合浦珠母贝幼贝生长性状的遗传参数估计

以广东徐闻和海南三亚2个地理群体合浦珠母贝(Pinctada fucata)为亲本, 采用人工授精方法构建了33个全同胞家系。在162日龄时从每个家系随机抽取50个个体, 共1 650个, 测量其壳长、壳高、壳宽和体质量4个生长性状, 利用动物模型对4个性状进行遗传参数分析。结果表明, 合浦珠母贝4个生长性状的总平均值分别为(16.28 ± 4.46) mm、(14.85 ± 4.39) mm、(4.58 ± 1.52) mm、(0.66 ± 0.67) g。4个性状的遗传力分别为0.202 ± 0.020、0.203 ± 0.020、0.200 ± 0.021和0.204 ± 0.020, 均属中等遗传力, 因此可以用选择育种进行遗传改良。4个性状间表型相关系数和遗传相关系数的范围分别为0.667~0.698和0.685~0.959, 其中壳长、壳高和壳宽之间的遗传相关系数均低于0.7, 与表型相关一致, 而三者与体重间的遗传相关系数较高(0.868~0.959), 其中壳宽与体质量间的遗传相关系数最高(0.959)。本研究中合浦珠母贝的4个性状为中等遗传力, 并可通过对壳宽的选育来改良体质量性状。上述结果为进一步开展合浦珠母贝选择育种研究奠定了基础。

     

合浦珠母贝不同生长阶段的基因型与环境互作效应

为了研究合浦珠母贝(Pinctada fucata)不同生长阶段基因型与环境的互作效应,从2012年1月在海南构建的家系中选取生长快、中、慢3个典型家系(分别编号为F1、F2和F3),于海南陵水黎安、广东湛江覃斗和广西防城白龙3个不同海区进行养殖试验,每个海区每个家系的放养数量为800个,养殖1年。采用方差和加性主效应乘积交互作用(additive main effects and multiplicative interaction,AMMI)模型对各家系不同生长阶段壳长、壳高、壳宽和体重4个性状的基因型与环境互作效应进行分析。结果显示,不同海区间合浦珠母贝的生长差异显著(P0.05),其中海南黎安的生长较快,广西白龙较慢;家系间,F2家系的壳长、壳高、壳宽生长速度最快,F1家系体重生长速度最快。整个试验阶段总体上4个性状的基因型方差占变异总方差的比例为72.47%~88.55%,环境方差占9%~24.13%,基因型×环境互作方差占3.32%~4.04%。随着生长的进行,4个性状的基因型方差所占比例由最早阶段的92.32%~98.66%下降到最后阶段的32.45%~68.10%,环境方差由0.11%~1.27%增加到24.41%~62.26%,家系×环境互作方差由0.79%~3.9%增加到3.84%~7.51%。AMMI模型双标图分析表明,F1家系与覃斗、F2家系与黎安存在一定的互作,而F3家系的稳定性最好。上述结果显示,基因型与环境互作效应较小,家系对表型变异的贡献较大,表明家系的选择非常重要。该结果对合浦珠母贝优良品系的选育与推广具有重要指导意义。     

马氏珠母贝选育群体4个世代的遗传变异

采用9个多态性微卫星位点分析马氏珠母贝(Pinctada fucata)壳高选育品系(JCS)4个世代的遗传多样性与遗传结构的变化。结果表明, 随着选育的进行,  4个世代每个位点的平均有效等位基因数(N_e)从2.096下降至1.435, 平均等位基因丰度(R_s)从3.556下降至 2.556, 平均观测杂合度从0.492渐次下降至0.269, 遗传多样性呈明显的下降趋势。随着选育世代的增加, 从等位基因频率的变化来看, 部分呈上升趋势, 部分呈下降趋势, 部分基本保持稳定, 其中, 一些低频基因甚至会趋于消失。在所有等位基因中, 大部分的频率呈随机变化趋势, 并在不同的世代间上下波动; HHM20- 158 bp的频率呈有规律的变化, 由中高频逐步上升至高频, 呈富集趋势。结论认为, 经过4代的选育, 马氏珠母贝选育群体遗传多样性呈降低趋势, 遗传结构趋于稳定。本研究旨在为后续的马氏珠母贝选育策略提供理论指导。

     

Estimating the heritability for growth-related traits in the pearl oyster,Pinctada fucata martensii (Dunker)

