鲤鱼（Cyprinuscarpio L．）体重和体长QTL的定位

利用265个AFLP标记、127个微卫星分子标记、37个EST-SSR标记和16个RAPD标记（共445个标记）对大头鲤／荷包红鲤抗寒品系的F2代雌核发育群体44个个体进行基因型检测，构建鲤鱼（CyprinuscarpioL.）遗传连锁图谱；利用软件WinQTLCart2.5采用复合区间作图法对体重和体长两个性状进行了QTL定位分析。结果共检测到与两个与体重性状相关的QTL，分别定位到LG24连锁群（qBWh-24-1）和LG20连锁群（qBWh-20-1）上，可解释的表型变异分别为7.98％和20.05％；检测到两个与体长性状相关的QTL，分别位于LG2连锁群（qFS-2-1）和LG20连锁群（qFS-20-1），可解释的表型变异分别为5.69％和12.69％，4个QTL的加性效应均为负值。     

鲤鱼(Cyprinus carpio L.)体重和体长QTL的定位

利用265个AFLP标记、127个微卫星分子标记、37个EST-SSR标记和16个RAPD标记(共445个标记)对大头鲤/荷包红鲤抗寒品系的F2代雌核发育群体44个个体进行基因型检测,构建鲤鱼(Cyprinus carpio L.)遗传连锁图谱;利用软件WinQTLCart2.5采用复合区间作图法对体重和体长两个性状进行了QTL定位分析。结果共检测到与两个与体重性状相关的QTL,分别定位到LG24连锁群(qBWh-24-1)和LG20连锁群(qBWh-20-1)上,可解释的表型变异分别为7.98%和20.05%;检测到两个与体长性状相关的QTL,分别位于LG2连锁群(qFS-2-1)和LG20连锁群(qFS-20-1),可解释的表型变异分别为5.69%和12.69%,4个QTL的加性效应均为负值。     

6个不同鲤群体的形态差异分析

对黄河鲤、荷包红鲤、高背荷包红鲤、兴国红鲤、建鲤和黑龙江野鲤等6个鲤群体的12个形态比例性状进行单因素方差分析、主成分分析和聚类分析。单因素方差分析结果表明,除眼后头长/头长外,各鲤群体间在其他比例性状上出现较明显的差异。主成分分析构建了3个主成分,其贡献率分别为35.316%、23.221%、10.146%,累计贡献率为68.683%,并明显可将6个鲤群体划分为2个簇,荷包红鲤和高背荷包红鲤明显与其他群体区分开。聚类分析结果与主成分分析一致。在体高/体长、头长/体长、体厚/体长和尾柄高/尾柄长等4个比例性状上,有些群体的差异系数大于1.28,说明这些群体在这4个指标上的差异可达到亚种水平。结果表明,6个鲤群体在形态上存在一定差异和分化,主要体现在体型和头部特征上。     

建鲤与黄河鲤的RAPD分子标记及其杂交优势的遗传分析

利用RAPD技术对建鲤、黄河鲤及其杂交子代进行了遗传分析,筛选的33个引物共扩增出155条带,其中多态性片段为105条,片段S18-1600、S472-300具有种的特异性,可作为鉴别建鲤与黄河鲤的分子遗传标记。建鲤、黄河鲤及其正、反交子代群体内的遗传相似系数分别为0.8240、0.7921、0.7920、0.8569,黄河鲤与正交F1具有较高的遗传变异水平,反交F1变异最小。正交F1、反交F1与亲本(建鲤、黄河鲤)的遗传距离分别为0.2233、0.2436、0.1749、0.2026,说明子代均继承了较多的建鲤的遗传物质。分析了子代与亲本的RAPD标记类型以及各类型的条带数。应用PIT标记系统测定了子代的杂种优势并探讨了优势产生的遗传机理。     

A Preliminary Genetic Linkage Map of Sinonovacula constricta(Lamarck, 1818) Based on Microsatellites Derived from RAD Sequencing

