Application of Karyotype and Genetic Characterization Analyses for Hybrid Breeding of Epinephelus Groupers

In this study, karyotypes and Cyt b gene sequences of seven different species of grouper including Plectropomus leopardus , Epinephelus coioides , E . flavocaeruleus , E . fuscoguttatus , E . lanceolatus , E . polyphekadion ,and E . tukula wereexamined.Allchromosomenumbersfromseven groupers were 2n =48with ahighnumberoftelocentric chromosomes (38 – 48) andfundamen-talarmnumbers(FNs)(48 – 54).ThemitochondrialCyt b genewasusedtoestablishthebarcodes of seven groupers andanalyzephylogenetic relationships among thesespecies. Wediscovered that Epinephelus groupers should be classified as monophyly. The minimum genetic distance expressed between E . coioides and E . tukula was 0.1276. From results of the cytogenetic and molecular analyses, it was demonstrated that Plectropomus is a relatively primitive genus of grouper,while Epinephelus isamore-modernderivedgenus.Resultsalsoshowedthat E . coioides and E . tukula have similar genetic characters and karyotypes, and should be foremost considered for artificial hybridization strategies. Furthermore, information on karyotypes of species within the Epinephelus is still insufficient, and further elucidation of karyotypes of Epinephelus willbeagreathelptofuturegeneticbreedingresearch. amplified using a PCR machine (BIO-RAD MJ Mini Gradient Thermal Cycler, Conmall Biotech-nology, Singapore) with initial denaturation at 95 (cid:1) C for 3 min; 35 cycles of 95 (cid:1) C for 1 min, 50 (cid:1) C for 1 min, and 72 (cid:1) C for 1 min; with a final extension of 72 (cid:1) C for 10 min. The reaction was cooled down to 25 (cid:1) C for 10 min. PCR products of the Cyt b gene were checked using 1% agarose gel electrophoresis and then stained with ethidium bromide (EtBr; 0.5 mg/mL). Target DNA fragments were eluted with a DNA Clean/Extraction kit (GeneMark, Taichung, Taiwan). Sizes of the purified DNA fragments were checked and then stored in a (cid:4) 20 (cid:1) C freezer. DNA fragments were directly sequenced on an Applied Biosystems (ABI, Foster City, CA, USA) automated ABI3730x1 DNA sequencer using a Bigdye sequencing kit (Perkin-Elmer, Wellesley, MA, USA). FOR or UnvH primers were used in the sequencing reaction, and the PCR cycle parameters for sequencing were 35 cycles of 30 s at 95 (cid:1) C, 30 s at 50 (cid:1) C, and 1 min at 72 (cid:1) C. Cyt b sequences study. Homologous sequences were aligned using ClustalW [21] and then manually checked. Interspecific genetic distances were analyzed using the Kimura-2-parameter (K2P) model [22], and numbers of different nucleo-tides with MEGA software best-fitting models of DNA substitution the lowest Bayesian Information Criterion (BIC) scores The phylogenetic trees of Cyt b using the Neighbor-joining (NJ) [25] and Maximum-likelihood (ML) bootstrap 1000