罗氏沼虾遗传多样性的RAPD研究

本采用RAPD技术,对70年代从马来西亚引进的七对罗氏沼虾后代的养殖群体（简称Ny）和90年代从新加坡引进的天然群体的F1与养殖群体交配繁殖的后代（简称Nx）的遗传多样性进行了研究。用20种随机引物对两个群体的基因组DNA进行分析的结果表明,扩增的DNA片段大小在0.1-1.5kb之间。Ny群体内的平均遗传相似底（similari ty,S)为0.8819,Nx群体内的平均遗传相似度（S）为0.5857。他们的遗传变异度（variability,V）分别是0.1181和0.4143.Ny群体的变异度明显小于Nx群体。这可能与Ny群体长时期的近亲间或在小群体内繁殖导致群种退化有关。     

罗氏沼虾缅甸野生群体和浙江养殖群体的遗传多样性比较

采用形态学方法、同工酶电泳技术和DNA分析方法对罗氏沼虾(Macrobrachium rosenbergii DeMan)缅甸野生群体(BNP种群)和浙江省养殖群体(ZCP种群)的遗传多样性进行比较分析。结果表明,两种群在外形、体色、生长等方面存在一定的差异;对罗氏沼虾11种酶的25个基因位点进行分析,得出BNP种群和ZCP种群的同工酶分析所得出的多态位点比例及平均杂合度分别为12％和:16％,0.0431和0.0501。根据同工酶的基因频率计算得两种群的遗传相似系数为0.9963,遗传距离为0.0037;22个经筛选的10bp随机引物共检测到139个RAPD位点,BNP种群的多态位点比例和遗传多态度(Ho)分别为33.81％和0.0940,高于ZCP种群的30.22％和0.0780,两个群体的平均遗传多态度(Hpop)为0.0860,90％以上的遗传变异是在群体内检测到的。根据RAPD扩增的基因频率得出两群体间的遗传距离为0.1206。无论是同工酶电泳结果还是RAPD分析结果,都表明罗氏沼虾缅甸野生群体的遗传多样性高于浙江养殖群体,说明人工养殖会导致罗氏沼虾遗传多样性的下降。结果还表明,罗氏沼虾群体遗传多样性水平欠丰富,因此需要对罗氏沼虾群体实行科学的管理。     

罗氏沼虾抗病选育群体的抗病性能及其遗传多样性分析

为深入了解罗氏沼虾抗病选育群体的抗病力和遗传信息,通过人工注射溶藻弧菌感染16个罗氏沼虾选育群体,并利用微卫星分子标记对选育群体进行遗传结构分析。结果显示,16个选育群体抗溶藻弧菌感染能力存在明显差别,并从中鉴定出抗病力强的群体3个(SCR11-6、SCR11-11和SCR11-16),其在感染溶藻弧菌感染后的成活率达80%;抗病力比较强的群体7个;抗病力一般的群体4个,成活率为65%～70%;抗病力差的群体2个,成活率为35%～50%。8对微卫星引物共检测到53个等位基因,每对引物的等位基因数为5～9个,平均为6.625个,多态信息含量(PIC)为0.629 4～0.829 4,平均为0.732 3。16个群体的平均PIC为0.493 2～0.695 6,平均观测杂合度为0.506 2～0.651 3,平均期望杂合度为0.551 9～0.733 2,遗传相似系数平均为0.655 2,遗传距离平均为0.434 4。并对DP和SP两群体罗氏沼虾个体扩增出的差异条带进行统计,分析其与罗氏沼虾抗病性状的相关性。结果表明,5个微卫星位点SUGbp8-103b、SUGbp8-101c、MRMB11、SUGbp8-137和MRMC2分别在203、263、185、335和96 bp等位基因与罗氏沼虾抗病性状存在一定的相关性,其中,位点MRMB11在185 bp等位基因跟抗病性有极显著的正相关性,相关系数为0.282。研究表明,16个选育群体中有4个群体的抗病力较强,同时与其它12个选育群体相比,这4个群体遗传多样性也比较丰富,这些罗氏沼虾群体的筛选为罗氏沼虾抗病新品种的培育奠定了重要基础。     