Sinonovacula constricta is one of the important economic aquaculture species in China. In this study, we constructed genetic linkage maps of S. constricta based on 300 microsatellite markers derived from RAD-seq using an F1 full-sib family. The female map contained 204 microsatellites assigned to 22 linkage groups, which covered 1529.5 cM with an average interval of 10.3 cM. The male consisted of 187 microsatellites in 19 linkage groups corresponding to the haploid chromosome number(n(28)19), which spanned 1429.3 cM with an average interval of 8.7 cM. The genome coverage was approximately 83.5% and 81.4%, respectively. An integrated map was constructed according to the common markers in parental linkage groups, which had a total length of 1683.8 cM with an average interval of 7.3 cM. The genome coverage of the integrated map was approximately 86.3%. The genetic linkage map would form the foundation for further studies on the quantitative trait loci(QTL), as well as accelerating the breeding process of this species.     

高淀粉水平下精氨酸对松浦镜鲤肠道形态与功能的影响

【目的】探讨高淀粉饲料中添加精氨酸对松浦镜鲤(CyprinuscarpioL.)肠道消化酶活性及肠道形态的影响。【方法】选用初始体质量(6.84±0.02)g的松浦镜鲤360尾,随机分为5组,每组4个重复,每个重复18尾鱼。实验饲料分为5组,分别在45%水平的高淀粉饲料中添加质量分数0%、0.6%、1.2%、1.8%、2.4%的精氨酸,实验周期为8周,进行肠道消化酶和形态的测定。【结果】前、中、后肠的淀粉酶活性,各组之间没有显著差异(P> 0.05);添加2.4%精氨酸组的前肠脂肪酶活性显著高于对照组(P <0.05),中肠和后肠的脂肪酶活性各组间没有显著差异(P>0.05);前、中、后肠蛋白酶活性随精氨酸添加量的增加而增加,添加1.8%和2.4%精氨酸组的前肠和中肠蛋白酶活性显著高于对照组(P<0.05),添加2.4%精氨酸组后肠的蛋白酶活性显著高于对照组(P<0.05)。高淀粉饲料中添加Arg显著影响了肠道皱襞高度,但对肠绒毛宽度和肌层厚度没有显著影响。2.4%精氨酸组前肠皱襞高度显著高于其他各组(P <0.05),1.8%精氨酸组中肠和后肠皱襞高度显著高于对照组(P <0.05)。1.2%、1.8%和2.4%组肠体指数显著高于对照组(P <0.05),2.4%精氨酸组肠长指数显著高于对照组(P <0.05)。各组前、中、后肠肠绒毛密度没有显著差异,且排列较整齐。【结论】高淀粉饲料中添加1.8%~2.4%精氨酸可以增加松浦镜鲤肠道消化酶活性,同时改善了松浦镜鲤的肠道形态结构。     

Preliminarily study on the maximum handling size,prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on gastropods

The present study preliminarily examined the differences in maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on four gastropods species( Bellamya aeruginosa, Radix auricularia, Parafossarulus sinensis and Alocinma longicornis) under laboratory conditions. In the maximum handling size trial, five fish from each age group(1-year-old and 2-year-old) and each genotype(transgenic and non-transgenic) of common carp were individually allowed to feed on B. aeruginosa with wide shell height range. The results showed that maximum handling size increased linearly with fish length, and there was no significant difference in maximum handling size between the two genotypes. In the size selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on three size groups of B. aeruginosa. The results show that the two genotypes of C. carpio favored the small-sized group over the large-sized group. In the species selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on thin-shelled B. aeruginosa and thick-shelled R. auricularia, and five pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on two gastropods species( P. sinensis and A. longicornis) with similar size and shell strength. The results showed that both genotypes preferred thin-shelled Radix auricularia rather than thick-shelled B. aeruginosa, but there were no significant difference in selectivity between the two genotypes when fed on P. sinensis and A. longicornis. The present study indicates that transgenic and non-transgenic C. carpio show similar selectivity of predation on the size-and species-limited gastropods. While this information may be useful for assessing the environmental risk of transgenic carp, it does not necessarily demonstrate that transgenic common carp might have lesser environmental impacts than non-transgenic carp.     

