鲫体长和体高相关的EST-SSR标记的筛选

鲫作为研究鱼类进化的独特材料,具有丰富的遗传多样性和多种不同倍性的亚种。采用一般线性回归模型（GLM）,对286个EST-SSR标记与鲫鱼自交F2代181尾个体的体长、体高及其比值进行单标记回归分析。Permutation检验（10 000次）结果显示：共有53个标记分别与体长、体高及体长/体高具有显著相关性,其中与体长显著相关的标记有24个（P〈0.05）,极显著相关的标记有6个（P〈0.01）;与体高显著相关的标记有30个（P〈0.05）,极显著相关的标记有9个（P〈0.01）;与体长/体高显著相关的标记有23个（P〈0.05）,极显著相关的标记有9个（P〈0.01）。本研究获得的53个与3种性状相关的标记为分子辅助育种（MAS）提供了理论基础。     

鲤的微卫星标记与体质量、体长、体高和吻长的相关分析

采用抑制消减杂交(suppression subtractive hybridization,SSH)技术研究皱纹盘鲍(Haliotis discus hannai Ino.)Vc缺乏诱导的差异表达基因.实验用皱纹盘鲍成鲍初始体质量为(74.32±0.43)g,初始壳长为(84.36±0.24)mm.配制了Vc缺乏(0.0 mg/kg)和高剂量添加(4 967.5 mg/kg)2个水平的人工饲料进行饲喂.经过170d的养殖后,分别构建肝胰腺和肾脏Vc缺乏消减cDNA文库.消减杂交效率分析显示,差异表达的基因分别被富集了大约25和25～210倍.从两个文库中随机挑选阳性克隆进行测序.在肝胰腺消减cDNA文库中获得63个片段,其中已知功能基因占54.0%,Gene ontology(GO)按照功能将其分为4类:代谢相关基因占15.9%,细胞代谢相关基因占30.2%.生物调节相关基因占4.8%,其他功能基因占3.2%.未知功能基因占46.0%.在肾脏消减cDNA文库中获得39个片段,其中已知功能基因占53.8%,GO将其分为3类:代谢相关基因占5.1%,细胞代谢相关基因占28.2%,生物调节相关基因占20.5%.未知功能基因占46.2%.其中,获得了包括细胞色素c氧化酶、葡萄糖-1脱氢酶、α-葡萄糖苷酶、β-1,3-D-葡聚糖酶、纤维素酶、藻酸盐裂解酶以及铁蛋白等在其他动物中被证明与Vc合成相关的基因,为进一步研究皱纹盘鲍Vc的合成和代谢奠定了基础.     

鲤改良品系与体质量、体长性状相关的TRAP标记筛选

应用TRAP标记对一个鲤(Cyprinus carpio)改良品系72个个体进行扩增,采用其中25个多态性较好引物组合用于该群体遗传多样性分析.运用卡方检验分析25对引物在体质量差异显著的2组鱼中频率差异显著的位点,初步筛选出与体质量和体长性状相关的TRAP标记.将初步筛选的TRAP标记用于随机群体132个个体进行位点性状间的关联分析.结果表明,25个TRAP分子标记共扩增出353个位点,其中多态性位点230个,平均多态性比率65％,平均多态性信息含量为0.28,Nei's遗传多样性指数平均值为0.219,Shannon信息指数平均值为0.329,表明该群体遗传多样性较为丰富.在初步筛选出的3个TRAP位点(ghlTrap04-140、4rTrap04-308、igf4Ga5-135)中,4rTrap04-308与体质量、体长呈极显著相关(P<0.01).本研究利用TRA这一新型分子标记对1个鲤改良群体进行遗传分析,并寻找出1个可能与鲤体质量,体长性状相关的功能基因位点,旨在为鲤体质量、体长性状的QTL定位和分子标记辅助育种奠定基础.     

