Poster Abstract
26. GENETIC DISCRIMINATION OF TWO WHITE HALFBEAK SPECIES HYPORHAMPHUS UNIFASCIATUS AND HYPORHAMPHUS ROBERTI BY RFLP-PCR
Hozana Leite Dantas, Fabio Magno da Silva Santana; Tatiane do Nascimento Medeiros; Willian Severi and Maria Raquel Moura Coimbra
Universidade Federal Rural de Pernambuco, Departamento de Pesca e Aquicultura – Laboratório de Genética Aplicada, Rua Dom Manoel de Medeiros s/n, CEP 52171-900
raquel@depaq.ufrpe.br
In Brazil, four out of seven species of Hemiramphidae family belong to halfbeak species, being black (Hemiramphus brasiliensis and Hemiramphus balao) and white types (Hyporhamphus unifasciatus and Hyporhamphus roberti). The white halfbeak species are overexploited due to their high value in local markets, which have threatened the sustainability of their stocks. These two species are morphologically and taxonomically distinguished by the number of gill rakers on the first lower gill arch and jaw length. Closely related species, such as these two, are quite difficult to differentiate, especially at larval stages. In restocking programs it is essential to identify nursery grounds in order to release hatchery-reared fingerlings into the wild. Among fish, most of the features used in taxonomical identification are descriptive, morphometric and meristic. This type of identification is not only time-consuming but also depends on some sort of subjectivity. Molecular biology techniques have brought new insights using DNA information. Here, we describe the genetic distinction between two halbeak species using PCR-RFLP in two mitochondrial genes, 4 NADH dehidrogenase (ND4) and 12S/16S rRNA.
A total of 40 individuals (20 of H. unifasciatus and 20 H. robertii) were sampled off Itamaracá island at the state of Pernambuco. Muscle tissue was removed and remaining material was preserved in formalin for taxonomic identification. Genomic DNA was extracted using phenol-chloroform protocol and the two mitochondrial genes were amplified by PCR. Amplicons were digested with BstnI and RsaI for ND4 and 12S/16S rRNA, respectively.
The amplification of ND4 gene generated a fragment of 1700 bp and the RFLP-PCR produced two bands for H. unifasciatus, one at 1100 and another at 600 bp, approximately. Yet for H. robertii, digestion showed three bands at 1000, 400 and 300 bp (Figure 1). Hemiramphus brasiliensis was used as an out-group and showed an RFLP-PCR banding pattern with fragments at 1200 and 500 bp. The 12S/16S rRNA amplification produced a fragment of 1500 bp. Likewise, the RsaI digestion for this gene showed a banding pattern of 600, 400, 300 and 200 bp for H. unifasciatus and 800, 300 and two of 200bp for H. robertii (Figure 2).
It is possible to conclude that RFLP-PCR of these two mitochondrial genes was capable of discriminating between the two white halfbeak species that occur off Brazilian coast. This approach will contribute in the location of nursery grounds, which is important to the success of restocking programs.
Figure 1 – Digestion with restriction enzyme BstnI of ND4 gene. Lanes 1, 2, 3 and 4: Hemiramphus brasiliensis (out-group) – 1200 and 500 bp; Lanes 5, 6 and 7: Hyporhamphus unifasciatus – 1100 and 600 bp; Lanes 8, 9 and 10: Hyporhamphus roberti – 1000, 400 and 300 bp. Lane 7: DNA ladder.
Figure 2 – Digestion with restriction enzyme RsaI of 12S/16S gene. Lanes 1, 2 and 3: Hyporhamphus unifasciatus – 600, 400, 300 and 200 bp; Lanes 4, 5 and 6: Hyporhamphus roberti – 800, 300 and two of 200 bp. Lane 7: DNA ladder.
Call For Papers