植物遗传多样性和系统学研究中的等位酶分析*

本文对“等位酶”的概念、等位酶分析方法在植物遗传多样性和系统学研究中应用的范围（包括种上和种下）进行了介绍和讨论,并对其优点和缺点进行了评论。认为等位酶分析是研究生物遗传多样性的重要方法,并为系统学和进化研究进入分子水平开辟了广阔而实用的前景,但使用分子资料并不意味着可以抛弃形态、细胞等其他方面的资料,它们的关系是互相补充,而决不是互相代替。     

植物遗传多样性和系统学研究中的等位酶分析*

本文对“等位酶”概念、等位酶分析方法在植物遗传多样性和系统学研究中应用的范围（包括种上和种下）进行了介绍和讨论,并对其优点和缺点进行了评论。认为等位酶分析是研究生物遗传多样性的重要方法,并为系统学和进化研究进入分子水平开辟了广阔而实用的前景,但使用分子资料并不意味着可以抛弃形态、细胞等其他方面的资料,它们的关系是互相补充,而决不是互相代替。     

Clonal diversity and genetic differentiation revealed by SSR markers in wild Vaccinium macrocarpon and Vaccinium oxycoccos

American cranberry (Vaccinium macrocarpon) is a perennial, woody plant species, native to North American bogs and wetlands. Cranberries represent one of the few agriculturally important native plants in which wild gene pools are still readily available within the undeveloped wetlands of the northern US and Canada. Earlier studies have reported low genetic variation in V. macrocarpon at the species and population level. However, in this study, we characterised 229 individuals of wild V. macrocarpon and V. oxycoccos (small cranberry) from Wisconsin and 22 accessions using microsatellite markers and observed substantial genetic variation and differentiation within and among populations and species. While V. macrocarpon was analysed using 108 alleles from 11 microsatellite loci revealing 42 unique genotypes, V. oxycoccos was analysed using 156 alleles from eight loci revealing 28 unique genotypes. There were a total of 182 alleles found in both species combined with 156 of those alleles present in V. oxycoccos and 84 alleles found in V. macrocarpon. All eight loci possessed species‐specific alleles with V. oxycoccos possessing 98 private alleles versus 26 private alleles found V. macrocarpon, and 58 alleles were found in common between both species. Our data will be valuable not only for future wild cranberry diversity and population genetics research, but for other cranberry breeding and genetics studies.     

Evolution in an autopolyploid group displaying predominantly bivalent pairing at meiosis: genomic similarity of diploid Vaccinium darrowi and autotetraploid V. corymbosum (Ericaceae)

The genomic relationship between V. darrowi Camp (2n = 2x = 24) and V. corymbosum L. (2n = 4x = 48) was examined using an interspecific tetraploid hybrid, US 75, and representatives of the parental species. Two features in the background of US 75 led to the prediction that it was an allopolyploid: (1) the parental species are quite distinct morphologically and geographically, and (2) the diploid genome was incorporated into US 75 via an unreduced gamete. However, US 75 recently was shown to display tetrasomic inheritance using molecular markers. In the present cytological study, US 75 was found to have a lower than expected number of multivalents for an autopolyploid, although it had a significantly higher number of quadrivalents than its autotetraploid parent, V. corymbosum. Normal chromosome distributions were observed at anaphase I and II, and pollen viability was high. Our findings suggest that little genomic divergence has developed between the Vaccinium species and that the polyploids may freely exchange genes with sympatric diploid species via unreduced gametes. This pattern of hybridization could be an important component of evolution in all autopolyploid groups, making them much more dynamic than traditionally assumed.     

