普通小麦细胞质雄性不育系及其保持系线粒体DNA和RAPD分析

应用随机扩增多态ＤＮＡ（Ｒａｎｄｏｍ ａｍｐｌｉｆｉｅｄ ｐｏｌｙｍｏｒｐｈｉｃ ＤＮＡｓ,ＲＡＰＤ）技术,对普通小麦细胞质雄性不育系（Ａ）及其保持系（Ｂ）线粒体基因组（ｍｉｔｏｃｈｏｎｄｒｉａｌ ｇｅｎｏｍｅ）的指纹图谱进行了分析。共使用引物４１个,其中２０个引物得到了扩增产物,部分引物扩增结果表现多态性。说明线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ＤＡＮ,ｍｔ,ＤＮＡ）序列在不育系和保持系之     

白鲢和镛鱼的扩增多态DNA分析

根据鱼类外周血细胞都有核的特点,采用从冷冻和低渗双重处理分离的细胞核的取基因组ＤＮＡ。以此法获得的白鲢和镛鱼的基因组ＤＮＡ为模板,和Ｏｐｅｒｏｎ公司生产的ＯＰＮ和ＯＰＭ两个组共４０个随机引物,对这两种鱼进行了随机扩增多态ＤＮＡ（ＲＡＰＤ）分析;确定了对这两种鱼基因组相关区域可进行随机ＰＣＲ扩增的有效引物,特别是哪些可产生种群内或群体的ＲＡＰＤ遗传标记即可产生个体特异性和群体特异性ＲＡＰＤ带谱的引物     

烟草黑胫病菌株亲缘关系的RAPD分析

从２２０个ＲＡＰＤ（Ｒａｎｄｏｍ Ａｍｐｌｉｆｉｅｄ Ｐｏｌｙｍｏｒｐｈｉｃ ＤＮＡｓ）随机引物中所选出的多态扩增性强的２１个引物对来源不同的３３个烟草黑胫病菌株进行全基因组ＤＮＡ遗传多样性分析和指纹构建。选用引物对受试菌株进行ＲＡＰＤ－ＰＣＲ扩增,共产生２４３条ＤＮＡ标记图带,其中１９１条为多态性图带,多态检测率为７８．６％。利用ＵＰＧＭＡ（Ｕｎｗｅｉｇｔｈｔｅｄ Ｐａｉｒ－ｇｒｏｕｐ Ｍｅｔｈ     

普通小麦细胞质雄性不育系及其保持系线粒体DNA的RAPD分析

应用随机扩增多态ＤＮＡ（ＲａｎｄｏｍａｍｐｌｉｆｉｅｄｐｏｌｙｍｏｒｐｈｉｃＤＮＡｓ,ＲＡＰＤ）技术,对普通小麦细胞质雄性不育系（Ａ）及其保持系（Ｂ）线粒体基因组（ｍｉｔｏｃｈｏｎｄｒｉａｌｇｅｎｏｍｅ）的指纹图谱进行了分析。共使用引物４１个,其中２０个引物得到了扩增产物,部分引物扩增结果表现多态性。说明线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ,ｍｔＤＮＡ）序列在不育系和保持系之间存在差异,进一步明确了小麦细胞质雄性不育（ｃｙｔｏｐｌａｓｍｉｃｍａｌｅｓｔｅｒｉｌｅ,ＣＭＳ）与ｍｔＤＮＡ的关系。     

E和St基因组特异RAPD片段在部分小麦族植物中的分布

两个Ｅ基因组（包括Ｅｅ和Ｅｂ）特异ＲＡＰＤ片段和两个Ｓｔ基因组特异ＲＡＰＤ片段的序列分析表明,４个片段均为新的ＤＮＡ克隆片段。染色体原位杂交显示ＯＰＤ１２４４４为区域化连续高度重复序列,而ＯＰＦ０３１２９６（Ｅｂ特异）、ＯＰＢ０８５２５（Ｓｔ特异）、ＯＰＮ０１８１７（Ｓｔ特异）为弥散性高度重复序列。研究还显示：大部分ＤＮＡ高度重复序列在亲缘关系较近的小麦族植物基因组间是共享的,差异可能主要是在重复次数及片段长度上,而能否用ＲＡＰＤ技术扩增主要决定于某一基因组的这些重复序列中有无与特定引物相匹配的区域。文中就这些重复序列在小麦远缘杂交后代外源遗传物质检测、多倍体物种染色体组组成研究中的潜在价值进行了讨论。     