Twelve paternal half-sib families (or 36 full-sib families) of the pearl oyster, Pinctada fucata martensii (Dunker), were produced according to the requirements of hierarchical genetic mating design. A total of 4320 individuals, aged 15 months, were measured for seven growth-related traits. Predicated upon the additive-dominance genetic analysis model, varying genetic variance components and then heritabilities of the growth-related traits of interest were estimated using analysis of variance. Results showed that seven growth-related traits had larger additive genetic variances (P<0.05); the dominance genetic variance of shell weight (SW) was smaller (P>0.05), the dominance genetic variances of other six traits were all larger (P<0.05). Narrow- and broad-sense heritabilities for the seven traits were, respectively, 0.64 ± 0.10 and 0.78 ± 0.12 for shell length, 0.49 ± 0.06 and 0.63 ± 0.09 for shell height, 0.38 ± 0.14 and 0.54 ± 0.16 for shell breadth, 0.41 ± 0.17 and 0.56 ± 0.11 for hinge length, 0.53 ± 0.11 and 0.68 ± 0.08 for body weight, 0.35 ± 0.07 and 0.55 ± 0.08 for tissue weight and 0.67 ± 0.10 and 0.75 ± 0.16 for SW. All heritability estimates were statistically significant (P<0.05). According to these results, the mass selection procedure is suggested for the breeding of P. martensii.     

合浦珠母贝4 种壳色选育系主要性状的比较分析

为评估合浦珠母贝(Pinctada fucata)白壳、黑壳、红壳和金壳色选育系F6代的育种性能,对养殖达18个月龄4种壳色合浦珠母贝的形态、质量、闭壳肌拉力和抗才女虫寄生病等12个性状指标进行了方差分析和多重比较。结果显示,4种壳色选育系间壳高、绞合线长、壳宽、体质量、壳质量、软体部质量、闭壳肌质量、闭壳肌拉力、右壳感染率和贝壳感染率均存在显著差异(P0.05)。综合相关分析表明,白壳色选育系可作为壳高和低才女虫感染率同时选育的最优育种群体,金壳色选育系可作为壳宽、体质量和闭壳肌拉力复合选育的最优育种群体。该研究结果为开展合浦珠母贝多性状复合选择育种工作提供了科学根据。     

Realized heritability and response to selection for shell height in the pearl oyster Pinctada fucata (Gould)

The common pearl oyster, Pinctada fucata (Gould), is the most important species that is cultured for production of marine pearls in China. Heavy mortality and the decline of pearl quality have resulted in a breeding programme being established in recent years. In this study, we conducted selective breeding for the second generation of pearl oyster P. fucata (JCS-2) by mass selection for shell height (SH) with a selection intensity of 1.614, and analysed the growth of the selected line (JCS-2) and the non-selected control line (JCC) during a 1-year grow-out period. The results show that the selected line grew faster than the control one in the SH and total weight (TW) ( P <0.05), and there were higher proportion of larger sized oysters. Coefficient of variation for SH of JCS-2 was smaller than that of JCC. The current genetic gains and realized heritability for JCS-2 averaged 16.03 ± 4.79% and 0.713 ± 0.208 at 3–15 months of age respectively. The findings indicated the selection response to faster growth for SH is markedly effective in the second generation, and there was a high correlated response of TW when selecting for SH.     

合浦珠母贝选育基础群体的遗传多样性评价

利用微卫星标记对2012年3月和6月分别构建的两批共17个合浦珠母贝(Pinctada fucata)选育基础群体进行了遗传多样性分析。6对微卫星引物在17个群体共680个个体中检测到29个等位基因,每个位点的等位基因数在2~7之间,平均有效等位基因数在2.06~3.07之间,平均观测杂合度在0.31~0.58之间,平均期望杂合度在0.46~0.65之间,平均多态信息含量在0.39~0.58之间,高度多态(PIC≥0.50)群体有11个(占64.7%)。Hardy-Weinberg平衡检验(卡方检验)的结果显示,在检测得到的102个数据结果中,有53个(占52.0%)极显著地偏离了Hardy-Weinberg遗传平衡(P0.01)。从遗传偏离指数(D)的结果可以看出,17个群体在不同的微卫星位点存在不同程度的杂合子缺失现象。本研究表明,两批群体的遗传多样性参数非常接近,每个群体的遗传多样性都较高,对群体之间的遗传距离和遗传分化格局有了初步的把握,为下一步交配设计构建下一代核心群体奠定了理论基础。     