Existence of Microsatellites in Expressed Sequence Tags of Common Carp （Cyprinus carpio L. ） Available in GenBank dbEST Database

Common carp expressed sequence tags （ESTs） were analyzed for the existence of microsatellites, or simple sequence repeats （SSRs）. In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2-6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were independent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a vari- ety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies （98/179） of hits on zebrafish sequences.     

复合酸化剂对鲤生长及消化酶活性的影响

在鲤鱼(Cyprinus carpio)基础饲料中,分别添加不同梯度的复合酸化剂(质量分数分别为0、0.2%、0.3%),研究复合酸化剂对其生长和消化酶活性的影响。结果表明:1)添加复合酸化剂显著(P<0.05)提高鲤鱼的相对增重率、特定生长率、饵料转化效率、饲料蛋白质效率以及饲料蛋白质沉积率等生长性能指标;2)添加质量分数0.3%的复合酸化剂时,鲤鱼肝胰脏、前肠和中肠的蛋白酶活性显著提高(P<0.05),酸化剂组的淀粉酶和脂肪酶活性有升高的趋势,但与对照组差异不显著;3)鱼体的粗蛋白质、粗脂肪、粗灰分、水分、肥满度及内脏比等品质指标在各组间差异不显著。综上所述,鲤鱼饲料中添加以柠檬酸、乳酸、磷酸等为主的复合酸化剂,可显著提高鲤鱼肝胰脏、前肠和中肠的蛋白酶活性,促进鲤鱼生长。     

A genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis based on AFLP, SSR, and RAPD markers

The Chinese shrimp Penaeus (Fenneropaeneus) chinensis is an important species in marine fishery and aquaculture in China. A female Chinese shrimp Penaeus (Fenneropaeneus) chinensis was captured from west coast of the Korean peninsula and mated with a “Yellow Sea No. 1” male to produce the first filial generation (F₁) 100 F₂ full-sib progeny from brother-sister crosses between F₁ families was used for the mapping study. A genetic linkage map of the Chinese shrimp was constructed, based on 354 markers, including 300 amplified fragment length polymorphism (AFLP) markers, 42 microsatellite (SSR) markers, and 12 randomly amplified polymorphism (RAPD) markers. Forty-seven linkage groups (LGs) were identified. The total map length was 4 580.5 cM, with an average spacing of 11.3 cM, covering 75.8% of the estimated genome size. The construction of this genetic linkage map was part of a genetic breeding program. This linkage map will contribute to the discovery of genes and quantitative trait loci (QTLs) in Chinese shrimp.     

异育银鲫气单胞菌病原菌鉴定和药敏试验

从患病异育银鲫体内分离到5株可疑菌株,经API 20NE细菌生化鉴定,YCS07-03和YZ07-11株鉴定率为99.2%和99.3%。注射感染中5×108 CFU.mL-1剂量组表现出较强的致病性,12 d死亡率100%。菌株在营养肉汤培养液中呈均匀混浊状;在营养琼脂平皿中呈灰白色半透明菌落;在血琼脂培养基中呈光滑灰白色菌落,YZ07-11株出现透明的β溶血环;在TCBS培养基呈黄色菌落;在SS和麦康凯培养基中形成无色透明的扁平菌落。经负染电镜观察,菌株两端圆,呈直杆状,无芽孢,极生单鞭毛,菌株大小(短径×长径)分别为1.53μm×2.71μm和0.80μm×2.12μm。菌株经生化实验鉴定为温和气单胞菌(Aeromonas sobria)和嗜水气单胞菌(Aeromonas hydrophila)。试验结果表明:菌株对丁胺卡那霉素、头孢噻肟、庆大霉素、左氟沙星、复合磺胺、氟嗪酸、洛美沙星、复方新诺明、链霉素和新霉素均产生高度敏感;对万古霉素为中度敏感;氨苄青霉素、羧苄青霉素、强力霉素和青霉素产生耐药。     

COMPARATIVE STUDIES ON THE FEEDING CAPACITY OF SILVER CARP AND BIGHEAD CARP

Study of the feeding capacity of silver carp and bighead carp by means of experimental ecologyshowed that the filtering frequency of silver carp was slightly greater than that of bighead carp,but thatthe latter's suction volume was much greater than the former's so the filtering rate(filtering frequency mul-tiplied by the suction volume)of silver carp was smaller than that of bighead carp.The filtering efficien-cy of silver carp for phytoplankton was greater than that of bighead carp for them.The removal ratesof silver carp for phytoplankton were greater than those of bighead carp,but for zooplankton the formerwere smaller than the latter.For food particles about 70 μm both removal rates were almost equal.The feeding habits of the two species in natural waters is also discussed in the paper.     

