The production of Brassica germplasm with a wider genetic base is essential for using them properly in the genetic improvement of rapeseed/mustard. During the present study, different RAPD (RandomAmplified Polymorphic DNA) primers were used to estimate the genetic distances among thirty different genotypes in all possible combinations. Level of genetic polymorphism at DNA level observed duringpresent study varied, but in general it was in the range of 21.54 to 59.36%. The dissimilarity coefficient matrix of these lines based on the data of four RAPD markers using UPGMA method was also used toconstruct a dendrogram. The dendrogram analysis indicated that the lines UCD-319/2, UCD-310/2 and P34/R3 were genetically apart from other lines that can be exploited in a synergistic way to create widergenetic base in local Brassica germplasm thus augmenting the Brassica breeding program

Ahmad, MA

Ahmed, N

Ali, S

Ali, W

Durrishahwar, N

Muhammad, W

Munir, I

Swati, ZA

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 6 (14), pp. 1634-1638, 18 July 2007     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals        Full Length Research Paper  Molecular characterization of some local and exotic Brassica juncea germplasm  Waqar Ali1, Iqbal Munir1, Mian Afaq Ahmad1, Wisal Muhammad1, Nisar Ahmed1, Durrishahwar2, Shahid Ali1 and Zahoor A. Swati1  1Institute of Biotechnology and Genetic Engineering (IBGE) NWFP Agricultural University, Peshawar, Pakistan. 2Department of Plant Breeding and Genetics, NWFP Agricultural University Peshawar, Pakistan.  Accepted 14 June, 2007  The production of Brassica germplasm with a wider genetic base is essential for using them properly in the genetic improvement of rapeseed/mustard. During the present study, different RAPD (Random Amplified Polymorphic DNA) primers were used to estimate the genetic distances among thirty different genotypes in all possible combinations. Level of genetic polymorphism at DNA level observed during present study varied, but in general it was in the range of 21.54 to 59.36%. The dissimilarity coefficient matrix of these lines based on the data of four RAPD markers using UPGMA method was also used to construct a dendrogram. The dendrogram analysis indicated that the lines UCD-319/2, UCD-310/2 and P34/R3 were genetically apart from other lines that can be exploited in a synergistic way to create wider genetic base in local Brassica germplasm thus augmenting the Brassica breeding program.   Key words: Rapeseed, Brassica juncea, genetic diversity, RAPD, molecular markers.   INTRODUCTION  Genetic variability is of prime importance for the improve-ment of many crop species including Brassica. There is increasing number of reports where Random Amplified Polymorphic DNAs (RAPDs) have been successfully used to estimate genetic variability in Brassica (Demeke et al., 1992; Wang et al., 2002, Dulson et al., 1998; Diva-ret et al., 1999), common wheat (Liu et al., 1999; Sivolap et al., 1999), maize (Zhang et al., 1998; Bernardo et al., 1997) and barley (Hamza et al., 2004). In Brassica and its related genera, RAPD markers have been used suc-cessfully for identification and phylogenetic relationship among and within the species (Ren et al., 1995). In gene-ral, polymorphism in amphidiploids is less than that obs-erved in diploid species. For example, the level of poly-morphism reported for B. napus is less than amphidip-loids 45% (Ferreira et al., 1994; Uzunova et al., 1995; Cheung et al., 1997), whereas in B. oleracea it can be higher  than  80%  (Cheung  et  al.,  1997). Similarly, in B.     *Corresponding author. E-mail: iqmunir@hotmail.com, waqarali04@yahoo.com. juncea a polymorphism of approximately 60% has been reported by Cheung et al. (1997). Lower levels of poly-morphism in are expected since they have also a lower level of out-crossing due to a weak and often non-existing self-incompatibility system. Molecular markers are the best tools for determining genetic relationships. A variety of molecular markers have been used to study the extent of genetic variation among the diverse group of important crops in the genus Brassica. These include restriction fragment length poly-morphism (RFLP), simple sequence repeats (SSR), ran-dom amplification of polymorphic DNA (RAPD) (Os-born and Lukens, 2003; Karp et al., 1997) and amplified frag-ment length polymorphism (AFLP) (Negi et al., 2001; Vos et al., 1995). These techniques differ in their principles and generate varying amounts of data. RFLP analysis is labour intensive, time consuming and expensive.  The purpose of present study was to characterize Brassica juncea lines from local and exotic sources at molecular level.  