Germplasm Identification Of Wild And Cultured Stock Of Large Yellow Croaker(Larimichthys Crocea)

Large yellow croaker（Larimichthys crocea）belongs to Perciformes,Sciaenidae,Larimichthys,is an important commercial marine fish species in China.However,with the deterioration of the environment and over-fishing,the wild large yellow croaker resources have been devastating damaged,already tended to endangered species.Artificially cultivated large yellow croaker appeared a series of problems such as germplasm degeneration and frequent disease.In the process of fishery production and management,identification of the large yellow croaker germplasm resources is lack of a systematic and relatively comprehensive research technique and method.In this study,the external morphology,scale features,annulation and color of otolith,combined with bone micro CT scanning microstructurethe and the mitochondrial D-loop region and the Cytb gene and microsatellites were used to compare and analyze the interspecific differences between wild and cultured stocks of large yellow croaker,in order to provide new technologies and methods for germplasm identification of large yellow croaker.Those studies are presented in detail as follows:（1）To collect the identification features between cultured stock and wild stock of large yellow croaker,morphometric traits,scales and otolith characteristics were compared in this study.Morphological traits comparison showed that the fatness,proportion of body height to body length,the body thickness to body length,length of caudel peduncle to tail length in cultured stock,were significantly higher than those in wild stock.The figure of cultured stock tended to be thick and short.The results of scales indicated that there was no significant difference between two groups,some scales had rings distribution and melt-erode phenomenon.The otolith wheel grain in wild stock was denser with inequality between light and shade.Otolith center nucleus and surrounding was yellow.There were 0-2 annuli rings observed in wild stock.Otolith in cultured stock exhibited thin wheel grain,light and shade was evenly spaced,otolith center nucleus and surrounding was colorless,with only one annuli ring.These characteristics reflected differences of food supply and living environment conditions between two groups,which can be helpful for stock identification and resource management of large yellow croaker.（2）To understand the differences in the spine micro structures of cultured and wild stocks of large yellow croaker,we used the micro CT scanning technique to capture the full fish of large yellow croaker.Furthermore,we compared the bone structure parameters of the dorsal and caudal vertebra in the cultured and wild stocks.The results show the head and tail to be directly connected with the trunk and 26 vertebrae,ribs were attached to the 1-11 th abdominal vertebrae,and the inter-muscular bones,pectoral fin,dorsal fin,ventral fin,anal fin,and caudal fin were not separated from the vertebrae.Bone mineral density,tissue mineral density,and bone volume fraction of the wild stock were all higher than those of the cultured stock,the trabecular spacing of the wild stock was lower than that of the cultured stock,and there was no difference in the wild and cultured stocks in their trabecular number,trabecular spacing,or structure model index.We attribute these differences to different nutritional,environmental,and movement elements of wild and cultured stocks.（3）By using 1028 bp and 788 bp of mtDNA Cytb gene and D-loop region,we analyzed the sequence variation and stock genetic structures of wild and cultured stocks of large yellow croaker.The result show,twenty variants were detected in the 1028 bp fragment of 105 samples of Cytb,there were 19 Variable sites in wild stock,15 for Breeding stock FY,14 and 8 for artificial breeding stock F3 and F4 generation.Genetic diversity index analysis show the haplotype diversity index（Hd）of wild stock was 0.842,and the nucleotide diversity index（π）was 0.00575,corresponding parameters of Breeding stock FY were 0.789,0.00491,artificial breeding stock F3 and F4 were 0.699,0.00355 and 0.607,0.00332.Haplotype diversity show that the wild stock contained 12 haplotypes,including 7 Specific haplotypes,which were higher than the corresponding parameters of cultured stocks.Thirty-three variants were detected in the 788 bp fragment of 109 samples of D-loop region,there were 29 Variable sites in wild stock,20 for Breeding stock FY,18 and 14 for artificial breeding stock F3 and F4.Genetic diversity index analysis show the haplotype diversity index（Hd）of wild stock was 0.901,and the nucleotide diversity index（π）was 0.01826,corresponding parameters of Breeding stock FY were 0.813,0.01778,artificial breeding stock F3 and F4 were 0.677,0.01391 and 0.727,0.1471.Haplotype diversity showed that the wild stock contained 15 haplotypes,including 11 Specific haplotypes,which were higher than the corresponding parameters of cultured stocks.The results revealed the genetic diversity of wild stock was higher than cultured stocks.According to the genetic differentiation index Fst and molecular variance analysis（AMOVA）show that the genetic variation of large yellow croaker was mainly from among the individuals in the stock,and the genetic differentiation within the cultured stocks was not significant,and there was a significant middle-level genetic differentiation between the wild stock of the large yellow croaker and the cultured stock.The UPGMA phylogenetic tree and the haplotype network diagram also support this view.As the D-loop region does not encode the protein and include more mutations,which has fast evolution rate is more suitable for interspecies stocks with different genetic differences can be more reliable for the germplasm identification of large yellow croaker.（4）Twelve microsatellite polymorphism markers were used to identify the differences between the wild and cultured stocks of large yellow croaker,the results show the number of alleles in 12 microsatellite loci have 4-10 were amplified,the average observed heterozygosity（HO）,expected heterozygosity（HE）and polymorphic information content（PIC）were 0.745,0.753 and 0.691,indicated that the loci in this study showed high polymorphism.Containing a specific site LYC08 can distinguish between the wild stock and cultured stocks.The wild stock have the largest observed heterozygosity（H_O=0.791）,expected heterozygosity（H_E=0.776）and shannon index（I=1.674）,the corresponding parameters of FY stock,F3 and F4 generation were 0.744,0.764,1.552;0.728,0.736,1.483;0.717,0.735,1.401,indicating that the wild stock has a high level of genetic diversity.The inbreeding coefficients showed that the highest F_IS values were found in F4 stock(F_IS=0.019),followed by F3 stock(F_IS=0.013),FY stock(F_IS=0.011),the results showed that there were heterozygous deletions in this three cultured stocks except the wild stock.Molecular variance analysis（AMOVA）show there are extremely significant between wild group and cultured group（P<0.01）,NJ tree based on Nei’s genetic distance,principal component analysis（PCA）and structural cluster analysis show that the wild stock and cultured stock have relatively distant relationship.