Isolation and characterization of microsatellite DNA markers in endangered Mekong giant catfish Pangasianodon gigas

ABSTRACT:   The Mekong giant catfish is one of the largest freshwater fish in the world, and is endemic to the Mekong basin. Recently, the number of Mekong giant catfish has drastically decreased. In this study, seven microsatellite DNA markers of the Mekong giant catfish were developed for evaluation of the species' genetic diversity. Mendelian inheritance of four of seven markers was confirmed using offspring (F1) produced from one pair. Number of alleles per locus in the founder population ranged 1–4 with a mean of 2.8, and that in F1 ranged 1–2 with a mean of 1.6. The expected heterozygosity in the founder population ranged 0–0.66 with a mean of 0.38, and that in F1 ranged 0–0.5 with a mean of 0.28. The mean expected heterozygosity in the founder population is apparently lower than that in Pangasius bocourti and other freshwater species, but higher than that in endangered species. In the future, the microsatellite data in this study will be used in minimal kinship approaches, because these individuals constitute the basic seed for release into wild in an effort to conserve the native population of Mekong giant catfish.     

Annual sex hormonal profiles, gonad development and age determination of the Mekong giant catfish (Pangasianodon gigas, Chevey)

Annual sex hormonal profiles, gonad development and age determination of 18 (13–14 kg body weight) and three (145–226 kg body weight) Mekong giant catfish (MGC) (Pangasianodon gigas, Chevey) reared in earthen ponds in Chiang Mai and from the Mekong River in Chiang Rai, Thailand, respectively, were investigated. The hormonal profiles were determined from blood samples of the fish by electrochemiluminescence immunoassay during May to August in 2000. The highest testosterone levels of 0.06 ng mL^?1 in both sexes and the highest oestradiol of 47.8 and 14.23 pg mL^?1 in females and males, respectively, were observed in May. The gonadosomatic index was found to be 0.07 for males and 0.38 for females from ponds in comparison with 2.27 for males and 8.29 for females from the Mekong River. Higher development stages of spermatocytes and oocytes of the cultured fish in May than in February and November were demonstrated. No mature germ cells were obtained from either the males or females, indicating no sexual maturity. The average age of fish was determined from the annual rings of the cross‐section of the pectoral fins by a stereomicroscope. The average age of fish in earthen pond determined from the annual ring was 8 years, which agreed with the actual culture records, while those from the Mekong River were 21 years. This information will be beneficial for the breeding programmes and conservation of the MGC.     

Habitat use and movement of hatchery-reared F2 Mekong giant catfish in the Mae Peum reservoir, Thailand, studied by acoustic telemetry

The horizontal and vertical movements of eight immature hatchery-reared (F2) Mekong giant catfish Pangasianodon gigas were monitored using acoustic telemetry in the Mae Peum reservoir, Thailand, from September to December 2005. All tagged fish were successfully monitored throughout the study period. All fish moved throughout the reservoir for approximately 1 month after release. Subsequently, their utilized areas became small, and the fish utilized deep areas of the reservoir. The fish displayed diel spatial movement patterns between deep areas in the day and shallow areas in the night. The vertical movements of the fish were related to the environment declination such as existence of hypoxic water and thermocline. Our results suggest that the establishment of a protected area in addition to conventional fisheries regulations may sustain the fish population in this reservoir.     

