RAPD引物的筛选及高粱基因组的多态性分析

以高粱的叶片为试材,采用CTAB法提取DNA,应用RAPD筛选技术对高粱基因进行分子标记.共筛选80个RAPD随机引物,其中有27个引物能对DNA扩增并能发现其中抗性基因与感性基因的区别.     

The detection of genome polymorphism in Stachys species using RAPD

Molecular genome analysis was for the first time carried out in the genus Stachys. RAPD analysis proved to be suitable for identifying the species-specific markers, studying the interspecific DNA polymorphism, and detecting the genetic changes that arise during in vitro culturing of Stachys sieboldii. In addition, RAPD was used for screening genetic variation in S. sieboldii regenerants obtained at various phytohormone concentrations. High cytokinin concentrations and multiple regeneration were shown to induce genetic changes detectable with RAPD patterns. High DNA polymorphism was demonstrated for two types of S. sieboldii callus cultures and for plants regenerated from a callus culture.     

山羊草属异源多倍体植物基因组进化的ＲＡＰＤ分析

和２４个随机引物对山羊草属（Ａegilops L.)异源多倍体物种对其祖先二倍体物进行ＲＡＰＤ分析,对扩增出的３１３条带进行聚类分析发现,含Ｄ基因组的多倍体与二倍体祖先Ａe.squarrosa(DD)在聚类图上聚为一支;除Ａe.juvenalis(DDMMUU)聚到上一支外,含Ｕ基因组的多倍 与二倍体祖先Ae.umbellulata(ＵＵ）在聚类图上聚为另一支;多倍体与其他二倍体均不聚在一起,表明多倍体分别与Ａe.squarrosa(DD)、Ae.umbellulata(UU)具有较近的亲缘关系,这说明多倍体开之后,Ｄ和Ｕ基因组变化较小,而其他基因组则发生了较大的变化。     

RAPD Analysis of the Genome in Species of the Genus Hordeum

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum.High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgaresensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between theHordeumspecies, between the H. spontaneumpopulations, and between regional H. vulgarecultivars and a dendrogram was constructed.     

HIV相关性口腔念珠菌RAPD多态性分析

运用RAPD技术对60株分离自HIV感染者口腔的念珠菌进行分析, 其中P2随机引物扩增条带数量0~5条, 大小300 bp~2 kb, 白色念珠菌以300 bp、400 bp和600 bp 3个主条带为主, 非白色念珠菌也存在类似条带。采用SPSS13.0聚类分析, 分为5个基因群, 14个基因型, 白色念珠菌主要为B群。其中P385和P403对5-氟孢嘧啶耐药, 聚为C1基因型(欧氏距离平方为0.115), P321和P522对两性霉素B耐药, 聚为D1基因型(欧氏距离平方为0.221)。表明HIV感染者口腔来源的念珠菌存在丰富的基因多态性, RAPD技术对于白色念珠菌的基因型鉴定具有重要意义; 念珠菌不同种间具有相对特征性的条带, 同种不同株间存在相似的条带特征; 某些条带特征可能与念珠菌的耐药性相关。     

Using RAPD for Estimating Genetic Polymorphism in and Phylogenetic Relationships among Species of the Genus Lycopersicon (Tourn.) Mill.

RAPD genome analysis of 53 species and cultivars of the genusLycopersicon (Tourn.) Mill. revealed their high genetic polymorphism (Tourn.) Mill., based on which their phylogenetic relationships were inferred. In total, 248 polymorphic DNA fragments were amplified. Intraspecific polymorphism was maximum (79%) in L. peruvianum and minimum (9%) in L. parviflorum. In general, genome divergence among cross-pollinating tomato species was substantially higher than in self-pollinating species. An UPGMA dendrogram constructed from the RAPD patterns was consisted with the Lycopersicon phylogeny inferred from the molecular data of RFLP, ISSR, and microsatellite analyses and with a classification based on morphological characters. The relationships of taxa within the genus Lycopersicon are discussed.     

