应用RAPD技术分析奥利亚罗非鱼和尼罗罗非鱼的基因多态性

以随机扩增多态DNA技术(RAPD）分析了奥利亚罗非鱼和尼罗罗非鱼两个养殖群体的群体内及群体间遗传关系,并探讨了该技术在种群鉴定中的应用。RAPD引物筛选结果表明,所测试的20个随机引物中(Table 1),除一个引物未扩增出任何片段外,其余19个引物均扩增出1～11个大小不等的片段,长度大部分在500—3000bp之间,共扩增出220个片段,平均每个引物产生55个片段。两群体间共有片段70条,大部分引物的扩增产物具有种间多态性,种群间相似系数为0.727。以筛选的引物对两种群不同个体(Fig．1,Table 2)及种群混合样品(Fig.2,Table 3)进行RAPD分析。结果表明,不同引物在扩增图谱上表现很大差异：奥利亚罗非鱼不同个体间表现为一致的扩增图谱,种内相似系数达1000,显示了其种群内遗传变异的缺乏;尼罗罗非鱼种内相似系数为0．827,个体间存在不同程度的多态性;两个种群间的相似系数分别为0．767和0.742,表明种间有较高的同源性,遗传距离为0．235,略低于国外的报道．此外,两个养殖群体间的扩增图谱比较也暗示了遗传渐渗现象的存在。实验表明,RAPD标记可以作为一种可靠的遗传标记,用于不同鱼类种群的鉴定,RAPD分析方法是一种快速,简便且行之有鼓的鉴定鱼类种群的方法。     

萨罗罗非鱼与尼罗罗非鱼正反杂交子代间的分子遗传学差异

用耐盐能力强的萨罗罗非鱼和生长性能优的尼罗罗非鱼进行人工正反杂交,对两者正反交F1代微卫星分子遗传学差异进行了研究,结果如下:(1)两亲本及其正反杂交子代间都有特异的微卫星分子标记.(2)群体遗传多样性指标大都是尼萨罗非鱼>尼罗罗非鱼>萨尼罗非鱼>萨罗罗非鱼.(3)群体内平均遗传相似指数为:萨罗罗非鱼>萨尼罗非鱼>尼罗罗非鱼>尼萨罗非鱼.(4)萨罗罗非鱼对杂交子代的影响要远大于尼罗罗非鱼;正交子代尼萨岁非鱼可能在生产上更有利用价值和具有更高的选育潜力.     

尼罗罗非鱼神经垂体的超微结构研究

尼罗罗非鱼神经垂体存在A_1、A_2和B型神经分泌纤维,其终端与腺细胞、毛细血管、组织间隔、基板或脑垂体细胞形成直接轴——腺突触联系、间接轴——腺联系和直接轴——脑垂体细胞突触联系,构成下丘脑对脑垂体腺细胞支配的三种联系形式。在神经垂体组织中还存在Ⅰ型和Ⅱ型两种结构和功能不同的脑垂体细胞。     

乌鳢一个养殖群体中性别连锁AFLP标记的筛选

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尼罗罗非鱼ghrelin基因的多态性及其与生长性状相关SNP位点的筛选

为了研究尼罗罗非鱼(Oreochromis niloticus)生长激素促分泌素基因(ghrelin)的多态性及其与生长的相关性, 研究以两个尼罗罗非鱼群体(快长群体和基础群体)的DNA样本各40份为模板, 通过PCR扩增和测序获得ghrelin基因序列。通过Dnasp v5和MEGA 5.0分析序列多态性、筛选有效SNP 位点; 采用Snapshot法对两个群体子代ghrelin基因中SNP位点进行基因分型, 然后分析SNP位点基因型与生长性状的相关性。结果表明, 快长群体ghrelin基因中的单核苷酸变异位点数(S)比基础群体要少, 而核苷酸多态性(Pi)和平均核苷酸差异数(K)要略高于基础群体。共筛得3个有效SNP 位点(S1、S2和S3), 均分布于第1个内含子中。遗传结构分析表明, 3个SNP 位点在两个群体的子代中均为低度多态性位点(PIC0.25), 但处于Hardy-Weinberg平衡(P0.05);快长群体子代中3个SNP 位点的观测杂合度、期望杂合度和多态信息含量等遗传多样性参数均小于基础群体子代的相应值, 3个SNP 位点的遗传多样性参数、基因型和基因频率在同一群体中高度一致, SNP 位点之间完全连锁。两个群体子代中3个SNP 位点处的优势基因型相同, 但快长群体子代中优势基因型频率要明显大于基础群体子代中相应基因型频率。对两个群体子代的生长性状与SNP基因型进行关联性分析的结果表明,尼罗罗非鱼个体的多项生长指标(体重、体长、体高、头长和尾柄高等)在不同基因型中存在显著差异(S1:GG AG, S2:TT AT, S3:AA AT)(P0.05)。D1双倍型(S1:GG, S2:TT, S3:AA)所对应的尼罗罗非鱼个体的多项生长指标(体重、体长、体高、头长和尾柄高等)显著高于D2双倍型(S1:AG, S2:AT, S3:AT)。以上结果表明, 尼罗罗非鱼ghrelin基因3个SNP 位点完全连锁, D1双倍型与快长性状密切相关, 可作为尼罗罗非鱼分子标记辅助育种的候选标记。     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

