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.     

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.     

凡纳滨对虾引进群体和2个养殖群体遗传变异的微卫星分析

利用8个微卫星标记对引进的SPF凡纳滨对虾G0和两个养殖群体(G1,G2)进行遗传多样性分析。8个座位共获得64个等位基因,位点的等位基因数在5~l3之间。多态信息含量PIC在0.405 2~0.869 3之间,其中有6个位点为高度多态位点,适合于多态性分析。8个座位丢失的和新产生的等位基因共30个,占总数的47%。3个群体的平均观测杂合度分别为0.193 8、0.196 1、0.232 5,说明3个群体的遗传多样性较低。对近交系数Fis分析显示3个群体中存在近交,通过对哈迪温伯格平衡检验,显示所有座位均显著偏离平衡,存在杂合子缺失。通过配对Fst和Ne i遗传距离分析,显示3个群体之间有明显的遗传分化,说明种群结构发生了明显的遗传变异,变异可能来自于突变、随机漂变和选择的共同作用。实验结果能够很好地解释经过若干群体选育后,子代群体发生种质退化的现状,由此建议综合采用遗传育种的方法从引进的亲虾中筛选出性状优良稳定的仔虾作为虾苗。     

Individual heterozygosity levels and relative growth performance in Oreochromis niloticus (L.) cultured under Fijian conditions

Tilapia (Oreochromis spp.) (Pisces: Cichlidae) are important freshwater fishes that are widely cultured worldwide and have been introduced to the Fiji islands as an aquaculture resource. An O. niloticus (L.) ‘Chitralada’ stock cultured under Fijian conditions was studied to assess the potential of using individual multilocus heterozygosity as an indicator of relative growth performance. Allozyme analysis was undertaken in each of three generations (n = 50/generation) of ‘Chitralada’ grow-out. Subsequent correlation and regression analysis of 25 allozyme loci with individual body weights demonstrated no significant association between individual heterozygosity and growth rate in any of the generations. Correlation analysis between heterozygosity based on nine microsatellite loci screened in a subset of ‘Chitralada’ individuals (n = 43, generation 2) and morphological measurements, demonstrated a significant relationship between heterozygosity at locus UNH146. Homozygous individuals at this locus were characterized by larger lengths and weights than heterozygotes. No significant correlations with either length or weight were observed at any of the other eight microsatellite loci. Selecting broodstock therefore based solely on individual allozyme or microsatellite heterozygosity levels is unlikely to increase relative growth performance in the Fijian cultured O. niloticus‘Chitralada’ stock.     

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.     

Development of microsatellite markers and evaluation of genetic diversity of the Amazonian ornamental fish Pterophyllum scalare

The objective of the present work was to develop species-specific microsatellite markers for P. scalare and to analyze the diversity and genetic structure of a wild population, from the Amazon River, and three commercial stocks (common, marble, and clown morphological variants), from farmers in Vieras-Minas Gerais. Through microsatellite-enriched genetic libraries, 11 microsatellite markers with adequate amplification patterns were characterized. Population genetic analysis identified eight polymorphic loci that generated 66 alleles ranging from two alleles (PSCA1B3) to nine (PSCA2H1). The polymorphic information content ranged from 0.031 to 0.827. High genetic differentiation was observed between the wild population and the stocks, and moderate differentiation between the three stocks. Deviation in the Hardy-Weinberg equilibrium was observed in one locus in the wild population, in five loci in the common morphological variant, in two in the marble, and in two in the clown morphological variant. Bayesian analysis of genetic structure revealed the existence of two clusters, one represented by the natural population and the other by the stocks. The developed microsatellite markers serve as a tool for the analysis of diversity and genetic structure and conservation studies of P. scalare.

     

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.     

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.     

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...     

Parentage Determination and Effective Population Size Estimation in Mass Spawning Pacific Oyster,Crassostrea gigas,Based on Microsatellite Analysis

Seven high polymorphic microsatellite loci were used to determine the pedigrees in a mass spawning of Pacific oyster, Crassostrea gigas, and to estimate the genetic variability between broodstock and offspring. Parental assignment was performed on a total of 155 individuals, including 141 offspring, 8 candidate mothers, and 6 candidate fathers. The assignment results of real offspring were generally in agreement with simulation with a success rate over 99% using only six of these loci. The allelic diversity and observed heterozygosity (H_o) exhibited similarity between parents and offspring populations, but the expected heterozygosity (H_e) had a significant decrease in offspring. Although all the males and females contributed to the next generation, the variances of reproductive success and unequal sex ratio resulted in a decline in effective population size (N_e = 11.42). The inbreeding rate of this small‐scale, mass spawning population was estimated at approximately 16.5% per generation. This gave us an insight that when designing breeding programs based on mass spawning for future oyster cultivation generations, the higher inbreeding and lower effective population size should be considered.     