镜鲤微卫星标记与体形性状的关联分析

应用荧光标记的通用引物法,利用100对微卫星标记对高背镜鲤(Cyprinus carpio L.)和低背镜鲤杂交所得F1190个个体的基因组DNA进行分型。利用SPSS软件对100个微卫星标记与鲤的体长、体高、体厚、头长、尾柄长及尾柄高等体形性状进行相关性分析,结果显示有22个微卫星座位与体长、体高、体厚等体形性状显著性相关(P<0.05)。其中CAFS110、CAFS159、CAFS624、CAFS907、CAFS2320、CAFS2321、HLJE319与体长,CAFS254、CAFS1119、HLJE310与体高,CAFS159、CAFS907、CAFS1119、CAFS1737、CAFS2137、CAFS2317与体厚,CAFS100、CAFS679、CAFS907、CAFS1119、HLJE319与头长,CAFS165、CAFS175、CAFS907、CAFS1127、CAFS1491、CAFS2322、HLJE284与尾柄长,CAFS907、CAFS1119、CAFS2137、CAFS2317、HLJE141与尾柄高的相关性达到显著水平(P<0.05)。这些标记将有助于镜鲤分子辅助育种的研究。     

建鲤、黄河鲤杂交后代微卫星标记多态性及其与体质量的关联性

为了解微卫星标记多样性与鲤杂交F1体质量的关联性,本研究以2个品种鲤(Cyprinus carpio)(建鲤、黄河鲤)双列杂交F1的4个群体为实验材料,应用25对微卫星引物进行遗传多样性分析,并检测不同基因型间体质量的差异。实验结果显示,不同组合单个位点的等位基因数为5.08～6.08,有效等位基因数为1.4～6.8,平均观测杂合度为0.62～0.77,平均期望观测杂合度为0.47～0.55,平均PIC为0.42～0.49。4个鲤群体中,黄河鲤纯繁群体(Hh)和黄建杂交群体(Hj)间的遗传相似性系数最大(0.979 5)。基于Nei’s遗传距离构建的UPGMA系统树显示,黄河鲤纯繁群体(Hh)和黄建杂交群体(Hj)亲缘关系最近。采用最小二乘法分析微卫星座位与体质量的关联性发现,HLJ13位点在纯繁组合不同基因型之间都检测到了显著性差异,Koi42位点只在建鲤纯繁组合内检测到基因型之间的差异;除此之外,只有Koi42AA型检测到的体质量值在杂交组合Hj和Jh中显著高于亲本建鲤和黄河鲤自繁组合。以上结果提示Koi42位点可能与杂种优势相关,这为下一步分子标记辅助选育奠定了基础。     

鲤头长、体厚、体高性状的QTL定位及遗传效应分析

用174个SSR、41个EST、345个SNP标记对以镜鲤良种后代为祖父母本所培育的杂交F2群体的68个个体进行基因型检测,运用JoinMap4.0软件包构建遗传连锁图。利用MapQTL5.0区间作图法(interval mapping,IM)和多QTL区间定位法(MQM mapping,MQM)进行QTL检测,通过置换实验(1000次重复)确定连锁群显著性水平阈值。在对体高、头长、体厚的区间定位中,共检测到6个与体高性状相关的QTLs区间,分布在LG1(SNP1339-SNP1490)、LG10(HLJE469-SNP1491)、LG12(SNP0922-HLJ1316)、LG13(SNP0937-HLJ328)、LG25(SNP1041-HLJ594)、LG35(SNP1425-SNP0389)等6个连锁群上,解释表型变异范围为20.0%～43.3%。其中,SNP1339-SNP1490区间LOD值最大为3.64,解释表型变异35.4%。6个与头长相关的QTLs,分布在LG1(SNP1339-SNP1490)、LG12(HLJ071-HLJ336)、LG13(SNP0937-HLJ328)、LG24(SN...     

