Study On Molecular Identification Marker Of The Major Exported Shrimp Species In China

Shrimp is an important marine animal with various kinds and more than 300 species of shrimp are of economic interest worldwide listed by FAO. According to FAO investigation, world production of shrimp, both captured and farmed, is about 6 million tones, and about 60% enters the international markets. Shrimp is now the most important internationally traded fishery commodity in terms of value. In china, shrimp also has an important postion in export trade, with the export weight nearly 300,000 tones per year and export value nearly $1.7 billion per year (both captured and farmed), including Penaeus momodon, Penaeus vanname, Penaeus chinensis, Penaeus japonica, Maeobrachium rosenbergii, Penaeus merguiensis, Solenocera crassicornis, Trachypenaeus Curvirostris, Procambarus clarkii mainly.With the requirement becoming more strictly, species identification has become one of technical trade barriers for export food and farm commodities. Traditionally, morphological characters are used to identify shrimp of different species. But the gross morphology of some closely related species are so similar that separation of them requires very detailed morphological knowledge known usually only to experts. Moreover, this method is difficult to be implemented for the investigation of processed shrimp products, in which the discriminable morphological characters are unavailable. Therefore, many efforts have been made recently to identify shrimp species using the more accurate molecular markers, such as the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), mitochondrial cytochrome c oxidase sununit I (COI) gene and the cytochrome b (Cyt b) gene.In this study, the highly conserved 18s,28s and 5.8s ribosomal genes of shrimp were used to design universal primer and amplify the ITS1 region of major exported shrimp species in China. According to the sequencing results, proper restriction enzyme was selected, to produce specific restriction endonuclease map as a molecular label to identify the species and origin of tested shrimp species, with strong specificity, good repeatability and low cost. Using the selected primers, length of the amplified ITS 1 sequences in this study ranged from 448 bp to 1491 bp, with a GC content of 40.95-66.83%. To our knowledge, the ITS1 of M. dalei and M. nipponense were the shortest and the longest sequences obtained in shrimp species up to date. Many microsatellite loci were found at 5' end and the middle region of ITS 1 sequences. For samples of the same species, these microsatellite loci seemed to be closely associated with intragenomic sequence variation and length polymorphisms, and they showed a higher degree of similarity in composition between species with closer relationship even in the family Penaeidae. This variation might obscure phylogenetic relationship between some shrimp populations, but the separation of five Penaeus species was well supported. In the combination with polymerase chain reaction-restriction fragment length polymerism methods (PCR-RFLP) analysis, ITS1 sequences from shrimp species belonging to different families and genera were also easily discernable. The results suggested that ITS1 was highly variable among different shrimp groups and could be an appropriate marker for species identification and molecular systematic studies.In this study, the fluorescence labeling techniques of AFLP was used to research the species identification and genetic variation level for China's major export shrimp species and analysis the systemic genetic relationships between different genuses of Penaeidae. The results of AFLP analysis indicated that the minimum genetic distance (0.214) and the maximum genetic identity (0.787) were between P. japonicus and P. monodon, and the maximum genetic distance (0.659) and the minimum genetic identity (0.341) were between S. crassicornis and T. Curvirostris. The results of genetic distance was similar to that by genetic identity, which show that the genetic distance obtained by AFLP labeling is stabilization relatively between different genuses of Penaeidae. Matrix of Jaccard's coefficients of genetic similarity was used to construct the dendrogram by UPGMA for all sample of Penaeidae. Using S. crassicornis as outgroup, all the tested shrimp species in the family of Penaeidae were clustered together. In the distance of 0.38 Jaccard, the Penaeus species were further subdivision as three obvious clades. M. ensis, M. dalei and P. hardwickii formed the first clade. The P. latisulcatus gathers with P. japonicus first before gathering with L. vannamei, forming the second clade. The group of T. curvirostris clustered to the third clade. These results demonstrate that the AFLP marker could reflect the genetic distances between shrimp species, and analysis their genetic relationships.In this study, a total of 841 DNA bands were detected and 601 bands were the polymorphic bands revealed by the 12 primer pairs, ranging from 50 to 550 base pairs (bp). The polymorphism rate was about 71.5%, the average bands and polymorphic bands amplified by each primer combination were 70.1 and 50.1, respectively. Comparison with the other reports about Penaeus, the polymorphism rate is higher in our AFLP analysis. Among the 12 primer pairs,4 primer combinations (E-ACG/M-CAA, E-AGG/M-CTA, E-AGG/M-CTC and E-AGG/M-CTT) can distinguish all the tested shrimp species. The average identification efficiency rate of 12 primer combinations is 92.8, which indicates that AFLP fingerprinting is effective in species identification, and means the genetic background was obvious different among the studied shrimps species.In this study, ITS sequence and AFLP were used to analysis genetic variation levels and diversity among different geographical populations of P. vannamei and P. japonica. The results indicate an obvious genetic divergence between the farmed P. vannamei populations in southeast China and the broodstock population from Hawaii, USA. Based on.the. obtained results,.we. regarded the established methods.could be used to evaluate the species degeneration and health degree of shrimp larvae, and would have important practical and prospective significance for promoting the development of aquaculture industry in China.