向日葵品种鉴定和纯度分析的AFLP研究

从杂交油葵A15及其亲本的1/2粒干种子中提取基因组DNA,选用17对引物组合进行AFLP分析,构建了它们的指纹图谱,17对引物在A系与R系当中共扩增出1125条扩增产物,其中144条带表现出多态性,平均每对引物扩增66条带。不同引物组合产生的DNA片段数目在50-70之间,大小分布于100bp-500bp,多态性比率为12.8%。从中筛选出的2对引物E-AAC/M-CTC和E-ACG/M-CTG可将亲本和子代区分开;引物对E-AAC/M-CTC在A系中扩增出440bp,190bp,160bp3条特征谱带,在R系中扩增出380bp,350bp,225bp,180bp4条特征谱带,E-ACG/M-CTG在A系中扩增出了2条特征带480bp和265bp,在R系中扩增出490bp,220bp,205bp,125bp4条特征谱带,且上述谱带均在子代中出现,用引物组合E-ACG/M-CTG对A15,双亲以及与A15外型十分相似的10个常用油葵杂交种进行AFLP分析,不仅表现出良好的多态性,并能够清楚地将它们加以区分,以其对50粒A15杂交种子进行纯度鉴定,得到与大田纯度检测一致的结果。说明使用AFLP标记检测油用向日葵的品种和纯度是可行的,对行种子纯度和品种鉴定的方法进行了讨论。     

向日葵品种鉴定和纯度分析的AFLP研究

从杂交油葵A15及其亲本的1/2粒干种子中提取基因组DNA,选用17对引物组合进 行AFLP分析,构建了它们的指纹图谱。17对引物在A系与R系当中共扩增出1125条扩增产物,其中144条带表现出多态性,平均每对引物扩增66条带,不同引物组合产生的DNA片段数目在50～70之间,大小分布于100bp～500bp,多态性比率为12.8%。从中筛选出的2对引物E_AAC/M_CTC和E_ACG/M_CTG可将亲本和子代区分开：引物对E_AAC/M_CTC在A系中扩增出440bp、190bp、160bp 3条特征谱带,在R系中扩增出380bp、350bp、225bp、180bp 4条特征谱带,E_ACG/M_CTG在A系中扩增出了2条特征带480bp和265bp,在R系中扩增出490bp、220bp、205bp、125bp 4条特征谱带,且上述谱带均在子代中出现。用引物组合E_ACG/M_CTG对A15、双亲以及与A15外型十分相似的10个常用油葵杂交种进行AFLP分析,不仅表现出良好的多态性,并能够清楚地将它们加以区分。以其对50粒A15杂交种子进行纯度鉴定,得到与大田纯度检测一致的结果,说明使用AFLP标记检测油用向日葵的品种和纯度是可行的。对现行种子纯度和品种鉴定的方法进行了讨论。     

大白菜杂交种''冠春''杂交率的RAPD分析

从春大白菜品种‘冠春’及其亲本中提取基因组DNA,用320个随机引物进行RAPD扩增,从中筛选出5 个可将亲本和子代区分的引物S4、S47、S73、S134和S194。S4产生父本特征带S4-370;S47和S134产生母本特征带S47-700 和S134-1200;S73和S494产生亲本互补的特征带S73-660、S73-730和S494-400、S494-1770,上述谱带均在子代中出现。以这5个引物产生的特征谱带建立杂交种‘冠春’及其亲本的RAPD特异指纹。通过对134个‘冠春’的种子进行纯度鉴定,结果表明2个父本和4个母本与大田检测结果完全一致。进一步验证了4种鉴定大白菜杂交种方法的可行性。     

基于SSR标记的海带、裙带菜的遗传构成分析

旨在通过对配子体克隆和孢子体幼苗的初代筛选,以培育出纯度更高、性状更优良的海带与裙带菜品种。以11个海带与裙带菜克隆组合为材料,在筛选出区分亲本♀与♂的数对引物的基础上,对其子代与亲本的遗传构成进行了分析。试验中共选出13对能够区分11个组合亲本♀与亲本♂的SSR标记,分析结果显示,2013年育苗的48号组合中所有子代都是其亲本的后代,49号组合中只有1个子代是其亲本的后代;2014年育苗的22号组合中所有子代都是其亲本的后代;2015年育苗的海带与裙带菜杂交组合中,36号、38号、39号与41号杂交成功,其他组合未杂交成功;2016年育苗的59号组合中有7个子代与亲本无亲缘关系。分析这11个组合中所有个体与亲本的遗传构成发现:部分个体是其亲本的后代,部分个体遗传构成与亲本不一致。     

甘蓝型油菜秦优10号杂交种纯度鉴定的SSR引物筛选

为了建立一套快速可靠的油菜杂交种纯度的鉴定方法,本文以秦优10号及其亲本、杂种ZZH2为试验材料,对前人开发的2对SSR引物(7号引物, 9号引物)进行了再次筛选.结果显示,7号引物不但能很好地区别出混入杂交种中的亲本,还能将杂交种子的同母异父组合种子从杂交种中分离出来,能够用于鉴别秦优10号杂交种子的真伪;9号引物能区分出杂交种和母本,但不能区分开杂交种和父本.同时,本试验利用人工制成的秦优10号杂交种标准样(纯度为100%)以及7份大田鉴定不同纯度梯度的杂交种子对7号引物鉴定结果的准确性进行了验证,鉴定结果与大田鉴定结果基本一致.本文结果将为鉴定秦优10号杂交种纯度提供更准确的技术资料.     

