Genetic relationship between broodstocks of olive flounder, Paralichthys olivaceus (Temminck and Schlegel) using microsatellite markers

For the first generation of a selective breeding programme, it is important to minimize the possibility of inbreeding. This mostly occurs by mating between closely related individuals, while proper mating can provide an opportunity to establish the base families with wide genetic variation from which selection for subsequent generations can be more effective. Genotyping with microsatellite‐based DNA markers can help us determine the genetic distances between the base populations. The genetic markers further facilitate the identification of the correct parents of the offspring (parentage assignments) reared together with many other families after hatching. We established a genetic analysis system with microsatellite DNA markers and analysed the genetic distances of three farmed stocks and a group of fish collected from wild populations using eight microsatellite markers. The averaged heterozygosity of the farming stocks was 0.826 and that of the wild population was 0.868. The hatchery strains had an average of 8.6 alleles per marker, which was less than a wild population that carried an average of 14.3 alleles per marker. Significant Hardy–Weinberg disequilibrium (HWDE) was observed in two farming stocks (P<0.05). Despite relatively low inbreeding coefficiency of the hatchery populations, the frequency of a few alleles was highly represented over others. It suggests that the hatchery stocks to some extent have experienced inbreeding or they originated from closely related individuals. We will develop a selective program using the DNA markers and will widen the usage of the DNA‐based genetic analysis system to other fish species.     

Genetic variation in farmed populations of the gilthead sea bream Sparus aurata in Greece using microsatellite DNA markers

Genetic variation in seven reared stocks of gilthead sea bream Sparus aurata, originating from Greek commercial farms, was assessed using five polymorphic microsatellite markers and was compared with that of two natural populations from the Ionian and the Adriatic Seas. The total number of alleles per marker ranged from 11 to 19 alleles, and hatchery samples showed the same levels of observed heterozygosity with samples from the wild but substantially smaller allelic diversity and expected heterozygosity. The global genetic differentiation for the cultivated samples was significant as indicated by F_st analysis, which might indicate random genetic drift and inbreeding events operating in the hatcheries. On the contrary, no significant difference was found between the two wild populations. Population pairwise tests between farmed and wild stocks were also significant, with the exception of one hatchery sample, the Central Greece 1, which was not significantly different from the two wild samples perhaps due to its recent use in aquaculture from wild‐caught animals. The UPGMA tree topology grouped the wild samples together with the Central Greece 1 stock, and showed a clear division between wild and farmed sample sets for the six remaining hatchery samples. Knowledge of the genetic variation in S. aurata cultured populations compared with that in the wild ones is essential for setting up appropriate guidelines for the proper monitoring and management of the stocks either under traditional practices or for the implementation of selective breeding programmes.     

Microsatellite genetic variation in wild and hatchery populations of the sea cucumber (Apostichopus japonicus Selenka) from northern China

Farming of the sea cucumber Apostichopus japonicus (Selenka) started 20 years ago and is still in rapid expansion in China. In order to assess the genetic status of both wild and cultivated stocks of this species, we used eight microsatellite markers to estimate the level of genetic diversity within five hatchery stocks and two wild populations of A. japonicus, and compared the degree of genetic differentiation between them. High levels of polymorphism were observed over all loci. The mean alleles and expected heterozygosities over the seven stocks were 10.4–12.3 and 0.735–0.783 respectively. The results of the microsatellite survey provide no evidence to show that hatchery practice of the sea cucumber in China to date has significantly affected the genetic variability of the cultured stocks. Significant differentiation was found between most pairs of the hatchery stocks and wild populations (F_st range: 0.008–0.036), and no obvious difference was detected between the wild populations (F_st=0.008). The information on the genetic variation and differentiation obtained in this study can be applied for future genetic monitoring of A. japonicus aquaculture stocks and will be useful for future genetic improvement by selective breeding, and for designing suitable management guidelines for these genetic materials.     

