Genetic diversity analysis with ISSR PCR on green algae <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Vitreoscilla hemoglobin gene (vgb) improves lutein production in Chlorella vulgaris

Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the efficiency of cell respiration and metabolism. In this study, we introduced a Vitreoscilla hemoglobin gene （vgb） into Chlorella vulgaris by Agrobacterium tumefaciens-mediated transformation （ATMT）. PCR analysis confirmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome. Analysis of biomass obtained in shake flasks revealed transformant biomass concentrations as high as 3.28 g/L, which was 38.81% higher than that of the wild-type strain. Lutein content of transformants also increased slightly. Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants, which was 36.77% higher than that of the wild-type strain. The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris, with applications to lutein production from Chlorella during fermentation.     

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 in two sea cucumber （Apostichopus japonicus） stocks revealed by ISSR markers

Sea cucumber Apostichopus japonicus samples were collected in Changdao, Penglai (PL), 27 individuals, and Lingshandao, Qingdao (QD), 30 individuals, in the Shandong Peninsula, China. Ten SSR primers were used to assess the genetic variation and relationship between and within the two stocks. Respectively, for each stock, the percentage of polymorphic bands was 85.2% and 86.9%; the gene diver- sity was 0.360 5 and 0.342 8; and the Shannon’s information index was 0.515 0 and 0.499 0. At species level, the percentage of polymorphic bands was 92.2%, the total gene diversity was 0.378 9 and the Shannon’s information index was 0.550 4. The coefficient of overall genetic differentiation and the ge- netic distances between the stocks were also calculated to be 0.073 0 and 0.079 6 using the POPGENE program. Results show that the genetic diversity of the two stocks is still large but the genetic distance between the two stocks is close. A dendrogram was constructed for the 57 individuals from the two stocks, showing that the genetic structure was unitary for PL stock but complex for QD stock.     

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.     

GENETIC DIVERSITY OF THE WILD AND REARED PSEUDOSCIAENA CROCEA

The genetic diversity of both wild and reared Pseudosciaena crocea (Richardson) col-lected from Guan-Jing-Yang in Ningde, China in May 1999 was investigated by random amplified poly-morphic DNA (RAPD) in the present study. The polymorphism and mean difference of the wild popula-tion as revealed by RAPD were 18.9% and 0.0960 respectively, and those of the reared stocks were rel-atively lower, with 16.7% in polymorphism and 0.0747 in mean difference. The genetic distance be-tween the two stocks was 0.0041. From the comprehensive investigation, the main reasons for the loss of genetic diversity were probably overilshing, small number of parents as broodstocks and the debatable arti-ficial ranching. Results from this study also showed that the large yellow croaker populations distributed along Fujian coastal waters including Guan-Jing-Yang still potentially wide genetic variability. It is sug-gested that genetic management and prevention should be scientifically conducted in order to maintain and improve the genetic diversity of the P. crocea population.     

Genetic diversity in two Japanese flounder populations from China seas inferred using microsatellite markers and COI sequences

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea (Qingdao population, QD) and East China Sea (Zhoushan population, ZS) using 10 polymorphic microsatellite loci and cytochrome c oxidase subunit I (COI) sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data (F_st =0.048 7, P<0.001) and mitochondrial DNA data (F_st =0.128, P<0.001) both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.     

胡子鲇3个养殖群体遗传结构的AFLP分析

应用AFLP方法,从64对引物中筛选出5对用于分析胡子鲇(Claris fuscus)厦门(XM)、江门(JM)、玉林(YL)等3个养殖群体共90个个体群体内、群体间遗传多样性。扩增共得到289条清晰谱带,引物多态性介于47.8%～63.8%,平均多态性比率为56.4%。胡子鲇群体总遗传杂合度(Ht)、遗传分化系数(Hs)、遗传分化度(Gst)和基因流(Nm)分别为0.161 1、0.153 6、0.051 1和8.501 8,表明胡子鲇多态性处于中等偏下水平,群体内部遗传分化系数较高,三个群体间无较大分化,群体间基因流动较好。其中,JM群体多态性比率、有效等位基因数Ne、Nei’s指数和Shannon指数都较XM群体和YL群体高,种内分化程度较好。对群体进行两两比较发现,XM群体和YL群体间分化程度最大,XM群体和JM群体次之,JM群体和YL群体最小。基于杂交优势,推断XM和YL群体繁殖所产生的后代将具有比亲代更为优良的性状。     

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.     

军曹鱼群体遗传结构的AFLP分析

筛选出10对扩增片段长度多态分析技术(Amplified fragment length polymorphism,AFLP)的选择性扩增引物(E-aga/M-cgt、E-aga/M-cga、E-agc/M-cga、E-agg/M-cga、E-act/M-cgc、E-aag/M-cgc、E-aca/M-cga、E-aac/M-cgc、E-aac/M-cac和E-aac/M-cat),分析了4个军曹鱼(Rachycentron canadum)全人工繁育群体海南1(HN1)、海南2(HN2)、湛江(ZJ)和流沙湾(LS)的遗传结构。结果表明:总体多态位点比例(83.9%)、分化指数(0.427 8)和Shannon信息指数(0.614 9)均处于较高水平,遗传多样性丰富;主坐标分析和邻位连接树中LS-HN2支与ZJ-HN1支明显分离,各群体内多样性水平存在差异(遗传多样性大小依次为HN1、HN2、LS、ZJ),但总体基因流仍然较大(Nm=10.327),分子方差分析(AMOVA)获得的群内方差(53%)也略大于群间(47%)。