HYBRIDIZATION AND CHLOROPLAST DNA VARIATION IN A PINUS SPECIES COMPLEX FROM ASIA

Heterologous hybridization of chloroplast DNA (cpDNA) involving 30 endonucleaseprobe combinations was used to analyze cpDNA variation in multiple individuals and populations of Pinus tabulaeformis (Carr.), Pinus yunnanensis (Franchèt) and Pinus massoniana (Lamb.). Restriction fragment patterns detected by several combinations distinguished among the three species. The obtained cpDNA markers were subsequently used to examine cpDNA variation of Pinus densata (Masters), a putative tertiary hybrid between P. tabulaeformis and P. yunnanensis. The analysis demonstrated that P. densata populations harbor three different haplotypes. Two of these haplotypes are characteristic of P. tabulaeformis and P. yunnanensis. However, the third haplotype found in P. densata appears to be absent in other extant Asian Pinus species. It is suggested that the observed cpDNA composition of P. densata populations is a result of past hybridization involving P. tabulaeformis, P. yunnanensis, and a third unknown or extinct taxon. Chloroplast DNA polymorphism in P. densata was much greater than that for nuclear allozyme markers in this and the other Pinus species. Population differentiation was also substantial in P. densata and exceeded that for allozyme markers. In contrast, no cpDNA polymorphism was detected in populations of P. tabulaeformis, P. yunnanensis, and P. massoniana. The study suggests that interspecific gene exchange may lead to the creation of stable cpDNA polymorphism in conifer hybrids.     

INTRASPECIFIC CHLOROPLAST DNA VARIATION AND BIOGEOGRAPHY OF NORTH AMERICAN LIRIODENDRON L. (MAGNOLIACEAE)

Restriction site variation in chloroplast DNA (cpDNA) was surveyed to analyze population dynamics in Liriodendron tulipifera L., a woody angiosperm found in eastern North America. Two cpDNA haplotypes, differing by the presence or absence of five restriction site changes (nucleotide sequence divergence estimated as approximately 0.15%) are geographically structured; 61 widespread populations possess the “northern” haplotype and three isolated populations of central Florida possess the “southern” haplotype. This geographic break in cpDNA distribution corresponds to patterns of geographic distribution revealed by a previous survey of allozyme variation, with the exception that analyses of allozyme data further divided the populations containing the northern cpDNA haplotype into two groups, a widespread upland group and a coastal intermediate group. Analyses of these two independent data sets together support the hypothesis that L. tulipifera survived the glacial advances of the Pleistocene in two distinct refugia, possibly as different taxa, and the intermediate coastal group was putatively formed from recent hybridizations between these entities.     

ARE POPULATIONS ISLANDS? ANALYSIS OF CHLOROPLAST DNA VARIATION IN AQUILEGIA

The degree to which conspecific populations are interconnected via ongoing gene flow remains an important focus of evolutionary biology. One major difficulty in distinguishing ongoing gene flow from historical subdivision is that either process can generate similar estimates of apparent gene flow. Thus, gene flow estimates themselves are insufficient to distinguish between these alternatives. However, genetic data coupled with additional information about demography and distribution do allow a distinction to be made. Here we address the specific question, does gene flow link populations of Aquilegia? In a survey of a 525 B.P. chloroplast DNA fragment sampled from 251 individual plants from 18 populations of three taxa, five haplotypes were identified. No significant relationship between geographic distance and apparent gene flow between population pairs existed. Further, the estimated level of gene flow was entirely compatible with a historical subdivision of Aquilegia populations during the late Pleistocene or early Holocene. Therefore, these patterns of variation are due not to ongoing gene flow, but rather to historical association among populations. Thus Aquilegia populations may be considered as distinct evolutionary entities with regard to seed-mediated processes. As a result, comparative analysis of ecological traits undergoing potentially rapid evolution (e.g., life histories, mating systems, inbreeding depression) should be possible in these taxa.     

