy and Siewy), differentiate among them and identify their genetic relationships using Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat (ISSR) and Sequence-Related Amplified Polymorphism (SRAP). The results of 15 RAPD, 15 ISSR primers and 40 SRAP primer combinations showed a total of 233, 239 and 574 reproducible bands, in which 66 (28.3%), 66 (27.6%) and 201 (35%), respectively were polymorphic and 37(15.9%), 65 (27.2%) and 257 (44.8%), respectively were specific markers. RAPD, ISSR and SRAP gave the highest percentages of polymorphism with Saedy genotype, which could be distinguished by one RAPD primer and two SRAP primer combinations. While four RAPD, three ISSR primers and 16 SRAP primer combinations distinguished Fallahy genotype, there were three RAPD, two ISSR primers and two SRAP primer combinations which distinguished Siewy genotype. The highest similarity indices were shown between Fallahy and Saedy genotypes (66%, 64%, 52% and 60%) using RAPD, ISSR, SRAP data and their combinations, respectively. The genetic relationships of the three genotypes as demonstrated by the phylogeny analysis, showed thatSiewy genotype was distinct from the other two genotypes, which were grouped together in one cluster. This implied that the genomic sequences of Siewy genotype varied at the genetic level and suggested that, it can be used as a genetic resource in the breeding programs to produce abiotic stress-tolerant genotypes. In conclusion, thesefindings not only provide a preliminary database for genetic biodiversity and can be useful in the breeding programs, but also are a necessity for production of Egyptian royalties of Moulokhyagenotypes.

Keywords:Corchorusspp., Moulokhya, phylogentic relationships, RAPD, ISSR, SRAP.

INTRODUCTION

Jew’s mallow (Corchorusolitorius L., 2n=14) is an annual herb, belongs to family Malvaceae, formerly underTiliaceae (Sinhaet al., 2011). It is an important leafyvegetable in Egypt (called Moloukhyia),since there are two major cultivars; Fallahy and Saedy, planted in the North and South, respectively. Besides that, there is a genotype which grows in Siwa Oasis, having special quality, taste, and high production (El-Wakilet al., 2010).The molecular markers are providing a quick and reliable method for estimating genetic relationships among plant genotypes (Thormannet al., 1994). Among the different types of molecular markers, Randomly Amplified Polymorphic DNAs (RAPDs) is useful for the assessment of genetic diversity (William et al., 1990) owing to its

1 3Hadia A. Heikal , 2Reham M. Abd El-Azeem and Hossam E. El-Wakil ,“ MOLECULAR FINGERPRINTING AND PHYLOGENETIC

RELATIONSHIPS AMONG THREE EGYPTIAN MOULOKHYIA GENOTYPES (CORCHORUS OLITORIUS L.) USING RAPD, ISSR AND SRAP MARKERS ” Indian Streams Research Journal | Volume 5 | Issue 1 | Feb 2015 | Online & Print

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simplicity, speed, and relatively low-cost (Rafalski and Tingey, 1993) when compared to other types of molecular markers. Inter-Simple Sequence Repeat (ISSR) markers have been successfully employed (also proven to be useful) for detecting genetic polymorphisms among accessions by generating a large number of markers that target multiple microsatellite loci distributed across the genome (Yang et al., 1996).Sequence-Related Amplified Polymorphism (SRAP) is a novel molecular marker technique designed to amplify open reading frames (ORFs) (Li and Quiros, 2001). The forward primers preferentially amplify exonic regions and the reverse primers preferentially amplify intronic regions and regions with promoters. The SRAP marker system has been used to investigate genetic diversity in plant species, including Brassica (Li and Quiros, 2001), Cucurbita (Ferriolet al., 2003b), cotton (Lin et al., 2004) and other plant species.

The objectives of this study aimed to identify the polymorphisms and determine genetic relationships among three Egyptian genotypes of Corchorus spp. (Fallahy, Saedy and Siewy) using random amplified polymorphic DNA (RAPD), Inter-simple Sequence Repeats (ISSR) and sequence-related amplified polymorphism (SRAP) markers.

MATERIALS AND METHODS

This study was done at the Department of Molecular Biology, Genetic Engineering & Biotechnology Research Institute, Sadat City University, Egypt; and Genetics Department, Faculty of Agriculture, Alexandria University, Egypt.

Plant materials:

In this study, three different genotypes of Corchorusolitorius(Moloukhyia) collected from three different locations in Egypt (Fallahy from Delta, Saedy from Upper Egypt and Siewy from Siwa Oasis) were used.

Genomic DNA Extraction:

A sample of about 0.1g of fresh young leaves tissues wasused from each genotype and ground into a fine powder using liquid nitrogen. Genomic DNA was extracted using the BioFlux Kit with adding 0.1gm PVP as a minor modification.

