兴安、长白及华北落叶松RAPD分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松（Larix gmelini）、长白落叶松（L.olgensis）和华北落叶松（L．principis—rupprechtii）的针叶及种子胚乳为研究材料,采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明,通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物,其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1500bp处扩增出特异条带,而在华北落叶松中没有：引物OPX-14在兴安落叶松基因组DNA中1200bp处扩增出特异条带,而在长白落叶松中没有：还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

Second meiotic division and polar body formation in mouse eggs fertilized in vitro

The second meiotic division and polar body formation in mouse eggs fertilized in vitro were observed by phase-contrast and polarizing microscopy, and recorded by time-lapse cinematography. Eggs were collected from oviducts of mice that had been superovulated by injections of PMS and HCG. Some eggs, inseminated with spermatozoa that had been collected from caudae epididymides of mature male mice and cultured for two to three hours before insemination, were observed continuously on a glass slide under a phase microscope. Other eggs were inseminated in Petri dishes in a 5% CO₂ incubator and examined every 20 minutes for 180 minutes. Compatible results in both sets of eggs showed that formation of the second polar body began 25–40 minutes after fusion of spermatozoon with the vitellus; it was completed 40–60 minutes later; anaphase II lasted approximately five minutes before the appearance of the furrow abstricting the second polar body. It is suggested that the furrowing associated with second polar body formation is guided by the same kind of forces that divide a cell mitotically.     

Dynamic rearrangement of surface proteins is essential for cytokinesis

Cytokinesis is a complex process that involves dynamic cortical rearrangement. Our recent time-lapse recordings of the mouse egg unexpectedly revealed a high motility of the second polar body (2pb). Experiments to address its underlying mechanism show that neither mechanical compression by the zona pellucida nor the connection via the mid-body is required for the 2pb movement. Time-lapse recordings establish that the 2pb moves together with the cell membrane. These recordings, in which cell surface proteins are labeled with fluorescent latex-microbeads or monovalent antibodies against whole mouse proteins, indicate that the majority of the surface proteins dynamically accumulate in the cleavage furrow at every cell division. Comparable dynamics of the cell surface proteins, and specifically of E-cadherin, are also observed in cultured epithelial cells. The surface protein dynamics are closely correlated with, and dependent on, those of the underlying cortical actin. The cortical actin network may form a scaffold for membrane proteins and thereby transfer them during contractile ring formation toward the cleavage furrow. Immobilization of surface proteins by tetravalent lectin-mediated crosslinking results in the failure of cleavage, demonstrating that the observed protein dynamics are essential for cytokinesis. We propose that dynamic rearrangement of the cell surface proteins is a common feature of cytokinesis, playing a key role in modifying the mechanical properties of the cell membrane during cortical ingression.     

植物RAPD标记的可靠性研究

随着ＲＡＰＤ技术应用范围越来越广,ＲＡＰＤ的优缺点也不断反映出来。相左实验结果的出现,对ＲＡＰＤ标记可靠性提出了置疑。本文从重复性、序列同源性和系统学价值三个方面,就ＲＡＰＤ标记的可靠性研究的现状进行了综述。并就怎样提高ＲＡＰＤ标记的可靠性进行了探讨     

Genetic Diversity and Molecular Markers of Cupped Oysters (Genera Crassostrea, Saccostrea, and Striostrea) in Thailand Revealed by Randomly Amplified Polymorphic DNA Analysis

Genetic diversity and species-diagnostic markers of 5 oysters in Thailand, Crassostrea belcheri (Sowerby, 1871), Crassostrea iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), Saccostrea forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819), were investigated by randomly amplified polymorphic DNA (RAPD) analysis. In a total, 135, 127, and 108 genotypes were observed from primers OPA09, OPB01, and OPB08 (Operon Technologies Inc., kits A and B), and 131 and 122 genotypes from primers UBC210 and UBC220 (University of British Columbia), respectively. Two hundred fifty-four reproducible and polymorphic fragments (200–2500 bp in length) were generated across the 5 investigated species. The average number of bands per primer varied between 12.4 and 32.2. The percentage of polymorphic bands within Crassostrea (53.23%–77.67%) was lower than that within Saccostrea and Striostrea oysters (86.21%–99.36%). Nine, species-specific markers were found in C. belcheri, 4 in C. iredalei, and 2 in S. cucullata. The mean of a ratio between the number of genotypes generated by each primer and the number of investigated specimens of C. belcheri (0.58) was lower than that of the remaining species (0.90–1.00). Genetic distances between pairs of oyster samples were between 0.105 and 0.811. A neighbor-joining tree indicated distant relationships between Crassostrea and Saccostrea oysters, but closer relationships were observed between the latter and Striostrea mytiloides. Received June 6, 2000; accepted September 12, 2000     