Test of Mendelian segregation among 10 microsatellite loci in the fourth generation of bester (Huso huso L. ×Acipenser ruthenus L.)

Ten microsatellite loci were tested on Mendelian segregation in the bester – hybrid of beluga, Huso huso L. and sterlet, Acipenser ruthenus L. in the fourth generation. All studied loci showed disomic inheritance and Mendelian segregation of their alleles. The molecular approach by microsatellite DNA analysis represents a reliable tool for interspecific characterization of sturgeons and their hybrids (different alleles of several loci are present in sturgeon fishes, and interspecific hybrids posesses both maternal and paternal specific alleles). This is the first report of the Mendelian segregation testing in interspecific hybrid of acipenserid fishes using microsatellite DNA markers.     

罗非鱼性别相关微卫星标记的初步筛选

为分析罗非鱼群体的遗传多样性以及筛选与罗非鱼性别相关的微卫星标记。应用24对微卫星引物,使用常规PCR及聚丙烯酰胺凝胶电泳的方法在两个尼罗罗非鱼(Oreochromis niloticus)群体和两个奥利亚罗非鱼(O.aureus)群体中初步筛选到UNH931、GM128、GM201、GM258、GM597以及UNH898共6个与性别相关的微卫星标记。然后使用降落PCR以及毛细管电泳的方法在两个尼罗罗非鱼群体、两个奥利亚罗非鱼群体以及ZY 1、WY 1、YY 1和YY 2型罗非鱼群体中进一步扩增这6个微卫星标记,统计各群体的遗传多样性参数:6个微卫星标记在上述群体共297个样本中检测到95个等位基因,其大小在97~302 bp之间,各位点在各群体等位基因数在1~13个之间,各群体平均观测等位基因数为2.167~9.333,平均有效等位基因数为1.624~4.966,平均观测杂合度为0.324~0.983,平均期望杂合度为0.329~0.782,平均多态信息含量为0.275~0.753;两个尼罗罗非鱼群体、WY 1和YY 2群体达到高度多态(PIC>0.5),两个奥利亚罗非鱼群体、ZY 1和YY 1群体为中度多态(0.25相似文献   

The impact of successive mass selection on population genetic structure in the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>) revealed by microsatellite markers

To evaluate the impact of mass selection on genetic structure in artificially closed populations of the Pacific oyster Crassostrea gigas, we performed mass selection over six generations on two stocks from Japan and Korea and analyzed their temporal genetic variation and structure using 18 microsatellite makers, which were compared with the base populations of the two selected lines and one wild population from China. The average numbers of alleles (Na), mean observed heterozygosities (Ho), and expected heterozygosities (He) varied over generations in the two selected lines (selected lines of Japan, Na = 10.7–14.9, Ho = 0.757–0.846, He = 0.778–0.871; selected lines of Korea, Na = 9.4–17.3, Ho = 0.736–0.865, He = 0.744–0.854). There was no significant reduction in heterozygosity in the two selected lines. However, the average number of alleles per locus was significantly lower in the fifth and sixth generations of the two selected lines compared with that in the base population and wild population (P < 0.05), suggesting that the successive mass selection in closed populations may increase the sensibility of rare alleles to genetic drift. Equalizing the sex ratio of parents and reducing the selection intensity properly with the increase of selective generations is recommended to minimize the deleterious effect of genetic drift and bottleneck caused by successive mass selection. The information obtained in this study is useful for the design of appropriate management strategies for selective breeding of C. gigas.     

Comparative assessment of the genetic variation in selectively bred generations from two geographic populations of ivory shell (Babylonia areolata)

Ivory shell, Babylonia areolata, is one of the commercially important mariculture species in southern China and South‐East Asia. The selection programme for growth trait has been conducted since 2010 in two selected lines for three generations, namely the Hainan (H) and Thailand (T) selected lines. Ten polymorphic microsatellite loci were used to monitor changes in their genetic diversity and structure during the selection process. A relatively high level of overall genetic diversity was observed, while the number of microsatellite allele declined slightly in the mass selection lines. Comparative diversity estimates indicated greater genetic diversity in H line than T line. Moreover, a relatively weak but significant genetic differentiation was found in both of the selected generations. In addition, the higher genetic variation observed in H line is most likely caused by artificial selection while selection bias on a growth advantage for the T population. These findings suggest no significant decline of genetic diversity was observed in the presently selected lines and that a potential exists for genetic improvement and development for B. areolata.     