长牡蛎3代人工选育群体的微卫星分析

进行群体选育时,因近交机率增加和有效亲本数的减少,可能导致选育群体的遗传多样性下降,进而引起选育群体的性状衰退。为监测长牡蛎人工选育群体在选育过程中的遗传差异,实验应用微卫星DNA标记对长牡蛎野生和人工3代选育群体及其基础群体的遗传多样性进行了研究。微卫星10个位点在所有群体中均表现出较高的多态性,6个群体的平均等位基因数范围为24.0～29.7个,期望和观测杂合度分别为0.925～0.956和0.724～0.809。与野生群体和基础群体相比,长牡蛎选育3代群体的平均等位基因数和等位基因丰富度略有下降,但杂合度水平未发生明显变化。哈迪—温伯格平衡(HWE)检验结果显示,60个群体—位点组合中47个群体—位点组合显著偏离HWE平衡(P<0.05)。Fis指数均为正值,平均范围0.152～0.233,表明各群体在10个位点上表现为一定程度的杂合子缺失。各群体间Fst值的范围为0.008～0.025,遗传分化程度较弱。结果表明,连续3代的人工选育尚未明显降低长牡蛎群体的遗传多样性,仍可以一定的选择压力对选育群体进行人工选育,从而保证长牡蛎的优良生长性状得到持续提高。     

大泷六线鱼放流群体与野生群体遗传多样性比较

基于线粒体DNA控制区高变区部分序列和4对微卫星标记,对大泷六线鱼放流群体及自然海域群体的遗传多样性与遗传差异进行了比较分析。线粒体DNA控制区序列分析的结果显示,413尾个体共检测到单倍型117种,其中仅Hap₃、 Hap₇和Hap₁7为共享单倍型,占总单倍型数目的 2.5%;放流、野生群体特有单倍型分别为20种和66种,分别占总单倍型数的17.09%和56.41%,放流群体特有单倍型数明显低于野生群体;放流群体和野生群体核苷酸多样性分别为0.005 1～0.006 7和0.005 8～0.007 5,单倍型多样性分别为0.856 7～0.949 9和0.883 1～0.954 9,遗传多样性均较高。微卫星标记分析结果显示,放流、野生群体平均等位基因数(Na)分别为13～44和13～27,平均多态信息含量为0.885 6和0.874 0,均具较高的遗传多态性;群体遗传结构分析结果表明,放流、野生群体间遗传分化水平较低。研究表明,山东近海大泷六线鱼放流群体与野生群体均具有较丰富的遗传多样性,且遗传结构未存在显著的群体分化...     

An evaluation of fish health‐management practices and occupational health hazards associated with Pangasius catfish (Pangasianodon hypophthalmus) aquaculture in the Mekong Delta,Vietnam

This study aimed to evaluate the current status on the use of probiotics, disinfectants and antimicrobials in hatcheries, nurseries and grow‐out farms producing Pangasius catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam. Semi‐structured interviews were conducted with 83 aquaculture enterprises (15 hatcheries, 32 nurseries and 36 grow‐out farms). Farmers reported use of a total of 24 different antimicrobials, e.g. for treatment of bacillary necrosis and motile aeromonad septicaemia, and a variety of disinfectants, probiotics and nutritional supplements. In contrast to small‐scale farmers, all large‐scale grow‐out farmers studied were certified and therefore had higher levels of formal education and specialized aquaculture training to diagnose and treat diseases. All farmers prepared their own medicated feed with a high risk of treatment failure, negative environmental impact from released antimicrobials and resistance development. Small‐scale farmers were at particular occupational health risks when handling antimicrobials and other chemicals, e.g. mixing medicated feed with bare hands. There is an urgent need to improve knowledge and use innovative approaches, e.g. private‐public partnerships, to assure a prudent use of chemicals, to improve capacity and access to disease diagnosis, particularly for small‐scale grow‐out farmers and nurseries. Efforts to control use of antimicrobials in aquaculture should be coordinated with the livestock and human health sectors taking an One‐Health approach.     