Development of Species-Specific Markers of the Tropical Oyster (Crassostrea belcheri) in Thailand

Randomly amplified polymorphic DNA (RAPD) analysis was used to identify species-specific markers of 5 oyster species in Thailand: Crassostrea belcheri, Crassostrea iredalei, Saccostrea cucullata, Saccostrea forskali, and Striostrea (Parastriostrea) mytiloides. Species-specific markers were found in C. belcheri, C. iredalei, and S. cucullata but not in S. forskali and S. mytiloides. Three C. belcheri–specific RAPD fragments were cloned and sequenced. A primer set was designed from each of the recombinant clones (pPACB1, pPACB2, and pPACB3). The polymerase chain reaction products showed expected sizes of 536, 600, and 500 bp, respectively, with the sensitivity of detection approximately 30 pg of C. belcheri total DNA template. The specificity of pPACB1 was examined against 135 individuals of indigenous oyster species in Thailand and against outgroup references S. commercialis (N= 12) and Perna viridis (N= 12). Results indicated the species-specific nature of primers developed from pPACB1. This primer set can be used for broodstock selection and determination of C. belcheri larvae to assist the selective breeding program for this commercially important species. Received December 8, 1999; accepted March 16, 2000.     

芽孢杆菌TS02特异RAPD标记的SCAR标记转化和验证

利用自主分离的蜡状芽孢杆菌菌株TS02,采用RAPD 方法对TS02及其同源性相近的5株芽孢杆菌(地衣芽孢杆菌、枯草芽孢杆菌、凝结芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌)进行了RAPD条带特异性分析,从TS02基因组中筛选获得了一个533 bp的特异RAPD标记TSR1.TSR1克隆、测序后,根据其序列设计出一对特异引物P1/P2进行扩增,结果只在TS02中扩增得到目的片段,而其余对照菌株扩增为阴性,从而证明试验得到了在种水平上对该菌种进行准确鉴定的特异SCAR标记.     

芽孢杆菌TS02特异RAPD标记的SCAR标记转化和验证

利用自主分离的蜡状芽孢杆菌菌株TS02, 采用RAPD方法对TS02及其同源性相近的5株芽孢杆菌(地衣芽孢杆菌、枯草芽孢杆菌、凝结芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌) 进行了RAPD条带特异性分析, 从TS02基因组中筛选获得了一个533 bp的特异RAPD标记TSR1。TSR1克隆、测序后, 根据其序列设计出一对特异引物P1/P2进行扩增, 结果只在TS02中扩增得到目的片段, 而其余对照菌株扩增为阴性, 从而证明试验得到了在种水平上对该菌种进行准确鉴定的特异SCAR标记。     

18种绣线菊分子身份证的构建和SCAR标记转化

取北方常见的18种绣线菊,用200条RAPD随机引物进行扩增,选出10条引物扩增出的清晰稳定、重复性好的图谱进行数据分析。10条引物共检测出51个位点。对凝胶电泳得到的谱带统计结果并赋值,用IDAnalysis 1.0软件进行数据分析的结果表明,仅需6条引物对应的7个位点可将18种绣线菊完全区分。在RAPD扩增过程中,找到三裂绣线菊的特异标记SL700,并将此标记转化成SCAR标记,这一特异标记在分子水平可将三裂绣线菊与其他17种绣线菊区分开来。     

黑木耳Au185菌株一个SCAR标记的建立

在对60个供试黑木耳菌株RAPD指纹图谱进行分析的基础上获得了黑木耳菌株Au185的RAPD特异性标记,将其克隆、测序,应用Primer5.0软件设计相应的特异引物对SC38-1,将RAPD特异性标记转化为黑木耳菌株Au185的稳定方便的SCAR标记,可快速、准确地鉴定出黑木耳菌株Au185。     

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.     

