天麻特异DNA序列的克隆及其在天麻鉴定中的应用

应用改进的RAPD方法测定了名贵中药材天麻基因组DNA指纹图谱;通过选择和回收各种天麻种群共有和优良种群特有的DNA片段,加以克隆、测序和生物信息学分析,证明其中5个DNA序列是未报道的,已被美国基因数据库收录,并运用高效液相色谱技术测定了天麻样本的有效成分天麻素含量。运用PCR技术研究了这些DNA序列在9个天麻种群中的分布及其与天麻素含量的关系。结果表明这5个DNA序列在这些天麻种群中的分布各不相同,其中DNA序列1是所研究的全部天麻种群共有、而其伪品没有的特异DNA分子标记;DNA序列2可能与天麻的天麻素含量高有关。这些DNA标记序列可用于天麻的真伪鉴别、品种鉴定和优选优育等。     

基于HPLC—MS的西藏天麻的化学成分分析与品质鉴定

目的：天麻具有许多药理作用和重要开发价值,通过探索天麻的未知化学成分和鉴定西藏天麻种群的品质,为深入研究、开发和利用西藏优良天麻种群提供科学技术支持。方法：本研究采用HLPC．MS（液质联用）技术测定西藏天麻的化学成分;采用高效液相色谱技术测定西藏6个天麻种群的生化指纹图谱,利用天麻素峰面积、数学公式和SPSS软件,计算和比较西藏6个天麻种群的平均天麻素含量和各种化学成分分离峰的总面。结果：从天麻块茎中发现了33个未报道的化学物质;西藏天麻的生化指纹图谱具有7个较大的共有特征分离峰,分别位于1．854、2．759、7．279、7．591、8．500、9．557、10．753min;根据生化指纹图谱特征可将它们分为三个主要类型;凡是带有一型生化指纹图谱的天麻个体和种群往往舍有更高的天麻素和更大的分离峰总面积。品质更优良。结论：西藏天麻种群3和种群6以一型生化指纹图谱为主,其天麻素含量最高,分离峰总面积最大,品质最优良,具有重要研究、开发和保护价值。本研究成果对于天麻的化学成分分析与鉴定、天麻种群和品质鉴定与评价以及西藏天麻的品种选优、资源保护与利用具有重要指导意义和科学价值。     

A molecular analysis of ground sloth diet through the last glaciation

DNA was extracted from five coprolites, excavated in Gypsum Cave, Nevada and radiocarbon dated to approximately 11 000, 20 000 and 28 500 years BP. All coprolites contained mitochondrial DNA sequences identical to a DNA sequence determined from a bone of the extinct ground sloth Nothrotheriops shastensis. A 157-bp fragment of the chloroplast gene for the large subunit of the ribulosebisphosphate carboxylase (rbcL) was amplified from the boluses and several hundred clones were sequenced. In addition, the same DNA fragment was sequenced from 99 plant species that occur in the vicinity of Gypsum Cave today. When these were compared to the DNA sequences in GenBank, 69 were correctly (two incorrectly) assigned to taxonomic orders. The plant sequences from the five coprolites as well as from one previously studied coprolite were compared to rbcL sequences in GenBank and the contemporary plant species. Thirteen families or orders of plants that formed part of the diet of the Shasta ground sloth could be identified, showing that the ground sloth was feeding on trees as well as herbs and grasses. The plants in the boluses further indicate that the climate 11 000 years BP was dryer than 20 000 and 28 500 years BP. However, the sloths seem to have visited water sources more frequently at 11 000 BP than at earlier times.     

大鼠高重复顺序DNA的核苷酸顺序分析及其与灵长类类α-DNA顺序的比较

 啮齿目动物高重复顺序DNA的结构特征,虽已有人进行过分析,并提出大鼠DNA中有酶切位点分布独特的高重复顺序,也有人认为在Sprague-Dawlay大鼠中有类似于α-DNA大小的高重复顺序,并命名为α型(α-type),是否大鼠中也含有类α顺序,并没有人讨论过。我们对Wistar大鼠高重复顺序DNA进行了分离、纯化、分子克隆及核苷酸序列分析,测定了全序列为370bp,它也是具有酶切点分布独特的高重复顺序,但与前者存在着18％的差异性。我们对此顺序与灵长类类α-DNA进行了比较分析,发现二者在几方面都不存在相似之处,如此序列G-C含量占全部碱基组成的37.3％,而类α顺序的G-C含量约在40％以上,一些酶切位点也与类α-DNA完全不相同,另外在此序列的相应位置上也不具有灵长类类α-DNA的三个“冷点区”等等,由此得出大鼠这一高重复顺序并非类α顺序,而类α顺序只是灵长类动物所特有的。     

