Analysis of RAPD loci in Indian mackerel (Rastrelliger kanagurta), as generated by the arbitrary primer OPA 07 (GAAACGGGTG), revealed a maximum within-region genetic variability for samples from the east coast of India. Dendograms did not show clear centre-specific clusters. Restricted intermixing among the individuals between the east and west coasts in suggested

Dharmalingam, K.

Jayasankar, P.

Analysis of RAP'D loci in R. kanagurta, as generated by the arbitrary primer OPA 07 (GAAACGGGTG), revealed maximum

within-region genetic variability for samples from the east coast of lndia. Dendograms did not show clear centre-specific

clusters. Restricted infennixing among the individuals between the east and west coasts is suggeste

Jayasankar, P

Dharmalingam, K

CMFRI Digital Repository

Analysis of RAPD Polymorphisms in Rastrelliger kanagurta off India P. Jayasankar and K. Dharmalingam Abstract . , Analysis of RAf'D loci in R. kanagurto, as generated by the arbitra"l"y primer OPA 07 (GAAACGGGTG), revealed maximum within-region g~netic variability for samples from the east coast oflndia. pendograms did not show clear centre-specific clusters. Restlicted infennixing among the in~ividuals between th~ east and we~t coa.sts is suzgest~. Introduction The Indian mackerel, Rastre/Jjger kanagurta (Cuvier) , is a pelag ic shoaling fi sh widely distribu ted in the Indo-West Pacific region. It is one of the major marine fi shery resources in Indian waters. About 70 % of mackerel ca tches are landed along the west coast and the rest along the east coast and the Nicobar Islands (Noble et al. 1992). Observations based on tag-g ing studies indicate two possible movemcn ts of mackerel in Indian waters, one showing limited mi -gration and the other showing long distance, north -south mig ration (Venkataraman 1970). Rao (] 962) indicated the possible existence of mackerel spawning g rounds in several areas a long the coast of peninsular India and the Andaman and Nicobar Islands. Some attempts have becn made to delineate the stock structure of Indian mackerel from the east and west coasts based on mor-phometry and meristic charac-teristi cs, and more recen tly by studying protein (allozyme) poly-morp hism s (Scshappa 19 85; In d i a \ Bangalore J~~ f Madras Ca llcut · • ) J 1\ ,,' ' -·0_ ,'",,,,,:r t"fY / , ) "'" <P Cochln \.~z / ) I~\ I ~ortK oc I) ) r'~ ~ . I Tnvandrum • \-~.J S r, \ ' Mand ap am lanka 01 ' --.--~.l..-- ----;c- __ __ _ ,,: c"'L--- --'--;;-- - - -.! 6 ·-~· 72° 74 0 76° 78° 80° 82 Fig. 1. Map of India showing samplmg locations (Mandapam, Manga/ore and Fort Koehi). 52 Menezes et al. 1990, 19 93 ; Verma et a l. 1994, 1996) . These studies reveal low genetic differ-en tiation in the species. DNA-level marker s have sev-eral advantages over morphomet-ric, meristic or protein (allozyme) markers for studying stock struc-ture in fishes (Fergusson ct al. 1995; O'Reilly and Wright 1995; ) ayasankar 199 7). Random Am-plified Polymorphic DNA (RAPD) is a DNA approach for detecting genetic polymorph isms (Welsh and McClelland 1990; Williams et al. 1990). Dispensability of prior knowledge of the target sequencc and tcchnical simplicity make RAPDs a ttractive for populati on genetic studies. Our carlier study (Jayasankar and Dharmalingam 199 7) il-lus tr ated th e gene r a ti o n of RAPD fingerpr ints in 30 mack-ere l specimens collected from commercial