微卫星DNA标记技术及其在遗传多样性研究中的应用

微卫星DNA的高突变率、中性、共湿性及其在真核基因组中的普遍性,使其成为居群遗传学研究、种质资源鉴定、亲缘关系分析和图谱构建的优越的分子标记。本研究系统介绍了微卫星DNA在结构和功能上的特点,并对微卫星DNA标记技术应用的遗传学机理和一般方法进行了扼要的阐述。另外,本研究还探讨了微卫星DNA标记技术在遗传多样性研究中的应用现状,并进一步提出其发展前景。     

Molecular Cloning of Growth Hormone Complementary DNA in Barfin Flounder (Verasper moseri)

The olive flounder (family Paralichthidae; Paralichthys olivaceus) growth hormone (ofGH) appears to be the most derived among known growth hormones, with the deletion of 14 consecutive amino acids in the carboxy-terminal region. To ascertain if this deletion is common to all flounders, growth hormone complementary DNA of the barfin flounder (bfGH) (family Pleuronectidae; Verasper moseri) has been cloned. It was amplified by polymerase chain reaction using single-strand cDNA from the pituitary gland. Excluding the poly(A) tail, the bfGH cDNA is 919 nucleotides long and contains a 609-bp open reading frame encoding a putative signal peptide of 17 amino acids and a mature protein of 186 amino acids. Northern blot analysis detected 1.0 kb of bfGH messenger RNA in the pituitary gland, which is a reasonable value considering the poly(A) tail. The deduced amino acid sequence of bfGH has 78% identity with the sequence of ofGH. A major difference is the presence of a 14 amino acid segment (140–153) in bfGH, as in other growth hormones, suggesting that this deletion in the olive flounder occurred after the divergence of the Pleuronectoidae. Received May 7, 1999; accepted July 13, 1999.     

Weak founder effect signal in a recent introduction of Caribbean Anolis

Species introductions provide a rare opportunity to study rapid evolutionary and genetic processes in natural systems, often under novel environmental pressures. Few empirical studies have been able to characterize genetic founder effects associated with demographic bottlenecks at the earliest stages of species introductions. This study utilizes prior mitochondrial DNA information which identifies the putative source population for a recently established ( c . 7 years between import and sampling) species introduction. We investigated the evidence for a founder effect in a highly successful introduction of a Puerto Rican Anolis species that has established itself on Dominica to the localized exclusion of the native, endemic anole. Five highly polymorphic microsatellite loci were used to explore the partitioning of genetic diversity within and between native source, native nonsource, and introduced populations of Anolis cristatellus . Group comparisons reveal significantly lower allelic richness and expected heterozygosity in introduced populations compared to native populations; however, tests for heterozygosity excess relative to allelic richness failed to provide consistent evidence for a founder effect within introduced populations. Significant levels of within-population genetic variation were present in both native and introduced populations. We suggest that aspects of the reproductive ecology of Anolis (high fecundity, sperm storage and multiple paternity) offer an important mechanism by which genetic variation may be maintained following demographic bottlenecks and founder events in some squamate taxa.     

Evaluation of the genetic variability of 13 microsatellite markers in native Indian pigs

We analysed polymorphism of 13 microsatellites in two Indian domesticated pig types (North Indian and Northeast Indian). Heterozygosity, polymorphism information content, and probability of identity of two random individuals were calculated for all microsatellites in both types. The number of alleles observed at a locus varied between five and 12. The evaluated microsatellites exhibited a very high heterozygosity and polymorphism information content. The probability of identity of two random individuals from different populations taking into account all the 13 microsatellites was as low as 3.51 × 10^-19. On the basis of these results, we propose that these microsatellite markers may be used with reliability for studying the genetic diversity and for identification of individuals in Indian pig types.     

The use of inter-specific hybrids in aquaculture and fisheries

Inter-specific hybrid fishes have been produced for aquaculture and stocking programmes to increase growth rate, transfer desirable traits between species, combine desirable traits of two species into a single group of fishes, reduce unwanted reproduction through production of sterile fish or mono-sex offspring, take advantage of sexual dimorphism, increase harvestability, increase environmental tolerances, and to increase overall hardiness in culture conditions. Hybrids constitute a significant proportion of some countries' production for certain taxa; for example, hybrid striped bass in the USA, hybrid clarid catfish in Thailand, hybrid characids in Venezuela, and hybrid tilapia in Israel. Despite its widespread use, there is a general impression that inter-specific hybridization is not a very useful tool for aquaculture. We believe this impression stems from inaccurate reporting of some useful hybrids, limited testing of strains used for hybrids, and from early work on salmonids that did not result in hybrids of commercial advantage.Experimentation with new hybrid fishes is ongoing, especially in marine culture systems where sterile fish may be preferred because of the concern that fish may escape into the marine and coastal environment.Hybridization has been used in tandem with polyploidization to improve developmental stability in hybrid progeny. The results of inter-specific hybridization can be variable and depend on the genetic structure (including the sex) of the parent fish. Inadvertent hybridization and backcrossing can lead to unexpected and undesirable results in hybrid progeny, such as failure to produce sterile fish, loss of color pattern, and reduced viability.Hybridization is only one tool to improve aquaculture production and will require knowledge of the genetic structure of the broodstock, good broodstock management and monitoring of the viability and fertility of the progeny. Hybridization does represent a genetic modification wherein genes are moved between different species; implications for biodiversity conservation and regulation of this type of modification are discussed.     

