Daily change of genetic variability in hatchery offspring of red sea bream during spawning season

ABSTRACT:   In order to assess a daily change of genetic variability during spawning season, hatched larvae of red sea bream sampled on different dates were assayed by polymorphic markers such as microsatellite DNA (msDNA) and mitochondria DNA (mtDNA) control region. Based on the microsatellite loci, the average number of alleles per locus ranged between 13.7 and 18.3. The expected heterozygosities ranged between 0.843 and 0.919. A total of 23 mtDNA haplotypes were detected via digestion of mtDNA D-loop sequences with five endonucleases: Taq  I, Alu  I, Mbo  I, Rsa  I and Hinf  I. Significant fluctuation of genetic variability during spawning season was detected by both types of DNA markers. It was suggested that the genetic variability was maintained by pooling the seed fish collected on different spawning dates in a hatchery.     

Relatedness structure estimated by microsatellites DNA and mitochondrial DNA polymerase chain reaction–restriction fragment length polymorphisms analyses in the wild population of kuruma prawn Penaeus japonicus

ABSTRACT: The genetic structure and variability of four wild populations of kuruma prawn in Japan were examined by estimating relatedness among individuals. The relatedness was estimated by five microsatellites (MS) DNA markers. Examination of relatedness showed that individuals were related significantly in Kumamoto and Kagoshima. In Kagoshima, some individuals showed full-sib level of relatedness. The analysis of mitochondrial (mt)DNA polymerase chain reaction–restriction fragment length polymorphisms (PCR-RFLP) was also performed, showing that the closely related individuals in Kagoshima tended to share a common haplotype. It is, therefore, supposed that there are many kins in Kumamoto and Kagoshima. However, the heterozygosities and allelic and genotypic frequencies in MS-DNA analysis were not significantly different among the localities. Moreover, the haplotype distributions of mtDNA in Kumamoto and Kagoshima were significantly different from other localities. Thus, it is suggested that no spatial differentiations occurred due to the geographic or historical effects between the localities and that there is the possibility of a mixture of hatchery populations in Kumamoto and Kagoshima.     

Genetic population evaluation of two closely related flatfish species, the rare barfin flounder and spotted halibut, along the Japanese coast

ABSTRACT:   Barfin flounder and spotted halibut have been selected as target species for stock enhancement in Japan. Understanding the genetic condition of the wild stock is a principal requirement in any stock enhancement program. The genetic variability of barfin flounder and spotted halibut, and the population structure of spotted halibut were evaluated using microsatellite DNA markers (msDNA) and the control region of the mitocondrial DNA (mtDNA). Barfin flounder and spotted halibut showed high genetic variability at the msDNA level. Barfin flounder A was 16.7 and H _e was 0.860; spotted halibut A _n ranged from 7.7 to 10.2 and H _e ranged from 0.710 to 0.774. At the mtDNA level, high haplotype ( h  = 0.922) and low nucleotide (π = 0.002) diversities were observed for barfin flounder; however, low haplotype and nucleotide diversities ( h  = 0.603–0.620 and π = 0.001–0.002), and very low haplotype and nucleotide diversities ( h  = 0.193 and π = 0.0003) were observed for spotted halibut in the north and south locations, respectively. Slight genetic differentiation among spotted halibut sampling locations was observed from the msDNA. MtDNA analyses showed genetic differentiation between north and south locations, but not within them. The designation of north-specific and south-specific management units in the future stock enhancement activities of spotted halibut is recommended.     

Evidence for genetic differentiation in the european conger eel Conger conger based on mitochondrial DNA analysis

ABSTRACT:   The European conger eel Conger conger is an important marine benthic fish in the North-East Atlantic and represents a valuable fishery resource. However, little is known about its reproductive biology. In an attempt to gain a better understanding of the conger eel population structure, mitochondrial DNA (mtDNA) sequences were examined. A region with 432 bp of the control region of the mtDNA was sequenced from 40 individuals from six different locations around the central and eastern North Atlantic Ocean. Thirty variable positions defined 28 distinct haplotypes. The average sequence difference within samples (1.3–4.2%) was comparable to those between samples (1.4–3.6%). MtDNA sequence-based statistical tests showed significant geographic differentiation between some local population samples, suggesting that the conger eel does not comprise a single panmictic population. However, given our sample sizes, these preliminary results should be interpreted with caution and more individuals from more sites, including the Mediterranean Sea, should be analyzed in detail. The genetic variability detected in this study is an initial step to elucidate the genetic background of the conger eel population structure.     

