| Oryzias curvinotus belongs to the order of Adrianichthyidae,Oryzias,which is broad-salinity,wide temperature,strong tolerance to hypoxia,large amount of eggs laid,short generation cycles,and easily raised in the laboratory.It is an indigenous fish distributed along the southern coast.It is closely related to the most clearly studied Oryzais latipes in the same genus,and both have an XX-XY gender control system and have excellent theoretical research value.The ecological investigation and genetic diversity analysis of the O.Curvinotus using mitochondrial DNA have enriched the research on its biological background and provide support for the development of model organisms for the development of O.curvinotus.The specific research results mainly include five parts:1.Ecological Investigation of the O.curvinotus.Physical and chemical indicators of water: salinity range is 0.14-33.57;water temperature range is 9.58-40.4℃;p H value range is 7.2-9.38;dissolved oxygen range is 2.11-17.12 ppm.The inhabitation characteristic of the O.curvinotus is that they are living in a gentle water stream with plants,once Gambusia affinis develops into a dominant species,the number of O.curvinotus will be greatly reduced.2.Effects of salinity on hatching of fertilized eggs.The fertilized eggs of the O.curvinotus in the Gaoqiao area of western Guangdong can be hatched within a range of fresh water to 45 of salinity.When the salinity is 25,the fertilized eggs have the highest hatching rate.With a 20 of salinity the hatching cycle and the cultivation cycle are the longest,while the low salinity(fresh water to 10 of salinity)and high salinity(40-45)cultivation cycle and hatching cycle are relatively short.3.The genetic sex identification of O.curvinotus.Developed a pair of primers that can quickly and accurately identify the genetic sex of the O.curvinotus.The primers can be determined by PCR amplification and agarose gel electrophoresis to detect the individual genetic sex.The males(XY)can amplify two bands(959 bp;656 bp),while females(XX)can only amplify a 656 bp band.4.Complete mitogenome sequence of the O.curvinotus(Teleostei:Beloniformes)and its phylogenetic analysis.The complete sequence of mitochondrial genome is determined and annotated.The results show that the length of the mitochondrial genomic sequence is 16676 bp,and the gene arrangement and composition are almost identical to those of other bony fish,including 37 genes and 1 control region.Of the 13 protein-coding genes,except for the COI gene,which starts with GTG,the rest of the encoded proteins use the ATG as the start codon.The secondary structures of the 22 t RNA genes are all typical of clover.The phylogenetic tree constructed using the complete sequence of the mitochondrial genomes of 9 species of Oryzias,and suggests that the genetic relationship between O.curvinotus and the Oryzias luzonensis is relatively close.5.Genetic diversity analysis of O.curvinotus along south coast based on mt DNA COⅠgene and D-loop region.The results of 136 COⅠgene sequences(579 bp)show that there are 77 variable sites in the COⅠgene sequences,and the base conversion / transversion ratio(TS /TV)is 5.42;The average number of nucleotide differences,nucleotide diversity,haplotype diversity and haplotype are 9.709,0.01677,0.967,52;AMOVA analysis reveals that genetic differences mainly occur between groups;Most individuals of phylogenetic trees constructed based on the CO Ⅰ gene sequences are clustered according to the sampling points.Based on 141 D-loop sequences(507 bp)analysis results: 37 variable sites are detected,the base conversion / transversion ratio(TS / TV)is 2.79;The average number of nucleotides,nucleotide diversity,haplotype diversity and haplotype are2.923,0.00577,0.911 and 29 respectively;AMOVA analysis reveals that genetic differences mainly occur between groups;Based on the D-loop region,the majority of individuals fail to cluster according to the sampling points.The results show that mitochondrial COⅠgene has a faster evolution rate than that of D-loop region,and it is more suitable for the study of genetic diversity and genetic differentiation in O.curvinotus.Based on the COⅠ gene and D-loop region sequences,the genetic diversity of O.curvinotus along south coast is at a high level.The population along south coast experienced a population expansion event and the genetic structure is clearly differentiated.Therefore,appropriate delineation of protection units is required. |
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