基于转录组平台的蛤仔微卫星标记筛选

以菲律宾蛤仔转录组测序所得拼接序列为基础,采用MISA软件进行微卫星分析,对其中的145个微卫星位点进行引物设计,得到具有清晰扩增条带的微卫星位点58个。对大连庄河野生蛤仔群体的扩增结果表明,18个位点显示单态性,40个位点表现为多态性。该群体40个多态性微卫星位点得到的等位基因数在2—6之间,平均等位基因数为3.4250±0.9718,观测杂合度和期望杂合度分别在0.0000—1.0000和0.0615—0.7996之间,平均值分别为0.2727±0.2272和0.4739±0.1902,群体平均Nei指数为0.4664±0.1872。多态信息含量(PIC)在0.0586—0.7529之间,平均值为0.4148±0.1707,其中16个微卫星位点的PIC值大于0.5,为高度多态性,15个位点0.25PIC0.5,为中度多态性,其余9个为低度多态性。经Sequential Bonferroni校正的Hardy-Weinberg平衡检验,有10个位点尚未偏离平衡。基于转录组平台筛选微卫星标记的方法,在很大程度上推动了DNA分子标记的开发。研究开发的微卫星标记可用于蛤仔群体遗传学、遗传连锁图谱构建及其他相关研究,为蛤仔分子标记辅助育种及群体种质保护等工作提供技术支持。     

虾夷扇贝遗传结构及微卫星标记与经济性状相关分析

选用30个多态性微卫星分子标记对大连大长山(96个)、大连獐子岛(96个)及日本青森县陆奥湾(96个)3个地区虾夷扇贝养殖群体(Patinopecten yessoensis)进行遗传多样性分析。结果显示,30个基因座共检测到198个等位基因(Na),平均等位基因数为6.63个,每个座位检测到的等位基因数为3-12,有效等位基因数(Ne)每个位点平均为4.70个,观测杂合度(Ho)平均值0.58,期望杂合度(He)的平均值为0.75,30个位点的平均多态信息含量为0.703,说明3个地区虾夷扇贝群体遗传多样性水平较高。运用统计软件SPSS11.5对30个微卫星标记与大长山群体生长性状相关性进行了分析。结果表明,位点DQ221714和FJ262378与壳长、壳高、壳宽和鲜重显著相关(P0.05)位点;位点FJ262372与壳宽、软体部重和闭壳肌重显著相关;位点FJ262369与软体部重及闭壳肌重显著相关。对差异显著的位点进行不同基因型间的多重比较,找到了与虾夷扇贝生长性状相关的基因型,进一步将所得显著性标记对大长山虾夷扇贝个体最大的17个和个体最小的13个基因组DNA进行检测,验证其准确性。结果表明,位点DQ221714在两组虾夷扇贝中存在特异性条带,可作为QTL定位候选标记。     

企鹅珍珠贝微卫星标记与生长性状的相关性研究

研究利用5个高度多态性的微卫星标记,对500个企鹅珍珠贝(Pteria penguin)的4个生长性状进行了关联分析。结果显示, QEB-D15和CL-232两个微卫星标记与企鹅珍珠贝的壳宽呈极显著相关(P<0.01);位点QEBD15基因型是239/263的个体壳宽为最大值,基因型是239/273的个体壳宽为最小值,推测263 bp等位基因与壳宽之间存在正相关关系,而273 bp等位基因与壳宽之间存在负相关关系;位点CL-232基因型为157/174的个体壳长、壳宽、总重的均值较同一位点的其他基因型均为最大值,该基因型推测为优势基因型;而基因型为177/192的个体壳长、壳宽、壳高和总重的均值较同一位点的其他基因型均为最小值,推测该基因型为劣势基因型,上述结果可为企鹅珍珠贝分子标记辅助选择育种提供理论依据和参考。     