Wild stock structure of <Emphasis Type="Italic">Girella punctata</Emphasis> in Japan revealed shallow genetic differentiation but subtle substructure in subsidiary distributions

Twelve populations of Girella punctata, from widespread locations of the species’ range in Japan and Korea, were screened for sequence variability within the mitochondrial DNA (mtDNA) control region (n = 128) and at five polymorphic microsatellite loci (n = 547) to determine the genetic structure maintaining population integrity. mtDNA variability of 132 variable sites within a 334-bp region reveals shallow genetic differentiation across populations. The weak differentiation of G. punctata was partly supported by the screening of five polymorphic microsatellite loci. However, hierarchical analysis of molecular variance and principal component analysis on the basis of allele frequencies in microsatellite loci extracted a subtle substructure in a subsidiary population and in near-subsidiary populations in the semi-enclosed Seto Inland Sea.     

Complete nucleotide sequence and variation of mitochondrial DNA from 10 individuals of walleye pollock, Theragra chalcogramma

ABSTRACT:   The complete mitochondrial genome sequence of 10 walleye pollocks, Theragra chalcogramma , from the Japan Sea and Bering Sea was determined. The 16 568–16 571 bp genome contains the same 37 mitochondrial structural genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in all other vertebrates analyzed, in an organization identical to that of other bony fish. The major non-coding region had several conserved sequence features. Nucleotide variations of ND1, ND5, and control region were high, and these regions appear to be good candidates for high-resolution markers in population studies.     

Genetic diversity of wild mekong giant catfish Pangasianodon gigas collected from Thailand and Cambodia

The Mekong giant catfish Pangasianodon gigas is endemic to the Mekong River and is a critically endangered species. The genotypes of the microsatellite DNA (msDNA) and mitochondrial DNA (mtDNA) markers (right domain of the control region) were detected to evaluate the present status of genetic divergence of this species from the Mekong River in Thailand and Cambodia. The observed and expected heterozygosity values of Mekong giant catfish in Thailand and Cambodia were relatively low in comparison with those of other nonendangered freshwater fish species. These two populations from Thailand and Cambodia showed similar levels of genetic diversity, as evaluated by the 384 nucleotides of the mtDNA control region with 13 haplotypes. The pairwise F _ST value between the two populations based on the genotype frequencies of msDNA and mtDNA markers suggested a close genetic relationship between the populations in Thailand and Cambodia. The results of this study support the conclusion that the Mekong giant catfish is critically endangered. Care should be taken to sustain the genetic diversity of this species, as the level of genetic variability has already decreased in the wild population. This species is a target species for an ongoing stock enhancement program in the Mekong River in Thailand. It is proposed to apply these markers for proper broodstock management, such as for minimal kinship selective breeding in the hatchery.     

Mitochondrial DNA variation and population structure of the Japanese mitten crab Eriocheir japonica

ABSTRACT:   The Japanese mitten crab Eriocheir japonica is a common grapsid species found throughout freshwater and estuarine regions in Japan. In order to obtain information on the genetic variation and population structure of this species, a polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis was conducted on the cytochrome oxidase subunit I (COI) of mitochondrial DNA, on 666 individuals from 19 sample sites covering the three main geographic regions of Japan (Main Islands, Okinawa, and Ogasawara). Genetic analysis using seven restriction enzymes produced an array of 61 composite haplotypes. Three regional groups corresponding to the three geographic regions were clearly identified by cluster and molecular variance model ( amova ) analyses. Each of the three groups showed dominant haplotypes that were almost completely absent in populations from the other geographic areas. Comparison with published information for other species indicates that the degree of genetic divergence between these three main groups is equivalent to the genetic distance between congeneric species. Thus, the population structure of the Japanese mitten crab, as inferred from mtDNA analysis, is formed by genetically distinct groups that closely reflect their geographic distribution in the Japanese archipelago as well as restricted gene flow.     