Genetic diversity and relatedness estimates for captive barramundi (Lates calcarifer,Bloch) broodstock informs efforts to form a base population for selective breeding

Aquaculture of barramundi or Asian seabass (Lates calcarifer) is growing in both Australia and Southeast Asia and there is substantial interest to improve production efficiency through selective breeding. The establishment of a large and genetically diverse base population is a prerequisite for a sustainable and long‐term productive breeding program. Before selective breeding programs can begin for Australian barramundi it is important to assess the overall genetic diversity of current captive broodstock populations. To address this question, 407 captive barramundi broodstock from eight separate Australian broodstock populations were genotyped using 16 polymorphic microsatellite DNA markers. A Bayesian STRUCTURE analysis indicated that captive Australian broodstock are broadly divided into two genetic stocks. Multivariate analysis between broodstock individuals and pairwise F_ST between broodstock populations also supported the existence of two stocks. Comparisons with data obtained from natural stocks suggested that hatchery individuals were either sourced from the two stocks or represented an admixture between them. Genetic diversity was low within each broodstock population (allelic richness ranged from 2.67 to 3.42 and heterozygosity ranged from 0.453 to 0.537) and relatedness estimates within hatcheries were generally low (average r was equal to 0.141). We recommend sourcing captive individuals according to high levels of neutral genetic diversity and low levels of relatedness for the establishment of a base population. We also make recommendations about including genetically diverse wild individuals.     

利用高通量测序开发的熊本牡蛎微卫星标记评价野生和养殖群体的遗传多样性及通用性

利用高通量测序的方法,从熊本牡蛎基因组中开发了20对具有多态性的微卫星标记,通过微卫星标记位点比较了野生群体和养殖群体的遗传多样性。野生群体中,所有位点共扩增出330个等位基因,等位基因数(N_a)范围为6～39,平均等位基因数为16.500 0;有效等位基因数(N_e)范围为1.352 9～33.361 7,平均值9.517 2;观测杂合度(H_o)范围为0.200 0～1.000 0,平均值0.671 5;期望杂合度(H_e)范围为0.265 6～0.987 7,平均值0.832 1;ShannonWeiner指数(Ⅰ)范围为0.648 3～3.585 8,平均值2.276 9;多态信息含量(PIC)范围为0.254 5～0.969 2,平均值0.803 5,共有16个位点符合Hardy-Weinberg平衡。养殖群体中,N_a平均值为10.250 0,N_e平均值为5.843 4,H_o平均值为0.639 1,H_e平均值为0.763 6,I平均值为1.791 4,PIC平均值为0.720 7。结果显示,熊本牡蛎养殖群体的遗传多样性低于野生群体,但仍然维持在高度多态水平。研究表明,在熊本牡蛎人工繁育过程中,使用大数量的亲本进行繁育,可有效防止选育群体的遗传多样性降低,但人工选育对选育群体的遗传多样性也产生了一定的影响。另外,分析了这些引物在近缘种葡萄牙牡蛎、长牡蛎、香港牡蛎、有明牡蛎、僧帽牡蛎、咬齿牡蛎以及舌骨牡蛎中的通用性情况,发现XB1-6、XB1-39和XB1-45 3个位点在8个物种中均能扩增出目的条带,XB1-41仅能在熊本牡蛎中扩增出目的条带。     

Genetic comparison of Iranian and Azeri populations of the oriental bream Abramis brama orientalis (Berg) using microsatellites

The aim of this study was to compare, levels of genetic polymorphism between Iranian and Azeri bream (Abramis brama orientalis) populations using five microsatellite loci. The reduced number of alleles in the Iranian populations compared with the Azeri population led us to infer that the genetic variability of the Iranian stocks has been reduced due to inbreeding and genetic drift. Significant population differentiation (F_st) was observed between the Azeri and the Iranian populations, which could be explained by the higher number of alleles in the Azeri population. Significant deviations from Hardy–Weinberg equilibrium were found at more loci in the Iranian populations than the Azeri population. Beyond the hypothesis of null alleles, the heterozygote deficiency may have arisen due to breeding between related individuals in the Iranian populations. This investigation represents the first approach to the knowledge of the genetic variability of Iranian and Azeri bream populations using microsatellite markers, and the reported results could be of interest for management and conservation programmes of this species in Iran.     

Genetic variability of cultured populations of the Pacific abalone (Haliotis discus hannai Ino) in China based on microsatellites

The genetic diversity of cultured populations of the Pacific abalone (Haliotis discus hannai Ino) from northern China was analysed using seven microsatellite markers. The microsatellite loci were polymorphic for all the populations, with an average of 8.7 alleles per locus (range 8.0–9.4). The mean observed and expected heterozygosities were 0.547 (range 0.500–0.596) and 0.774 (range 0.754–0.787) respectively. The allelic diversity in terms of number of alleles per locus was considerably lower than that previously found in wild populations (range 21.8–23.0), indicating that bottleneck effects occurred when each population was founded. Significant genetic differentiation among the five cultured populations was shown using F_st and R_st values, and pairwise comparison based on allelic distribution. A neighbour‐joining analysis of the genetic distance did not show a consistent relationship between the geographic and the genetic distances, suggesting the existence of exchanges of breeds and eggs between the hatcheries. The results obtained in this study are useful for a number of areas of interest for fisheries management and the aquaculture industry, especially with regard to breeding programmes.     

