Weighted Correlation Network Analysis(WGCNA) of Japanese Flounder(Paralichthys olivaceus) Embryo Transcriptome Provides Crucial Gene Sets for Understanding Haploid Syndrome and Rescue by Diploidization

Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.

Effects of Dietary Soy Isoflavones on Feed Intake,Growth Performance and Digestibility in Juvenile Japanese Flounder(Paralichthys olivaceus)

An 8-week feeding trial was conducted to investigate the effects of dietary soy isoflavones on feeding intake,growth performance,and digestion of juvenile Japanese flounder(Paralichthys olivaceus).Four isonitrogenous(49% crude protein) and isoenergetic(20.1 MJ kg-1) diets were formulated to contain four graded levels of soy isoflavones,namely,0,1,4 and 8 g soy isoflavones in 1 kg of diet.Each diet was randomly fed to triplicate tanks of fish(Initial average weight:2.58 g ± 0.01 g),and each tank was stocked with 35 fish.No significant difference was observed among diets with levels of 0,1 and 4 g kg-1 soy isoflavones in feed intake,weight gain,feed efficiency ratio(FER),proximate composition of fish whole body and apparent digestibility coefficients(ADC) of nutrients and energy(P>0.05).However,high dietary soy isoflavones level(8 g kg-1) significantly depressed weight gain,FER,whole-body crude lipid content of fish and ADC of nutrients(P<0.05).These results indicate that high level of dietary soy isoflavones(above 4 g kg-1) significantly depresses growth responses and FER of Japanese flounder.However,as the content of soy isoflavones in soybean meal is around 1 to 3 g kg-1,the adverse effects might be neglected when soybean products are used as a fish feed ingredient.

Molecular Cloning and Expression Analysis of Toll-like Receptor 1 cDNA in Japanese Flounder,Paralichthys olivaceus

[Objective] The paper aimed to clone the full length gene of Toll-like recep- tors （TLRs） in Japanese flounder （Paralichthys olivaceus）, and analyze their structural features and expression regularity. [Method] The full length cDNA sequence of Toll like receptor 1（TLR1） gene was identified from Japanese flounder head kidney by ho- mologous cloning and rapid amplification cDNA ends （RACE）. The bioinformatics and expression model of this gene was analyzed. [Result] The TLR1 cDNA was 2 947 bp, a 2 418 bp open reading frame （ORF）, encoding 805 amino acid （aa） residues, including signal peptide, six leucine-rich repeat（LRR） motifs, two transmembrane zones and one TolI/IL 1 receptor （TIR） domain. The molecular weight of the deduced protein was 91.15 KDa, and the isoelectric point was 6.49. The amino acid sequence of Japanese flounder TLR1 possessed 69%-35% identity with the TLRls of other verte- brates, further analysis showed that the TIR domain of Japanese flounder TLR1 shared 84%-62% identities with TIR domains in other vertebrates. Japanese flounder TLR1 protein firstly clustered with TLRls in Epinephelus coioides in the phylogenetic analysis. The transcription of Japanese flounder TLR1 was examined by real-time quantitative PCR, and its mRNA was mainly detected in liver, heart and spleen. [Conclusion] The results lay a foundation for further studying the functions of TLR1 and developing immune potentiator in Japanese flounder.

Single Nucleotide Polymorphism of FSHβ Gene Associated with Reproductive Traits in Japanese Flounder (Paralichthys olivaceus)

Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development.This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder.We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals.We identified only an SNP (T/C) in the coding region of exon3 of FSHβ.The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene.Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) (P<0.05).Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI (P<0.05) than that of genotype CC.Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.

Effects of Calanoid Copepod Schmackeria poplesia as a Live Food on the Growth, Survival and Fatty Acid Composition of Larvae and Juveniles of Japanese Flounder, Paralichthys olivaceus

Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.

