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Genetic Variation and Differentiation of Wild Stocks of Critically Endangered Puntius Sarana (hamilton) and Their F1 Crossbreed Through Allozyme Electrophoresis

Imran Parvez
Department of Fisheries Biology and Genetics, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur, Bangladesh

Mohammad Ashraful Alam
Department of Fisheries Biology and Genetics, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur, Bangladesh

A.K.M. Rohul Amin
Department of Fisheries Biology and Genetics, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur, Bangladesh

Mohammad Rashidul Islam
Department of Fisheries Biology and Genetics, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur, Bangladesh

Mohammad Mukhlesur Rahman Khan
Departments of Fisheries Biology and Genetics, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh

Abstract/ Executive Summary:

The lower genetic variation and differentiation in wild parental stocks of critically endangered Puntius sarana and slight genetic improvement in their F1 crossbreed were inferred by horizontal starch gel electrophoresis with nine allozyme markers. Out of fifteen loci, four loci (EST-1*, GPI-2*, G3PDH-2*, PGM*) were polymorphic in F1 crossbreed progeny and in maternal parent, and only two loci (EST-1*, G3PDH-2*) in paternal parent. A rare allele *c at locus EST-1*, the highest mean number of polymorphic loci (26.6), mean number of alleles per locus (1.33) and observed heterozygosity (0.066) were observed in F1 generation. The higher fixation index (FIS) value indicated heterozygote deficiency in parental stocks, and the negative FIS value at locus GPI-2* and PGM* indicated excess of heterozygote in F1 crossbreed progeny. The overall genetic differentiation (FST), gene flow (Nm), maximum genetic distance (D) were 0.0690, 3.3755 and 0.0183 respectively, showed very low genetic differentiation among them. Crossbreeding of geographically isolated endemic wild stocks of critically endangered P. sarana could be potential for the revival of the species from the being extinction.

Keywords: Puntius sarana, Allozyme marker, Crossbreed, Genetic variation

Location: Kangsha river of Netrokona district and Sukhair haor (natural depression) of Sunamganj district of Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Fish

Objectives:

The current study current study was aimed to test the hypothesis whether the genetic variability and differentiation in F₁ cross-breed increased or decreased in compare to two wild parental stocks of P. sarana using horizontal starch gel electrophoresis.

Methodology:

The fish stock - Fish tissue samples for allozyme electrophoresis were collected from the stocks maintained in rectangular sized earthen ponds of Field Complex, Bangladesh Agricultural University, Mymensingh, Bangladesh. In brief, two wild stocks P. sarana were collected from the Kangsha river of Netrokona district and Sukhair haor (natural depression) of Sunamganj district of Bangladesh. The broods were reared in rectangular size pond for a period of 6 months. Three breeding line were developed where in line-1 both male and female P. sarana broods from Sukhair stock, in line-2 female from Sukhair stock and male from Kangsha stock and in line-3 both female and male brood were breed form Kangsha stock. The F₁ crossbreed of line-1, line-2 and line-3 were named as SS, SK and KK respectively and reared in earthen ponds of the same field complex for a period of 4 month under same feeding regime ). As the SK line showed higher growth and survival rate, we selected the SK line and their parents as the samples for the allozyme electrophoresis. Collection and preservation of fish tissue Muscle samples from 30 individual of SK line and their parental stocks (female from Sukhair stock and male from Kangskha stock) were collected by using sterilized scalpel and scissors, and immediately after collection were stored in -80ºC for electrophoresis. Horizontal starch gel electrophoresis Horizontal starch gel electrophoresis technique was used in this study. In brief, the inoculation of protein extracts from collected muscle were done by using paper wicks, followed by loading the inoculated wicks in between two pieces of gel from left to right serially. Ethylene blue containing two wicks were placed as dye marker at both end of the gel to determine the rate of protein migration. The gel was placed in the electrophoresis chamber and electricity was run from power supply at 100V (EPS 301, Sweden) for 30 minutes and at 150V for 5-6 hrs after removing the wicks. Staining gene with allozyme markers and scoring of alleles After running, the gel was sliced horizontally into 1.0 mm sized five sections. The sliced gels were stained with 10 different enzymes. The components of staining solution with some co-factors that were used in this experiment are listed in Table 2. All staining were routinely accomplished in an oven set at 45-55^oC. Some of the staining (EST) was accomplished in the dark, due to the presence of light sensitive components in the enzyme staining reactions. Loci and alleles were designated. The individual fish genotype at each allozyme locus was determined to assess the genetic variation between parents and offspring. A locus was considered polymorphic if the frequency of the most common allele was ≤0.99. Data analysis Allelic frequencies, polymorphic loci (P), and fixation index (FIS) were estimated using GeneAlEx version 6.0. The deviations of expected genotypes from the observed genotypes were calculated according to Hardy-Weinberg equilibrium. The mean number of polymorphic loci per population, the mean number of alleles per locus and the heterozygosities (observed, Ho and expected, He) were estimated with the help of POPGENE version 1.31 computer package programs. The genetic differentiation (FST) and gene flow (Nm) among populations were also estimated by using GeneAlEx computer program version 6.0. Genetic distance (D) was measured with the help of POPGENE, (version 1.31) and G-Stat, (version 3.1).

Source of Information: International Journal of Biosciences | IJB |; ISSN: 2220-6655 (Print), 2222-5234 (Online); Vol. 7, No. 5, p. 47-57, 2015

Article Link (Online Journal): http://www.innspub.net; http://dx.doi.org/10.12692/ijb/7.5.47-57

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In Conclusion, the wild stocks of the critically endangered P. sarana showed very lower levels of genetic diversity and differentiation. The crossbreeding between two wild stocks produced F1 progeny that showed higher genetic variation. The higher genetic variation in SK population may be due to using of broods from two different sources. In the previous study we observed that this crossbreed progeny performed a higher survival and growth rate compared to two other intra-breed progeny. This higher growth and survival may be due to slight genetic improvement in SK population. For the conservation of this critically endangered fish species long term research initiatives with a massive collection of P. sarana wild stocks from all the reported region of the country is required. After collection, a well planned crossbreeding program needed to be design and implement.

Knowledge Management: Journal