The Use of Random Amplified Polymorphic DNA (RAPD) to Identify Largemouth Bass Subspecies and Their Intergrades

D. JODY WILLIAMS (Texas Parks and Wildlife Department, San Marcos, Texas 78667)

STEVEN KAZIANIS (University of Texas - M.D. Anderson Cancer Center Science Park -
Research Division, Smithville, Texas, 78957)

RONALD B. WALTER (Southwest Texas University, San Marcos, Texas 78667)

Texas Parks and Wildlife Department (TPWD) began producing and stocking Florida largemouth bass Micropterus salmoides floridanus in the early 1970’s to help support the largemouth bass recreational fishery. To ensure that only pure Florida largemouth bass are stocked, potential broodstock are evaluation using agarose gel electrophoresis, followed by histochemical staining of two diagnostic isozymes to identify those with pure Florida largemouth bass genotype. A limitation of this technique is that later generation or backcross intergrades can exhibit a wide variety of genotypes and may not be distinguishable from pure Florida parental types.

Random amplified polymorphic DNA (RAPD) is a genetic technique based upon the amplification of genomic DNA with a single primer of arbitrary sequence. These primers detect polymorphisms in DNA sequence that can be used as genetic markers and are inherited in a Mendelian fashion. The rationale for RAPD PCR is that at low PCR stringency conditions (i.e. low annealing temperature and/or high magnesium concentrations), a primer is likely to find many sequences with the template DNA, to which it can anneal with mismatches. If there is a high frequency of these sequences in the template DNA, it is likely that pairs of these sequences will be arranged inversely to one another and within about two kilobases of each other. Given this, PCR will give amplification of these fragments which can vary in size when different species, subspecies, etc. are analyzed. Since there are an unlimited number of primers that can be used, the number of polymorphisms that can be identified and used for subspecies identification are also unlimited. The gives RAPD PCR a distinct advantage over agarose gel electrophoresis when only two loci are examined.

Nineteen 25-30-mer primers were screened for polymorphisms and five were chosen for further evaluation. Twenty fish from each group (Florida-wild type, Northern-wild type, and fish of unknown origin (Fx)) were analyzed using RAPD PCR. First generation intergrades were produced and used to detect Mendelian inheritance of polymorphic bands. Evidence is presented for the usefulness of RAPD PCR in distinguishing between the different subspecies and intergrades of largemouth bass. Because of the increased sensitivity and the relative ease of this technique, it is shown to be far superior to agarose gel electrophoresis for broodstock evaluation.

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