罗氏沼虾种群SSR分析中样本量及标记量对遗传多样性指标的影响

利用60对微卫星分子标记分别对罗氏沼虾3个养殖群体进行遗传多样性分析,通过比较样本量及标记量对遗传多样性指标的影响,探讨最适宜的样本量及标记量。结果显示,样本量和标记量的大小均对遗传多样性指数有较大的影响,其中样本量与平均等位基因数和平均有效等位基因数呈高度正相关,与杂合度和Nei氏遗传多样性指数分别呈中度、高度相关,样本量为10~30时,遗传参数变化差异显著,样本量大于30,变化差异不显著;标记量与遗传参数也存在不同程度的相关性,标记量为5~25时,各遗传多样性指数变化有明显差异,标记量大于25时,各遗传参数变化较小,多态信息含量不同的标记直接影响到群体遗传参数。性别对群体遗传多样性指标无显著性影响。研究表明,应尽可能选择多态信息含量高的微卫星分子标记开展罗氏沼虾群体遗传多样性分析,可不考虑样本性别,样本量不宜小于30,标记量不宜少于25。     

日本沼虾微卫星引物筛选及群体遗传多样性分析

采用(CT)15、(CA)15两种生物素标记及磁珠富集法构建了太湖日本沼虾基因组微卫星富集文库。120个微卫星克隆中83个位点的核心序列重复在10个以上,长度介于191～580bp,平均为311bp;重复类型中,(CA/GT)n及(AG/TC)n分别占59.09%及20.45%,还检测到(GC)n、(AAG)n、(ACAA)n和(GGCAGA)n等17种类型,包括25.75%完美型、20.45%非完美型和53.80%复合性。用其中12条微卫星引物扩增吴江、滨湖、宜兴和吴兴4个群体240个个体的基因组DNA,各位点表现出较高的遗传多样性,除Mni86、Mni103和Mni114外,其它群体位点大都表现出显著的杂合子缺失,4个群体的遗传分化为低度分化(FST<0.05),4个群体中97.58%遗传变异存在于群体内,仅有2.42%的遗传变异来自于群体之间;吴江和吴兴群体亲缘关系最近,与宜兴和滨湖群体亲缘关系渐远。     

罗氏沼虾不同群体杂交效果分析

对罗氏沼虾3个群体:缅甸群体(AA)、浙江养殖群体(BB)和广西养殖群体(CC)及其6个杂交组合子一代5月龄的4个生长性状进行了测定。计算了各项指标的杂种优势率,并对各性状进行了方差分析和多重比较。结果表明,除BB♀×AA♂杂交组合的子一代在腹长和体重上没有表现出杂种优势,CC♀×BB♂杂交组合的子一代在头胸甲长指标上表现出杂种劣势外,5月龄的杂交子一代在4个生长指标中均表现出一定程度的杂种优势(0.11%～10.18%)。不同杂交组合间和不同性状间的杂种优势存在差异,AA♀×CC♂杂交组合子一代4个指标的杂种优势率均高于其他杂交组合子一代,体重的平均杂种优势率均高于其他3个指标的平均杂种优势率。方差分析结果表明,自繁群体后代间在4个生长指标上不存在显著差异;杂种子一代与该组合的自繁后代相比较,只有缅甸群体、广西养殖群体及其杂种子一代的体长指标在群体间存在显著差异;6个杂交子一代的体重指标差异不显著,其他3个指标均存在显著差异。LSD多重比较结果显示,AA♀×CC♂杂交子一代体长与自繁(CC♀×CC♂)后代的体长存在显著差异;在头胸甲长、腹长和体长3个指标中,CC♀×BB♂杂交子一代与AA♀×BB♂杂交子一代和AA♀×CC♂杂交子一代均存在显著差异,并且CC♀×BB♂杂交子一代在3个指标中的平均值均低于这两个杂交组合。     

