杉木种子随体染色体SCAR标记的建立

回收、纯化由引物OPB07(5’-GGTGACGCAG-3’) OPB18(5’-CCACAGCAGT-3’)扩增而得的杉木(Cunninghamia lanceolata(Lamb.)Hook)种子随体染色体特异性RAPD(随机扩增的DNA多态性分析)片段OPB07-18907,将该片段克隆至pUCm-T载体,转化受体菌后筛选出阳性克隆,并对插入片段进行测序,根据序列特点设计两对SCAR(序列特异性扩增区)引物,PCR结果显示,这两对引物的4种组合都可以扩增出属于随体染色体的特征带,适宜退火温度为57℃。成功将特异RAPD标记OPB07-18907转化为稳定的SCAR标记。开发随体染色体SCAR标记目的是:一方面能在分子水平上鉴定微分离的杉木随体染色体,另一方面,也可以将杉木已构建的遗传图谱中连锁群与染色体进行对应。探讨了该SCAR标记对杉木核型分析的作用。     

染色体特异DNA文库的构建与鉴定

为构建人类21号染色体特异DNA文库, 以应用于人类遗传疾病的鉴定和研究, 文章采用循环温度梯度法溶解释放微分离的人外周血细胞21号染色体DNA, 将其进行简并寡核苷酸引物PCR(Degenerate oligo nucleotide primer-PCR, DOP-PCR)扩增后, 利用100~500 bp和500~2 000 bp分段回收纯化的两种不同片段大小的DOP-PCR产物构建染色体特异DNA文库, 并分别采用荧光原位杂交(Florescence in situ hybridization, FISH)和斑点杂交对DOP-PCR产物的来源和随机取样的文库克隆进行检测以评估所构建DNA文库的特异性。结果表明: 循环温度梯度法能有效溶解释放微分离的21号染色体DNA; 通过对DOP-PCR产物的分段回收纯化和克隆, 增加了大片段DNA的连接效率; 利用FISH技术和斑点杂交双重鉴定实验证明了文库的特异性, 从而构建了21号染色体特异的DNA文库, 并建立了构建染色体特异DNA文库及检测其特异性的方法, 为21号染色相关遗传疾病的鉴定和研究奠定了基础。     

菜蛾对杀虫双和杀螟丹抗药性遗传的DNA随机扩增多态性研究

利用室内选育119代的抗杀虫双和抗杀螟丹小菜蛾Plutellaxylostella品系和敏感品系,通过反复回交建立近等基因系.用Operon公司合成的80个引物对抗杀虫双近等基因系和抗杀螟丹近等基因系小菜蛾基因组DNA进行PCR扩增;在抗杀虫双近等基因系中有3个引物分别产生了1条特异扩增带,2个引物分别产生了2条特异扩增带,1个引物产生3条特异扩增带;在抗杀螟丹近等基因系中,有4个引物分别产生了1条特异扩增带,1个引物产生了2条特异扩增带.在培育近等基因系的多次回交过程中,与抗性有关的遗传因子被逐步置换到敏感品系的基因组中,因此可以认为这些特异带与小菜蛾对杀虫双和杀螟丹的敏感性有关.     

膜荚黄芪SCAR标记的建立

用RAPD方法对膜荚黄芪和蒙古黄芪进行指纹图谱的研究。采用BSA法从120个10碱基随机引物筛选出7个在膜荚黄芪基因池和蒙古黄芪基因池中表现多态性的引物。单株检测表明,引物OPD14具有膜荚黄芪特异性,在检测的膜荚黄芪个体中均能各自扩增出一条300 bp左右的特异带,而在蒙古黄芪的单株中则未见,将该膜荚黄芪特异性片断命名为OPD14_-300。获得的RAPD标记OPD14_-300经克隆、测序、重新设计一对特异性引物转化成更稳定的SCAR标记;该SCAR标记只在膜荚黄芪个体中出现,达到了在分子水平上快速、准确地鉴定膜荚黄芪和蒙古黄芪的目的。     