棉花纤维发生相关AFLP标记

以徐州142及其无绒无絮（无纤维）突变体(fl)为材料,利用AFLP标记技术对该近等基因系进行DNA多态性分析。在64对引物产生的6360多条扩增带中,发现1条差异带（CF1）稳定出现在有纤维和无纤维之间。进一步用徐州142无绒无絮突变系与纤维发育正常的渝棉一号的杂交后代F2、F3分离群体以及几个陆地棉品种作材料验证。结果表明,差异带（CF1）与纤维的产生呈共分离,该差异带是与棉花纤维发生相关的分子标记。进而对CF1进行回收、克隆和序列测定,CF1长205bp,通过6个开放阅读框推断的氨基酸序列,与酚羟化酶α-亚基、外表皮蛋白C、NADH脱氢酶亚基1、NADH－CoQ氧化还原酶、2－氧代－铁氧化蛋白－氧化还原酶以及假拟14．5kD蛋白（hypotheitcal 14.5kD protein）有较高的相似性。     

尼罗罗非鱼消化道肥大细胞的组化性质

实验采用改良甲苯胺蓝(MTB)、阿利新蓝-沙黄(AB/SO)、甲基绿-派洛宁(MG-P)、天青Ⅱ-伊红-瑞氏混合液和硫堇5种组化染色法,对尼罗罗非鱼(Nile tilapia)消化道组织中的肥大细胞(Mast cell,MC)组化性质进行研究。尼罗罗非鱼的食管、胃及小肠壁内均显示有肥大细胞,在食管和胃的切片标本上肥大细胞主要分布在黏膜固有层和胃腺体之间。在肠道中的肥大细胞主要分布在黏膜固有层和肠上皮下方,少量肥大细胞存在于黏膜下层结缔组织中。细胞呈圆形、椭圆形,也有长梭形的。而且肥大细胞有沿血管分布的特点。5种组化染色结果表明:AB/SO、MTB和MG-P显示的MC效果较好,尤其AB/SO染色效果最好,肥大细胞轮廓清楚,胞质颗粒较清晰;尼罗罗非鱼肥大细胞胞浆颗粒都呈红色,即肥大细胞胞浆主要含肝素,不含组胺。天青Ⅱ-伊红-瑞氏混合液染色效果也很好,但被染的肥大细胞较少;80%乙醇硫堇染色,在尼罗罗非鱼消化道各段组织中均未能鉴定出肥大细胞。尼罗罗非鱼消化道肥大细胞大多分布于浅层的黏膜或血管、腺体周围的结缔组织等易表露于环境抗原的位点。罗非鱼消化道黏膜层结缔组织中的肥大细胞与大多数脊椎动物的肥大细胞一样,具有沿血管分布的特性,说明硬骨鱼的肥大细胞如哺乳动物肥大细胞一样与血管有着密切的关系。       