异型雄性罗氏沼虾遗传多样性的微卫星分析

利用微卫星DNA标记研究3种近亲交配形态型雄性罗氏沼虾遗传多样性,所用虾样均采自国家级(南宁)罗氏沼虾良种场,共150尾成年近亲交配的雄虾,其中蓝色长臂型、橙色长臂型、小个体型各50尾。研究结果表明,3组样品的生长发育差异显著,蓝色长臂型、橙色长臂型、小个体型各组中微卫星位点的总等位基因分别为24、28和22,3组样品所有位点的平均观察杂合度为0.474～0.556。其平均群体内近交系数为-0.0189,说明杂合子过剩,对比分析和群体间分化系数表明,3组雄虾遗传差异明显,最大遗传间距位于蓝色长臂型和小个体型,最小的遗传间距位于蓝色长臂型和橙色长臂型。研究结果将为罗氏沼虾优质父本选育和调控雄性遗传发育提供基础性遗传信息。     

3个不同群体罗氏沼虾线粒体16SrRNA基因序列分析及遗传差异

利用PCR技术扩增获得罗氏沼虾线粒体16SrRNA基因的部分片段,通过序列测定和用限制性内切酶MboI、AluI和TaqI分别对上述PCR产物进行限制性片段长度多态分析(RFLP),分析了取自缅甸引进种子代、广西养殖群体及江苏养殖群体3个不同群体罗氏沼虾的遗传差异。结果表明:在3个群体间序列同源性对比无变异,PCR-RFLP未发现有片段多态性,3个群体间不存在遗传结构差异。可能表明这3个群体的罗氏沼虾起源同一个种群,且分化较低。     

罗氏沼虾养殖技术

罗氏沼虾又名马来西亚大虾,原产于东南亚国家,属热带河口性淡水虾。具有生长快、个体大、食性广、疾病少、养殖周期短和商品价值高等特点。70年代世界各地广泛引种进行试养。 1.罗氏沼虾的生活习性罗氏沼虾是生活在热带地区的淡水虾,天然水域中的罗氏沼虾具有生殖洄游习性,     

罗氏沼虾的工厂化育苗

罗氏沼虾Macrobrachium rosenbergii Aeman,又名马来西亚大虾,是一种大型淡水虾,原产于热带、亚热带一些国家和地区,其适温范围与罗非鱼基本相同.因具有生长快、个体大、食性广、营养价值高、养殖周期短等特点,引起了世界上许多国家的重视.我国于1976年引进,1977年在广西人工繁育成功。为探讨在北方地区育苗和养殖的可行性,作     

罗氏沼虾淡化养殖的现状与展望

为了解超氧化物歧化酶 (SOD)分子生物学特性和免疫功能,实验克隆了罗氏沼虾胞质锰SOD基因 (MrcMnSOD),制备多克隆抗体,并分析在嗜水气单胞菌感染下该基因的表达模式。结果显示,嗜水气单胞菌感染可显著诱导MrcMnSOD在转录水平和蛋白质水平进行高表达。为探究MrcMnSOD参与免疫应答的机制,进一步的抑菌实验表明,该蛋白质可显著抑制3种革兰氏阴性细菌 (大肠杆菌、副溶血性弧菌、嗜水气单胞菌)和2种革兰氏阳性细菌 (无乳链球菌、金黄色葡萄球菌)的生长,且抑制作用与蛋白质浓度的关系不显著。研究表明,MrcMnSOD可能作为一种免疫相关分子参与免疫应答反应。本研究初步探讨了MrcMnSOD的免疫生物学功能,旨在为深入研究罗氏沼虾SOD的功能奠定相关基础。

     

Genetic diversity of cultured and wild populations of the giant freshwater prawn Macrobrachium rosenbergii (de Man, 1879) based on microsatellite analysis

