Investigating the Genetic Bases of Growth Regulation by E2F3 in Dwarf Surf Clams Mulinia lateralis

Bivalve aquaculture plays a crucial role in the aquaculture industry due to the economic value of many bivalve species.Understanding the underlying genetic basis of bivalve growth regulation is essential for enhancing germplasm innovation and ensuring sustainable development of the industry.Though numerous candidate genes have been identified,their functional validation remains challenging.Fortunately,the dwarf surf clam(Mulinia lateralis)serves as a promising model organism for investigating genetic mechanisms underlying growth regulation in bivalves.The GWAS study in the Yesso scallop(Patinopecten yessoensis)has pinpointed the E2F3 gene as a key regulator of growth-related traits.However,the specific role of E2F3 in bivalve growth remains unclear.This study aimed to further confirm the regulatory function of the E2F3 gene in the dwarf surf clam through RNA interference experiments.Our results revealed several genes are associated with individual growth and development,including CTS7,HSP70B2,and PGLYRP3,as well as genes involved in lipid metabolism such as FABP2 and FASN.Functional enrichment analysis indicated that E2F3 primarily modulates critical processes like amino acid and lipid metabolism.These findings suggest that E2F3 likely regulates growth in the dwarf surf clam by influencing amino acid and lipid metabolism.Overall,this study advances our understanding on the function of E2F3 gene in growth regulation in bivalves,providing valuable insights for future research in this field.

双壳贝类积累转化麻痹性贝毒的研究进展

麻痹性贝毒(Paralytic shellfish toxins,PST)是一类分布广、危害大的海洋毒素。滤食性双壳贝类在摄食、消化产毒单胞藻和细菌等过程中积累代谢PST,并通过食物链进行传递,给人类生命健康和水产业带来不利影响。随着贝类基础生物学和养殖产业的发展,以及基因组学和毒素检测技术的不断进步,近年来,各国学者对贝类吸收、转运和代谢PST的规律有了更深入的认识,为养殖贝类食品安全风险防控提供了理论参考。为更全面了解贝类积累和利用PST的研究进展,本文从PST在双壳贝类中的分布、积累转化特征与分子机制等方面进行了综述。

高效自动化HD-Marker文库构建流程的建立及评估

针对HD-Marker (High-throughput, sequencing-based GoldenGate approach)技术应用于育种研究时面临的短时间内快速完成大批量样品基因分型的挑战,本研究基于自动化移液工作站建立了HD-Marker文库自动化构建体系并评估了此方法在文库构建中的可靠性。本研究基于自动化移液工作站建立了HD-Marker文库自动化构建流程,并评估了此方法在文库构建中的可靠性。结果表明扩增产物具有较高均匀性,浓度范围在23.1~38.7 ng/μL之间;90.63%的文库可获得清晰文库条带;文库位点捕获效率均在97%以上,分型位点比例能达到100%,与标准WGS文库数据相比,位点分型准确性也在97%~100%之间。此自动化文库构建流程可并行处理96个样品的文库制备,整体运行时间仅为人工操作的1/7。本研究首次实现了标准化的自动化HD-Marker基因分型文库制备,为大规模育种群体的基因分型效率的提高提供依据,并为全基因组选择育种的实施提供了具有实用价值的高效率技术平台。

东星斑BCO基因家族鉴定及其表达对体色的影响

为了探究β-类胡萝卜素裂解加氧酶(Beta-carotene oxygenase family,BCO)家族在东星斑(Plectropomus leopardus)体色形成中的作用。本研究在东星斑基因组中鉴定得到4个BCO家族基因,通过生物信息学方法对该家族基因的系统进化、基因结构、理化性质、蛋白结构以及保守性进行预测和分析。结果显示东星斑4个BCO基因都包含一个主要的RPE65功能域,并且其二级结构、超二级结构和三级结构都具有高度保守性。定量分析结果表明Bco2l和Bco2b在类胡萝卜素的主要代谢组织肝脏中有较高的表达,Bco1和Bco2l在肠道中表达较高。另外,红、黑体色差异个体中BCO基因的mRNA表达水平在多个组织中存在显著差异,其中Bco2l的表达量和东星斑红色体色具有较高的相关性,表明Bco2l可能是影响东星斑体色的关键调控基因。

AFLP analysis revealed differences in genetic diversity of four natural populations of Manila clam (Ruditapes philippinarum) in China

