用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较

由Ralstonia solanacearum E.F.Smith引起的青枯病是若干亚洲和非洲国家花生生产的重要限制因子,利用抗病品种是防治这一病害最好的措施。虽然一大批抗青枯病花生种质资源材料已被鉴定出来,但对其遗传多样性没有足够的研究,限制了在育种中的有效利用。本研究以31份对青枯病具有不同抗性的栽培种花生种质为材料,通过简单序列重复(SSR)和扩增片段长度多态性(AFLP)技术分析了它们的遗传多样性。通过78对SSR引物和126对AFLP引物的鉴定,筛选出能显示抗青枯病种质多态性的SSR引物29对和AFLP引物32对。所选用的29对多态性SSR引物共扩增91条多态性带,平均每对引物扩增3.14条多态性带;32对多态性AFLP引物共扩增72条多态性带,平均扩增2.25条多态性带。在所筛选引物中,4对SSR引物(14H06,7G02,3A8,16C6)和1对AFLP引物(P1M62)检测花生多态性的效果优于其他引物。SSR分析获得的31个花生种质的遗传距离为0.12-0.94,平均为0.53,而AFLP分析获得的遗传距离为0.06～0.57,平均为0.25,基于SSR分析的遗传距离大于基于AFLP分析的遗传距离,疏枝亚种组的遗传分化相对大于密枝亚种组。基于两种分析方法所获得的聚类结果基本一致,但SSR数据聚类结果与栽培种花生的形态分类系统更为吻合。根据分析结果,对构建青枯病抗性遗传图谱群体的核心亲本和抗性育种策略提出了建议。     

濒危植物绵刺8个种群遗传多样性的AFLP分析

利用8对AFLP引物对我国绵刺的8个种群240份材料的基因组DNA进行扩增,得到大小在65～530bp之间的397条清晰显带,其中296(74.56%)条呈多态性,平均每对AFLP引物得到37条多态性带;用PopGen32软件将AFLP多态性数据进行分析,不同种群的Nei's基因多样性指数和Shannon信息指数变化范围分别在0.0845～0.1779和0.1280～0.2377之间,其中遗传多样性最高为上沙窝种群,最低为银根种群,可将上沙窝种群作为种质遗传中心之一进行保护.绵刺遗传变异有68.31%存在种群内,31.69%种群之间,说明变异主要存在于种群内部.8个种群的平均遗传距离为0.1341,按UPGMA进行聚类分析,结果表明绵刺种群具有明显的地域相关性和遗传类型趋同性,说明不同的种群可能有共同的起源,随机遗传漂变不是影响绵刺种群遗传多样性的主要过程.建议在迁地保护和取样时,不仅要在每个种群中取足够多的个体,而且要在尽可能多的种群中取样,最大限度地保护绵刺的遗传多样性,为进一步地系统演化研究奠定基础.     

利用AFLP标记鉴定小豆种质遗传多样性

利用11对AFLP引物对106份小豆种质的基因组DNA进行扩增,共扩增出603条清晰可辨的带型,其中208条具有多态性,比例为34.5%,平均每对引物扩增出18.9条多态性带.基于AFLP标记,把106份小豆种质聚类划分为4个组群,该组群的划分与小豆的生态地域性存在一定的相关性.     

利用AFLP标记鉴定小豆种质遗传多样性

利用11对AFLP引物对106份小豆种质的基因组DNA进行扩增,共扩增出603条清晰可辨的带型,其中208条具有多态性,比例为34．5％,平均每对引物扩增出18．9条多态性带。基于AFLP标记,把106份小豆种质聚类划分为4个组群,该组群的划分与小豆的生态地域性存在一定的相关性。     

60份优质番茄自交系遗传多样性AFLP分析

本文以AFLP技术对60份优质番茄自交系的遗传多样性进行了分析。17对AFLP引物组合共扩增得到905条带,其中多态性条带251条,平均每对引物检测到约15个多态性位点,平均多态率为27.7%;仅用E7M4和E7M10两对引物组合就可以绘制60份番茄自交系的指纹图谱。60个自交系间的Jaccard遗传相似系数变幅为0.259-0.952,平均值为0.664。采用UPGMA方法聚类,遗传相似系数变幅为0.54-0.57时,60份番茄材料可以划分为7个类群。聚类结果与来源、果实大小、果形等性状未表现出明显的相关性。     