白梭吻鲈3个群体的遗传结构比较

为了加强良种选育工作,切实做好亲本遗传管理,防止近交衰退的发生,利用20个微卫星分子标记对白梭吻鲈(Sander lucioperca)新疆野生群体及山东和苏州2个养殖群体的遗传结构进行检测。结果表明,20个微卫星标记中18个有扩增产物,14个呈现多态性;每个位点的等位基因数为2~6个,平均等位基因数为3.6个,3个群体的平均等位基因数为2.57~3.36,平均观测杂合度为0.5085~0.5621,平均多态性信息含量为0.3931~0.4764,表明3个白梭吻鲈群体的遗传多样性处于中等偏低水平,遗传多样性大小为:新疆群体苏州群体山东群体。群体的Fst为0.1798,表明群体间有一定的遗传分化。在白梭吻鲈人工繁殖与养殖过程中,必须加强亲本遗传结构监测并维持一定数量的亲本规模,以利于其产业的持续发展。     

棘头梅童鱼七个野生群体遗传多样性的微卫星分析

为研究中国沿海地区棘头梅童鱼群体的遗传多样性,利用微卫星标记技术,采用9对微卫星引物对中国连云港(LYG)、大丰(DF)、崇明(CM)、舟山(ZS)、温州(WZ)、宁德(ND)、厦门(XM)棘头梅童鱼7个野生群体的遗传多样性和遗传结构进行了分析。结果显示,实验检测到63个等位基因,各位点等位基因数为3~13个,有效等位基因数为1.7510~8.0317;各位点的观测杂合度(Ho)为0.3596~0.7854,期望杂合度(He)为0.4300~0.8780;多态信息含量(PIC)为0.3604~0.8631,其中有2个位点表现为中度多态(0.25PIC0.5),7个位点表现为高度多态(PIC0.5),具有较丰富的遗传多样性水平。Hardy-Weinber平衡分析显示,7个群体的大部分位点未偏离平衡。基于群体间Nei氏标准遗传距离构建的7个野生群体UPGMA系统进化树结果显示,ND和WZ群体遗传关系最近,ZS和WZ群体遗传关系最远,WZ和ND聚为一支,但总体上没有显著的遗传分化。     

Genetic diversity and structure of Northern Sheatfish (Silurus soldatovi) assessed by newly developed and cross‐species microsatellites

Twenty‐five polymorphic microsatellite markers, including 13 newly isolated markers and 12 existing cross‐species markers, were used to assess the genetic variation of 141 individuals from three cultured stocks of northern sheatfish, Silurus soldatovi, in China. Samples were collected from Heilongjiang (H), Tongerpu (T) and Liaoning (L). Allelic richness, expected and observed heterozygosity among 25 loci were calculated, and the mean A_e, H_o and H_e were estimated in three stocks. In low polymorphic locus HLJcf19 and HLJcf38, most individuals in all three stocks were homozygous and had only one same allele, while only few heterozygous individuals had the other allele in one certain stock. And all the three stocks showed excess of heterozygotes, because of significant negative F_IS values. Genetic distance, UPGMA dendrogram, as well as structure analysis, all indicated that Stock H and L were closer among three stocks. The results revealed that though all stocks had no reduction in recent time, their effective population size were significantly lower to the ideal value.     

微卫星分析四个大黄鱼群体的遗传多样性

为保护人工养殖大黄鱼的种质资源,用15对微卫星标记对来自浙江沿海地区的四个大黄鱼养殖群体共171个个体进行了遗传多样性分析.结果显示,这些标记在四个群体中均表现出丰富的多态性;共检测到206个等位基因;各基因座观测等位基因数7～23个,平均等位基因数13.73,大小在88～318bp之间.四个群体的平均观测杂和度(Ho)为0.5981～0.6893,期望杂和度(He)为0.7319～0.7944,多态信息含量(PIC)0.7138～0.7836,表明四个养殖群体在所检测位点均具有较好的多态性.对遗传距离的计算结果表明闵粤东群体与反交群体较近,聚类结果中首先聚到一起.本研究首次选择微卫星标记评估人共选育大黄鱼群体的遗传多样性,并采用高精度的377 DNA测序仪进行检测,将对大黄鱼的种质资源保护提供科学依据.     

