基于SSR-seq技术评估中间球海胆多代选育群体和普通养殖群体的遗传多样性

为明确不同选育群体中间球海胆的遗传多样性和遗传结构,利用SSR-seq技术和15个微卫星位点,对1个家系选育群体(FP)、1个群体选育群体(IP)和1个未经选育的普通养殖群体(CP)的遗传多样性及遗传结构进行了分析。结果显示,15个微卫星位点共检测出112个等位基因,FP、IP、CP 3个群体的平均观测等位基因数(N_a)分别为5.077、5.133和6.133个,平均有效等位基因(N_e)分别为2.816、2.873和3.638个,平均观测杂合度(H_o)分别为0.522、0.441和0.501,平均期望杂合度(H_e)分别为0.595、0.599和0.667,平均多态性信息含量(PIC)分别为0.546、0.543和0.623。家系选育群体(FP) H_e与H_o的差值(0.073)低于IP (0.158)和CP (0.166),平均固定指数(F)(0.115)低于IP (0.248)和CP (0.246)。3个群体间遗传分化系数(F_st)介...     

福建沿海试养中间球海胆的初步研究

为明确不同选育群体中间球海胆的遗传多样性和遗传结构,利用SSR-seq技术和15个微卫星位点,对1个家系选育群体 (FP)、1个群体选育群体 (IP)和1个未经选育的普通养殖群体 (CP)的遗传多样性及遗传结构进行了分析。结果显示,15个微卫星位点共检测出112个等位基因,FP、IP、CP 3个群体的平均观测等位基因数 (N_a)分别为5.077、5.133和6.133个,平均有效等位基因 (N_e)分别为2.816、2.873和3.638个,平均观测杂合度 (H_o)分别为0.522、0.441和0.501,平均期望杂合度 (H_e)分别为0.595、0.599和0.667,平均多态性信息含量 (PIC)分别为0.546、0.543和0.623。家系选育群体 (FP) H_e与H_o的差值 (0.073)低于IP (0.158)和CP (0.166),平均固定指数 (F) (0.115)低于IP (0.248)和CP (0.246)。3个群体间遗传分化系数 (F_st)介于0.018~0.176,为中低等程度的遗传分化。分子方差分析 (AMOVA)结果显示,3个群体的遗传变异主要源于个体间。主成分分析 (PCoA)和聚类进化树结果均显示,3个群体之间的亲缘关系较近,其中IP群体遗传分化程度最高。研究表明,3个中间球海胆群体均具有较高的遗传多样性,多代家系选育和群体选育均未明显降低群体的遗传多样性,家系选育更有利于保持群体的杂合度和控制群体的近交水平,群体选育则会提升群体的遗传分化程度。     

光棘球海胆和中间球海胆早期发育的形态差异

球海胆属（Sh。楷叫优。ntrotu抓9海胆世界上共有十几种,光棘球海胆（S.n。;d u s）和中间球海胆（S.fll。。。。巾l。）是我国北方主要的经济品种,其营养价值和经济价值高,国际市场需求量大,开展海胆人工育苗和增养殖是海胆产业可持续发展的重要举措。而掌握其胚胎和幼虫生长发育的区别,不仪对苗种生产有帮助,而且对海胆资源的保护利用十分有利。 1卵及胚胎 光棘球海胆的卵径为106 pITI左右,黄色;中间球海胆90 pill左右,橙色,其卵径比光棘球诲胆小一点。受精膜举起后,光棘球海胆的卵径为     

光棘球海胆(♀)×中间球海胆(♂)杂交育苗试验

以光棘球海胆为母本,中间球海胆为父本进行杂交试验及苗种培育.试验结果表明,通过筛选同时得到自然成熟的父母本精、卵,在比正常海胆自交精子量高40倍以上的精子作用下,杂交受精率为2.5%.在适宜的水温、光照及饵料等的培养条件下,杂交海胆能够生长附着变态,变态时间与母本相近.培育出的杂交海胆外部颜色及棘色、棘长介于两亲本之间.杂交海胆经过6～7个月的中间暂养,出苗量为0.6×10~4个/m~3,个体壳径3～30 mm,平均壳径8.8 mm.     

