| The Chinese sucker,Myxocyprinus asiaticus（Bleeker）belonging to Catostomidae,is China ’ s second-class protected animal species and so far the single species and genus of Catostomidae in China,and is mainly distributed in Yangtze River and Min River and theirs tributaries.In recent years the wild populations of the species have dramatically declined as a result of over-fishing,water pollution and dam construction.In Min River area it is difficut to find the Chinese sucker.Artificial releasing is one of the most effective ways in the respect of increasing the resources of M.asiaticus in Yangtze River.In order to know the genetic diversity of artificial feeding suckers,assessing the quality of sucker’s progeny is an important step to realize the protection of M.asiaticus resources.Our study used Mitochondrial DNA,microsatellite and MHC Ⅱ B molecular markers to assess the genetic diversity of artificial feeding suckers so as to provide the scientific evidence for the protection of M.asiaticus resources.The main content and results are as follows: 1.Genetic diversity analysis of M.asiaticus based on mitochondrial DNABased on Mitochondrial DNA（D-loop+Cytb）the whole length 2994 nucleotide sequence are obtained.In Mitochondrial sequence of 164 M.asiaticus 87 variable sites,83 parsimony informative sites are detected and 18 haplotypes are defined.The range of haplotype diversity（h）were 0.483-0.835,the range of nucleotide diversity（π）were 0.02351-0.03121.In the four artificially propagated populations,WZ population displayed the highest haplotype diversity 0.835,and MS population the lowest 0.486.While WZ showed the lowest nucleotide diversity（π）0.10052.Fst of four artificially propagated populations ranges from 0.03274-0.39849,in which Fst between MS and WZ is 0.39849,and Fst between MS and LZ 0.33294,suggesting a clear differentiation between MS and WZ and LZ.While Fst between LZ and WZ is 0.03274,showing a very low level of genetic differentiation between this two populations.Haplotype network diagram and phylogenetic tree show no relationship with geographic population.The main haplotype is H1 surrounded by some unique haplotypes of YB,MS and LZ.Some overhaplotype which are never detected are found.AMOVA analysis shows that the intra-species variation is bigger than inter-species variation and the former is 77.97%.2.Genetic diversity analysis of M.asiaticus based on microsatellite markerBased on the 454 sequencing method,the polymorphic microsatellite loci are developed.1/8 "454" Sequencing for M.asiaticus,after screening primitive sequence,choosing core area as the tri-nucleotide and tetranucleotide from 6976 sequences which can be used for primer design to design microstallite primers,36 pairs of polymorphic microsatellite loci were finally obtained.Ten pairs of primers are selected from pairs of polymorphic microsatellite loci,and 86 alleles are detected,in 4 cochineal populations.The total number of alleles in the WZ population was 72,while the number of alleles in the YB population was the least,and the number of alleles was 62.The largest distribution of the number of private alleles and the number of rare alleles is still the WZ population,7 and 8,respectively.But the LZ is the least,2 and 3,respectively.Comparising the genetic diversity index of 4 species of M.asiaticus,we found that the heterozygosity of WZ population was the largest and that of YB and MS population were smaller.The genetic differentiation between populations is Fst and Nei ’s genetic distance.The genetic differentiation of WZ and MS is the largest among the four populations.The genetic differentiation of LZ and MS is larger,YB and MS are moderately differentiated.It is speculated that WZ and LZ form a genetic population,and that the other two are genetic groups 3.Genetic diversity analysis of M.asiaticus based on MHC molecular markerFirstly,Cloning MHC Ⅱ B gene and obtaining 1690 bp,the full length of cDNA sequence.Open reading frame（ORF）of MHC Ⅱ DAB was 759 bp,which is predicted to encode 252-amino acids.There are detected 67 variable sites,including 54 parsimony-informative sites,in the whole nucleotide sequence of MHC Ⅱ B gene.The proportion of nucleotide variable sites of MHCⅡ B gene is about 8.83% and that of amino acid variable sites of MHCⅡ β gene are 15.48%.The variable rate of nucleotide and amino acid are 20.60% and 37.08% in the β1 domain respectively,which is much higher than in the β2 domain.A constructed phylogenetic tree showes that 8 alleles of MHC class II β gene in M.asiaticus are clusted and also clusted with Triplophysa rosa,and that M.asiaticus has a close relationship with Carp and zebrafish and far relationship with guppy.The analysis of selective pressure of this gene during evolution indicated that there are 22 positive selected sites and in the 13 antigen binding sites of β domain 7 was positively selected,which was predicted to be one of reasons maintaining high polymorphism of MHC ⅡB.Secondly,primers were designed at the exon 2 of the amplified MHC ⅡB gene,sequence tag are added at both ends,and then constructed a cDNA gene bank for four populations（162）.Illumina PE250 was used to carry out high-throughput sequencing in four populations,and 50 261bp-long alleles were obtained.From the analysis of genetic diversity of four populations of M.asiaticus showed haplotype H,and haplotype diversity h is highest is MS（45,0.926）,the lowest is LZ（41,0.91）;the nucleotide diversity index,π is from big to small are WZ（0.37722）,MS（0.33598）,YB（0.34358）and LZ（0.31847）.The largest genetic distance between four species of carmine is WZ and MS（0.03389）,and the smallest is WZ and LZ（0.02174）.The Fst value of genetic differentiation index WZ and MS is maximum（0.45261）,LZ and WZ Fst are 0.03010.The results of genetic variation in molecular variance analysis are mainly from intra-specific individuals.In conclusion,the genetic diversity of artificial feeding suckers population from WZ is among the highest level and contains the most richest private haplotypes.The results play an important role in judging the genetic quality of artificial feeding suckers and moreover to provide supporting theories for the scientific management. |