吉丽罗非鱼(尼罗罗非鱼♀×萨罗罗非鱼♂)及其两亲本遗传变异的微卫星分析

吉丽罗非鱼是由耐盐性较强的萨罗罗非鱼做父本与生长速度较快的尼罗罗非鱼做母本进行杂交、杂交后代自交产生,2009年全国水产原良种审定委员会审定为养殖新品种。为了分析吉丽罗非鱼及其两亲本遗传特性,选择有代表性的6对微卫星引物,对这3种罗非鱼遗传变异进行研究分析。研究结果表明:(1)6对微卫星引物扩增产物片段大小为180～350bp,共发现21个等位基因,鱼类群体间、微卫星座位间及等位基因间都存在极显著差异。(2)有效等位基因数(Ne)、Nei's基因多样指数(H)和多态信息含量(PIC)值等群体遗传多样性指标都是吉丽罗非鱼>尼罗罗非鱼>萨罗罗非鱼,吉丽罗非鱼PIC值达到了0.657,属于高度多态性。(3)吉丽罗非鱼与萨罗罗非鱼的遗传距离要比与尼罗罗非鱼的近,萨罗罗非鱼对吉丽罗非鱼的遗传影响要大于尼罗罗非鱼。     

LHRH-A对尼罗罗非鱼生长及生长轴相关基因表达的影响

促性腺激素释放激素(GnRH)的主要作用是刺激脑垂体促性激素(GtH)的释放, 亦可促进鱼类生长激素(GH)的释放。促黄体素释放激素类似物(LHRH-A)是哺乳类GnRH的类似物, 为了分析LHRH-A对尼罗罗非鱼生长调节的作用, 设计了长期和短期2个实验, 采用腹腔注射(剂量0.1 μg/g体重)方法, 分析LHRH-A对尼罗罗非鱼绝对生长率、特定生长率、肝体系数和肥满度的影响, 并应用荧光实时定量PCR方法检测在注射LHRH-A后不同时段(6h、12h、24h、2周)尼罗罗非鱼垂体GH、肝脏GHR和肝脏IGF-I基因的表达变化。结果表明, LHRH-A组尼罗罗非鱼的绝对生长率、特定生长率、肝体系数、肥满度均显著高于对照组(P<0.05); 注射LHRH-A后12h、24h垂体GH mRNA表达水平均显著升高(P<0.05), 2周后恢复到对照组水平; 注射LHRH-A后24h和2周肝脏GHR mRNA表达水平显著上升(P<0.05); 注射LHRH-A后6h肝脏IGF-I mRNA表达水平显著升高(P<0.05), 12h、24h和2周恢复到对照组水平。以上结果提示, LHRH-A可显著上调尼罗罗非鱼生长轴相关基因的表达, 从而促进鱼类的生长。     

Mitotic chromosomal abnormalities and DNA polymorphism in the Nile tilapia (Oreochromis niloticus,Linnaeus, 1758) as a biomarker for water pollution by heavy metals

Mitotic chromosomal aberrations and DNA polymorphism (RAPD marker) were carried out on the Nile tilapia Oreochromis niloticus collected from five sites in Minia governorate, Egypt to test their applicability as biomonitors for heavy metal contaminants of water. The diploid chromosome number of O. niloticus population was 2 n = 44. Different types of chromosomal aberrations were recorded (e.g., deletion, ring, centromeric attenuation, end-to-end association, dicentric chromosome, stickiness chromosomes, endomitosis, fragments and chromatid gap). The chromosomal aberrations varied between O. niloticus population collected from five sites, and the most common type was ring (R) chromosomes. Samples obtained from Bahr Yousef and Irrigation drain exhibited the highest aberration frequency. The frequency of chromosomal aberration was positively correlated with the concentration of heavy metals where their concentration in the surface water of Irrigation drain and Bahr Yousef exceeded the limits defined by WHO as well as the concentration of Pb in muscles. The RAPD marker was also used to identify genetic variation among Nile tilapia samples collected from five different water sources. It created polymorphic and unique bands that can be used as genetic markers to track DNA variations. The dendrogram also revealed that exposure to heavy metal pollution causes gradual accumulation of variance, whereas areas subjected to environmental stress showed higher genetic variation and clustered together.     

