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Abstract

We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species.

Procedimentos ômicos aplicados em qualidade e segurança de pescado

“…The PCR-RAPD technique has been used to develop markers for sex differentiation in many fish species such as in Nile tilapia (Oreochromis niloticus) (BARDAKCI, 2000), African catfish (Clarias gariepinus) (KOVÁCS et al, 2000), and blowfish (Tetradon nigroviridis) (LI et al, 2002). This method is simple and quick, only a small amount of DNA is needed, it targets many sequences in the DNA of the sample allowing simultaneous comparison, and, most importantly, no prior knowledge of the genetic make-up of the fish is required (WILLIAMS et al, 1990).…”

Genomic Approaches to Study Genetic and Environmental Influences on Fish Sex Determination and Differentiation

“…In this context, it is not surprising that, because of such diversity of sex-determining mechanisms, the isolation of master sex-determining genes in fish is so far limited to the dmy gene of the medaka, Oryzias latipes (Matsuda et al 2002; Nanda et al 2002). However, since sex-linked markers and/or the sex-determining region is known already in several species, including aquaculture-relevant species such as the catfish— Clarias gariepinus (Kovacs et al 2000), Nile tilapia— Oreochromis niloticus (Ezaz et al 2004), rainbow trout (Felip et al 2005), turbot (Martínez et al 2009), and the half-smooth tongue sole— Hippoglossus stenolepis (Shao et al 2010), the application of NGS (Next Generation Sequencing) techniques will help in the eventual identification of the sex-determining genes.…”

FluoMEP: A new genotyping method combining the advantages of randomly amplified polymorphic DNA and amplified fragment length polymorphism

“…Fin clips, tail clips, or epidermis sections were collected under anesthesia and stored in absolute ethanol until use. Genomic DNA was isolated from the tissues according to a standard method described earlier [3]. DNA samples were aliquoted and stored at 2807C until use.…”

“…Purified PCR products were codigested with the two restriction endonucleases and cloned into pBS-SK cloning vector predigested with the same two enzymes by using the Rapid Ligation kit (Roche, Basel, Switzerland). At least 96 colonies were picked from each clone-set, cultures in liquid LB were grown overnight, and colony PCR was performed according to [3].…”

“…One of the most frequent tasks of that kind is to PCRamplify differences between two varieties of the same species at the genomic level, including searches for sex-specific markers (see e.g., ref. [1][2][3]), isolation of sequences related to disease resistance (see e.g., ref. [4,5]), and identification of differences among strains, subspecies, or species (see e.g., ref.…”