中间球海胆性腺氨基酸组成研究

试验结果表明,中间球海胆鲜味氨基酸含量占总氨基酸含量的43%以上,尤其是含有较高的甘氨酸、谷氨酸.雄性性腺鲜味氨基酸含量高于雌性,因此,雄性味更美.在生产中采集中间球海胆时期最好选在早期成熟阶段,在海胆成熟后期自然排放之前采收完毕.     

中间球海胆饥饿代谢研究

于 2 0 0 2年 4月至 5月测定了饥饿状态下中间球海胆 (Strongylocentrotusintermedius)耗氧率和排氨率的变化情况 ,得出海胆饥饿后耗氧率、排氨率及氧氮比变化曲线 ,并对海胆性腺主要成分进行组织化学分析。结果表明 :随饥饿时间的延长 ,中间球海胆的耗氧率呈“稳定、急速下降、再稳定”的阶段性变化 ,在整个饥饿期间内下降了 4 5 .6 % ;排氨率经短暂稳定后 ,先上升再阶段性下降 ,实验结束时共下降了 32 .33% ;氧氮比则表现出平稳、下降、上升的趋势。组织化学的测定结果是海胆饥饿前后性腺的主要变化成分为蛋白质和糖类 ,综上可认为 ,海胆饥饿后能源物质利用顺序为 :蛋白质 糖类、蛋白质、蛋白质 糖类     

中间球海胆与光棘球海胆规模化杂交育苗技术

海胆规模化杂交育苗,受精率为33%,适宜的水温、光照及饵料等培养条件下,杂交海胆能够正常生长和附着变态,变态时间与母本相近。用0.1mol/L的KCl诱导5 min,杂交海胆全部变态。杂交海胆壳径>2~3 mm剥离进入网箱培育,有利于快速生长。稚胆培养温度以12~15℃为宜。     

中间球海胆(♀)×光棘球海胆(♂)F1代的生殖腺特征

对中间球海胆(♀)与光棘球海胆(♂)杂交F1代的生殖腺特征进行了研究,并与母本中间球海胆进行了比较。试验结果表明,中间球海胆的生殖腺颜色一般呈橙色或橘黄色(不同发育时期有所不同),结构致密;杂交海胆中有约90%表现为橙色和橘黄色,约10%呈黄色和浅棕色,约20%的个体生殖腺结构疏松,且和发育周期及色泽无关。杂交海胆的生殖腺指数与中间球海胆的变化基本一致,并且雌雄个体差别不明显。杂交海胆在生殖腺发育的整个过程中,生殖腺指数虽然也出现周期性的变化,但组织切片观察发现杂交海胆生殖腺发育不能象母本一样有明显分期,只有个别雌性个体能产生成熟卵细胞,雄性个体未见成熟精子。     

鲤野生和养殖群体遗传多样性分析中的微卫星(SSLP)分析

采用7个微卫星标记,研究了鲤(Cyprinus carpio)、高寒鲤和荷包红鲤抗寒品系(Cyprinus carpio var.wananensis)三个群体的遗传变异。结果显示,鲤野生群体、高寒鲤、荷包红鲤抗寒品系的平均杂合度分别为0.276、0.147、0.0952,平均无偏倚杂合度期望值为0.431、0.355、0.305。鲤野生群体的杂合度明显高于其他两个养殖品种,说明其较其他两个养殖品种的遗传多样性丰富。     