罗氏沼虾转录组SSR标记筛选及其与体质量相关性分析

研究通过转录组测序对罗氏沼虾(Macrobrachium rosenbergii)SSR标记进行了发掘, 获得SSR位点18592个。随机选取了63个SSR位点设计引物并利用10个个体进行多态性位点筛选, 共获得31个具有多态的SSR标记, 利用其中19个SSR标记对全同胞家系群(FS)119尾虾及孟加拉F3代野生群(MJL)199尾虾的群体遗传多样性进行分析, 并对SSR位点与体质量性状进行关联性分析。结果表明: FS群体共检测到40个等位基因, 平均等位基因数为2.1053, 平均观测杂合度0.4525, 平均期望杂合度0.3804, 平均多态信息含量0.3076, 处于中度遗传多态水平; MJL群体共检测到65个等位基因, 平均等位基因数为3.4211, 平均观测杂合度0.4105, 平均期望杂合度0.4496, 平均多态信息含量0.3882, 处于中度遗传多态水平。 19个SSR位点与FS群体体质量没有相关性(P>0.05), 而在MJL群体中, 4个SSR位点与体质量显著相关(P<0.05)。对差异显著的位点进行不同基因型体质量性状的多重比较, MR28位点277/285基因型体质量均值极显著高于277/289和285/285基因型(P<0.01), 显著高于285/289、273/289、270/273和285/293基因型(P<0.05); MR32位点266/266和266/270基因型体质量均值显著高于270/270基因型(P<0.05); MR34位点210/214基因型体质量均值显著高于210/210和214/214基因型(P<0.05); MR45位点174/190基因型体质量均值显著高于182/182和182/190基因型(P<0.05)。研究结果为罗氏沼虾分子标记辅助育种奠定了基础。     

两个镜鲤半同胞家系的遗传多样性及经济性状分析

在两个镜鲤半同胞家系中,各随机选取47尾作为实验鱼,测量体重、体长、全长等数量性状,利用24个微卫星分子标记对其进行遗传检测,共检测到57个等位基因,每个基因座的等位基因数为1－6个不等,平均等位基因3.21个,片段长度在134－371bp之间,有效等位基因数Ne为1.00－2.89, 平均观察杂合度Ho为0.00－0.83,平均期望杂合度He为0.00－0.66,平均多态信息含量PIC为0.00－0.58。结果表明：2个家系的遗传多样性处于中度水平,但连锁不平衡分析表明这两个家系在较大的选择压力下,已严重偏离Hardy-Wenberg平衡。利用SPSS程序下的GLM过程对24个微卫星位点与主要经济性状的相关性进行分析,结果发现：HLJ519,HLJ848、HLJ855、HLJE8 4个微卫星位点对镜鲤体重显著影响(p<0.05),其中,位点HLJ519,HLJ848、HLJ855还对体长和全长存在显著影响(p<0.05)。对这些位点基因型所对应的表型均值进行了多重比较,找到了一些对主要经济性状有利的基因型。     

