利用AFLP分析西南特色猕猴桃的遗传多样性

为分析西南地区特色猕猴桃(Actinidia Lindl.)的遗传多样性,建立并优化了猕猴桃DNA 多态性分析的AFLP 体系,AFLP 标记体系为300 ng 基因组DNA 用EcoR Ⅰ/Mse Ⅰ (15 U/5 U)于37℃下酶切2 h,加接头后的混合物稀释5 倍用于预扩增,预扩增产物再稀释10 倍后进行选择性扩增.结果表明,筛选的22 对引物进行扩增反应,共得到979 条条带,其中多态性条带为649 条.采用优化的AFLP 体系,对西南地区的10 种猕猴桃50 个样本进行了遗传多样性分析,包括普通品种中华猕猴桃、美味猕猴桃,及特色品种硬毛猕猴桃、毛被猕猴桃、革叶猕猴桃、四萼猕猴桃、狗枣猕猴桃、葛枣猕猴桃、京梨猕猴桃和紫果猕猴,结果表明种内和种间的聚类关系明显;斑果组(sect. Maculatae)和净果组(sect. Leiocarpae)的种间有聚类交叉,并呈现地理区域性分布.     

橡胶树AFLP银染体系的建立和优化

目的:扩增片段长度多态性(AFLP)为遗传图谱的构建及育种的辅助选择提供了有力的工具。建立一套适合橡胶树的AFLP技术优化体系。方法:以197个GT1×IAN873橡胶树杂交群体为材料,通过对影响AFLP的多种关键因素如模板DNA质量、酶切连接体系、酶切连接反应时间、预扩增体系、选择性体系的分析进行研究。结果与结论:找出一套适于热带植物基因组DNA提取及橡胶树AFLP技术。用改进的CTAB法,经多次抽提纯化,用细玻璃棒挑出DNA得到高质量的模板DNA;酶切连接时DNA模板为250ng,反应体系采用各3U的EcoRⅠ/MseⅡ/T4连接酶,反应时间为9h;预扩增模板为稀释1/2的酶切连接产物,用量为1μL;选择性扩增要用稀释至1/20的预扩增产物。     

天麻AFLP分析技术体系的建立

目的:建立一个适于天麻研究用的AFLP分析技术体系。方法:以11份天麻种质资源为试材,构建供试天麻的AFLP指纹图谱,同时对AFLP分析过程中DNA提取、酶切、连接、预扩增、引物筛选、选择性扩增、电泳和银染等多种因素进行分析。结果:AFLP分析体系要求DNA模板质量高,酶切连接可同时进行,37℃、9h效果较好,预扩增要求高质量DNA聚合酶,产物15倍稀释作为选择性扩增模板效果好,制胶前玻璃板的处理和银染时间的掌控对获得较好结果非常关键。P-GGA/M-GT引物构建的指纹图谱拥有可清晰辨认的扩增条带45条。结论:AFLP指纹技术具有稳定性高、重复性好等优点,可用于天麻DNA分析。     

斜带髭鲷高质量DNA提取及AFLP分析体系建立

目的:提取斜带髭鲷高质量基因组DNA并建立适用于AFLP分析的体系。方法:以斜带髭鲷(Hapalogenys nitens)为材料提取高质量基因组DNA,并进行AFLP扩增。对AFLP分析体系中几个关键环节(基因组双酶切、连接、预扩增及选择性扩增、变性聚丙烯酰胺凝胶电泳和银染)做了优化。结果:提取的斜带髭鲷全基因组DNA纯度高,无拖尾。采用9组选择性引物共检测出350个不同的扩增位点,其中多态位点为175个,多态性比例为49.58%。聚丙烯酰胺凝胶电泳图像染色均匀,条带清晰且无背景干扰。结论:该分析体系的建立为斜带髭鲷的群体遗传多态性、种质资源、遗传图谱、遗传育种等研究奠定基础。     

蚕豆AFLP技术体系的建立与优化

对蚕豆DNA提取质量和浓度、DNA双酶切与连接、酶切连接产物的预扩增和选择性扩增等AFLP技术体系中的关键技术进行了优化处理,构建了蚕豆AFLP银染技术体系。酶切与连接可在12.5μl体系中一步完成,酶切连接温度为37℃,反应时间12~14 h;预扩增体系为20μl,选择性扩增体系为10μl。采用该技术体系应用8对引物构建的蚕豆种质资源AFLP指纹图谱,扩增条带多、多态性强且质量好,可满足遗传多样性分析要求。     

中华稻蝗AFLP反应体系的建立与优化

以中华稻蝗为研究材料,提取到高质量的总DNA.通过优化酶切连接、预扩增、选择性扩增等实验条件,确定了AFLP银染技术方法,从而建立了中华稻蝗AFLP分子标记研究体系,得到了清晰的AFLP指纹图谱,为探讨稻蝗种间遗传学关系提供了新的技术手段.     

