Molecular Cytogenetic Analysis In Female Half-smooth Tongue Sole (Cynoglossus Semilaevis)

In this study, we did some researches about molecular cytogenetic analysis in female half-smooth tongue sole (Cynoglossus semilaevis), including the character analysis of genome structure in female C. semilaevis, fluorescent in situ hybridization (FISH) on chromosomes with the clones of female C. semilaevis fosmid library, laser microdissection and amplification of chromosome W from C. semilaevis, construction and analysis of chromosome W library, and analysis of female specific fosmid clones. Moreover, to actualize the all female production of C. semilaevis, two technical problems should be solved: one is how to identify ZZ and ZW, viz. how to distinguish the sex reversion ZW female ones; the other is how to identify ZW and WW, viz. how to screen out the WW female ones. In this study, we developed a double primer PCR method and female-specific FISH probes which could solve these two questions, respectively. The major results are as follows:1. Analysis of female half-smooth tongue sole genomeGenome sizes of C. semilaevis, Paralichthys olivaceus, Kareius bicoloratus and Verasper variegates were estimated via flow cytometry method and compared. The male and female C. semilaevis genome sizes were estimated to be 586.80 Mb and 606.36 Mb, respectively. Additionally, we found that we could not distinguish the male and female C. semilaevis fishes via flow cytometry method.1,152 individual clones were sequenced, resulting in 2,247 sequences. A total of 1,921,341 bp of genomic sequences was generated, representing approximately 3.17‰of the female C. semilaevis genome. Using Tandem Repeats Finder (TRF) software to analyze the sequences, a total of 889 tandem repeats were found, including 303 microsatellites, 586 minisatellites, accounting for 34.09% and 65.91% of all tandem repeats, respectively. No satellites were found. The total length of tandem repeats was 105,307 bp, in which the length of microsatellites and minisatellites was 19,363 bp and 85,944 bp, accounting for 18.39% and 81.61% of the total length of tandem repeats, and 1.01% and 4.47% of the total length, respectively.RepeatMasker analysis showed that a total of 38 interspersed repetitive sequences were found, which account for 0.26% of the total length. There are 4 types in these sequences: DNA transposons, LTR retrotransposons, LINE retrotransposons and SINE retrotransposons. Among them, the type of DNA transposons is the most frequent and length abundant.2. Chromosome assignment of fosmid clones in C. semilaevisBased on the analysis of end sequences, we selected 8 clones for FISH. Of the 5 clones which were likely to be low or single copy sequences, 4 clones represent single locus and the other one showed signals on almost all chromosomes. Hybridization with 3 clones contained rRNA gene sequences, 2 showed signals on chromosome W and other 3 couples of euchromosomes and 1 showed signals only on one couple of euchromosomes. These results will undoubtedly be useful for many aspects of cytogenetic research in C. semilaevis including chromosomal assignment of genes, sex chromosome evolutions, and chromosomal rearrangements.3. Laser microdissection, amplification and chromosome library construction of C. semilaevis chromosome WLaser microdissection was performed on the chromosome W of female C. semilaevis using Leica LMD system. The isolated chromosomes were successfully amplified and reverse painted on the sex chromosomes of C. semilaevis. Chromosome W library was constructed and 288 clones were sequenced and analyzed for tandem repeats, interspersed repetitive sequences and BLAST. As a result, we found 52 tandem repeats containing 12 microsatellites and 40 minisatellites, and only 2 interspersed repetitive sequences. Additionally, we found that 9 clones contained almost the same 540 bp fragments, which indicated that the sex chromosomes of C. semilaevis might be enriched for repetitive DNA elements and high or moderate in copy number.4. The obtainment and analysis of sex-specific fragment in C. semilaevisPrimers were designed from chromosome W library sequences and 2 female-specific primers were selected using 5 female and 5 male individuals as PCR templates. After appended another positive primer, we developed a double primer PCR method which could distinguish the male and female C. semilaevis fishes truly and rapidly. After library screening from fosmid library, we got 2 female-specific fosmid clones. These clones were both mapped on the middle region of chromosome W without any sequence comparability. The female-specific FISH probes prepared by these two clones will help us screen out the WW female ones. These results would be great promotion for the all-female production and the culture benefits of C. semilaevis.