用RAPD分析四种无尾类的系统演化关系

利用RAPD技术检测了分属无尾目3个科（雨蛙科、蟾蜍科、蛙科）的黑眶蟾蜍（Bufomelanostictus)、中国雨蛙（Hyla chinensis）、泽陆蛙（Rana limnocharis）、沼水蛙（R.guentheri)的系统发生关系。经19个随机引物对4个物种基因组DNA进行扩增,选择其中扩增谱带清晰的16个引物进行分析,计算不同科间及同一科内不同种间的遗传距离,结果表明：16个引物获得的RAPD谱带均表现出不同程度的多态性;泽陆蛙与沼水蛙间的亲缘关系最近,而黑眶蟾蜍与中国雨蛙之间的亲缘关系较黑眶蟾蜍与蛙科的泽陆蛙、沼水蛙之间以及中国雨蛙与泽陆蛙、沼水蛙之间的亲缘关系近,从基因组DNA水平上也说明雨蛙科与蟾蜍科间的亲缘关系更近,与蛙科的亲缘关系更远,这与形态学、染色体和线粒体DNA多态性研究的分析结果一致,从而进一步从分子水平上为无属目这3科的系统演化提供了新的证据。     

Genetic Diversity Analysis of Starchy Curcuma Species Using RAPD Markers

The genetic variability in starchy Curcuma species was assessed using Random Amplified Polymorphic DNA (RAPD) technique. The RAPD pattern generated by 20 primers revealed a high degree of polymorphism. A total of 274 bands were generated of which 264 were polymorphic. All the species were separated into 3 clusters using UPGMA. C. aromatica, C. leucorrhiza, and C. brog formed a cluster within which C. longa and C. zedoaria formed a subgroup. C. harita was genetically distinct from all the other Curcuma species. Since it is difficult to distinguish different species by leaf morphology, the RAPD pattern has high utility in identification of starchy Curcuma species.     

白杨派树种亲缘关系的RAPD分析

运用RAPD技术对白杨派中7个树种和3个人工杂交种共计50个无性系的亲缘关系进行分析.结果表明:筛选出的26条随机引物共扩增产生222条谱带,其中200条为多态性谱带,多态性带达90.09%,且种间遗传变异大于种内无性系间的遗传变异.聚类分析结果显示,参试的50个材料可分为2大类:第一类由山杨、响叶杨、河北杨组成;第二类由毛白杨、银毛杨、84K杨、银白杨、银灰杨、新疆杨和银新杨组成;3个人工杂交种与白杨亚派树种的亲缘关系较近;人工杂交种银毛杨与84K杨间亲缘关系最近.     

RAPD and ISSR analyses of species and populations of the genus Stachys

Molecular analysis of the genome was performed for 14 species of the genus Stachys. RAPD and ISSR analyses of the Stachys genome revealed 574 polymorphic fragments, including genus- and species-specific markers. Based on the patterns, UPGMA and the Jacquard coefficient were used to estimate the genetic distances between Stachys species and populations and to construct dendrograms reflecting the phylogenetic relationships among the Stachys species. Molecular analysis of the Stachys genome refined the phylogenetic positions of some species and revealed synonymous species.     

RAPD and ISSR analyses of species and populations of the genus Stachys

Molecular analysis of the genome was performed for 14 species of the genus Stachys. RAPD and ISSR analyses of the Stachys genome revealed 574 polymorphic fragments, including genus-and species-specific markers. Based on the patterns, UPGMA and the Jacquard coefficient were used to estimate the genetic distances between Stachys species and populations and to construct dendrograms reflecting the phylogenetic relationships among the Stachys species. Molecular analysis of the Stachys genome refined the phylogenetic positions of some species and revealed synonymous species.     