A comparative histological and histochemical study of the post-gastric alimentary canal from three species of pleuronectid, the Atlantic halibut, the yellowtail flounder and the winter flounder

The histology and mucus histochemistry of the pleuronectid post-gastric alimentary canal was examined using light and electron microscopy. Distinct differences in goblet cell mucus histochemistry were observed between species, with the two closest taxonomic species, the winter flounder and the yellowtail flounder showing the most diversity and the halibut showing regional variation. Numbers of goblet cells within post-gastric regions did not differ significantly between species, but were significantly different between regions within species increasing toward the rectum. The post-gastric region was divisible into two areas based upon the ultrastructural features of lipid digestion and absorption in the intestine and pyloric caeca, and of exogenous protein in the rectum. The combination of species-specific histochemical differences in mucus and general histological and ultrastructural differences within the post-gastric regions between these species suggest a correlation between lumenal environmental conditions/histology and natural prey preference.     

Genetic characterization of 18 novel microsatellite loci in northern pike (Esox lucius L.)

The northern pike (Esox lucius L.), an important predatory freshwater species, is undergoing significant population decline. In this study, 18 novel polymorphic microsatellite loci were isolated and used for assessing genetic variation in the Chinese Ulungur and Hungarian Balaton populations of the species. The number of alleles ranged from 2 to 13, observed heterozygosity from 0.154 to 0.920 and expected heterozygosity from 0.145 to 0.921, thereby indicating the specific usefulness of these suites of markers for investigating genetic variability.     

Evaluation of the genetic variability of 23 bovine microsatellite markers in four Belgian cattle breeds

The polymorphism of 23 microsatellites in the four main cattle breeds in Belgium (Holstein Friesian, Belgian Blue, Belgian Red Pied and East Flemish) was analysed. Heterozygosity, polymorphism information content, the effective number of alleles, exclusion probability and the probability of genotypic identity for two random individuals were calculated for all microsatellites and all breeds. The Belgian Blue breed is generally a little less polymorphic in comparison with the other three breeds. Estimates of the genetic distances between these breeds confirmed the widely accepted proposition that the Belgian Blue is the most genetically distinct of these breeds. The three other breeds are likely to become one population, given current breeding strategies. Exclusion probabilities in parentage control cases are >0·9999 in all four breeds when all 23 microsatellites are used and >0·98 with only the two most polymorphic multiplexes.     

利用线粒体COI和微卫星标记分析文蛤7个地理群体的遗传变异

利用线粒体细胞色素氧化酶亚基I(COI)和微卫星标记分析了文蛤7个地理群体(朝鲜新义州,辽宁丹东、蛤蜊岗和盘山,山东东营,江苏如东和启东)的遗传多样性和群体分化。PCR扩增获得142条602 bp的COI核苷酸片段,比对到13个变异位点,包括11个转换和2个颠换,定义了22个单倍型,共享单倍型12个,新义州、丹东和启东群体分别拥有特有单倍型。单倍型多样性最高的是如东群体(h=0.900),最低的是东营群体(h=0.600);核苷酸多样性最高的是丹东群体(π=0.00350),最低的是蛤蜊岗群体(π=0.00115)。基于COI数据的Fu's Fs中性检验和核苷酸不配对分析揭示文蛤种群历史上曾经历过群体扩张事件。分子变异分析(AMOVA)表明,群体内遗传变异占71.64%,群体间遗传变异占28.36%,群体间发生显著的遗传分化(P0.05)。7个微卫星标记扩增280个个体共获得54个等位基因,平均等位基因数为7.7个,平均观测杂合度和期望杂合度分别为0.3878和0.7996。江苏群体具有较高的遗传多样性,但7个群体间遗传多样性不存在显著差异(Kruskal-Wallis检验,P0.05)。Hardy-Weinberg平衡检验结果显示49个群体-位点组合中有18个偏离平衡(P0.05),表现为杂合子缺失。单倍型邻接(NJ)树显示聚类未展示地域性特色,但某几个同一或者相近地理群体的单倍型具有聚类现象(如东和启东部分单倍型出现地理聚类)。依据群体间遗传距离以Kimura 2-parameter为模型建立UPGMA系统发育树,显示丹东群体和江苏的如东和启东群体聚为一支,暗示江苏苗种的异地养殖已经污染丹东文蛤的遗传背景。     