Survey of genetic variation at three microsatellite loci in captive populations of endangered Japanese minnow <Emphasis Type="Italic">Aphyocypris chinensis</Emphasis> with implications for reduction of inbreeding

The Japanese population of the cyprinid minnow Aphyocypris chinensis is nearing extinction in the wild. The genetic diversity of three microsatellite loci in five captive populations was investigated, and an effective breeding strategy to reduce inbreeding from pairwise relatedness (R _xy ) between each captive line is discussed. The average number of alleles ranged 2.33–4.67 and the average heterozygosity ranged 0.283–0.602. The pairwise relatedness observed in most combinations showed a significant decrease between the populations. It is suggested that exchange of individuals between different breeding lines should effectively stop inbreeding. Studies show that the effective population size (N _e ) estimated from the number of parental individuals was 8.54 in one captive population, which is insufficient to maintain genetic diversity. It is recommended that more parental individuals should be used, and to exchange fish in a rotating mating mode between institutions participating in captive breeding of A. chinensis.     

Use of microsatellite loci and AFLP markers to verify gynogenesis and clonal lines in Nile tilapia Oreochromis niloticus L.

To develop an effective system for parentage analysis in gynogenetic and clonal progeny of Nile tilapia, Oreochromis niloticus L., polymorphic microsatellite loci and amplified fragment length polymorphisms (AFLPs) were investigated in several gynogenetic families and clonal lines. Six microsatellite loci were screened in two meiotic gynogenetic families to look for loci with high gene–centromere recombination rates, which can be used to discriminate meiotic from mitotic gynogenetics. Microsatellite loci UNH189 and UNH211 showed 96.7% and 92.0% heterozygosity, respectively, in these families, while other loci showed lower recombination frequencies. Scoring both UNH189 and UNH211 would give a very low probability of an individual meiotic gynogenetic being homozygous for both loci. Multiplex polymerase chain reaction of microsatellite loci was used to verify parentage in four families of mitotic gynogenetics and five fully inbred clonal lines. The genotype of each clonal line should serve as a unique identifier. Twelve AFLP primers were also investigated and 26 diagnostic AFLP bands were identified to follow inheritance in mitotic gynogenetic individuals. Amplified fragment length polymorphisms were found to be effective for this purpose but microsatellites were more appropriate since they are co‐dominant, while AFLPs are dominant markers. A multiplex of the microsatellite loci used in this study would be useful for general parental assignment as well as for the analysis of the products of chromosome set manipulations.     

Genetic Introgression Between Arctic Charr (Salvelinus Alpinus) and Brook Trout (Salvelinus Fontinalis) in Bavarian Hatchery Stocks Inferred from Nuclear and Mitochondrial DNA Markers

Nuclear insulin-like growth factor 2 gene (IGF-2), growth hormone 1 gene (GH-1) and internal transcribed spacer 1 (ITS-1) of the ribosomal DNA as well as the mitochondrial NADH-3 and NADH-4 dehydrogenase genes (ND-3/4) exhibited species-specific restriction fragment patterns and three microsatellite loci (Sfo18, Ssa85 and Ssa197) had non-overlapping allele size ranges in Arctic charr and brook trout and were used as diagnostic markers for testing genetic purity of hatchery stocks and wild populations of Arctic charr and brook trout in Bavaria, Germany. Screening of four wild populations (three in Arctic charr and one in brook trout) revealed only a single hybrid (back-cross to brook trout) individual in L. Starnberg. In contrast, in three (out of five) hatchery stocks of Arctic charr and in both hatchery stocks of brook trout hybrids were detected with the frequency from 3 to 100%. Three hatchery stocks (SS2, SA and BS1) represent a hybrid swarm because they contained a very high proportion of hybrids (from 83 to 100%) and most or all hybrid individuals had alien alleles at only one or a few of six unlinked diagnostic loci, indicating that post-F₁ hybrids represent the majority of individuals in these stocks and introgression has taken place. Release or escape of introgressed individuals from hatcheries into natural water bodies should be avoided in order to protect the biological diversity and genetic integrity of native fish populations.     