镜鲤多家系体长和体质量QTL定位分析

为了克服单个家系数量性状位点(QTL)检测效率低、假阳性高等缺点,实验利用250对微卫星(SSR)标记对镜鲤8个全同胞家系的522尾子代进行基因组扫描,采用半同胞家系的分析策略对镜鲤体长(SL)和体质量(BW)性状进行QTL分析。结果显示,基于父系的QTL分析,共检测到4个QTL区间,其中,3个体长的QTL中,1个为95%基因组水平(genome-wide)显著性,位于LG24,可解释表型变异率为20.3%;其余2个均为95%染色体水平(chromosome-wide)显著性,分别位于LG6和LG30,可解释表型变异率分别为11.9%和11.6%。1个体质量的QTL达到99%基因组水平,位于LG24,可解释表型变异率达到38.3%,且与体长QTL区间重叠。基于母系的QTL分析,共检测到8个QTL区间,其中,5个体长的QTL中,1个为99%染色体水平,位于LG8,可解释表型变异率为16.6%;其余4个均为95%染色体水平,分别位于LG24、LG30、LG31和LG45,可解释表型变异率为9.6%~14.2%,且位于LG24和LG30上的QTL为父母本共有;3个体质量的QTL均与体长QTL区间重叠,1个为95%染色体水平,位于LG24,其余2个均为99%染色体水平,位于LG30和LG45,可解释表型变异率分别为14.1%和13.6%。进一步分析发现,位于LG24上的体长和体质量QTL区间重叠且均为父母本共有,体质量的3个QTL均与体长QTL存在重叠区域且呈现成簇分布的特点。本研究结果不仅可以为鲤分子育种提供更可靠的标记,而且为家系和品种间QTL变异规律的探索提供基础数据。     

源于鳞被相关基因的微卫星标记与鲤 4 种生长性状的相关性分析

选用来源于鲤基因组中的鳞被相关基因(ant、eda、edar、fgfr)及其上下游序列中的155个微卫星标记,对德国镜鲤(Cyprinus carpio L.)与黑龙江野鲤(Cyprinus carpio haematopterus)杂交的F2 116尾个体的遗传多样性进行检测,以卡方检验估计群体Hardy-Weinberg平衡.结果表明,36个微卫星标记表现为多态,各标记的等位基因数在2～4个浮动,共检测到86个等位基因,平均每个标记2.388 9个;平均有效等位基因数为2.209 4;平均观测杂合度为0.624 5;平均期望杂合度为0.529 2;平均多态信息含量为0.432 1,其中23个微卫星标记表现为中度多态(0.25≤PIC＜0.5),13个微卫星标记表现为高度多态(PIC≥0.5),说明这个群体属于中度多态性水平.Hardy-Weinberg平衡检验结果显示,61％标记显著偏离平衡,人工选择压力和自交对家系造成了严重的影响.SPSS 17.0分析发现分别有10(28％)、7(19％)、7(19％)和11个(31％)标记与体质量、体长、体高和体厚存在显著相关性,并发现11个优势基因型.源于鳞被相关基因的微卫星标记与生长性状连锁的比例较高,以上结果从分子水平上提示鳞被基因与所研究4种生长性状存在一定的相关性.     

镜鲤体质量和体长的QTL定位研究

以镜鲤(Cyprinus carpio L.)全同胞家系为材料,用940对微卫星(SSR)标记进行基因组扫描,采用半同胞家系的分析策略对体质量(BW)和体长(SL)进行QTL定位分析。QTL检测显示:基于父系的QTL分析,共检测到8个QTL区间。5个与体质量相关的QTL区间中,1个QTL为95%基因组水平(genome-wide)显著性,位于LG26连锁群,其他4个QTL均为95%染色体水平(chromosome-wide)显著性;3个与体长相关的QTL区间与体质量的QTL区间重叠,其中,1个QTL为99%基因组水平显著性,其余2个QTL均为95%染色体水平显著性。基于母系的QTL分析,共检测到11个QTL区间。6个与体质量相关的QTL区间中,1个QTL为99%基因组水平显著性,位于LG26,2个QTL为99%染色体水平显著性,其余3个QTL均为95%染色体水平显著性;5个与体长相关的QTL中有4个与体质量QTL区间重叠,其中,1个QTL为99%基因组水平显著性,1个QTL为99%染色体水平显著性,其余3个QTL均为95%显著性染色体水平。结果表明,在LG26上,存在着与体质量和体长都显著相关的QTL区间,且均达到基因组显著性水平,最小置信区间为3 cM。此QTL结果可以应用于鲤鱼分子标记辅助育种。     