The amplified fragment length polymorphism （AFLP） technology was used to analyze the genetic diversities in four natural populations of Manila clam （ Ruditapes philippinarum）, distributed in four sea areas of China, i.e. , the Bohai Sea, the Huanghai Sea, the East China Sea and the South China Sea. Two hundred and sixty-four AFLP loci were analysed in 195 individuals and revealed high levels of genetic diversity. The percentage of polymorphic loci ranged from 92.13% to 96.06% and the Shannon＇ s information index was from 0.256 8 to 0. 275 6. By analyzing molecular variance （ AMOVA）, it was found that there were high levels of genetic differentiation between populations of Qingdao and the other three sea areas. Cluster analysis by Nei＇ s pairwise distances grouped specimens by geographical origin, except the population of Qingdao. A conclusion can be drawn that there are high genetic diversities in the four natural populations of Manila clam in China and some distinct differences existed among and between the four populations. The results also indicated that human cultivation activities will have great influence on the genetic structure of the population of Qingdao.

Assessing genetic diversity of wild populations of Japanese flounderusing AFLP markers

Amplified fragment length polymorphism （AFLP） analysis was used to evaluate the genetic diversity of four wild geographical populations of Japanese flounder （Paralichthys olivaceus）. A total of 775 loci （58.32% of which was polymorphic） in the range between 100 and 1 300 base pairs were detected from 110 individuals using seven primer combinations. The percentage of polymorphic loci detected by single primer combination for each population was calculated, ranging from 19.59% to 53.33%. Genetic similarities within and among the populations were calculated from the binary matrices of presence - absence. Phylogenetic tree of four populations was constructed by using the UPGMA method using PHYLIP Version 3.5. According to intrapopulation genetic similarities, CW population displayed the highest genetic diversity value and KY population had the lowest genetic diversity value. The distance between CW and CF populations was the farthest, which was possibly resulted from the farthest distance of Weihai of Shandong and Fujian of China compared with the geographical distance between other locations of populations. The subpopulation differentiation value （G.,） is 0. 356 5, showing a certain extent of differentiation among the four geographical populations. AFLP technology was confirmed to be an effective tool to assess within- and among-population genetic diversity of Japanese flounder. The present survey provided significant insights for research in the Japanese flounder breeding program.

Mapping Toll-Like Receptor Signaling Pathway Genes of Zhikong Scallop(Chlamys farreri) with FISH

Toll-like receptor(TLR) signaling pathway plays a pivotal role in the innate immune system. Studies on TLR signaling pathway genes in Zhikong scallop(Chlamys farreri) have mainly focused on sequence analysis and expression profiling, no research has been carried out on their localization. The chromosomal position of TLR signaling pathway genes can be valuable for assemblying scallop genome and analysizing gene regulatory networks. In the present study, five key TLR signaling pathway genes(Cf TLR, Cf Myd88, Cf TRAF6, Cf NFκB, and Cf IκB) containing bacterial artificial chromosomes(BACs) were isolated and physically mapped through fluorescence in situ hybridization on five non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of these key immune genes of C. farreri will aid to the research on innate immunity, assignment of interested genes to chromosomes, and integration of physical, linkage and cytogenetic maps of this species.

Amplicon-Based Illumina Sequencing and Quantitative PCR Reveals Nanoplankton Diversity and Biomass in Surface Water of Qinhuangdao Coastal Area, China

Aureococcus anophagefferens caused brown tides for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dy- namics of nanoplankton during the brown tides, sequences of the V9 region of the 18S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spa- tial variation in the 18S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July, 2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July 2011. The highest 18S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from 2011 to 2013. Redundancy analysis (RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other sta- tions for its more selective feeding.

Association of myostatin Variants with Growth Traits of Zhikong Scallop(Chlamys farreri)

Scallop is a popular sea food and an important aquaculture shellfish.Identification of genes and genetic variants relating to scallop growth could benefit high-yielding scallop breeding.Myostatin(MSTN) is a conservative regulator of muscle growth,and has become one of the most important target genes for genetic improvement of the production of farmed animals.In this study,four single nucleotide polymorphisms(SNPs) were identified in the 5' flanking region of MSTN gene(Cf MSTN) in Zhikong scallop(Chlamys farreri).The association of these SNPs with scallop growth traits,including shell length,shell height,body weight and striated muscle weight was analyzed.The SNP g-1162G>T was found to associate with shell length,shell height,and striated muscle weight.The TT type scallops showed significantly higher trait values than those of GT type,and the GG type individuals exhibited median values.On the contrary,significantly more Cf MSTN transcripts were detected in the striated muscle of GT type scallops than in those of TT and GG type ones.Our results suggested that Cf MSTN might regulate the scallop muscle growth negatively,and SNP g-1162G>T can be used as a candidate marker for the selective breeding of high-yielding scallop.