Freshwater prawn (Macrobrachium rosenbergii) culture in the Western Hemisphere is primarily, if not entirely, derived from 36 individual prawns originally introduced to Hawaii from Malaysia in 1965 and 1966. Little information is available regarding genetic variation within and among cultured prawn stocks worldwide. The goal of the current study was to characterize genetic diversity in various prawn populations with emphasis on those cultured in North America. Five microsatellite loci were screened to estimate genetic diversity in two wild (Myanmar and India‐wild) and seven cultured (Hawaii‐1, Hawaii‐2, India‐cultured, Israel, Kentucky, Mississippi and Texas) populations. Average allelic richness ranged from 3.96 (Israel) to 20.45 (Myanmar). Average expected heterozygosity ranged from 0.580 (Israel) to 0.935 (Myanmar). Many of the cultured populations exhibited reduced genetic diversity when compared with the Myanmar and the India‐cultured populations. Significant deficiency in heterozygotes was detected in the India‐cultured, Mississippi and Kentucky populations (overall F_is estimated of 0.053, 0.067 and 0.108 respectively) reflecting moderate levels of inbreeding. Overall estimate of fixation index (F_st = 0.1569) revealed moderately high levels of differentiation among the populations. Outcome of this study provide a baseline assessment of genetic diversity in some available strains that will be useful for the development of breeding programmes.     

养殖密度对罗氏沼虾生长、性别分化与性腺发育的影响

通过比较4个养殖密度(7.50×104、12.75×104、42.00×104、97.50×104尾/hm~2)条件下罗氏沼虾的生长、存活、性别分化、性腺发育等指标,研究养殖密度对罗氏沼虾生长、性别分化和早期性腺发育的影响。结果显示,在实验密度范围内,养殖密度显著影响罗氏沼虾的生长、规格整齐度、性别分化与性腺发育,随着实验时间延长,密度对生长的抑制效应越明显;7.50×104尾/hm~2组罗氏沼虾体质量增速加速度与97.50×104尾/hm~2组的倍数由养殖10 d时的2.3上升到80 d时的3.4;低密度组罗氏沼虾性别分化与性腺发育比高密度组早约10 d,但规格显著大于高密度组;7.50×104尾/hm~2组雌虾初始平均体长(4.158±0.592)cm比97.50×104尾/hm~2组大1.09 cm;养殖密度对性别比没有影响,但对雌虾成活率影响较大。研究表明,养殖密度显著影响罗氏沼虾生长和性腺发育,合适的养殖密度是预防罗氏沼虾性早熟的有效措施。     

Serotonin induces ovarian maturation in giant freshwater prawn broodstock, Macrobrachium rosenbergii de Man

This study investigated the effects of serotonin (5-hydroxytryptamine or 5HT) on ovarian development in Macrobrachium rosenbergii de Man. Adult female prawns at the ovarian stage I (spent) were injected with 5HT at 1, 5, 10, 20 and 50 μg g^− 1 body weight (BW) intramuscularly on days 0, 5 and 10, and sacrificed on day 15. The doses as related to the effect could be categorized into three levels: low (1 and 5 μg g^− 1 BW of 5HT), medium (10 and 20 μg g^− 1 BW of 5HT) and high (50 μg g^− 1 BW of 5HT). The low-dose, especially at 1 μg g^− 1 BW, caused prawns to exhibit a significant increase in ovarian index (ovarian weight/body weight × 100) (5.79 ± 0.09%) as compared to the control (1.49%). The ovaries of most of these prawns could develop to stage IV (mature) and contained synchronously mature oocytes while most of the control ovaries remained at stage I and II (proliferative), and contained only oogonia to previtellogenic (Oc1, Oc2) and early vitellogenic oocytes (Oc3). The medium- and high-dose treated prawns exhibited ovaries that could reach stages III and IV and contained various types of oocytes of different maturity. Pretreatment with 5HT receptor antagonist, cyproheptadine (CYP), at 10 μg g^− 1 BW before 5HT injection significantly suppressed the effect of 5HT. Intramuscular injection of the 5HT-primed thoracic ganglion culture medium into CYP-pretreated prawns resulted in the increase of ovarian index about 5–6 times more than in the control, and in the groups injected with 5HT-primed media from muscle strip, eyestalk and brain. The ovaries of most prawn could develop up to stage IV and contained synchronously developed vitellogenic (Oc4) and mature oocytes (Oc5). These findings suggest that 5HT indirectly induces ovarian development and oocytes maturation in M. rosenbergii, probably via a putative ovarian stimulating factor released from the thoracic ganglia.     