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27份我国不同地区分布的羊草(Leymus chinensis (Trin.) Tzvel)材料进行了基因组DNA多态性分析,8对AFLP引物组合在27个不同羊草基因型中共扩增出537条带,产生出的DNA片段大小分布在75 bp-530 bp之间.其中单态性带89条,占16.6%,多态性带329条,占61.3%.平均每对引物组合扩增的DNA带数为66.13,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0.216,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537个AFLP标记为原始数据,根据Nei和Li的方法对27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;27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.     

SRAP技术研究烟粉虱遗传多样性

采用AFLP、SRAP2种标记方法分别对2个烟粉虱Bemisia tabaci Gennadius种群(一品红、甘蓝)的多态性进行分析。结果表明,(1)2种方法平均每对引物组合产生的条带数分别为29.4和21.8。(2)AFLP法每对引物组合产生10～23条多态性带,平均17.20条,多态性带的比例平均为57.93%。SRAP法每对引物组合产生5～18条多态性带,平均13.3条,多态性带的比例平均为60.59%。(3)前者的基因多样性范围为0.1503～0.2838,平均为0.2297;后者的基因多样性范围为0.0977～0.2911,平均为0.2332。证明利用SRAP技术和AFLP技术研究烟粉虱的遗传多样性是有效的。     

40份玉米自交系的ISSR遗传多样性分析

利用ISSR分子标记技术对40份玉米自交系进行亲缘关系分析。从59条ISSR引物中筛选出10条重复性高、多态性好的引物,分别对全部供试材料进行扩增,共扩出90条清晰谱带,其中多态性条带81条,多态性比率为90.00%,表明供试材料基因组DNA的多态性较高。用 NTSYSpc-2.10软件中的 UPGMA进行聚类分析,40份玉米自交系的遗传相似系数变化范围在0.65~1.00之间,在此基础上构建聚类分析树状图,揭示了玉米各自交系间的亲缘关系。本研究为今后分子水平上玉米优良自交系的改良与选育提供了一定的参考依据。     

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。     

猕猴桃AFLP分析体系的建立

从64对AFLP分析引物中随机选取了19对引物组合。经过跑小板的聚丙烯酰胺凝胶,银染显带AFLP条带,从中筛选出可做荧光的8对引物。再从中选取了扩增位点丰富,带型质量好,分辨率较高,条带信号强度一致性好,条带分布均匀,且条带较完整的4对引物：E-AAC＋M-CAC、E-AAG＋M-CTG、E-AAC＋M-CAG、EAAC＋M-CTA进行荧光跑带、读带。共在156个位点上扩增出条带,4对引物共扩增出多态性条带132条,多态性比例平均为84．62％,4对引物对10份猕猴桃材料的区分率达100％。说明该4对引物用于猕猴桃属植物的AFLP分析是可行的。     

利用向日葵重组自交系构建遗传图谱

以向日葵自选系K55为母本、K58为父本杂交组合,通过单粒传得到的187个F_5：6代重组自交系群体为作图材料,联合应用SSR和AFLP标记构建遗传连锁图谱。经过78对SSR引物和48对AFLP引物组合选择性扩增,分别得到341和1119条带,共1460条,分别获得多态性条带184条和393条,共577条多态性条带,占所有条带的39.52%。SSR和AFLP标记各有84个和108个多态性标记偏离孟德尔分离比例(P=0.05),共192个偏分离标记。采用JoinMap4.0软件进行连锁分析,构建了1张总长度为2759.4 cM、包含17个连锁群、连锁495个多态性标记的遗传图谱,其中偏分离标记170个,标记间的平均图距为5.57 cM。每个连锁群上分布有5~72个标记,长68.88~250.17 cM。本图谱为向日葵永久性图谱,为向日葵重要性状QTL定位和基因克隆奠定基础。     