landings a t two lo-ca tions on the wes t coast ( 11 fr om Manga lore a nd 8 fr om Fort Kochi) a nd one on the cast coas l (Man dap am, 11 sp cci-m e n s) of In dia (9 ° 1 7'-12 ° 52'N, 74 ° 52'- 7 9° 15 'E). This paper analyzes the finger -prints to evalu a te g enetic variation. Naga, The ICLARM Quarterty Materials and Methods Details of sampling sites, sam-pling methods, extraction of ge-nomic DNA and optimization of DNA amplification by RAPD -PCR are given elsewhere (Jayasankar and Dharmalingam 1997). Briefly, total genomic DNA was extracted from frozen/alcohol-preserved muscle by a modified phenol-chloroform procedure and was amplified in 25~1 reaction mix-tures containing 10 mM Tris HCI pH 8.3, 50 mM KCI, 0.001 %gela -tin, 2.4 mM MgCI" 0.03 mM each of dATP, d°rfP, dGTP and dCTP, 0.64 ~M random primer and 1 U Taq DNA polymerase. PCR was programmed for 1 initial cycle of 30 sec denaturation (94°C), 30 sec annealing (36°C) and 1 min ex-tension (72°C) followed by 45 cyclcs of 30 sec denaturation, 30 sec annealing and 2 min exten-sion. A final extension for 7 min was performed at the end. Am-plified products were resolved in 1.4% agarose (Sigma) gel electro-phoresis ill 1 X TAE. They were stained in ethidium bromide solu -tion and polaroid photographs were taken under UV illumina-tion. Of the RAPD markers gen-erated by 35 arbitrary primers from kits A, F and G of Operon Technologies, Inc., those produccd by OPA 07 were considered to in -tcrpret genctic relationships in view of their high percentage of amplification (amplification refers to thc presence of at least one polymorphic band per gel) and better reproducibility. Data sets gencratcd by two commercia l brands of Taq DNA polymerase enzymes, Taq 1 (Amresco) and Taq 2 (Rama Biotechnologies) were analyzed. Molecu lar weights of RAPD bands (= loci) were estimated us-ing a simple BASIC program based on the relative mobility of the fragments with respect to a stan-dard set of DNA size markers (I Hind III or I Hind III + EeaR!). This was followed by cluster analysis. A distance measure , Si mpl e Matching Coefficient (SMC) was calculated by pairwise compari -son of loci . SMC is given as the ratio of total number of matches for two individuals compared (i.e., both loci absent or present) to the total number of possible loci. SMC value of 1 indicates that two indi -viduals have completely similar patterns. The genetic di stance between the two individuals is 1-SMC. The distance matrix was ana-lyzed using the Fitch-Margoliash program vcrsion 3.56c of the PHYLIP package. Trees (dendo-g rams) were produced using Saitou and Nei's (1987) Neigh-bor Joining Analysis. RAPD poly-morphisms were scored as a "1" for the presence of a fragment and "0" for its absence. Results and Discussion Taq 1 generated more loci with wider range (Tables 1 and 2). Within-region variability was maximum for Mandapam (east coast) samples while Mangalore and FOli Kochi (west coast) samples Table 1. RAPD markers generated using Amresco Taq DNA polymerase in R. kanagurta col/p-cted from three locations in India. "1" denotes presence of a locus and "0" denotes the absence of the same. M, Mandapam; ML, Mangalore; F, Fort Kochi. lndividualslloci (kb) SHe 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.3 1.4 1.5 2.0 M1 1 1 0 0 1 0 0 1 0 1 1 0 1 0 M2 1 0 0 0 1 1 0 1 0 1 1 0 1 0 MJ 1 1 0 0 0 l ' 0 0 0 0 0 0 0 0 