RAPD fingerprinting of the ornamental fish Badis badis (Hamilton 1822) and Dario dario (Kullander and Britz 2002) (Perciformes, Badidae) from West Bengal, India

Abstract

We used random amplification of polymorphic DNA (RAPD) to generate species-specific diagnostic fragment patterns for the molecular identification of the ornamental aquarium fish species Badis badis and Dario dario. Seven arbitrary oligodecamer primers produced a total of 116 bands of which 98.23% were polymorphic. The size of the amplified products was in the range 340 bp to 2170 bp. Intraspecies genetic similarity was 0.879 ± 0.023 for B. badis and 0.840 ± 0.014 for D. dario while interspecies genetic similarity was 0.602 ± 0.017, with cluster analysis displaying separate taxonomic and evolutionary status for these fish. The results show that RAPD was useful for the molecular identification of aquarium fish species, with morphological traits also being important.Key words: Badis badis, Dario dario, diagnostic markers, RAPD molecular identification, West Bengal.Received: July 11, 2007; Accepted: November 26, 2007. India is endowed with a vast fish biological resource representing more than 10% of world fish diversity (Das and Pandey, 1998). However, most indigenous aquarium fish have not been genetically characterized and documented and are being indiscriminately caught from their natural environments leading to their depletion and probable extinction. Taxonomic methods of fish identification and characterization are primarily based on morphological characters which may sometimes be insufficient to identify a species, particularly in the early stages of development when only a few morphological characters are observable (Ayoma et al., 2000). The ornamental freshwater fish Badis badis (Hamilton, 1822) The objective of the study described in this paper was to use random amplified polymorphic DNA (RAPD) markers to develop genetic baseline data for these fish and also provide genetic data to clarify the taxonomic status of B. badis and D. dario which have undergone recent changes in nomenclature and systematic position.Specimens of B. badis and D. dario were collected from sites in the Nadia district (22°56'11.36" N, 88°29'36.35" E), North 24 Parganas district (22°45'25.79" N, 88°25'4.59" E) and Cooch Bihar region (26°18'18.48" N, 89°26'51.71" E) of the Indian state of West Bengal. A total of 20 specimens were collected for each species and preserved in 90% ethanol. For each fish, Genomic DNA was extracted from muscle following the protocol of Sambrook and Russel (2001) with minor modifications. The polymerase chain reaction (PCR) was carried out in a 25 μL reaction mixture consisting of 100 ng DNA, 100 pM of decamer primer, 10 mM dNTP mixture (Dynazyme), 10X PCR buffer (Dynazyme), 1.5 mM MgCl 2 and 0.05 units of Taq polymerase (Dynazyme). Reactions were carried out using a GeneAmp PCR system 2400 (Applied Biosystems) programmed for one step of 4 min at 94°C, 40 cycles of 1 min at 94°C, 1 min at 34°C and 2 min at 72°C, followed by a final extension step of 10 min at 72°C. All primers used were 10 base oligomers obtained from Operon Technologies (Alameda, USA). Twenty oligodecamer primers from Kit Geneti...

Study of the Genetic Diversity of the Ornamental FishBadis badis(Hamilton-Buchanan, 1822) in the Terai Region of Sub-Himalayan West Bengal, India

“…The obtained results showed that the analyzed decamer primers permitted the detection of polymorphic fragments in Badis badis, revealing lower levels of genetic variability within and between two populations of major river streams of the study region. In the present study the number of amplified fragments generated by primer OPB12 was 6-10, 8-9 by primer OPB17, and 3 by primer OPB18, which were comparable with the amplified fragments observed by Brahmane et al [24] (OPB12 = 4-6 bands, OPB17 = 3-6 bands, and OPB18 = 3-4 bands) in Badis badis populations collected from three districts of West Bengal namely, Nadia, North 24 Parganas, and Coochbehar. While there is an absence of comparable data on Badis badis and other related species from other Indian rivers, we have compared our results with some evolutionary related species as well as some vulnerable species found in geographically related areas.…”

“…RAPD-PCR amplifies DNA segments of variable lengths and such length polymorphisms are inherited in a Mendelian fashion and thus can be used as genetic markers [10]. RAPD analysis has been extensively used to evaluate genetic diversity in Brycon lundii, Astyanax altiparanae and Prochilodus marggravii [11][12][13], flounder [14], Labeo rohita [15], catfish [16,17], mud eel [18], ornamental reef fishes [19], crocodile [20], black rat snake [21], and hilsa [22]; and in subspecies identification of tilapia [23], Badis badis, Dario dario [24], and lamprey [25]. A very limited genomic research has been carried out in Badis badis species till date and insufficient genomic information is available to perform other sophisticated fingerprinting techniques where whole genomic sequence is necessary; therefore, RAPD fingerprinting is the best choice for genetic diversity analysis in this species.…”

Two genetic stocks of Steindachneridion melanodermatum living in sympatry in nature and genetic variability of wild parents and F1 generation

“…In other groups, RAPD Genetic variability in Steindachneridion melanodermatum markers have been combined with methodologies of a less arbitrary nature in order to generate better defined DNA patterns, i.e., DNA fingerprinting, for the purpose of molecular characterization (Aggarwal et al, 2008;Demir et al, 2010), for analysis of genetic diversity (Brahmane et al, 2008;Rabello et al, 2008) to provide practical information for selection of parentals in planning breeding strategies (Ahmad et al, 2010), and to determine phylogenetic relationships (Marouelli et al, 2010) besides population differentiation (Klinbunga et al, 2010) between wild and cultivated stocks (Sesli and Yegenoglu, 2010). Although the RAPD method does not provide accurate information regarding the specific genotype of the specimens analyzed or the dominant character of the marker, it is a fast, inexpensive tool that allows access to information on general genetic variability within and between populations, even when considering that the observed variation may be underestimated.…”

Morphological Characterization and genetic Diversity of Macrobrachium rosenbergii (De Man, 1879) Crossbreeding Result from Samas, Bone, and Sintetis Populations

“…Analisis keragaman genetik suatu organisme mampu dijadikan sebagai landasan persilangan untuk menghasilkan benih yang unggul (Kilawati, 2006). Analisis diversitas genetik dengan metode RAPD telah dilakukan pada beberapa jenis udang dan ikan, di antaranya pada Badis badis dan Dario dario (Brahmane et al, 2008), Tenualosa ilisha (Brahmane et al, 2006), beberapa jenis Macrobrachium (Guerra et al, 2010), serta beberapa jenis ikan air tawar dan ikan laut (Ali et al, 2004). Berdasarkan penelitian yang dilakukan oleh Cabezas et al (2010), Indeks Similaritas dengan primer RAPD yang menunjukkan beberapa populasi merupakan satu spesies yang sama adalah lebih besar atau sama dengan 50%.…”