半滑舌鳎生长性状的微卫星标记筛选

结合分离群体标记关联分析法与随机选择群体标记关联分析法,利用69个微卫星位点,检测半滑舌鳎(Cynoglossus semilaevis)半同胞家系的极端大群体和极端小群体,筛选出12个与全长、体高、体重显著或极显著相关的微卫星位点。验证群体的平均等位基因数为5.167个,群体平均有效等位基因数为3.319,平均观测杂合度为0.516,期望杂合度0.670,多态信息含量0.621。分析结果显示:在雌性群体中,微卫星位点csou4、cyse123、ghrh-ssr、cyse72、hncyse160与全长显著相关,ghrh-ssr与体高显著相关,csou4、ghrh-ssr、cyse72、hncyse160与体重相关;在雄性群体中,微卫星位点hncyse129、hncyse160、sox9-1与全长相关,csou34、cyse22、cyse123、hncse67、sox9-1与体高显著相关,未发现与体重相关位点。     

半滑舌鳎微卫星标记遗传连锁图谱的构建

利用全基因组测序方法筛选出微卫星标记,以渤海近海野生个体和人工养殖的半滑舌鳎(Cynoglossus semi-laevis)为亲本交配产生的F1全同胞家系为作图群体,构建了半滑舌鳎雌、雄微卫星标记遗传连锁图谱。用320对引物对父母本和92个F1个体进行遗传分析,共得到288个分离标记,其中包含112个偏分离标记(P<0.05)。其中雌性框架图包含242个标记,分布在21个连锁群上,总长度1 311.9 cM,标记间平均距离为4.9 cM,图谱覆盖率为83.3%;雄性框架图定位标记218个,21个连锁群,总长度1 316.2 cM,标记间平均距离为5.5 cM,覆盖率为82%。半滑舌鳎遗传连锁图谱的构建为半滑舌鳎重要经济性状QTL定位、分子标记辅助育种和性别控制奠定了重要基础。     

半滑舌鳎抗哈维氏弧菌病相关微卫星标记筛选及QTL定位

为了筛选出与抗哈维氏弧菌病相关的微卫星标记并进行QTL定位,以2014年感染实验中存活率为52.22%的半滑舌鳎(Cynoglossus semilaevis)家系F1412(Family+年份+家系号)为材料,采用混合分离子分析法(bulked segregant analysis,BSA)对169个微卫星标记(SSR)进行筛选,得到1个可能与哈维氏弧菌病相关的微卫星标记scaffold479_23523。用scaffold479_23523所在的连锁群LG18上的37个SSR对94尾抗病、感病个体进行基因分型,得到3个图谱:整合图谱(LG18)、雌性图谱(LG18F)和雄性图谱(LG18M),并用两种不同的模式进行单标记分析和QTL定位。模式一得到3个显著性相关标记(P0.05)和1个极显著性相关标记(P0.01),图谱LG18F定位出一个区间qE-F1;模式二得到4个显著相关标记(P0.05)和1个极显著相关标记(P0.01),图谱LG18M定位出两个区间qE-M1、qE-M2。在全基因组序列的分布范围上,区间qE-F1包含了qE-M1及qE-M2,因此qE-F1更可能与抗病性状相关。本研究开发出了抗哈维氏弧菌病性状相关微卫星标记及QTL区间,为抗病新品种的培育奠定了基础。     

半滑舌鳎养殖群体和减数分裂雌核发育群体的微卫星标记遗传多样性分析

利用24对微卫星分子标记对半滑舌鳎Cynoglossus semilaevis的养殖群体和减数分裂雌核发育群体进行遗传多样性分析。结果表明,两个半滑舌鳎群体平均等位基因数分别为7.0和4.8,平均有效等位基因数分别为3.935和2.411,平均观测杂合度(HO)分别为0.713 5和0.586 5,养殖群体的遗传多样性明显高于减数分裂雌核发育群体。有1个位点在养殖群体中偏离哈代-温伯格平衡,13个位点在减数分裂雌核发育群体中偏离哈代-温伯格平衡。群体间基因分化系数(GST)为0.093 6,遗传距离为0.420 0,表明两群体间遗传分化显著。     