M4 0 0 0 0 0 0 0 0 0 0 0 1 0 0 M5 1 1 0 0 0 1 0 0 1 0 0 0 1 0 M6 0 0 1 0 0 0 0 0 0 0 0 1 0 0 M7 0 1 0 0 0 0 0 1 0 0 1 1 0 0 M8 0 1 1 0 1 1 0 1 1 0 1 1 0 0 ML1 1 1 1 1 0 0 0 0 1 0 0 0 1 0 ML2 0 1 1 0 0 0 0 1 0 0 0 1 0 0 ML3 0 1 1 0 0 0 0 0 0 0 0 1 0 0 F1 1 0 0 0 0 0 0 1 0 1 0 0 1 0 F2 1 0 0 0 1 1 1 1 0 1 1 0 1 1 FJ 1 0 0 0 1 0 0 1 0 0 0 0 1 0 F4 1 1 1 0 0 0 0 0 0 0 0 0 0 0 F5 1 0 0 0 0 0 0 1 0 0 0 0 1 0 F6 0 1 0 0 0 1 0 1 0 0 0 0 1 0 F7 1 1 0 0 0 0 0 0 0 0 0 0 1 0 F8 1 1 0 0 0 0 0 1 0 1 1 0 1 0 -F9 0 1 0 0 1 0 0 1 0 0 1 0 1 0 F10 0 1 0 0 1 1 0 0 1 0 0 1 0 0 F11 1 1 1 1 1 1 0 1 1 1 0 1 0 0 F12 0 0 0 1 1 1 0 1 0 0 0 1 0 0 July·December 1997 53 Table 2. RAPD markers generated using Rama Biotechnologies Taq DNA polymerase in Indian mackerel collected from three locations in India. "1" denotes presence of a locus and "0" denotes the absence of the same. M, Mandapamj ML, Mangalorei F, Folt Kochi. Site 0.6 M1 a M2 0 M3 0 M4 0 M5 0 M6 0 M7 1 MB 1 M9 a Ml0 0 Mll 0 ML 1 0 ML2 0 ML3 0 ML4 a ML5 a ML6 1 ML7 0 ML8 1 ML9 0 ML10 0 MLll 0 Fl 0 F2 0 F3 0 F4 0 F5 a F6 1 F7 0 F8 0 showed relatively more homoge-neity (Table 3). A perusal of the dendograms (Figs. 2 and 3) based on l-SMC matrices shows an ab-sence of clear center-specific clus-ters in both data sets. Observation of greater branch lengths be-tween Fort Kochi and Mandapam individuals (Fl 1 and M8 in Fig. 2) and those between Mangalore and Mandapam individuals (MLG and M8 in Fig. 3) as well as uniformity of mean SMC values in the indi-viduals from the two cenlers of the west coast suggest restricted inter-mixing of mackerels between the east and west coasts of India. It is noted in this context that samples Individuals/loci (kb) 0.7 0.9 1.0 1.1 1.3 1.4 a 0 0 0 1 a a a a a a a 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 a 1 0 1 0 1 0 1 0 0 0 a 0 1 0 0 a a 0 0 0 0 0 a 0 0 0 1 0 a 0 0 0 0 0 0 a 0 0 1 0 a 0 0 0 0 0 0 1 a a 1 1 1 0 0 1 1 1 1 0 1 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 a 0 a 0 0 0 0 1 a 0 0 0 0 0 a a 1 0 a a 1 a a 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 of orange roughy from two distinct New Zealand spawning sites did not reveal obvious differences, al-though the authors conclude that the degree of intersample identity would be premature (Baker et al. 1992). In our earlier work (J ayasankar and .Dharmalingam 1997), RAPD loci produced by the same random primer showed markedly differ-ent patterns between Indian mackerel (N. kanagurta) and king seer (Scomberomus commerson). Similarly, arbitrary primers of dif-ferent sequences yielded different banding patterns in the same tem-plate of both seombroid fishes, in-Table 3. Simple Matching Coefficients (S MC) of RAPD loci in R. kanagurta collected from three locations in India. 54 Center Mandapam Mangalore Fort Kochi Pooled Amresco Range 0.18· 0.82 0.50 . 0.75 0.29 . 0.93 0.09 . 0.93 Taq data set Mean + sd 0.47 ± 0.18 0.67 ± 0. 14 0.62 ± 0.16 0.60 " 0.17 Rama Biotechnologies Taq data set Ranqe Mean + sd 0.33-1.00 0.62±0.18 0.27 . 1.00 0.33 . 0.89 0.36 . 