用RAPD标记研究对虾属六个种间的亲缘关系

用ＲＡＰＤ技术对对虾属中六种对虾基因组ＤＮＡ的多态性进行研究。在事先优化的反应条件下用２０个随机引物扩增,共得到３６４条清晰稳定的多态性片段,片段长度在２３０￣２８００ｂｐ之间。统计这些片段,根据扩增片段的共享度计算出相对遗传距离指数,然后用ＵＰＧＭＡ和ＮＪ等聚类方法对其进行分析,构建系统树,确定了它们相互间的亲缘关系。从聚类分析的结果可以看到,外部形态比较相似的种类在基因组ＤＮＡ上表现出较大的相     

应用原位杂交及RAPD技术标记抗黄矮病小麦一中间偃麦草染色体异附加系

以生物素（Ｂｉｏｔｉｎ－１６－ｄＵＴＰ）标记中间偃麦草基因组 ＤＮＡ为探针,与抗黄矮病小麦－中间偃麦草染色体异附加系Ｚ６进行原位杂交,鉴定出附加的１对中间偃麦草染色体。对异附加系 Ｚ６和 Ｌ１及它们的小麦亲本进行了 ＲＡＰＤ分析,从 １２０个随机引物中,筛选出 ２个引物可以扩增出附加染色体的特异ＤＮＡ片段,可作为鉴定寻人小麦的中间偃麦草染色质的分子标记。     

鹅膏菌属真菌RAPD分析及亲缘关系的研究

本文对采自湖南莽山的２６种鹅膏菌属（Ａｍａｎｉｔａ）真菌进行了随机扩增多态性ＤＮＡ（ＲＡＰＤ）分析,４０个随机引物中筛选出扩增效果较好的６个引物,每个引物能产生１～１０条ＤＮＡ条带,获得的ＲＡＰＤ谱带清晰并呈现多态性ＯＰＧ１５、ＯＰＨ０４两个引物扩增的ＲＡＰＤ谱带能将２６种鹅膏菌完全区分开来,通过６个引物的ＲＡＰＤ分析获得的平均相似性系数表明种与种之间的相关系数在２０－６０％之间,平均链锁聚类分析     

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增,共获扩增片段１０３ 条,分子量在０．３ － ３ｋｂ 之间,其中５ 种引物ＯＰＪ－ １ ,７ ,９,１１ ,１２ 扩增出差异片段１２ 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变;ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响,以及氮离子注入诱变效应的可能机制。     

烟草黑胫病菌株亲缘关系的RAPD分析

从２２０个ＲＡＰＤ（ＲａｎｄｏｍＡｍｐｌｉｆｉｅｄＰｏｌｙｍｏｒｐｈｉｃ　ＤＮＡｓ）随机引物中所选出的多态扩增性强的２１个引物对来源不同的刀个烟草黑胫病菌株进行全基因组ＤＮＡ遗传多样性分析和指纹构建。选用引物对受试菌株进行ＲＡＰＤ－ＰＣＲ扩增,共产生２４３条ＤＮＡ标记图带,其中１９１条为多态性图带,多态检测率为７８．６％。利用ＵＰＧＭＡ（ＵｎｗｅｉｇｔｈｔｅＰａｉｒ－ｇｒｏｕｐ　ＭｅｔｈｏｄＷｉｔｈＡｒｉｔｈｍｅｔｉｃ　Ａｖｅｒａｇｅ）软件对受试菌株间的遗传距离进行聚类分析构建系统树状图,受试对个菌株被划分为５个遗传聚类组即（Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ）。结果证明供试菌株具有丰富的遗传多样性,虽各自的遗传背景差异显著,但其亲缘关系相近,遗传聚类组的划分与菌株的地理来源未有明显的相关性。     

Genomic changes at early stages of formation of allopolyploid Aegilops longissima x Triticum urartu

Using the model of synthetic allopolyploid Aegilops longissima TL05 x Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing of PCR spectra obtained while amplifying genome DNA of allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for subtelomere DNA sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some PCR fragments typical for parental genome in RAPD specters of allopolyploid TL05 x TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomere primers such changes in PSR specters were shown only for one of 66 pays of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in specter of synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.     

Genome- and species-specific markers and genome relationships of diploid perennial species in Triticeae based on RAPD analyses.