RAPD-PCR analysis:

RAPD-PCR was performed using 15 decameroligo-nucleotide primers (Table 1) that were selected from the Operon kit (Operon Technologies Inc., Alabameda, CA). Each 25µl PCR reaction mixture were containing;50ng genomic DNA. (1µl) 25pmole primer, 12.5µl MaximoTaq DNA polymerase (GeneOn, Germany) which containing

+25mM of each dNTP, 50mM Mg , 1.25U of Taq DNA polymerase and, PCR buffer (20mMKCl, 16mM (NH ) SO , 4 2 4

20mM Tris-HCl, pH 8.8). Amplification was performed using Biometra T gradient Thermalcycler which was 1

programmed according to Solimanet al. (2003); de-naturation step 94 °C for 3 min, followed by 40 cycles of 94°C for 1min, 36°C for 1min and 72°C for 1min with a final extension at 72°C for 10min. The PCR products were electrophoresed in 1.4% ethidium bromide stained agarose gels with 0.5XTris-borate-EDTA (TBE) buffer and visualized with UV transilluminator.

ISSR-PCR analysis:

Fifteen primers based on dinucleotide, tetranucleotide or pentanucleotide repeats were used in ISSR analysis (Table 1).The PCR reaction mixture consisted of 20ng genomic DNA, 5X PCR buffer (Promega), 25mM/L MgCl (Promega), 100µM/L of each dNTP (promega), 66ng/µl primer and 5 U/µl Taq polymerase in a 25µl volume. 2

The amplification protocol was carried out according to Heikalet al. (2007).The pre-reaction began with an initial denaturation at 94°C for 5min, followed by 5 cycles of 92°C for 30sec, 35°C for 2min and 72°C for 90 sec, followed by 35 cycles of 92°C for30sec, 40°C for 30sec and 72°C for 90sec, with a final extension at 72°C for 5min, and eventually stored at 4°C. The amplified products were electrophoresed in 1.4% ethidium bromide stained agarose gel with 0.5X TBE buffer and visualized with UV transillumintor.

SRAPanalysis:

In this study, Ninety oligo-primer combinations (9 forward primers and 10 reveres primers, Table 2) were applied for SRAP analysis of 3 jew’s mallow genotypes. The SRAP-PCR reaction mixture with a total volume of 15µl consisted of (1µl) 0.3mmol-1 primers, 8µl MaximoTaq DNA Polymerase (GeneOn, Germany) which

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-1containing (1.2mmol-1 dNTPs,32.0mmol Mg+2, 0.8U Taq DNA Polymerase, PCR buffer (20mM KCl, 16mM (NH ) SO , 20mM Tris-HCl, pH 8.8) and30ng of genomic DNA. DNA amplifications were performed with 4 2 4

Biometra T gradient Thermacycler. SRAP-PCR program was conducted according to Daweiet al. (2010)as follow: 1

94°C for 5min followed by 5 cycles of 94°C for 60s, 35°C for 60s, and 72°C for 60s; followed by 35 cycles of 94°C for 60s, 50°C for 60s, 72°C for 60s; and final extension at 72°C for 7min. The amplified products were electrophoresed in 2% ethidium bromide stained agarose gel (Liu et al., 2008) with 0.5X TBE buffer and visualized with UV transilluminator.

Data analysis:

Patterns of the studied genotypes using RAPDs, ISSRs and SRAPs primers were scored as presence (1), or absence (0) bands by using of the Phoretix 1D image analysis system (PhoretixInternational, London) to integrate the data. Similarity indices were calculated and consensus tree was developed based on the RAPD, ISSR and SRAP banding patterns of the three Corchorus genotypes using SPSS statistical analysis program (Version11.5). The genetic relationships among the 3 genotypes, at the molecular level, were determined.

Table 1: Code and sequences of the fifteen different random primers (10-mer oligonucleotides) and fifteen different ISSR primers used in the present study.

RAPD Primers ISSR Primers

No Oligo Name

Primer code

Sequence (5’-3’) No Oligo Name Primer

code Sequence (5’-3’)