Uncovering Cryptic Diversity in Two Amoebozoan Species Using Complete Mitochondrial Genome Sequences

The Amoebozoa are a major eukaryotic lineage that encompasses a wide range of amoeboid organisms. The group is understudied from a systematic perspective: molecular tools have only been applied in the last 15 yr. Hence, there is an undersampling of both genes and taxa in the group especially compared to plants, animals, and fungi. Here, we present the complete mitochondrial genomes of two ubiquitous and abundant morpho‐species (Acanthamoeba castellanii and Vermamoeba vermiformis). Both have mitochondrial genomes of close relatives previously available, enabling insights into recent divergences at a genomic scale, while simultaneously offering comparisons with divergence estimates obtained from traditionally used single genes, SSU rDNA and cox1. The newly sequenced mt genomes are significantly divergent from their previously sequenced conspecifics (A. castellannii 16.4% divergence at nucleotide level and 10.4% amino acid; V. vermiformis 21.6% and 13.1%, respectively), while divergence at the small subunit ribosomal DNA is below 1% within both species. Morphological analyses determined that these lineages are indistinguishable from their previously sequenced counterparts. Phylogenetic reconstructions using 26 mt genes also indicate a level of divergence that is comparable to divergence among species, while reconstructions using the small subunit ribosomal DNA (SSU rDNA) do not. In addition, we demonstrate that between closely related taxa, there are high levels of synteny, which can be explored for primer design to obtain larger fragments than the traditional barcoding genes. We conclude that, although most systematic work has relied on SSU, this gene alone can severely underestimate diversity. Thus, we suggest that the mt genome emerges as an alternative for unraveling the lower level phylogenetic relationships of Amoebozoa.     

Evaluation of Genome Sequencing Quality in Selected Plant Species Using Expressed Sequence Tags

Background

With the completion of genome sequencing projects for more than 30 plant species, large volumes of genome sequences have been produced and stored in online databases. Advancements in sequencing technologies have reduced the cost and time of whole genome sequencing enabling more and more plants to be subjected to genome sequencing. Despite this, genome sequence qualities of multiple plants have not been evaluated.

Methodology/Principal Finding

Integrity and accuracy were calculated to evaluate the genome sequence quality of 32 plants. The integrity of a genome sequence is presented by the ratio of chromosome size and genome size (or between scaffold size and genome size), which ranged from 55.31% to nearly 100%. The accuracy of genome sequence was presented by the ratio between matched EST and selected ESTs where 52.93% ∼ 98.28% and 89.02% ∼ 98.85% of the randomly selected clean ESTs could be mapped to chromosome and scaffold sequences, respectively. According to the integrity, accuracy and other analysis of each plant species, thirteen plant species were divided into four levels. Arabidopsis thaliana, Oryza sativa and Zea mays had the highest quality, followed by Brachypodium distachyon, Populus trichocarpa, Vitis vinifera and Glycine max, Sorghum bicolor, Solanum lycopersicum and Fragaria vesca, and Lotus japonicus, Medicago truncatula and Malus × domestica in that order. Assembling the scaffold sequences into chromosome sequences should be the primary task for the remaining nineteen species. Low GC content and repeat DNA influences genome sequence assembly.

Conclusion

The quality of plant genome sequences was found to be lower than envisaged and thus the rapid development of genome sequencing projects as well as research on bioinformatics tools and the algorithms of genome sequence assembly should provide increased processing and correction of genome sequences that have already been published.     

Clinical applications of Genome Polymorphism Scans

Applications of Genome Polymorphism Scans range from the relatively simple such as gender determination and confirmation of biological relationships, to the relatively complex such as determination of autozygosity and propagation of genetic information throughout pedigrees. Unlike nearly all other clinical DNA tests, the Scan is a universal test – it covers all people and all genes. In balance, I argue that the Genome Polymorphism Scan is the most powerful, affordable clinical DNA test available today.     