长江流域4个野生大眼鳜群体的遗传多样性分析

为探讨长江流域大眼鳜(Siniperca kneri)野生群体的遗传多样性和遗传结构,为长江大眼鳜野生群体资源的有效管理和合理开发利用提供基础科学数据支撑,本研究基于线粒体细胞色素 b (cytochrome b, Cyt b)基因及控制区(D-loop)全序列,对 4 个群体(赤水河群体-CS、南京群体-NJ、岳阳群体-YY、宜昌群体-YC)共计 79 尾个体进行了分析。结果如下:(1)获得了长 1141 bp 的 Cyt b 基因全序列,共检测到 26 种单倍型。单倍型多样性(haplotype diversity,Hd)指数为 0.685 (CS)~0.924 (YC),核苷酸多样性(nucleotide diversity,π)指数为 0.176%(CS)~0.285%(YY)。群体内与群体间的遗传距离均在 0.002~0.003。(2)获得了长度为 834~840 bp 的 D-loop 全序列,在 79 尾个体中共检测到46 种单倍型。单倍型多样性指数为 0.754 (CS)~0.990 (NJ),核苷酸多样性指数为 0.548%(CS)~1.412%(YC)。群体内遗传距离在 0.005 (CS)~0.014 (YC),群体间遗传距离在 0.008~0.012,提示 4 个野生群体均尚未达到亚种分化水平。分子方差分析(AMOVA)显示群体内的变异是总变异的主要来源,长江流域大眼鳜野生群体无显著遗传结构差异。     

Current status of farming practices of striped catfish, Pangasianodon hypophthalmus in the Mekong Delta, Vietnam

Aquaculture of catfish, Pangasianodon hypophthalmus (Sauvage), locally known as “ca tra”, and commonly referred to as striped catfish, river catfish and sutchi catfish, in Vietnam, having recorded a production of 683,000 tonnes in 2007, valued at about 645 million US$ is one of the largest single species based farming system, restricted to a small geographical area, in the world. The product is almost totally exported to over 100 countries as frozen fillets, as an acceptable alternative to white fish. Catfish is farmed mostly in earthen ponds, up to 4 m deep, in nine provinces in the Mekong Delta in South Vietnam. The results of the grow-out system of catfish farming in the Mekong Delta from a survey of 89 farms are presented. The farm size ranged from 0.2 to 30 ha with a mean of 4.09 ha. The frequency distribution of the yield in tonne/ha/crop and tonne/ML/crop corresponded to a normal distribution curve, where 75% of the farms yielded 300 tonnes/ha/crop or more. It was found that the yield per crop was significantly correlated (p < 0.05) to stocking density, pond depth and volume but not to pond surface area. Yields per crop was significantly different (p < 0.05) between upper and lower provinces of the Mekong Delta and water source (river versus channels), amongst others. It was evident that diseases and/or symptoms were observed to occur mostly in accordance with the onset of rains. In this paper the history of the catfish farming in the Mekong Delta is briefly traced, and current harvesting and marketing procedures as well as pertinent social elements of the farming community are dealt with.     

长江上游特有鱼类红唇薄鳅线粒体控制区遗传多样性研究

红唇薄鳅(Leptobotia rubrilabris)是长江上游特有鱼类,近年来资源量不断下降。本研究利用线粒体控制区序列片段(896 bp)分析了长江上游红唇薄鳅的遗传多样性及遗传结构,为其资源保护提供科学依据。共采集了119尾样本,来自长江上游江津、四川南溪、岷江下游蕨溪等。结果显示,共检测到58个单倍型,平均单倍型多样性为0.967,平均核苷酸多样性0.006 7,表明红唇薄鳅种群具有较高的遗传多样性。单倍型NETWORK网络关系图和分析系统树结果显示红唇薄鳅单倍型不按地理分布聚类,但是明显分成了2个谱系,表明红唇薄鳅群体发生了同域遗传分化。AMOVA分析结果显示,采样点和采样时间的群体间没有发生遗传分化。中性检验、错配分析及BSP(Bayesian skyline plot)分析表明红唇薄鳅Lineage 1种群在距今0.007 5~0.055 Ma(百万年)期间发生过种群选择或扩张事件。     