不同年龄段大连群体菲律宾蛤仔EST-SSR多样性

为查明年龄结构对菲律宾蛤仔同一群体内遗传多样的影响,采用14个SSR分子标记对大连石河不同年龄段的野生蛤仔进行了检测。结果表明:不同年龄段(1龄-Age1、2龄-Age2、3龄-Age3)蛤仔均维持着较高的遗传多样性。根据POPGENE 1.31和SPSS16.0统计分析显示,位点Rp-11、Rp-12、Rp-19对3个年龄段蛤仔的等位基因数差异极显著(P<0.01);位点Rp-20、Rp-24、Rp-27、Rp-30对其差异显著(P<0.05);剩余7个位点表现为差异不显著(P>0.05)。在平均水平上,每位点等位基因数目Na为4.3095,有效等位基因数目Ne为2.3729,多态位点百分数P(%)为14。观察杂合度和期望杂合度都比较高,观察杂合度平均为Ho=0.2335,期望杂合度平均为He=0.5140。而且,Ne和He随年龄的变化表现出Age2>Age3>Age1的趋势。各年龄段蛤仔——Age1、Age2、Age3的平均观察杂合度(Ho)和平均期望杂合度(He)分别为0.2357、0.2546、0.2159和0.4951、0.5286、0.5184。Age2的遗传多样性指数高于Age1及Age3,遗传分化相对较低。其中,Age1与Age3蛤仔遗传距离最小,D为0.0195,即变异很小;而Age1与Age2遗传距离较大,D为0.0437,变化范围不大(0.0195—0.0437)。从遗传一致度的数值上看了3个年龄段蛤仔的遗传相似程度很大,平均为0.9655。Age1与Age3遗传相似程度高达0.9807,而Age1与Age2相似程度较小为0.9572。说明不同年龄段蛤仔相似程度非常高。根据不同年龄段蛤仔的遗传距离,采用UPGMA平均聚类方法对其进行聚类可知,Age3与Age1蛤仔间遗传距离较小,与Age2蛤仔差异较大。通过对等位基因频率进行卡方检验发现,随着年龄结构的变化,部分基因基因频率减小;同时随着年龄的增长,有部分等位基因得到了纯化。大连群体蛤仔总的遗传分化较低,其遗传分化指数Fst为0.0248(Fst<0.05),遗传分化系数为0.02,说明总的遗传变异中有2%来自于不同年龄段的蛤仔之间。遗传距离和遗传一致度均值分别为0.035和0.9655,基因流(Nm=9.8238)相对流畅,进一步表明年龄结构对蛤仔种群内遗传分化的影响较小。     

红壳色文蛤F1代养殖群体多样性分析

运用形态学标记和分子标记对江苏省文蛤良种场红壳色文蛤F1代养殖群体(父母本为江苏野生红壳色群体)进行遗传多样性分析。用文蛤壳长、壳宽、壳高和体重4个可量性状进行形态学数据的聚类分析,显示可量性状变异系数在38.48%-82.95%。运用7个引物微卫星基因座的多态性进行了养殖群体F1代的分子标记评估,结果表明,7个微卫星基因位点的平均等位基因数3.857 1,平均有效等位基因数2.583 0,平均观察杂合度0.565 3,平均期望杂合度0.565 3,Shannon指数平均数1.050 1,多态信息含量平均数0.522 5。综合形态学数据和分子标记的研究结果表明,红壳色文蛤江苏养殖群体F1代的遗传多样性处于中度偏高水平,具有较高的遗传改良潜力。     

SSR分子标记在山茶属观赏资源遗传多样性研究中的应用

采用微卫星(SSR)分子标记的方法,利用20对SSR引物对33份山茶属资源(含种和品种)的DNA样品进行了PCR扩增,从中筛选出10对扩增效果较好的SSR引物,并对33份资源进行了遗传多样性分析。结果显示:10对引物在33个植物材料中共检测出123个等位基因,每个SSR位点的等位基因数为3~22个,SSR2引物检测到的等位基因数量最多,达22个,平均每对引物含有12.3个等位基因,平均多态性信息量为0.74,变化范围为0.06~0.96。利用遗传距离矩阵按UPGMA方法进行聚类,结果表明33份植物材料可分为9个分支,很好的区分了品种间的亲缘关系,可为杂交育种的组合选择提供理论基础。不同花型的山茶属植物在10个微卫星位点上共发现42个特异等位基因,可能与不同的花型性状有关。     

五个家系吉富罗非鱼的遗传多样性分析

吉富罗非鱼是用家系选育方法获得的优良品系.该方法可避免近亲交配引起的衰退.从罗非鱼的第二代遗传图谱上选取10个微卫星标记,对吉富罗非鱼5个家系共121尾鱼进行遗传多样性分析.结果表明:各位点的等位基因数为2～6个,平均等位基因数为4;有效等位基因数1.3920～3.6689,平均有效等位基因数为2.4733;平均杂合度观测值0.5984,平均杂合度期望值0.5642.5个家系的多态信息含量从小到大以依次为22家系、25家系、59家系、49家系、31家系,均为中度多态性.家系间基因分化系数(GST)为0.1676,各家系之间存在一定遗传分化.根据遗传距离采用UPGMA法对5个家系进行聚类,49家系和59家系遗传距离最小聚为一类, 它们与31家系遗传距离最远.对10个微卫星位点的基因型进行分析,结果发现在GM578位点,22家系呈其他几个家系没有的BB基因型,有望成为22家系的标记.     