罗氏沼虾性成熟前后形态性状对体质量的通径分析

为探明罗氏沼虾(Macrobrachium rosenbergii)养殖群体在性成熟前后表型性状间的相互关系,本研究随机选取人工养殖的罗氏沼虾1106尾,对其体质量、全长、体长等15个性状进行测量,并采用相关分析和通径分析阐明形态性状对体质量的影响。结果表明：在性成熟前后及同一阶段雌雄群体之间,影响体质量的关键指标以及所构建的关键表型性状多元回归方程是截然不同的;在性成熟后,雌雄个体呈现出明显的性二态现象,除第二步足长和第二步足重雄性极显著大于雌性(P<0.01),雌性全长、体长、额剑长、头胸甲宽、头胸甲高、腹长、腹宽、腹高和腹部重极显著大于雄性(P<0.01),这可能与雌性为提升其繁殖能力有关。统计结果显示被保留的性状与体质量的复相关系数大于0.85,表明本研究已确定影响各群体体质量的主要性状。因此在选育过程中,建议将罗氏沼虾的性成熟状态和雌雄群体的优良表型作为选育依据,并有效利用影响体质量的关键性状指标。本研究可以为罗氏沼虾的良种选育提供参考依据。     

Classification of differentiating oocytes during ovarian cycle in the giant freshwater prawn, Macrobrachium rosenbergii de man

Based on the light microscopic observations of cells' sizes, chromatin patterns, amount of lipid droplets and yolk granules, the female germ cells could be classified into four different phases, which include 1) oogonia (Oog), 2) primary oocytes (pOc), 3) secondary oocytes (sOc), and 4) mature oocyte (mOc). Oog are small oval-shaped cells with irregular-shaped nuclei sizing 4–6 μm in diameter. They rest on the connective tissue germinal cord at the tip of each ovarian pouch (lobule). Oogonia increase their number through mitotic division, and the daughter cells move into ovarian pouch where they undergo first meiotic division to become primary oocytes, which have various steps of 1st meiotic prophase accumulating at the innermost zone of the ovarian pouch. The primary oocytes are small oval-shaped cells (8.5–10 μm in diameter) with large nuclei containing chromatin in various states of condensation that finally transform into chromatids. Their nuclei are surrounded by thin rim of faint blue-stained cytoplasm. The secondary oocytes derived from 2nd meiosis and comprise five steps: Oc1 and Oc2, classified as previtellogenic oocytes, Oc3 and Oc4, classified as vitellogenic oocytes, and mature oocyte (mOc) The zones of ovarian pouch are defined based on the accumulation of various steps of developing oocytes, namely, oogenic, previtellogenic, vitellogenic and mature zones, respectively. The ovarian cycle is divided into five stages based on the number and types of oocytes present in each stage. Stage 0 and I are spawn and spent stages. Stage II and III are proliferative and premature stages, while stage IV is mature stage. During ovarian stage I, each ovarian pouch contains primarily oogonia, primary oocytes, Oc1 and a few Oc2. In stage II, the pouch contains mainly Oc2 and Oc3, while in stage III the predominant cells are Oc4. Mature oocytes appear synchronously, in stage IV. The ovulating mature oocytes pass through the thin disrupted wall of ovarian pouch into subcapsular space, that leads into the oviduct situated on the ventro-lateral side of the ovarian lobe. At spawning, the ovarian pouches break down and only connective sheaths and hemolymph sinuses remain. The germinal cords and islets of oogonia remain in the central area of stage 0 ovary. The ovarian capsule, including the muscular layer, becomes attenuated as the ovary progresses from stage 0 to IV. The hemolymph vessels become highly convoluted in the central area of the ovary, and they branch radially into smaller hemolymph sinuses around each oogenic pouch.