半滑舌鳎生长相关基因的微卫星及其在种群遗传结构分析中的应用

利用在生长激素(GH)、生长激素释放激素(GHRH)和垂体腺苷酸环化酶激活多肽(PACAP)基因中发现的7个微卫星位点,分析了半滑舌鳎两个野生群体(渤海群体和黄海群体)和1个养殖群体间以及各群体内雌雄个体间的遗传多态性差异。结果表明,7个位点中有4个位点表现出多态性,在3个群体中的等位基因数的分布范围为2～37,平均为9.5;有效等位基因数分布范围为2～28.9,平均为8.4。各位点的平均观测杂合度、平均期望杂合度和平均多态信息含量分布范围分别为0.5145～0.7738、0.5690～0.8671和0.4829～0.8314。群体间的成对FST值及个体分配分析的结果表明,半滑舌鳎野生群体和养殖群体之间存在显著性遗传差异,而在两个野生群体之间差异不显著。此外,等位基因分布和双倍体基因型分布的差异性检测结果表明,这4个多态性位点在3个群体的雌、雄性别间均不存在显著性差异。     

半滑舌鳎精子冷冻保存

半滑舌鳎精子冷冻保存对于人工繁殖育苗、杂交育种、雌核发育及其性别控制研究具有重要的意义,为此,本文对半滑舌鳎精子冷冻保存方法进行了研究。分别利用2．8mol／L的二甲基亚砜（DMSO）、甘油（Gly）和1,2-丙二醇（PG）冷冻保存该鱼精子。结果显示,DMSO冷冻保存精子的活力较高。利用MPRS＋2．8mol／L DMSO以1：0．5、1：1、1：1．5和1：2的比例稀释并冷冻精子,1：1比例在冻前能够抑制精子的运动,冻后活力可达82．50±3．54％,显著高于其他稀释比例（P〈0．05）。分别利用冷冻保存液A（MPRS＋2．8mol／L DMSO）和B（TS-2＋2．8mol／LDMSO）稀释平衡精子,精子在A中的冻前快速运动时间、寿命分别为37．75±6．45S和145．00±78．98S,与鲜精无显著差异（P〉0．05）。利用以上两种冷冻稀释液冷冻保存精子,精子在A液中的冻后活力和寿命分别可达53．50±6．69％和98．00±13．51s,冷冻效果优于B液（P〈0．05）。冷冻后精子的受精率和孵化率分别为55．00±5．00％和35．00±13．23％,受精率与鲜精无显著性差异（P〉0．05）,因此认为MPRS＋2．8mol／L DMSO可用于半滑舌鳎精子的冷冻保存。     

半滑舌鳎土池养殖试验

2006年6月,从山东引进半滑舌鳎鱼苗950尾于面积0.2 hm2的单养池塘进行试养.经过8个月的养殖,至2007年2月,共捕获半滑舌鳎810尾,起捕率85%,最大舌鳎鱼体长达到36 cm,体质量405 g,平均体长30.5 cm,体质量340 g.试验过程中发现在33 ℃高温和10 ℃的低温季节,半滑舌鳎均可生长,月平均增长率甚至高达28%,在此温度范围内,半滑舌鳎的生长与温度的相关关系不显著;但对温度快速下降的耐受性较差,易钻泥导致窒息死亡.     