Changes in the methylated sequences and molecular population of wheat DNA during germination

The fractions of unique (Cot less than 405), moderately (Cot=0.13--405) and highly reiterated (Cot less than 0--0.13) sequences were isolated from DNA of wheat seeds and 3 day old seedlings, and GC content, amount of 5-methylcytosine and its distribution among various pyrimidine isostichs in the fractions isolated were studied. Different in Cot value DNA fractions from seeds or from seedlings are similar in GC content and in all other characteristics studied. Seed DNA differs from DNA of seedlings in the content of pyrimidine isostichs from the respective fractions of reiterated sequences. Pronounced differences in the amount of pyridmidine clusters with various base composition in the corresponding fractions of DNA from seeds and seedlings were found. These differences in the frequencies of respective pyrimidine clusters from DNA of seeds and seedlings may be considered as being a result of changes in the molecular population of wheat DNA on germination. The seed and seedling DNA differ significantly in the 5-methylcytosine content in the respective pyrimidine isostichs isolated from unique sequences. In the seedling DNA some other nucleotide sequences are to be methylated as compared to DNA of dormat seeds. Thus, on germination some changes occur in DNA methylation as well as in the genome organization.     

Genome size and numerical polymorphism for the B chromosome in races of maize (Zea mays ssp. mays, Poaceae)

Twenty-one native populations (1120 individuals) of maize from Northern Argentina were studied. These populations, which belong to 13 native races, were cultivated at different altitudes (80-3620 m). Nineteen of the populations analyzed showed B chromosome (Bs) numerical polymorphism. The frequency of individuals with Bs varied from 0 to 94%. The number of Bs per plant varied from 0 to 8 Bs, with the predominant doses being 0, 1, 2, and 3. Those populations with varying number of Bs showed a positive and statistically significant correlation of mean number of Bs with altitude. The DNA content, in plants without Bs (A-DNA)(2n = 20), of 17 populations of the 21 studied was determined. A 36% variation (5.0-6.8 pg) in A-DNA content was found. A significant negative correlation between A-DNA content and altitude of cultivation and between A-DNA content and mean number of Bs was found. This indicates that there is a close interrelationship between the DNA content of A chromosomes and doses of Bs. These results suggest that there is a maximum limit to the mass of nuclear DNA so that Bs are tolerated as long as this maximum limit is not exceeded.     

DNA structures associated with autonomously replicating sequences from plants

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.Supported by Grant 1RO1-GM41708-O1 from the National Institute of Health.     

Patterns of population structure at microsatellite and mitochondrial DNA markers in the franciscana dolphin (Pontoporia blainvillei)

The franciscana dolphin, Pontorporia blainvillei, is an endemic cetacean of the Atlantic coast of South America. Its coastal distribution and restricted movement patterns make this species vulnerable to anthropogenic factors, particularly to incidental bycatch. We used mitochondrial DNA control region sequences, 10 microsatellites, and sex data to investigate the population structure of the franciscana dolphin from a previously established management area, which includes the southern edge of its geographic range. F‐statistics and Bayesian cluster analyses revealed the existence of three genetically distinct populations. Based on the microsatellite loci, similar levels of genetic variability were found in the area; 13 private alleles were found in Monte Hermoso, but none in Claromecó. When considering the mitochondrial DNA control region sequences, lower levels of genetic diversity were found in Monte Hermoso, when compared to the other localities. Low levels of gene flow were found between most localities. Additionally, no evidence of isolation by distance nor sex‐biased dispersal was detected in the study area. In view of these results showing that populations from Necochea/Claromecó, Monte Hermoso, and Río Negro were found to be genetically distinct and the available genetic information for the species previously published, Argentina would comprise five distinct populations: Samborombón West/Samborombón South, Cabo San Antonio/Buenos Aires East, Necochea/Claromecó/Buenos Aires Southwest, Monte Hermoso, and Río Negro. In order to ensure the long‐term survival of the franciscana dolphin, management and conservation strategies should be developed considering each of these populations as different management units.     