3个鲤群体的微卫星标记与生长性状相关性分析

利用筛选出鲤（Cyprinus carpio）的15对多态性较高的微卫星引物,分析其与黄河鲤（C．carpio haematopterus Temminck et Schlegel）、建鲤（C．carpio var．jian）和黑龙江野鲤（C．carpio haematopterus）群体中90个个体的体质量、体长、体厚和体高性状的相关性．利用SAS的一般线性模制（GLM）对15个微卫星标记与3个鲤群体的生长性状进行显著性检验。结果表明,Mfw5与黄河鲤的体高相关（P〈0．05）;Hlj013、Cca09和Mfw7均与建鲤的体质量、体长和体高相关（P〈0．05）;Mfw2与建鲤的体质量和体厚相关（P〈0．05）;Mfw29与建鲤的体质量相关（P〈0．05）;Mfw6与黑龙江野鲤的体质量、体长、体厚和体高均相关（P〈0．05）;Mfw4与黑龙江野鲤的体质量、体长和体高相关（P〈0．05）;Mfw11与黑龙江野鲤的体厚和体高相关（P〈0．05）。对同一性状不同基因型间进行多重比较,找到3个群体中与生长性状相关的基因型。     

鲤与生长性状相关的EST-SSRs标记筛选

以大头鲤(Cyprinus pellegrini Tchang)(母本)与荷包红鲤(Cyprinus carpio wuyuanensis)抗寒品系(父本)杂交产生的F1作为亲本,以其自交F2作为分离群体,结合"拟测交"策略,用EST-SSRs标记对其中92个个体进行基因型检测.利用Windows Map Manager 2.0的标记回归法对符合拟测交分离的70个EST-SSRs标记进行单标记回归分析,检测出8个与鲤体长、体高、体厚性状相关的标记.对同一标记不同基因型个体间生长性状进行显著性比较,通过t检验,找到与生长性状相关的基因型.将上述8个EST序列与GenBank数据库进行BLAST比对,有3条EST序列与蛋白库中已知功能的序列高度同源.其中HLJE225与编码斑马鱼(J9enio rerio)冷诱导基因RNA结合蛋白的基因同源性水平高达97%,HLJE222与编码斑马鱼DAZ相关蛋白1基因同源性水平也高达97%,HLJE599与编码斑点叉尾鲴(Ictalurus punctatus)核糖体蛋白L6基因的同源水平为87%.本研究鉴定的与鲤生长性状相关的功能基因及有利用价值的基因型,为鲤重要生长性状的QTL(数量性状基闪座)定位和分子标记辅助育种提供了一定的依据.[中国水产科学,2009,16(1):15-22]     

Genetic characterization of wild Dutch common carp (Cyprinus carpio L.)

Six male carp, caught in the water system surrounding the Anna Paulowna (AP) Polder in The Netherlands, were characterized using allozyme and microsatellite markers. At the sMDH‐A1,2* loci an allele was found, which has previously only been found in wild River Rhine and wild Vietnamese common carp. Microsatellite allele frequencies showed that these AP carp were significantly different from a group of carp originating from several different domesticated strains. Based on both allozyme and microsatellite data, the AP carp probably originated from a wild or feral self‐sustaining population.     

Developmental quantitative genetic analysis of body weight and morphological traits in red common carp,Cyprinus carpio L.