Development of 101 Novel EST-Derived Single Nucleotide Polymorphism Markers for Zhikong Scallop (Chlamys farreri)

Zhikong scallop(Chlamys farreri) is an important maricultured species in China.Many researches on this species,such as population genetics and QTL fine-mapping,need a large number of molecular markers.In this study,based on the expressed sequence tags(EST),a total of 300 putative single nucleotide polymorphisms(SNPs) were selected and validated using high resolution melting(HRM) technology with unlabeled probe.Of them,101(33.7%) were found to be polymorphic in 48 individuals from 4 populations.Further evaluation with 48 individuals from Qingdao population showed that all the polymorphic loci had two alleles with the minor allele frequency ranged from 0.046 to 0.500.The observed and expected heterozygosities ranged from 0.000 to 0.925 and from 0.089 to 0.505,respectively.Fifteen loci deviated significantly from Hardy-Weinberg equilibrium and significant linkage disequilibrate was detected in one pair of markers.BLASTx gave significant hits for 72 of the 101 polymorphic SNP-containing ESTs.Thirty four polymorphic SNP loci were predicted to be non-synonymous substitutions as they caused either the change of codons(33 SNPs) or pretermination of translation(1 SNP).The markers developed can be used for the population studies and genetic improvement on Zhikong scallop.

A Population Genetic Analysis of Continuously Selected Chlamys farreri Populations

This study applied an optimized one-step 2b-RAD library construction strategy and performed simplified genome sequencing of 539 individuals from three continuously selected Chlamys farreri populations. SNP screening was performed using RAD typing software and population genetic parameters for the continuously selected populations from three generations(G1, G2, G3) were determined. The results showed that the optimized one-step 2b-RAD library construction strategy greatly simplified the experimental process, making it suitable for efficiently constructing a large number of libraries. A total of 18450 SNP markers were identified, which evenly distributed throughout the genome. Population genetic analysis of these three generations showed that the mean value of observed heterozygosity was 0.275 ± 0.177, 0.272 ± 0.181 and 0.275 ± 0.166, respectively. Meanwhile, the mean value of expected heterozygosity was 0.275 ± 0.141, 0.274 ± 0.145 and 0.280 ± 0.133, respectively. The Wright's fixation index(F) was 0.04291, 0.04976 and 0.06685, respectively. Markers deviated from Hardy–Weinberg equilibrium accounted for 10.34%, 12.64%, and 23.11%, and the Shannon diversity index was 0.0999 ± 0.0404, 0.0921 ± 0.0388 and 0.0733 ± 0.0308. FIS(also known as the inbreeding coefficient) of the three populations was 0.0256, 0.0323 and 0.0468, respectively. We suggested that the 2b-RAD method is well suited to population genetic studies of aquacultured organisms. Moreover, our results indicated that the continuous selection affected the population genetic structure of the cultured Penglai-Red scallop, but the change was not significant; therefore, population selection should continue.

Identification of Monitoring Organ in Bivalves for Early Warning of Paralytic Shellfish Toxins Accumulation

Bivalve farming plays a dominant role in mariculture in China.Paralytic shellfish toxins(PSTs)can be accumulated in bivalves and cause poisoning the consumers.A sensitive detection of PSTs can provide early warning to decrease poisoning events in bivalve consuming.PSTs are traditionally examined using the whole soft-tissues.However,PSTs accumulation varies dramatically in different tissues of bivalves.Some tough tissues/organs(such as mantle),which account for a large proportion of the total soft body,exhibit a lower accumulation of PSTs and make the toxin extraction time-and reagent-consuming,potentially decreasing the accuracy and sensitivity of PSTs monitoring in bivalves.To develop a sensitive and cost-effective approach for PSTs examination in massively farmed bivalves,we fed three commercially important bivalves,Yesso scallop Patinopecten yessoensis,Pacific oyster Crassostrea gigas,and blue mussel Mytilus edulis with PSTs-producing dinoflagellate Alexandrium catenella,and detected PSTs concentration in different tissues.For all three bivalve species,the digestive gland accumulated much more PSTs than other tissues,and the digestive gland’s toxicity was significantly correlated with the PSTs toxicity of the whole soft-tissues,with r^(2)=0.94,0.92,and 0.94 for Yesso scallop,Pacific oyster,and blue mussel,respectively.When the toxicity of the whole soft-tissues reached 80μgSTXeq(100g)^(−1),the regulatory limit for commercial shellfish,the digestive gland’s toxicity reached 571.48,498.90,and 859.20μgSTXeq(100g)^(−1) in Yesso scallop,Pacific oyster,and blue mussel,respectively.Our results indicate that digestive gland can be used for the sensitive and cost-effective monitoring of PSTs in bivalves.