西南地区麻疯树天然种群遗传多样性的等位酶变异

为了揭示麻疯树(Jatropha curcas)天然种群的遗传多样性和遗传结构, 采用聚苯烯酰胺凝胶垂直平板电泳技术, 对采自四川、云南、贵州3个省的10个麻疯树天然种群的叶片样本进行了同工酶分析。7个酶系统10个位点的检测结果表明: 麻疯树种群水平上的遗传多样性较高, 每位点平均等位基因数为2.428 6, 多态位点百分率为97.14%, 平均期望杂合度为0.396 4。种群间遗传分化系数为0.041 3, 种群间总的基因流较高, 为5.808 9, 种群间遗传一致度较高(Shannon信息指数为0.921 7- 0.995 3)。非加权类平均法(UPGMA)聚类结果显示, 10个种群的遗传距离与地理距离相关性不显著。麻疯树天然种群具有较低程度的遗传分化、较高的基因流, 种内及种群内多样性丰富, 这为麻疯树优良品种的选育提供了良好的遗传基础。     

Allozyme and RAPD analysis of the genetic diversity and geographic variation in wild populations of the American chestnut (Fagaceae)

Genetic variation among 12 populations of the American chestnut (Castanea dentata) was investigated. Population genetic parameters estimated from allozyme variation suggest that C. dentata at both the population and species level has narrow genetic diversity as compared to other species in the genus. Average expected heterozygosity was relatively low for the population collected in the Black Rock Mountain State Park, Georgia (He = 0.096 +/- 0.035), and high for the population in east central Alabama (He = 0.196 +/- 0.048). Partitioning of the genetic diversity based on 18 isozyme loci showed that ~10% of the allozyme diversity resided among populations. Cluster analysis using unweighted pair-group method using arithmetric averages of Rogers' genetic distance and principal components analysis based on allele frequencies of both isozyme and RAPD loci revealed four groups: the southernmost population, south-central Appalachian populations, north-central Appalachian populations, and northern Appalachian populations. Based on results presented in this study, a conservation strategy and several recommendations related to the backcross breeding aimed at restoring C. dentata are discussed.     

Allozyme diversity in the tetraploid endemic Thymus loscosii (Lamiaceae)

BACKGROUND AND AIMS: Thymus loscosii (Lamiaceae) is a tetraploid perennial species endemic to the Ebro river basin (north-eastern Spain), which is included in the National Catalogue of Endangered Species. It is a tetraploid species (2n = 54), presumably an autotetraploid originated by the duplication of a 2n = 28 genome and the subsequent loss of two chromosomes. Allozyme electrophoresis was conducted to survey the levels and distribution of genetic diversity and to test the previous autopolyploid hypothesis for its origin. In addition, both in situ and ex situ conservation measures are proposed. METHODS: Eight populations were sampled for analysis by standard methods of starch gel electrophoresis, and six putative enzymatic loci were resolved (five consistently and one only partially). KEY RESULTS: Banding patterns exhibited no evidence of fixed heterozygosity and showed both balanced and unbalanced heterozygotes. In addition, most individuals showed a pattern consistent with the presence of three or four alleles at a single locus. High levels of genetic variability were found at population level (P = 85 %, A = 3.0, He = 0.422), in addition to a trend of an excess of heterozygotes. CONCLUSIONS: Allozyme data support the hypothesis that T. loscosii is an autotetraploid, and the high number of alleles at some loci may be due to repeated polyploidization events. The high values of genetic variation found in this species agree with those expected for tetraploids. The excess of heterozygotes may be due to some barriers to inbreeding (e.g. occurrence of gynodioecy) and/or selection for heterozygosity.     

中国沿海中部珊瑚菜居群等位酶变异及其遗传多样性

利用聚丙烯酰胺凝胶电泳方法,分析了我国沿海中部（江苏、山东和浙江）海滨沙滩珊瑚菜（Glehnia littoralis Fr.Schmidt ex Miq.)7个居群8种酶系统19个位点的等位基因遗传变异特征,结果表明居群内多态位点比率平均为82．4％,每一位点平均等位基因数为2．77,有效等位基因数为2．24,固定指数F的平均值为－0．091。珊瑚菜居群基因多样性80．9％产生于居群内,19．1％产生于居群间。居群间的遗传距离平均为0．317,遗传一致性为0．728,居群内维持着较高水平的遗传多样性。据此,对珊瑚菜的渐危机理进行了讨论。     

Low genetic diversity and allozymic evidence for autopolyploidy in the tetraploid Pyrenean endemic larkspur Delphinium montanum (Ranunculaceae)

