| The golden pompano Trachinotus ovatus is a most important marinefish in South China because of its delicious taste, simple diet, rapid growthand easy feeding. However, theinbreeding depression of T. ovatus could becaused in the breeding process by randomly selecting parents withoutartificial breeding. Hence it is urgent need to evaluate the level of geneticdiversity and the genetic structure of breeding population to provide areference for the choice of parents and formulation of pairing strategy inthe genetic breeding. In the present study,21polymorphic microsatelliteloci from T. ovatus were isolated and screened in the Microsatellite(Microsatellite locus, SSR) enrichment library by using FIASCO. Andgenetic diversity and genetic structure of the Daya Bay and the Sanyabreeding population were evaluated using the polymorphic microsatellitemarkers repectively screened from genomic DNA in this study.Meanwhile,the mitochondrial DNA complete sequence of T. ovatus were successfulsequenced, and the phylogenetic relationships were studied withmitochondrial DNA complete sequence of other6Carangidae fish from theGenBank database in this study. It is difficult todistinguish between maleand female of theT. ovatus from the external form, therefore, it brings greatdifficulties on choosing of parents in process of genetic breeding. So forestablishing molecular recognition of male and female individuals, thisresearch attempted to employ AFLP and RAPD for isolating thesex-specific markers in T. ovatu by genome-wide scanning. The results ofresearch as follows：(1) The screening of T. ovatu microsatelliteThe microsatellite enriched fragment of T. ovatu was enriched usingmagnetic beads, after the establishment of the microsatellite sequences enriched library, about450colony forming units of microsatelliteenrichment library were acquired, then320ready clons were got. Then219library positive clones were screened and sequenced, about150clones(68.5﹪) contain repetitions of microsatellite sequences more than5.137pairs primers were designed from those sequences, however, only108pairsof microsatellite were successful amplificated. Then48parents of T.ovatuwere used to evaluatethe polymorphic primers. Results indicated thatTwenty-one polymorphic microsatellites were isolated and characterized,the number of alleles(Na) ranged from2to10. The observed(HO) andexpected heterozygosities (HE)ranged from0.083to0.792and from0.081to0.886, respectively, with an average of0.585and0.589. Thepolymorphism information content (PIC) value ranged from0.0767to0.8623. Six loci (TO33, TO47, TO67, TO80, TO132-1, TO145) significantdeviated from Hardy-Weinberg equilibrium(P <0.01).(2) Genetic polymorphism analysis of2breeding populations of T. ovatus11of21polymorphism microsatellite loci were used to investigate thegenetic diversity analysis of2breeding populations of T. ovatus. The resultindicated that the average number of alleles (Na) in each microsatellitelocus of the Sanya and the Daya Bay breeding populations are5.and3.6,respectively; the average number of valid alleles (Ne) are2.965and2.244,respectively; the observed heterozygosity (Ho) of the two populationsrange from0.292to0.814, and from0.207to0.840, respectively; and theexpected heterzygosity (He) range from0.307to0.813and from0.189to0.761, respectively; The PIC value ranges from0.014to0.719in Sanyabreeding population, with an average of0.509, and the PIC value of theDaya Bay breeding population ranges from0.168to0.711, with an averageof0.436. Five loci deviate from Hardy-Weinberg equilibrium in Sanyabreeding population (P<0.05), while only two loci deviate fromHardy-Weinberg equilibrium in Daya Bay breeding population. The Nei’sgenetic similarity coefficient of these two populations is0.561and theirgenetic distance is0.578. Genetic differentiation index of the two breeding populations (Fst) is0.152.It indicates that two populations of T.ovatus areclear divided into two groups by Structure analysis and cluster analysis,respectively, and have a higher genetic differentiation, so two breedingpopulations can be managed as two breeding populations, and there willhave a good genetic progress by hybridizing between the two breedingpopulations.(3) sex-specific markers detected in T. ovatusIn order to obtainsex-specific markers, AFLP technology and RAPDtechnology were employed for isolating the sex-specific markers in T.ovatuby genome-wide scanning. A total of2322loci were successfulamplified by used64primer combinations, of which eighteen loci (26.6％)primer combinations yielded sex-associated amplification. However, thereis no finding any sex-specific DNA markers for T. ovatu, meanwhile,238primers of RAPD were used to isolate sex-specific markers, only26primers of RAPD yielded sex-associated amplification, there is also nofinding any sex-specific DNA markers for T. ovatu.(4) Mitochondrial genome cloning ofT. ovatus and phylogenetic analysis ofCarangidaeAccording to the homology of Carangidae mitochondrial sequence todesign10set of primers by PCR amplification and sequencing analysis, themitochondrial DNA sequence of T. ovatus were successful sequenced,Themitochondrial genome is16564bp length and consists of13protein-coding genes,2rRNAgenes (12SrRNA and16SrRNA),22tRNAgenes and a control region.With the exception of ND6and eight tRNAgenes(Gln，Ala， Asn，Cys，Try，Glu，Pro， Ser), all other mitochondrialgenes are encodedon the heavy strand, Comparing our sequence with thatof another NCBI accessionno.(KF356397), the homology of two completemitochondrial genome sequences was99%, and there were higher valuesofnucleotide sequence variations in16S rRNA (0.17%) and Control region(1.7%), due to its high sequence variations, this gene can be used to study thepopulation genetic structure of T. ovatus. The molecular phylogeneticrelationship of Carangidae was analyzed using the neighbor-joining (NJ)method for mitochondrial DNA sequence of T. ovatus we got and6otherCarangidae fish from the Genbank database, the results showed thatTrachinotini and Seriolinae formed a sister group, Trachurus andDecapterusformed a sister group, and Carangoides with two sister groupwere clustered into one, then clustered with Alepes. Caranginaewerelocated in the base of the tree. |
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