应用微卫星分型方法进行大熊猫父亲鉴定

1979年我们利用水稻花粉单倍体植株的茎节、幼穗、叶鞘和叶片等组织进行离体培养,诱导出二倍体植株。     

Rapid Isolation of DNA from Fresh and Preserved Fish Scales for Polymerase Chain Reaction

We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence. Received November 6, 2000; accepted February 26, 2001.     

贵州紫云刺葡萄自然发酵中野生酿酒酵母基因型多样性分析

[目的] 研究贵州紫云县刺葡萄自然发酵过程中野生酿酒酵母的基因型多样性,分析不同基因型酵母在不同发酵时期的动态变化,为优良酿酒酵母资源的开发利用提供理论依据。[方法] 采用Interdelta指纹图谱分析方法和微卫星分子标记法,研究贵州紫云县刺葡萄自然发酵中野生酿酒酵母的基因型多样性,并通过DPS软件分析不同基因型之间的遗传关系。[结果] 贵州紫云县刺葡萄自然发酵中共分离野生酿酒酵母75株,经Interdelta指纹图谱分析方法和微卫星分子标记法鉴定为10个基因型,其中基因型6、9、10、11、14、15、16为野生酿酒酵母独有的7个基因型,7、17和18为野生与商业酿酒酵母共有的3个基因型,此外,本研究所用其他商业酿酒酵母另有独有的9个基因型（1、2、3、4、5、8、12、13和19）。75株野生酿酒酵母中基因型17的占比最高为36%,其次为基因型10占比为13.3%。在自然发酵过程中不同基因型呈现此消彼长的变化,每一种基因型的菌株细胞密度在10⁴-10⁷ CFU/mL之间。[结论] 贵州紫云县刺葡萄自然发酵样品展现了丰富的酿酒酵母菌株基因型多样性,其中基因型10和17为主导基因型,该研究为贵州刺葡萄优良野生酿酒酵母资源的开发奠定了基础。     

Loggerhead turtle eggshells as a source of maternal nuclear genomic DNA for population genetic studies

Tagging studies on nesting beaches are commonly used to estimate nesting frequency, remigration interval and nesting population size for marine turtle rookeries. Estimates of these demographic parameters from tagging projects may be biased because of the small scale of tagging efforts relative to female nest site fidelity and the logistical difficulty of intercepting all nesting females. Therefore, alternative and supplemental means of individual identification of nesting females are required. We demonstrate that maternal nuclear microsatellite DNA can be isolated from unincubated eggshells of the loggerhead sea turtle (Caretta caretta) through comparison of DNA extracted from 59 eggs collected within 15 h of oviposition and DNA derived from skin samples from respective nesting females. Scorable microsatellite genotypes were produced in 897 of 994 (90.2%) single-locus egg amplifications attempted. Among eggs from known females, 730 of 748 (97.6%) single-locus, egg-derived genotypes matched the respective skin-derived genotypes. Allelic dropout was the most common type of error, followed by the presence of nonmaternal, presumably paternal, alleles. Genotypes derived from unincubated eggshells permit individual assignment of nests and therefore demographic parameter estimates for loggerhead turtle nesting populations, despite genotyping errors that require further optimization. Although sampling unincubated eggs is destructive, this technique is noninvasive to nesting females and is applicable in marine turtle population genetics studies when individual resolution is required but direct interception of nesting females is undesirable or logistically infeasible.     

Differentiation of the Italian wolf and the domestic dog based on microsatellite analysis

The Italian wolf is in the process of regaining the Alpine region which comes into conflict with the extensive sheep keeping practiced in Switzerland during the summer. As in Switzerland, the wolf is a protected species, the government reimburses losses caused by wolves. Therefore we wanted to know whether the Italian wolf could be distinguished from the domestic dog by microsatellite analysis if DNA samples of the predators could be secured. The evaluation of combined genotypes for the microsatellites CanBern6, CPH4, CPH7, CPH9, CPH12, CPH22 and ZuBeCa1 made it possible to identify an individual as either a domestic dog or an Italian wolf. The assignment of an individual to either one of the two populations is based on the logarithm of the likelihood ratio of an individual being an Italian wolf rather than a domestic dog, given a specific combined genotype. The distribution of the Italian wolf combined genotypes (n = 42) is clearly distinct from the distribution of the domestic dog combined genotypes (n = 90). The likelihood ratio for the "worst" Italian wolf combined genotype was 2.3 E+5 and for the "worst" domestic dog combined genotype was 3.8 E-5.     

Sympatric wolf and coyote populations of the western Great Lakes region are reproductively isolated

Interpretation of the genetic composition and taxonomic history of wolves in the western Great Lakes region (WGLR) of the United States has long been debated and has become more important to their conservation given the recent changes in their status under the Endangered Species Act. Currently, the two competing hypotheses on WGLR wolves are that they resulted from hybridization between (i) grey wolves (Canis lupus) and western coyotes (C. latrans) or (ii) between grey wolves and eastern wolves (C. lycaon). We performed a genetic analysis of sympatric wolves and coyotes from the region to assess the degree of reproductive isolation between them and to clarify the taxonomic status of WGLR wolves. Based on data from maternal, paternal and bi‐parental genetic markers, we demonstrate a clear genetic distinction between sympatric wolves and coyotes and conclude that they are reproductively isolated and that wolf–coyote hybridization in the WGLR is uncommon. The data reject the hypothesis that wolves in the WGLR derive from hybridization between grey wolves and western coyotes, and we conclude that the extant WGLR wolf population is derived from hybridization between grey wolves and eastern wolves. Grey‐eastern wolf hybrids (C. lupus × lycaon) comprise a substantial population that extends across Michigan, Wisconsin, Minnesota and western Ontario. These findings have important implications for the conservation and management of wolves in North America, specifically concerning the overestimation of grey wolf numbers in the United States and the need to address policies for hybrids.     