Allozyme electrophoresis was conducted to survey the levels and distribution of genetic diversity in the tetraploid perennial larkspur Delphinium montanum (Ranunculaceae), which is endemic to the eastern Pyrenees of Spain and France and considered to be endangered. Seven populations were sampled, resolving 14 putative enzymatic loci belonging to eight enzymes. Banding patterns stained in gels revealed several enzymatic duplications attributable to autotetraploidy, such as the presence of both balanced and unbalanced heterozygotes and the lack of fixed heterozygosity. However, variability in D. montanum ( P  = 23.8%, A  = 1.48, and H _e = 0.082) was lower than that expected for an autotetraploid species. This, in addition to the scarcity of loci showing three or four different alleles, could indicate that this species has suffered genetic erosion through population bottlenecks, or, alternatively, that it is undergoing diploidization.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 211–222.     

Comparison of genetic diversity between narrowly endemic shrub Menziesia goyozanensis and its widespread congener M. pentandra (Ericaceae)

Menziesia goyozanensis, a Japanese kindof fool's huckleberry, is a shrub speciesendemic only to Mt. Goyo in northern Japan.This species is listed as `criticallyendangered' in the Red Data Book of wild plantsin Japan, and only a single population has yetbeen discovered. The allozyme diversity andRAPD variation of Menziesia pentandrawere eight- and threefold greater than those ofM. goyozanensis, respectively, althoughwe examined only five populations representinga part of the distribution of M.pentandra. Because the environment surroundingthe population of M. goyoznaensis seemsto be stable at present, it is not necessary toplan an urgent ecological conservation practicefor the species. However, considering that thehabitat of M. goyozanensis is very smalland large vegetative disturbances occasionallyoccur in cool temperate forests, in which thespecies dwells, ex situ conservation isessential.     

Allozyme variation and genetic inter-relationships between seven flatfish species (Pleuronectiformes)

A total of 11 enzymes coded by 14 loci were assayed for each of 17 populations (from the north east Atlantic and Mediterranean) of seven flatfish species, including representatives of three of the four European families of the order Pleuronectiformes. Diagnostic alleles were observed for each species and there were fixed differences between the species at many loci. Thus all species were genetically distinct, although there were some common alleles. H_obs was higher than the average of a range of fish species and was also higher than that of vertebrate species as a whole. It seems that flatfish as a group may show higher levels of genetic variation than other fish. Values of genetic identity for all pairwise comparisons fell comfortably within the ranges expected. The data support the grouping of plaice and flounder into a single genus, Pleuronectes , but it is concluded that the retention of the dab in a separate genus, Limanda , is justified. A high level of genetic divergence was found between Dover and thickback soles. Genetic divergence data support the hypothesis that Pleuronectes flesus luscus in the Aegean Sea is a distinct subspecies of Pleuronectes flesus (flounder). The data show a clear separation of the Mediterranean Dover sole population from those in the Atlantic. Low genetic divergence was observed between the Aegean Sea and Atlantic brill populations. It is speculated that about 5 Mya the families Pleuronectidae and later Soleidae evolved from the ancestral Scophthalmidae.     

Allozyme diversity in Pinus virginiana (Pinaceae): intraspecific and interspecific comparisons

Two of the four members of subsection Contortae of the genus Pinus occur in the southeastern United States: Pinus virginiana, which ranges throughout the southern and central Appalachian Mountains, and P. clausa, which is restricted to Florida and southern Alabama. We examined allozyme variation within P. virginiana and genetic relationships between this species and the two varieties of P. clausa (var. clausa and var. immuginata). P. virginiana maintains more genetic diversity at both the species (Hes = 0.139) and population (Hep = 0.128) levels than the other three species in the subsection, which may reflect the combination of its widespread distribution and the absence of cone serotiny. Genetic differentiation among populations in P. virginiana was relatively low (GST = 0.053), but significant contrasts in allozyme frequencies and genetic diversity were apparent between populations to the northwest vs. outheast of the Appalachian Mountains. These regional differences likely resulted initially from historical processes that occurred during the Pleistocene and early Holocene, and have been reinforced by modern selective pressures and barriers to gene flow. The mean genetic distance between populations of P. virginiana and P. clausa (D = 0.071) was greater than that between populations of the two varieties of P. clausa (D = 0.012), which suggests that the two varieties diverged at some point after the separation of the two species.     

