Study On The Development And Genetic Diversity Of Microsatellite In The Important Maricultural Fish,Platichthys Stellatus

Starry Flounder(Platichthys stellatus) is a benthic fish and gradually become an important economical marine fish in north China with high nutritional value and commercial value. In this study, Platichthys stellatus as the main research object, information on microsatellite locus in Platichthys stellatus was analyzed based on transcriptome sequencing. The genetic diversity of two groups Platichthys stellatus and other Pleuronectidaes as well as their hybrids were analyzed by microsatellite markers and Mitochondrial genes. This study has laid a theoretical foundation for fish farming and wild fish resources protection witch summarized as follows:1. The pituitary gland and hypothalamus organizations of Platichthys stellatus R were as material for the transcriptome sequencing based on HiSeq2000 transcriptome sequencing method. The SSR library of Platichthys stellatus was built through using the software MIS A to search for SSR loci in Unigene sequences of transcriptome sequencing.20095 SSRs were found in 30640 unigenes while the total sequence length is 82619097 bp. The percentage of dinucleotide repeats (63.64%) was the largest in all SSR loci with (AC)n lagest, followed by tri-nucleotide repeats (33.10%), petra-nucleotide repeats(2.36%) and hexa-nucleotide repeats(0.60%) and penta-nucleotide repeats (0.30%). Furthermore, there were 100 types of repeat units in total. The type of dinucleotide repeat units was the least, with four kinds being found. The type of hexa-nucleotide repeat units was the most, with 37 kinds being found. Trinucleotide, petranucleotide, pentanucleotide repeat unit types were 10,25,24, respectively.2. Twenty pairs of fluorescent primers associated with growth or immune function were selected and applied in Platichthys stellatus R, Platichthys stellatus J, Kareius bicoloratus, Pseudopleuronectes yokohamae, and the hybrids (P.stellatus♀ X K.bicoloratus ♂). The PCR results were separated by capillary electrophoresis using ABI3730XL DNA Analyzerm. The results showed that there were 19 loci amplified successfully in Platichthys stellatus R,18 loci in Platichthys stellatus J,12 loci in Pseudopleuronectes yokohamae,15 in Kareius bicoloratus and 18 in the hybrids. The effective number of alleles were as follows:Platichthys stellatus R (2.07), Platichthys stellatus J (1.93),Kareius bicoloratus (1.86), Pseudopleuronectes yokohamae (1.59), the hybrids (1.81). The average observed heterozygosity were Platichthys stellatus R (0.350), Platichthys stellatus J (0.278), Kareius bicoloratus (0.274), Pseudopleuronectes yokohamae (0.247), the hybrids (0.525), respectively. The proportion of highly polymorphic loci (PIC> 0.5) in total sites were Platichthys stellatus R (26.31%), Platichthys stellatus J (22.22%), Kareius bicoloratus (26.67%), Pseudopleuronectes yokohamae (16.67%), the hybrids (22.22%), respectively. Nei’s genetic diversity index were Platichthys stellatus R (0.420), Platichthys stellatus J (0.387), Kareius bicoloratus (0.302), Pseudopleuronectes yokohamae (0.226), the hybrids (0.336), respectively. The experimental results indicate that the genetic diversity of Platichthys stellatus, Pseudopleuronectes yokohamae, Kareius bicoloratus and the hybrids were not high that we should strengthen the fishery management, protection of the genetic diversity of wild fish.3. Mitochondrial DNA cytochrome b gene (Cytb) and mitochondrial cytochrome c oxidase subunit I gene (COI) fragment of the five fishes were preliminarily studied by PCR amplification and direct sequencing. The results showed that the content of base (A+T) was higher than that of the base (C+G) both in all the five fishes. The genetic distance calculated by Kimura 2-parameter model was consistent with the UPGMA and NJ phylogenetic trees that both show that Platichthys stellatus, Kareius bicoloratus and their brids were closer than Pseudopleuronectes yokohamae in genetic relationship. Almost no difference between Platichthys stellatus R and Platichthys stellatus J. The hybrids (P. stellatus♀×K.bicoloratus♂) and P. stellatus were closer than the genetic distance between the hybrids and Kareius bicoloratus.4. The hybrids appeared in two characters:eyes on the left side and eyes on the right side and with the number ratio of almost 1:1. No differences were found between two kinds hybrids whether using microsatellite marker or mitochondrial gene Cytb. But in the NJ phylogenetic tree based on gene COI, the big branch of F1 hybrids and P. stellatus is divided into two:P. stellatus and F1 hybrids with the left eyes clustered to one branch, F1 hybrids with the right eyes gathered for another small branch alone. Mitochondrial COI gene may participate in eyes shift in hybrids according to the result.