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The project entitled "Understanding Hybrid Incompatibility and Reproductive Isolation of Channel Catfish and Blue Catfish" will have three objectives, based on what has been already done with the project at Syracuse University, and what we feel most productive based on the progress to date:1. Development of sex-linked markers of blue catfish and validation of their application to the identification of genetic sex of blue catfish.2. Comparative genomic and epigenomic analysis of sex determination of blue catfish and channel catfish by whole genome methylation and RNA-Seq analysis.3. Comparative epigenomic and expression analyses of reciprocal interspecific F1 hybrids (CXB hybrid and BXC hybrid) to understand postzygotic reproductive isolation, particularly allelic methylation and expression, in relation to embryonic development and sex differentiation.These revised objectives are the same set of objectives as we proposed in the original proposal, with minor modifications: The revised objectives 2 and 3 were the same as original objective 1 and 3, but the revised objective 1 is new, which replaced the original objective 2. This revision is necessary because we found that the sex-linked microsatellite markers we identified for channel catfish were not applicable to blue catfish. In channel catfish, many microsatellite loci within the sex determination region (SDR) are fixed, such that they have the same size of PCR products when amplified from X chromosome, without any individual variations. Similarly, they are fixed when on Y chromosome such that only one PCR band was produced when female (XX) samples are used, but two bands were produced when male (XY) samples were used. These fixed sex markers were tested now in blue catfish and they were not applicable because they were not fixed markers in blue catfish as found with channel catfish. Therefore, sex markers need to be developed from blue catfish to allow identification of genetic sex during early sex differentiation, as we proposed for all the objectives.To identify the sex determination locus, we will conduct GWAS analysis. We have already collected 128 samples, 64 samples from male blue catfish and 64 samples from female blue catfish. Because these fish were one-year old, their sex can be identified from phenotypes. The blood samples were collected, and genomic DNA was extracted from these 128 fish. The male and female samples were pooled and subjected to Illumina sequencing. We have obtained genomic sequences, and GWAS analysis is under way. As soon as we obtain information of sex determination locus in blue catfish, closely linked microsatellite markers will be developed for the identification of genetic sex.This new objective replaced original objective 2. This change will not reduce the overall goals of the project but is both necessary and productive. In the original proposal, only one strain each for compatible and incompatible blue catfish was proposed, which will not provide significant information because it is not a qualitative trait. Blue catfish strains have not been well defined, so detailed analysis at the molecular level without concrete phenotypic support is not productive. Therefore, we concluded that we need to replace the original objective 2, with the new objective 1. Development and validation of blue catfish sex markers will have practical applications because for hybrid production, only male blue catfish are needed. Availability of sex markers will allow differentiation of genetic males and females at early stages of life, reducing rearing of brood stocks containing both females and males.

Liu, J.; Dunham, RE, .
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