松毛虫属（Dendrolimus) 部分种类亲缘关系与遗传分化的等位酶分析

采用等位酶聚丙烯酰胺凝胶电泳技术对松毛虫属5个种和亚种的野生种群进行了亲缘关系和遗传变异的研究.8种等位酶系统(乳酸脱氢酶LDH、苹果酸脱氢酶MDH、苹果酸酶ME、乙醇脱氢酶ADH、甲酸脱氢酶FDH、谷氨酸脱氢酶GDH、过氧化物酶POD、过氧化氢酶CAT)共检测到12个基因位点,其中6个位点为多态位点,检测到15个等位基因.松毛虫属5个种和亚种的总体水平多态位点比率P＝50%,平均有效基因数A = 1.917,平均期望杂合度He =0.267,平均遗传距离为0.0730～0.5701.遗传参数表明松毛虫属昆虫种间存在较高程度的遗传变异,聚类图和遗传距离数据表明赤松毛虫与马尾松毛虫亲缘关系最近,落叶松毛虫与思茅松毛虫亲缘关系最远.     

Allozyme variation and population genetic structure of Betula alnoides from Guangxi,China

Allozyme variation and population genetic structure of Betula alnoides Buch. Ham. ex D. Don in 11 natural populations from Guangxi Zhuang Autonomous Region, China, were investigated by starch gel electrophoresis. Variation at 15 loci from 10 enzyme systems was analyzed. Allozyme analysis revealed a high level of genetic variation in this species, with percentage of polymorphic loci (P _p ), the average number of alleles per locus (A _p ), and the expected heterozygosity (H _ep ) being 55.2%, 2.0, and 0.204, respectively, which exceeds the average level among out-crossing wind-pollinated woody species at the population level. At the species level, P _s , A _s , and H _es were 60.0%, 2.67, and 0.206, respectively.The observed heterozygosity (H _op ) was higher than H _ep , indicating the existence of natural selection against homozygotes. The negative fixation index (F = –0.216) implied a significant excess of heterozygosity at the population level. Among-population differentiation (F _ST ) accounted for 4.0% of the total variation. No significant correlation was detected between the genetic distance and geographic distance among populations. Extensive gene flow was inferred, based on the allozyme data )N _m = 6.000 from F _ST , N _m = 5.605 from the private allele method). The results demonstrated that the fragmentation status of B. alnoides had no remarkable effects on the population genetic structure of this species. Some populations are recommended for both in situ genetic conservation and germplasm collection for breeding programs.     

樟叶越桔熊果苷合成酶基因VdAS1的克隆及序列分析?

根据樟叶越桔( Vaccinium dunalianum)叶芽转录组测序获得的熊果苷合成酶基因VdAS1部分EST序列设计引物,结合RACE￣PCR技术获得VdAS1基因全长1577 bp cDNA序列。其完整的开放阅读框推测编码由475个氨基酸残基组成的VdAS1蛋白,理论相对分子量为52?22 kD,等电点pI＝5?74,负电荷残基( Asp＋Glu)总数为53个,正电荷残基( Arg＋Lys)总数为45个,不稳定系数为37?93,属稳定性蛋白质。生物信息学分析表明VdAS1与枸杞的糖基转移酶相似率高达74％,其二级结构主要构件为α￣螺旋和随机卷曲,不存在跨膜区,属于亲水性蛋白质,并结合信号肽预测结果推测VdAS1直接锚定在细胞基质中行使功能。该研究为后期VdAS1的异源表达和功能研究奠定了基础。     

Low diversity but high differentiation: the population genetics of Aglaope infausta (Zygaenidae: Lepidoptera)