半滑舌鳎营养需求研究进展

概述了国内外关于半滑舌鳎对营养物质需求的研究进展,以期对半滑舌鳎营养学研究的完善起到一定的引导作用,为开发适合半滑舌鳎养殖的配合饲料提供理论依据。     

半滑舌鳎微卫星标记的开发及其在F1家系中分离方式分析

利用富集文库-菌落原位杂交的方法筛选半滑舌鳎( Cynoglossus semilaevis)微卫星标记,构建富含(AC)和(AG)序列重复的半滑舌鳎微卫星富集文库,随机从文库中挑选1060个克隆,筛选得到883个(83.3％)阳性信号,菌落PCR检测742个阳性克隆片段的长度在500～1 200 bp范围内,随机挑选其中50个克隆进行测序,共设计引物33对,进行退火温度优化、多态性检测后,共得到20个多态的半滑舌鳎微卫星标记.对其进行分离方式分析,其中有17个位点符合孟德尔分离定律,共表现5种分离方式,可用于半滑舌鳎遗传连锁图谱的构建;另外有3个位点偏离孟德尔分离定律(P＜0.05).结果同时证明富集文库-菌落原位杂交是半滑舌鳎微卫星标记大量筛选和高密度遗传连锁图谱构建的一种行之有效的方法.     

半滑舌鳎染色体核型分析

The tonguefish Cynoglossus semilaevis (Giinther) is a rare fish species in Chinese offing, inhabiting in the warm water bottom. The metaphase chromosome preparation of the fries has been got from their fins by hot air drying methods, while the metaphase chromosomes of one year old young fish which has physiologically sex differentiation has teen got from their renal tissues by the method of PHA and colchicine injection. The karyotypes were examined. The result shows that there are 42 acrocentric chromosomes in diploid and their karyotype formula is 2n = 42t, and there existed heterotypic sex chromosome which belongs to ZW/ZZ type. The sex ratio in artificial bred stock is nearly 1 : 1.     

不同脂肪含量的配合饲料对半滑舌鳎苗种生长的影响

通过不同脂肪含量饲料的对比喂养试验,初步了解半滑舌鳎（Cynoglossus semilaevis）对饲料中脂肪含量的需求,使用脂肪含量分别为10.9％、14.2％和16.6％的配合饲料对半滑舌鳎进行了60d的喂养试验.结果表明,用脂肪含量为10.9％的配合饲料喂养的半滑舌鳎生长情况优于其他两种更高脂肪含量的配合饲料....     

半滑舌鳎胚胎发育过程中孵化腺细胞动态特征

应用组织切片法对半滑舌鳎早期胚胎发育过程中的孵化腺细胞的发生、分布及动态过程进行了观察。半滑舌鳎的孵化腺细胞最初由胚胎眼晶体期在头部腹面及其与卵黄囊连接处出现。孵化腺起源于外胚层,为单层细胞腺体。随着胚胎的发育,孵化腺进一步成熟,孵化腺细胞数量急剧增多,细胞体积有所增大,仍以单层细胞分布于胚体和卵黄囊外表面。胚胎孵出前,孵化腺细胞达500～800个,分布于胚胎头部两侧、腹面及其与卵黄囊连接处和卵黄囊的前腹面。孵化腺细胞大多呈椭圆形,短径6～8μm,长径7～11μm,H.E染色呈粉红色。仔鱼孵出约48 h后,孵化腺细胞从表皮中消失。     

Influence of larval co-feeding with live and inert diets on weaning the tongue sole Cynoglossus semilaevis