粤东海域褐菖鲉种群线粒体DNA控制区序列变异及其在平鲉亚科中的分类地位

对捕自粤东海域的47尾野生褐菖鲉Sebastiscus marmoratus线粒体DNA控制区序列进行了扩增和序列变异分析,并结合GenBank中10种其他平鲉亚科的同源序列,分析了平鲉亚科的分子系统。结果表明,所获序列长度在538～544bp之间,具69个碱基替换位点和36个插入/缺失位点;控制区碱基组成中,A、T、C、G含量分别为34.5%、29.0%、15.8%、20.7%。共检测到38个单倍型。该种群的单倍型多样度(Hd)和核苷酸多样性(Pi)分别为0.978和0.0192。以鲉亚科棘鲉Hoplosebastes armatus作为外群构建的分子系统树显示,褐菖鲉为11种平鲉亚科中较早分化出的种,位于进化树的基部;平鲉亚科为单系群,分为4个分支,分别为平鲉属Sebastes、菖鲉属Sebastiscus、眶棘鲉属Hozukius和无鳔鲉属Helicolenus,与传统的分类地位一致。研究结果表明,控制区序列不仅适合褐菖鲉种群遗传多样性研究,同样也适合分子系统发育研究。     

淤泥湖、梁子湖、鄱阳湖团头鲂mtDNA序列变异及遗传结构分析

测定了淤泥湖、梁子湖及鄱阳湖团头鲂(Megalobrama amblycephala)共53尾样本的线粒体DNA(mtDNA)控制区序列。结果显示:在获得的411 bp长度控制区序列中,检测到3个突变位点、5个单倍型。遗传多样性分析显示,3个种群的遗传多样性水平比较低,且淤泥湖种群最低。分子方差分析(AMOVA)表明,淤泥湖种群与其它两个种群有明显遗传分化,但梁子湖与鄱阳湖种群间未出现分化。     

Low genetic variability in an endangered population of fiddler crab <Emphasis Type="Italic">Uca arcuata</Emphasis> on Okinawajima Island: analysis of mitochondrial DNA

ABSTRACT:   Restriction fragment length polymorphism analysis was performed on polymerase chain reaction-amplified DNA fragments containing the D-loop , ND2 , and CO I genes of fiddler crab Uca arcuata mitochondrial DNA. In total, 316 individuals from six populations in Japan and two populations in Taiwan were analyzed using five restriction endonucleases ( Afa I, Bcn I, Cfr 13I, Hae III and Hin fI), yielding 85 haplotypes. Samples were taken from Nakagusuku Bay, Okinawajima Island, which is the only known distribution of U. arcuata in the Ryukyu Archipelago. The Okinawajima Island population is isolated geographically from others and showed a marked low genetic variability ( h  = 0.2539, π  = 0.0005) and significant differentiation from other population samples in haplotype composition. We suggest that a substantial decrease in the genetic variability of the Okinawajima Island population was caused by genetic drift under the conditions of small population size and low gene flow from other populations. It is important to conserve the intertidal zone in Nakagusuku Bay for the maintenance of this endangered population.     

Random amplified polymorphic DNA markers for three Japanese laminarian species

ABSTRACT: Random amplified polymorphic DNA (RAPD) was studied to detect genetic markers for three economically important Japanese laminarian species, Laminaria japonica Areschoug, L. religiosa Miyabe and L. ochotensis Miyabe, which were sampled from their representative localities on the coasts from Tsugaru Straits to the Sea of Japan. DNA templates for RAPD were extracted from lamina using a DNA extraction kit and were purified with glassmilk. Reproducible RAPD markers for these three species were detected using three random primers from a total of 15 tested. In L. japonica and L. religiosa , these RAPD markers were confirmed to be useful for populations from other localities. The three species studied showed high intraspecific and interspecific band sharing index (BSI) values. Like annual typical L. religiosa from other localities, biennial individuals identified as putative L. religiosa from Atsuta could be discriminated from L. japonica but not from L. ochotensis by any of the RAPD genetic markers studied so far.     

Genetic variation and population structure of the Pacific cod <Emphasis Type="Italic">Gadus macrocephalus</Emphasis> in Korean waters revealed by mtDNA and msDNA markers

To investigate the extent of genetic differentiation among wild populations of the Pacific cod Gadus macrocephalus, we have examined genetic polymorphism at five locations within Korean waters [Boryeong in the West Sea (WC-BR); Jinhae Bay in the South Sea (SC-JH); Jumunjin (EC-JM), Jukbyeon (EC-JB), and Bangeojin (EC-BJ) off the eastern coast of Korea] using mitochondrial DNA (mtDNA) control region and microsatellite DNA (msDNA) markers. Nucleotide sequence analysis of 584 bp in the variable portion of the 5′ end of the mtDNA control region revealed 27 variable nucleotide sites among 184 individuals, which defined eight, three, and 11 haplotypes in the western, southern, and eastern coast populations, respectively. The mtDNA analysis revealed a low variability but significant local differentiation among populations from these three areas within Korean waters. msDNA analysis also revealed moderate polymorphism in the wild populations, with a mean of 13.8–22.6 alleles per locus for the five msDNA markers and observed (and expected) heterozygosities of 0.755 (0.825) for the WC-BR, 0.793 (0.810) for the SC-JH, 0.920 (0.905) for the EC-BJ, 0.783 (0.865) for the EC-JB, and 0.804 (0.812) for the EC-JM populations. Analysis of msDNA loci indicated that Pacific cod sampled at the WC-BR, SC-JH, and EC-JB sites belong to genetically distinct populations. However, no significant difference was found between the Pacific cod population from SC-JH and that from EC-BJ. Consequently, three genetically distinct populations, namely, WC-BR, SC-JH and EC-BJ, and EC-JB, were identified using msDNA analysis. These results indicate that genetically distinct populations of Pacific cod are present in Korean coastal waters where spawning aggregations occur.     