Analysis of New Microsatellite Markers Developed From Reported Sequences of Japanese Flounder Paralichthys olivaceus

The expressed sequence tags（ESTs）of Japanese flounder,Paralichthys olivaeeus,were selected from GenBank to identify simple sequence repeats（SSRs）or microsatellites.A bioinformatic analysis of 11111 ESTs identified 751 SSR-containing ESTs,including 440 dinucleotide,254 trinucleotide,53 tetranueleotide,95 pentanucleotide and 40 hexanucleotide microsatellites respectively.The CA/TG and GA/TC repeats were the most abundant microsatellites.AT-rich types were predominant among trinucleotide and tetranucleotide microsatellites.PCR primers were designed to amplify 10 identified microsatellites loci.The PCR results from eight pairs of primers showed polymorphisms in wild populations.In 30 wild individuals,the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average PIC value was 0.8.These microsatellite markers should prove to be a useful addition to the microsatellite markers that are now available for this species.

Genetic diversity in two Japanese flounder populations from China seas inferred using microsatellite markers and COI sequences

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea （Qingdao population, QD） and East China Sea （Zhoushan population, ZS） using 10 polymorphic mierosatellite loci and cytochrome c oxidase subunit I （COI） sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data （Fst=0.048 7, P〈0.001） and mitochondrial DNA data （Fst=0.128, P〈0.001） both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.

Ontogeny of the Lymphoid Organs of Japanese Flounder,Paralichthys olivaceus

Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40 d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells were observed within a week after hatching. The spleen was first seen after 8 d of hatching. The thymus appeared after 15 d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40 d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.

Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV （lymphocystis disease virus）-infected Japanese flounder （Paralichthys olivaceus）, serum proteins from self-healing and sick Japanese flounder were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Protein spots demonstrating changes greater than two-fold in the expression level were digested and further identified in capillary liquid chromatography tandem mass spectrometry （LC-MS/MS）. Two immunityrelevant proteins were thus identified as transferrin and the complement component C3 of Japanese flounder. These findings suggest that the two proteins may play important roles in the self-healing of lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing in LCDV-infected Japanese flounder by improving their innate immunity.

Genome-wide identification and transcriptome-based expression analysis of sox gene family in the Japanese flounder Paralichthys olivaceus

Sox genes are transcription factors that ubiquitously exist in animal species, and share a conserved high mobility group(HMG) box. They play important biological roles in diverse developmental processes, such as sex determination and differentiation, chondrogenesis, neurogenesis, and early embryonic development. In this study, we identified 25 sox genes from genome and transcriptome of Japanese flounder Paralichthys olivaceus. These s ox genes could be mainly classified into seven subfamilies(B1, B2, C, D, E, F, and K), and each subfamily exhibited a relatively conserved gene structure. Besides, subfamilies A and G were found exclusively in human and mouse, and sox 32 in subfamily K only existed in teleosts. Compared with other mammals, some s ox genes in teleosts had two duplicates. The loss, duplication, and divergence of sox genes during evolution provided an evidence for whole-genome duplication that occurred in the radiation of teleosts. The expression of Japanese flounder sox genes was also analyzed by FPKM value. Our results showed that certain s ox genes exhibited obviously tissue-specific and spatio-temproal expression. Especially, gonal-basied expression analysis uncovered that s ox7 and s ox2 were ovary-biased, and s ox8 b was testis-biased. Moreover, sox10 a was expressed specifically in ovary, and sox8 a in testis. Therefore this study provide a solid foundation for future functional and evolutionary analysis of sox genes in Japanese flounder.

DEVELOPMENT OF RETINAL STRUCTURE AND VISUAL ACUITY IN JAPANESE FLOUNDER(PARALICHTHYS OLIVACEUS)

The retinal structure and visual acuity in Japanese flounder Paralichthys olivaceus at different stages of development were examined by light microscopy. The resolving power of the retina, the visual axis and the best visual field were estimated based on the distribution of cone cells in the retina. The visual system of the larvae appears poorly developed at hatching. The larvae with total length (TL) of less than 10 mm, have single cones only and the eyes were well pigmented. At 10-11 mm TL, most single cones fused to form double cones, with the single and double cones forming a mosaic pattern. From larvae to early juvenile the retina stretches, the cones increase in diameter and rods increase in number. Based on the highest density of the cones in the ventro temporal region, the visual axis was orientated up forward. The resolving power of the retina in 40-530 mm TL Japanese flounder was found to range from 25.1 to 11.5 min. The results indicated continual improvements in the visual system of the growing fish towards higher resolving power, visual acuity and sensitivity.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder(Paralichthys olivaceus)