The tongue sole Cynoglossus semilaevis, an inshore fish in China, has showed great potential in aquaculture recently. However, poor survival was recorded during the period of weaning from live Artemia to artificial diets. In this paper, the influence of co‐feeding larvae with live and inert diet on weaning performance was described. The C. semilaevis larvae were reared at 21 ± 1 °C and fed four different feeding regimes from 6 days post‐hatching (dph): A, Artemia (10 individuals mL^?1); B, Artemia (5 individuals mL^?1); C, mixed diet (10 Artemia individuals mL^?1 and 12 mg L^?1 inert diet); and D, mixed diet (5 Artemia individuals mL^?1 and 12 mg L^?1 inert diet). Rotifers were also supplied in all cases during the first days of feeding. Mixed diets of commercial formulated feed and live prey (rotifers and Artemia) allowed larvae to complete metamorphosis, achieving similar specific growth rate (SGR) (18.5 ± 1.4% and 18.7 ± 1.6%) and survival (40 ± 7.6% and 48.5 ± 6.8%) compared with larvae fed on live feed alone (SGR of 18.3 ± 1.2%, 19.3 ± 1.9% and survival of 41.2 ± 11.3%, 38 ± 4.9%). However, in metamorphosed fish, when live feed was withdrawn on 31 dph, there was significant difference (P < 0.05) in survival and growth among treatments. Metamorphosed fish, previously fed mixture diets during larval stages, had similar survival (62.1 ± 7.6% and 62.8 ± 3.9% for regimes C and D, respectively) but higher than that obtained for fish that previously fed on live feed (49.3 ± 2% and 42.1 ± 3.9% for regimes A and B, respectively) after weaning (day 60). The SGR of weaned fish previously fed live feed was similar (3.1 ± 0.6% and 2.92 ± 0.6% for regimes A and B, respectively) but lower than that recorded for fish that was fed from day 6 to day 30 on the mixed diet (4.5 ± 1.1% and 4.9 ± 0.3% for regimes C and D, respectively). It is suggested that weaning of C. semilaevis from early development would appear to be feasible and larval co‐feeding improves growth and survival.     

半滑舌鳎的循环水养殖模式及经济效益分析

2009年3月至10月进行了半滑舌鳎(Cynoglossuss semilaevis Guanther)生产性养殖试验,期间对一套循环水养殖系统的水处理效果及半滑舌鳎养殖模式进行了深入的研究。养殖水体经系统处理后,养殖池内水温18～21℃,pH 7.0～8.0,DO≥6.6 mg/L,养殖池进水氨氮0.017～0.178 mg/L,亚硝酸氮0.012～0.064mg/L。文中详细阐述了放苗前养殖车间消毒、苗种选择与运输、生物滤池的培养、苗种投放、养殖过程中系统的日常维护、饵料投喂、光照控制、水质因子控制等内容,并对养殖半滑舌鳎8个月的经济效益进行了分析。以期为国内工厂化循环水养殖半滑舌鳎提供技术支持和经济效益分析方面的参考。     

土霉素对半滑舌鳎后肠好氧菌耐药性影响与药物残留分析

在（22±1）℃海水温度下,以初始体重（180～220）g的半滑舌鳎为研究对象,分别在配合饲料中以土霉素浓度为0mg·kg^-1和125mg·kg^-1拌料投喂,研究半滑舌鳎后肠中好氧菌耐药率的变化情况,同时使用高效液相色谱检测半滑舌鳎肌肉中土霉素的残留量。结果表明：在添加16mg·kg^-1土霉素的选择性培养基上,半滑舌鳎后肠好氧菌其耐药率从开始投喂时的（12．32±2．36）％增加到停药期间最大值（43．15±4．20）％;而在160mg·L^-1浓度土霉素的培养基上,其耐药率从开始的（5．6±2．94）％增加到（18．64±0．45）％。土霉素残留实验表明,在投喂药期间,土霉素在肌肉中的残留量处于（2．23±0．40）～（3．14±0．15）mg·kg^-1之间,但投药期结束后,残留量迅速下降,直到第6天降到检测限以下。本研究结果表明,在土霉素作用下,实验组耐药率出现较大的变化,但在停药16天时基本恢复到正常水平。     

Effects of Dietary Binders on Survival and Growth Performance of Postlarval Tongue Sole, Cynoglossus semilaevis (Günther)