1 OPA-05 A5 5’-AGG GGT CTT G­3’

1 ISSR 814 S1 5’-CTC TCT CTC TCT CTC TTG-3’

2 OPA-10 A10 5’-GTG ATC GCA G­3’

2 ISSR 844A S2 5’-CTC TCT CTC TCT CTC TAC-3’

3 OPA-15 A15 5’-TTC CGA ACC C­3’

3 ISSR 844B S3 5’-CTC TCT CTC TCT CTC TGC-3’

4 OPB-07 B7 5’- GGT GAC GCA G-3’

4 ISSR 17898A S4 5’- CAC ACA CAC ACA AC -3’

5 OPC-08 C8 5’- TGG ACC GGT G-3’

5 ISSR 17898B S5 5’- CAC ACA CAC ACA GT -3’

6 OPC-12 C12 5’- TGT CAT CCC C­3’

6 ISSR 17899A S6 5’- CAC ACA CAC ACA AG-3’

7 OPC-16 C16 5’- CAC CAT CCA G-3’

7 ISSR 17899B S7 5’- CAC ACA CAC ACA GG-3’

8 OPD-04 D4 5’- TCT GGT GAG G-3’

8 ISSR HB-8 S8 5’- GAG AGA GAG AGA GG -3’

9 OPD-11 D11 5’- AGC GCC ATT G-3’

9 ISSR HB-9 S9 5’- GTG TGT GTG TGT GG -3’

10 OPD-19 D19 5’- CTG GGG ACT T-3’

10 ISSR HB-10 S10 5’- GAG AGA GAG AGA CC -3’

11 OPH-11 H11 5’- AGC GCC ATT G-3’

11 ISSR HB-11 S11 5’- GTG TGT GTG TGT CC -3’

12 OPH-18 H18 5’- GAA TCG GCC A-3’

12 ISSR HB-12 S12 5’- CAC CACCAC GC -3’

13 OPR-01 R1 5’- CTT CCG CAG T­3’

13 ISSR HB-13 S13 5’- GAG GAGGAG GC -3’

14 OPR-02 R2 5’- GGT GCG GGA A-3’

14 ISSR HB-14 S14 5’- CTC CTCCTC GC -3’

15 OPR-05 R5 5’- GAC CTA GTG G-3’

15 ISSR HB-15 S15 5’- GTG GTGGTG GC -3’

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Table 2: Forward and reverse SRAP primers information.

Forward Primers Reverse Primers

No Oligo Name

Sequence (5’-3’) No Oligo Name

Sequence (5’-3’)

1 Me1 5’- TGA GTC CAA ACC GGA TA ­

3’ 1 Em1 5’- GAC TGC GTA CGA ATT AAT -3’

2 Me2 5’- TGA GTC CAA ACC GGA GC ­

3’ 2 Em2 5’- GAC TGC GTA CGA ATT TGC -3’

3 Me3 5’- TGA GTC CAA ACC GGA AT ­

3’ 3 Em3 5’- GAC TGC GTA CGA ATT GAC -3’

4 Me4 5’- TGA GTC CAA ACC GGA CC ­

3’ 4 Em4 5’- GAC TGC GTA CGA ATT TGA -3’

5 Me5 5’- TGA GTC CAA ACC GGA AG ­

3’ 5 Em5 5’- GAC TGC GTA CGA ATT AAC -3’

6 Me6 5’- TGA GTC CAA ACC GGA CA ­

3’ 6 Em6 5’- GAC TGC GTA CGA ATT GCA -3’

7 Me7 5’- TGA GTC CAA ACC GGA CG ­

3’ 7 Em7 5’- GAC TGC GTA CGA ATT CAA -3’

8 Me8 5’- TGA GTC CAA ACC GGA CT ­

3’ 8 Em8 5’- GAC TGC GTA CGA ATT CAC -3’

9 Me9 5’- TGA GTC CAA ACC GGA GG ­

3’ 9 Em9 5’- GAC TGC GTA CGA ATT CAG -3’

10 Em10 5’- GAC TGC GTA CGA ATT CAT -3’

RESULTS

1-RAPD Results:

