两个地区东方田鼠基因组RAPD分析比较研究

目的　从DNA的水平分析比较两个地区东方田鼠的分子遗传特征,探讨以RAPD标记鉴别两个地区的东方田鼠。方法　筛选6条10bp的随机引物对洞庭湖和青铜峡地区的东方田鼠基因组进行了随机扩增多态DNA(RAPD)分析,并对这两个地区的东方田鼠的基因组DNA进行了比较。结果　①两个地区东方田鼠的所有受试个体中共有的片段数为20条,这是两个地区东方田鼠的共性所在;②两个地区东方田鼠各有其特异性扩增片段;③引物S17和S80可作为鉴别两个地区东方田鼠的特异性引物;④不同地区的东方田鼠其不同个体之间的共享度较低,且存在较大差异;两个地区东方田鼠的遗传背景均呈非均一性。结论　运用RAPD方法可以作为鉴别不同地区东方田鼠的基因多态性的标记。     

东方田鼠特异DNA片段的克隆及核苷酸序列分析

目的获得东方田鼠的特异DNA序列.方法Aβ基因使用PCR,基因克隆,斑点杂交,DNA序列分析,生物信息学技术.结果根据小鼠MHCⅡ外显子2及其两侧序列,合成引物并扩增东方田鼠基因组DNA,将PCR产物回收、测序后,分别设计内引物扩增东方田鼠基因组DNA,其中一对引物可得到特异性扩增带,将得到的DNA片段插入PGEM-Teasy载体,进行序列分析.用这对引物扩增人、昆明小鼠、BALB/c小鼠及C57BL/6J小鼠基因组DNA,均无扩增产物.以东方田鼠特异性扩增产物为探针进行斑点杂交,除东方田鼠基因组DNA外,其他几种动物基因组DNA均为阴性结果.进一步对该DNA片段进行了BLAST同源性搜索和外显子预测,在Genbank中没有发现高度同源序列,并且找到一个可能的外显子,该外显子由69个氨基酸组成.结论获得的DNA片段为东方田鼠的特异片段,这将为从分子水平深入研究东方田鼠的遗传背景、生物进化规律以及东方田鼠抗日本血吸虫的机理奠定基础.     

湖南等三地区东方田鼠遗传特性的分析比较

用染色体G带核型分析、生化位点、随机扩增多态性DNA(RAPD)标记等方法,对湖南洞庭湖湖滨、宁夏青铜峡市农田和黑龙江伊春市金山屯草甸3个地区的东方田鼠遗传特性进行了分析。结果表明,湖南和宁夏地区东方田鼠染色体数均为2N＝52,黑龙江东方田鼠染色体数则为2N＝42;生化位点结果显示3个地区的东方田鼠均呈遗传非均一性;湖南、宁夏和黑龙江鼠的个体间RAPD遗传距离分别为0．244（0．143—0．353)、0．226(0．161—0．294)、0．541(0.357—0．692)。湖南和宁夏两地区鼠种群间的遗传距离为0．367,湖南和宁夏鼠杂交一代与其亲代的RAPD遗传距离在0．310以下;但黑龙江鼠与湖南和宁夏鼠种群问的遗传距离分别为0．619和0．633。总的表明,3个地区的东方田鼠均呈遗传非均一性,但湖南与宁夏鼠在染色体、生化位点和RAPD标记等方面都具有相似性,并可杂交,而黑龙江鼠与其它两地的鼠不能杂交,且黑龙江鼠在遗传特性方面与前二地鼠有很大差异,因而后的“种”级分类地位值得进一步研究。     

用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD方法扩增出的高变异产物含有重复序列.用含重复序列的个体特异性RAPD产物作探针,与无关个体鸡基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变异性较高的DNA指纹图谱.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