芒AFLP反应体系的建立

以芒DNA为材料,对AFLP分子标记分析中的基因组酶切体系选择性扩增中Mg2+、dNTP和Taq酶浓度等4个因素进行了比较。结果表明20μL基因组双酶切体系中,使用1UEcoRⅠ和1UM seⅠ酶切3 h能够实现完全酶切;选择性扩增的PCR 10μL反应体系中1.4 mmol.L-1Mg2+,0.4 mmol.L-1dNTP及0.6 U Taq酶是进行芒AFLP分析的最佳反应条件,能够得到丰富稳定的带纹。该体系的构建为AFLP技术在芒相关研究中的应用奠定了基础。     

应用AFLP技术对不同山羊种群进行遗传多样性检测的方法初探

7种不同山羊品种或种群的基因组DNA经限制性内切酶酶切 ,连接特异性接头 ,用 5条人工设计的与接头序列相识别的AFLP选择性引物 ,进行AFLP -PCR扩增 ,以琼脂糖凝胶电泳检测扩增结果。不同山羊种群基因组DNA的扩增结果具有差异。从而得出结论 :AFLP技术是一种适宜于山羊的遗传检测方法。     

短蛸AFLP分子标记分析体系的优化与建立

本研究构建了短蛸扩增片段长度多态性(AFLP)分析体系,对DNA提取、双酶切反应、连接反应、预扩增反应、选择性扩增反应和银染等步骤进行了分析。得到了一种适于短蛸AFLP技术分析的优化体系,该体系中各优化因素为:模板DNA浓度为200 ng/μL;酶切体系中,MseI和EcoR I各加入5 units,缓冲液使用MseI buffer Tango,反应时间为3-4 h;连接最适反应时间为12 h;预扩增产物最适稀释倍数为20倍。该体系的构建为AFLP技术在短蛸分子遗传多样性研究中的应用奠定了基础。     

裸燕麦AFLP反应体系的优化

影响裸燕麦AFLP反应的关键因素包括基因组DNA提取过程中氯仿-异戊醇的抽提次数,酶切时间,预扩增产物稀释倍数,选择性扩增中Mg2+、dNTP、引物浓度等.本研究对这些影响因素进行了优化,初步建立了适合裸燕麦的AFLP反应体系.并将该体系应用于引物筛选,在12份材料中,共筛选出20对条带清晰、多态性好的引物组合,为裸燕麦遗传多样性分析提供了基础.     

应用AFLP标记对江西省猕猴桃种质资源的鉴别及其分类意义

 对江西省猕猴桃种质资源进行扩增片段长度多态性(AFLP)标记来鉴定分析.首先从64对引物筛选出4对引物,对31份种质材料的DNA进行检测,得到190个扩增基因位点,其中多态性位点179个,多态性比例为94.2%,对31份种质材料的区分率达到100%.然后,对扩增结果进行UPGMA聚类分析,谱系图显示,31份种质材料之间的相似系数在0.50～0.85之间,表明猕猴桃种质之间遗传关系相对来说不是很近.在相似系数0.56的水平上,可以将31份种质大致分为4个类群：净果组和斑果组为一类群;糙毛组为一类群;星毛组为一类群;中华猕猴桃和美味猕猴桃为一类群.从树状图中看,原为中华猕猴桃的一个变种的“赣猕5号”,现与中华猕猴桃并列,有对其作进一步分类方面深入研究的必要.本研究从分子角度鉴定分析了江西省猕猴桃种质资源及其遗传关系,其结果在很大程度上与传统分类是一致的,同时也为江西省猕猴桃种质资源分类学研究提供了新的证据.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi

Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potential to alleviate this technical bottleneck. In the present work, we have sought to investigate the suitability of genomic DNA synthesized using Phi29 based WGA for AFLP analysis. We first used genomic DNA from Saccharomyces cerevisiae to optimize the protocol for the use of SDA-amplified DNA for AFLP analysis. Based on the optimized protocol we obtained AFLP fingerprints which were indistinguishable from the non-amplified genomic DNA. Finally, AFLP analysis was performed using SDA synthesized genomic DNA from single spores of various species of arbuscular mycorrhizal (AM) fungi. Unique and highly reproducible fingerprints for each species were obtained. The present study introduces the application of WGA-mediated AFLP to AM fungal biology; similarly, our protocol could be useful for other microbial genomes currently not amenable to genetic analysis owing to the paucity of starting template.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Strain typing of shiitake (<Emphasis Type="Italic">Lentinula edodes</Emphasis>) cultivars by AFLP analysis,focusing on a heat-dried fruiting body

