Construction Of Genetic Linkage Maps Of Guppy And Isolation Of Sex-specific AFLP Markers In Guppy(Poecilia Reticulata)

Guppy fish (Poecilia reticulata) belongs to class Actinopterygii (ray-finned fish), order Cyprinodontiformes, family Poeciliidae and genus Poecilia. It mainly inhabits the coastal streams and rivers of Venezuela, Guyana, Surinam and several of the Lesser Antilles including Trinidad and Tobago. Guppy has become popular among aquarists and hobbyists and played an important role in the freshwater ornamental fish industry worldwide. Guppy is also a model fish for studying the genetic bases of heterosis, aging and inbreed depletion crucial for aquaculture because of its relatively short life cycle, easiness of breeding and reproduction, availability of commercial strains homologous for the traits such as body color and tail shape. In the present study, we constructed linkage maps for guppy using microsatellite DNA and AFLP markers, and isolated a sex-specific AFLP marker by screening the genomes of guppy using AFLP technique. This study provides strong support for the study of sex determination and sex differentiation in fish.1. Construction of linkage maps for guppyGenetic linkage maps were constructed for using AFLP and microsatellite markers in an F1 family. A total of 667 polymorphic loci were produced with the 120 AFLP primer pairs. Among all the polymorphic markers,282 dominantly inherited bands were from the female parent,385 were from the male, and 62 markers showed a significant distortion from the expected 1:1 ratio(P<0.05),then 649 markers were used for linkage analysis. Among the 65 informative microsatellites ,29 and 43 markers valued for the female and the male map, respectively.Separate linkage maps were constructed for male and female parents. The male map contained 25 linkage groups (including 1 triplets and 1 doublets), to which 332 markers (31microsatellites and 301AFLPs) were assigned. The map spanned a length of 1120.5cM, covering 84.1% of the estimated genome size (1333.1cM). The size of linkage groups ranged from 9.2cM to 108.7cM with an average of 44.8cM. The number of markers per group varied from 2 to 35 with an average of 13.3. The female parent map contained 25 linkage groups (including 1 triplets and 2 doublets), to which 233 markers (20microsatellites and 213AFLPs) were assigned. The map spanned a length of 901.2cM, covering 77.3% of the estimated genome size (1059.8cM). The maximum and average spaces between 420 loci were 22.9cM and 4.3cM respectively. The size of linkage groups ranged from 14.1cM to 97.8cM with an average of 36.0cM. The number of markers per group varied from 2 to 26 with an average of 9.3.2. Isolation of sex-specific AFLP markersThe differences of genomic DNA between females and males of guppy were studied by using AFLP technique. As a result ,a male-specific maker were found to be only present in all males but absent in all females. The marker was excised, cloned and sequenced. A pair of PCR primers was designed, which could amplify sex-specific bands in male genome DNA. So we converted the AFLP marker into a PCR marker, SCAR. Selection in identifying the sex of guppy with this SCAR marker will be more efficient and less expensive than with AFLP markers.