菲律宾蛤仔EST-SSRs标记开发及不同地理群体遗传多样性

利用13对微卫星引物对大连、莆田、青岛3个地理群体蛤仔遗传多样性进行了检测。结果表明:13个基因座共检测到154个等位基因,每个座位检测到的等位基因数在2-7个之间,平均有效等位基因数为2.7657;3个群体平均观测杂合度分别为0.4387、0.4194、0.2383,平均期望杂合度分别为0.6488、0.6484、0.5526;群体间的遗传多样性差异不显著(P＞0.05)。NJ聚类结果显示大连和莆田群体的蛤仔亲缘关系较近,二者与青岛群体关系较远。3个群体均有不同程度的偏离Hardy-Weinberg平衡现象,表明各群体基因频率和基因型频率的稳定性相对较低。本研究所获得的微卫星标记的多态信息含量(PIC)>0.5,说明这些微卫星位点的多样性较高,可为下一步遗传图谱构建研究提供参考。     

Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum populations.     

Molecular cloning and characterization of Y-box gene (Rpybx) from Manila clam and its expression analysis in different strains under low-temperature stress

Manila clam, Ruditapes philippinarum, is an economically important marine bivalve species. Y-box proteins are members of the cold shock proteins family and highly conserved from bacteria to humans. In this study, a novel Y-box gene (Rpybx) was cloned from the Manila clam and gene expression profiling was performed on three shell color strains (white, zebra and white zebra) and two wild populations (Southern and Northern) of R. philippinarum. The complete ORF length of Rpybx is 1367 bp, encoding 253 amino acids residues. Based on the amino acid sequence analysis and phylogenetic analysis, the Rpybx gene was identified as a member of the invertebrate Y-box proteins family. Rpybx has a similar tertiary structure to human Y-box protein YB-1. The Rpybx mRNA levels were analyzed by qPCR under acute and gradually varied cold stress. Under acute low-temperature stress, the expression of Rpybx mRNA in gills and hepatopancreas was significantly increased in all selected strains and populations (P < 0.05). The Northern population showed the lowest relative expression level of Rpybx. The expressions of Rpybx were greatly upregulated in gills and hepatopancreas of different stains and populations at 5 or −2°C under gradually varied temperature stress (P < 0.05). The results shed light on the biological function of the Rpybx gene in defending against low-temperature challenge and further exploring the molecular mechanism of cold tolerance and resistance in R. philippinarum.     

Genetic Mapping and QTL Analysis of Growth-Related Traits in Pinctada fucata Using Restriction-Site Associated DNA Sequencing

The pearl oyster, Pinctada fucata (P. fucata), is one of the marine bivalves that is predominantly cultured for pearl production. To obtain more genetic information for breeding purposes, we constructed a high-density linkage map of P. fucata and identified quantitative trait loci (QTL) for growth-related traits. One F1 family, which included the two parents, 48 largest progeny and 50 smallest progeny, was sampled to construct a linkage map using restriction site-associated DNA sequencing (RAD-Seq). With low coverage data, 1956.53 million clean reads and 86,342 candidate RAD loci were generated. A total of 1373 segregating SNPs were used to construct a sex-average linkage map. This spanned 1091.81 centimorgans (cM), with 14 linkage groups and an average marker interval of 1.41 cM. The genetic linkage map coverage, Coa, was 97.24%. Thirty-nine QTL-peak loci, for seven growth-related traits, were identified using the single-marker analysis, nonparametric mapping Kruskal-Wallis (KW) test. Parameters included three for shell height, six for shell length, five for shell width, four for hinge length, 11 for total weight, eight for soft tissue weight and two for shell weight. The QTL peak loci for shell height, shell length and shell weight were all located in linkage group 6. The genotype frequencies of most QTL peak loci showed significant differences between the large subpopulation and the small subpopulation (P<0.05). These results highlight the effectiveness of RAD-Seq as a tool for generation of QTL-targeted and genome-wide marker data in the non-model animal, P. fucata, and its possible utility in marker-assisted selection (MAS).     