Inheritance Characteristics of Microsatellite DNA Loci in Experimental Families of Japanese Flounder Paralichthys olivaceus

Allele segregating patterns of microsatellite DNA loci in 5 experimental families of Japanese flounder Paralichthys olivaceus and genotype frequencies for deviation from Hardy-Weinberg expectations (HWE) in natural P. olivaceus populations were studied to assess inheritability. Of the 12 microsatellite loci examined, 1 locus had a possibility of scoring errors of heterozygous individuals caused by unreproducible polymerase chain reaction amplifications of a particular allele. At the remaining 11 loci, almost all of alleles were segregated according to Mendelian transmission, and observed genotype frequencies in natural populations were consistent with HWE. The results demonstrated here would provide useful information supporting the suitability of these microsatellite loci as inheritable P. olivaceus genetic markers. Received September 25, 2000; accepted March 1, 2001     

Genetic variation analysis within and among Chinese indigenous swine populations using microsatellite markers

The genetic structure of five types of Taihu pig (Erhualian, Middle Meishan, Small Meishan, Mizhu and Shawutou), Jiangquhai and Dongchuan pigs in China were investigated, by means of 27 microsatellite markers proposed by the Food and Agriculture Organisation, International Society for Animal Genetics (FAO-ISAG). The Hardy-Weinberg equilibrium test indicated that genetic drift had occurred in these populations, which may be caused by founder effects, intensive selection and close breeding. Genetic heterozygosity and the effective number of alleles per population were calculated, and showed that the genetic variability of the Jiangquhai pig was the largest, while the Small Meishan had the lowest. Genetic differentiation was within each population as shown by the fixation index (F(ST)=0.18). Both a neighbour-joining (NJ) tree constructed from Nei's standard genetic distance and principal component analysis based on allele frequencies can distinguish types of Taihu pig from the other two breeds. Among Taihu pig populations, Erhualian and Mizhu grouped into one branch, while Middle Meishan, Small Meishan and Shawutou clustered as another branch. By including previously published data on European pigs reported, we confirmed that Chinese indigenous pigs and European pigs have diverged into two distinct groups.     

Application of Microsatellite Markers to Population Genetics Studies of Japanese Flounder Paralichthys olivaceus

We examined population genetic structure by means of microsatellite analysis among 7 Japanese flounder (Paralichthys olivaceus) populations collected from coastal sea areas around Japan. As was expected, all of the 11 microsatellite loci examined were variable in all populations (number of alleles per locus, 15.2–18.2; average of expected heterozygosity, 0.74–0.78). Eleven population pairs in 21 possible pairwise comparisons showed significant genetic heterogeneity associated with allele frequency distributions or fixation index (F _ST ). Modified Cavalli-Sforza chord distance (D _A ) and Nei's standard genetic distance (D _ST ) ranged from 0.051 to 0.090, and from 0.000 to 0.025, respectively. There was evidence that the populations assessed in this study were not drawn from a single panmictic population; however, it appears that Japanese flounder populations around Japan are not well-structured, as an estimate of the fixation index value among the 7 localities was very low (F _ST = 0.0025). Received April 27, 2001; accepted June 27, 2001.     

Development and characterization of nine polymorphic microsatellite markers in the Chilean kelp Lessonia nigrescens

A total of nine microsatellite loci were isolated and characterized in the Chilean kelp Lessonia nigrescens Bory. Using two different enriched libraries, we observed 1-14 alleles per locus in two samples of 21 kelp individuals each. The observed heterozygosities ranged from 0.05 to 0.80 and all loci are in Hardy-Weinberg equilibrium for one or both samples. Seventeen samples collected from different sites showed high allele diversity along the species distribution. The variation detected at these markers is currently being used for the study of populations of Lessonia nigrescens at different geographical scales.     

Specific microsatellite loci for brook charr reveal strong population subdivision on a microgeographic scale

We have isolated specific microsatellite loci from a partial genomic library of brook charr Salvelinus fontinalis. Their usefulness was investigated by measuring intra- and inter-population genetic diversity at four loci among 20 individuals from each of five lakes located 3 to 22 km apart in La Mauricie national park (Canada). These markers were moderately to highly polymorphic. A total of five, six, 16 and 18 alleles per locus were detected, and their overall expected heterozygosity was 0.53, 0.58, 0.86 and 0.87. Strong inter-population diversity was detected. Highly significant differences in allelic frequencies were found in all but two pairwise χ² permutation tests between populations at all loci. Numerous population unique alleles were observed in all five lakes. Consequently, a highly significant component of total genetic diversity was due to interpopulation variance, as exemplified by G _ST values of 0.33, 0.42, 0.47 and 0.84 for each individual locus. Altogether, the results indicated that these loci should be valuable in addressing fine scale population genetics questions in brook charr. To our knowledge, they also represent the first available microsatellites developed in the genus Salvelinus.