Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0-0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.     

Genetic diversity and population structure of Eleutheronema rhadinum in the East and South China Seas revealed in mitochondrial COI sequences

Eleutheronema rhadinum is a potential commercial fisheries species and is subject to intense exploitation in China. Knowledge on the population structure of E. rhadinum in Chinese coastal waters, which is important for sustainable exploitation and proper resource management, is lacking. In the present study, the genetic diversity and population structure of E. rhadinum were evaluated using a 564-base pair fragment of the mitochondrial cytochrome c oxidase subunit I （COl） gene. A total of 76 specimens were collected from three localities around the East （Qidong and Zhoushan） and South China Seas （Zhuhai）. Among these individuals, nine polymorphic sites were detected and 1 l distinct haplotypes were defined. High levels ofhaplotype diversity （h=0.759i0.035） and low levels of nucleotide diversity （re=0.001 98i0.003 26） were observed in these populations, Hierarchical analysis of molecular variance （AMOVA） indicated that 96.72% of the genetic variation occurred within the populations, whereas 3.28% occurred among populations. No significant genealogical branches or clusters were recognized on the neighbor-joining tree. Intra-group variation among populations was significant （~0~t=0.032 85, P〈0.01）. These results suggest that E. rhadinum populations in the East and South China Seas have developed divergent genetic structures. Tests of neutral evolution and mismatch distribution suggest that E. rhadinum may have experienced a population expansion. The present study provides basic information for the conservation and sustainable exploitation of this species.     

Analysis of genetic diversity and differentiation of seven stocks of Litopenaeus vannamei using microsatellite markers

Seven microsatellite markers were used to evaluate the genetic diversity and differentiation of seven stocks of Litopenaeus vannamei, which were introduced from Central and South America to China. All seven microsatellite loci were polymorphic, with polymorphism information content(PIC) values ranging from 0.593 to 0.952. Totally 92 alleles were identified, and the number of alleles(Na) and effective alleles(Ne) varied between 4 and 21 and 2.7 and 14.6, respectively. Observed heterozygosity(Ho) values were lower than the expected heterozygosity(He) values(0.526–0.754), which indicated that the seven stocks possessed a rich genetic diversity. Thirty-seven tests were detected for reasonable significant deviation from Hardy-Weinberg equilibrium. Fis values were positive at five loci, suggesting that there was a relatively high degree of inbreeding within stocks. Pairwise Fst values ranged from 0.0225 to 0.151, and most of the stock pairs were moderately differentiated. Genetic distance and cluster analysis using UPGMA revealed a close genetic relationship of L. vannamei between Pop2 and Pop3. AMOVA indicated that the genetic variation among stocks(11.3%) was much lower than that within stocks(88.7%). Although the seven stocks had a certain degree of genetic differentiation and a rich genetic diversity, there is an increasing risk of decreased performance due to inbreeding in subsequent generations.     

Development and validation of 89 novel expressed sequence tag-derived microsatellite markers in blood clam, <Emphasis Type="Italic">Tegillarca granosa</Emphasis>

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

军曹鱼全人工繁殖群体遗传特征的SSR分析

利用8个微卫星DNA位点分析南海海域5个军曹鱼全人工繁育群体(HN1、HN2、ZJ、FJ和LS)子代的遗传多样性特征和群体间遗传分化。结果显示,军曹鱼养殖群体与天然群体的遗传结构特征基本一致:1)平均有效等位基因数为3.910±0.440,观测杂合度为0.595±0.049,分子方差分析(AMOVA)表明,个体内分化占主导(46%),军曹鱼养殖群体整体遗传多样性较高;2)群体间基因流明显(N_m=2.5959,F_(st)=0.0878),整体分化程度较低。各养殖群体表现出不同于天然大群体的特征:1)绝大部分位点均明显偏离哈温平衡,杂合子缺失或过剩现象普遍存在;2)聚类和群体分配分析等表明HN2与另四个养殖群体(HN1、ZJ、FJ和LS)分化明显。     

Population genetics of Sargassum horneri (Fucales, Phaeophyta) in China revealed by ISSR and SRAP markers