Conversion of an RAPD marker to an STS marker for barley variety identification

Barley (Hordeum vulgare L.) variety identification is important to the malting and brewing industries. Because many new malting cultivars (varieties) are closely related, new and more effective identification techniques are needed. We report on a series of techniques used to convert an RAPD marker to a more stable STS marker that can identify barley Stander from Robust, an important distinction for the American malting and brewing industries. The techniques included DNA extraction, RAPD amplification, random cloning of all amplified fragments, selection of clones by insert size, DNA sequencing of select inserts, design of a barley-based primer pair, and detection of a single nucleotide polymorphism using restriction endonucleaseAlu I. The barley-based primer pair was used to further sequence the RAPD fragment. Five single nucleotide polymorphisms between Robust and Stander exist, one of which was detected by electrophoresing DNA fragments differentially restricted byAlu I. The conversion technique was different from ones previously reported in that it did not require manual extraction of DNA fragments from a gel. This could be applied to other situations in which RAPD marker conversion would be desirable.     

RAPD分子标记及其在作物遗传育种中的应用

ＲＡＰＤ分子标记是一种新型的分了遗传标记,其原理是采用１０个碱基的随机引物,以基因组ＤＮＡ为模板进行ＰＣＲ随机扩增。其优越性在于其方法简单;分析中的花费少,用时短;分析所需的ＤＮＡ用量也不多等。本文着重介绍以电泳技术和ＰＣＲ扩增技术为核心的分子标记以及在农作物遗传育种中的应用。     

内蒙古四个民族耳垂基因频率

本文在调查内蒙古汉族、蒙古族、回族、朝鲜族耳垂性状的基础上,计算出上述4个民族的基因频率,并进行了4个民族之间、 4个民族与赫哲族、柯尔克孜族之间耳垂显性基因频率的比较, 研究结果提示:我国北方地区民族群体的耳垂显性基因频率由西向东有逐渐降低的趋势。     

异源四倍体鲫鲤雌雄差异的RAPD标记

异源四倍体鲫鲤是从红鲫和湘江野鲤的杂交后代选育出来的,已经形成了一个遗传性状稳定的四倍体鱼新种群。用异源四倍体鲫鲤(雄性)和二倍体白鲫(雌性)生产的三倍体湘云鲫已经在全国推广应用。因此,如果能够了解异源四倍体鲫鲤的性别分化机制,人为地控制异源四倍体鲫鲤的性别分化,生产出大量的超雄鱼,这对于三倍体湘云鲫的产业化生产有重要的意义。刘少军等对异源四倍体鲫鲤的染色体组型进行了分析,并没有发现异源四倍体鲫鲤有明显的特化的性染色体,这说明通过细胞遗传学研究异源四倍体鲫鲤的性别遗传机制是有困难的。     

粘类小麦雄性不育恢复基因的遗传分析及RAPD标记

利用RAPD分子标记对粘类小麦雄性不育系ms（Kots）-90-110的恢复系Rk5451的恢复基因进行了标记定位。选取具有高恢复力的恢复系康本材料Rk5451和Rk5253为父本与ms（Kots）-90-110杂交．F1代再与保持系90-110回交;以90-110／／ms（Kots）-90-110／Rk5451的BC1F1代分离群体为研究对象．利用分离群体分组分析法（Bulked Segregant Analysis．BSA）．以350个随机引物对Rk5451的主效恢复基因进行RAPD分析．筛选到27个可在亲本间扩增出多态性的引物．其中引物S120经多次重复能在亲本间及不育和可育池间扩增出稳定的多态性片段S120-1745。     

Genetic Diversity and Species-Diagnostic Markers of Mud Crabs (Genus Scylla) in Eastern Thailand Determined by RAPD Analysis

Genetic diversity of three mud crab species, Scylla serrata (Forsk?l), S. oceanica (Dana), and S. tranquebarica (Fabricius), collected from two locations in eastern Thailand (Chanthaburi and Trat) was examined by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Ninety-one reproducible RAPD fragments, generated by UBC456, UBC457, and YNZ22, were polymorphic. The percentage of polymorphic bands within populations ranged from 47.92% to 77.59%. Species-specific RAPD markers were also observed and used to construct a molecular diagnostic key in these taxa. Large genetic differences between species were found (D(ij) = 0.425 to 0.751), whereas those between populations within each species were much lower (D(ij) = 0.171 to 0.199). The neighbor-joining tree based on genetic distances among pairs of individuals indicated three distinct groups, corresponding to S. serrata, S. oceanica, and S. tranquebarica. No genotypes were shared among these three species. This suggests the absence of genetic exchanges between sympatric mud crab species in eastern Thailand. Therefore, mud crabs in this area should be recognized as three different species rather than a single panmictic species exhibiting different morphs.