Large extent of mitochondrial DNA transfer from Oreochromis aureus to O. niloticus in West Africa

Introgressive hybridization has an important evolutionary significance in terms of gene diversity and speciation. Among the major groups of vertebrates, fish show a strong propensity to hybridize. In order to highlight the possible occurrence of gene flow between two tilapia species, Oreochromis niloticus and O. aureus, a comparison of allozyme and mitochondrial DNA (mtDNA) polymorphism was performed on sympatric and allopatric populations of these two species. Nuclear data were congruent with the morphological identification of O. niloticus and O. aureus populations. In opposition, the mtDNA analysis resulted in two strictly differentiated groups which did not follow the morphological and nuclear DNA classification. The first group consisted of East African O. niloticus populations and the second included all the O. aureus populations and the West African O. niloticus populations. Moreover, in some cases, the same sequences were detected in both species. These data strongly support a differential introgression of mtDNA from O. aureus to O. niloticus involving all the West African area. This work points out the risk of misinterpretation of mtDNA or nuclear DNA data when only one single class of marker is used.     

Effects of sodium chloride on water quality and growth of Oreochromis niloticus in earthen ponds

A 2 × 2 factorial experiment was conducted between April and August 2001 to evaluate the effects of NaCl on Oreochromis niloticus growth and water quality in twelve 0.015 ha limed ponds. The design involved fertilizer and salt as factors with two treatments for each factor. Each salt‐fertilizer combination was replicated three times and fish were not offered external food during the 98 days culture. Growth of O. niloticus was significantly enhanced by salt at higher fertilizer level but not at the lower fertilization level. Although salt had no direct effect on fish growth, a significant salt‐fertilizer interaction was demonstrated. Water quality variables, with a few exceptions, were similar among the salted treatments. Total ammonia increased significantly with fertilization level, but the values were similar in salted and unsalted treatments at the same fertilizer level. Total nitrogen was higher in the salted than unsalted treatments while the organic matter content was lower in the salted treatments. The reasons for the better growth of O. niloticus, are discussed with respect to water quality variables. The present results suggest that fertilization rates of 20 kg N ha^?1 may have negative effects on fish growth. However, presence of sodium chloride seems to reduce these negative effects.     

Growth characteristics of skeletal muscle tissue in Oreochromis niloticus larvae fed on a lysine supplemented diet

The effect of lysine amino acid supplementation on the growth characteristics and morphological pattern of skeletal muscle tissue in Nile tilapia Oreochromis niloticus larvae was evaluated. There were four treatments (T) with increasing levels of lysine supplement (T1 = 0·0%; T2 = 1·1%; T3 = 1·7%; T4 = 4·0%) and one treatment with a commercial diet (T5). In all treatments, morphological and histochemical muscle tissue analyses were similar. Two distinct layers were identified: a superficial red layer, more developed in the lateral line region, formed by fibres with intense to moderate NADH‐TR reaction and strong acid‐stable mATPase activity, and a deep white one, most of the muscle mass, formed by fibres with weak NADH‐TR reaction and strong alkali‐stable mATPase activity. There was an intermediate layer between these two layers with fibres exhibiting either weak acid‐stable or acid‐labile mATPase activity. Body mass increase was significantly higher in T5 than in the lysine treatments (T1–T4). There was no difference in number and diameters of muscle fibres between lysine treatments. In T5, muscle fibre diameter and number were higher. The frequency of red fibres with diameters ≤8 μm was higher in the lysine treatments, and with diameters between 16 and 24 μm, was higher in T5. Most white fibre diameters in T5 were significantly larger than 24 μm and in T1–T4 were between 8 and 16 μm. Cell proliferation was higher in the lysine treatments and muscle growth in T5 was mainly by fibre hypertrophy.     

Otolith microstructure pattern in Oreochromis niloticus (L.)

The microstructure of otoliths from young and old Oreochromis niloticus (L.) were studied. Otoliths were prepared histologically except for those from newly hatched fish. Hatching results in the formation of a check in the otoliths, which appeared 1 day later. Other checks are rare in juvenile otoliths but common in adult otoliths. Faint and non-daily increments were observed within the hatching check. After hatching, increments were deposited daily. Sub-daily increments were faint and narrow, they were present in the area along the dorso-ventral axis of the otolith but did not continue into the lateral region. Discontinuous zones in the medial area appeared different from those in the lateral area. New growth centres were not only found in the juvenile fish otoliths, but also in adult fish otoliths.     