Agarose gel electrophoresis for the 15 RAPD-PCR amplified DNA products of three Moulokhya genotypes studied are shown in Fig. (1) and Table (3). The results revealed that, the 15 RAPD primers produced a total of 233 reproducible fragments ranging from 10 (primer A10) to 27 fragments (primer R1), that ranged in size from 215 to 371bp with an average of 15.5 bands for each primer. Table (3) showed that 138 (59.2%) DNA amplified fragments were common fragments (monomorphic) in the three genotypes and 66 (28.3%) amplified fragments werepolymorphic bands. The highest percentage of common fragments was 85.7% using primer H18, while the lowest percentage of common fragments was 31.6% with primer A5.No common fragments were showed with primer H11, which gave the highest percentage of polymorphism (90.9%). The lowest polymorphic percentage (14.3%) was recorded using primer H18, whereas no polymorphism was revealed with primers A15, C8 and D19. Table (3) indicated that the amplification product of Fallahy genotype produced 74 reproducible fragments, from which 15 (20.3%) were polymorphic. Furthermore, the amplification product of Saedy genotype produced 79 reproducible fragments, from which 25 (31.7%) were polymorphic and the amplification product of Siewy genotype produced 80 reproducible fragment, from which 18 (22.5%) were polymorphic. While primer R1 gave the highest number of bands (9 and 10 bands) with Fallahy and Saedy genotypes, respectively. Primers R1and C12 gave the highest number of bands (8 bands) with Siewy genotype. The lowest number of bands (3 bands) was revealed when primers A10, B7 and C16 were used with Fallahy genotype, primers A10, A15 and C8 used with Saedy genotype and primer A15 was used with Siewy genotypes. Primer H11not revealed any reproducible fragments with Fallahy genotypes. Primer R1 gave the highest percentage (55.6%) of polymorphism when used with Fallahy genotype, while primer H11 gave the highest percentages (100% and 83%) with Saedy and Siewy genotypes, respectively. The lowest percentage of polymorphism (12.5%) was recorded using primer C12 with Fallahy and Siewy genotypes, whereas primers D4 and H18 gave the lowest percentage (20%) of polymorphism with Saedy genotype. There were no polymorphism revealed using 8 primers (A10, A15, B7, C8, D11, D19, H11 and H18), 4 primers (A15, C8, D19 and R5) and 4 primers (A15, C8, D4 and D19) with Fallahy, Saedy and Siewy genotypes, respectively.

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Genotype-specific markers are shown in Table (3). Overall, 37 (15.9%) positive specific markers out of 233fragments were scored for the presence of unique bands among the three genotypes of moulokhya, 13 of them for Fallahy, 8 for Saedy and 16 for Siewy. There were 29 (12.5%) negative specific markers for the absence of a common fragment. The number of positive specific bands per primer varied (1-5) as with size range of fragments (262­3714bp). The highest total number of specific markers was obtained with primers A15, D4 and A5 (5 fragments). The primers C16, D11 and H11 produced the lowest number of specific markers (one fragment), while no specific markers were revealed with primers B7 and H18. The highest number of specific markers was scored for the genotype Fallahy (3 markers), when primer A15 was used. Primers C16 and H11, as a positive specific markers, and primer D4, as a negative specific marker were identified that could discriminate the Siewy genotype from the other two genotypes. While primer R5 was identified that could discriminate the Saedy genotype as a negative specific marker, primer D11 was identified that could discriminate the Fallahy genotype from the other two genotypes, as a positive and negative specific markers, besides primers A10, B7 and H18, as a negative specific markers.

In conclusion, 15 RAPD primers used could produce polymorphic and specific markers, to discriminate and identify the different genotypes. Moreover, RAPD are considering a universal marker, which can be used in different species.

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2-ISSR Results:

To quantify ISSR polymorphism, the data obtained were constructed as a matrix of the binary character states. The presence or absence of amplified fragments of certain size in ISSR patterns was considered as state 1 or 0, respectively. Table (4) indicated that, the 15 ISSR primers amplified 7 to 28 bands with primers S9 and S6, respectively that ranged in size between 160 and 2613bp (Fig. 2). The 15 ISSR primers produced a total of 239 reproducible fragments, with an average of 15.9 bands for each primer. There were 66 (27.6%) polymorphic bands and 108 (45.2%) common fragments. The highest percentage of polymorphism was 62.5% using primer S14, which with primer S15 did not shown any common bands. The lowest percentage of polymorphism was 13.3% with primer S4, while no polymorphism were revealed with primers S2 and S3, which gave the highest percentage of common fragments (100%). The results showed that, the amplification product of Fallahy, Saedy and Siewy genotypes produced 83, 75 and 81 reproducible fragments, from which 21 (25.3%) fragments, 29 (38.7%) and 16 (19.8%) were polymorphic bands, respectively. The primer S6 gave the highest number of bands (10, 8 and10 bands) with Fallahy, Saedy and Siewy genotypes, respectively, while primer S9 gave the lowest number of bands (3, 2 and 2 bands) with Fallahy, Saedy and Siewy genotypes, respectively. The highest percentages (50%, 75% and 66.7%) of polymorphism were revealed by 3 primers (S5, S14 and S15), 2 primers (S14 and S15) and primer S14 when used with Fallahy, Saedy and Siewy genotypes, respectively. Primers S1, S4 and S6 gave the lowest percentages (12.5%, 20% and 10%) of polymorphism with Fallahy, Saedy and Siewy, respectively. There were no polymorphism revealed using primers S2, S3 and S9 with Fallahy, Saedy and Siewy genotypes, beside primer S4 with Fallahy genotype and S11 with Siewygenotype.