Enhancing capacity for freshwater conservation at the genetic level: a demonstration using three stream macroinvertebrates

1. Species diversity is declining more rapidly in freshwater ecosystems than in any other, but the consequences for genetic diversity, and hence evolutionary potential, are poorly understood. In part this reflects limited use and development of modern molecular tools and genetic approaches to address conservation questions in rivers, lakes and wetlands. As widespread, diverse and functionally important organisms, freshwater macroinvertebrates are ideal candidates for genetic approaches to reveal, for example, the conservation consequences of demographic histories and past disturbances. However, the availability of microsatellite markers for this group is very limited.
2. Using next generation sequencing, microsatellite markers were developed for Isoperla grammatica (Poda, 1761), Amphinemura sulcicollis (Stephens, 1836) and Baetis rhodani (Pictet, 1843) to enable conservation genetic investigations of these widespread invertebrate species. Fifty‐two robust microsatellite loci were developed (18, 21 and 13 per species), all with high levels of allelic diversity (7–27, 3–16, 5–13 alleles per loci, respectively).
3. These tools will allow assessment of genetic structure, dispersal and demographic resilience in these model species as a function of environmental change and variation, thereby aiding freshwater monitoring and conservation. The authors urge further capacity building to support genetic applications to the conservation biology of other aquatic organisms.

Copyright © 2016 John Wiley & Sons, Ltd.     

Can genetic diversity be maintained during mass selection of the Chinese mitten crab,Eriocheir sinensis?

The Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species and food source in Eastern Asia. This study assesses the changes in genetic diversity in successive generations of early‐ and late‐maturing strains of E. sinensis using 30 microsatellite markers with high polymorphism. The mean average number of alleles (N) in the founder population (G0), first generation (G1), second generation (G2) and third generation (G3) of the early‐maturing strain were 18.367, 14.800, 16.400 and 16.533, respectively; while in late‐maturing strain the values were 18.500, 16.267, 14.367 and 16.533 respectively. Likewise, there was a slight decline in average allelic richness (R_s) in the three successive generations. In both strains, the mean observed heterozygosity (H_o) remained relatively constant for the early‐maturing strain and the values were 0.655, 0.667, 0.685 and 0.705, respectively; and for the late‐maturing strain these were 0.665, 0.672, 0.688 and 0.702 respectively. Similarly, the expected heterozygosity (H_e) remained constant, ranging from 0.823 to 0.854. There was a decrease in effective population sizes (N_e) of the early‐maturing strain with successive generations: values were 492.2, 35.0, 134.7 and 193.2, respectively; while the values in the late‐maturing strain were 1268.5, 75.6, 111.5 and 97.2 respectively. All pairwise population distances were very close in both strains. In conclusion, these results suggest that mass selection of E. sinensis did not significantly diminish genetic diversity although there was a decline for the N_e. Therefore, it is important to maintain sufficient broodstock numbers and a large effective population when following a selective breeding programme.     

三倍体牙鲆(Paralichthys olivaceus)群体遗传多样性的微卫星分析

采用微卫星遗传标记技术对三倍体牙鲆群体及二倍体对照群体进行比较分析。取2012年冷休克诱导和培育至11月龄的三倍体及同期二倍体对照牙鲆个体各32尾,通过高盐法提取肌肉组织总DNA;利用筛选得到的21对微卫星引物进行PCR扩增,并经14%聚丙烯酰胺凝胶电泳分型后,进行人工判读和分析。结果显示,21个微卫星位点均为多态,二倍体和三倍体群体的平均等位基因数(A)、基因型总数、平均有效等位基因数(ae)、平均观测杂合度(HO)和平均期望杂合度(HE)分别为5.6和5.0、190和142、3.7和2.8、0.613和0.868、0.681和0.629。二倍体和三倍体群体的多态信息含量(PIC)分别为0.642和0.589,个体识别率(DP)为0.660和0.609,非父排除率(PPE)为0.482和0.399,其累积个体识别率和非父排除率均达到0.999,表明所选座位均属中高识别力的遗传标记,可以将它们应用于今后牙鲆雌核发育群体的遗传变异分析以及进一步的遗传育种的研究中。二倍体和三倍体群体间的遗传距离(D)为0.312,遗传相似系数(I)为0.732,基因分化系数(GST)为0.971。以上结果说明,三倍体诱导对牙鲆的遗传结构造成了一定影响,导致三倍体遗传多样性水平相对二倍体有所下降,二倍体和三倍体牙鲆的基因型也存在一定差异。