Genetic diversity and structure of Manila clam (Ruditapes philippinarum) populations from Liaodong peninsula revealed by SSR markers

Manila clam (Ruditapes philippinarum) is an important commercially marine bivalve, and its wild populations have been severely declining in the coast of China during the last decade. In this study, a set of 7 genomic simple sequence repeat (SSR), and 5 expressed sequences tag (EST)-derived SSR markers were analyzed on eight wild R. philippinarum populations to assess the genetic diversity and population differentiation. A total of 114 alleles were detected on 12 loci, and the number of alleles per locus in each population ranged from 2 to 11, and allelic richness per locus varied from 2.00 to 10.88 for each sample. The average of observed and expected heterozygosity ranged from 0.386 to 0.550, and from 0.533 to 0.707, respectively. Pairwise F_ST values indicated that all population pairs had significant genetic differentiation (overall F_ST = 0.242, P < 0.01). Cluster analysis using unweighted pair group method with the arithmetic mean (UPGMA) separated the eight populations into two groups. This study will shed light on the domestication and cultivation on population genetic diversity of R. philippinarum, and also provide the foundation for conservation of R. philippinarum germplasm resources in clam breeding practices.     

Gymnophallid digenean Parvatrema duboisi uses Manila clam as the first and second intermediate host

To identify the metacercariae of a gymnophallid trematode in the Manila clam Ruditapes philippinarum from the Ariake Sea, experimental infection and molecular analysis were conducted. Based on the morphology of adult worms obtained from experimentally infected mice, the parasite was identified as Parvatrema duboisi. Comparison of internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) sequences for metacercariae and sporocysts from Manila clams and adult worms collected from wild wigeon Anas penelope showed minor differences ranging from 0 to 0.8%. These data strongly suggest that in the Ariake Sea, the parasite has a lifecycle using the Manila clam as the first and second intermediate hosts and wigeon as the definitive host.     

Reproduction of an alien Ruditapes clam (Bivalvia: Veneridae) on recreational clam-gathering grounds in Tokyo Bay,Japan

The Manila clam Ruditapes philippinarum is an important fisheries resource and popular recreational native species in Japan. To counter the domestic shortage of Manila clams caused by depleted catches, similar clams were introduced into Japan from China and the Korean peninsula. The introduced clam is an unnamed morph closely related to R. philippinarum that originates from southern China. Its shell is morphologically different from that of the typical Manila clam and there are small but distinct genetic differences. There is evidence to suggest that the Chinese morph may hybridize with the Manila clam in the Ariake Sea, but not in Tokyo Bay. We carried out studies to determine the degree of overlap of the breeding seasons of both taxa by collecting samples of the Manila clam and the alien clam released onto recreational clam-gathering grounds in Tokyo Bay and their gonadal maturity was examined histologically. Spawning-stage individuals of the Manila clam appeared from April to August and those of the southern Chinese morph from July to August, indicating overlapping spawning periods and thus the potential for hybridization. We discussed the forming condition of the hybridized clam population in terms of reproduction and recruitment process of the clams.     