日本蟳4个野生群体遗传多样性的微卫星分析

利用10对微卫星引物对我国大连黑石礁(DL)、莱州湾(LZ)、青岛鳌山湾(QD)、海州湾(HZ)4个日本蟳野生群体的遗传多样性进行了分析。结果表明,不同引物获得的等位基因数从11～17不等,10个位点其平均等位基因数为13.600 0,平均有效等位基因数为8.592 0;各位点的平均观测杂合度(H_o)为0.318 2～0.858 4,平均期望杂合度(H_e)为0.846 6～0.923 9;平均多态信息含量(PIC)为0.828 0～0.914 0,说明各位点在4个日本蟳野生群体内均表现出很高的遗传多样性水平。各群体间遗传分化较大,基因分化指数(F_ST)为0.032 74～0.088 03。群体间遗传相似性系数、遗传距离及UPGMA 聚类分析表明,鳌山湾与海州湾群体亲缘关系最近,而海州湾和莱州湾群体亲缘关系最远。     

基于微卫星评估草鱼放流亲本对野生群体遗传多样性的影响

利用筛选的13对草鱼多态性微卫星标记,开展了2011至2015年长江中游草鱼亲本增殖放流对野生群体遗传多样性的影响评估。通过对各位点的遗传多样性分析,13个微卫星位点的多态信息含量为0.8622(0.657~0.950),基因多样度为0.8555(0.675~0.936)。15个群体的有效等位基因数为7.4503~10.1536,等位基因丰度为11.483~15.204,说明15个草鱼群体的遗传多样性水平总体较高。遗传分化指数分析表明,群体间不存在显著遗传分化(FST5%)。通过贝叶斯聚类分析和主成分分析可将草鱼群体分为4个组群,根据分组结果以及来源划分分别对草鱼群体进行AMOVA分析,发现遗传变异大部分来自于群体内个体间,组间及组内群体间的分化水平较低(FCT5%,FSC5%),与FST分析结果一致。研究表明,当前草鱼亲本增殖放流模式对野生群体遗传结构影响不明显。     

福建牡蛎选育群体的遗传多样性

利用8个微卫星标记对福建牡蛎(Crassostrea angulata)基础群体、‘金蛎1号’选育系F6和野生群体进行遗传多样性分析。结果表明,每个位点在各群体的等位基因数为7~24个,各群体在所有位点的平均等位基因数为10.3~17.6个,平均等位基因丰度为9.8~16.8。平均观测杂合度和平均期望杂合度分别为0.655~0.662和0.788~0.872。经邦弗朗尼校正,哈迪–温伯格平衡检验结果显示,在24个群体–位点组合中18个群体–位点组合显著偏离平衡(P0.01)。群体内近交系数F_(is)值介于0.0095~0.2874,平均值为0.1992,遗传分化系数F_(st)介于0.0224~0.1627,平均值为0.0767,暗示选育群体中存在较低水平的非随机交配现象,属于中度偏低分化。研究表明,连续的选育对群体的遗传分化产生了一定的影响,但是,选育群体仍然具有较高水平的遗传多样性。     

长牡蛎中国群体和美国群体杂交效应与三倍体的优势

将长牡蛎中国群体二倍体分别与美国群体二倍体和四倍体进行杂交,实验共设置4组,分别为杂交二倍体组、杂交三倍体组、中国二倍体组和美国二倍体组,比较了各实验组卵裂率、D幼率、D形幼虫大小及幼虫期、稚贝期的壳高生长、存活率等生物学指标,并估算杂交二倍体的杂种优势率和杂交三倍体的三倍体优势率。结果表明,杂交二倍体幼虫壳高生长的杂种优势率不明显,平均杂种优势率为1.21%,幼虫的存活率及稚贝的壳高生长表现出明显的杂种优势,平均杂种优势率分别为34.47%和20.39%。杂交三倍体的D形幼虫大小、幼虫和稚贝的壳高生长、存活率均表现出三倍体优势,D形幼虫大小三倍体优势率为5.19%,幼虫期壳高生长和存活的平均三倍体优势率分别为4.00%和19.92%,稚贝壳高生长和存活的平均三倍体优势率分别为30.18%和54.43%,200日龄,杂交三倍体鲜体质量的三倍体优势率为202.96%,存活三倍体优势率为73.60%。此外,稚贝期的杂交二倍体生长性状的杂种优势率和杂交三倍体的三倍体优势率均高于幼虫期的优势率。研究表明,中、美两地理群体杂交获得的三倍体长牡蛎子代生长和存活性状都比二倍体优良。杂交三倍体的优良性状主要是三倍体优势,杂交优势的贡献率还有待进一步实验证实。     

