“薄皮”ד垂枝”板栗RAPD遗传连锁图构建

使用20组共400条RAPD随机引物,在"薄皮"和"垂枝"两个板栗亲本无性系及其5个杂交后代进行筛选,共有187条引物获得了清晰、重复性好的多态性条带,占供试引物的46.75%.应用筛选出的多态性引物在两亲本及其60个杂交后代中进行扩增,获得的多态性条带在后代中的分离比用卡方测验(α=0.05)分析,结果共有143个标记符合1:1测交分离比,其中来源于母本"薄皮"板栗的有88个,来源于父本"垂枝"的有55个.采用作图软件Mapmaker和回交群体模型分别对符合测交分离比的标记进行亲本特异的标记分群和连锁分析,设定LOD值为3和最大重组值0为0.50作为可信统计度与最大连锁标记数量之间的最佳组合,其中母本的标记定位了10个连锁组,父本的标记定位了7个连锁组.母本"薄皮'板栗遗传连锁图共包含88个标记,覆盖了板栗基因组总长约823.1 cM(Kosambi,以下同),占基因组的66.7%;父本"垂枝"板栗遗传连锁图共包含55个标记,覆盖了板栗基因组总长约720.8 cM,占基因组的41.7%.母本"薄皮"板栗遗传连锁图上标记间的遗传距离为1.6～26.5 cM,平均9.4 cM,连锁组的大小从15.5 cM到166.3 cM,平均为82.3 cM;父本"垂枝"板栗遗传连锁图上标记间的遗传距离为2.1～39.9 cM,平均13.1 cM,连锁组的大小从53.5 cM到139.6 cM,平均为103.0 cM.     

安徽野生香菇遗传多样性及杂种优势的RAPD分析

采用随机扩增多态性DNA(RAPD)技术,利用17个引物对26个安徽野生香菇菌株和6个香菇栽培品种进行了遗传多样性分析,并构建了遗传相关聚类图。在128个RAPD标记中,多态性标记为104个,占81.3%。聚类分析显示,当以相异距离0.25为阈值,32个菌株被划为5个RAPD遗传组,多数菌株之间遗传相似性较低。表明供试菌株在DNA水平上存在比较显著的遗传变异,具有较丰富的遗传多样性。杂种优势的检测表明亲本间遗传距离较大的组合其杂种优势也较强。     

DNA指纹图谱技术及其在作物品种资源中的应用

随着各种新型DNA分子标记的出现,带动了DNA指纹图谱技术的快速发展。本文简要阐述了几种常见DNA指纹鉴定技术,如限制性片段长度多态性(restrictionfragmentlengthpolymorphism,简称RFLP)、随机扩增多态性DNA(randomamplifiedpolymorphicDNA,简称RAPD)、扩增片段长度多态性(am-plifiedfragmentlengthpolymorphism,简称AFLP)、简单重复序列或微卫星标记(simplesequencerepeat,简称SSR)、内部简单重复序列(inter-simplesequencerepeat,简称ISSR)和单核苷酸多态性(singlenucleotidepoly-morphisms,简称SNPs)等的基本原理、在技术上的优缺点及其在作物品种资源中的应用,包括品种的鉴定、纯度的检测、品种亲缘关系与分类的研究及品种专利权的正当维护等。同时,对DNA指纹图谱技术在应用中的存在问题及相应的解决途径进行了简单的讨论,如目前DNA的提取方法与育种应用的大规模群体不相适应和大多数DNA指纹图谱检测技术仍比较繁琐,限制了该技术在育种中的大规模应用等。     

Assessment of genetic diversity among selected groundnut germplasm. I: RAPD analysis

Assessment of genetic diversity in a crop species is prerequisite to its improvement. The use of germplasm with distinct DNA profiles will help to generate genetically diversified breeding populations. The aims of the present experiment were to study molecular diversity among selected groundnut accessions and identify those with distinct DNA profiles for mapping and genetic enhancement. Twenty‐six accessions and eight primers of a 10‐mer were selected for random amplified polymorphic DNA assay. The genetic similarity (S_ij) ranged from 59.0% to 98.8%, with an average of 86.2%. Both multidimensional scaling and unweighted pair‐group method with arithmetic averages (UPGMA) dendrograms revealed the existence of five distinct clusters. However, this classification could not be related to known biological information about the accessions falling into different clusters. Some accessions with diverse DNA profiles (ICG 1448, 7101, and 1471, and ICGV 99006 and 99014) were identified for mapping and genetic enhancement in groundnut.     