An environmentally induced tidal periodicity of microgrowth increment formation in subtidal populations of the clam Ruditapes philippinarum

The periodicity of increment formation in the shell of the Manila clam Ruditapes philippinarum was investigated in the subtidal zone of the Auray River estuary (South Brittany, France). Calcein markings were performed at different periods between May and October 2007 using in situ benthic chambers tented by scuba divers. This study shows that shell microgrowth increments were well-defined and deposited with a tidal periodicity in the subtidal zone, providing the calendar base for high-resolution ecological studies and environmental reconstruction from these R. philippinarum shells. Endogenous rhythmicity in shell microgrowth increment formation and oxygen consumption was previously documented in this species from intertidal flats. Our study suggests that, in the subtidal zone, Manila clams' rhythmic activity may be controlled by such an endogenous process, synchronized by tidal cues. As in other bivalves, R. philippinarum is an osmoconformer euryhaline bivalve. The tidal rhythmicity of shell microgrowth increments in subtidal specimens of this species could be explained by a behavioral adaptation of valve closure at low tide to protect the clam from low salinities and/or to synchronize with food availability. Finally, large inter-individual variability in tidally associated growth rates and asynchronous growth breaks were observed, and could be due to genetic variability between individuals, asynchronous partial spawning events or predation.     

Impact of the infaunal Manila clam, <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>, on sediment stability

The aim of this study was to examine the impact of bioturbation by the Manila clam, Ruditapes philippinarum, on sediment stability. A laboratory benthic annular flume system (AFS) was deployed to evaluate the relationship between sediment stability of a subtidal mudflat and density of the infaunal clam under the influence of different current velocities. There was a significant correlation between mean erosion rate and current velocities in all treatments with clams (p < 0.001). There was also a significant correlation between mean erosion rate and R. philippinarum density (p < 0.001), reflecting bioturbation-enhanced sediment erosion. The effects of clam density on sediment erodability were more marked at the lower current velocities. In the control, the critical erosion velocity (Ū_crit) was about 32 cm s⁻¹. With increasing R. philippinarum density, Ū_crit decreased down to the minimum value of about 20 cm s⁻¹ at a density of 206 clams m⁻². This study demonstrated that the burrowing activity of R. philippinarum reduces sediment stability, particularly at relatively low current velocities (25 cm s⁻¹) and at densities below those found in the clam cultivation areas within the Sacca di Goro lagoon.     

Short-term hypoxia modulates Rapana venosa (Muricidae) prey preference in Adriatic lagoons

We studied the effect of short-term hypoxia on predation by the non-native rapa whelk (Rapana venosa) on the three most common bivalve prey in northern Adriatic lagoons: the non-native ark shell (Scapharca inaequivalvis) and Manila clam (Tapes philippinarum), and the native cockle Cerastoderma glaucum. In mesocosm experiments, bivalves were offered to predator as single prey, and as multiple choice of prey. Bivalve prey showed behavioral responses when exposed to short-term hypoxia. Under normoxia, prey were consumed in almost equal numbers when offered separately, but when choice was offered to the predator R. venosa showed a marked preference for S. inaequivalvis. Short-term hypoxia increased the vulnerability of T. philippinarum, and, consequently, rapa whelks modified their predatory behavior and switched their preference to T. philippinarum. We hypothesize this to be a case of environmentally-mediated, non-reciprocal indirect interaction between the two non-native prey species, whose causes are based upon differences in specific traits of prey. Hypoxia may facilitate the coexistence of the two non-native bivalves via predator switching. The native cockle seems the net loser of the game.     

Development of EST-SSRs in scallop (Patinopecten yessoensis) from sequence database

Patinopecten yessoensis is a kind of cold water shellfish, and is an important economic species in China. In our study, we developed and evaluated simple sequence repeat (SSR) markers of P. yessoensis. Characteristics of 11 EST-SSR loci were investigated using 40 P. yessoensis individuals. The number of alleles per locus ranged from two to five. The observed heterozygosity ranged from 0.1026 to 0.9487, while the expected heterozygosity ranged from 0.1865 to 0.7433. All loci except P4 departed from Hardy–Weinberg equilibrium (P < 0.05) significantly. There was no LD observed between all pairs of EST-SSRs loci. These loci and markers will be useful for population genetics and systemic evolution of scallop.