Sargassum horneri is a common brown macro-alga that is found in the inter-tidal ecosystems of China. To investigate the current status of seaweed resources and provide basic data for its sustainable development, ISSR (inter simple sequence repeat) and SRAP (sequence related amplified polymorphism) markers were used to analyze the population genetics among nine natural populations of S. horneri. The nine studied populations were distributed over 2 000 km from northeast to south China. The percentage of polymorphic loci P% (ISSR, 99.44%; SRAP, 100.00%), Nei’s genetic diversity H (ISSR, 0.107-0.199; SRAP, 0.100-0.153), and Shannon’s information index I (ISSR, 0.157-0.291; SRAP, 0.148-0.219) indicated a fair amount of genetic variability among the nine populations. Moreover, the high degree of gene differentiation G st (ISSR, 0.654; SRAP, 0.718) and low gene flow N m (ISSR, 0.265; SRAP, 0.196) implied that there was significant among-population differentiation, possibly as a result of habitat fragmentation. The matrices of genetic distances and fixation indices (F st ) among the populations correlated well with their geographical distribution (Mantel test R=0.541 5, 0.541 8; P=0.005 0, 0.002 0 and R=0.728 6, 0.641 2; P=0.001 0, 0.001 0, respectively); the Rongcheng population in the Shandong peninsula was the only exception. Overall, the genetic differentiation agreed with the geographic isolation. The fair amount of genetic diversity that was revealed in the S. horneri populations in China indicated that the seaweed resources had not been seriously affected by external factors.     

The loss of genetic diversity during captive breeding of the endangered sculpin, Trachidermus fasciatus, based on ISSR markers: implications for its conservation

Inter-simple sequence repeat (ISSR) markers were used to determine the genetic variation and genetic differentiation of cultured and wild populations of Trachidermus fasciatus, an endangered catadromous fish species in China. Six selected primers were used to amplify DNA samples from 85 individuals, and 353 loci were detected. Relatively low genetic diversity was detected in the cultured population (the percentage of polymorphic loci PPL = 73.80%, Nei’s gene diversity h = 0.178 2, Shannon information index ...     

Genetic variation assessed with microsatellites in mass selection lines of the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>) in China

Four successive mass selection lines of the Pacific oyster, Crassostrea gigas, selected for faster growth in breeding programs in China were examined at ten polymorphic microsatellite loci to assess the level of allelic diversity and estimate the effective population size. These data were compared with those of their base population. The results showed that the genetic variation of the four generations were maintained at high levels with an average allelic richness of 18.8–20.6, and a mean expected heterozygosity of 0.902–0.921. They were not reduced compared with those of their base population. Estimated effective population sizes based on temporal variances in microsatellite frequencies were smaller to that of sex ratio-corrected broodstock count estimates. Using a relatively large number of broodstock and keeping an equal sex ratio in the broodstock each generation may have contributed to retaining the original genetic diversity and maintaining relatively large effective population size. The results obtained in this study showed that the genetic variation was not affected greatly by mass selection progress and high genetic variation still existed in the mass selection lines, suggesting that there is still potential for increasing the gains in future generations of C. gigas. The present study provided important information for future genetic improvement by selective breeding, and for the design of suitable management guidelines for genetic breeding of C. gigas.     

Significant population genetic structure detected in the rock bream <Emphasis Type="Italic">Oplegnathus fasciatus</Emphasis> (Temminck &amp; Schlegel, 1844) inferred from fluorescent-AFLP analysis

Oplegnathus fasciatus (rock bream) is a commercial rocky reef fish species in East Asia that has been considered for aquaculture. We estimated the population genetic diversity and population structure of the species along the coastal waters of China using fluorescent-amplified fragment length polymorphisms technology. Using 53 individuals from three populations and four pairs of selective primers, we amplified 1 264 bands, 98.73% of which were polymorphic. The Zhoushan population showed the highest Nei’s genetic diversity and Shannon genetic diversity. The results of analysis of molecular variance (AMOVA) showed that 59.55% of genetic variation existed among populations and 40.45% occurred within populations, which indicated that a significant population genetic structure existed in the species. The pairwise fixation index F _st ranged from 0.20 to 0.63 and were significant after sequential Bonferroni correction. The topology of an unweighted pair group method with arithmetic mean tree showed two significant genealogical branches corresponding to the sampling locations of North and South China. The AMOVA and STRUCTURE analyses suggested that the O. fasciatus populations examined should comprise two stocks.     