To validate strain typing by amplified fragment length polymorphism (AFLP) analysis in shiitake (Lentinula edodes) cultivars, the reproducibility of AFLP markers with DNA extracted from the heat-dried fruiting body was evaluated. DNAs were extracted from three different portions of the heat-dried fruiting body – the stipe, pileus, and gill – and AFLP analysis of all parts was carried out using two combinations of selected amplification primer pairs. AFLP profiles of DNA from the gill tissue of heat-dried fruiting body were almost identical to those of cultured mycelia in the same strains, although it was difficult to detect reproducible AFLP profiles from stipe and pileus DNA. These results indicated that AFLP analysis would be applicable for strain typing with heat-dried fruiting bodies of L. edodes by using the DNA extracted from gills.Contribution No. 364 of the Tottori Mycological Institute     

High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer

Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70,000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure.     

一种高效提取猕猴桃DNA和RNA的方法

在总核酸提取方法(PS法)的基础上,经多次实践改进,得出一种以高盐低pH的HAc-NaAc缓冲体系提取总核酸的简便方法,可以从富含多糖、多酚时猕猴桃叶片和花蕾中提取同时含有DNA和RNA的总核酸.所得的总核酸在LiCl溶液中选择性沉淀RNA,从而有效地分离出DNA和RNA样品.紫外分光光度法和琼脂糖凝胶电泳分析表明,所提取的DNA和RNA具有较高的纯度和完整性.通过样品DNA的PCR和样品RNA的RT-PCR,认为所提取的DNA样品和RNA样品能够满足分子生物学试验的基本要求.     

Multicolour fluorescent detection and mapping of AFLP markers in chicken (Gallus domesticus)

We describe the mapping of amplified restriction fragment polymorphism (AFLP) markers in chicken (Gallus domesticus) using a multi-colour fluorescent detection system. DNA was used from a population consisting of four families with a total of 183 F2 individuals. The enzyme combination EcoRI/TaqI was used for double digestion, and fluorescently labelled fragments were analysed on an ABI PRISM 377 DNA sequencer. Polymorphic signals in the range of 50-500 bp were genotyped with the ABI PRISM Genotyper 2.0 software, which enabled the analysis of both dominant and incomplete dominant markers (with respect to AFLP, often referred to as codominant). In 19 sets consisting of 3 EcoRI/TaqI primer pair combinations each, a total of 475 polymorphic markers was detected. From these polymorphisms 344 markers could be mapped on the Wageningen linkage map. Fourteen markers were length polymorphisms of the same fragment and 28 markers Z-linked and uniformative; 64 AFLP markers appeared to be unlinked and 25 AFLP markers could not be accurately mapped on the basis of the genotyping results. The resulting AFLP/microsatellite linkage map is comprised of 33 linkage groups with a total of 835 loci.     

羊草种质基因组DNA的AFLP多态性研究

羊草是禾本科牧草之王 ,在当前我国西部生态建设和草原畜牧业发展中发挥着重要作用。用AFLP方法对2 7份我国不同地区分布的羊草 (Leymuschinensis (Trin .)Tzvel)材料进行了基因组DNA多态性分析 ,8对AFLP引物组合在 2 7个不同羊草基因型中共扩增出 5 37条带 ,产生出的DNA片段大小分布在 75bp - 5 30bp之间。其中单态性带 89条 ,占 16 .6 % ,多态性带 32 9条 ,占 6 1.3%。平均每对引物组合扩增的DNA带数为 6 6 .13,总的多态性比率为 78.84%。AFLP多态信息含量PIC值分布于 0 .0 - 0 .5之间 ,平均PIC值为 0 .2 16 ,出现的PIC最大值 (0 .5 )约占AFLP标记的 8.5 % ,说明羊草基因组DNA的多态性比较丰富。以 5 37个AFLP标记为原始数据 ,根据Nei和Li的方法对 2 7份羊草材料进行遗传变异和聚类分析的结果表明 :羊草种内有高频率的遗传变异发生 ,且与地理分布和生态环境密切相关 ;2 7份羊草不同基因型被划分为四大类群 ,不同类群相互间的遗传距离相对较大 ,在树状图中表现为较远的亲缘关系。对羊草种内遗传变异发生的原因和品种的形成进行了初步讨论。     

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE)

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.