(S.richardsonii) Fish Species GKSivaraman

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Naylor et al, 2000) and habitat modification due to industrialization, river valley ... from Kumaon Himalayan rivers such as Gola near Kathgodam (515m above ...
Title: Assessment Population Genetic Structure and Diversity among Indian Snow Trout (S.richardsonii) Fish Species

G.K.Sivaraman1, A.Barat2, S.Ali3, K.D.Joshi4 and P.C.Mahanta5 Directorate of Coldwater Fisheries Research (ICAR), Bhimtal -263 136, India.

------------------------------------------------------------------------------------------------------1

Corresponding author:

Address: Dr. G.K.Sivaraman, Senior Scientist, Research Centre of Central Institute of Fisheries Technology, Matsyabahvan, Bhidia Plot, Veraval– 362 269. Gujarat, India. Mobile: +91-9037762685; E-mail: [email protected].

2

A.Barat:Principal Scientist,Directorate of Coldwater Fisheries Research (ICAR), Bhimtal -263

136,Uttarakhand, India. 3

S.Ali: Scientist, Directorate of Coldwater Fisheries Research (ICAR), Bhimtal -263

136,Uttarakhand, India. 4

K.D.Joshi: Principal Scientist & SIC, CIFRI Centre, Allahabad, UP.

5

P.C.Mahanta: Director, Directorate of Coldwater Fisheries Research (ICAR), Bhimtal -263

136,Uttarakhand, India.

Electronic copy available at: http://ssrn.com/abstract=2261713

ABSTRACT

In this study, the levels of genetic variability were estimated in S.richadsonii fish populations from three different locations viz., Chirapani stream (pop 1), Gola (pop 2) and KosiRiver (pop 3) by using OPY Operon primers and 10 primers were produced distinct and repeatable amplification as well polymorphic loci. The higher proportion of polymorphic loci was observed in Champawat population as compared to other two populations and with the overall % polymorphisms were 14.76. The OPY 01, OPY 04, OPY 11 and OPY 16 primers were found to be the population specific banding patterns among these three populations. The Nei's genetic diversity (h) was found to be highest in pop1 andpop2 with 0.24 and 0.2 in pop3. The total gene diversity (Ht) in the population was 0.2962, within sample gene diversity (Hs) of 0.2321 and the genetic differentiation (GST) among the populations was 0.2164. The largest genetic distance (0.1565) was obtained between pop1 and pop2, whereas the smallest genetic distance was between pop1 and pop3 (0.1058), followed by pop3 and pop2 (0.1385). Further it is suggested that the levels of genetic identity and diversity obtained was very low and might be due to the less number of polymorphic loci and migration rate.

Key words: Coldwater Fish; RAPD; Genetic Diversity, Markers, Population.

Electronic copy available at: http://ssrn.com/abstract=2261713

INTRODUCTION Recent years, decrease in the biodiversity of fish population has been reported in all aquatic environments due to over fishing, destructive fishing methods, indiscriminate fishing of brood stock and juveniles by poachers, aquatic pollution, degradation of ecological conditions of aquatic system, spread of diseases, uncontrolled introduction of exotic fishes (Kumar, 1999, Naylor et al, 2000) and habitat modification due to industrialization, river valley projects (Natrajan and Sehgal, 1982). Variation at population level can provide an idea about genetic classes, genetic diversity, evolutionary relationship with wild relatives and is extremely useful to gather the information on individuals identify, breeding patterns, degree of relatedness and disturbance of genetic variation among them (Schierwateret al., 1994).The RAPD marker is a potential tool for monitoring genetic variation at species/ population level and as well as its simplicity and low cost, it can be employed as a complementary technique in reproductive management strategies with the aim to minimize loss of genetic variability in the progeny.In the present study, RAPD-PCR analysis developed for the assessment of the genetic diversity among three populations of the Indian snow trout S.richardsonii. MATERIALS AND METHODS Fish Samples Indian snow trout (Scizothoraxrichardsonii)fin tissue samples (n= 24) were collected from Kumaon Himalayan rivers such as Gola near Kathgodam (515m above mean sea level, asl) and Kosi near Ratighat (1000m asl) in Nainital district and Chirapani stream (1620m asl) in Champawat district using cast nets.