RESTRICTION ENDONUCLEASE ANALYSIS OF RIBOSOMAL DNA SEQUENCE VARIATION IN LAMINARIA (PHAEOPHYTA)1

The taxonomy and evolutionary relationships of species in the genus Laminaria are poorly understood. Previous studies have demonstrated significant plasticity of morphological characters used to describe taxa, and interfertility has been reported among putative species. We analyzed nuclear ribosomal DNA (rDNA) sequence variation in eight species of Laminaria (L. agardhii Kjell., L. digitata (Huds.) Lamour., L. groenlandica Rosenv. [sensu Druehl 1968], L. longicruris De la Pyl., L. longipes Bory, L. saccharina (L.) Lamour., L. setchellii Silva, and L. yezoensis Miyabe) to elucidate evolutionary relationships in this genus. Restriction maps were constructed using a small subunit rDNA probe from Costaria costata (Turn.) Saunders, an rDNA repeat from the nematode Caenorhabditis elegans, and 11 hexameric restriction endonucleases in an annealing analysis of genomic DNA. Laminaria rDNA restriction maps were compared to each other and to that of the outgroup taxon, C. costata. rDNA restriction maps of Laminaria species and C. costata were similar. Restriction fragment length polymorphisms mapped to both the coding regions and the nontranscribed spacer of rDNA. Laminaria species were distinguished with this method. The restriction maps of L. agardhii, L. saccharina, and L. longicruris were identical, supporting a previous hypothesis that these species are conspecific. Comparison of restriction maps of Laminaria species suggested that the generic subdivision of Sections Simplices and Digitatae may be invalid.     

懒猴属的核糖体DNA变异及其种间分化关系

用１５种限制性内切酶和人２８Ｓ、１８ＳｒＤＮＡ探针构建了懒猴属各物种核糖体ＤＮＡ重复单位的限制性内切酶图谱。在进化速率较高的非转录间隔区,在大、中、小懒猴中分别定位了２３、２４、２４个酶切位点。大懒猴与中懒猴有１２个位点不同,与小懒猴有１４个位点不同,而中、小懒猴间则只有一个位点的差异。经过计算,大懒猴与中懒猴的遗传距离值为１２．６５％,与小懒猴的差异为１４．２４％,中、小懒猴间的差异则仅为０．７     

PHYLOGEOGRAPHIC PATTERNS IN COASTAL NORTH AMERICAN TIGER BEETLES (CICINDELA DORSALIS SAY) INFERRED FROM MITOCHONDRIAL DNA SEQUENCES

Genetic variation was assessed in the tiger beetle, Cicindela dorsalis, by sequencing of three regions of the mtDNA genome. Populations of four morphologically distinguishable subspecies were sampled from 28 representative locations covering almost the entire geographic range of the species in coastal North America. In 78 individuals analyzed for 656 base pairs from four different genes, 17 different haplotypes could be distinguished. A cladistic analysis grouped the haplotype sequences into two main lineages, one from the Atlantic Ocean and one from the Gulf of Mexico. Haplotypes within the two clades were very similar to each other. Most of the characters that distinguished these closely related haplotypes were homoplastic. The geographic distribution of haplotypes did not coincide with the distribution of morphological subspecies, but no evidence for hybridization between two subspecies could be inferred from this observation. The implications of these findings for the evolution of gene sequences at and below the species level are discussed.     

黑麦属种间叶绿体DNA的变异性和种系发生的关系

为了获得有关黑麦属种间关系、黑麦属与小麦属和山羊草属种系发牛关系的新资料,应用Ban HI等8种限制性核酸内切酶酶解黑麦属5个种的叶绿体DNA,进行琼脂糖凝胶电泳,分析其酶解图谱。结果表明,黑麦属种叶绿体基因组的大小与小麦属和山羊草属的叶绿体基因组非常相似。根据黑麦属5个种遗传距离的估算,并与小麦属和山羊草属已知叶绿体基因组间所观察到的遗传距离相比,黑麦属叶绿体基因组的分化很少;这些资料进一步证实黑麦属的近代起源;并表明黑麦属、小麦属和山羊草属间的亲缘关系是非常密切的。     