Polite DNA: Functional density and functional compatibility in genomes

Summary Certain as yet poorly defined functions of DNA appear to involve collectively domain-sized sequences. It is proposed that most sequence segments within a domain may be either functionally superfluous or instrumental, depending on how many related sequences are present in the domain. When redundant and functionally dispensable, such DNA segments presumably still have to conform to compositional or sequence-motif patterns that characterize the domain. In its relations with neighboring sequences, such DNA is required to be polite. Polite DNA is DNA that, without being crucially involved in function, is subject to constraints of conformity and, through its base composition, respects a function for which it is not required. This concept is developed by contrasting the distribution of specific and general functions over DNA with this distribution as found in proteins and by distinguishing functional compatibility from pivotal functionality. The sequence constraints to which heterochromatin as well as, apparently, long interspersed repetitive sequences are known to be subject seem to imply that DNA, even when it does not carry out a pivotal function, is indeed, at the very least, required to be polite.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza

Large variation in genome size as determined by the nuclear DNA content and the mitotic chromosome size among diploid rice species is revealed using flow cytometry and image analyses. Both the total chromosomal length (r_0.939) and the total chromosomal area (r_0.927) correlated well with the nuclear DNA content. Among all the species examined, Oryza australiensis (E genome) and O. brachyantha (F genome), respectively, were the largest and smallest in genome size. O. sativa (A genome) involving all the cultivated species showed the intermediate genome size between them. The distribution patterns of genome-specific repetitive DNA sequences were physically determined using fluorescence in situ hybridization (FISH). O. brachyantha had limited sites of the repetitive DNA sequences specific to the F genome. O. australiensis showed overall amplification of genome-specific DNA sequences throughout the chromosomes. The amplification of the repetitive DNA sequences causes the variation in the chromosome morphology and thus the genome size among diploid species in the genus Oryza.     

Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

野生天麻DNA导入马铃薯实现遗传转化的研究

采用浸苗法将野生天麻总DNA导入马铃薯试管苗,对筛选得到的转化植株进行蛋白及药用成份的分析。结果显示:(1)在200株转化的马铃薯中有21株的紫外扫描图谱与正常对照组有显著差异,且在220nm有明显吸收峰。(2)5株经PCR扩增出野生天麻抗真菌蛋白(GAFP)基因。(3)转基因马铃薯与正常马铃薯的蛋白表达有明显差异,并且在转基因马铃薯中有一条与野生天麻抗真菌蛋白(GAFP)相同的条带。而正常马铃薯中无此条带。(4)通过薄层层析法检测出3株转基因马铃薯表达野生天麻的有效药用成份天麻素。说明采用浸苗法进行外源总DNA导入是可行的。     

不同菌材对天麻(Gastrodia elata)产量的影响

以速生树种亮叶桦（Betula luminifera Winkler）和响叶杨（Populus adenopoda Maxim）、经济树种核桃（Juglans regia Linn）为菌材,壳斗科菌材白栎（Quercus fabri Hance）为对照,分别设置不同的菌材用量和麻种用量水平,仿野生栽培红杆天麻（Gastrodia elata Bl.f.elat）和乌杆天麻（Gastrodia elata Bl.f.glauca S.Chow）,分析不同菌材对天麻产量的影响,筛选出高产的菌材种类、用量和天麻变型及麻种用量的组合,探讨亮叶桦、响叶杨和核桃代替壳斗科植物栽培天麻的可行性。结果表明：不同菌材对红杆天麻和乌杆天麻产量的影响有差异。两种天麻变型对菌材的偏好不同,红杆天麻在白栎和亮叶桦菌材处理下的平均产量高于响叶杨和核桃菌材处理下的产量,而乌杆天麻在亮叶桦和核桃菌材处理下的产量高于响叶杨和白栎菌材处理下的产量。两种天麻变型的最高产量的组合均出现在亮叶桦菌材伴栽的处理中。响叶杨伴栽天麻,效果不及亮叶桦和核桃。4种菌材对天麻的折干率影响不明显。在实际生产中,以亮叶桦、核桃和响叶杨作为替代菌材是完全可行的。从72个处理中筛选出高产的组合,单位面积菌材和麻种用量分别如下：12 kg亮叶桦菌材搭配0.8 kg红杆天麻麻种,12 kg核桃菌材搭配0.6 kg红杆天麻麻种,14 kg响叶杨菌材搭配0.8 kg红杆天麻麻种;14 kg亮叶桦菌材搭配0.6 kg乌杆天麻麻种,12 kg亮叶桦菌材搭配0.6 kg乌杆天麻麻种,10 kg核桃菌材搭配0.8 kg乌杆天麻麻种。     