日本对虾养殖群体两个世代遗传结构的微卫星分析

利用8对微卫星引物对日本对虾养殖群体两个世代的遗传结构进行了分析,共检测到108个等位基因,等位基因长度为196-528 bp,各位点等位基因数为6-23个。亲本和子代群体每个位点平均等位基因数目分别为8.5个和11.5个,平均观测杂合度分别为0.721和0.632,平均期望杂合度分别为0.775和0.764,平均多态信息含量分别为0.732和0.729。根据标记信息,利用最大似然法对两个世代的个体分别进行系谱结构推断,亲本群体划分为6个群体,群体间遗传距离为0.277-2.356,均值为1.510;子代群体划分为17个群体,群体间遗传距离为0-2.593,均值为1.113。亲本和子代分群体间的遗传距离变化范围为0-3.089,均值为1.238。根据各分群体间的Nei's遗传距离,用非加权组平均法(UPGMA)构建了日本对虾两个世代的遗传进化树。其中18个分群体(4个亲本群体和14个子群体)经过聚类后形成3个分支,其余5个分群体(两个亲本群体和3个子群体)明显偏离于主支。研究发现,子代的观测杂合度明显低于亲本,说明子代在继承亲本遗传信息的过程中已经丢失了一定的杂合性。     

虾夷马粪海胆溶菌酶基因全长cDNA的克隆与表达分析

本实验采用RT-PCR和cDNA末端快速扩增(RACE)技术克隆得到了虾夷马粪海胆(Strongylocentrotus intermedius)溶菌酶(LYZ)基因的全长cDNA序列。结果表明, 虾夷马粪海胆LYZ基因全长为912 bp, 含有1个480 bp的开放阅读框(ORF), 编码159个氨基酸, 其中第1−20个氨基酸为信号肽, 蛋白计算分子量为17.69 kD, 等电点为7.75。氨基酸比对分析表明, 虾夷马粪海胆LYZ基因与紫球海胆(Strongylocentrotus purpuratus)和刺参(Apostichopus japonicus)的i型LYZ基因相似百分比分别为91.4%和59.3%, 并且含有i型LYZ基因的保守序列DVGSLSCGP (Y)Y(F)QIK, 所以推断本实验克隆的溶菌酶为i型。采用实时定量PCR方法, 以β-actin为内标, 对其在虾夷马粪海胆各组织中的表达进行研究, 发现LYZ基因在围口膜中表达量最高, 其次是齿间肌、管足、肠、体腔液、雄性性腺和雌性性腺。利用脂多糖(LPS)刺激虾夷马粪海胆, 取刺激后不同时间的海胆体腔液, 对该基因的表达差异进行分析。结果表明, 虾夷马粪海胆的LYZ基因在LPS刺激后8 h时表达量最高, 12 h时开始逐步回落, 至36 h时回落至对照组相近水平。本结果可为虾夷马粪海胆免疫学研究及抗病相关分子标记的开发提供参考依据。

     

虾夷马粪海胆体腔细胞的类型及功能

取平均壳径分别为1．9cm和4．4cm虾夷马粪海胆(Strongylocentrotus intermedius)进行实验观察。结果表明，海胆体腔有2种类型的细胞，即变形吞噬细胞和色素细胞。变形吞噬细胞形状不定，能伸出伪足，核较大，线粒体、溶酶体等细胞器丰富。色素细胞具突起，内有紫红色颗粒，颗粒溶于酒精等多种溶剂中，使得电镜下细胞内含有大量空泡，细胞核很少见，细胞器较少。变形细胞离体后可凝集，具吞噬酵母的能力，吞噬能力与温度成正相关。色素细胞具有辅助的免疫功能。     