Table (4) showed a total of 65 (27.2%) positive specific markers out of 239 fragments among the three genotypes used in this study, 26 (31.3%) of them for Fallahy, 10(13.3%) for Saedy and 29 (35.8%) for Siewy genotypes. There were 33 (13.8%) negative specific markers for the absence of a common fragment. The number of positive specific bands per primer varied (1-9) with size range of fragments (160-2613bp). The highest total number (9 fragments) of specific markers was obtained with primer S12. The primers S7 and S9 produced one specific fragment for each, while no specific markers were revealed with primers S2 and S3. Considering to each genotype, the highest number of specific markers were 4 fragments when used one primer (S12) and 2 primers (S11 and S15) with Fallahy and Seiwy genotypes, respectively. Saedy genotype revealed2 fragments as the highest number of specific markerswhen used primers S11 and S12. Primers S7 and S9 as a positive specific markers and primer S4 as a negative specific marker were identified that could discriminate the Fallahy genotype from the other two genotypes, while primers S10 and S11 were identified as a positive and negative specific markers that could discriminate the Siewy genotype from the other two genotypes.

In conclusion, 15 ISSR primers used could produce polymorphic and specific markers, to discriminate and identify the different genotypes.

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3-SRAP Results:

Sequence-related amplified polymorphism (SRAP) was the first time used with the Egyptian moulokhya genotypes. DNA from fresh leaves of the three genotypes was screened with 90 primer combinations, of which 40 primer combinations (Table5) gave stable and reproducible amplification patterns (Fig. 3). Among the total 574 amplified fragments, 201 (35%) were polymorphic with an average of 14.4 fragments for each primer combination. Table (5) showed that 117 (20.4%) DNA amplified fragments were common fragments (monomorphic) in the three genotypes. The highest percentage of common fragments was 63.2% using Me6-Em1and Me6-Em3 primer combinations. The lowest percentage of common fragments was 15.8% with Me6-Em9 primer combination.

The highest percentage of polymorphism (72.7%) using Me4-Em3 primer combination, whereas the lowest percentage (14.3%) was recorded using Me4-Em4 primer combination. No polymorphism was revealed with primer combinationMe2-Em8. Table (5) indicated that the amplification product of Fallahy genotype produced 157 reproducible fragments, from which81 (51.6%) were polymorphic. Furthermore, the amplification product of Saedy genotype produced 207 reproducible fragments, from which 130(62.8%) were polymorphic and the amplification product of Siewy genotype produced 210 reproducible fragments, from which 106 (50%) were polymorphic. While primer combination Me6-Em2 gave the highest number of bands (9) with Fallahy and Saedy genotypes, primer combination Me6-Em4 gave the highest number of bands (8) with Siewy genotype. The lowest number of bands (1, 2 and 3 band) were revealed when primer combinations Me5-Em5, Me5-Em9 and Me7-Em2 were used with Fallahy genotype, Me2-Em10 when used with Saedy genotype and Me2-Em8, Me4-Em3, Me5-Em6, Me7-Em3, Me7-Em4 and Me7-Em5when used with Siewy genotype, respectively. The highest percentage (100%) of polymorphism was recorded using 4 primer combinations (Me4-Em2, Me4-Em3, Me5-Em9 and Me7-Em2), 8 primer combinations (Me1-Em4, Me1-Em6, Me1-Em8, Me3-Em8, Me3-Em9, Me4-Em2, Me5-Em8 and Me6-Em1) and 4 primer combinations (Me5-Em6, Me5-Em7, Me6-Em1 and Me6-Em2) with Fallahy, Saedy and Siewy, respectively. The lowest percentage of polymorphism (20%, 16.7% and 14.3%) were recorded using primer combinationsMe9-Em7, Me7-Em3 and Me1-Em5 with Fallahy, Saedy and Siewygenotypes, respectively. There were no polymorphism revealed using7 primer combinations (Me2-Em8, Me4-Em5, Me5-Em5, Me7-Em3, Me7-Em4, Me7-Em5and Me9­Em6), oneprimer combination (Me2-Em8) and3primer combinations (Me2-Em8, Me2-Em10 andMe7-Em9) with Fallahy, Saedy and Siewy genotypes, respectively.

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Genotype-specific markers are shown in Table (5). Overall, 257 (44.8%) positive specific markers out of 574 fragments were scored for the presence of unique bands among the three genotypes of moulokhya, 76 of them for Fallahy, 77 for Saedy and 104 for Siewy. There were 101 (17.6%) negative specific markers for the absence of a common fragment, 58 of them for Fallahy, 10 for Saedy and 32 for Siewy. The number of positive specific bands per primer combination varied (2-15), which obtained with primer combinations Me4-Em2 and Me2-Em8, respectively. The highest number of specific markers was scored for the genotype Saedy (7 markers) when primer combination Me2–Em8 was used. There were one primer combination (Me6-Em1) as a positive specific marker and 16 primer combinations as a negative specific markers (Me3-Em8, Me4-Em5, Me5-Em2, Me5-Em5, Me5-Em6, Me5-Em7, Me5-Em8, Me6-Em1, Me6-Em3, Me6-Em4, Me6-Em5, Me7-Em3, Me7-Em4, Me7-Em5, Me9-Em6 and Me9-Em7) identified that could discriminate the Fallahy genotype from the other two genotypes. While 2 primer combinations (Me1-Em9 and Me4-Em4) as a negative specific markers were identified that could discriminate the Saedy genotype, Siewy genotype could be discriminate by primer combination Me4-Em2 as a positive and negative specific markers besides Me2-Em10 primer combination as a negative specific marker.