玉米CMS-S小孢子败育过程中的细胞程序性死亡

以玉米（Zea mays L.）S型细胞质雄性不育系(CMS-S)的一对近等基因系S-Mo17^Rf3Rf3 和S-Mo17^rf3rf3为材料，采用TdT介导的dUTP DNA末端标记（TUNEL）、细胞色素C免疫原位杂交和DNA寡聚核小体片段电泳等方法，分别在细胞学水平和DNA水平上研究了玉米CMS-S小孢子败育的细胞程序性死亡（PCD）过程。结果表明，在花粉母细胞减数分裂后的四分体解离时期，不育花药的绒粘层细胞较可育花药提前裂解；在不育系S-Mo17^rf3rf3花药和花粉S-rf3中均明显出现PCD过程的DNA片段化以及线粒体细胞色素C外渗的现象，证明了玉米CMS-S的花粉败育与花药绒粘层细胞的提前凋亡和小孢子细胞的程序性死亡有关。     

甘蓝型油菜Pol CMS育性恢复基因的RAPD标记

采用恢、 保回交群体和集团分离分析(BSA), 筛选了860个10 mer随机引物, 找到了与甘蓝型油菜波里马细胞质雄性不育系(Pol cms)育性恢复基因(Rf)连锁的两个RAPD标记AH19690和AI16830。 它们位于Rf的一侧,与该基因的遗传图距分别为5.9 cM和13.6 cM,两标记间的遗传图距为7.8 cM。这两个RAPD标记的发掘,为进一步利用分子标记     

Induced mutagenesis in Jatropha curcas L. using ethyl methanesulphonate (EMS) and assessment of DNA polymorphism through RAPD markers

Ethyl methanesulphonate (EMS) has been employed in a number of genotoxic studies in plants as a model alkylated agent that readily reacts with DNA-producing alkylated nucleotides. Therefore, the present study was aimed at assessing DNA polymorphism induced by different concentrations (control, 1, 2, 3, and 4%) of EMS through a Randomly Amplified Polymorphic DNA (RAPD) marker analysis. The improved agronomic traits such as germination, flowering, maturity, seed traits, and oil content were recorded in 1% EMS-treated plants, while the corresponding parameters reduced significantly (P > 0.05) in 4% EMS-treated plants as compared to the control. Among 25 random primers used, 19 primers produced polymorphic bands. The number of amplicons varied from 1 to 8 with an average of 3.68 bands, of which 2.12 were polymorphic. The highest polymorphic bands (6) and percentage of polymorphism (85.71) were produced by the primer OPAK-20. In a dendrogram constructed based on Jaccard’s coefficient similarity, the treated plants and control were grouped into three clusters: (a) control and 2 and 3% concentrations of EMS-treated plants merged together; (b) 1% concentration of EMS-treated plants clustered alone; (c) 4%concentration of EMS-treated plants also clustered alone. We conclude that the effect of EMS could change the pattern of germination, flowering, seed yield, and oil content of J. curcas. DNA polymorphism detected by RAPD marker analysis offered a useful biomarker assay for the evaluation of effects of chemical mutagens.     

苏丹草RAPD反应条件的优化与应用

为研究不同因素对苏丹草随机扩增多态性DNA（RAPD）反应体系的影响，建立并优化反应体系；再利用优化体系绘制了DNA指纹图谱以区分两个高丹草品种。提取苏丹草的基因组DNA，分别设置Mg2+浓度为1.0 mmol/L、1.5 mmol/L、2.0 mmol/L、2.5 mmol/L、3.0 mmol/L；退火温度为34℃、35℃、36℃、37℃、38℃；Taq酶含量为0.6U、0.8U、1.0U、1.2U、1.4U进行PCR扩增。结果表明：Mg2+2.0mmol/L，退火温度36度，Tap酶活性值1.2U是进行苏丹草RAPD分析的最优反应条件。利用最优反应条件绘制了皖草2号和皖草3号的DNA指纹图谱，在该图谱中有A、B两条特征带，可以区分皖草2号和皖草3号。     

Novel approach for DNA sequence preprocessing with intelligent detection

A novel approach for DNA sequence preprocessing by merging intelligent detection is proposed. This approach can automatically find and locate contaminants using statistical analysis methods, random search and graph-theoretic operations, while no extra background information, such as vector sequence, splice site and clone adapter are needed during preprocessing. Experiments on Zebrafish DNA show that the approach can significantly improve the efficiency and accuracy of DNA sequence preprocessing and provide more stable performance than the conventional methods do, particularly in high-throughput DNA sequence preprocessing.     