Developmental genetic analysis was conducted for body weight, total length, standard length and body height in red common carp by a mixed genetic model with additive–dominance effects, based on complete diallel crosses made by Xingguo red common carp (Cyprinus carpio var. singuonensis), purse red common carp (Cyprinus carpio var. wuyuanensis) and Oujiang color common carp (Cyprinus carpio var. color). Unconditional genetic analysis revealed that the traits of body weight, total length and standard length were mainly controlled by dominance effects before June but by additive effects after June, which suggests a high potential for heterosis during the early developmental period and an opportunity for selection during the late developmental periods for common carp. Through conditional genetic analysis, lots of new expression of genes of additive effects for body weight, total length and standard length existed during April to August and significant new expression of genes from dominance effects for these three traits at the other period. New expression from additive and dominance effect genes for body height occurred intermittently during the ontogeny. The conditional genetic procedure is a useful tool to understand the expression of genes controlling developmental quantitative traits at a specific developmental period (t − 1 → t) during ontogeny. It is also important to determine the appropriate developmental period (t − 1 → t) for trait measurement in developmental quantitative genetic analysis in fish.     

Immunomodulatory effect of cimetidine in common carp (Cyprinus carpio L.)

The main indication of cimetidine is being H₂-receptor antagonist, but studies suggest that cimetidine may also act as a non-specific stimulant of cell-mediated immunity and immunomodulator. In order to determine the immunomodulatory effect of dietary intake of cimetidine in the common carp (100 ± 10 g), subjects were fed diets containing 0 (control), 50, 100 and 200 mg cimetidine kg^?1 of dry diet for a period of 6 weeks. TLC and NBT assays were significantly (P < 0.05) stimulated in cimetidine-supplemented groups displaying the highest value in 200 mg kg^?1 group. A decrease (P < 0.05) in cortisol and ACH50 value was recorded in fish treated with cimetidine. Serum protein, albumin and serum globulin levels were not significantly changed. The findings of the present investigation suggest that the incorporation of cimetidine in the diet of common carp enhances the non-specific immunity.     

Artificial spawning of carp, Cyprinus carpio (L.)

The effect of reproduction was investigated on females of Hungarian strain W, French strain F, and their cross‐breed 1X whose ovulation was stimulated with carp pituitary (0.3 mg kg^?1and, after 12 h, 2.7 mg kg^?1) or Ovopel (one‐fifth of a pellet per kg and, after 12 h, one pellet per kg). It was found that in the case of Ovopel treatment, the percentage of spawning females of strain F and the cross‐breed 1X was higher than in the hypophysed fish compared. The applied ovulation stimulators did not significantly affect the weight of obtained eggs, whereas the significant (P ≤ 0.01) effect was recorded with respect to the quality of eggs after 12‐, 24‐, 36‐ and 48‐h of incubation. After Ovopel stimulation, the quality of eggs was better. The origin of the females had no statistically significant effect on the weight of eggs although the yield of eggs from fish of strain W was much smaller than that from females of strain F and the 1X cross‐breed. The interaction between the ovulation stimulator and the provenance of the females was significant (P ≤ 0.05) for the percentage of live embryos after 48‐h of incubation of eggs. Eggs of the best quality (and highest weight) were obtained from fish of strain F and cross‐breed 1X treated with Ovopel. In females of strain F that spawned within 6 and 10 h after the second Ovopel injection, the effect of the ovulation time on the weight of eggs was non‐significant. It was significant with respect to the percentage of egg fertilization and of live embryos after 36‐h of incubation (P ≤ 0.01 and P ≤ 0.05 respectively). The better quality of eggs (and their higher weight) was recorded when this time was shorter.     

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpio L.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late‐shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late‐shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M‐C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M‐C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late‐shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.     