Follistatin （Fst） is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns ofFst gene at five different development stages of Japanese flounder （stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months）. The muscle tissue of Japanese flounder was obtained at different development stages in dais experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Genome-Wide Identification of heat shock protein 10/60 Genes in Japanese Flounder (Paralichthys olivaceus) and Their Regulated Expression After Bacterial Infection

Heat shock proteins 10/60(hsp10/60)are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions.Besides the chaperone and housekeeping functions,they are also known to be involved in immune response during bacterial infection.In this study,we identified and annotated 10 hsp10/60 genes through bioinformatic analysis in Japanese flounder(Paralichthys olivaceus).Among them one member of hsp10(hspe)family and nine members of hsp60(hspd)family were identified.Phylogenetic and selection pressure analysis showed that the hsp10/60 genes were evolutionarily constrained and their function was conserved.Besides,hsp10/60 genes were involved in different embryonic and larval stages and acted as the sentinel role in an unchallenged organism.In addition,we also observed the expression patterns of hsp10/60 genes after Edwardsiella tarda infection,for the first time in Japanese flounder.Eight out of 10 genes were differentially expressed after bacterial challenges,the significantly regulated expressions of flounder hsp10/60 genes after bacterial infections suggested their involvement in immune response in flounder.Our results provide valuable information for clarifying the evolutionary relationship,and early insights of the immune functions of hsp10/60 genes in Japanese flounder.

A potential germ cell-specific marker in Japanese flounder,Paralichthys olivaceus：identification and characterization of lymphocyte antigen 75(Ly75/CD205)

Some germ cell marker genes, such as vasa, nanos, and dead end( dnd), have been identified in fish. Recently, lymphocyte antigen 75(Ly75/CD205) has been identified as a mitotic germ cell-specific cell-surface marker in several fish species. In this study, the Japanese flounder Paralichthys olivaceus ly75 homolog( ly75) was cloned and its expression pattern in gonads was analyzed. The full-length c DNA of ly75 was 7 346 bp, with an open reading frame(ORF) of 5 229 bp. The ORF encoded a protein containing 1 742 amino acids with a predicted molecular mass of 196.89 k Da. In adult tissues, ly75 transcripts were detected in all analyzed tissues but abundantly in the testis. In in-situ hybridization analyses, ly75 mRNA was predominantly localized in oocytes in the ovary and spermatogonia in the testis, but ly75 mRNA was not detected in oogonia, spermatocytes, spermatids, or spermatozoa. These results indicated that ly75 could be a potential germ cell-specific marker in P. olivaceus, as in other fishes.

Mutations in Exons of the CYP17-Ⅱ Gene Affect Sex Steroid Concentration in Male Japanese Flounder(Paralichthys olivaceus)

As a specific gene of fish,cytochrome P450c17-Ⅱ(CYP17-Ⅱ) gene plays a key role in the growth,development and reproduction level of fish.In this study,the single-stranded conformational polymorphism(SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-Ⅱ gene in a population of 75 male Japanese flounder(Paralichthys olivaceus).Three single nucleotide polymorphisms(SNPs) were identified in CYP17-Ⅱ gene of Japanese flounder.They were c.G594A(p.G188R),c.G939A and c.G1502A(p.G490D).SNP1(c.G594A),located in exon 4 of CYP17-Ⅱ gene,was significantly associated with gonadosomatic index(GSI).Individuals with genotype GG of SNP1 had significantly lower GSI(P < 0.05) than those with geno-type AA or AG.SNP2(c.G939A) located at the CpG island of CYP17-Ⅱ gene.The mutation changed the methylation of exon 6.Indi-viduals with genotype AA of SNP2 had significantly lower serum testosterone(T) level and hepatosomatic index(HSI) compared to those with genotype GG.The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder.How-ever,the SNP3(c.G1502A) located in exon 9 did not affect the four measured reproductive traits.This study showed that CYP17-Ⅱ gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

