InDel-RFLPs Of Silver Carp (Hypophthalmichthys Molitrix) And Their Application To Analyzing The Genetic Diversity And Constructing The Linkage Map

Silver carp (Hypophthalmichthys molitrix) is one of the most intensively pond-cultured fish species in China. Other popular cultured pond fish species in China include bighead carp (Aristichthys nobilis), grass carp (Ctenopharyngodon idella) and black carp (Mylopharyngodon piceus). The cultivation of these four fish species dates back more than 1000 years to the Tang Dynasty. They are the main sources of protein for humans in mainland China. They have been introduced into more than 20 countries and regions of the world either for human consumption or for either their ability to aid in water quality control. In order to initiate the genetic study of silver carp, a combined SSR and AFLP sex average linkage map has been constructed. However, the map should be densified with more sequence tagged markers. In order to detour the limitations faced in the development of SSR markers, our interest in this study was to develop InDels based restriction fragment length polymorphism (InDel-RFLP) markers for silver carp.Microsatellites (or simple sequence repeats, SSRs) and insertion-deletions (InDels) are the common variations across eukaryotic genomes. In order to detour the limitations faced in the development of SSR markers, 133 randomly sequenced genomic fragments were used to develop InDel based restriction fragment length polymorphism (InDel-RFLP) markers for silver carp (Hypophthalmichthys molitrix). Of them, 65 were polymorphic in a wild silver carp population containing 31 individuals collected from Jingzhou fragment (the middle reach) of Yangtze River. In total, 161 alleles were identified. The allele number per locus varied from 2 to 4 with an average of 2.5. Thirty five InDel-RFLPs were assigned to the sex average SSR-AFLP (amplified fragment length polymorphism) linkage map of silver carp. It was found that ca.2kb fragments were more useful to developing InDel-RFLP markers than ca.1kb fragments. The fragments were amplified from the genomic DNA with the PCR products digested with one of the four restriction endonucleases (BsuR I, Hha I, Taq I and Vsp I) into shorter ones which were separated via polyacrylamide gel electrophoresis (PAGE) and visualized with silver staining. It avoided searching for the fine position of InDels and integrated the high resolution of PAGE, which will provide a rich source of molecular markers for linkage map construction and traits mapping of silver carp.1. Genetic diversity analysisOf 133 pairs of primers, 65 detected polymorphisms in a wild silver carp population. The allele number per locus varied from 2 to 4 with an average of 2.5. The observed and expected heterozygosities ranged from 0.036 to 1.000 and 0.070 to 0.701, respectively. Five loci (ZP02, ZP26, ZP47, P06 and P17) deviated significantly from HWE, and 2 loci (ZP26 and ZP81) were in linkage disequilibrium. Given that 31 individuals represented a wild population of silver carp, the InDel-RFLPs may serve as the markers for evaluating the genetic diversity and determine the genetic structure.2. Constructing the linkage mapOf 133 InDel-RFLPs, 39 were found to be polymorphic between the 2 parents of an intraspecific hybrid mapping panel used by Zhang et al. Of them, 35 were assigned to the sex average linkage map of silver carp. It is also interesting to note that the percentages of loci polymorphic in intraspecific mapping panel, assigned to the linkage map and diverse in the wild silver carp population yielded by ca.2kb fragments were significantly higher than those yielded by ca.1kb fragments. The fragments of about 2kb in size can be easily amplified with the normal PCR, and the selected cutters can digest the PCR products into an appropriate number of short fragments suitable for separation via PAGE. We prefer that the 2kb fragments should be selected for developing InDel-RFLP markers.3. InDel-RFLP markers applied to 3 closely related species of silver carpThe existences of InDel-RFLPs in the closely related species of silver carp were also determined. It was found that 55 (45 detected polymorphism), 22 (10 detected polymorphism) and 30(14 detected polymorphism) crossly amplified the genomic DNA of bighead carp, grass carp and black carp, respectively. Only the numbers of alleles detected in different fish species were compared; the number of individuals each closely related species was very limited. We may conclude that bighead carp is the closest species of silver carp; the number of loci crossly amplified by silver carp primers and the number of alleles they detected were much more than those of grass carp and black carp.