PHYLOGENY OF THE BATRACHOSPERMALES (RHODOPHYTA) INFERRED FROM rbcL AND 18S RIBOSOMAL DNA GENE SEQUENCES

The sequence data from the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbc L) gene and 18S ribosomal DNA (small subunit) of taxa in the freshwater rhodophyte order Batrachospermales were used to construct phylogenetic hypotheses. Taxa examined in this study represent four families, eight genera, and six sections of the genus Batrachospermum . In addition, Rhododraparnaldia oregonica Sheath, Whittick et Cole, was included in the analysis because it shares particular ultrastructural, reproductive, and morphological characteristics with members of the Batrachospermales and Acrochaetiales. The trees generated from each gene, as well as a combined data set, were largely congruent. Rhododraparnaldia consistently occurs on an early branch within the Acrochaetiales – Palmariales clade and does not appear to be a member of the Batrachospermales. In addition, Thorea violacea Bory de St. Vincent was not closely related to the other taxa of the Batrachospermales in all trees and hence the family Thoreaceae does not appear to be a natural grouping within this order. All other taxa analyzed, which are presently classified within this order, formed a monophyletic clade in most analyses. Psilosiphon scoparium Entwisle was not closely allied with the taxa of the Lemaneaceae, lending support to the newly proposed family Psilosiphonaceae. Sequence data from the remaining taxa of the Lemaneaceae support the concept of a derived monophyletic clade. The genus Batrachospermum appears to comprise many morphologically similar but distantly related taxa, which will need further investigation to resolve their taxonomic status. Tuomeya, Sirodotia and Nothocladus are retained at the generic level until further data are obtained.     

RESTRICTION ENZYME ANALYSIS AND CLONING OF HIGH MOLECULAR WEIGHT GENOMIC DNA ISOLATED FROM CHLORELLA SOROKINIANA (CHLOROPHYTA)1

High molecular weight (50–70 kb) genomic DNA was isolated from the eukaryotic green alga, Chlorella sorokiniana spec. nov, (formerly Chlorella pyrenoidosa Chick, strain 7-11-05), for restriction endonuclease digestion studies and for preparation of a genomic DNA library. Twenty restriction endonucleases were examined for their abilities to digest this DNA. Nine of the endonucleases gave nearly complete digestion of the DNA, whereas 11 gave only partial digestion. Additional purification steps to remove possible contamination by proteins, RNAs, or polysaccharides did not improve digestion. Digestion studies with pairs of endonuclease isoschizomers, of which one member was sensitive to base methylation, suggested that 5-methylcytosine might be responsible Jor inhibition of certain endonucleases. Analysis of the DNA showed it to contain 63% GC and to have a high content (5.1 mol %) of 5-methylcytostne but no other methylated or unusual bases. Evidence indicates that this high 5-methylcytosine content, which is a characteristic of higher plant genomic DNA rather than of eukaryotic microorganisms, interfered with the cloning of restriction fragments (or fragments produced by mechanical shearing) of C. sorokiniana genomic DNA in standard bacterial host-strains. Escherichia coli strain K803, which is a permissive host for cloning highly methylated DNA from higher plants, also permitted the cloning of a complete genomic library of 15–20 kb Mbol restriction fragments inserted into the BamHI site of the γ vector, EMBL 3. This C. sorokiniana genomic library appears to be the first genomic-library constructed for any species of Chlorella.     