河南省小黄黝鱼群体遗传多样性

小黄黝鱼[Micropercops swinhonis (Günther, 1873)]是中国特有的一种小型淡水虾虎鱼类,广泛分布于长江及以北各水系,其中在河南省各水系均有分布。为评估河南省小黄黝鱼种质资源现状及其遗传多样性,本研究对河南省境内黄河、长江、淮河和海河4个水系77尾小黄黝鱼样品进行线粒体COI基因的扩增。结果显示,河南省小黄黝鱼的平均单倍型多样性(H_d)为0.89088,平均核苷酸多样性(π)为0.00361,其中淮河水系华行村种群(HXC)核苷酸多样性最高(π=0.00433),黄河水系马颊河种群(MJH)核苷酸多样性最低(π=0.00078);系统发育树和单倍型网络图结果均显示小黄黝鱼并未按照水系形成明显的地理遗传支系;根据4个水系进行分组, AMOVA分析显示仅16.00%的变异来自于水系间,而78.43%遗传变异来自各群体内部;同样地,未预先设定分组情况的SAMOVA分析也得到了相似的结果,不支持小黄黝鱼具有明显的地理遗传结构的假设;种群历史动态分析显示,河南省小黄黝鱼种群呈逐步增长趋势; Fu’s FS中性检验与错配分析表明,河南小黄黝鱼种群可能经历过近期群体扩张事件,且该过程可能与气候变为暖湿型有关。     

利用微卫星荧光多重PCR技术分析壳白长牡蛎3代人工选育群体的遗传多样性

为了探讨壳白长牡蛎人工选育对群体遗传变异的影响,实验利用4个多重PCR组合共10个微卫星标记分析了连续3代壳白长牡蛎人工选育群体和野生群体及基础群体的遗传多样性。结果发现,6个群体的平均等位基因数量为7.2~12.6,等位基因丰度为6.8~11.0,期望和观测杂合度分别为0.672~0.769和0.486~0.542;与野生群体相比,3代选育群体的平均等位基因数显著降低,但平均期望杂合度并无显著差异。哈迪—温伯格平衡检验结果显示,在60个群体—位点组合中有39个群体—位点组合显著偏离哈迪—温伯格平衡,近交系数F_(is)范围为0.215~0.342。群体间遗传分化指数F_(st)范围为0.005~0.076,处于中—低等的遗传分化水平。研究表明,虽然连续选育对群体的遗传多样性和遗传分化造成了一定程度的影响,但人工选育群体依然表现为较高的遗传多样性,仍可以一定的选择压力对选育群体进行人工选育。     

Genetic differentiation of the bronze featherback Notopterus notopterus between Mekong River and Tonle Sap Lake populations by mitochondrial DNA analysis

ABSTRACT:   Although the bronze featherback Notopterus notopterus is an important fish in South-East Asia, the population structure has not been investigated. In this study, genetic diversity and population structure of the bronze featherback were examined using nucleotide sequence analysis of the mitochondrial DNA control region for 332 fish collected from Tonle Sap Lake and the Mekong River in Cambodia. The average nucleotide diversity (π) of all samples was 0.033, and the Mekong River samples had higher nucleotide diversity (0.034) than Tonle Sap Lake samples (0.028). The nucleotide diversity between the lake and river samples varied from 0.029 to 0.037. The genetic differentiation between the river and lake populations was also supported by the pairwise F -statistic values and hierarchical analysis of molecular variance, indicating that the Tonle Sap Lake population is genetically isolated from the population in main stream of the Mekong River.     