1.00 0.63 ± 0.21 O.63±0.16 0.69 ± 0.16 1 .5 1.6 1 .7 1.9 2.0 . a a 1 a a a 0 1 0 0 1 0 1 0 0 0 0 1 0 0 0 1 0 0 0 a 1 0 0 0 1 0 0 0 0 0 1 0 0 a 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 1 0 0 1 0 1 0 0 0 0 1 0 a 0 a 1 0 0 1 a 1 a a a 1 0 0 0 0 1 0 0 0 0 1 0 0 1 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 1 0 a 1 0 1 0 0 0 0 1 0 0 0 0 1 0 a a 1 a a a 1 0 0 1 0 0 1 0 0 0 1 a a a a dicating uti lity of RAPD's manipu-lation of primer sequence to gen-erate amplification products of de-sired complexity to suit different purposes like genetic mapping or genotyping. Concentrations of DNA, MgCI, and Taq DNA po lymerase, primer choice and gel staining methods can influence the num-ber and intensity of amplification products (Hadrys et al. 1992). RAPDs of Indian mackerel are sensitive to the concentrations of primer, MgCI, and the brand of 'l"Elq DNA polymerase enzyme (] ayasankar and Dharmalinzam 1997). RAPD-based analyses of Orcochro117is have shown that wh ile primers differed in the leve l of information they af-forded, general consensus be-tween each of the primers was supported statist ically (Bardakci and Skibinski 1994). Southern-Naga, The tCLARM Quarterty blo tting o f RAPDs wit h o ligo -nucleotide r ep eat probes ca n bc more in fo rma tive (Cifare lli e t a l. '199 5). We suggest a combination of g r eater sample size, hig her num-ber of in formative primers and a Taq D NA p olym e rase e n zy m e which can yield more loci for gen -erating adequa te data for inter -preting genetic stock structure in fi sh populations. Acknowledgment The present work was done un-der a National Associateship Scheme (BT/ MP/ 04 /Z0/ 9 5) awarded by the Department of Biotechnology, Governmen t of India, to P. J ayasankar of the Schoo l of Bioechnology, Mad urai Kamaraj University, Madurai, India. References Baker, C.S., M. MacCarthy, PJ Smith, A. P. Perry and G.K. Cham bers. 1992. DNA fingerprints of orange roushy, H opiostelhu5 :It/an /icu:,:' a population comparison. Mar. BioI. 113:56 1-567. Bardakci , F. and DO.F. Skibinski. 1994 . The application of the RAPD tech-nique in ti lapia fish: species and subspecies ident ifica tion. Hered-ity 73: 11 7- 123. Cifare lli , R.A ., 1\1. Gallitc lli and F. Celli ni. 1995. Random amplified hyb r idiz ation l11 ic ros ate ll i tes (RAHM): isolalion of a ncw class of microsatellite -contai ll ing DNA c lones . Nu cleic Acids Res. 2 3: 3802-3803 Fergusson, A.,J- B. Taggart, FA Prodhol, 0. McMeel, C. Tllompsol1,c. Stone, July·December 1997 _____ _ _ _ _____ ,---- --- Fll L-____ _ _ _ MB -I~------ Fl2 i-L ---.-- FlO '-[ - ----Ml1 I _-f-- MLJ Lrl ~ : 1 I MI2 '----- - - - -- M7 --L_~__.J~~ _ _'_~_.-L _ _ --1 _ _ , ----"-_~__',F I 0.24 0.20 0.16 0.1 2 0.08 0.04 0 I·SM C Va lue Fig. 2. Dendrograms illustrating the genetic relationship among different mackerel specimens collected from three locations in India based on 1·SMC matrix using Neighbor Joining Analysis. RAPDs were generated by Amresto Taq polymerase. fA, Mandapam; ML , Mangafore; F, Fort Kochi. P. McGinnity and R. A. Hynes. 1995. The applicalion of molecu · lar markers to the study and con-servation offish populutions, w ith special reference to Sal111o. J. Fish BioI. 47 (Supplemen t A): 103 -1 26. Had rys, A. , M. Bati ck and B. Schicrwatcr. 1992. Application, of ra ndom ampli fied polymorphic DNA (RAFD) in molecular ecology. Mol. Ecol. I: 55 · 64. Jayasankar, P. 199 7. DNA fingerprml-inz fosters zenetk research in fish-eries and aqu aculture. f ishing Chimcs 17(2): 11-15. Jayasankar, P. and K. Dharmalinz am . 