| In this study, we identified a sex-specific DNA marker using randomly amplified polymorphic DNA (RAPD) fingerprinting, simple sequence repeat(SSR) and inter-simple sequence repeat (ISSR) in Yellow River carps (C. carpio from the Yellow River). Two hundred twenty random primers for RAPD fingerprinting, ten primes for SSR and one hundred primes for ISSR were used in pooled DNA samples and individual DNA samples from male and female fish. The results of SSR-PCR showed one candidate band which were different between male and female pools, however, no sex-specific band was found when was tested repeatedly in male and female individuals by dot boltting hybridization. The results of ISSR-PCR showed as SSR, no sex-linked suspected bands were found among all the amplification bands with 10 primers selected.In RAPD-PCR, Five candidate bands, which were different between male and female, were tested five times repeatedly in male and female individuals to verify their stability. Ultimately we found that one of the primers, S2107 (CACCATCGTG), produced a stable sex-specific band in the DNA fingerprints of all males tested, but not females. DNA sequencing revealed that this 909 bp long DNA fragment has a low similarity to a repetitive sequence in zebrafish, which was named as Ccmf1. The BLAST search revealed that the sequence of the sex-specific fragment in Yellow River C.carpio had a low similarity to a repetitive sequence (DKEY-24 H 22) located in linkage group 1 of zebrafish.Furthermore, a sex subtractive genomic DNA library was successfully constructed using suppression subtractive hybridization (SSH) between male and female Cyprinus carpio. To eliminate the false positives from self-ligations, we screened 150 clones from the sex subtractive genomic DNA library by PCR with the nested primers and identified 88 out of 150 clones carrying the distinguishable insertions. These clones selected were dotted onto two duplicate nylon membranes and hybridized separately with DIG-labeled forward or reverse subtracted DNA. 22 clones exhibited distinct differences. These positive clones were the candidate sex-specific fragments. Specific primers were designed for the 22 clones. PCR was used to further confirm the SSH results by using specific primers to amplify the fragments from the individual male and female genomic DNA. Only the DNA fragments in clones f9 and f10 could be amplified in genomic DNA samples from all males but not in any of the females. which were named as Ccmf2 (387 bp) and Ccmf3 (183 bp) respectively. BLASTN and BLASTX search results showed there are no homologous sequences at the DNA level and no significant similarity (E-value>10-4) at the protein level in other species for the two sequences. As the three fragments isolated were linked to male sex, we speculate that the chromosome type of the Yellow River carp is XX / XY.To confirm male-specificity of Ccmf1, Ccmf2 and Ccmf3 sequences, we tested an additional 50 male and 50 female Yellow River C. carpio. The Ccmf1&2 reached 99% and 98% overall efficiency respective; the Ccmf3 was 100% accuracy. So they have the low number of presumed recombinants among the individuals tested as Ccmf1 and Ccmf2 markers in the Yellow River strain of C. carpio, and can be used to rapidly and accurately identify the gender of Yellow River C. carpio.Three sex-specific markers were used as probes in an attempt to search for homologous sequences in the genome of other common carp strains and C. idellus by a dot blotting hybridization experiment. in the three tested common carp strains the molecular sexing efficiency for Ccmf1, Ccmf2, and Ccmf3 was 67%, 80%, and 93% in the females and 67%, 100% and 73% in the males, respectively. The results revealed that the molecular diversity exists on the Y chromosome of common carps. We may provide a very efficient selective tool for practically breeding monosex female populations in aqua-cultural production, and the sex-specific chromosome region may be characterized and used to study mechanisms of chromosome evolution in this fish species.To comprehensive understand the mechanisms of gonadal development in Yellow River carp, the two-way cDNA subtractive libraries from mature male and female gonads was constructed. 280 clones from male libraries and 240 from female’s carrying the distinguishable insertions were identified by PCR with the nested primers. 180 clones (90 of each library for male and female) were screened with dot bltting, 64 positive clones were obtaind, of which 21 from male libraries and 43 from famales were sequenced. Blast anaysis was performed to find some differentially expressed genes related to gonad development in Yellow River carp. The results showed that of 14 ESTs of 21 sequenced clones shared significant similarities in Genbank datdbase. In accordance with their function, 14 ESTs belong to structural protein genes, the protein translation mechanism related genes, ionic channels proteins and transporters genes, tumor-related and tumor suppressor genes, etc. 4 ESTS of 21 had no significant similarities in Genbank datdbase, whose function were unknown. 32 differences ESTs from the female libraries represented 27 known genes and 5 unknown function genes. In the known genes, the occurence frequencies of respiratory chain-related and protein synthesis-related genes were higher than others. Reference to the gene function of zebra fish and other vertebrates, the known function genes from female EST library belong to regulatory factors, structural proteins, enzymes, transcription factors, signal control, etc.According to semi-quantitative expression in sperm and ovarian of the Yellow River carp, we found 10 differentially expressed gene fragments in the male libraries: 5 of which are testis-specific genes (Including 1 unknown gene discovered firstly); the others are highly expressed genes in the testis (Including 1 unknown gene discovered firstly). In the female libraries, we found 8 differentially expressed gene fragments: 2 of which are ovarian-specific gene; the others are highly expressed genes in the ovarian (Including 1 unknown gene discovered firstly).3 genes (Hmwt1a, HmSetd6, HmPsmb2) which may be involved in gonadal development were chosen to amplify their full-length sequences by RACE-PCR. The results shown Hmwt1a is 2173 bp long, HmSetd6 is 1763bp long, and HmPsmb2 is 884 bp long; Semi-quantisative RT-PCR and fluorescent quantitation RT-PCR were employed to analyse the expression of three genes in the different organizations of Yellow River carps at sexual maturity stages, and the result indicated that Hmwt1a was expressed at highest levels in the testis than ovary, so we speculated that the Hmwt1a gene may be important regulatory genes in male sexual development in Yellow River carp. On the contrary, the Hmsetd6 gene whose expression was higher in the ovary than testis, The HmPsmb2 gene differentially expressed genes in ovarian. Base on the comparison of gene function associated with gonadal development in vertebrates,we speculated that the Hmsetd6 gene involved in the female character development as epigenetic regulation of genes in the Yellow River carp and the HmPsmb2 gene maintained mainly the female characters in the physical or behavioral as sex-bias genes in the Yellow River carp. The discovery of these genes and the construction of gonadal differential EST library, which will help us to learn about of the molecular mechanism gonad development and sex differentiation in the Yellow River carp, as well as provide rich resources of genetic identification for understanding the mechanism of sex control in the Yellow River carp. |
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