A microsatellite genetic linkage map of black rockfish (Sebastes schlegeli)

Ovoviviparous black rockfish (Sebastes schlegeli) is an important marine fish species for aquaculture and fisheries in China. Genetic information of this species is scarce because of the lack of microsatellite markers. In this study, a large number of microsatellite markers of black rockfish were isolated by constructing microsatellite-enriched libraries. Female- and male-specific genetic linkage maps were constructed using 435 microsatellite markers genotyped in a full-sib family of the fish species. The female linkage map contained 140 microsatellite markers, in which 23 linkage groups had a total genetic length of 1334.1 cM and average inter-marker space of 13.3 cM. The male linkage map contained 156 microsatellite markers, in which 25 linkage groups had a total genetic length of 1359.6 cM and average inter-marker distance of 12.4 cM. The genome coverage of the female and male linkage maps was 68.6% and 69.3%, respectively. The female-to-male ratio of the recombination rate was approximately 1.07:1 in adjacent microsatellite markers. This paper presents the first genetic linkage map of microsatellites in black rockfish. The collection of polymorphic markers and sex-specific linkage maps of black rockfish could be useful for further investigations on parental assignment, population genetics, quantitative trait loci mapping, and marker-assisted selection in related breeding programs.     

An AFLP Genetic Linkage Map of Pacific Abalone （Haliotis discus hannai）

A genetic linkage map of Pacific abalone （Haliotis discus hannai） was constructed using AFLP markers based on a two-way pseudo-testcross strategy in a full-sib family. With 33 primer combinations, a total of 455 markers （225 from the female parent and 230 from the male parent） segregated in a 1 ： 1 ratio, corresponding to DNA polymorphism： heterozygous in one parent and null in the other. The female framework map consisted of 174 markers distributed in 18 linkage groups, equivalent to the H. discus hannai haploid chromosome number, and spanning a total length of 2031.4 cM, with an average interval of 13.0 cM between adjacent markers. The male framework map consisted of 195 markers mapped on 19 linkage groups, spanning a total length of 2273.4cM, with an average spacing of 12.9cM between adjacent markers. The estimated coverage for the framework linkage maps was 81.2% for the female and 82.1% for the male, on the basis of two estimates of genome length. Fifty-two markers （11.4%） remained unlinked. The level of segregation distortion observed in this cross was 20.4%. These linkage maps will serve as a starting point for linkage studies in the Pacific abalone with potential application for marker-assisted selection in breeding programs.     

Quantitative trait loci detection of <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> resistance in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese flounder (Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. olivaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis (BSA) and quantitative trait loci (QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333 (♀: F0768 ×♂: F0915) (Nomenclature rule: F+year+family number) were used to detect simple sequence repeats (SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers (Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group (LG)-1 exhibited a significant difference between DNA, pooled/bulked from the resistant and susceptible groups (P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of significance in LG1, where 17 and 18 SSR markers were identified, respectively. Each model found three resistance-related QTLs by composite interval mapping (CIM). These six QTLs, designated qE1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, qE-2 and qE-4, were located at the 66.7 cM region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese flounder in the future.     