Characteristics of nuclear DNA in the genus Oryza

Summary DNAs have been isolated from various Oryza species and studied using physical techniques. The percent of guanine plus cytosine has been determined by thermal denaturation. While the base composition varied between the species, no heterogeneity in the base pair distribution was observed. Renaturation kinetics data of DNAs from different species show that the proportion of repeated DNA sequences vary considerably depending on the DNA content per cell, whereas the nonrepetitive DNA component remains relatively constant. These results suggest that in addition to a small range of DNA variation between the species, changes in the base composition and proportion of repeated sequences have accompanied divergence of the species within the genus.     

Geographic population structure and sequence divergence in the mitochondrial DNA control region of the Japanese wild boar (Sus scrofa leucomystax), with reference to those of domestic pigs

Mitochondrial DNA (mtDNA) control regions from 40 Japanese wild boars were examined by direct sequencing after amplification by PCR. From the DNA sequences obtained, we found eight haplotypes, whose differences arose via transitions. The geographical distribution of these different haplotypes indicated that wild boar populations inhabited limited areas and that there was some restricted gene flow between local populations. Eight mtDNA haplotypes from Eastern and Western domestic pigs and the Ryukyu wild boar were also analyzed as references to those from Japanese wild boars. The cluster analyses of the control-region sequences showed that those from Japanese wild boards belong to the Asian type as do those from Eastern domestic pigs and the Ryukyu wild boar, which differed from the European type (Western domestic pigs).     

RAPD条件优化及天麻基因组DNA多态性分析

建立了RAPD扩增条件快速优化程序与方法．并应用于天麻基因组DNA扩增条件的优化及多态性的测定：获得了天麻基因组DNA的RAPD扩增优化条件和DNA指纹图谱;分析了模板DNA、引物、dNTP、Taq DNA聚合酶等的浓度和退火温度对RAPD扩增的影响．结果表明：天麻基因组DNA用引物S1扩增的片段具有更明显的多态性,这种指纹图谱更适合于天麻遗传分化研究;而用引物S12扩增的DNA指纹图谱具有更大的相似性,这种指纹图谱更适合于天麻真伪鉴别．该方法使RAPD扩增条件优化过程实现了程序化和数量化,是获得RAPD优化条件的简便快速、经济实用方法．应用该方法进行RAPD扩增,可获得图谱清晰、稳定可靠的实验结果．     

Incorporation of mitochondrial fragments in the nuclear genome (Numts) of the longhorned beetle Monochamus galloprovincialis (Coleoptera, Cerambycidae)

A mitochondrial DNA (mtDNA) study, based on 43 European populations (33 of them sampled in France) of Monochamus galloprovincialis , vector of the pinewood nematode, and 14 populations of its sister species Monochamus sutor was realized. Sequencing of 792 bp of the cytochrome oxidase I (COI) and 521 bp of the COII genes revealed numerous ambiguities on multiple nucleotide sites for half of M. galloprovincialis specimens studied (44.8%). Hypotheses of heteroplasmy and pseudogenes ( Numts ) were examined. The mtDNA isolation by alkaline lysis and cloning (for three successfully used individuals) both support the hypothesis that the ambiguous sequences amplified were not of mtDNA nature and validate the presence of Numts in the nuclear genome of M. galloprovincialis . Multiple copies of mtDNA-like sequences were found paralogous to COI, tRNA leucin and COII regions. Phenetic analysis placed different recently diverged mtDNA-like sequences as a close relative of mtDNA sequences, and grouped 10 closely related mtDNA-like sequences as a more basal clade, closer to ancestral states and to M. sutor . This result supports that this nuclear family of pseudogenes arose independently of the other events and may represent mitochondrial haplotypes sampled from M. galloprovincialis ancestral populations. This is the first time that Numts are proved for a longhorned beetle, whereas no Numts were found within its sister species M. sutor. The incorporation mechanism of Numts in unknown for M. galloprovincialis , however, excess of ambiguous sites corresponding to synonymous mutations placed on third codon position as well as the absence of Numts in M. sutor , conducted to the hypothesis of a recent transfer of these Numts in the nuclear genome of M. galloprovincialis .     

