| The South China Sea is located in the Indian-Pacific central area, one of the world's most abundant marine germplasm resources .The South China Sea is an important aquatic resource base in China,the fish species of South China Sea is much higher than 3000 kinds according to the survey data. And they are involved into 3 classes, 35 orders and 236 families and 822 genera. The identification of fish species requires a wealth of professional knowledge and experience, especially the similarity of the similar species, but also from the morphological difficult to distinguish. In recent years, the development of DNA bar code technology fish species identification provides a fast and effective method, and the establishment of a sound DNA bar code database is the use of DNA barcoding technology for fish,eggs and larvae identification basis. With the support of the national basic work, the collection and preservation of the fish samples of the South China Sea and the construction of the DNA barcoding database were carried out,and the identification of the fish eggs was carried out using the established DNA bar code technology.The main results were as follows:1. South China Sea fish certificate collection and preservation of DNA barcodingdatabase.The collected samples 518 samples were obtained by morphological identification, belonging to 26 orders, 91 families, 152 genera and 219 species .This research totally obtained 419 COI sequences of South China Sea fishes,and they belong to 19 orders, 75 families, 126 genera and 169 species respectively.Each species contains 1?8 individuals,and 2.47 individuals in average. Among them,the number of species who contain 2 or more than 2 individuals is 93. Using the specific amplification sequence with COI gene and matching with GenBank for sequence analysis, we performed the similarity retrieval and species identification for fish eggs COI gene sequences and BOLD system database. The remaining 12 samples belong to 5 species, and there is no corresponding bar code sequence in the BOLD database. Using 419 samples of COI sequence for cluster analysis, these results show that the mitochondrial COI gene sequences don't present any insertion and deletion in bases.The average position of base composition in these 19 sequences: T: 29.7%, C:28.3%,A: 23.3%,G: 18.3%,The average A+ T content (53.0%) is significantly higher than G + C content (46.6%), which showed the AT base bias. The GC content in the third codon site is the highest (48.5%), the first site takes second place (46.5%), and the second site is the lowest (43.8%). The difference of A + T content in all three codons isn't big. The A + T content in the first codon site up to 53.4% on average. We also use Molecular Evolutionary Genetics Analysis (MEGA) 5.0 software to calculate the genetic distance of K2P calculation model. The calculation based on COI sequences of intraspecific genetic distance and genetic distance varied from 0.0000 to 0.0208 respectively (average rate is 0.0027) and from 0.0012 to 0.4812 (average rate is 0.2298), and the fold change of genetic distance to interspecific genetic distance is 85. The largest genetic distance (0.0208) was found in Sphenanthias tosaensis; The lowest intraspecific genetic distance (0.0012) was found between Centroberyx druzhinini and Centroberyx rubricaudus. Among them, 163 species (96.4%) can be clustered into an independent branch, and has a high support degree. The species that we can't separate are Cephalopholis spiloparaea?Cephalopholis sonnerati?Scarus rivulatus?Scarus quoyi. And Cephaloscyllium isabellum with Cephaloscyllium umbratile; Centroberyx druzhinini with Centroberyx rubricaudus; Scarus dimidiatus with Brama orcini, which all can cluster into one group. The reason that causes this phenomenon may be the fast speciation and introgressive hybridization (ntrogressive hybridization), etc. To sum up, DNA barcoding COI sequences can be used to identify the fish species resources of South China Sea and can be effective to distinguish them.So it can provides a reference basis for the germplasm resources protection?development and utilization.2. Identification of Fish Eggs from lagoon of Yongshu Reef.According to the PCR amplification experiments, 71 eggs samples were analyzed and 30 COI sequence were obtained. And the success rate of sequencing is 42.3%. All the 71 collected egg samples are preliminaryly divided into two kinds, round and oval round according to the external morphological characteristics. Round eggs individuals are 44 and 27 of them can be successfully sequenced (the sequencing success rate is 61.4%); Oval round eggs are 27 individuals and only 3 eggs can be successfully sequenced (the sequencing success rate is 11.1%). The mitochondrial COI sequences of 30 eggs has been performed comparison analysis with BOLD database respectively and the results showed the sequence similarity with the BOLD database varied from 99.6% to 100%. All these 30 eggs can be identified into species level,which belonged to 1 section, 7 families and 13 species. The families of Acanthuridae have 5 species and 19 eggs totally, which occupied 63.3% rate in all the identified eggs. The families of Scaridae and Labridae all have 2 species;The families of Carangidae?Siganidae?Lutjanidae and Zanclidae all just have 1 species.Ctenochaetus binotatus and White tail ctenochaetus striatus have 10 and 4 eggs respectively. These two kinds of species have been the advantage species in this area and the rest of species only have 1?2 eggs.The K2P (Kimura 2-parameter) genetic distance between barcode sequences within species was 0.2%, whereas interspecific divergences among species within genera was0.004?0.132,and intergeneric divergences among genera within families was 0.117?0.185. The evolutionary tree was constructed according to the K2P model using genetic distance of adjacent system, which showed that the same kind of species can be divided into a branch.The Acanthurus and Ctenochaetus striatus of Acanthuridae can be clustered into a branch, but Acanthurus can't form a single branch with unicornis.3. The eggs were collected from Jiangmen sea area, and the eggs were identified by DNA bar code technique. In this study, 317 individuals were obtained .217 effective mitochondrial COI sequences were obtained, and the success rate was 68.5%. The genetic distance was 0 ?0.006, the average genetic distance was 0.002, the interspecific genetic distance was 0.149 ? 0.325, the average genetic distance was 0.255,The fold change of interspecific genetic distance over intraspecific genetic is 128 .The genetic distance of the phylogenetic tree based on the COI sequence of the eggs shows that all species can be clustered as independent branches and have high support. The results show that DNA barcode technology can be used as an effective method for the identification of eggs, and can be widely used in the identification of fish eggs in coastal waters. |
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