Genetica 110: 267–276, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 267 Male-specific DNA markers from African catfish (Clarias gariepinus) Bal´ azs Kov´ acs 1 , S´ andor Egedi 1 , Rich´ ard B´ artfai 1,2 & L´ aszl´ o Orb´ an 1,2,∗ 1 Laboratory of Aquatic Molecular Biology, Agricultural Biotechnology Center, POB 411, Gödöllö, H-2101 Hungary; 2 Laboratory of Fish Biotechnology and Reproduction, Institute of Molecular Agrobiology, National University of Singapore, 1 Research Link, The NUS, Singapore 117 604; ∗ Author for correspondence (Phone: 65-872-7413; Fax: 65-872-7007; E-mail: orban@ima.org.sg) Accepted 21 August 2001 Key words: Clarias, RAPD, sex determination, siluroid, teleost Abstract We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gar- iepinus (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 2.6kb 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. Abbreviations: AFLP – amplified fragment length polymorphism; CgaK – sex-independent internal control from African catfish genome; CgaY1 – sex-specific DNA marker #1 from African catfish; CgaY2 – sex-specific DNA marker #2 from African catfish; DAF – DNA amplification fingerprinting; RAPD – random amplified polymorphic DNA; SCAR – sequence characterized amplified region. Introduction The family of air-breathing or labyrinth catfishes (Clariidae) is one of the largest among Siluriformes. It has over 100 representative species native to most of Africa and Southeastern Asia (Burgess, 1989). Due to their hardiness and adaptability, they were also in- troduced to many other regions, like Europe, Florida or even Hawaii. The African catfish, Clarias gar- iepinus (Burchell, 1822; formerly sharptooth catfish, Clarias lazera Valenciennes 1840 or Clarias mos- sambicus Peters 1822; for ref. see Teugels, 1984) is one of the most prominent species of the family and it is thought to be ‘the most widely distributed fish species in Africa’ (Skelton, 1993). Besides being farmed in a number of African, Asian, and European countries (Huisman & Richter, 1987) the African catfish is also an important object for biological research. It has been used extensively for genome manipulation experiments: gynogenesis (Volckaert et al., 1994, 1997; Varadi et al., 1999; Galbusera, Volckaert & Ollevier, 2000) and andro- genesis (Bongers et al., 1995). Hybrids between C. gariepinus and related catfish species (see e.g., Hecht & Lublinkhof, 1985; Legendre et al., 1992) as well as triploid (Henken, Brunink & Richter, 1987) and