Use of Microsatellite DNA Profiling to Identify Japanese Flounder,Paralichthys olivaceus of Hatchery Origin

Japanese flounder, Paralichthys olivaceus, is an economically important marine fish species in Asia. A suite of 18 microsatellite markers chosen from published genetic linkage maps was used to carry out parentage assignments of 188 hatchery‐reared juveniles from a small number of breeders. The probabilities of exclusion for the 18 microsatellite markers were 0.604–0.913, and the effectiveness of combined probability of exclusion reached 100% when using the eight microsatellite markers with higher Excl 1 probabilities. The cultured and wild stocks (WSs) were differentiated in a release‐recapture population based on these markers. Of the 321 recaptured offspring, 28.34% were assigned to their parental pairs in our broodstock, whereas the remaining offspring could not be traced back to a possible sire or dam. Significant reduction in genetic diversity of the cultured stock (CS) had not been found compared with that of the WS. The results suggest that CSs released into the wild will not adversely affect the genetic structure of natural populations. Our results demonstrate that these markers provide an efficient tool for parentage assignments and genetic analysis of Japanese flounder.     

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.     

Genetic characteristics of Pacific cod populations in Korea based on microsatellite markers

Pacific cod (Gadus macrocephalus) is a commercially important species in Korea. However, overfishing in the 1990s caused a strong decline in catch, and in response, artificially cultured juveniles have been released annually to enhance the stocks. Cod fishing has recently increased in Korea, and the variation within the cod population necessitates the study of their genetic characteristics. In this study, we utilized two newly developed and eight previously reported microsatellite markers to examine the genetic characteristics of Pacific cod populations. We compared the genetic diversity and genetic structure of three cod populations from the Korean coasts with three populations from the North Pacific Ocean. Genetic differences were identified in the eastern sea cod population from Korea. These genetic differences included a large number of alleles per locus, a large number of unique alleles, higher frequencies of the most common alleles, and lower gene diversity compared to the other Korean populations. Additionally, the eastern sea cod population was genetically significantly differentiated from the southern and western sea populations in Korea, in which no significant genetic differentiation was detected (F _ST = 0.033 and 0.031, respectively, P < 0.005).     

Residual genetic variability in domesticated populations of the Pacific blue shrimp (Litopenaeus stylirostris) of New Caledonia,French Polynesia and Hawaii and some management recommendations

The Latin American shrimp Litopenaeus stylirostris was introduced in three different Pacific islands (Tahiti, New Caledonia via Tahiti, and Hawaii) and hatchery-propagated for 7–25 generations to develop shrimp farming based on these domesticated stocks. Three microsatellite markers have been used in an attempt to assess the genetic bases of the populations available to start a selective breeding program. The comparison of eight hatchery stocks (five New Caledonian, two Hawaiian and one Tahitian stocks) and one wild Ecuadorian population showed a much lower variability in the domesticated stocks than in the wild population, especially in New Caledonia and Tahiti (2–3.7 vs. 14–27 alleles per locus; 20–60% vs. 90% expected heterozygosity). The Tahitian and the New Caledonian stocks share the same alleles, suggesting that the loss of alleles occurred during the common past of these populations. On the contrary, New Caledonian and Hawaiian populations share only one common allele at the three loci studied. Although the low genetic variability and the resulting inbreeding of the New Caledonian stocks do not seem to affect their present performance, the results of this study demonstrate the usefulness of the introduction of new stocks in order to increase the potential responses to new controlled or uncontrolled selective pressures. The introduction in New Caledonia of the Hawaiian domesticated stocks, which would provide the local shrimp industry with 40% of the allelic diversity of the species, is advised and preferred to the one of wild animals in order to take advantage (i) of the spontaneous selection which occurred during domestication and (ii) of their favourable sanitary “specific pathogen free” status (no presence of four viruses: WSV, YHV, IHHNV, TSV) which limits the risk of introduction of pathogens.     