中间球海胆卵膜与受精膜去除方法及染色体核型分析

去除卵膜及受精膜是成功制备棘皮动物染色体的关键步骤。为找到高效去除海胆卵膜与受精膜的方法,使用三氮唑（2 g/L）、盐酸海水溶液（pH 4.75）、二硫苏糖醇（3×10^-3 mol/L）和对氨基苯甲酸（3×10^-3 mol/L）等4种化学试剂分别对中间球海胆（）卵子及各时期胚胎进行处理,比较了不同试剂、处理时间或处理时期的去膜率、去膜后受精率和胚胎畸形率等指标的差异;利用常规空气干燥法对经去膜处理的囊胚期胚胎制备染色体,并进行核型分析。结果表明,三氮唑、盐酸海水溶液、二硫苏糖醇和对氨基苯甲酸等4种试剂对中间球海胆的卵膜和受精膜均有去除效果,其中2 g/L三氮唑处理未受精卵30 min的去膜率为85.50%,去膜后受精率为97.25%,畸形率为1.75%,去膜效果优于其余处理组。利用经该方法去膜后的早期囊胚进行染色体制备效果较好,获得了61个分散良好、形态完整的染色体中期分裂相。核型分析表明,中间球海胆的二倍体染色体数为2n=42,核型公式为：2n=20m+20sm+2st,NF=84,即有10对中部着丝粒染色体,10对亚中部着丝粒染色体,1对亚端部着丝粒染色体,染色体臂数为NF=84。本研究可为海胆染色体制备及染色体操作育种提供技术参考。     

Genetic variation of wild and cultured populations of the Kuruma prawn Marsupenaeus japonicus (Bate 1888) using microsatellites

The microsatellite DNA technique was used to detect the genetic variations between wild and cultured populations of Kuruma prawn Marsupenaeus japonicus Bate 1888. All the six microsatellite loci screened in this study showed high polymorphism for their PIC (0.6701–0.8989), which was much more than the standard value of 0.5. A total of 73 alleles were observed over six loci from 93 shrimps. The mean number of allele locus ranged from 9.83 (cultured) to 11.83 (wild). The number of effective alleles varied from 6.86 (cultured) to 8.58 (wild). The average of observed heterozygosity (H_o) of populations varied from 0.6935 (cultured) to 0.7370 (wild), and that of expected heterozygosity (He) was 0.8169 (wild) and 0.8209 (cultured). Tests of Hardy–Weinberg showed that these loci deviated significantly or highly significantly in one or both populations. Compared with the wild population, the cultured population showed little reduction in genetic variation. The total number of alleles (71, 59) was not significantly (P=0.296) different between wild and cultured populations. The paired‐samples t test of observed heterozygosity and expected heterozygosity implied that there was no significant difference (P=0.572 and 0.891 respectively) between wild and cultured populations. However, some rare allele loss might have occurred in the cultured population. A total of 14 unique alleles were found in the wild population, but only two unique alleles were observed in the cultured population. Therefore, there is a need to monitor genetic variability of cultured population, and to improve the hatchery program for the conservation of wild Kuruma prawn resources.     

Heritability and phenotypic correlations of gonad sweetness in the sea urchin Strongylocentrotus intermedius

Gonad is the only edible part of sea urchins. Thus, a number of studies have been focusing on how to improve both quantity and quality of their gonads. However, as far as our knowledge, the genetic basis of gonad flavor remains totally unknown in sea urchins. In the present study, we found that the heritability of gonad sweetness was at a high level of 0.56, clearly indicating that it is, to a large extent, under genetic control. Gonad sweetness was significantly positively correlated with gonad weight (P < 0.05), a* (P < 0.01) and b* (P < 0.01), while significantly negative correlated with L* (P < 0.01), ΔE ₁ (P < 0.01) and ΔE ₂ (P < 0.01). The present study provides valuable information into the genetic basis of gonad sweetness and evidences that gonad sweetness is potential to be improved in sea urchin genetic breeding programs.     

Effect of feed deprivation and refeeding on the MYP gene expression of the sea urchin (Strongylocentrotus intermedius)