Top-crossing with evaluation of slaughtering value in common carp (Cyprinus carpio L.) offspring

A three-year test of growth performance andoffspring survival from top-crossing of commoncarp hybrids in low altitude (350 m above thesea level, middle European climate) and in highaltitude (750 m above the sea level, middleEuropean climate) was terminated by assessmentof slaughtering value of edible parts in therespective strains. A recently establishedHungarian synthetic mirror carp strain (HSM)was chosen for testing as maternal strain. TheHSM, as well as wild Amur carp (AC), Ropshacarp (ROP) and Tata carp (TAT) were used aspaternal strains.The evidential strain*altitude interactioneffect was demonstrated as significant forweight of fish (0.0005), fillet without skin(0.0001), index of highbackedness (0.005),index of head length (0.0404),Fulton's coefficient (0.0497),index of widebackedness (0.0315) andgonadosomatic index (0.0082). The interactioneffect was demonstrated as insignificantbetween altitude*sex and betweenstrain*sex.The comparative test of analysis of variance(ANOVA) revealed significantly (P < 0.05)higher weight of fish after killing on lowaltitude (1611.6 g) than on high altitude(1090.3 g), but processed body yield andpercentage of fillet without skin was notsignificantly different (64.0 and 34.2 for lowaltitude, 64.2 and 33.4 for high altitude). Thepercentage of processed body weight andpercentage of fillet without skin in analysisboth altitudes were significantly better infemales (65.5 and 34.6%) than in males (62.5and 33.00%), respectively. The highestsignificant differences in weight of fish afterkilling were found between HSM (890.8 g) andHSM × AC (1283.3 g) in high altitude. In lowaltitude, it was between HSM (1527.2 g) andHSM × AC (1706.9 g) and HSM × ROP (1693.0 g).     

微卫星分子标记辅助镜鲤家系构建

用微卫星标记辅助进行镜鲤(Cyprinus carpio L.)家系的建立及选育,通过对亲本遗传结构及遗传差异的分析,预测产生优良家系的最佳配组。用28个微卫星分子标记评估松浦镜鲤亲本群体的遗传潜力,结果显示,群体的多态信息含量(PIC)达到0.5627,处于高度的遗传多样性水平,具备进一步繁育筛选优良群体的潜质。用x2检验和遗传偏离指数(d)估计Hardy-Weinberg平衡,结果表明群体处于平衡状态,但在16个位点表现出杂合子缺失,这可能跟长期的人工选择有关。依据个体间的遗传距离,连续2年共建立1对1亲本的繁育家系70个,用网箱分别养殖,2月龄家系体长均值为7.24～10.60cm,体质量均值为14.02～40.63g,方差分析显示家系间体长、体质量差异均极显著;对其中4个家系1龄鱼种生长情况及遗传结构分析也表明家系间的遗传分化较大,近交系数(Fst)达到0.1537,家系的多态信息含量在0.3056～0.4077之间,保持中度多态水平。由子代家系的生长和遗传结构两方面的实验结果证实了微卫星在辅助家系建立尤其是亲本选配方面具有较好的预测性,所获得的家系差异较大,具备进一步选育优良家系的潜力,从而证实了用此方法对现有镜鲤种质进行遗传改良是可行的。     

利用SSR﹑EST-SSR、SNP标记对鲤食物转化率、体厚、体质量3种性状的分析

利用Windows Map Manager2.0的标记回归法对鲤(Cyprinus carpio L.)F2群体进行单标记定位分析.用91个SSR标记、33个EST标记、364个SNP标记对鲤进行全基因组扫描,结果得到与食物转化率、体厚、体质量3个性状显著相关(P＜0.05)的标记,分别为27、35、27个,其中与食物转化率相关的标记SNP0041、SNP0044,其相关性达到极显著水平(P＜0.001),贡献率分别达到15%、16%.这些标记可作为分子标记辅助育种的参考标记.将得到的与食物转化率显著相关的EST座位HLJEl4、HLJE253和与体质量显著相关的HLJE253座位在NCBI上进行BLAST比对,结果显示HLJE253与斑马鱼上编码ORF2结构蛋白的基因相关,相关程度达61%.将得到的与3种性状相关的SNP标记在NCBI上进行序列查找,通过Blast比对找到相关的基因,并对可能的功能作了注释.