小菜蛾对杀虫双和杀螟丹抗药性遗传的DNA随机扩增多态性研究

利用室内选育119代的抗杀虫和抗杀螟丹小菜蛾Plutella xylostella品系和敏感品系,通过反复回交建立近等基因系。用Operon公司全盛的80个引物对抗杀虫比近等基因系和抗杀螟丹近等基因系小菜蛾基因组DNA进行PCR扩增;在抗杀虫双近等基因系中3个引物分别产生了1条特异扩增带,2个引物分别产生了2条特异扩增带,1个引物产生3条特异拉增带;在抗杀螟丹近等基因系中,有4个引物分别产生了1条特异扩增带,1个引物产生了2条特异扩增带,在培育近等基因系的多次回交过程中,与抗性有关的遗传因子被逐步换到敏感品系的基因组中,因此可以认为这些特异带与小菜蛾对杀虫双和杀螟丹的敏感性有关。     

黄鳝单条染色体的显微分离及特异性检测

建立了显微分离黄鳝单条染色体及检测其特异性的方法;在Olympus倒置显微镜下用毛细玻璃微针手工分离黄鳝减数分裂Ⅰ终变期3号染色体,将期DNA作模板进行DOP-PCR扩增后,分别以α^-32P-dCTP和Biotin-11-dUTP标记的单条染色体DNAPCR扩增产物及Biotin-11-dUTP标记的大豆18SrRNA基因作探针进行Southern杂交,FISH和Dot杂交来检测其特异性,结果表明：（1）减数分裂Ⅰ终变期染色体标本是进行染色体显微操作的理想材料;（2）DOP-PCR扩增产物片段在200-1000bp之间,平均600bp左右;（3）杂交结果显示,本研究所获得的单条染色体是黄鳝3号染色体;（4）与显微操作仪和微激光分离相比较。该方法不需要昂贵仪器,在常规实验室即可操作,具有广泛的普及应用意义。     

甜菜夜蛾抗高效氯氟氰菊酯近等基因系的RAPD标记

用随机引物扩增的方法对近等基因系抗高效氯氟氰菊酯种群和敏感种群甜菜夜蛾进行比较分析,从160条引物中筛选,引物S42(GGACCCAACC)和OPH13(GACGCCACAC)在抗性种群中扩增分别得到了1条特异扩增带,带型清晰、重复性较好,所以S42和OPH13产生的两条特异扩增带是分别与甜菜夜蛾对高效氯氟氰菊酯抗性相关的RAPD分子标记。     

SCAR标记在茶树种质资源鉴定中的应用

SCAR标记是一种在RAPD技术的基础上发展起来的新型分子标记技术,提高了分子标记辅助选择育种的效率,在茶树种质资源的合理开发与利用中具有广阔的应用前景.运用优化后的RAPD反应体系对10个茶树品种的基因组DNA进行遗传差异分析,随机引物S89、S4分别在白毫早和福云6号中扩增得到长度为498 bp、1 622 bp的差异片段,命名为BHZ498、FY1622.根据它们的测序结果分别设计了一对特异引物,BHZ498的特异引物为SB1/SB2;FY1622的特异引物为SC1/SC2,用这两对特异引物对10个茶树品种的基因组DNA进行扩增.引物SB1/SB2和SC1/SC2分别在白毫早和福云6号中扩增出唯一的一条扩增带,而这两对引物在其他供试茶树材料中均无相应的扩增带,结果表明已将BHZ498、FY1622标记成功转化成SCAR标记.     

粘类小麦细胞质雄性不育系的RAPD分析

利用250条10-聚寡核苷酸随机引物对具粘果山羊草(Aegilops kotschyi)、易变山羊草(Ae.variabilis)、偏凸山羊草(Ae.ventricosa)和二角山羊草(Ae．bicornis)细胞质不育系及其保持系5-1的总DNA进行了RAPD多态性分析,其中31条引物对4种不育系及其保持系总DNA均无扩增,217条引物扩增条带完全相同。有2条随机引物在2种不育系之间有特异的扩增片段,其中引物S22在偏凸山羊草细胞质雄性不育系基因组DNA中扩增出分子量约为1600bp的特异带,引物S202在粘果山羊草细胞质雄性不育系基因组DNA中扩增出约1300bp特异带。线粒体基因组DNA的RAPD分析表明,4种不育系及其保持系mtDNA存在明显的差异。证明了S22—1600为偏凸山羊草细胞质不育系及其mtDNA基因组DNA的RAPD特异片段．S202—1300可能为粘果山羊草细胞质不育系及其ctDNA基因组DNA的RAPD特异片段。     