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。     

Parentage assignment of stocked black sea bream <Emphasis Type="Italic">Acanthopagrus schlegelii</Emphasis> in Hiroshima Bay using microsatellite DNA markers

The genetic contribution of 51 broodstock, comprising 29 females and 22 males, reared at Hiroshima City Marine Products Promotion Center for the production of stocked black sea bream was monitored during two consecutive years using seven microsatellite DNA loci. The high discrimination ability of these markers was reflected in the polymorphic identification content (PIC=0.831), the exclusion probability (Q≈1), and the low probability of identity index (I=3.635⁻¹⁰). The total number of breeders contributing to the mating process was estimated at 32 (62.7%) in 2000 and 30 (58.8%) in 2001. On pedigree reconstruction, 69.3% of the offspring were successfully assigned to a single broodstock pair. Loss of alleles accounted for 16.9% during seed production; nevertheless, 90.9% of males and 69.0% of females participated in the mating process. Based on microsatellite genetic tagging, 58.9% of the fish sampled during the two months after release were identified as hatchery stock, presenting no significant differences from wild conspecifics in either fork length or body weight.     

基于微卫星标记对长江江苏段鳙增殖放流效果评估

为评估长江江苏段鳙(Aristichthys nobilis)增殖放流效果,本研究基于微卫星标记的亲子鉴定技术,选用 10对扩增效果稳定的微卫星荧光标记引物,结合毛细管电泳对江苏省内良种场 563 尾鳙亲鱼和长江江苏段 687 尾回捕鳙进行基因分型,并对位点多态性及亲子鉴定结果进行了分析。结果表明,各位点等位基因介于 19~55 之间,其观测杂合度为 0.530~0.909(平均值为 0.748),期望杂合度为 0.818~0.929 (平均值为 0.882),多态性信息含量为0.797~0.924 (平均值为 0.871);Cervus3.0 模拟结果显示,当双亲未知且置信度为 95%时, 10 个微卫星位点的累积非亲权排除率为 99.996%。亲子鉴定结果发现,所有回捕鳙中有 42 尾与亲本之间存在亲子关系,被确认来源于鳙增殖放流群体,并由此推算此次评估亲本的放流子代对长江江苏段野生群体的贡献率为 6.11%。结果显示该研究进行的鳙亲子鉴定技术可为长江鳙增殖放流效果评估工作提供可靠的数据支撑,为长江渔业资源管理提供参考资料。     

Genetic changes in Oreochromis shiranus (Trewavas) associated with the early stages of national aquaculture development in Malawi

A study was carried out to investigate the genetic diversity during domestication of Oreochromis shiranus (Trewavas) and to see if it could be associated with events in the known history of aquaculture development in Malawi. Five polymorphic microsatellite loci were scored in 14 populations of O. shiranus and one population of O. mossambicus (Peters). The mean number of alleles per locus ranged from 4.4 ± 1.03 to 13.2 ± 3.31 and was higher in the wild populations than in the domesticated populations. Other measures of genetic diversity were also lower in the domesticated compared with the wild populations, and the decline in diversity was correlated with the time elapsed since the founding of the farm stocks. Ordination analysis grouped domesticated populations into three: (1) those that trace their genealogy from Lakes Chiuta and Chilwa populations and are now spread all over the country; (2) those that come from Lakes Malawi and Malombe; and (3) hybrids between O. shiranus and O. mossambicus. Genetic differentiation among farms was strongly influenced by the pattern of known exchanges among the farmers and introgressive hybridization that had occurred between O. shiranus and O. mossambicus in the farmers’ ponds. Thus, the process of genetic changes in the species subsequent to domestication are best explained and predicted by socio-economic factors that influence the behaviour of farmers, rather than by the time-and-distance models of standard population genetics.     

基于新开发微卫星标记评价番红砗磲两个野生群体的遗传多样性及近缘种通用性检测

采用磁珠富集和PCR筛选相结合的方法,得到番红砗磲的19个多态性微卫星标记。利用新开发微卫星标记对西沙群岛2个野生群体的遗传多样性进行比较分析,七连屿海域野生群体和永兴岛海域野生群体的平均观测等位基因数(Na)分别为11.105、11.895,平均有效等位基因数(Ne)分别为6.274、6.173,平均期望杂合度(He)分别为0.776、0.788,平均多态性信息含量分别为0.730、0.744,发现2个野生群体的遗传多样性都处于高度多态水平,说明其有效群体大小仍然保持在较高水平。Bonferroni校正后,在2个群体中各有4个位点偏离Hardy-Weinberg平衡。另外分析了这些引物在近缘种中的通用性情况,发现鳞砗磲中有7对微卫星引物具有通用性,6对具有多态性;无鳞砗磲中有3对微卫星引物具有通用性,1对具有多态性;诺瓦砗磲中有5对微卫星引物具有通用性,5对具有多态性;长砗磲中有9对微卫星引物具有通用性,8对具有多态性;砗蚝中有2对引物具有通用性,2对具有多态性。