高产王浆西蜂DNA分子中的相关基因标志筛选及其鉴定

为了探讨西蜂与高产王浆相关特异基因标记 ,用 12种随机引物 (P1～P12 )对产王浆量不同的4品系西蜂的基因组DNA进行了RAPD PCR分析 ,分别获得了产王浆量高、低不同西蜂的DNA多态性图谱 ,并从P2 引物的DNA多态性图谱中筛选出一差异DNA片段P2 316bp .将P2 316bp差异DNA片段用地高辛标记制备成探针 ,进行Southern杂交鉴定 .实验显示 ,探针与高产王浆西蜂基因组DNA的扩增产物出现了阳性杂交信号 ,而与低产王浆西蜂基因组DNA的扩增产物未出现阳性杂交信号 .结果表明 ,该差异性基因片段P2 316bp是西蜂高产王浆优良性状相关的遗传标记 ,序列为 30 5个核苷酸 .     

白鲢和鳙鱼的随机扩增多态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提取基因组DNA.以此法获得的白鲢和鳙鱼的基因组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.讨论了RAPD遗传分子标记在鱼类遗传,特别是遗传多样性研究,和鱼类种质资源评估和管理中的应用前景问题.     

洞庭湖日本血吸虫疫区东方田鼠生化与分子遗传学标记的初步研究

目的建立东方田鼠生化与分子遗传学标记检测法.方法参考近交系小鼠、大鼠生化遗传标记检测方法,对东方田鼠某些同功酶进行活性测定.根据东方田鼠特异DNA序列,合成引物,扩增东方田鼠基因组DNA,将PCR产物回收、序列分析并进行生物信息学分析.结果东方田鼠Trf、Es-3和Ce-2三个生化位点呈现遗传多态性,而Id1、Mod-1、Car-2、Gpd-1、Gpi-1、Hbb、Es-1七个生化位点无遗传多态性.用合成的特异引物扩增东方田鼠基因组DNA,得到了丰富的多态性资料.对其中的20只东方田鼠的扩增产物进行测序并进行多序列比较,发现10个等位基因以及16个单核苷酸多态性(SNP)位点.结论初步建立了东方田鼠生化及分子遗传学标记.分子遗传学标记与生化遗传标记相比,具有杂合率高、重复性好、易于操作等优点.这些结果为深入研究东方田鼠的遗传背景、生物进化规律积累了基础资料.     

东方田鼠长江亚种和指名亚种基因组DNA序列比较分析

根据东方田鼠基因组DNA序列片段(CenBank登录号：AF277394),通过PCR方法扩增东方田鼠长江亚种(Microtus fortis calamorum)和宁夏指名亚种(Microtus fortis fortis)基因组DNA,得到-670bp左右的特异扩增片段。将PCR扩增产物克隆到pGEM-Teasy裁体,进行DNA序列分析,并用生物信息学方法比较东方田鼠长江亚种与指名亚种之间该序列的差异。结果表明：在东方田鼠两个亚种中共发现19个不同的等位基因,不同的个体在该序列存在广泛的单个核苷酸多态性(SNP),多态性位点多达25个;变换类型包括：转换(G→A、A→G、T→C、C→T)、颠换(G→T、A→T、T→A、C→A)、插入(CA)和缺失(TGTTTT)。东方田鼠长江亚种和指名亚种两个种群之间存在明显差别,尤其是在146、192、223、224、235位,但两种群间同源性仍高达98％。同时采用系统发育树(phylogenetic tree)分析方法,对两个亚种的亲缘关系进行了分析和比较,结果显示,东方田鼠长江亚种和宁夏指名亚种基因组DNA明显的分为两大组别。     

布氏田鼠封闭群遗传结构的随机扩增多态DNA分析

目的使用随机扩增多态DNA标记建立标准化的布氏田鼠封闭群遗传质量控制分子标记库。方法使用高盐沉淀法从鼠尾中提取布氏田鼠基因组DNA。采用40条PRAD引物对布氏田鼠封闭群进行PCR扩增,琼脂糖电泳分离条带,参考标准分子量标记计算条带大小,并使用多态位点数、单态位点数以及多态位点比率评价种群的遗传多样性。结果筛选出8个能获得清晰稳定扩增条带的RAPD标记。这8个RAPD标记检测到的多态位点数存在明显差异。8个引物得到的遗传多态位点的数据之和能揭示种群的遗传结构。结论本实验建立了检测布氏田鼠封闭群遗传结构的RAPD标记。     