合浦珠母贝对不同种类及浓度的单胞藻摄食与消化效果研究

从单胞藻粒径大小、表面结构、生化组成及饵料浓度等角度开展了合浦珠母贝（Pinctada fucata）对不同种类及浓度的单胞藻摄食与消化效果研究。投喂粒径大小相近的牟氏角毛藻（Chaetoceros muelleri）和湛江叉鞭金藻（Dicrateria zhanjiangensis）混合藻,粒径大小不同的亚心形扁藻（Platymonas subcordiformis）和海水小球藻（Marine chlorella）混合藻,用细胞计数法检测剩余混合藻中各单胞藻浓度。分别投喂以上4种不同浓度梯度的单胞藻,观察比较粪便中单胞藻消化状况。结果显示,合浦珠母贝对粒径大小相近的单胞藻没有表现出明显的选择性摄食（P>0.05）,对粒径较大的单胞藻摄食率明显高于粒径较小的单胞藻（P<0.05）;随着投喂单胞藻浓度的升高,合浦珠母贝对单胞藻的消化程度降低。 

     

合浦珠母贝DNA的抽提和RAPD反应体系的优化

分别从保存于-70℃和95%乙醇的合浦珠母贝组织提取总DNA。结果表明,乙醇保存标本用双蒸馏水预处理后所提的DNA与-70℃保存的标本所提的DNA质量均较好。用抽提的DNA为模板优化RAPD条件,得到适合于合浦珠母贝的RAPDPCR反应条件为:20μL反应体系中包括20ngDNA模板,1×PCRBuffer,0.1mmol·L-1dNTPs,2.5mmol·L-1MgCl2,0.2μmol·L-1引物,1UTaqDNA聚合酶。最佳退火温度40℃。PCR产物用非变性PAGE银染和琼脂糖EB染色检测所得到的主要带型相同,但PAGE能检测到更多的多态带。     

微胶囊饲料对马氏珠母贝育珠性能、生长基因和矿化基因表达的影响

本研究分析了自主研发的微胶囊饲料替代微藻对马氏珠母贝(Pinctada fucata martensii)育珠性能、生长相关基因和矿化相关基因表达的影响。共设置了3个实验组,其中,EG1组投喂亚心形扁藻(Platymonas subcordiformis),EG2组投喂微胶囊饲料+亚心形扁藻,EG3组投喂微胶囊饲料。养殖170 d后,比较马氏珠母贝的育珠性能、闭壳肌生长相关基因EGFR、FGF18、GHITM和TβRI以及外套膜的中央膜和边缘膜矿化相关基因pearlin、DPT、pif177和N19的表达。结果表明:(1)各组间马氏珠母贝育珠贝的存活率、留核率不存在显著性差异(P0.05),所采收珍珠的珍珠层厚度及平均质量也不存在显著性差异(P0.05);(2)各实验组间马氏珠母贝闭壳肌中EGFR、FGF18、GHITM和TβR I的相对表达量不存在显著性差异(P0.05);(3)各实验组间马氏珠母贝中央膜中DPT和N19的相对表达量差异不显著(P0.05),EG2和EG3组pearlin的表达量显著性高于EG1组(P0.05),pif177在EG3组中的表达量显著性高于EG1和EG2组(P0.05);(4)各实验组间马氏珠母贝边缘膜中pearlin、DPT和N19的相对表达量差异不显著(P0.05),EG2和EG3组pif177的表达量显著高于EG1组(P0.05)。研究结果说明,该微胶囊饲料可以替代部分微藻进行育珠生产,为进一步研发马氏珠母贝的人工配合饲料及开展工厂化育珠积累了参考资料。     

合浦珠母贝3个家系的AFLP标记分离与遗传多样性研究

对AFLP标记在合浦珠母贝(Pinctada fucata)印度家系(印度贝♀×印度贝♂,PII)、杂交家系(三亚贝♀×印度贝♂,PSI)和三亚家系(三亚贝♀×三亚贝♂,PSS)的遗传分离及3个家系的遗传多样性进行了分析。3对引物共产生57个位点,分离位点比例为57.4%~87.7%,符合孟德尔规律的分离位点为70.0%~71.0%;1:1分离位点占总分离位点的24.0%~45.2%,其中杂交家系的比例最高。3个家系的多态位点比例为93.0%~100.0%,总基因多样性为0.460。家系内的基因多样性为PII0.434,PSI0.331,PSS0.366,平均为0.377±0.030。家系间的遗传分化显著(GST=0.180)。遗传距离分析表明,PSI与PSS之间的遗传距离最大(0.157),PII与PSS之间的遗传距离最小(0.121)。UPGMA系统树表明,PII和PSS的亲缘关系较近,而PSI与这2个家系较远。上述结果表明,第一代家系的遗传多样性仍很高,但家系之间的遗传分化较大。该研究结果对育种过程中遗传资源管理具有一定指导作用。     