Eight different genomes (E, H, I, P, R, St, W, and Ns) represented by 22 diploid species of the tribe Triticeae were analyzed using the random amplified polymorphic DNA (RAPD) technique. The genome relationships were obtained based on 371 RAPD fragments produced with 30 primers. The four species of the genus Psathyrostachys (having various Ns genomes) were closely related. The genomes Ee and Eb had a similarly close relationship and were distinct from all other genomes analyzed. Genomes P, R, and St were grouped in one cluster and genomes H and I in another. Genome W had a distant relationship with all other genomes. These results agree with the conclusions from studies of chromosome pairing and isozyme and DNA sequence analyses. Twenty-nine and 11 RAPD fragments are considered to be genome- and species-specific markers, respectively. One to six genome-specific markers were identified for each genome. These RAPD markers are useful in studies of genome evolution, analysis of genome composition, and genome identification.     

Genomic changes at early stages of formation of allopolyploid <Emphasis Type="Italic">Aegilops longissima × Triticum urartu</Emphasis>

Using the model of synthetic allopolyploid Aegilops longissima TL05 × Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing PCR fragments of the allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for these chromosomal regions sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some RAPD fragments typical for parental genome in allopolyploid TL05 × TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomeric primers the changes in PCR fragments were shown only for one of 66 pairs of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in the synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.     

Identification of RAPD markers linked to A and B genome sequences in Musa L.

Plantains and bananas (Musa spp. sect. eumusa) originated from intra- and interspecific hybridization between two wild diploid species, M. acuminata Colla. and M. balbisiana Colla., which contributed the A and B genomes, respectively. Polyploidy and hybridization have given rise to a number of diploid, triploid, and tetraploid clones with different permutations of the A and B genomes. Thus, dessert and highland bananas are classified mainly as AAA, plantains are AAB, and cooking bananas are ABB. Classification of Musa into genomic groups has been based on morphological characteristics. This study aimed to identify RAPD (random amplified polymorphic DNA) markers for the A and B genomes. Eighty 10-mer Operon primers were used to amplify DNA from M. acuminata subsp. burmannicoides clone 'Calcutta 4' (AA genomes) and M. balbisiana clone 'Honduras' (BB genomes). Three primers (A17, A18, and D10) that produced unique genome-specific fragments in the two species were identified. These primers were tested in a sample of 40 genotypes representing various genome combinations. The RAPD markers were able to elucidate the genome composition of all the genotypes. The results showed that RAPD analysis can provide a quick and reliable system for genome identification in Musa that could facilitate genome characterization and manipulations in breeding lines.     

小麦与高冰草体细胞杂种后代的核基因组和叶绿体基因组的遗传特征

以小麦(Triticum aestivum L.)与高冰草(Agropyron elongatum(Host)Nevski)体细胞杂种同一个克隆来源的F2-F6自交系Ⅱ-2、Ⅱ-Ⅰ-8以及由Ⅱ-Ⅰ-8 F2分离形成的8-1(F3-F6)为材料,利用小麦叶绿体基因组的微卫星(Microsatellite)特异引物及随机扩增多态性DNA(RAPD)引物进行分析.结果表明,杂种株系的叶绿体基因组组成一致,均以小麦叶绿体基因组为主,仅在rpl14和rpl16基因的间隔序列中检测到双亲的特征带,表明有高冰草的叶绿体DNA在杂种中存在,并稳定遗传至第六代.RAPD分析表明,不同杂种株系中存在不同的高冰草核DNA片段,核基因组在传代中基本稳定.     

RAPD-based analysis of introgression of barley genetic material into the genome of alloplasmic wheat lines (Hordeum geniculatum All./ Triticum aestivum L.)

Genomes of three alloplasmic wheat lines obtained on the basis of barley--wheat hybrid Horderum geniculatum All. (2n = 28) x Triticum aestivum L. (2n = 42)(Pyrotrix 28) were examined using random amplified polymorphic DNA (RAPD) analysis. Line L-29 was obtained after first backcross of the initial hybrid with the wheat variety Pyrotrix 28 and ten subsequent self-pollinating generations. This line was represented by euploid plants with typical to the common wheat chromosome number (2n = 42), as well as by aneuploids, which contained an additional telocentric chromosome in the main karyotype (2n = 42 + t). Lines L-26 and L-27 were obtained by two backcrosses of one BC1 plant with the wheat variety Novosibirskaya 67 and one subsequent self-polination of one BC3 plant. Chromosome number in all these plants corresponded to 2n = 40 + 4t. RAPD analysis was carried out using seven primers, which were previously proved to be effective for identification of the barley genome fragments within hybrid genomes of alloplasmic lines. The presence of barley genome fragments in line L-29 was revealed by use of five primers, while in lines L-26 and L-27 these fragments were detected by use of one primer. The significant difference in the number of barley RAPD fragments in the genomes of alloplasmic lines obtained at different backcrossing stages suggests more intense displacement of barley genome during backcrossing compared to self-pollination in BC1 plants.     