Sequence variability in the mitochondrial DNA control region of five Sebastes species

ABSTRACT: Sequence variability of the control region of mitochondrial DNA in five Sebastes species ( S. thompsoni , S. joyneri , S. inermis , S. schlegeli and S. owstoni ) was investigated. Of 324 nucleotide sites in the control region of S. thompsoni , 56 sites (17.3%) varied, and all 20 individuals had different haplotypes. The other four species had between five and 17 sites (1.5–5.9%), which was fewer than that observed in S. thompsoni . The nucleotide diversity of S. thompsoni was highest (3.45%) among the five species, and that of S. schlegeli was the lowest (0.19%). The results demonstrated that sequence variability exists in the control region of the five Sebastes fishes investigated, and that sequence data in the control region may be suitable for stock structure analysis. Also, the extent of genetic variablitiy in the control region differed among these species. In particular, the mitochondrial DNA control region in S. thompsoni was characterized by high sequence variability, which may indicate a large effective population size.     

半滑舌鳎线粒体 DNA 含量测定方法的建立与优化

本研究旨在应用实时荧光定量PCR技术,建立半滑舌鳎(Cynoglossus semilaevis)线粒体DNA(mtDNA)含量测定方法并进行优化.通过质粒标准品的线性化处理、模板DNA的酶切和超声处理、mtDNA和核DNA(nDNA)基因引物的筛选实验,建立半滑舌鳎mtDNA含量测定方法.结果表明,质粒标准品的构象对荧光定量PCR标准曲线影响较大,线性化的质粒更适合用作标准品;酚-氯仿方法提取的模板DNA适用于mtDNA含量测定,无需进行预处理;用D-loop和ND1这2对引物所得的拷贝数较小且结果一致,适用于mtDNA拷贝数的测定;以不同核基因为参照所得的mtDNA含量可能存在差异,当以单拷贝核基因ENC1和MYH6为参照时,可以计算出单个细胞中mtDNA含量,若以多拷贝基因GAPDH为参照,mtDNA含量测定值则较小.采用本方法分别对半滑舌鳎肝、肾、脾和肌肉组织的mtDNA含量进行重复性检测实验,结果表明,相同组织的mtDNA含量显示出良好的重复性(P＞0.05),而不同组织中mtDNA含量具有差异性,可见该方法稳定可靠,能为海洋鱼类mtDNA含量检测提供借鉴.     

辽东湾斑海豹线粒体苏氨酸和脯氨酸及控制区部分序列分析

通过PCR产物测序法,测定了46只辽东湾斑海豹线粒体DNA的一段包括部分苏氨酸和脯氨酸tRNA基因及部分控制区的717 bp序列,得到17个序列单元型。与港海豹相比,所有辽东湾斑海豹17个单元型在控制区序列中都有2处缺失,是区分港海豹和斑海豹的种间分子标记。与日本海和鄂霍次克海区斑海豹m tDNA相同片段进行比较研究,发现辽东湾斑海豹的线粒体控制区DNA的单元型比例相对较少,遗传多样性水平相对较低。辽东湾斑海豹m tDNA的16296位点即苏氨酸tRNA基因序列的最后一位有1个C碱基的插入,16607位点存在T/C转换,这两个变异位点是鉴别辽东湾斑海豹与日本及鄂霍次克海区斑海豹的重要分子标记。初步表明,辽东湾斑海豹与日本海、鄂霍次克海斑海豹是属于不同的地理种群,或者辽东湾斑海豹同这两个海区斑海豹未发生基因交流或者雌性个体的相互交流。     

Genetic identification of native populations of fluvial white-spotted charr <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis> in the upper Tone River drainage

