Ploidy detection, genetic diversity, and quantitative trait loci mapping in molly fish (Poecilia spp)

A study was conducted to construct a preliminary linkage map for molly, Poecilia and to test  association between markers and QTL traits. In I inkage map construction, three major steps were  involved. First is the development of mapping population. Background genetic information of the  parental species such as occurrence of triploidy and genetic variability were crucial before any  attempt to develop a mapping population. Triploid detection was carried out in this study by using  highly polymorphic microsatellite (SSRs) primer pairs. Ten SSRs primer pairs could clearly  differentiate among one, two and three alleles which could be used to infer different ploidy  levels. The presence of two alleles indicates a diploid at that particular locus and evidence of  three alleles could be inferred as triploid. If such association was direct, the occurrence of  triploidy in molly fish sampled in this study was extremely low and negligible. The genetic diversity among  the  four  species  of  live-bearer  fishes  namely  Xiphophorus  maculatus, Xiphophorus  helleri, Poecilia reticulata and Poecilia latipinna were revealed using 15 polymorphic SSRs primer  pairs. A total of 131 samples which comprised of these four species of live-bearers were collected  in the middle of October 2009 from Aquatic International, Subang Jaya, Malaysia. Results showed  that the clustering pattern of both Xiphophorus and Poecilia as revealed by these molecular markers seems to be in accordance with their taxonomy. In P. latipinna, the occurrence of inbreeding or outbreeding was absent as indicated by the level of heterozygosity. Majority of alleles were  homozygous within  P. latipinna and  this seems to resemble  the genome characteristic of a  purebred fish. Purebred fish is an ideal parental material in any mapping population. A mapping population developed  through  backcross and testcross strategy was successfully attempted. A parallel study on the mode of inheritance of background body colour had  also been initiated by evaluating body colour of progenies derived from crosses between two different colour variants of molly, [P. latipinna (non-black) and P. sphenops (black)]. Non-black body colour was found to be completely dominant over black and was not sex-linked. Multiple genes interaction which acted nonadditively was also found  to be influencing this phenotypic trait. However, such interaction effect was restricted in crosses generated between genetically related fish. The second step in linkage map construction is identification of polymorphism using molecular markers. A total of 142 SSRs primer pairs which includes those that were developed for Xiphophorus maculatus (123 primer pairs) and Poecilia reticulata (19 primer pairs)  were screened and 95 primer pairs were able to be amplified on the DNA of molly, Poecilia. However, only 29 SSRs primer pairs were polymorphic when genotyped on 77  progenies derived from two backcrosses (n = 56) and a testcross (n = 21). The fish samples used for  the development of mapping population were collected from Aquatic International Subang Jaya, Malaysia. The final step is linkage analysis of polymorphic markers. Among the 29 polymorphic SSRs primer  pairs identified, only 18 were informative and could be used to construct the linkage map. The  constructed linkage map consists of four linkage groups covering a map size of 516.1 cM. One  potential QTL, dorsal fin length was successfully associated between marker regions ofMsd021, Msb069 and Msb068 on linkage group 2. The major drawback found in this study was low percentage of polymorphic SSRs primer pairs. Such  condition not only greatly restricted the number of markers that could be positioned on the linkage  map but also minimizes the number of linkage group that could be generated. Map enrichment effort  using both amplified fragment length polymorphism (AFLP) markers and development of new SSRs primer  pairs for this species are strongly suggested.