玉米花粉粒直接PCR技术研究

利用成熟花粉粒制成悬液作为玉米基因组DNA模板直接进行PCR扩散,研究了不同花粉悬液浓度、花粉上清液对RAPD-PCR的影响,结果表明：花粉悬液浓度在4μg/50μl以上,用10碱基随机引物均能扩增出较清晰的PCR条带,且与叶片按改良Guidet法提取的DNA模板扩增的RAPD带型无明显差异,利用花粉悬液能有效地进行基因组DNA变异的RAPD分析和基因SCAR标记的检测。玉米单株花粉量可用于数百次以上的PCR反应,较叶片等植物组织用常规法提取DNA快速、简便、廉价,可有效地应用于以PCR为基础的植物基因组DNA变异,农艺性状的RAPD标记和分子标记辅助育种的研究。     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD及SRAP两种分子标记技术对苦瓜杂交种纯度的鉴定分析

以F1代苦瓜杂交种如玉11号及其亲本为材料,利用RAPD及SRAP两种分子标记技术对这3种苦瓜基因组DNA进行比较分析,以获得该杂交种及其亲本（或母本）差异目的基因片段。经过多次对该3种苦瓜叶片DNA提取,PCR扩增及其PCR产物的琼脂糖凝胶电泳分析,在供试的46个RAPD引物及121对SRAP引物中,筛选出1个RAPD引物及1对SRAP引物能区分该苦瓜杂交种及其母本种子,通过进一步验证分析,证明该两种分子标记的特异引物可作为如玉11号苦瓜杂交种子的纯度鉴定之用。     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

利用RAPD分子标记对三组三系杂交水稻及亲本的遗传分析和鉴定

利用RAPD技术,从248个随机寡聚核苷酸（10bp）中筛选出13个引物能在供试的三组三系杂交水稻及亲本间扩增出43条稳定性较好的多态性片段,其中6个引物能在供试材料间扩增出20个强的多态性标记。利用这些标记能有效地区分各组合中不育系、保持系、恢复系和F1,并能看出各组合中不育系与保持系、不育系与恢复系、F1与亲本间的遗传关系。 Abstract:A total of 248 arbitrary 10-mer oligonucleotide primers were screened using RAPD (random amplified polymorphic DNA) techniques with the genome DNA of three groups of three-line hybrid rice and their parents.Thirteen primers produced 43 polymorphism fragments.Six primers of them produced 20 obviously repeatable polymorphic markers among rice lines tested.Using this RAPD markers,the hybrid rice combinations (sterile-line,maintainer-line,restorer-line and F1)can be effectively identified,and the genetic relationship among them can be shown.     

Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa.

Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

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.     

Identification of broccoli and cauliflower cultivars with RAPD markers

Summary RAPD (Random Amplified Polymorphic DNA) markers generated by 4 arbitrary 10-mer primers, discriminated 14 broccoli and 12 cauliflower cultivars (Brassica oleracea L.) by banding profiles. The size of the amplified DNA fragments ranged from 300 to 2600 base pairs. Twenty-eight percent of the markers were fixed in both broccoli and cauliflower, whereas 12.5% were specific to either crop. The rest were polymorphic in either or both crops. The markers generated by two and three primers were sufficient to distinguish each of the broccoli and cauliflower cultivars, respectively. The average difference in markers was 14.5 between broccoli and cauliflower markers, 5.8 between two broccoli cultivars and 7.9 between two cauliflower cultivars. Larger differences for each crop were found between cultivars from different seed companies than within the same company. RAPD markers provide a quick and reliable alternative to identify broccoli and cauliflower cultivars.