育珠对合浦珠母贝N19和Prismalin-14基因表达水平的影响

为探讨育珠对壳基质蛋白基因表达的影响,开展了合浦珠母贝（Pinctada fucata）N19和Prismalin-14基因在育珠贝和非育珠贝中的荧光定量表达研究。结果显示,N19和Prismalin-14在育珠贝与非育珠贝的闭壳肌、肝胰脏、性腺、肠、鳃等组织中均无表达,在外套膜均有表达;N19在珍珠囊表达,而Prismalin-14则不表达。N19勺Prismalin．14在育珠贝外套膜的表达量均大于非育珠贝,表明育珠可能对某些壳基质蛋白的表达具有一定的促进作用。其中Prismalin-14在育珠贝外套膜的表达量最高（P〈0．01）,是非育珠贝外套膜Prismalin-14的1．23倍,是育珠贝外套膜N19的25．04倍,是非育珠贝外套膜N19的41．04倍,推测在珍珠贝生长过程中,对Prismalil7-14的需求量可能比N19大。N19在育珠贝珍珠囊中表达量最高（P〈0．01）,是育珠贝N19外套膜的11．58倍,是非育珠贝外套膜的18．97倍,推测在珍珠形成的过程中,对N19的需求量可能比贝壳自身生长对N19的需求量大。     

合浦珠母贝遗传连锁图谱的构建

用AFLP标记构建了印度合浦珠母贝(Pinctada fucata)全同胞家系的遗传图谱。用经过筛选的36对引物组合对父母本和62个子代个体进行遗传分析,共得到1 547个标记,包括581个1∶1分离标记。母本分离标记294个,其中178个符合1∶1孟德尔分离规律;父本分离标记287个,其中182个符合1∶1孟德尔分离规律。雌性框架图包括33个遗传标记,分布在14个连锁群中,标记间平均间隔24.3 cM,有2个3联体,11个连锁对,图谱总长度为488.5 cM。雄性框架图包括53个遗传标记,分布在19个连锁群中,标记间平均间隔30.5 cM,有4个3联体,10个连锁对,图谱总长度为1 035.5 cM。雌雄两个框架图中AFLP标记的分布都较均匀。雌雄基因组估算长度分别为1 168.4 cM和2 037.1 cM,图谱覆盖率分别为41.8%和50.8%。本研究为进一步构建高密度遗传连锁图谱及QTL定位分析奠定了基础。     

Genetic parameters of growth and survival in the Pacific oyster Crassostrea gigas

The Pacific oyster Crassostrea gigas is a representative bivalve mollusc that is widely cultured in the world and is the largest molluscan group cultured in China. In order to assess the feasibility of improving survival of C. gigas through genetic selection, the heritability and genetic correlations for growth and survival traits between different life stages were examined. Genetic parameters were estimated based on intraclass correlations of 49 full‐sib families (29 half‐sib families) in larvae (4 and 20 days after fertilization) and spat (140 days after fertilization) stages. The heritability for growth traits in larvae and spat was 0.30–0.86 and 0.53–0.59, respectively, and varied with ages. The heritability of survival was low in larvae (0.13 ± 0.05 and 0.17 ± 0.04, respectively for 4 and 20 days after fertilization) but medium (0.39 ± 0.07) in spat, suggesting that selection for increasing spat survival was feasible. The genetic correlation between growth traits within age was medium to high and positive (ranging from 0.47 to 0.96, respectively, between shell length (SL) and shell height (SH) at 20 days and between SL and SH at 140 days after fertilization), suggesting that selection to improve single growth trait will cause positive response in another growth traits in C. gigas. The genetic correlations between survival and growth traits at 140 days were low but positive (ranging from 0.23 to 0.27, respectively, between survival and SH and between survival and SL at 140 days after fertilization), suggesting that selection for survival may not have a negative response in growth. Overall, this study suggests that survival traits should be taken as improving target of next selection breeding programme in C. gigas.     