In conclusion, 40 SRAP primer combinations used could produce polymorphic and specific markers, to discriminate and identify the different genotypes.

4-Genetic similarity and phylogenetic relationship:

The genetic similarity and relationships among the three Egyptian moulokhya (Corchorusolitorius) genotypes were estimated by SPSS statistical analysis program (Version 11.5) using separate data of RAPD, ISSR and SRAP techniques (Figure4and Table6). The results showed that the highest similarity index (66%, 64% and 52%)were observed between Fallahy and Saedy genotypes according to RAPD, ISSR, SRAP data, respectively, while the same data recorded the lowest similarity index (52%, 38% and 44%), respectively between Fallahy and

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. Molecular Fingerprinting And Phylogenetic Relationships Among Three Egyptian Moulokhyia Genotypes .............

Siewy genotypes. The dendrogram for phylogentic relationships revealed that, genotype Siewy distinct from the other two genotypes, which they were grouped together, according to the data from RAPD, ISSR and SRAP. Genotypes distribution on the consensus tree according to the banding patterns of RAPD, ISSR and SRAP were the same. Therefore, it was better to use the banding patterns combination of the three techniques to use more segments of the genome that will increase the validity of the consensus tree and confirm it. Table (6) exhibited that the most two closely related genotypes were Fallahy and Saedy with highest similarity index (60%), which were shown also in the consensus tree (Fig.4). On the other hand, the most two distantly related genotypes were between Fallahy and Siewy with the lowest similarity index (50%) and the Siewy genotype was distinct separately in the consensus tree.

Table 6: Similarity indices for the three Egyptian moulokhya genotypes on the base of their banding patterns with RAPD, ISSR, SRAP and their combined data.

Case

Matix File Input based on RAPD data

Matix File Input based on ISSR data

Matix File Input based on SRAP data

Matix File Input based on

Combined RAPD, ISSR

and SRAP data

Fallahy Saedy Fallahy Saedy Fallahy Saedy Fallahy Saedy

Saedy 0.658 0.642 0.519 0.597

Siewy 0.523 0.649 0.383 0.558 0.438 0.505 0.496 0.564

DISCUSSION

Genetic information plays a significant role in determining the effectiveness of work in many areas, and molecular markers now provide an excellent tool of obtaining large amounts of genetic data to inform the conservation process (Heikalet al., 2007). There are a few studies on molecular diversity among Egyptian moulokhya genotypes. However, very few efforts were made in the past to develop molecular markers to study the genetic variability among the Corchorus spp. (Hossainet al., 2002 &2003; Qi et al., 2004; Basuet al., 2004; Roy et al., 2006; Haqueet al., 2007; Mir et al., 2008b). In this study, RAPD, ISSR and SRAP techniques were used to study the genetic diversity and relationships between the three Egyptian genotypes of moulokhya. It is the first time that SRAP technique used with the Egyptainmoulokhya genotypes. The results indicated that, Saedy genotype gave the highest percentage of polymorphism (31.7%, 38.7%% and 62.8%) using RAPD, ISSR and SRAP, respectively compared with the other two genotypes, Fallahy and Siewy. Considering to specific markers, Fallahy genotype gave the highest percentage (36.5% and 85.4%) with RAPD and SRAP, respectively. While the Siewy genotype showed the highest percentage of specific markers (56.8%) when ISSR was used, the Saedy genotype showed the lowest specific marker percentage (15.2%, 18.7% and 42%) with RAPD, ISSR and SRAP, respectively. These results demonstratedthat the highest similarity index was between Fallahy and Saedy genotypes using RAPD, ISSR and SRAP data, separately and its combined data. The same data showed the lowest similarity index between Fallahy and Seiwy. This may be due to that the two genotypes, Fallahy and Saedyare cultivated in the delta of Egypt, while Siewy genotype is cultivated in the Siwa Oasis using well water, and may be consider this area as an isolated area, which make Siewy genotype having a gene pool thatmay be differed from the other genotypes. These results agree with the results of Hamzaet al. (2013), where the results showed that the similarity index of the two genotypes Fallahy and Saedy grouped together in one group and Siewy genotype was separate using RAPD and ISSR combined data.