Effect of Epinephrine on the Settlement and Metamorphosis of Manila Clam Larvae

Chemical inducement and DDRT-PCR （differential display reverse transcription PCR） are adopted to investigate the effect of epinephrine （EPI） on the settlement and metamorphosis of Manila clam larvae. Chemical inducement shows that EPI has an effect to some extent on the metamorphosis of Manila clam larvae at all concentrations and in all treatments designed. The most significant result of inducement is obtained at the concentration of 10^-6 tool L^-1 and for 4 h. DDRT-PCR using six primer pairs shows that the gene expression pattern is quite different between EPI treatment and the control. Three hundred and forty-three amplification bands are obtained in total, among which, 67 （19.53%） are differentially appeared. Therefore, EPI has an effect on the gene expression of the eye spot larval Manila clam. It can be hypothesized that EPI is a settlement and metamorphosis inducer for Manila clam. EPI may lead to larvae settlement and metamorphosis by binding to the receptors on the membrane and then changing the gene expression of larvae cells.

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica Sporophyte via AFLP Technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng,China.Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis.The parameters of the method were optimized.Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer.Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers.A total of 21 loci were obtained and 17 of them were polymorphic.The mean percent age of polymorphic loci of this population was 80.95%.The Nei's gene diversity (H) within this population was 0.3028 and the average Shannon's Information index (I) was 0.4498.A genetic distance matrix among different individuals was constructed as well.Through this study,an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established.The results of this research also revealed a high level of genetic diversity within the studied population.

An Efficient Procedure for Isolating Microsatellite DNAs from Sea Cucumber(Apostichopus japonicus)

The construction of enrichment library proves to be one of the efficient approaches for isolating microsatellites in this study. The genomic DNA of sea cucumber was digested with HaeIII and size-selected DNA fragments (250-700 bp) were ligated to an adaptor. Microsatellite-containing sequences were captured by using a combination of GA and CA probes, which were attached to a nylon membrane. The microsatellite enrichment library constructed in this study consisted of approximately 700 clones. Two hun-dred and thirty-two clones reacted positively after the library screening procedure. Of the 50 clones sequenced, all contained at least one microsatellite and one duplicate clone was found. Approximately 86% of the sequenced fragments permitted to design primers for sequence tagged microsatellite site (STMS).

Genetic diversity and specific markers in four scallop species, Patinopecten yessoensis, Argopecten irradians, Chlamys nobilis and C.farreri

The AFLP （amplified fragrnent length polymorphism）technique was used to analyse the genetic diversity in four scallop species, Patinopecten yessoensis, A rgopecten irradians, Chlamys nobilis and C. farreri. The genetic similarity indexes of these four species are 0.8415, 0.7863, 0.7190 and 0.6731, while Shannon diversity indexes are 43.52, 58.87, 80.16 and 92.83, respectively. As analyzed, the genetic diversities in two native species, i.e., C, farreri and C. nobilis, are higher than those in other two introduced species, A. irradians and P. yessoensis. The results also showed that C. nobilis and C, farreri shared the most common loci. The genetic distance indicated that C. nobilis and C. farreri are closely related. Moreover, out of 510 AFLP markers, 21 specific bands are found to distinguish the four species scallops and these markers may be applied to the specific germplasm characterization and molecular assistant classification in scallops.

Development and Characterization of Microsatellite Markers for the Pacific Abalone (Haliotis discus) via EST Database Mining

The EST database of the Pacific abalone (Haliotis discus) was mined for developing microsatellite markers. A total of 1476 EST sequences were registered in GenBank when data mining was performed. Fifty sequences (approximately 3.4%) were found to contain one or more microsatellites. Based on the length and GC content of the flanking regions, cluster analysis and BLASTN, 13 microsatellite-containing ESTs were selected for PCR primer design. The results showed that 10 out of 13 primer pairs could amplify scorable PCR products and showed polymorphism. The number of alleles ranged from 2 to 13 and the values of Ho and He varied from 0.1222 to 0.8611 and 0.2449 to 0.9311, respectively. No significant linkage disequilibrium (LD) between any pairs of these loci was found, and 6 of 10 loci conformed to the Hardy-Weinberg equilibrium (HWE). These EST-SSRs are therefore potential tools for studies of intraspecies variation and hybrid identification.