用RAPD和染色体原位杂交检测Elymus rectisetus染色体附加到普通小麦中

报道小麦×Elymusrectisetus属间杂种BC2F236个衍生系,通过染色体原位杂交和RAPD分析,检测Elymusrectisetus染色体及其特异染色体DNA标记。原位杂交结果表明,36个衍生系中大多数系含1对异源Erectisetus染色体,少数系含3条以上Ereclisetus染色体;RAPD结果表明,有13个随机引物（OPBA-08、OPB－14OPEA-09、OPF-05、OPF－09、OPJ-05、OPK-03、OPN-12、OPP-20、OPS－12、OPT-20、OPZ-09、OPZ-11）能够在这36个衍生系中分别扩增出普通小麦所没有的Erectisetus特异的染色体DNA片段。其中Erectisetus特异染色体DNA片段OPF－05480bp、OPF－09650bp和OPZ-11350bp只能够在“1048”系统的全部8个株系中扩增出;OPN-12350bp只能够在“1057”系统的全部6个株系中扩增出;OPB-14900bp和OPM-05420bp只能够在“1034”、“1026”2个系统的全部22个株系和“1057”－5－3株系中扩增出。直接获得了3个类型不同的异附加系,省去常规通过测配和附加系两两杂交F1PMCMI细胞学鉴定来确证异附加系之间的异同。由此说明染色体原位杂交和RAPD分析有机结合是鉴定异附加系（异代换系）快速有效的方法,且方法简单、易行．     

DNA markers for sex: Molecular evidence for gender dimorphism in dioecious Mercurialis annua L.

Male specific Random Amplified Polymorphic DNA (RAPD) markers, OPB01-1562 and OPC07-303, were identified and sequenced in dioecious Mercurialis annua. Sequence Characterized Amplified Region (SCAR) primers were designed. Several internal segments of OPB01-1562 were amplified as male specific SCAR markers. These markers were PCR amplified from strong, intermediate and weak male subtypes selected according to their resistance to feminization by cytokinin. Nucleotide sequence of OPB01-1562 isolated from three male subtypes were near identical. The OPB01-1562 and derived SCAR markers were absent in females as well as hexaploid Mercurialis male and monoecious individuals. The gender relationship of the markers was maintained in all ecotypes tested. There were 2 internal fragments of OPB01-1562, which were PCR amplified from all genotypes of diploid and hexaploid Mercurialis. It is argued that identification of gender specific DNA suggests a dimorphic differentiation of the genome of dioecious Mercurialis annua.     

Genetic Diversity Analysis of Elite Pearl Millet Inbred Lines using RAPD and SSR Markers

Pearl millet [Pennisetum glaucum (L) R Br] is one of the widely grown cereal crops in the arid and semi-arid regions of Africa and India. We undertook a study to ascertain the genetic diversity in 21 elite inbreds (parental lines of 13 pearl millet hybrids in India) using 20 Random Amplified Polymorphic DNA (RAPD) and 21 Simple Sequence Repeat (SSR) markers. Based on Polymorphism Information Content (PIC) and unique banding profiles, 6 RAPD primers OPD12, OPA16, OPB6, OPA19, OPB5 and OPB1, and 3 SSR markers Xpsmp2208, Xpsmp2223 and Xpsmp2220, were found to be highly discriminative. The PIC values ranged from 0.28 to 0.48 for the RAPD and from 0.24 to 0.60 for the SSR markers. Cluster analysis and principal component analysis of the combined dataset of RAPD and SSR markers indicated moderate genetic divergence among the elite pearl millet germplasm, besides unraveling the genetic relationships among the male sterile lines and the restorers.     

DNA amplification fingerprinting as a tool for checking genetic purity of strains in the cyanobacterial inoculum

Summary The electrophoretic patterns for 17 different cyanobacterial cultures derived from 6 different decamer primers were analysed to provide diagnostic fingerprints for each culture and their genetic distances based on RAPD markers.The primer OPB 09 produced a maximum of 24 amplified products. The primers OPB 09, OPG 04 and OPAH 02 generated markers specific for Nostoc cultures. Westiellopsis was found to be distinct from other cyanobacterial cultures in the RAPD profile obtained with the primer OPAH 02. The primer OPF 03 generated specific markers for Tolypothrix tenuis. Fischerella cultures could be identified with the primers OPB 09, OPAG 03 and OPF 05. The study revealed that these RAPD markers could be further used to identify and establish the genetic purity of the strains in the cyanobacterial inoculum. There was a similarity of 60–90% within Westiellopsis cultures. Nostoc cultures shared 50–80% similarity with Westiellopsis cultures. Anabaenacultures were similar to Westiellopsiscultures by 60–70. The markers produced for each culture were also applied to phylogenetic analysis to infer genetic relatedness in this group of prokaryotes. The dendrogram analysis clearly revealed that free-living cyanobacterial cultures are closely related to each other and are distinct from the symbiotic forms.     