Corresponding to comparison between the different molecular techniques with each genotype, the results showed that, the polymorphism percentages were 28.3%, 27.6% and 35% using RAPD, ISSR and SRAP, respectively. For each genotype, the polymorphism percentages of Fallahy genotype were 20.3%, 25.3% and 51.6% using RAPD, ISSR and SRAP, respectively. While the polymorphism percentages with Saedy genotype were 31.7%, 38.7% and 62.8% using RAPD, ISSR and SRAP, respectively, the percentages with Siewy were 22.7%, 19.8% and 50% using RAPD, ISSR and SRAP, respectively.

According to the specific marker percentages (positive and negative), results were 28.3%, 41% and 62.4% using RAPD, ISSR and SRAP, respectively. For the results of each genotype, it indicated that, Fallahy genotype had total specific marker percentages 36.5%, 45.8% and 85.4%, while the total of specific marker percentages with Saedy genotype were 45.2%, 18.7% and 42%, these percentages with Seiwy genotype were 33.8%, 56.8% and 65.2% using RAPD, ISSR and SRAP, respectively.

On the other hand, the Results revealed the number of primers that could be used as molecular specific marker (positive and negative) with each genotype. There were 4 RAPD primers (D11, A10, B7 and H18), 3 ISSR primers (S4 and S7 and S9) and 16 SRAP primer combinations (Me3 -Em8, Me4-Em5, Me5-Em2, Me5-Em5, Me5­Em6, Me5-Em7, Me5-Em8, Me6-Em1, Me6-Em3, Me6-Em4, Me6-Em5, Me7-Em3, Me7-Em4, Me7-Em5, Me9­

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. Molecular Fingerprinting And Phylogenetic Relationships Among Three Egyptian Moulokhyia Genotypes .............

Em6 and Me9-Em7) could be discriminate Fallahy genotype from other two genotypes. While there were one RAPD primer (R5) and 2 SRAP primer combinations (Me1-Em9 and Me4-Em4) could be discriminate Saidy genotype from the other two genotypes, there was no ISSR primer could be discriminate it. Genotype Siewy could be discriminated by 3 RAPD primers (C16, H11 and D4) and 2 ISSR primers (S10 and S11) and 2 SRAP primer combinations (Me2-Em10 and Me4-Em2). This shows clear that the RAPD and SRAP can be used more broadly with Fallahy genotype to discriminate it from other two genotypes, whereas, it preferred to use ISSR with Siewy genotype.

The results proved that SRAP, which gavehigher percentages of polymorphism, positive and negative specific bands, is more specified than RAPD and ISSR. These could be explained because of SRAP primers designed to amplify ORFs. This means that, the techniques used in this study were given increased polymorphisms with the developed technique (SRAP > ISSR > RAPD), and this result applies with Budaket al. (2004),who compared the four marker systems in buffalo grass and figured out the values of average discriminating power as: SRAP >SSR >ISSR > RAPD. A possible explanation is that SRAP marker is based on two-primer amplification of PCR, and its primer combination is more efficient than arbitrary primer of ISSR (Li and Quiros, 2001).Since, Siewy was the most salt resistant genotype (Hamzaet al., 2013), it can be used as a genetic resource in the breeding programs.

In conclusion, Moulokhya is a very important vegetable crop in Egypt with unknown range of diversity. Here, we reported valuable molecular tools. The information of polymorphism and specific markers using RAPD, ISSR and SRAP in the three Egyptian genotypes of moulokhya is useful in the assessment of its genetic diversity. The Siewy genotype proved of value in breeding moulokhya for salt-tolerant based on the fact it is distantly-related with the other two studied genotypes (Fallahy and Saedy). Further study is planned for a wider screening of moulokhya in Egypt.

