| The Varicorhinus sp. is a kind of precious and wild fish in mountain Tai, it's meat have high nutritional value and special remedy value. In order to preserve this rare species, such as improving its germplasm resource and increasing its yield, we observed characteristics of Varicorhinus sp.embryos during the embryonic development process. We found that the eggs were sinkable, slightly sticky and with color of orange or yellowish. Before swelling, eggs were 1.65 mm～1.82 mm in diameter, and the diameter of swelled eggs were 2.38 mm～2.45 mm. Under 22.5℃～24.5℃of water, it took 46 hours and 50 minutes for the fertilized eggs to hatch. The embryonic development process could be divided into seven stages, including blastoderm formation, cell cleavage, blastula, gastrula, neural, organ forming and hatching stage. The total length of newly hatched larvae were 6 mm～6.5 mm and the myomeres were 46～48, and it had heartbeat and blood circulation, but no pigment in body.The sequence of BMP11 gene was cloned from Varicorhinus sp. cDNA, and its expression in different stages of embryonic development and of different tissues were examined. The Varicorhinus sp. BMP11 (643bp) has a sequence homology of 92.34% to zebrafish BMP11,which demonstrated the gene was highly conserved among different species. Eleven different stages of embryonic development were selected to study the expression regularity of BMP11, the result showed the gene was expressed during the whole stages of embryonic developing. Although its expression amount was not stable, it was an important gene for embryonic developing. In addition, the expression of BMP11 were different from ages, and the expressing quantities were more in muscles , gills, ovary and brain, less in spleen, kidney and intestines.To make sure the classification status of Varicorhinus sp. and the evolutionary relationship between Varicorhinus sp. and other fish species in the same Barbinae, we cloned mtDNA cytb and D-loop sequence. The results showed that there were 828 monophyletic sites and 312 polymorphic sites(27.37%) in the whole 1140 bp of Varicorhinus sp. cytb,without insertion and deletion. The results of homologous fragment analysis showed that the mean contents were T= 27.3%,C=29.1%,A=29.1% and G=14.5%, the content of (T+A) (56.4%) was significantly higher than that of (G+C)(43.6%). The times of transition were much higher than transversion, and the ratio between Transition and transversion was 4.6. Phylogenetic tree of cytb showed that the Varicorhinus sp. and Varicorhinus macrolepis clustered together, which demonstrated they were the same species. Based on the Phylogenetic tree of cytb,we obviously concluded that all of the fishes clustered to five branches, which belonged to five genus of Barbinae—Varossocheilus, Spinibarbus, Acrossocheilus, Barbodes and Sinocylocheilus. Firstly, Varicorhinus sp. and Varicorhinus macrolepis clustered together, then they were clustered with V.simus, which showed the relationships among them were closer than to the others. Species of A.elongatus and A.labiatus clustered firstly, then two subgenus clustered together. S.caldwelli and S.denticulatus belonged to the subgenus of Spinibarbus. The two species of Sinocylocheilus clustered together. B.laticeps was a group alone. The above results were in accord with their geographical distributions. The relationship between Varicorhinus sp. and the other genus as followed: the relationship between Varicorhinus sp. and Acrossocheilus was closest, next is Spinibarbus and Barbodes, and the relationship between Varicorhinus sp. and Sinocylocheilus was the farthest. According to the findings of this paper, we inferred the divergency time of Barbinae from the molecular evolution rate. The divergence of Sinocylocheilus (15.2%～16.5%) occurred about 32 Mya before, Barbodes(14.2%～15.0 %)occurred about 29 Mya before, Spinibarbus(13.0%～14.6%)occurred about 28 Mya before, and Acrossocheilu (10.7%～13.8%)occurred about 24 Mya before.The whole sequence of Varicorhinus sp. D-loop included 1024 bp, in which there was a microsatellite causing the polymorphism among individual, and there was 11 variation sites ( percentage 1.74%) in it. The homologous fragment analysis showed that the average content of T,C,A,G were 32.4%,20.8%,33.6%,13.3%, respectively, T+A content (66.0%)was higher than G+C(34.0%). Transition was higher lightly than transversion, and the ratio between Transition and transversion was 1.5. Based on the whole sequence of Varicorhinus sp. D-loop, we analyzed the genetic distance(0～0.5%) between Varicorhinus sp. and Varicorhinus macrolepis, which was same almostly to the genetic distance between individual of Varicorhinus sp. (0.1%～0.6%) and Varicorhinus macrolepis(0～0.5%). The genetic distance between the two species and Cirrhinus molitorella was same almostly (20.3%～20.8% and 20.2%～20.5%). Phylogenetic tree of D-loop also showed that the Varicorhinus sp. and Varicorhinus macrolepis clustered together. The results above demonstrated they were the same species.In addition,we also analyzed the gene frequency, Hardy– Weinberg equilibrium,genetic distance,genetic similarity of the two group using microsatellite marker. The result revealed the polymorphism of Varicorhinus sp. and Varicorhinus macrolepis was very poor,the genetic distance between two group was vey small (0.0113), and the genetic similarity was high (1.9887).So, we could conclude that Varicorhinus sp. and Varicorhinus macrolepis are one species by the sequence analysis of mtDNA cytb,D-loop and microsatellite marker. |