中国沿海拟穴青蟹群体遗传多样性的微卫星分析

利用17对微卫星引物对我国沿海7个拟穴青蟹野生群体(杭州湾、三门湾、闽江口、东山湾、珠江口、北部湾、清澜港)进行了遗传多样性分析。结果表明,17个位点在所有青蟹群体中均为高度多态(PIC>0.5),共检测到253个等位基因;7群体的平均等位基因数(A)为9.41～10.94,平均有效等位基因数(N_e)为5.42～6.87,平均杂合度(H)为0.511～0.563,群体遗传多样性水平较高。分子方差分析(AMOVA)结果显示,94.13%的遗传变异存在于群体内,5.87%的遗传变异存在于群体间,两两群体间F_ST值为0.035 5～0.081 7(P<0.01),表明群体间遗传分化水平中等偏低。Hardy-Weinberg平衡检测表明,7群体普遍存在杂合子缺失现象。青蟹群体间遗传距离为0.253 0～0.571 9,UPGMA聚类分析表明,杭州湾与三门湾群体首先聚在一起,再与闽江口群体、东山湾群体聚为一支;珠江口群体与清澜港群体聚为另一支;两分支最后与北部湾群体聚类在一起。     

用微卫星标记分析不同形态变异类型日本囊对虾的遗传多样性

选用9个多态性微卫星分子标记,分析我国近海浙江舟山(ZS)、福建厦门(XM)、广东惠来(HLQ及HLB)、海南陵水(LS)及广西北海(BH)日本囊对虾6个群体2种形态变异类型群体间的遗传变异,探讨了日本囊对虾的种质资源状况。结果显示,9个位点在6个日本囊对虾群体中均为高度多态(PIC>0.5),共检测出235个等位基因;6个群体平均等位基因数在13.2～17.7;PIC平均值在0.851 2～0.903 5;平均观测杂合度(Ho)在0.729 6～0.788 9,平均期望杂合度(He)在0.880 5～0.925 1;表明群体遗传多样性水平较高。Hardy-Weinberg平衡检测显示,6个群体普遍存在杂合子缺失现象。分子变异方差分析(AMOVA)结果表明,遗传变异6.94%来自群体间,93.06%来自群体内。由ZS、XM和HLQ群体组成的形态变异类型Ⅰ与由HLB、LS和BH群体组成的形态变异类型Ⅱ之间的Fst均大于0.05,发生了中等程度的遗传分化;相同形态变异类型各群体间的Fst均小于0.05,遗传分化不明显。日本囊对虾群体间的遗传距离(DA)为0.178 5～0.621 8;UPGMA聚类分析表明,聚类关系符合距离隔离模式,BH群体和LS群体遗传距离最小,亲缘关系最近,它们首先聚在一起,然后与HLB群体聚为一支;XM群体和HLQ群体先聚在一起,再与ZS群体聚为另一支。     

香鱼野生群体和养殖群体遗传多样性比较

香鱼是分布于中国、日本和朝鲜的一种珍稀名贵经济鱼类,本实验比较分析了香鱼养殖和野生群体的遗传多样性。研究结果显示,在长度为445 bp的控制区部分序列上,鳌山卫养殖群体的单倍型多样度h(0.198 4±0.092 4)和核苷酸多样度π(0.000 8±0.000 9)显著低于东张水库野生群体(h=0.810 5±0.067;π=0.002 6±0.002 0),两群体产生了较大的遗传分化(F st=0.447,P=0);单倍型邻接关系树的拓扑结构简单,未呈现明显的地理谱系结构,日本香鱼个体与中国香鱼亲缘关系较远;东张群体的历史动态分析结果表明其可能经历过近期的群体扩张事件。无论是养殖群体还是野生群体,中国香鱼群体的遗传多样性现状不容乐观。