RAPD Fingerprint to Appraise the Genetic Fidelity of In Vitro Propagated <Emphasis Type="Italic">Araucaria excelsa</Emphasis> R. Br. var. glauca Plantlets

Randomly amplified polymorphic DNA (RAPD) was used as a tool to assess the genetic fidelity of in vitro propagated Araucaria excelsa R. Br. var. glauca with explants taken from orthotropic stem along with their related mother plants after treatment with kinetin, 2iP, BA (0.02–0.26 mg/l) and TDZ (0.001–1 mg/l) to produce axillary shoots. TDZ and kinetin induced more shoot and higher length per explant. Results showed a total of 1,676 fragments were generated with 12 RAPD primers in micropropagated plants and their donor mother plants. The number of loci ranged from 6 in OPB 12–18 in OPY 07 with a size ranging from 250 bp in OPH 19–3500 bp in OPH 11. Cluster analysis of RAPD data using UPGMA (unweighted pair group method with arithmetic average) revealed more than 92% genetic similarities between tissue cultured plants and their corresponding mother plant measured by the Jaccard’s similarity coefficient. Similarity matrix and PCoA (two dimensional principal coordinate analysis) resulted in the same affinity. Primers had shown 36% polymorphism. However, careful monitoring of tissue culture derived plants might be needed to determine that rooted shoots are adventitious in origin.     

Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 pat-1) on chromosome 1 of B. patellaris

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 ^pat-1. Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11₈₀₀, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11₈₀₀, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11₈₀₀ loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 ^pat-1 locus. Two copies of the middle-repetitive OPX2₁₁₀₀ marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis

The random amplified polymorphic DNA (RAPD) technique was employed in the haplo-diploid dioecious species Gracilaria gracilis to identify sex-linked PCR markers. Sixty-nine decamer oligonucleotide primers were tested on two bulks of DNA, one from five haploid males and the other from five haploid females. One of these primers (OPD13) generated a 430-bp fragment specific to males and a 620-bp fragment specific to females. The diploid individuals (tetrasporophytes) showed the co-occurrence of these two fragments. In order to verify the linkage between the sexual phenotypes and these markers, a progeny array of 59 haploid individuals (male and female) born on a diploid individual was analysed, in all of which the two markers produced by the OPD13 primer segregated perfectly with sex.     

Genetic relationships among wild pomegranate (Punica granatum) genotypes from Coruh Valley in Turkey

The pomegranate has been used traditionally in Coruh Valley in Turkey for a long time; fruits are harvested from wild, semi-domesticated and cultivated trees. In the valley, it occurs in general along with olive trees. We sampled 23 wild-grown pomegranate genotypes sampled from different parts of Coruh Valley and compared them using RAPD primers to determine genetic variability. Eighty-six RAPD primers were used for molecular characterizations, among which 12 gave reliable polymorphic patterns. These primers generated 145 RAPD bands of which 91% were polymorphic. The highest polymorphism ratio was observed with primers OPY06, OPY13, OPBA03, OPBB03, OPBB07, and OPBB09 (100%), while the lowest was with OPBB09 and OPBB10 (75%). The band size was between 250 and 2400 bp. There were five main clusters in the dendrogram; the highest genetic similarity was 0.24 and the lowest was 0.08.     

Determination of the site of origin of Pinellia ternata roots based on RAPD analysis and PCR-RFLP

Analyses of randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) were performed in an effort to distinguish between two different origins of Pinellia ternata. To determine whether the origin of Pinellia ternata is in China or Korea. RAPD analysis was carried out using ten 20-mer random primers. Although the coefficients of similarity between the DNA of Chinese and Korean accessions of Pinellia ternata were high, distinguishable band patterns were observed in the reaction performed using primer numbers 3, 6 and 10. Primer 3 produced one (410 bp) and primer 6 produced four (410 bp, 350 bp, 300 bp, 250 bp) Chinese Pinellia-specific fragments. Primer 10 produced one (900 bp) Korean Pinellia rhizome-specific fragment. In addition, using PCR-RFLP analysis, different fingerprints were obtained from Korean and Chinese Pinellia ternata respectively. These results suggest that the analyses with RAPD and PCR-RFLP can be used to authenticate the relevant Chinese and Korean herbal medicines.