Such analyses, which are not previously available for these Brassica lines, can be utilized for selecting better parents for breeding programs. For this purpose  thirty Brassica lines were analyzed at molecular     Table 1. Description of all 30 B. juncea lines used in the current study.  S. No Genotypes S. No Genotypes 1 UCD – 635 16 RL – 16 2 UCD - 310/2 17 T – 42 3 UCD - 310/2 18 500 – 2 4 UCD - 6/15 19 PR 79 R6 5 UCD - 10/8 20 PR – 78 6 UCD - 5/12 21 P34 – R3 7 UCD - 8/10 22 P105 - R7 8 UCD - 40/1 23 P118 - R8 9 UCD - ¾ 24 P 27 - R 2 10 UCD - 46/1 25 P107 - R8 11 UCD - 6/24 26 P – 35 12 UCD - 319/2 27 P 269 – 1 13 K – 424 28 PR57/72 14 CV - 54 – 102 29 Raya Anmol 15 SONG – 86 30 BSA    Table 2. PCR thermal profile.  Step Temperature Duration Hot Start 94oC 4 min. Denaturation 94oC 1 min. Primer Annealing 36oC 1 min. Extension 72oC 2 min Total Cycles (40)   Final Extension 72oC 10 min    level using random amplified polymorphic DNA (RAPD) primers.   MATERIALS AND METHODS  DNA isolation  Thirty B. juncea genotypes of local and exotic origin were cha-racterized during the present study using molecular approach (Table 1). For genomic DNA isolation, about 10 cm of fresh leaf material from 3 - 4 weeks old seedling was grind to fine powder in a 2 ml eppendorf tube under liquid nitrogen, following the method of Weining and Langridge (1991). The powder was homogenized with 500 L of DNA extraction buffer (4% SDS, 0.1 M Tris-Hcl, 10 mM EDTA, pH 8.0) and an equal volume of phenol:chloroform:isoamyl alcohol in the ratio of 25:24:1 respectively, was added to it. The whole mixture was vigorously shaken for 20 - 30 s and aqueous phase was recovered by centrifugation at 5000 rpm for 5 min. The supernatant was transferred to a fresh tube and the DNA was precipitated from it by adding 1/10th volume of 3 M sodium acetate (pH 5.0) with an equal volume of isopropanol. The DNA was pelleted by centrifugation for 7 min, washed twice with ice cold 70% ethanol, dried at 37oC and dissolved in 40 - 45 l of TE buffer (10 mM Tris-Hcl, 1mM EDTA, pH 8.0) containing 40 g/ml RNase A. The concentration of DNA was estimated by comparing its intensity with that of the  DNA of known  concentration on  a  0.8%  agarose  Ali et al.        1635    gel ith Tris borate EDTA (TBE) buffer. The DNA was diluted with double distilled, autoclaved and de-ionized water at the ratio of 1:5 concentrations for use in PCR.   Polymerase chain reaction  The Polymerase Chain Reaction was carried out using protocols of Prasad et al. (2000) with modifications in thermal profile given in Table 2. The Random Amplified Polymorphic DNA markers synthe-sized from the Gene Link Technology, USA were employed for genetic diversity analyses (sequence information for the primers are given in Table 3). DNA amplification was carried out in PCR tubes containing 17 l reaction mixtures, having 1 l template DNA, 0.5 l RAPD primer, 9 l of dd H2O and 5 l of PCR mix (composed of 460 l H2O, 500 l buffer, 10 l each of dNTPs and 300 l MgCl2) (Table 5).  The PCR reaction mixture, except DNA and primer in the above mentioned volume was pooled to sterilized PCR tubes and was mixed thoroughly by gentle pipetting. DNA and primer were added to the reaction mixture and centrifuged mildly to collect all constit-uents and were subjected to the thermal profile given above. The reaction was carried out in the Thermocycler (GeneAmp 2700) (Table 4).   Data analysis   For statistical analysis, all the scorable bands were considered as single locus/allel. The loci were scored as present or abscent. Bi-variate 1-0 data matrix was generated. Genetic distances were cal-culated using Unweighted Pair Group of Arithmetic Means (UPGMA) procedure as follows (Nei and Li, 1979):  GD= (1- dxy)/(dx+dy-dxy)  where GD = Genetic distance among two genotypes, dxy = total number of common loci (bands) in two genotypes, dx = Total num-ber of loci (bands) in genotype 1, and dy = total number of loci (bands) in genotype 2. The 1-0 bivariate data matrix for each set of wheat lines based on the data of four RAPD primers was used to construct a dendrogram using computer program “Popgene32” ver-sion 1.31 (http://www.ualberta.ca./~fyeh/fyeh)   RESULTS AND DISCUSSION    Random Amplified Polymorphic DNAs (RAPDs) analyses are widely used for detecting genetic polymorphism bet-ween genotypes at molecular level in many crop species. During the present study four RAPD primers (GLA05, GLA07, GLA09 and GLA10) were used to estimate gene-tic diversity in B. juncea genotypes. Level of genetic poly-morphism (estimated as percent genetic distance) obser-ved during present study varied, but in general it was in the range of 21.54 to 59.36%. Size range of scorable bands (alleles) was from 100 -7000 bp range of scorable bands (alleles) was from 100 -7000 bp (an example of PCR amplification using RAPD primer GLA10 is presen-ted in Figure 1). Maximum genetic distance (86%) was observed between UCD-6/15 and P269-1, closely follo-wed by UCD-6/15 and P118-R8 (84%). Minimum genetic distance (8.3%) was observed between P107-R8 and Raya Anmol. Similar results were reported by Das et al. (1999) and Cansian and Echeverrigaray (2000), who obs-1636           Afr. J. Biotechnol.    