Reconciling patterns of genetic variation with stream structure, earth history and biology in the Australian freshwater fish Craterocephalus stercusmuscarum (Atherinidae)

We examined the consequences of barriers, stream architecture and putative dispersal capability on levels of genetic differentiation among populations of the freshwater fish Craterocephalus stercusmuscarum. Seven polymorphic allozyme loci and sequences of a 498-bp fragment of the ATPase 6 mitochondrial DNA (mtDNA) gene were used to assess patterns of genetic variation among 16 populations from upland and lowland streams of five drainages in northern Queensland, Australia. Concordant patterns at both genetic markers revealed that there were significant levels of genetic subdivision among all populations, while an analysis of molecular variation showed that the distribution of genetic diversity was not consistent with contemporary drainage structure. There were reciprocally monophyletic mtDNA clades and fixed or large frequency differences at allozyme loci either side of instream barriers such as waterfalls. This implied barriers were effective in restricting gene flow between upland and lowland populations separated by waterfalls. However, there were two genetically distinct groups in upland areas, even within the same subcatchment, as well as high levels of genetic subdivision among lowland populations, suggesting barriers alone do not explain the patterns of genetic diversity. The data revealed a complex phylogeographic pattern, which we interpreted to be the result of one or more invasion events of independent lineages to different sections of each drainage, possibly mediated by well documented geomorphological changes. Our results highlight the importance of earth structure and history in shaping population genetic structure in stream organisms where dispersal capability may be limited, and reveal that the contemporary structure of drainages is not necessarily a good indicator of genetic relationships among populations.     

Ploidy levels among species in the 'Oxalis tuberosa alliance' as inferred by flow cytometry

The 'Oxalis tuberosa alliance' is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa Molina (Oxalidaceae), commonly known as 'oca'. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C-values in the alliance by estimating nuclear DNA contents of these accessions using chicken erythrocytes as internal standard. Ten Bolivian accessions of cultivated O. tuberosa were confirmed to be octoploid, with a mean nuclear DNA content of approx. 3.6 pg/2C. Two Peruvian wild Oxalis species, O. phaeotricha and O. picchensis, were inferred to be tetraploid (both with approx. 1.67 pg/2C), the latter being one of the putative progenitors of O. tuberosa identified by chloroplast-expressed glutamine synthetase data in prior work. The remaining accessions (from 78 populations provisionally identified as 35 species) were DNA diploid, with nuclear DNA contents varying from 0.79 to 1.34 pg/2C.     

Population genetic structure and the effect of founder events on the genetic variability of introduced sika deer, Cervus nippon, in Germany and Austria

The relationships among 214 wild-living sika deer from five locations in Germany and two in Lower Austria were examined using mitochondrial DNA (mtDNA) control region sequence. A total of 18 haplotypes are grouped consistently into two major divergent clades, A and B, which differ by a mean of 8.4% sequence divergence. Recently introduced sika deer showed a complex pattern of population structuring, which probably results from historical vicariance in at least two unknown source populations from southeastern Asia (as previously described by morphological and mtDNA findings), and subsequent population admixture as a result of human-mediated restocking. A strong genetic differentiation among populations was indicated by a global _ST value of 0.78 reflecting mainly the differential distribution of clades A and B haplotypes. There was no association between related haplotypes and their distribution among local populations. These indicate that genealogy is a better predictor of the genetic affinity among most sika deer populations than their present-day locations. The abundant mitochondrial divergence we observed, may reflect a subspecies differentiation and could be associated with phenotypic differences among the introduced sika deer.An erratum to this article can be found at