Application Of Fluorescence In Situ Hybridization (FISH) In Chinese Shrimp, Fenneropenaeus Chinensis And Zhikong Scallop, Chlamys Farreri: Gene Mapping And Map Integration

Chinese shrimp (Fenneropenaeus chinensis) and Zhikong scallop (Chlamys farreri), distributing mainly around China, are economically important marine aquaculture species. Diseases and degeneration of cultured breeding lines are the main obstacles hampering the culture of the two species in the past severl years. Many efforts were devoted to resolve these problems.The chromosome studies in F. chinensis and C. farreri have been progressed very slowly because the chromosomes are difficult to obtain and to analyze. In this study, Fluorescence in situ hybridization (FISH) was employed to promote the studies on chromosomes of F. chinensis and C. farreri. Some genes were mapped to the chromosomes, and SNP markers were developed among these genes. The potential applications of these results were discussed.1. FISH technology platform was constructed in F. chinensis and C. farreri successfully. One test method for FISH was developed, through which researchers can catch the factors which had interfered with FISH experiments. The test method would facilitate the applications of FISH to other marine invertebrates.2. 5S rDNA was localized on one pair of homologous chromosomes of F. chinensis, which made it an ideal chromosome-specific probe. To further verify that, FISH attempts were carried out on chromosomes obtained from triploid F. chinensis. The results showed that three 5S rDNA-bearing chromosomes were identified from 132 chromosomes, which certified the hypothesis that 5S rDNA can be used to identify chromosomes of F. chinensis. Besides, terminal synapsis was observed to occur in the meiosis of spermatocytes based on the analysis of signal distribution of 5S rDNA on chromosomes obtained from testis.3. 18S rDNA and histone gene h3 were mapped to the chromosomes of C. farreri simultaneously through double-color FISH. The two probes were revealed to localize on short arms of each pair of homologous chromosomes. Single locus of 18S rDNA and h3 in the genome of C. farreri indicates their potential applications in chromosome identification. On the other hand, the chromosomal localization of 18S rDNA and h3 can provide useful information for evolutional analysis of bivalve.4. BAC-FISH were developed in C. farreri with the aid of BAC genomic DNA libraries, which ensure the chromosomal localization of low-copy functional genes. Three BAC clones containing immune-related genes, including heat shock protein 70 gene (hsp70), serine protease gene (sp) and lipopolysaccharide and beta-1,3-glucan binding protein gene (lgbp), were mapped to the chromosomes of C. farreri. The results revealed that the three clones located on long arms of each pair of homologous chromosomes respectively.Two-color BAC-FISH were developed for mapping more than one BACs to explore the comparative localizations of these BACs. The results revealed that six lgbp-containing BACs co-localized with each other because merged signals were detected for every probe combination. The localizations of immune-related genes would promote researches on immune system of C. farreri. And these BACs can also be used as chromosome-specific probes for chromosome identification in C. farreri. Optimizations were carried out to employ M-FISH technology in C. farreri. Five probes mapped previously, namely 18S rDNA, histone gene h3 and three immune gene-containing BACs (CFB123C08 for hsp70, CFB040H03 for sp and CFB094J04 for lgbp) were mapped to the chromosomes simutaneously. Although the results revealed a dramatically decrease for positive signals, nine of ten signals were detected in an incomplete metaphase, which can be distinguished from each other basing on the colors of the signals and their locations on the chromosomes. The distribution characters of the signals were consistent with which had been imaged according to the experiment design, indicating that M-FISH was feasible in C. farreri with further optimizations. On the other hand, a cytogenetic map would be constructed accordingly when M-FISH technology was developed successfully.5. Several SNP markers were developed in immune genes of C. farreri to promote the construction of integration maps. Both sequencing of PCR products and multi-alignment were used to develop SNPs in hsp70, sp, and lgbp. Six base mutations in hsp70, six in sp and seven in lgbp were detected. The 13 base mutations detected in sp and lgbp were believed to be SNPs because high consistencies on the mutation sites were revealed in three DNA samples either from single or mixed individuals. And the six base mutations detected in hsp70 should be further verified before they were taken as SNPs because false positives can be caused by mismatching in PCR reaction and sequencing, which can not be avoided in multi-alignment. The SNPs can be localized to the genetic linkage map when a proper family of C. farreri is available, leading to the integration of genetic linkage map and cytogenetic map (chromosomal mapping). Besides, the abundance of SNP revealed in this work would make it possible to develop SNPs in any given region of the genome of C. farreri.