Molecular Ecology Notes (2001) 1, 115–116 © 2001 Blackwell Science Ltd Blackwell Science, Ltd PRIMER NOTE Microsatellite markers from the Indian major carp species, Catla catla S. K. J. M c CONNELL,* J. LEAMON* D. O. F. SKIBINSKI* and G. C. MAIR,*† *School of Biological Sciences, University of Wales, Swansea, Singleton Park, Swansea, SA2 8PP, UK, †DFID Fish Genetics Program at AIT, Aquaculture & Aquatic Resources Management (AARM), Asian Institute of Technology, PO Box 4, Klong Luang, Pathumthani 12120, Thailand Abstract The Indian major carp Catla catla is indigenous to the major river systems of Northern India, Pakistan and Bangladesh, and is an important contributor to aquaculture production in India and Bangladesh. Five polymorphic microsatellite loci, developed from Catla catla are described and their utility in other major carp and cyprinid species is tested. Keywords: Catla, Indian major carp, microsatellite Received 9 November 2000; revision accepted 30 November 2000 The Indian major carp Catla catla is indigenous to the major river systems of Northern India, Pakistan and Bangladesh. However extensive transfers of this species for the stock- ing of natural water bodies and for aquaculture have hampered efforts to delineate the exact species range. Three major carp species Catla (C. catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala) are the main components of composite fish farming in the subcontinent. Of these, Catla is the fastest growing and, in 1998, contributed 34% of the total aquaculture production of 1.85 million metric tonnes of major carp in India and Bangladesh (FAO 2000). Here we describe the development of five microsatellite markers for C. catla that will be of use in studies of genetic diversity and population structure, and in pedigree analysis in this commercially important species. Total genomic DNA (100 μg) from a single C. catla indi- vidual, was digested with the restriction enzyme Sau3A. The restricted DNA was run out on a low melting point agarose gel, and gel containing the size fraction from 300 to 700 bp was excised and purified on a spin column (Costar). DNA was re-suspended at 150 ng/μL and ligated into the pBluescript SK + vector (Gibco BRL). Vector was then transformed into XL1 blue electrocompetent cells (Gibco BRL) following recommended procedures and the result- ing recombinant cells were plated onto 24.5 × 24.5 cm LB agar plates (Corning) containing 100 μg/mL ampicillin and grown overnight at 37 °C. Colonies were transferred to a 22 × 22 cm nylon membrane (Biodyne A 1.2 μm) follow- ing the manufacturer’s recommendations. Membranes were hybridized overnight at 62 °C to a mix of repeat motif oligonucleotides (10 pmol each of GT 10 , CT 10 , TTTC 5 ), end- labelled with [γ 32 ]-ATP. Positive colonies were confirmed by reprobing. Cycle-sequencing was performed on plas- mid DNA using standard M13 sequencing primers and products were run on a LICOR 4200 sequencer. Seventeen clones were sequenced and locus-specific polymerase chain reaction (PCR) primers were designed for seven loci. PCR reactions were performed using a Hybaid Omni- gene thermal cycler, under the following conditions: 180 s at 95 °C, then 30 cycles of 60 s at 95°, 30 s at the primer spe- cific annealing temperature, 60 s at 72 °C, followed by 180 s at 72 °C. The reaction mix contained 50 ng of DNA, 1.5 mm MgCl 2 , 1× Reaction Buffer [20 mm (NH 4 ) 2 SO 4 , 75 mm Tris- HCl pH 9.0, 0.01% (w/v) Tween], 0.2 mm of each dNTP (Promega), 10 pmol of each primer, 0.2 U Taq polymerase (ABGene), in a final reaction volume of 15 μL. Amplified products were resolved on 6% nondenaturing polyacryla- mide gels (19:1 acrylamide:bisacrylamide) stained with silver (Naish & Skibinski 1998). Alleles at each locus were sized against a size standard ladder using the program dnafrag (Nash 1989). PCR primers, optimal PCR conditions and a preliminary assessment of variability for five microsatellite loci are shown in Table 1. Observed heterozygosity values (0.26 – 0.0.82) were high at all the loci and the number of alleles Correspondence: S.K.J. McConnell. Fax: + 44 (0) 1792 295447; E-mail: s.k.mcconnell@swansea.ac.uk