Table 3. Detailed description and Sequence information of primers used.  Primer Sequence Size TM (oC) Mol. Wt. (Da) A05 primer (AGGGGTCTTG ) 10 bp 29.5 2987.9 A07 primer (GAAACGGGTG) 10 bp 29.5 3037.0 A09 primer (GGGTAACGCC) 10 bp 33.6 3012.9 A10 primer (GTGATCGCAG) 10 bp 33.6 3012.9  TM = Melting temperature of primer, Mol. Wt = Molecular weight of the primer.                        Figure 1. PCR profile of 30 genotypes of Brassica juncea using RAPD primer GLA10. (=marker, 1.UCD – 635, 2. UCD - 310/2, 3. UCD - 310/2, 4. UCD - 6/15, 5. UCD - 10/8, 6. UCD - 5/12, 7. UCD - 8/10, 8. UCD - 40/1, 9. UCD - ¾, 10. UCD - 46/1,  11. UCD - 6/24, 12. UCD - 319/2, 13. K – 424, 14. CV - 54 – 102, 15. SONG – 86, 16. RL – 16, 17. T – 42, 18. 500 – 2, 19. PR 79 R6, 20. PR – 78,  21. P34 - R3,  22. P105 - R7, 23. P118 - R8, 24. P 27 - R2, 25. P107 - R8, 26. P- 35, 27. P 269 – 1. 28. PR57/72, 29. Raya Anmol, 30. BSA).    Table 4. PCR reagents.  Reagents Volume H20 460 l Buffer 500 l dATP (100 mM)   10 l dCTP (100 mM)`   10 l dGTP (100 mM)   10 l dATP (100 mM)   10 l MgCl2  (25 mM) 300 l Total 1.3 ml    erved more or less similar ranges of genetic dissimila-rities in different Brassica lines. Table 5. Reagents for single reaction.  Reagents Volume H2O 9 µl PCR Mix 5 µl Primer 1 µl Taq Polymerase 0.5 µl DNA 1 µl Total 16.5 µl    For individual RAPD primers, higher level of genetic polymorphism among the B. juncea lines was found in case of RAPD primer GLA07 (not shown), where higher levels  of  genetic  variability were observed among differ-                     1     2    3    4    5    6    7    8    9    10  11  12  13  14  15   16  17  18  19      1       1                                       20      21      22     23      24      25      26      27     28        29       30                          20      21      22     23      24      25      26      27     28        29       30                    bp 23137   9416                  6667  4361   2322  2027                                                 bp 23137   9416                                                                                   6667  4361   2322  2027                                                                                                                Ali et al.        1637      Figure 2. Dendogram constructed for 30 Brassica juncea lines based on genetic distances using four RAPD primers.    ent comparisons, indicating its power for the identification of individual genotypes. In Brassica and its related genera, RAPD markers have been used successfully for identification and phylogenetic relationship among and within the species (Ren et al., 1995). RAPD analysis has also been extensively used to document the genetic variation in Brassicas (Zhu and Wu, 1998; Demeke et al., 1992; Jain et al., 1994; Bhatia et al., 1995; Thormann et al., 1994). PCR-based fingerprinting techniques, is very efficient both in cost and time to identify RAPD markers associated with a trait. The RAPD markers are easier and quicker to use and are preferred in applications where the relationships between closely-related breeding lines are of interest (Hallden et al., 1994). The 1-0 bivariate data dissimilarity coefficient matrix for thirty B. juncea lines based on the data of four RAPD primers using UPGMA method was used to construct a dendrogram (Figure 2). Based on the dendrogram analy-sis the thirty B. juncea lines can be categorized into 3 major groups i.e. A, B and C. Brassica lines UCD-319/2 was most distantly related among the 30 lines used in the study. The other two lines, showing higher genetic dissi-milarities, were UCD-310/2 and P34/R3.  It is recommended that genetically distant lines obser-ved among the 30 B. juncea genotypes should be used in future breeding program for improving yield and quality characteristics of Brassica. Further it was observed that PCR based assays like RAPD can be used effectively to estimate genetic variability in B. juncea and considering easy handling of the technique, they are especially suita-ble for breeding programs where large number of lines / accession have to be analyzed.   1638           Afr. J. 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Molecular characterization of some local and exotic Brassica juncea germplasm

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Molecular characterization of some local and exotic Brassica juncea germplasm

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