Isolation of DNA The extraction of DNA was undertaken as per the method of Sivaramanet al., (2010) from the fin tissues. The concentration of the DNA was estimated by measuring the absorbance at 260 and 280 nm in a UV-visible spectrophotometer and the good quality DNA was subjected to PCR amplifications. Polymerase Chain Reaction (PCR) The RAPD-PCR was carried out in a total volume of 25 µl containing 50ng of genomic DNA, 100pM of random primer, 200 µM of each dNTP, 2.5 µl 10 X PCR reaction buffer, 1.5 mM Mgcl2 and 1U of Taq DNA polymerase with an initial denaturation at 95°C for 5 min. and 35 cycles consist of 94°C for 1 min, 36°C for 1 min and 72°C for 1 min and final extension at 72 ° C for 5 min.

The amplified products were resolved on 1.2 % submarine agarose gel

electrophoresis and visualized in the Gel Doc system. Scoring of the RAPD profile and estimation of molecular sizes Genetic diversity was calculated as observed number of alleles (na), effective number of alleles (ne) (Kimura and Crow, 1964), the number of polymorphic bands, Nei's gene diversity 'h' (Nei, 1973), Shannon's information Index (Lewontin, 1972), total genetic diversity in population (Ht), within sample gene diversity (Hs) for RAPD data. GST was calculated using the Nei method, from the total genetic diversity in the pooled populations (Ht) and mean diversity within each population (Hs) as: Gst = 1- Hs/Ht. Nei’s unbiased genetic identity (I) and genetic distance (D) (Nei, 1978) was used to estimation of gene frequency divergence among the populations.Cluster analysis was then performed to create dendrogram-using UPGMA by SAS Software (version 6.12).

RESULTS AND DISCUSSION Genetic polymorphism among S.richardsonii populations The genetic polymorphisms withinthe populationswere6.56, 4.92 and 3.28 in Pop1, pop2 andpop3, respectively with the average of 14.76 %. The pop1 is having higher proportion as compared to other two populations, which is in accordance with the result of Das et al.,(2005) observed the varied range of 42.6, 31.7, 30, 19.2, 16.8 and 14.3% polymorphic loci in different carp species. Similarly, Hatanka and Galetti, (2003), Dergamet al. (2002), Arnishi and Okimoto (2004) and Grapputoet al (2006) also employed 3 to 5 random primers in different fish populations and could amplified 31 to 74 numbers of fragments with the size ranging from 300bp to 1500bp. Whereas a higher proportion of polymorphisms were observed by Liu and Chu-Wu (2007) of 86.00 to 92.11% in Hiroshima and Arnishi and Okimoto (2004) of 92.29 to 93.32% in Goseong populations. Genetic diversity among populations The OPY 01, OPY 04, OPY 11 and OPY 16 primers were found to be the population specific among these three populations. The fragment size of 986, 553 and 1232 bp with OPY 01, OPY 11 and OPY 16 was found to be specific for pop3 and 1655, 1419 and 1281bp was specific to the pop1 with OPY 04 and 870 bp sizes with OPY 01 in pop2 samples, respectively. The primers from OPY series were found to be effective in developing species-specific RAPD markers in Indian snow trout fish populations. Similar to the present findings, Barman et al. (2003), Callejas and Ochando (2002) and Yoon and Kim (2001) also found 406 (0.25-1.50 kbp), 19 (413-1155 bp) and 36 (0.19kb-1.35 kbp) also identifiedspecies-specific markers by employing up to 34 primersin different fish populations.Based on the polymorphic loci, the amount of

genetic variation among these 3 populations wereRanibag (pop3) pop3, of 0.2466, 0.2406 and 0.2091 respectively.Similar to the present study, Lui and Chu-Wu (2007), Bardakciet al. (2004) and Grapputoet al. (2006) also found the genetic diversity index ranging from 0.1022-0.1634 (0.122), 0.0579 and 0.1563 among different fish population respectively. Whereas Brahmaneet al. (2006) observed the lowest genetic distance (0.213) between the Allahabad and Lalgola populations and highest (0.394) in between the Allahabad and Bhadbhud populations. In present study the genetic diversity and distance is very low as compared to above studies and itmight be attributed to the less number of polymorphic loci and further suggested that the low rate of gene flow among these populations. Genetic distances and dendrogram The largest genetic distance (0.1565) was obtained between pop1 and pop3, whereas the smallest genetic distance was between pop1 and pop2 (0.1058) followed by pop2 and pop3(0.1385). The dendrogram showed two clusters, one consisted of pop1 and pop2 and the other one was pop3 (Fig.1) that is the separation of pop3 from the other two. Similar to the present investigation, Bardakci and Skibinski(1994), Takagi and Taniguchi (1995),Cominciniet al.,(1996), Callejus and Ochando(2002), Barman et al., (2003), Bardakciet al.,(2004),Das et