Accurate methods of DNA extraction and PCR-based genotyping for single scallop embryos/larvae long preserved in ethanol

Marine scallops are sessile as adult but have a long planktonic larval phase showing great possibility to migrate in marine realm lacking of obvious barriers. Genetic analysis of scallop embryos/larvae based on molecular markers is very essential to clarify the spatial and temporal gene flow and the unique population and community structure. However, the technical challenges, such as single embryos/larvae isolation and low quantity and poor quality of DNA extracted, make genotyping for a single embryo/larva long preserved in ethanol to be a really difficult task. In this study, we analysed the factors that might affect the DNA quantity and quality for simple sequence repeat‐based genotyping for single embryos/larvae. Based on the factors analysed, we developed a LoTEPA buffer‐based method, of which the accuracy, stability and reproducibility were evaluated by controlled inter‐ and intraspecies and self‐fertilized scallop families. The genotyping results showed the high success rate of more than 90% in total for embryos/larvae preserved in ethanol for 1–5 years. Furthermore, the successful genotyping for the larvae sampled from a natural habitat well demonstrated the potential use of this method in practical ecological analysis.     

Juvenile habitat partitioning and relative productivity in allochronically isolated sockeye salmon (Oncorhynchus nerka)

Allochronic divergence, like spatial isolation, may contribute to population diversity and adaptation, however the challenges for tracking habitat utilization in shared environments are far greater. Adult Klukshu River (Yukon, Canada) sockeye salmon, Oncorhynchus nerka, return as genetically distinct "early" and "late" runs. Early and late adult spawning populations (1999 and 2000) and their subsequent fry (sampled at 7 sites in 2000 and at 8 sites in 2001 throughout Klukshu Lake and River) were genotyped at eight microsatellite loci. Bayesian assignment was used to determine the spatial distribution of early versus late fry; although intermixed, the distribution of fry significantly differed in Klukshu Lake and in the Klukshu River in 2001, based on crosstab analyses. Late-run fry predominated in Klukshu Lake at all sites, while early-run fry were most common in the north and south of Klukshu Lake and in Klukshu River. Early-run spawners had significantly higher relative productivity (early life survival) than late-run fish (2.9 times more fry produced per early-run adult in 2000, and 9.2 times more in 2001). This study demonstrates spatial habitat partitioning and differences in the contribution of allochronically isolated populations to fry abundance, and highlights annual variability that likely contributes to recruitment variation.     

Polymorphisms of GA/GT Microsatellite Loci from Anguilla japonica

Six novel microsatellite loci, containing (GA)_15\N17 or (GT)_10\N19 perfect tandem repeats, were isolated and characterized for the catadromous eel Anguilla japonica. The allelic size of the 6 loci ranged from 79 to 226 bp in length. All loci were polymorphic with a mean number of 14.7 alleles per locus and a mean heterozygosity of 0.67, suggesting higher polymorphism than that of freshwater and anadromous fishes, but lower than that of marine fishes. Genotype diversity of the 6 loci ranged from 0.22 to 0.61 with a mean value of approximately 0.5. Cross-species amplification showed that 5 of the 6 microsatellite primers proved to be useful in addressing questions of population genetics for all Anguilla species. Received October 11, 2000; accepted December 29, 2000.     

Multilocus genotypes from Charles Darwin's finches: biodiversity lost since the voyage of the Beagle

Genetic analysis of museum specimens offers a direct window into a past that can predate the loss of extinct forms. We genotyped 18 Galápagos finches collected by Charles Darwin and companions during the voyage of the Beagle in 1835, and 22 specimens collected in 1901. Our goals were to determine if significant genetic diversity has been lost since the Beagle voyage and to determine the genetic source of specimens for which the collection locale was not recorded. Using ‘ancient’ DNA techniques, we quantified variation at 14 autosomal microsatellite loci. Assignment tests showed several museum specimens genetically matched recently field-sampled birds from their island of origin. Some were misclassified or were difficult to classify. Darwin''s exceptionally large ground finches (Geospiza magnirostris) from Floreana and San Cristóbal were genetically distinct from several other currently existing populations. Sharp-beaked ground finches (Geospiza difficilis) from Floreana and Isabela were also genetically distinct. These four populations are currently extinct, yet they were more genetically distinct from congeners than many other species of Darwin''s finches are from each other. We conclude that a significant amount of the finch biodiversity observed and collected by Darwin has been lost since the voyage of the Beagle.     

MICA基因微卫星多态在中国13个群体中的分布

通过对中国13个群体（云南汉族、广东汉族、山东汉族、白族、傣族、拉祜族、黎族、纳西族、撒拉族、畲族、土族、佤族和云南藏族）共577例无亲缘关系的研究对象的DNA样本进行MICA基因微卫星扫描分型,获得了该微卫星的不同等位基因在各群体中的遗传数据。结果表明,该微卫星在不同群体中的分布存在差异,并有较高的多态信息含量（PIC）,是一个有用的遗传标记,在人类进化研究、个体识别、亲子鉴定、基因作图与定位以及疾病诊断方面可能有较大的潜在应用价值。