1997 . Potenlia l applica ti on of RAPD and RAHM markers in ge-nome analysis of scombroid fi shes. Curro Sci. 72(6): 383-390. Menezes , M.R., S. Naik and M. Mar-tins. 1990. Genetic and morpho-logical variations in the Indiflll mackerel RflsfrciJig er k:lIU~'{Urt:l (Cuvier 1817) from the Goa re-gion. Proc. Indian Acad. Sci. (Anim. Sci.) 99: 457 -465 . Menezes, M .R. ) S. Naik Ilnd .1\1. Ma r-tins. 1993. Gene tic divergence in the Indian mackerel i?aslrciliger knm{gllrtH (Cuv.) from the coastal waters of peninsular India and the All dam an sea. India n J. Fi sh. 40: 135-t4 1. 55 Noble, A., G. Gopakumar, N. Gopalakrishnapillai, G.M. Kulkarni, K. Narayanakurup, S. Reuben, M. Sivadas and T.M. Yohannan. 1992. Assessment of mackerel stock along the Indian coast. Indian). Fish. 39:11-124. O'Reilly, P. and J.M. Wright. 1995. The evolving technology of DNA fingerprinting and its applica-tion to fisheries and aquaculture. J. Fish BioI. 47 (Supplement A): 29-55. Rao, K.V. 1962. Observations on the bio-nomics of the Indian mackerel , Ros/relliger kOl1ogur/o (Cuv.) caught in the Lawson's Bay, near Waltail", Andhra coast. Proc. Symp. Scombroid Fishes. Mar. BioI. Assoc. India 2:574-585. Sailou, N. and M. Nei . 1987. The neighbor-joining: method: a new method for reconstructing phy-logenetic trees. Mol. BioI. Ivol. 4: 406 -425. Seshappa, G. 1985. On the homogene-ity of the mackerel population al Calicul during the years 1969 10 1976 as determined on the basis ofC/ L, C/ W and TLiSL ratios. In-dian) . Fish. 32:359-374. Venkalaraman, G. 1970. The Indian mackerel IV. Bionomics and hfe history. Bull. Central Mar. Fish. Res. Ins!. 24: 17-40. Verma, N.K., J.D. Gupta, P.e. Thomas and M.K. George. 1994. Bio-chemical genetic polymorphism in the Indian mackerel RllstrelJjger k8l1agur/a from Mangalore region. Mar. Fish. Info. Service: Tech. Ext. Ser. 12:5-7. Verma, N.K., P.C. Thomas and M.K. 56 George. 1996. Biochemical ge-netic polymorphism in the Indian mackerel Ros/relfJger kal1Bgurta of mud bank and post mud bank period. Mar. Fish. Info. Service: Tech. Ext. Ser. 142: 9- 10. ML7 -r F7 M6 M5 [ F8 ML 1 I ML9 I ML 10 M9 ~ F3 MI .- ML3 M2 c: M4 g Ii ~ -~ F4 .l!l en ML4 -F2 ~ en M3 ML5 MLl ML8 ML2 FI MIl y MIO M7 F6 ML6 ! I I M8 0.20 0.16 0.12 0.08 0.04 0 I-SMC Valve Fig. 3. Dendrograms illustrating the genetic relationship among different mackerel specimens collected from three locations in India based on 1·SMC matrix using Neighbor Joining Analysis. RAPOs were generated by Rama Biotechnologies Taq polymerase. M, Mandapami ML, Manga/ore; F, Fort Kochi. Welsh, J. and M. McClelland. 1990. Fingerprintinggenomes using PCR with arbitrary primers. Nucleic Acids Res. 18: 72 13-72 18. Wil1iams, G.K'1 A.R. Kubelik, K.L. Livar, J.A. Rafalski and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as ge-netic markers. Nucleic Acids Res. 18:6531 -6535. P. JAYASANKAR is Scientist (Senior Scale) from Central Marine Fisher-ies /{esearch Institute., PH No. 1603J Kochi-682 014, lJ1dio (e-mail: mdc-cmfri@ x400.nicgw:nic.in). K. DIiARMALI NGAM is Head and Caw-dil1%r, School of Biotechl1ology, Madurai Kamaraj Universitlj Madurai-625, India (e-mail: dharm%bic-mku@dbt.ernet.in). Naga, The ICLARM Quarterly 

Analysis of RAPD Polymorphisms in Rastrelliger kanagurta off India

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Analysis of RAPD polymorphisms in Rastrelliger kanagurta off India

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Analysis of RAPD polymorphisms in Rastrelliger kanagurta off India

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