| Sciaenid fishes (family Sciaenidae) are very important marine fish resource and attractive species for aquaculture. There are a number of species in this family in China Sea, with about 30 species in 17 genera. Due to a prolonged period of overfishing, the resources of sciaenid fishes have been remarkably declined, some species even was listed as endangered, such as Larimichthys crocea, it used to be one of the most important commercial fishes in China, but the wild populations have been collapsed since 1970s.The contents of this study includes the following four parts:phylogenetic estimation of Sciaenidae in the East China Sea inferred from nuclear DNA sequence; New microsatellite markers for L.crocea and cross-amplification in closely related species; Genetic diversity and population identification of L. crocea revealed by the mitochondrial and nuclear DNA sequence and development and application of SNP markers in genome of L.crocea, the main results are as follows:(1) Phylogenetic estimation of Sciaenidae in the East China Sea inferred from nuclear DNA sequenceFish of the family Sciaenidae (croaker or drum fish, family Sciaenidae) are highly important commercial fishery resources. They are mainly distributed along the Pacific Ocean, Atlantic Ocean, Indian Ocean and south of East Africa. The systematic and phylogenetic relationships of the Sciaenidae have been widely debated.Morphologic disparity among the sciaenids has prompted numerous conflicting phylogenetic hypotheses because of a lack of clear synapomorphies, the ability of morphology to resolve such phylogenetic conflict is limited.Here we describe a phylogenetic analysis of sciaenids of the East China Sea based on nuclear exon-primed intron-crossing genes (EPIC) and a mitochondrial gene (COI). Separate analyses of the two data partitions resulted in mostly congruent trees. Although there were some differences in the classification of these species, the main difference between trees obtained by the mitochondrial gene (COI) and nuclear DNA sequences was the position of Miichthys miiuy and Johnius belangerii. In the mitochondrial phylogeny, J. belangerii was placed at the most basal position forming an individual clade, while other species formed another large cluster. M. miiuy formed an independent basal sub-clade grouped with Larimichthys and Collichthys. Collichthys lucidus was grouped with Pseudosciaenacrocea and Larimichthys polyactis. Trees based on the nuclear genes differed somewhat from those based on the mitochondrial gene COI. In this analysis, two groups resulted, the Larimichthys and Collichthys clade, and another clade including a total of five species:J. belangerii, Nibea albiflora, Pennahia argentata, Sciaenops ocellatus, and Argyrosomus japonicus; J. belangerii clustered with N. albiflora. M. miiuy was placed at the basal position of the other cluster because it was an independent basal sub-clade grouped with J. belangerii, N. albiflora, P. argentata, S. ocellatus, and A.japonicus. Many aspects of the phylogeny of the Sciaenidae remain unresolved, and further analysis based on more molecular information and extensive taxon sampling is necessary to elucidate the phylogenetic relationships among the major lineages within Sciaenidae.(2)New microsatellite markers for L. Crocea and cross-amplification in closely related speciesThe large yellow croaker (L.crocea), included in the suborder Percoidei, family Sciaenidae, and genus Pseudosciaena, is one of the most economically marine fish in China, which is mainly limited to coastal waters of continental East Asia. Due to various of factors including over wintering aggregations, poor stock management, heavy exploitation habitat pollution and marine ecosystem shift, the wild resources of the large yellow croaker has almost collapsed.Management and artificial cage culture were introduced by the Chinese governmental organizations since 1980s in order to restore the wild stocks and the artificial culture of this species got successful. However, there is still a long way to go to restore the wild stocks. At the same time, an understanding of the genetic background of the large yellow croaker populations is significantly and urgent for resource conservation and breeding. We identified and characterized 27 microsatellites from L. crocea. We cross-amplified the 27 microsatellite loci in J. belangerii, N. albiflora and A. japonicus. The results show that 24,26, and 25 of 27 primers pairs, respectively, amplified stably with rich polymorphism. The products were screened for SSR. The number of alleles among these loci ranged from 6 to 14. The observed and expected heterozygosity ranged from 0.078 to 0.372 and 0.515 to 0.905, respectively. Most of the SSR markers enabled amplification of alleles in morphologically similar species of the Sciaenids family. These results prove that the SSR markers that we developed are useful for cost-saving and determining genetic diversity and structure among natural populations of Sciaenids, as well as identifying the hybrid offspring and their parents. This study could contribute to the better understanding of genetic diversity of the large yellow croaker and related species.(3) Genetic diversity and population identification of L.crocea revealed by the mitochondrial and nuclear DNA sequenceThe geographical population identification of large yellow croaker has been controversial for decades because of artificial releasing and breeding. In this study, mitochondrial cytochrome oxidase-1 (COI) and two genomic DNA sequences (exon-primed intron-crossing markers) were used to identify the geographical populations (three wild and two cultured populations) of this croaker. Phylogenetic analysis suggested that three populations were resulted as per two nuclear genes; however, this result is different from that of the COI gene. This identification might reflect the geographical populations of this croaker. Moreover, this study provided new insights into the genetic divergence, population identification, and genetic background of large yellow croaker populations. The results of this study can be used as reference to aid the resource conservation and artificial breeding of this fish.(4) Development and application of SNP markers in genome of L. CroceaA draft genome of Lcrocea containing 619 Mb with 88% of the estimated genome size was sequenced and assembled using next-generation sequencing at 82 x depth.250 single nucleotide polymorphisms (SNPs) loci were developed and Sequenom MassARRAY was introduced to investigate genotype within 4 different geographicalpopulations. The resulted showed that the effective number of alleles ranged froml.328 to1.341, with an average value ofl.335, the average values of expected and observed heterozygosities of the SNPs ranged from 0.273-0.320 and 0.190-0.235, respectively. Polymorphism information content (PIC) values ranged from 0.215-0.247, FST was ranged from 0.00781 to 0.2703, FIS was ranged from 0.1738 to 0.2786. The 60 loci were developed successfully and showed polymorphism. A total of AA, CA, CC, CT, TT, GG, GA, GT, AT, GC 10 genotypes were found, Among the 60 SNPS, there were 29 transformation and 31 transversion,147 loci were detected.There are some unique markers for the opulations, scf117, scf708, scf247 were unique for Zhoushan wild population, scf559 was unique for Zhangpu wild population, scf100 and scf166 were unique for Hainan wild population; scf305 and scf664 were unique for Ningde farm population. The tag of scf117, scfl87and scf511were in the protein-coding area, the proteins were ATRAID, FAM210B and NCKAP5, respectively. In summary, the SNPs study of large yellow croaker is effective and suggesting that SNP markers have been useful for population genetic assessment and breeding studies. |
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