一种纯化高等植物叶绿体DNA的有效方法

１９８４年,Ｂｏｏｋｊａｎｓ ｅｔ ａｌ．建立了高盐介质纯叶绿体ＤＮＡ的方法。在我们进行的高粱叶绿体光系统基因研究中发现,用Ｂｏｏｋｊａｎｓ的方法提取的ｃｔＤＮＡ酶切效果欠佳,产率太低。应用我们反进的高盐低ＰＨ方法,纯化了高粱、小麦、水稻和速效豌豆等植物的ｃｔＤＮＡ。结果表明,这种改进的方法具有有得率高,酶切效果好和操作简便等优点。     

RESTRICTION ENZYME ANALYSIS OF DNA FROM SPECIES OF BULINUS (BASOMMATOPHORA: PLANORBIDAE) USING A CLONED RIBOSOMAL RNA GENE PROBE

A ribosomal RNA gene probe (pSM889) has been used to study restrictionenzyme digests of various species of Bulinus. In order to minimiseproblems of DNA shearing associated with snail tissues a methodof extracting nucleic acids from material embedded in agaroseblocks has been used. Restriction enzyme digests with Bgl IIand Bam HI hybridised to pSM889 showed clear differences betweenB. truncatus, B. wrighti, B. africanus and B. forskalii, representingthe four species groups of Bulinus. No differences were observedbetween samples of B. tropicus and B. truncatus digested withBam HI, Bgl II and Pst I. Intra-specific variation was observedbetween samples of B. forskalii from Säo Tomé andAngola digested with Bgl II and Hind III although restrictionprofiles for Bam HI, Pst I and Bst EII digests were similar.Intra-specific variation was also observed between two differentpopulation samples of B. wrighti from South Yemen using BamHI and Bgl II digested genomic DNA hybridised to pSM889. (Received 5 December 1989; accepted 19 April 1990)     

香菇栽培种线粒体DNA和核糖体DNA多态性研究初探

本文应用RFLP技术研究了10个香菇主栽品种的线粒体DNA（mtDNA）和核糖体DNA（rDNA）的部分小区段,利用PCR技术扩增了rDNA5．8＋ITS区段及mtDNA的小区段,分析这些片段的限制性酸切图谱,并进行菌株间的遗传相似系数的估算。结果显示：菌株间的rDNA在5．8＋ITS区段差异很小,表明同一种内菌株间的rDNA具有相对的遗传稳定性;不同菌株间未检出mtDNA的差异,表明菌株间在所研究的区段具有很高的遗传相似性。     

GENETICAL POPULATION STRUCTURE IN PLANTS: GENE FLOW BETWEEN DIPLOID SEXUAL AND TRIPLOID ASEXUAL DANDELIONS (TARAXACUM SECTION RUDERALIA)

Levels and distribution of genetic variation were studied in central and western European populations of Taraxacum section Ruderalia containing differing mixtures of sexual diploid and asexual triploid plants. All sexual populations were panmictic with their variation partitioned mainly among populations. Genotypic diversity in triploid samples was very high with few clones widespread and many clones restricted to one or a few populations. Extensive amounts of gene (pollen) flow between the diploid and triploid components of a population were inferred from the following data: (1) the two ploidy levels share all major allozyme polymorphisms; (2) the intrapopulational homogeneity in genic variation between diploids and triploids contrasts strongly with the geographic differentiation at each ploidy level separately; (3) population-unique alleles simultaneously occur at the two ploidy levels; (4) not only sexuals but also asexuals generally simulate Hardy-Weinberg expectations. Most likely, intrapopulational gene exchange occurs bidirectionally by mechanisms such as reductional pollen meiosis in apomictic plants, facultative apomixis, and formation of unreduced gametes in sexuals. Thus, diploid and triploid Taraxacum section Ruderalia are less genetically isolated than has previously been supposed and probably form a cohesive evolutionary unit with the level at which gene pools are shared differing by population.     

ARE CHLOROPLAST AND MITOCHONDRIAL DNA VARIATION SPECIES INDEPENDENT IN OAKS?

Extensive introgression of cytoplasmic genomes across oak species is now a well-established fact. To distinguish between ancient hybridization events and ongoing introgression, a direct test for the existence of local exchanges is proposed. Such local exchanges must be comparatively recent, that is, contemporaneous with or later than the last postglacial recolonization. The test is applied to an extensive set of data comprising 377 pure or mixed populations (1744 individuals) of four white oak species in southern France. After demonstrating that local exchanges have occurred frequently between all species pairs, another test is performed to check if species status does nevertheless play some role in restricting cytoplasmic gene flow. The results vary according to the species pairs considered, and the observed pattern may be related to the ecology and/or compatibility of interspecific crosses. It is also shown that, for some of these oak species, the presence of related species in a population significantly influences the intraspecific diversity. Altogether, the results demonstrate that (1) intraspecific cytoplasmic gene flow varies according to the species, (2) interspecific cytoplasmic gene flow varies according to the species pair, and (3) both components of gene flow are at least partly related.     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.     