Genetic Variation Among White Croaker Populations

To investigate the genetic structures and differentiation of different wild populations of white croaker （Pennahia argentara）, horizontal starch gel electrophoresis was performed on 133 individuals collected from five different locations in China and Japan. The eleven enzyme systems revealed 15 loci, of which eleven were polymorphic. The percentage ofpolymorphic loci of white croaker populations varied from 6.67% to 53.3.3%; the mean observed and expected heterozygosity ranged from 0.0033 to 0.0133 and 0.0032 to 0.0191, respectively. The expected heterozygosity revealed a low genetic variability for white croaker in comparison with other marine fishes. The genetic distances between populations ranged from 0.00005 to 0.00026. A weak differentiation was observed within each clade and between clades; and no significant differences in gene frequencies among populations were observed in white croaker. Among the five populations, three Chinese populations showed more genetic diversity than that in Japanese populations.     

Development and Validation of 89 Novel Expressed Sequence Tag-Derived Microsatellite Markers in Blood Clam,Tegillarca granosa

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

Genetic diversity of populations and clones of Rhopilema esculentum in China based on AFLP analysis

Amplified fragment length polymorphisms(AFLP) markers were developed to assess the genetic variation of populations and clones of Rhopilema esculentum Kishinouye(Scyphozoa,Rhizostomatidae).One hundred and seventy-nine loci from 56 individuals of two hatchery populations and two wild populations were genotyped with five primer combinations.The polymorphic ratio,Shannon’s diversity index and average heterozygosity were 70.3%,0.346 and 0.228 for the white hatchery population,74.3%,0.313,and 0.201 for the red hatchery population,79.3%,0.349,and 0.224 for the Jiangsu wild population,and 74.9%,0.328 and 0.210 for the Penglai wild population,respectively.Thus,all populations had a relatively high level of genetic diversity.A specific band was identified that could separate the white from the red hatchery population.There was 84.85% genetic differentiation within populations.Individual cluster analysis using unweighted pair-group method with arithmetic mean(UPGMA) suggested that hatchery populations and wild populations could be divided.For the hatchery populations,the white and red populations clustered separately;however,for the wild populations,Penglai and Jiangsu populations clustered together.The genetic diversity at the clone level was also determined.Our data suggest that there are relatively high genetic diversities within populations but low genetic differentiation between populations,which may be related to the long-term use of germplasm resources from Jiangsu Province for artificial seeding and releasing.These findings will benefit the artificial seeding and conservation of the germplasm resources.     

基于近缘EST-SSR引物的企鹅珍珠贝养殖和野生群体的遗传标记

利用近源物种马氏珠母贝的EST-SSR引物对企鹅珍珠贝DNA进行扩增,获得7对可扩增出目标条带的近缘分子标记,分析野生群体(YS)和养殖群体(YZ)企鹅珍珠贝的遗传多样性。结果显示,7个位点共获得26个等位基因;野生和养殖群体的等位基因数分别为3~5和3~4,平均期望杂合度(He)分别为0.623 5、0.496 8;平均观测杂合度(Ho)分别为0.431 8、0.302 8;HNUPM047位点的多态信息含量较低,为0.446 7,其他位点均在0.5以上,说明该遗传标记可提供比较丰富的遗传信息。表明养殖群体遗传多样性水平低于野生群体。     

Inbreeding and genetic diversity analysis in a hatchery release population and clones of Rhopilema esculentum based on microsatellite markers

Ten microsatellite markers were used to analyze the levels of genetic diversity and inbreeding in a hatchery release population of Rhopilema esculentum Kishinouye(Scyphozoa: Rhizostomatidae). A total of 85 alleles were detected in 600 individuals. Within-population levels of observed( H o) and expected( H e) heterozygosity ranged from 0.152 to 0.839(mean=0.464) and from 0.235 to 0.821(mean=0.618), respectively. The polymorphism information content(PIC) of each marker ranged from 0.207 to 0.795 with an average of 0.580, indicating that the hatchery population maintained a high level of genetic diversity. Inbreeding levels were estimated in the hatchery population and the inbreeding coefficient was 0.203. This result revealed that a certain level of inbreeding occurred within the population. Meanwhile, we also determined genetic diversity at the clone level. Several polyps from the same scyphistomae were genotyped at the ten microsatellite loci and there was virtually no difference in their genotypes. Furthermore, we calculated the probabilities of exclusion. When both parents were known, the average exclusion probability of ten loci was 99.99%. Our data suggest that the ten microsatellite markers can not only be used to analyze the identity of individuals but they can also be applied to parentage identification. Our research provides a theoretical basis and technical support for genetic diversity detection and reasonable selection of R. esculentum hatchery populations. These findings support the use of releasing studies and conservation of R. esculentum germplasm resources.     