al.,(2005),Aranishi and Okimoto(2004)and Li et al.,(2007) has been used for phylogenetic relationship in different fish populations. The present study revealed that the formation of cluster pop1 with pop3 with least genetic distances among them and might be attributed to the geological events as well as man-made interventions of fish species for their genetic differentiation and dispersal. CONCLUSION In present investigation, the resultsrevealed the highest level of genetic diversities within population as compared to between populations might be attributed to very low rate of gene flow and little migration among these populations. It is further suggested that the OPY Operon series primers were found to be effective in developing species-specific markers in Indian snow trout fish populations. ACKNOWLEDGEMENTS We duly acknowledge the financial assistance provided by the Directorate of Coldwater Fisheries Research Bhimtal, and Indian Council of Agricultural Research,New Delhi India to carry out this research work. REFERENCES Aranishi, F. and Okimoto, T. 2004.Genetic relationship between cultured populations of Pacific oyster revealed by RAPD analysis. Journal applied Genetics,45(4): 435-443. Baradakci, F. and Skibinski, D.O.F. 1994. Application of the RAPD technique in Tilapia fish: species & subspecies identification. Heredity, 73: 117-123.

Bardakci,F., Tatar.,N. and Hrbek., T. 2004. Genetic relationships between Anatolian species and sub- species of AphanuisNarda, 1827 (Pisces, Cyprinodontiformes) based on RAPD markers.Biologia Bratislava, 59(5) :559 – 566. Barman, H.K., Barat, A., Yadav, B.M., Banerjee, S., Meher, P.K., Reddy, P.V.G.K., Jana, R.K. 2003. Genetic variation between four species of Indian Major Carps as revealed by Random amplified polymorphic DNA assay. Aquaculture,217: 115 – 123. Brahmane, M.P., Das, M.K., Sinha, M.R., Sugunan, V.V., Mukherjee, A., Singh, S.N., Prakas, S., Maurye, P. and Hajra. 2006. Use of RAPD fingerprinting for Delineating population Hilsa shad Tenualosailisha (Hamilton,1822). Genetic Molecular Research,5(4): 643 – 652. Callejas, C. and Ochando, M.D. (2002) Phylogeneticrelationships among Spanish Barbus species (Pisces, Cyprinidae)shown by RAPD markers. Heredity, 89(1): 36-43. Comincini, S.M.,Sironi, C.,Bandi, C.,Giunta, M.,Rubini, F. and Fontana. 1996. RAPD analysis of systematics relationships among the Cervidae.Heredity,76:215–221. Das, P., Prasad, H., Meher, P.K., Barat, A., and Jana.R.K. 2005. Evaluation of genetic relationship among six Labeo species using Random amplified polymorphic DNA (RAPD). Aquaculture Research, 36: 564-569. Dergan, J.A., Paiva, S.R. and Schaeffer, C.E. 2002. Phylogeography and RAPD-PCR variation in Hopliasmalabaricus (Blanch, 1794) (pisces, teleostei) in southeaster Brazil. Genetics and Molecular Biology, 25(4):379-387.