The composition and nutritional status of the diet of Oreochromis niloticus in Lake Chamo, Ethiopia

The stomach contents of Oreochromis niloticus from L. Chamo were microscopically examined and chemically analysed in order to study the relationship between food quality, growth and condition. More than 20 genera of phytoplankton plus Copepoda, Rotifera and nauplii were present in the stomach contents. The diatom, Melosira , was the most dominant alga in the diet, contributing about 30% of the organic matter. The composition of the food in terms of organic nutrients was dominated by carbohydrate (20.3% dry weight). Both protein and fat made low contributions to the diet; 10% and 5.8% dry weight, respectively. The overall performance of the fish in assimilating food was high: 43.3% for ash free dry weight, 61.2% for protein, 57.2% for lipid, 29% for carbohydrate and 55.7% for energy. The digestible protein-energy ratio was 15.7 mg kJ⁻¹, suggesting that the food was qualitatively good both for fast growth and for maintaining good condition. It was concluded that the small amount of good quality food ingested, the low fish population density in the lake and the high temperature of the water are the major factors responsible for making O. niloticus a fast-growing fish in Lake Chamo.     

Isolation and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus L.)

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.     

Observations on limnological conditions associated with a fish kill of Oreochromis niloticus in Lake Chivero following collapse of an algal bloom

Possible causes of deaths of Oreochromis niloticus in Lake Chivero were examined in relation to changes in limnological conditions monitored over a 25‐month period. The fish deaths coincided with the collapse of an algal bloom that had developed and builtup in the lake for 8 months. Chlorophyll a and dissolved oxygen increased to average concentrations of 42.4 μg l^?1 and 10.9 mg l^?1 respectively prior to the collapse of the bloom. Dissolved oxygen decreased when the bloom started to die off and coincided with the fish deaths when the average surface dissolved oxygen concentration in the lake was 3.9 mg l^?1 and was at a depth of 5 m <2 mg l^?1. Mortality probably resulted from depressed oxygen levels caused by the high oxygen demand from the massive algal die‐off and released algal toxins. This is the first time that die‐off of algae has been linked to fish‐kills in Lake Chivero as occurs in other hypereutrophic systems.     

Genetic characterization of four strains of Nile tilapia (Oreochromis niloticus L.) using microsatellite markers

Four domesticated strains of Nile tilapia (Oreochromis niloticus L.) were genetically characterized using 14 microsatellite markers and 64 animals per strain. Two strains, Chitralada (AIT) and International Development Research Centers (IDRC) were obtained from the AIT institute, Bangkok, Thailand. The GIFT strain (5th generation) came from NAGRI, Thailand, and the GÖTT strain was supplied by the University of Göttingen, Germany. The average numbers of alleles per marker were 5.0 (GÖTT), 5.4 (AIT), 5.6 (IDRC) and 7.5 (GIFT). Private alleles were found at all markers with the exception of two. No fixation of alleles was found at any marker. Population differentiation, F_ST, was 0.178 (great genetic differentiation) and confirmed grouping of the animals in strains. The expected level of heterozygosity ranged from 0.624 to 0.711, but the observed level of heterozygosity significantly deviated from the expected level in three strains. This was probably because of small population size. Moderate to great genetic differentiation was found between strains. A phylogenetic tree reflected the strains known histories. Application of the Weitzman approach showed that all strains have added value for the total genetic diversity and thus should be retained.     

微囊藻毒素对尼罗罗非鱼原代肝细胞致毒机理的探讨

采用离体细胞培养诱导方法,研究微囊藻毒素-LR(microcystin-LR,MC-LR)对尼罗罗非鱼(Oreochromis niloticus)原代肝细胞的毒性效应.尼罗罗非鱼原代肝细胞经10、50、150、500 μg/L MC-LR体外诱导24h后,单细胞微量凝胶电泳(SCGE)检测显示,与对照组相比处理组出现明显的彗星拖尾现象,说明MC-LR可引起尼罗罗非鱼肝细胞DNA的损伤,并随着剂量的增加,DNA的损伤程度增大.PI/Annexin V双染色流式细胞仪(FCM)检测表明MC-LR能明显引起肝细胞凋亡,与SCGE结果一致,且DNA损伤程度越大,细胞早期凋亡率越高,呈现明显的时间、剂量依赖性.本研究为进一步从分子、细胞水平阐明MC-LR的毒性以及致毒机理提供重要的理论依据.