编码金属标签对牙鲆(Paralichthysolivaceus)苗种标记的效果

为了开发适宜牙鲆(Paralichthys olivaceus)苗种放流的标志技术,利用编码金属标签(CWT)对不同规格牙鲆苗种进行了标记实验,并从标记死亡率、脱标率、适宜标记鱼规格等方面评价了CWT的标记效果。结果显示,CWT标记3种规格的牙鲆苗种后,脱标均发生在标记后的4 d内,其后未再发生脱标现象。小规格苗种[全长为(5.92±0.41)cm]死亡率较高(13%),中规格苗种[全长(8.92±0.36)cm]死亡率为2%,大规格苗种[全长为(12.06±0.62)cm]死亡率为1%。小规格、中规格和大规格苗种脱标率分别为3.3%、2.4%和0.7%。建立了标记死亡率(M)与苗种全长(TL)、体厚度(BT)的关系模型:M=0.7254 TL^2–15.3220TL+79.4260(R^2=0.9601);M=1.3627 BT^2–15.5610 BT+44.4330(R^2=0.9645),为适宜苗种规格选择与标记效果评价提供了依据。今后利用CWT标签进行牙鲆苗种标志放流时,建议选择全长6 cm以上的苗种进行背部肌肉标记,标记对苗种游泳行为和生长无影响,表明CWT是一种适宜在牙鲆大规模标志放流中应用的理想标志方式。

Genome-Wide Identification,Characterization,and Expression Analyses of the Mucin Genes in Japanese Flounder(Paralichthys olivaceus)

Mucin family members play an indispensable role in immunity against pathogen invasion.Currently,there are very few studies on members of the mucin family in Japanese flounder Paralichthys olivaceus.In this regard,we characterized the mucin members in Japanese flounder as well as their involvement in response to pathogen infection.In our results,a total of 9 mucin genes were identified based on the whole genome database of flounder.Among them,MUC2-1,MUC2-2,MUC5AC-1,MUC5AC-2 and MUC5B are secreted mucins,while MUC3A,MUC3B,MUC13 and MUC15 are membrane binding mucins.The collinearity results showed that the adjacent MUC genes of flounder had collinearity relationship with many other teleosts.Phylogenetic tree results showed that the mucin gene of flounder was divided into several subfamilies.In addition,the expression patterns of flounder mucin family members were examined in 11 healthy tissues.The expression changes of mucin gene were also detected in six immune tissues(gill,intestine,skin,liver,kidney,spleen)after pathogen infection,revealing their role in disease resistance.Collinear analysis indicates the adjacent mucin genes.According to the protein interaction network(PPI)results,the mucin genes interact with the galnt genes.The results provide a solid foundation for further research on the function of mucins in mucosal immunity of flounder.

一种利用Tol2转座子快速获得牙鲆稳转细胞的方法

脂质体转染是分子生物学中研究基因功能和基因表达调控的常规手段。然而,在海水硬骨鱼细胞中,脂质体转染效率低下且很难通过转染后直接筛选的方式获得稳转细胞。本研究以pminiTol2为模板,在末端反向重复序列中间添加了CMV启动子、EGFP荧光蛋白、新霉素抗性基因等功能区域,构建了CMV-EGFP-pminiTol2重组载体。CMV-EGFP-pminiTol2和pCS-TP质粒经脂质体包裹共转染至牙鲆(Paralichthys olivaceus)卵巢颗粒细胞后,仅需2~3周的新霉素(G418)筛选即可获得稳定转染的细胞。本实验首次探究了利用Tol2转座子系统快速筛选获得牙鲆稳转细胞的方法,其研究成果可应用于牙鲆分子生物学研究,并为其他硬骨鱼类稳转细胞构建及筛选提供新思路。

牙鲆野生群体与养殖群体的遗传多样性分析

利用AFLP技术对荣成野生群体和养殖群体牙鲆进行遗传多样性分析和比较。实验采用 7对引物组合在 2个群体中共扩增出 797个位点 ,其中多态位点数为 43 3个 ,占总位点数的 5 4.2 7%。各引物组合在 2个群体中的扩增位点数目和多态位点比例有较大不同 ,但养殖群体的总扩增位点数和多态位点比例均低于野生群体 ,野生群体和养殖群体的平均多态位点率分别为 46.18%和 40 .0 7% ,其中 ,E3 8M 5 0引物组合在两群体中扩增的多态位点比例差异显著 (P <0 .0 5 )。养殖群体中低频位点明显减少而隐性纯合基因位点显著增加。群体遗传结构分析表明 ,两群体间的遗传距离比较小 ,群体遗传结构相似 ,说明养殖群体尚没有形成自己独立的遗传结构。