Grapputo, A., Bisazz,a A. and Pilastro, A. 2006. Invasion success despite reductionof genetic diversity

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(Gambusiaholbrooki).Italian Journal of zoology,73(1):67-73. Hatanaka, T. and GalettiJr, P.M. 2003. RAPD markers indicate the occurrence of structured population in a migratory fresh water fish species. Genetic Molecular biology,26(1): 1415– 4757. Kimura, M. and Crow, J.F. 1964.The number of alleles that can be maintained in finite populations.Genetics, 49: 725-738. Kumar, K., 1988. Gobindsagar reservoir, a case study on the use of carp stocking for fisheries management.FAO Fisheries Technical Report,405 (Suppl): 46-70. Rome, FAO. Lewontin, R.C. 1972.The apportionment of human diversity. Evolutionary Biology,6: 381–398. Liu Lia.and Liu Chu-Wu. 2007. Genetic diversity and molecular markers of five snapper species.Chinese Journal of Agricultural Biotechnology, 4(1): 39-46. Li, C., Zhang, G.O.G.and Lu, G. 2007. A practical approach to phylogenomics: the phylogeny of ray-finned fish (Actinopterygii) as a case study. BMC Evolutionary Biology 7(44): 1-11. Natrajan, A.V. and Sehgal, K.L. 1982. State of art report on biological behavior of migratory fishes in the context of river velly projects.Report. CIFRI, Barreckpore. 42pp. Nayler, R.L., Goldberg, R.J., Primavera, J.H., Kautsky, N., Beveridge, M.C.M., Clay, J., Folke, C., Lubchenco.J., Mooney, H. andTroell, M. 2000. “Effect of Aquaculture on World fish supplies”. Nature, 405: 1017-1024.

Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci., USA, 70: 3321-3323. Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals.Genetics, 89: 583-590. Sivaraman, G.K., Barat, A., Ali, S.,Pandey, N.N.,Joshi., K.D. and Mahanta, P.C. (2010). An analysis of genetic diversity among Indian coldwater fishes (Pisces: Cyprinidae) using RAPD markers. The Icfai University Journal of Genetics & Evolution, 3(2): 31-40. Schierwater, B., Streit, B., Wagner, G. P. and De Salle R. 1994. MolecularEcology and Evolution: Approaches and Applications. BirkhauserVerlag, 622 pp. Takagi, M. and Taniguchi, N. 1995. Random amplified polymorphic DNA(RAPD) for identification of three species of Anguilla, A. japonica,A. australis, and A. bicolor,Fisheries Science, 61: 884-885. Yoon, J.M. and Kim, G.W. 2001. Random amplified polymorphic DNA-Polymerase Chain Reaction analysis of two different populations of cultured Korean Catfishes Silurusasotus. Indian Academy of Sciences, 26: 641-647.

Table 1: Primers and their code used for PCR amplication Sl.No. Primer name

Primer Sequence

Length

G+C content (%)

1

OPY-01

5’-GTGGCATCTC-3’

10 mer

60

2

OPY-02

5’-CATCGCCGCA-3’

10 mer

70

3

OPY-04

5’-GGCTGCAATG-3’

10 mer

60

4

OPY-07

5’-AGAGCCGTCA-3’

10 mer

60

5

OPY-10

5’-CAAACGTGGG-3’

10 mer

60

6

OPY-11

5’-AGACGATGGG-3’

10 mer

60

7

OPY-13

5’-GGGTCTCGGT-3’

10 mer

70

8

OPY-14

5’-GGTCGATCTG-3’

10 mer

60

9

OPY-16

5’-GGGCCAATGT-3’

10 mer

60

10

OPY-20

5’-TGAGGGTCCC-3’

10 mer

70

Table 2:Genetic variability within and between the populations of S.richardsonii na ne h (Nei's I Populations Sample (observed (effective gene (Shannon's Nos. no. of no. of diversity) Index) alleles) alleles) Population 1

Population 2

Population 3

Over All Mean

24

24

24

24

Poly. loci

1.6230

1.4327

0.2466

0.3610

38

(0.4887)

(0.3892)

(0.2094)

(0.2983)

(62.3%)

1.7049

1.4098

0.2406

0.3619

43

(0.4599)

(0.3723)

(0.1952)

(0.2738)

(70.5%)

1.6066

1.3585

0.2091

0.3133

37

(0.4926)

(0.3786)

(0.2022)

(0.2867)

(60.6%)

1.9508

1.4945

0.2962

0.4523

39.3

(0.2180)

(0.3355)

(0.1596)

(0.2059)

(64.46%)

Table3:Nei's genetic identity (above diagonal) and genetic distance (below diagonal) Pop ID

Population 1

Population 2

Population 3

Population 1

****

0.8996

0.8551

Population 2

0.1058

****

0.8706

Population 3

0.1565

0.1385

****