Rapd fingerprinting of diploid Lolium perenne × hexaploid Festuca arundinacea hybrid genomes

We tested the application of RAPD technology for identification of hybrid genomes originated from a maternal clone of Lolium perenne L. (2n = 2x = 14) bearing cytoplasmic male sterility, which was pollinated separately by five clones of Festuca arundinacea Schreb. cv. Barocco (2n = 6x = 42). Six classes of RAPD markers were recognized, specific to: 1) Festuca genome and inherited into F1 hybrid genomes, 2) Lolium genome inherited into F1 hybrid genomes, 3) Lolium-specific bands not found in F1 progeny, 4) Festuca-specific bands not found in F1 progeny, 5) new bands found only in F1 hybrid profiles, 6) bands common to all parental and F1 hybrid genotypes. RAPD data were shown to have full potential a) to serve as an unequivocal proof of genome recombination in perennial ryegrass × tall fescue hybrids, b) to identify hybrid genomes, c) to reveal phenetic relationships of the accessions from crossing families, d) to enhance, by fingerprinting, the selection of superior hybrid material for further breeding. RAPD data were found to be consistent with the festucoid phenotype of F1 hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genome and species relationships in genus<Emphasis Type="Italic"> Avena</Emphasis> based on RAPD and AFLP molecular markers

Species and genome relationships among 11 diploid (A and C genomes), five tetraploid (AB and AC genomes) and two hexaploid (ACD genome) Avena taxa were investigated using amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers. The two primer pairs used for the AFLP reactions produced a total of 354 polymorphic bands, while 187 reproducible bands were generated using ten RAPD primers. Genetic similarities amongst the entries were estimated using the Jaccard and Dice algorithms, and cluster analyses were performed using UPGMA and neighbor joining methods. Principle coordinate analysis was also applied. The highest cophenetic correlation coefficient was obtained for the Jaccard algorithm and UPGMA clustering method (r=0.99 for AFLP and r=0.94 for RAPD). No major clustering differences were present between phenograms produced with AFLPs and RAPDs. Furthermore, data produced with AFLPs and RAPDs were highly correlated (r=0.92), indicating the reliability of our results. All A genome diploid taxa are clustered together according to their karyotype. The AB genome tetraploids were found to form a subcluster within the A_s genome diploids (AFLPs), indicating their near-autoploid origin. The AC genome tetraploids are clustered to the ACD genome hexaploids. Finally, the C genome diploids form an outer branch, indicating the major genomic divergence between the A and C genomes in Avena.Communicated by J.S. Heslop-Harrison     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

应用RAPD特异标记分析拟鹅观草属部分物种的基因组组成

选用小麦族中8个基本基因组(E、H、I、P、St、W、Ns、R)的特异RAPD引物进行PCR扩增检测,分析Pseudoroegneriagracillima、P.kosaninii、Roegneriaalashanica和R.magnicaespes这4个四倍体物种的基因组组成。结果表明:P.gracillima、P.kosaninii、R.alashanica和R.magnicaespes中除了含有St或经修饰的St基因组外,都不含E、H、I、P、W、Ns和R基因组,由此推断P.gracillima和P.kosaninii至少含有一个St或经修饰的St基因组,另一个基因组是否为Y基因组,需要进一步研究证实。结合前人细胞遗传学研究的结果,推断R.alashanica和R.magnicaespes为同源四倍体或部分同源四倍体,其基因组组成为StStStSt或St1St1St2St2。因此,结合外部形态特征以及前人细胞遗传学、分子标记研究和核型分析的结果,推断R.alashanica和R.magnicaespes与P.elytrigioides一样,也可能是在中国分布的四倍体拟鹅观草属物种,为系统整理和研究国产拟鹅观草属物种及其地理分布提供了DNA分子水平上的资料。