棉花纤维伸长发育期的基因表达分析

利用cDNA芯片技术分析陆地棉显性单基因突变的李氏超短纤维突变体（Li₁li₁）和其野生型（li₁li₁）纤维发育4 DPA（day post anthesis）的基因差异表达情况。RT-PCR验证4DPA芯片结果，得到15个差异表达的EST（expressed sequence tag），其中9个下调表达，即野     

花粉介导法获得油菜转基因植株研究

以甘蓝型冬油菜品种晋油7号为受体，载有GUS基因的质粒pBI121为供体，在7.5％的蔗糖等渗溶液中，通过花粉介导转化方法将GUS基因导入油菜花粉，随着花粉管的萌发进入胚囊，参与有丝分裂，将外源质粒转入受体材料。经田间植株性状比较、GUS组织化学定位检测、PCR扩增检测及PCR-Southern杂交检测，证明外源GUS基因已整合到油菜基因组中。     

LAMP检测无乳链球菌方法的建立及应用

以无乳链球菌纤连蛋白fbs基因为主要研究对象,采取环介导等温扩增技术(Loop-Mediated Isothermal Amplification,LAMP),针对fbs基因的6个区域设计4条特异性引物,利用一种链置换DNA聚合酶(Bst DNA polymerase)在63℃保温1 h,通过荧光显色即可完成对无乳链球菌的检测工作.结果显示,LAMP方法能够特异性检测fbs基因,其检测灵敏度是常规PCR方法的100倍,并与实时荧光定量PCR方法相当.所建立的针对无乳链球菌fbs基因的LAMP检测方法具有高度的特异性及稳定性,结果可靠,非常适合无乳链球菌的快速检测.     

刺五加成熟种子的愈伤组织诱导及其RAPD分析

以刺五加的成熟种子为材料，通过离体培养诱导产生愈伤组织，利用RAPD分子标记方法，在DNA水平上分析无菌苗和愈伤组织的遗传变异。10个10碱基引物中筛选出4个引物，对它们的PCR扩增结果进行了分析。结果表明刺五加愈伤组织与无菌苗相比出现了DNA水平的变异。     

Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties

Summary In this study, two PCR-based methodologies were evaluated for potential use in the determination of DNA diversity between 20 commercial sugarcane hybrids and 6 outgroup varieties of S. spontaneum, S. officinarum and hybrids from early in the genealogy. The first method involved PCR amplification of sugarcane DNA in the presence of random, decamer primers (RAPDs), while the second protocol utilized specific microsatellite and telomere sequences as primers. A total of 41 RAPD primers (356 loci) were screened across the varieties of which 15 (160 loci) were used in the calculation of DNA diversity (expressed% similarity). This varied from 61 to 95%, with most of the commercial varieties showing more than 80% similarity in their DNA. The RAPD data indicated that there had been a gradual decline in DNA diversity (84% reduction) from the early inter-specific crosses to the commercial hybrids, probably as a result of backcrossing and in-breeding strategies used in the previous 5 to 6 generations of sugarcane breeding. The microsatellite and telomere data produced a much greater range in DNA similarity values (25–91%), probably due to the fact that these primers detect highly variable regions of the genome. It is suggested that these specific primers would not be suitable for determination of DNA diversity, but could be used more effectively in the development of a methodology for routine, rapid identification of sugarcane varieties.     

Monitoring genetic fidelity vs somaclonal variation in Norway spruce (Picea abies) somatic embryogenesis by RAPD analysis

Summary Somaclonal variation, which is a welcome source of genetic variation for crop breeding, is unwanted when direct regenerants have to be used in tissue culture mass propagation (eg. in many forest trees), or in the regeneration of genetically transformed plants. Random amplified polymorphic DNA (RAPD) was used to analyse somatic embryos and plants regenerated from embryogenic cell lines in Norway spruce, Picea abies (L.) Karst. RAPD facilitated the identification of clones, as material from the same cell lines shared identical patterns of amplified fragments, whereas regenerants from different cell lines were easily distinguishable by their respective patterns. For comparisons with explant donor genotypes, cell lines were initiated from cotyledons. Some of the seedlings that had parts of their cotyledons removed were grown on as control plants. Somatic embryos regenerated from cotyledon cell lines showed no aberrations in RAPD banding patterns with respect to donor plants. We conclude that gross somaclonal variation is absent in our plant regeneration system.Abbreviations ESM embryogenic suspensor mass - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - (2,4-dichlorophenoxy)acetic acid 2,4-D - 1-naphthaleneacetic acid NAA     