Cryopreservation of Veliger Larvae of Trumpet Shell, Charonia sauliae: an Essential Preparation to Artificial Propagation

Trumpet shell, Charonia sauliae, is an endangered and valuable species, but its artificial propagation protocol has not been successfully established. To estimate the possibility of cryopreservation for larvae of C. sauliae, which is a potential preparation for its artificial reproduction and further research, in this study a protocoi for the cryopreservation of veliger larvae of trumpet shell was optimized. Through a two-step cryopreservation procedure, four kinds of cryoprotectants （ethylene glycol, 1, 2-propanediol, dimethyl sulfoxide and glycerol） were employed at three concentrations （1.0, 1.5 and 2.0molL^-1） respectively and survival rates of larvae were determined after a storage of lh. The larvae frozen with these four cryoprotectants after 1 h storage were cultured, and then survival rates were determined at 24, 72 and 120 h after thawing. Dimethyl sulfoxide at a concentration of 1.5 molL^-1 showed the best protective effect in all experiments （p〈0.05）. And survival rates of larvae frozen with dimethyl sulfoxide were determined after 1, 7 and 15 d of storage. The survival rates of larvae frozen with 1.5 molL^-1 dimethyl sulfoxide after 1 h, 1 d, 7 d and 15 d of storage were 80.77% ±7.51%, 80.34%±11.28%, 83.10%±9.14% and 77.23%±6.22% respectively. No significant differences in survival rates of larvae frozen with dimethyl sulfoxide were observed after various storage periods （p〉0.05）.

Verify the Function of a Potential Growth-Regulating Gene in Marine Bivalve Using a Candidate Model Organism Mulinia lateralis

A better understanding of genetic bases of growth regulation is essential for bivalve breeding,which is helpful to improve the yield of the commercially important bivalves.While previous studies have identified some candidate genes accounting for variation in growth-related traits through genotype-phenotype association analyses,seldom of them have verified the functions of these putative,growth-related genes beyond the genomic level due to the difficulty of culturing commercial bivalves under laboratory conditions.Fortunately,dwarf surf clam Mulinia lateralis can serve as a model organism for studying marine bivalves given its short generation time,the feasibility of being grown under experimental conditions and the availability of genetic and biological information.Using dwarf surf clam as a model bivalve,we characterize E2F3,a gene that has been found to account for variation in growth in scallops by a previous genome-wide association study,and verify its function in growth regulation through RNA interference(RNAi)experiments.For the first time,E2F3 in dwarf surf clam,which is termed as MulE2F3,is characterized.The results reveal that dwarf surf clams with MulE2F3 knocked down exhibit a reduction in both shell size and soft-tissue weight,indicating the functions of MulE2F3 in positively regulating bivalve growth.More importantly,we demonstrate how dwarf surf clam can be used as a model organism to investigate gene functions in commercial bivalves,shedding light on genetic causes for variation in growth to enhance the efficiency of bivalve farming.

Performance Comparison of Two Efficient Genomic Selection Methods(gsbay & MixP ) Applied in Aquacultural Organisms

Genomic selection is more and more popular in animal and plant breeding industries all around the world, as it can be applied early in life without impacting selection candidates. The objective of this study was to bring the advantages of genomic selection to scallop breeding. Two different genomic selection tools Mix P and gsbay were applied on genomic evaluation of simulated data and Zhikong scallop(Chlamys farreri) field data. The data were compared with genomic best linear unbiased prediction(GBLUP) method which has been applied widely. Our results showed that both Mix P and gsbay could accurately estimate single-nucleotide polymorphism(SNP) marker effects, and thereby could be applied for the analysis of genomic estimated breeding values(GEBV). In simulated data from different scenarios, the accuracy of GEBV acquired was ranged from 0.20 to 0.78 by Mix P; it was ranged from 0.21 to 0.67 by gsbay; and it was ranged from 0.21 to 0.61 by GBLUP. Estimations made by Mix P and gsbay were expected to be more reliable than those estimated by GBLUP. Predictions made by gsbay were more robust, while with Mix P the computation is much faster, especially in dealing with large-scale data. These results suggested that both algorithms implemented by Mix P and gsbay are feasible to carry out genomic selection in scallop breeding, and more genotype data will be necessary to produce genomic estimated breeding values with a higher accuracy for the industry.