Microsatellite variation in the oyster <Emphasis Type="Italic">Crassostrea plicatula</Emphasis> along the coast of China

Genetic diversity and differentiation of the oyster Crassostrea plicatula populations from China’s coast were studied based on seven microsatellite loci. All loci showed high polymorphism for all five C. plicatula populations, with an average number of allele per locus of 19.3–27.9 and an average expected heterozygosity of 0.889–0.952. Significant departures from Hardy-Weinberg equilibrium and deficits of heterozygotes were observed over most populations at each locus, which were fully explained by null alleles. Microsatellite analysis revealed significant subdivision in the C. plicatula populations. According to the neighbor-joining tree constructed on the basis of the D _A distance, the five populations fell into three regional groups, showing a relatively homogeneous genetic structure in geographically close populations. Assignation tests correctly assigned high percentages of individuals to their original populations and groups, and also confirmed the existence of genetic differentiation among C. plicatula populations. The results obtained in this study will facilitate the formulation of appropriate fisheries management programs, stock identification and conservation of biodiversity for the species.     

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus： karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish. Supported by National Natural Science Foundation of China (No. 30571445), National High-Technology Research and Development Program (863 Program, No. 2006AA10A404), and Project from the Ministry of Science and Technology of China (No. 2006DKA30470-017)     

Genetic variation of natural and cultured stocks of Paralichthys olivaceus by allozyme and RAPD

Population genetics of the left-eyed flounder, Paralichthys olivaceus, including natural and cultured stocks distributed in the coastal waters near Qingdao of eastern maritime China, was analyzed in allozyme and RAPD. The results showed that among total 29 gene loci of 15 isozymes, 9 and 7 were po- lymorphic in natural and cultured stocks, respectively. The status of genetic diversity in P. olivaceus is low in terms of polymorphic loci in chi-square test and genetic departure index of Hardy-Weinberg equi- librium. More alleles in IDHP, CAT, GDH and Ldh-C allozymes were found in the fish, which could be used as markers in assortive breeding and distinguishing stock, population or species evolution. Total 88 and 86 RAPD bands ranging from 200 to 2 500 bp were recognized individually in average of 7.8–8.0 bands per primer. The genetic diversity in cultured stock is lower than that in natural ones showing an ob- viously decreasing genetic divergence. Therefore, effective countermeasures must be taken to protect ge- netic resources of marine cultured fishes. The 2 markers have their own pros and cons. Combining the 2 markers to investigate the genetic variation of populations is suggested. The results provide basic data of this flounder and they are useful for studying genetic improvement and genetic resources of the fish.     

Tissue Expression and Stock Variation of Isozymes of Stone Flounder （Kareius bicoloratus）

Tissue expression and stock variation of isozymes of stone flounder (Kareius bicoloratus) were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes ex- amined were obviously tissue-specific. The expressions of SOD*, GDH*, G3PDH-2* and ADH-2* were detected only in liver, SDH-1*, MDH-1* and ADH-1* only in muscle, and LDH-B* and LDH-C* only in eyes. In comparison, MDH-2*, GPI-3* and SDH-2* were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were se- lected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percent- ages of polymorphic loci (P0.99) were 29.17% and 25.00%, the observed heterozygosities (H0) were 0.028 ± 0.014 and 0.040 ± 0.019, and the expected heterozygosities (He) were 0.039 ± 0.017 and 0.052 ± 0.022 in Qingdao and Weihai stock, respectively. The coeffi- cient of gene differentiation (Fst) and genetic distance (D) between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymor- phic loci and the mean heterozygosity of K. bicoloratus were at a middle level.     

Inheritance pattern of microsatellite loci and their use for kinship analysis in the Japanese scallop Patinopecten yessoensis

The inheritance mode of seven microsatellite markers was investigated in Patinopecten yessoensis larvae from four controlled crosses, and the feasibility of using these markers for kinship estimation was also examined. All the seven microsatellite loci were compatible with Mendelian inheritance. Neither sex-linked barriers to transmission nor major barriers to fertilization between gametes from the parents were evident. Two of the seven loci showed the presence of null alleles in two families, suggesting the need to conduct comprehensive species-specific inheritance studies for microsatellite loci used in population genetic studies. However, even if the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance, offspring from four families were all unambiguously discriminated in the neighbor-joining dendrogram. This result indicates that the microsatellite markers used may be capable of discriminating between related and unrelated scallop larvae in the absence of pedigree information, and of investigating the effective number of parents contributing to the hatchery population of the Japanese scallop.