To determine the main expression site of major yolk protein (MYP) gene and the mechanisms for adaptation to starvation and refeeding in Strongylocentrotus intermedius, MYP mRNA expression amounts were analysed using a real‐time RT‐PCR. The results showed that MYP could be transcribed in the intestine, stomach, gonad and coelomocytes, and that the intestine was the main expression site of MYP gene in non‐starved urchins. The MYP synthesis in the intestine decreased during 15 days of starvation (67.70%, 52.58% and 71.35% of the control at 5, 10 and 15 days of fasting respectively) and then increased dramatically by different amounts (the peaks were 2.71‐, 12.16‐ and 7.89‐fold that of the control respectively) during the refeeding stages. Nevertheless, the expression amounts in the gonads did not decline, but increased continuously during all periods of fasting (2.66‐, 3.72‐ and 13.19‐fold that of the control at 5, 10 and 15 days of starvation respectively) and during the refeeding stages. At the end of the recovery feeding experiment, the levels reached 9.58‐, 17.48‐ and 100.69‐fold that of the control. These data suggested that the ‘priority’ strategy for the sea urchin is to reduce MYP expression amounts in the intestine if food is limited and to increase MYP gene expression in the gonad to protect reproductive function.     

温度和盐度对中间球海胆胚胎发育早期进程联合效应

为探讨高温和低盐对中间球海胆(Strongylocentrotus intermedius)早期胚胎发育进程的影响, 本研究利用中心复合设计(CCD)和响应曲面分析法(RSM), 开展不同温度(12~26 ℃)和盐度(22~34)对中间球海胆胚胎发育早期进程的联合效应研究, 旨在建立温度和盐度对中间球海胆胚胎发育进程的定量关系模型, 并通过统计优化方法得出温度和盐度的最佳组合。结果显示, 在实验设定的温度和盐度范围内, 随着温度的升高, 中间球海胆早期胚胎发育时间呈现出先缩短后延长的趋势; 随着盐度的降低, 中间球海胆胚胎发育早期时间延长。温度的一次效应、二次效应和盐度的一次效应均显著影响(P<0.05)中间球海胆胚胎发育早期进程; 温度一次项系数的绝对值均大于盐度的一次项系数; 温盐的联合效应对中间球海胆胚胎发育早期进程的影响不显著(P>0.05)。实验建立的 2 细胞期、8 细胞期、16 细胞期、囊胚期、上浮期和四腕幼虫期发育进程模型方程决定系数分别为 0.9576、0.9508、0.9689、0.9932、 0.9681 和 0.9763。模型优化和验证试验得出, 温度 20.47 ℃和盐度 31.46 时, 中间球海胆 2 细胞期、8 细胞期、16 细胞期、囊胚期、上浮期、四腕幼虫期的发育时间最短, 分别为 1.28 h、2.07 h、3.31 h、4.14 h、11.28 h 和 47.31 h。 研究结果表明高温和低盐会延长中间球海胆早期胚胎发育时间。     

Estimates of genetic parameters for growth traits of the sea urchin,Strongylocentrotus intermedius

Heritabilities and genetic and phenotypic correlations were estimated for body weight, test diameter, and test height of the sea urchin from measurements on progeny resulting from 11 sires and 33 dams by artificial fertilization of 3 females by single males, and measurements at 8, 10, and 12 months after metamorphism. Point estimate for heritabilities based on the sire components of variance were moderate to high for body weight (0.21–0.49), test diameter (0.21–0.47), and test height (0.22–0.37). Genetic correlations were significant for body weight with test diameter (0.30∼0.65) and test height (0.30∼0.54) and test diameter with test height (0.31∼0.65). Genetic correlation estimates, derived the nested design and half-sib correlation analysis used in this study, appear to provide reliable estimates. Significant phenotypic correlations were found for body weight with test diameter (0.82∼0.86) and test height (0.49∼0.83), and test diameter with test height (0.47∼0.84). The phenotypic correlations for test height with body weight (0.491) and test diameter (0.467) at 12 months' of age were smaller than those earlier sampling periods.     

温度对中间球海胆幼胆生存、生长和行为的影响

底播增殖是一种提高中间球海胆(Strongylocentrotus intermedius)产量的有效方法.底播增殖受到多种内在和外在因素的影响,其中温度发挥着十分重要的作用,因此确定中间球海胆适宜增殖温度范围至关重要.本实验将大(23.29±0.27)mm、小(18.78±0.19)mm两种规格的中间球海胆放置在3个...