Aim Aglaope infausta is a thermophilous Zygaenid of Atlanto-Mediterranean origin, distributed in Portugal, Spain, France and north-western Italy reaching its north-eastern distribution limit in western Germany. The local, regional and inter-regional genetic structure of this species is studied in this analysis. Location and methods The allozymes of 456 individuals from 12 populations (11 from western Germany and one from southern Portugal) were studied by electrophoresis. Results Six of the 19 loci analysed were monomorphic. Genetic differentiation between populations was high (F_ST: 0.404), while the mean genetic diversity was low (H_e: 3.4%). Most (96.5%) of the genetic variance between populations was between the Portuguese and the German samples, but also the differentiation within Germany was considerable (F_SR: 0.101). In Germany, A. infausta occurs in two major regions (middle Rhine and Nahe) that are geographically separated, and 55.5% of the genetic variance was found between these two regions. The populations of both areas do not differ in their genetic diversity, but those of the middle Rhine have significantly higher genetic distances among them than the Nahe populations (0.020 and 0.015, respectively). F_ST was also higher in the middle Rhine region than in the Nahe region (0.089 and 0.045, respectively). Main conclusions Aglaope infausta shows a very low level of genetic heterogeneity for a lepidopteran species. However, this genetic poverty is not affecting the species’ viability. During the ice ages, differentiation into two genetic lineages occurred, most probably in a south-western and a south-eastern differentiation centre in Iberia. The gap in the distribution range in Germany is clearly reflected in the genetic structure. This differentiation must have developed relatively quickly because western Germany most probably was colonized during the climatic optimum 6000 years ago.     

RAPDs identify varietal misclassification and regional divergence in cranberry [Vaccinium macrocarpon (Ait.) Pursh]

The majority of cultivated cranberry varieties were selected from native populations in the 1800s and early 1900s from sites in Massachusetts, New Jersey, and Wisconsin. Since their initial selections 100–150 years ago, varietal identities have become increasingly confused; primarily the result of there being a paucity of qualitative markers to effectively distinguish among varieties. Random amplified polymorphic DNA (RAPD) technology has the potential for allowing a more definitive classification of varieties and was used in this study to characterize 22 cranberry varieties. Twenty-two decamer primers amplified 162 scorable DNA fragments, of which 66 (41%) were polymorphic. On the basis of these 66 silver-stained RAPDs (ssRAPDs), 17 unique profiles were identified rather than the expected 22. Fourteen varieties had unique ssRAPD profiles, while the remaining 8 were represented by 3 ssRAPD profiles. Permuational analyses of the data suggest that the observed ssRAPD profile duplications are examples of varietal misclassification. Further analyses identified 2 ssRAPD markers that were found only in Eastern varieties (from Mass. and N. J.) and not in Wisconsin varieties. With varieties differing on average by 22 bands, ssRAPDs are shown to be effective in varietal identification and the assessment of genetic diversity in cranberry.New Jersey Agricultural Experiment Station, Publ No. D-12163-8-93, Supported by State funds.     

Temporal variation of the population structure and genetic diversity of Farfantepenaeus notialis assessed by allozyme loci

Population genetic studies carried out on penaeid shrimps have disclosed different patterns of population subdivision, revealing new aspects of shrimp biology as well as the effects of historical contingency molding those patterns. However, the stability of observed allele frequencies over time still remains untested. The objective of this article is to show the analysis of the temporal variation of allozymes in a shrimp species inhabiting Cuba which proves that the genetic structure of this species could significantly change in time. The study involves four populations of Farfantepenaeus notialis sampled in a period of 8 years. The significant statistics obtained from partitions observed in 1995 were not detected in 2003 (as suggested by AMOVA and F(ST)), whereas temporal genetic differentiation and heterozygosity became highly significant. The results strongly suggest that the effect of migrations could be the cause for the loss of F. notialis genetic structure in 2003. It is therefore imperative to call attention on the vulnerability of these populations when facing unstable environmental and habitat conditions.     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae):implications for its conservation

Omphalogramma souliei Franch. Is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. Souliei in NW Yunnan, China. The genetic diversity at the species level is low with P= 42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenog-amy predominated and autogamy played an assistant role in O. Souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.