不同温度下哲罗鲑幼鱼生长性状的遗传参数估计

采用每4个雄性亲本与1个雌性亲本交配的巢式设计方法,建立哲罗鲑(Hucho taimen)9组母系半同胞、34个父系全同胞家系.19个家系置于(13±1.5)℃(高温),15个家系置于(9±1.5)℃(低温)条件下饲养,计算幼鱼各月龄的体质量和体长性状的遗传力和遗传相关.结果表明,哲罗鲑幼鱼在低温条件下体质量遗传力为0.413～0.675,体长遗传力为0.297～0.777;高温条件下体质量遗传力为0.396～0.558,体长遗传力为0.194～0.624,均属于中高遗传力.在2个温度条件下母本间遗传方差组分均大于父本间遗传方差组分,存在较大的母本效应或显性效应,而根据父本间遗传方差组分估计的遗传力较为无偏.低温条件下体长、体质量遗传力估计值高于高温条件下的估计值,表明基因和环境互作效应较为明显,在低温条件下对体长和体质量进行选择能达到更好的效果.体长和体质量在不同生长时期、不同温度条件下都具有显著的遗传正相关和表型正相关(P＜0.05),表型相关系数为0.815～0.939,遗传相关系数为0.794～0.939,表明通过体质量或体长进行选育均能达到改良生长性状的目的.     

Effects of glycopeptides on development,growth and non‐specific immunity of pearl oyster Pinctada fucata (Gould)

The effects of glycopeptides, prepared from pearl oyster Pinctada fucata, on embryonic development, larval and juvenile growth and adult non‐specific immunity of P. fucata were investigated in this study. Glycopeptides had a pronounced stimulatory effect on embryonic development and larval and juvenile growth of P. fucata. enhancing with increased glycopeptide concentrations. All of haemocytes, phagocytosis, aggregation, serum microbiostatic activity and bacteriocidal activity all showed significant increases after 60‐day feeding, relative to unfed controls. The major conclusion is that glycopeptides had a pronounced stimulatory effect on the non‐specific immunity of pearl oysters.     

Evaluation of the progeny of third-generation Sydney rock oyster Saccostrea glomerata (Gould, 1850) breeding lines for resistance to QX disease Marteilia sydneyi and winter mortality Bonamia roughleyi

A breeding program for Sydney rock oysters Saccostrea glomerata (Gould, 1850) has been selecting oysters for resistance to QX disease (Marteilia sydneyi) and winter mortality (Bonamia roughleyi) for three generations at three sites in Georges River, New South Wales, Australia. The experimental sites are located at the upper, middle and lower reaches of oyster growing areas in the estuary. QX disease mainly occurs in the middle and upper reaches and is most severe at the latter. Winter mortality on the other hand occurs mainly at the lower and middle reaches and is most severe at the former. Progeny of third‐generation Sydney rock oyster breeding lines were evaluated for resistance to both QX disease and winter mortality against a non‐selected control. Line 1, selected for QX disease resistance at the upper estuary site, had excellent resistance to one season of exposure to disease, but suffered high mortality during the second season of exposure. However, these oysters had already reached market size of 50 g whole weight, with low mortality at 2 years of age, before the second episode of QX disease. Line 2 showed good improvement in resistance to both diseases, whereas Line 3, was the most resistant to winter mortality. Selection for resistance to QX disease did not appear to confer resistance to winter mortality and the converse also applied.     

Comparative growth and survival of spat of the Caribbean pearl oyster,Pinctada imbricata cultivated indoor with microalgae diets and outdoor with natural diet

We report the results of survival and growth in size and dry mass of spat of the Caribbean pearl oyster Pinctada imbricata cultivated under outdoor (field culture) and indoor (Laboratory) conditions. Field group fed on environmental seston. Laboratory groups were fed with mono, binary and ternary mixtures of three cultivated algae: Isochrysis galbana (Ig), Tetraselmis chuii (Ig) and the Chaetoceros sp. (Ch‐A, isolated from north‐eastern Venezuela). After 30 days of trial, fatty acid profiles of spat were determined along with growth in length and height shell, adductor muscle and soft tissue dry mass. During the field grow‐out phase (field culture), samplings were performed at days 1, 15 and 30 to measure environmental variables of phytoplankton biomass (chlorophyll a), dissolved oxygen, seston, temperature and salinity. A significant increase in size and soft tissue mass occurred in spat fed the diets including the tropical diatom (Chaetoceros sp.). In contrast, monoalgal diets of Tc and Ig yielded no significant differences in size and mass of spat, compared with the field culture. These results suggest that nutritional requirements of cultivated spat for specific fatty acids of physiological importance for marine bivalves, such as: 16:0, 16:1n‐7, 18:2n‐6, 20:4n‐6, 18:3n‐3 and 20:5n‐3, were satisfied from microalgal diets with Ch‐A, alone or in combination, compared with spat fed from the field culture.