Preliminary genetic linkage map of the abalone Haliotis diversicolor Reeve

Haliotis diversicolor Reeve is one of the most important mollusks cultured in South China. Preliminary genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) markers. A total of 2 596 AFLP markers were obtained from 28 primer combinations in two parents and 78 offsprings. Among them, 412 markers (15.9%) were polymorphic and segregated in the mapping family. Chi-square tests showed that 151 (84.4%) markers segregated according to the expected 1:1 Mendelian ratio (P<0.05) in the female parent, and 200 (85.8%) in the male parent. For the female map, 179 markers were used for linkage analysis and 90 markers were assigned to 17 linkage groups with an average interval length of 25.7 cm. For the male map, 233 markers were used and 94 were mapped into 18 linkage groups, with an average interval of 25.0 cm. The estimated genome length was 2 773.0 cm for the female and 2 817.1 cm for the male map. The observed length of the linkage map was 1 875.2 cm and 1 896.5 cm for the female and male maps, respectively. When doublets were considered, the map length increased to 2 152.8 cm for the female and 2 032.7 cm for the male map, corresponding to genome coverage of 77.6% and 72.2%, respectively.     

Location of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> resistance-associated trait loci in half-smooth tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis> at its microsatellite linkage map

A cultured female half-smooth tongue sole (Cynoglossus semilaevis) was crossed with a wild male, yielding the first filial generation of pseudo-testcrossing from which 200 fish were randomly selected to locate the Vibrio anguillarum resistance trait in half-smooth tongue sole at its microsatellite linkage map. In total, 129 microsatellites were arrayed into 18 linkage groups, ≥4 each. The map reconstructed was 852.85 cM in length with an average spacing of 7.68 cM, covering 72.07% of that expected (1 183.35 cM). The V. anguillarum resistance trait was a composite rather than a unit trait, which was tentatively partitioned into Survival time in Hours After V. anguillarum Infection (SHAVI) and Immunity of V. Anguillarum Infection (IVAI). Above a logarithm of the odds (LOD) threshold of 2.5, 18 loci relative to SHAVI and 3 relative to IVAI were identified. The 3 loci relative to IVAI explained 18.78%, 5.87% and 6.50% of the total phenotypic variation in immunity. The microsatellites bounding the 3 quantitative trait loci (QTLs) of IVAI may in future aid to the selection of V. anguillarum-immune half-smooth tongue sole varieties, and facilitate cloning the gene(s) controlling such immunity.     

Comparison of intestinal bacterial communities in grass carp, Ctenopharyngodon idellus, from two different habitats

The intestinal bacteria of vertebrates form a close relationship with their host.External and internal conditions of the host,including its habitat,affect the intestinal bacterial community.Similarly,the intestinal bacterial community can,in turn,influence the host,particularly with respect to disease resistance.We compared the intestinal bacterial communities of grass carp that were collected from farm-ponds or a lake.We conducted denaturing gradient gel electrophoresis of amplified 16S rRNA genes,from which 66 different operational taxonomic units were identified.Using both the unweighted pair-group method with arithmetic means clustering and principal component analysis ordination,we found that the intestinal bacterial communities from the two groups of pond fish were clustered together and inset into the clusters of wild fish,except for DF-7,and there was no significant correlation between genetic diversity of grass carp and their intestinal bacterial communities(Mantel one-tailed test,R=0.157,P=0.175).Cetobacterium appeared more frequently in the intestine of grass carp collected from pond.A more thorough understanding of the role played by intestinal microbiota on fish health would be of considerable benefit to the aquaculture industry.     

地衣芽孢杆菌在异育银鲫日粮中的应用

在异育银鲫基础日粮中分别添加质量分数0.01%、0.02%、0.03%、0.04%的地衣芽孢杆菌,测定了鱼体的生长及食糜植酸酶活性等。结果表明:与对照组相比,添加量0.01%、0.02%、0.03%组的增重率分别显著提高了12.46%、18.59%、18.84%;添加量0.02%、0.03%组的磷表观消化率分别提高37.63%、35.03%(P<0.05);饵料系数降低了0.52、0.57(P<0.05),其他组差异不显著。与对照组相比,0.03%组食糜植酸酶活性提高50.79%、0.04%组肝体比降低20.91%(P<0.05),其他组差异不显著。因此在异育银鲫日粮中添加地衣芽孢杆菌提高了机体酶活性,促进了鱼体生长。     

Genome-wide mining,characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats(SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class(44.08%), followed by mononucleotides(29.67%), trinucleotides(18.96%), tetranucleotides(5.66%), hexanucleotides(1.07%), and pentanucleotides(0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci(90.0%) were successfully amplified with specific products and 24(80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17(with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.