ABSTRACT:   Stocking of exogenous, hatchery-reared white-spotted charr Salvelinus leucomaenis has been conducted throughout much of their range in Honshu Island, Japan, to increase angling opportunities. Although the native charr populations are thought to have declined because of hybridization with introduced fish, their distribution and genetic status have been uncertain. Fine population structures of charr in the upper Tone River drainage were examined using mitochondrial DNA and microsatellite analyses so as to clarify the presence of native populations. One common mtDNA haplotype was detected in all populations in the Ohashi River and Watarase River, and four and one tributary populations were monomorphic for such haplotypes, respectively. However, several haplotypes, considered to have originated from stocked hatchery fish, were observed in the stocked and the remaining populations. Judging from the genetic integrity over a fine geographic scale, the former were considered as indicative of native populations and the latter as admixtures with hatchery fish. Comparisons of genetic diversity, deviations from the Hardy–Weinberg equilibrium, principal component analysis, and relatedness estimations based on microsatellite DNA can also provide evidence for distinguishing native populations from those influenced by hatchery fish.     

Australian sperm whales from different whaling stocks belong to the same population

1. Understanding the factors driving population structure in marine mammals is needed to evaluate the impacts of previous exploitation, current anthropogenic threats, conservation status, and success of population recovery efforts.
2. Sperm whales are characterized by a worldwide distribution, low genetic diversity, complex patterns of social and genetic structure that differ significantly within and between ocean basins, and a long history of being commercially whaled. In Australia, sperm whales from the (International Whaling Commission assigned) southern hemisphere ‘Division 5’ stock were very heavily exploited by whaling.
3. The present study assessed the potential effects of whaling on the genetic diversity of sperm whales in Australia and the population genetic structure of these whales within a global context. A combination of historical and contemporary sperm whale samples (n = 157) were analysed across six regions, from south-eastern Australia (‘Division 6’ stock in the Pacific Ocean) to south-western Australia (‘Division 5’ stock in the Indian Ocean).
4. Sperm whales sampled from the ‘Division 5’ and ‘Division 6’ stocks belong to the same population based on nuclear and mitochondrial DNA (mtDNA) analyses. Four novel sperm whale mtDNA haplotypes were identified in animals from Australian waters. Levels of genetic diversity were low in Australian sperm whales but were similar to those previously reported for populations in the Indian and Pacific Oceans.
5. Given the genetic distinctiveness of sperm whales in Australian waters from other regions in the Pacific and Indian Oceans, and the lack of recovery in population numbers, further scientific studies are needed to increase our understanding of population dynamics and the effectiveness of threat management strategies in this species.

     

三倍体湘云鲫及其亲本线粒体DNA的比较研究

用人工选育的异源四倍体鲫鲤（♂）与白鲫（♀）杂交获得具有明显生长优势的三倍体湘云鲫。采用差速离心和核酸酶处理等方法，从三倍体、白鲫和异源四倍体鲫鲤肝组织中提取线粒体DNA，并用9种限制性内切酶进行单酶酶切分析。经琼脂糖凝胶电泳后计算出各酶切片段的大小，测得三倍体、白鲫和异源四倍体鲫鲤mtDNA的分子大小分别为16．24kb、16．60kb和16．20kb。根据各单倍型间的酶切片段共享度，估算出3个群体间的遗传距离，说明了mtDNA母系遗传的特性。     

Population structure of anchovy Engraulis encrasicolus L. in the Mediterranean Sea inferred from multiple methods

Population structure of anchovy, Engraulis encrasicolus L., in the northern Mediterranean was investigated using multiple methods. Variation in body shape, otolith shape and growth was investigated in samples from the central Aegean and Ionian Seas. Intron length polymorphism from nuclear DNA, and mitochondrial DNA variation was investigated for samples from these basins, as well as northern Adriatic (ADR) and Gulf of Lions (LION). Two lineages (clades) of mtDNA that co-occur in the populations of anchovy throughout the region were compared with respect to growth, body shape, otolith shape, and intron variation, to check for any reproductive isolation within areas between the clades. The two clades were not different in any of the investigated parameters and appear to be completely crossbreeding. There were significant differences among areas with regard to mtDNA clade proportions, nuclear allele frequencies, body shape and otolith shape, indicating that several reproductively isolated populations of anchovy exists in the northern Mediterranean. There was an overall fair to good agreement between population structures inferred from the different classes of markers. The mitotype variation indicated a larger population differentiation than the two nuclear DNA loci, which may be related to the uniparental transmission and the lack of recombination of mtDNA.