RAPD法检测番茄种子纯度的研究

以番茄F1代杂交品种“强选1号”的干种子为材料,用RAPD方法对种子的纯度进行了初步的鉴定。所用RAPD引物是从110个RAPD随机引物中筛选出的BA1072号引物。使用这种方法不需进行田间种植即可完成F1代要种的纯度鉴定工作。     

冬枣×宁梨巨枣的子代SRAP鉴定及遗传多样性分析

应用SRAP分子标记方法对冬枣×宁梨巨枣的子代进行了分子鉴定及遗传多样性分析。采用构建基因池的方法对SRAP分子标记引物进行筛选,从88对引物中筛选出15对多态性好、主带清晰的引物,并对子代进行了真实性鉴定及多态性分析。结果表明:(1)15对引物共产生95个多态性条带,平均每对引物产生6.3个多态性条带,显示了较高的多态性比率。(2)80个子代中44个具有父本特征带,鉴定为真杂种。子代遗传多样性及UPGMA聚类分析表明,子代个体与亲本间的遗传相似系数在0.55~0.98之间,个体差异明显。该研究结果为枣树杂交育种提供了重要的分子证据。     

杂交小麦''''西杂一号''''种子纯度鉴定的研究

以杂种小麦新品种‘西杂一号＇及其亲本为试验材料,利用A-PAGE和RAPD技术,对其进行了研究与分析.A-PAGE分析表明,亲本西农Fp-1和西农Mp-1有4条醇溶蛋白差异带,并在‘西杂一号＇中表现出3对互补条带.从128个具有多态性随机引物中筛选出1个扩增带稳定、清晰且为双亲互补型的特征引物.并将这两种方法对‘西杂一号＇种子纯度鉴定的结果与田间鉴定相比较,结果表明两种方法都可以用于杂种小麦杂交种及其亲本种子纯度的鉴定.     

杂交小麦‘西杂一号’种子纯度鉴定的研究

以杂种小麦新品种‘西杂一号’及其亲本为试验材料,利用A-PAGE和RAPD技术,对其进行了研究与分析.A-PAGE分析表明,亲本西农Fp-1和西农Mp-1有4条醇溶蛋白差异带,并在‘西杂一号’中表现出3对互补条带.从128个具有多态性随机引物中筛选出1个扩增带稳定、清晰且为双亲互补型的特征引物.并将这两种方法对‘西杂一号’种子纯度鉴定的结果与田间鉴定相比较,结果表明两种方法都可以用于杂种小麦杂交种及其亲本种子纯度的鉴定.     

杂交油菜种子纯度的RAPD检测研究

在简化油菜DNA提取方法的基础上,建立了适用于杂交油菜种子纯度检测的RAPD技术分析系统,从814条随机引物中筛选获得了2条可同时稳定区分CMS杂交油菜品种秦优7号及其亲本的引物,经过大量的不同来源油菜个体的验证,证明了该方法具有很好的重复性,准确性和可靠性,与酯酶同工酶法比较,发现利用RAPD技术能够检测酯酶同工酶法难以鉴别的杂交种及其亲本系,因此,选择合适引物,建立良好的反应体系,简化DNA提取程序,降低分析成本等,可使RAPD技术实用化。     

曲霉菌的RAPD分析及其在酿造工业中的应用

以米曲霉沪酿3.042(AS3.951)、黄曲霉GIM3.18、酱油曲霉AS3.495为参照,利用RAPD分子标记技术对16株曲霉菌进行系统发育分析。通过改进提取方法,获得了质量较好的模板DNA,凝胶电泳结果和分光光度法检测结果表明其适合用于进一步的RAPD-PCR试验。从9个待选引物中筛选到3个扩增产物谱带多、特征好、覆盖面广的引物:Primer1、Primer2、Primer5,重复实验证明其RAPD-PCR扩增图谱具有较好的稳定性,扩增产物谱带一般8~14条,各试验菌株主带4~9条,次带丰富。由此构建的系统进化树较好地吻合了传统的形态分类学,证实了RAPD分子标记技术在此类微生物系统发育分析中应用的可行性,也为酿造工业中检出产黄曲霉毒素的污染菌株提供了理论基础。     

Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens

Summary The identification of somatic hybrids between Solanum tuberosum and S. brevidens can be carried out using polymerase chain reaction (PCR) and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. Five commercial primers have been tested. Each primer directed the amplification of a genome-specific fingerprint for the fusion parents and S. brevidens. The size of the amplified DNA fragments ranged from 100 to 1800 base pairs. The somatic hybrids showed a combination of the parental banding profiles with four of the five primers surveyed, whereas regenerants from one of the parents had the same or a similar banding pattern to that of the parent. Thus RAPD markers provide a quick, simple and preliminary screening method for putative somatic hybrids.Abbreviations EDTA ethylenediaminetetraacetic acid, - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphisms - TBE Tris-borate-EDTA buffer - Tris trizma base     

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.     

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。     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

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。