Detection of Intra-Clonal Genetic Variability in Vegetatively Propagated Tea Using RAPD Markers

The role of random amplified polymorphic DNA (RAPD) markers in detecting intra-clonal genetic variability in vegetatively propagated UPASI-9 clone of tea (Camellia sinensis) was studied. Twenty five decamer primers were used, of which three did not amplify, three gave single bands and the rest of nineteen primers generated upto twelve bands (an average of 6.3 bands per primer). Twenty one primers exhibiting amplified products gave monomorphic banding patterns. Only one primer (OPE-17) gave a unique extra band of similar size in four plants.     

低能N~+离子束注入香瓜种子引起的变异及后代基因组的RAPD分析

用低能N+离子束对香瓜种子进行不同能量和剂量组合的处理。从各处理组与对照组当代和第三代苗期形态特征观察、考种中发现 :在一定能量和剂量组合下 ,处理组香瓜叶型和果肉厚度发生了明显的变化 ,这种形态学变化是可遗传的。应用随机引物扩增多态性DNA (RandomamplifiedpolymorphicDNA即RAPD)技术分析对照组和注入低能N+离子束处理组的香瓜总DNA的结果显示 :10 0种随机引物中的 2 4种引物扩增出 44条多态性片段 ,且其中一种引物所扩增的条带与叶型变化相连锁。表明低能N+注入香瓜种子可引起其基因组DNA发生变异 ,一定注入剂量和能量的组合 ,可导致特异性的变异。     

两种泥鳅RAPD标记遗传稳定性分析

用从20个随机引物中筛选出的7个引物对泥鳅（Misgurnus anguillicaudatus）和大鳞副泥鳅（Paramisgurnus dabryanus）及其自交与杂交子代进行了RAPD遗传稳定性分析。结果表明,RAPD谱带主要呈孟德尔式遗传,该类带纹明亮稳定,在泥鳅子代中占99．11％;在大鳞副泥鳅子代中占100％;在杂交子代中占99．36％。也存在非孟德尔式遗传谱带,该类带纹较暗、稳定性差,在泥鳅子代中为0．89％;在杂交子代中为0．64％;在大鳞副泥鳅中未见此类带纹。实验结果说明RAPD谱带具有很高的遗传稳定性。     

RAPD Analysis of DNA Polymorphism in Turkish Sheep Breeds

The genetic diversity and phylogenetic relationships of 108 individual sheep from three Turkish sheep breeds (K?v?rc?k, Gökçeada, and Sak?z) were studied using RAPD analysis. Polymorphisms within and between populations were assayed using 15 random primers, and 82 loci were amplified ranging from 250 to 2,500 bp. The percentage of polymorphic loci was found to be 80.49, 78.05, and 73.17%, for K?v?rc?k, Gökçeada, and Sak?z sheep breeds, respectively. Total genetic diversity was 0.2265, and the average coefficient of genetic differentiation was 0.1181. Genetically, the Gökçeada breed was more closely related to the Sak?z breed than to the K?v?rc?k breed.     

Variation and Morphogenetic Characteristics of Different Stachys Species during Microclonal Propagation

Morphogenesis of different Stachys species introduced in in vitro culture have been compared. The frequency of altered forms have been demonstrated to be related to the plant genotype. All regenerants of S. sieboldii, which reproduces in vivo only vegetatively, are phenotypically normal, irrespective of the concentrations of plant growth regulators at which they have been obtained. Only changes in isozyme patterns have been observed in the regenerants grown in media containing at least 10 mg/l benzyl aminopurine (BAP); most of these changes are the absence of a particular component of the pattern. The cross-pollinating species Stachys ocymastrum, which typically reproduces by seeds, has yielded morphologically altered forms even in phytohormone-free media; its isozyme patterns often contained a new component. Analysis of the isoperoxidase patterns of regenerants of both Stachys species obtained with the use of high phytohormone concentrations has demonstrated qualitative and quantitative changes suggesting the appearance of somaclonal variants even in the course of plant regeneration directly from nodal segments, bypassing callus formation. Changes have also been found in Stachysplants regenerating from the callus tissue.