A 30‐d feeding experiment was conducted in tanks to investigate the effects of four different binders (2% in microdiet) on the survival, growth performance, and specific activity of digestive enzymes in tongue sole, Cynoglossus semilaevis Günther, postlarvae (24 d after hatching, with initial average wet weight of 22.4 ± 6.65 mg [mean ± SD]). Five approximately isonitrogenous and isoenergetic microbound diets were formulated with carrageenan (Car), sodium carboxymethylcellulose (CMS), sodium alginate (SA), and gelatin (Gel) as binders, and a diet with no special binder as a control. A commercial diet (RQ Com., manufactured by Marubeni Nisshin Feed Co., Ltd., Chita, Japan) was used as another tested diet, and Artemia nauplii was used as live prey control. The results showed that the survival of the fish fed SA diet (31.3%) was significantly higher than that of fish fed CMS (21.8%), Car (10.8%), and control (21.8%) diets (P < 0.05), but significantly lower than that of the fish fed the live prey (54.5%) diet (P < 0.05). The results of growth followed the similar pattern as that of survival, although there were no significant differences in specific growth rate (SGR) among fish fed SA, CMS, Gel, the control, and RQ diets (7.2, 6.8, 7.0, 6.9, and 7.3% per day; P > 0.05). However, fish fed Car diet had a significantly lower SGR (5.9% per day) than fish fed other diets. Percentages of secreted amylase in fish fed artificial diets were not significantly different, but fish fed artificial diets had significantly higher (P < 0.05) secreted amylase than live prey group. Percentage of secreted trypsin in fish fed SA diet (54.9%) was relatively higher compared with other binders, and not significantly different from RQ Com. and live prey treatments. However, trypsin in fish fed Car diets (29.3%) was significantly lower than in other groups. Specific activities of alkaline phosphatase (AP) in the intestine and AP and leucine aminopeptidase N in brush border membranes in fish fed SA diet (264.6, 1882.8, and 187.2 mU/mg protein) were relatively higher compared with other binders. These results indicate that SA is one of suitable binders compared with carrageenan, CMS, and gelatin in microdiet of postlarval tongue sole.     

翘嘴鳜微卫星标记及其与主要经济性状的相关分析

利用筛选出的7对具有较高多态性的微卫星标记,检测106尾翘嘴鳜Siniperca chuatsi选育个体的基因组DNA,分析这些微卫星标记与翘嘴鳜体长、体质量和体高的相关性。结果获得67个等位基因,各位点的等位基因数为3~19,片段大小在147~530bp之间;期望杂合度0.5092~0.9207,均值为0.7574;各位点的多态信息含量在0.4639~0.9101之间,均值为0.7197,表明所选择的SSR标记识别力较高,适用于翘嘴鳜选育群体遗传分析和标记辅助育种研究。相关性分析结果表明,G4位点中含有的片段为219bp等位基因的基因型(229/219或219/219)个体的体质量、体长和体高的表型效应显著高于其他的基因型,G10位点中246/246基因型的体质量和体高表型效应显著高于其他基因型,可作为未来翘嘴鳜分子标记辅助育种的重要参考位点。     

Effects of Protein and Lipid Levels in Practical Diets on Growth and Body Composition of Tongue Sole,Cynoglossus semilaevis Gunther

A 9‐wk feeding experiment was conducted to estimate the optimal dietary protein and lipid levels for tongue sole, Cynoglossus semilaevis Gunther (initial average weight of 43.8 ± 0.18 g). Six practical test diets were formulated to contain three protein levels (45, 50, and 55%, respectively) at two lipid levels (12 and 16%, respectively) with P/E ratios ranging from 87.1 to 110.5 mg protein/kcal. Each diet was randomly fed to triplicate groups of 20 fish per tank (1000 L). The results showed that fish fed the diet with 55% protein and 12% lipid (P/E ratio of 110.5 mg protein/kcal) had the highest thermal‐unit growth coefficient (TGC), feed efficiency ratio, protein productive value, and energy retention. TGC was significantly increased with increasing dietary protein levels irrespective of dietary lipid levels (P < 0.05). However, fish fed the diet with 16% lipid showed significant lower growth than fish fed the diet with 12% lipid. These results suggest that the diet containing 55% protein and 12% lipid with P/E of 110.5 mg protein/kcal is optimal for tongue sole and the increase of dietary lipid level has no effective protein‐sparing effect.