粒毛盘菌属种间关系的分子系统学初探*

粒毛盘菌属包括晶杯菌科中子囊盘表面具有毛状物,毛状物全表面具有颗粒状纹饰以及外囊盘被结构为矩胞组织的部分种类,种间形态解剖结构多样,研究采用ITS15.8S rDNAITS2序列分析的方法探讨该属种间的系统发育关系。在供试的粒毛盘菌15个分类单元中,14个以89%的支持强度值相聚在一起,其中Lachnum clandestinum与 L. fuscescens关系最近,其支持强度值为100%;其次为L. controversum 与L. spartinae,其支持强度值为97%;而姊妹群L. euterpes,L. pteridophyllum,L. singerianum和L. lushanense彼此关系较远,其支持强度值均小于50%;L. sclerotii位于系统发育树的最外侧,为供试15个分类单元中距离最远的一个,结果表明粒毛盘菌属可能是多起源的。     

光裸方格星虫野生与养殖群体线粒体控制区序列的遗传差异分析

基于线粒体控制区序列对光裸方格星虫（Sipunculus nudus Linnaeus,1766）的2个养殖群体（营盘YP、竹林ZL）和4个野生群体（防城港FC、钦州QZ、大冠沙DG和越南海防YN）的91个个体进行遗传差异分析,研究光裸方格星虫养殖和野生群体的遗传变异情况。结果显示:获得的514 bp DNA序列中,野生与养殖群体的多态性位点数分别为82和60,均显示出对AT的偏倚性。共定义85个单倍型,共享单倍型4个,其中共享单倍型Hap5为原始单倍型,营盘群体均为独享单倍型。各群体的单倍型多样性（H_d）相同,野生群体的平均核苷酸多样性（P_i）（0.01531）略高于养殖群体（0.01514）,6个群体的遗传多样性水平依次为YN > YP > QZ > FC > ZL > DG。各群体间的遗传分化并不显著（P>0.05）,光裸方格星虫的遗传变异主要来自群体内个体间（99.08%）,同时未发现明显的地理谱系结构。研究表明,光裸方格星虫野生群体的遗传多样性水平总体略高于养殖群体;滩涂底播养殖方式较池塘养殖更利于维持光裸方格星虫遗传多样性;各群体间不存在显著的遗传分化,养殖群体正逐渐积累遗传变异,但尚未足够以形成其独立的遗传结构。     

猪獾和黄鼬mtDNA物理图谱及位点变异性初探

本实验用ＡｐａⅠ,ＢｇｌⅠ,ＢｇｌⅡ,ＣｌａⅠ,ＥｃｏＲⅠ,ＥｃｏＲⅤ,ＨｉｎｄⅢ,ＨｐｅⅠ,ＰｓｔⅠ,ＰｖｕⅠⅡ,ＳａｃⅠ,ＳａｌⅠ等１２种限制性内切酶分析猪獾和黄鼬的ｍｔＤＮＡ限制性片段,并用双酶解法构建限制性内切酶图谱。结合以往积累的资料,我们对哺乳动物ｍｔＤＮＡ限制性位点在远缘物种间的保守性和变异性进行了初步讨论。     

几种转基因植物体细胞克隆变异的多样性研究

分析了杨树、水稻和甘蔗转基因植株体细胞克隆的表型变异和基因组DNA多态性。探讨了以下问题:转基因植株体细胞克隆的1)表型多样性, 2)基因组DNA多样性, 3)二者的相关性, 4)表型变异和DNA变异的可遗传特性, 5)产生的可能原因,以及6)在农业生产上的应用。