遗传标记及其在园艺植物研究中的应用

评述了形态学标记（morphological markers）、细胞学标记(cytological markers)、生化标记(biochemical markers)、DNA分子标记(DNA molecular markers)等技术的发展现状,着重介绍了近年来DNA指纹图谱技术,即限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)、随机扩增片段长度多态性 (Random Amplified Polymophismic DNA,RAPD)、简单重复序列 (simple sequence repeats,SSR)、扩增片段长度多态性(Amplified Fragment Length Polymorphism,AFLP)、单核苷酸多态性 (single nucleotide polymorphism,SNP) 等标记技术的特点及其在园艺植物品种的分类鉴定、纯度的分析检测及品种间亲缘关系等研究中的应用。     

甜菜细胞质雄性不育系及保持系叶绿体DNA的RAPD分析

对细胞质雄性不育甜菜的不育系及其保持系的叶绿体DNA(ctDNA)进行了RAPD分析,结果表明：在所选的70个引物中,甜菜细胞质雄性不育系和保持系叶绿体DNA之间不存在差异性。与此同时,对叶绿体DNA的提取方法进行了改进,在不需密度梯度离心条件下即可获得高纯度的DNA;优化了甜菜叶绿体的RAPD的反应条件。     

A genetic map of macadamia based on randomly amplified DNA fingerprinting (RAF) markers

The first genetic linkage map of macadamia (Macadamia integrifolia and M. tetraphylla) is presented. The map is based on 56 F₁ progeny of cultivars ‘Keauhou’ and ‘A16’. Eighty-four percent of the 382 markers analysed segregated as Mendelian loci. The two-way pseudo-testcross mapping strategy allowed construction of separate parental cultivar maps. Ninety bridging loci enabled merging of these maps to produce a detailed genetic map of macadamia, 1100 cm in length and spanning 70–80% of the genome. The combined map comprised 24 linkage groups with 265 framework markers: 259 markers from randomly amplified DNA fingerprinting (RAF), five random amplified polymorphic DNA (RAPD), and one sequence-tagged microsatellite site (STMS). The RAF marker system unexpectedly revealed 16 codominant markers, one of them a putative microsatellite locus and exhibiting four distinct alleles in the cross. This molecular study is the most comprehensive examination to date of genetic loci of macadamia, and is a major step towards developing marker-assisted selection for this crop. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular markers and their applications in wheat breeding

In recent years, considerable emphasis has been placed on the development of molecular markers to be used for a variety of objectives. This review attempts to give an account of different molecular markers—restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), sequence-tagged sites (STS), DNA amplification fingerprinting (DAF), amplified fragment length polymorphisms (AFLPs) and microsatellites (STMS)—currently available for genome mapping and for tagging different traits in wheat. Other markers, including microsatellite-primed polymerase chain reaction (MP-PCR), expressed sequence tags (ESTs) and single nucleotide polymorphisms (SNPs) are also discussed. Recent information on synteny in cereal genomes, marker-assisted selection, marker validation and their relevance to cereal breeding in general and wheat breeding in particular are also examined.     

Molecular characterization of taro (Colocasia esculenta) using RAPD markers

Forty-four taro (Colocasia esculenta), two tanier (Xanthosoma species) and one Colocasia gigantea accessions were evaluated for genetic diversity using random amplified polymorphic DNA (RAPD) primers. Seventy-three of 112 primers amplified PCR DNA products used to fingerprint the accessions. Thirty-two primers were considered highly informative because they amplified more than 5 bands or amplified one or more polymorphic bands that distinguished between accessions. RAPDs showed high genetic diversity in taro accessions from Indonesia, were capable in distinguishing between Hawaiian accessions, and could separate triploid from diploid accessions. UPGMA cluster analysis of genetic similarity estimates (Jaccard's coefficient), separated the accessions into 3 main groups with C. esculenta divided into 5 subgroups. These primers will be useful for future genetic analysis and provide taro breeders with a genetic basis for selection of parents for crop improvement. Polymorphic markers identified in the DNA fingerprinting study will be useful to screen a segregating population which is being generated in our laboratory aimed at developing a taro genetic linkage map. This revised version was published online in July 2006 with corrections to the Cover Date.