REFERENCES

1.Basu, A., Ghosh, M., Meyer, R., Powell, W., Basak, S.L.,Sen, S.K., 2004. Analysis of genetic diversity in cultivated jute determined by means of SSR markers and AFLP profiling. Crop Sci., 44: 678-685.2.Budak,H., Shearman,R.C.,Parmaksiz, I. 2004. Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs, SSRs, RAPDs, and SRAPs. TheorAppl Genet., 109: 280-288.3.Dawei, X., Feng, S., Zhao, H., Jiang, H., Shen, B., Shi, N., Lu, J., Liu, J., Wang, H. 2010. The linkage maps of Dendrobium species based on RAPD and SRAP markers. J. Genet. Genomics, 37: 197-204.4.El-Wakil, H.M., Soliman, A.,Khaled, A.E., Abdelsalam,N.R.,Nassar, A.M.K. 2010. Genetic description of jew’s mallow (Corchorusolitorius L.) in Egypt. Midwest ASPB meeting AMES, Candia (Poster).5.Ferriol, M., Pico, B., Nuez, F. 2003b. Genetic diversity of some accessions of Cucurbita maxima from Spain using RAPD and SRAP markers. Genet. Res. Crop Evol., 50: 227-238.6.Hamza, A.H., El-Wakil, H.E., Khaled, A.E., Fayed, A., Abdelsalam, N.R.,Hebatullah, M.Rady. 2013. Biochemical and molecular studies on three local genotypes of jew’s mallow (Corchorusolitorius L.). Journal of the Advances in Agricultural Researches, 18(4): 30-47.7.Haque, S., Begum, S., Sarker, R.H., Khan, H. 2007. Determining genetic diversity of some jute varieties and accessions using RAPD markers. Plant Tiss. Cult. Biotechnol., 17: 183-191.8.Heikal, A.Hadia, Mabrouk, Y., Badawy, O.M., El-Shehawy, A.,Effat, A. Badr, 2007. Fingerprinting Egyptian Gramineae species using random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) markers. Res J Cell& Mol Bio.,1(1): 23-30.9.Hossain, M.B., Haque, S.,Khan, H. 2002. DNA fingerprinting of jute germplasm by RAPD. J BiochemMol Biol., 35:414-419.10.Hossain, M.B., Awal, A., Rahman, M.A., Haque, S., Khan, H. 2003. Distinction between cold-sensitive and –tolerant jute by DNA polymorphisms. J. Bio. Mol. Bio.,36(5): 427-432.11.Li, G.,Quiros, C.F. 2001. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor. Apple. Genet., 103: 455-461.12.Lin, Z.X., Zhang, X.L.,Nie, Y.C.2004. Evaluation of application of a new molecular marker SRAP on analysis of F2 segregation population and genetic diversity in Cotton. Acta Gent Sin., 31: 622-626 (in Chinese with English abstract). 13.Liu, L.J., Xiang, P.D.Bo, W. 2008. Genetic relation analysis on Ramie (Boehmerianivea L. Gaud) Inbred Lines by SRAP markers. Agric. Sci. in China, 7(8): 944-949.14.Mir, R.R., Rustgi, S., Sharma, S., Singh, R., Goyal, A., Kumar, J., Gaur, A., Tyagi, A.K., Sinha, M.K., Balyan, H.S., Gupta, P.K. 2008b. A preliminary genetic analysis of fibre traits and the use of new genomic SSRs for genetic diversity in jute. Euphytica., 161: 413-427.15.Qi,J.M., Zhou, D., Wu, W.R., Wu, W., Lin, L., Fang, P., Wu, J.M., Wu, J. 2004. A comparison between RAPD and ISSR technology in detection of genetic diversity of Jute. Sci. Agric. Sin., 37: 2006-2011.

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. Molecular Fingerprinting And Phylogenetic Relationships Among Three Egyptian Moulokhyia Genotypes .............

16.Rafalski, J.A.,Tingey, S.V. 1993. Genetic diagnosis in plant breeding: RAPDs, microsatellites and machines. Genetic., 9: 275-280.17.Roy, A., Bandyopadhyay, A., Mahapatra, A.K., Ghosh, S.K., Singh, N.K., Bansal, K.C., Koundal, K.R.,Mohapatra, T. 2006. Evaluation of genetic diversity in jute (Corchrusspesies) using STMS, ISSR and RAPD markers. Plant Breed., 125: 292-297.18.Sinha, M.K., Kar, C.S.,Ramasubramanian, T.,Kundu, A.,Mahapatra, B.S. 2011. Wild crop relatives: Genomic and Breeding Resources, Industrial Crops. In: C. Kole (ed.), Corchorus. DOI 10.1007/978-3-642-21102-7_2, Springer-Verlag, Berlin Heidelberg 2011.19.Soliman, S.S., Bahy, A.A., Mohamed, M.M. 2003. Genetic comparisons of Egyptian date palm cultivars (Phoenix dactylifera L.) by RAPD-PCR. African Journal of Biotechnology, 2: 86-86.20.Thormann, C.E., Ferreira, M.E., Camargo, L.E.A., Tivang, J.G., Osborn, T.C. 1994. Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species. TheorAppl Genet., 88: 973-980.21.Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalsky, J.A.,Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as gentic markers. Nucl Acids Res., 18: 6531-6535.22.Yang, W., Oliveira, A.C., Godwin, I., Schertz, K.,Bennetzen, J.L. 1996. Comparison of DNA marker technologies in characterizing plant genome diversity: variability in Chinese sorghum. Crop. Sci., 36: 1669-1676.

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