Genetic Diversity and Population Structure of Yellow Camellia (Camellia nitidissima) in China as Revealed by RAPD and AFLP Markers

Camellia nitidissima, a rare plant but a useful genetic resource for commercial cultivation of ornamental camellias, is distributed in a narrow region of South China and North Vietnam. In this study, RAPD and AFLP markers were used to assess the genetic diversity and population structure of six natural populations of C. nitidissima from Guangxi in South China. Twenty RAPD primers amplified 183 bands, of which 143 bands were polymorphic, and 8 AFLP primer pairs produced 502 bands, of which 364 were polymorphic. Independent as well as combined analyses of the cluster analyses of the RAPD and AFLP fragments showed that the six populations could be classified into two major genetic groups corresponding to the Nanning and Fangcheng areas. The Mantel test revealed significant correlation between the genetic and geographic distances of C. nitidissima populations (r = 0.953, p = 0.036). AMOVA analysis allowed the partitioning of the genetic variation between groups (36.09%), among populations within groups (25.78%), and within populations (38.14%). An understanding of both the genetic diversity and the population structure of C. nitidissima in China can also provide insight into the conservation and management of this endangered species.     

中国马铃薯晚疫病菌AFLP遗传多样性分析

应用AFLP分子标记检测了我国部分马铃薯主要产区马铃薯晚疫病菌的遗传多样性及不同地区菌株间的亲缘关系。在200对引物组合中,利用6个菌株筛选出12对多态性好、带型清晰的引物组合。利用这12对引物组合对1997-2002年间采自我国黑龙江、河北、四川和云南4省的50株菌株进行了PCR扩增,共扩增出922条谱带,其中多态性标记530条,占57.5%。利用NTSYSpc软件中UPGMA算法构建了我国马铃薯晚疫病菌的亲缘关系树状图,聚类分析结果表明我国马铃薯晚疫病菌的遗传多样性与病原菌的地理来源有一定的相关性,而与交配型、生理小种和对甲霜灵的抗性无明显的相关性。用POPGENE软件计算了各群体间的遗传多样性参数,结果表明我国马铃薯晚疫病菌的遗传多样性程度不高,不同地区种群间分化不明显。     

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.     

用扩增片段的长度多态性分子标记探讨盾叶薯蓣的遗传多样性

用扩增片段的长度多态性(amplified fragment length polymorphism,AFLP)标记分析研究了中国5个盾叶薯蓣居群30个个体的遗传多样性。筛选出9对AFLP引物,从中检测到14698条清晰可见的条带,其中多态性带12628条,多态性比率85.92%。Shannon信息指数(I)为0.3656±0.1721,物种水平的Nei基因多样性(H)为0.2322±0.2200。遗传变异分析表明,物种水平的遗传分化系数Gst为0.4827,说明其群体间存在一定的遗传分化,居群间的基因流Nm为0.5358,居群间遗传交换较小。聚类分析结果显示5个居群盾叶薯蓣有较为丰富的遗传变异,且与地理分布有相关性。     

基于AFLP标记的中国西藏近缘野生大麦遗传多样性分析

选取7对引物组合, 构建了36份西藏近缘野生大麦和4份栽培大麦的AFLP指纹图谱, 共获得清晰可辨的条带227条, 其中多态性带194条, 占85.46％, 从DNA分子水平显示出所试材料遗传多样性较为丰富。计算得各样品间的遗传距离(欧氏距离)介于2.646~10.488之间, 应用离差平方和法对供试材料的AFLP数据结果进行聚类, 建立了40份大麦材料的AFLP树状图, 聚类结果将40份材料分为5类, 进一步揭示出供试材料间遗传背景的相似性和复杂性。结合Nei’s遗传一致性分析结果, 发现近缘六棱野生大麦较之近缘二棱野生与栽培大麦的亲缘关系更近, 支持栽培大麦是从野生二棱大麦起源, 而野生六棱大麦是进化过程中的过渡类型的大麦系统发生观点。     

Polymorphism in ornamental and common carp strains (Cyprinus carpio L.) as revealed by AFLP analysis and a new set of microsatellite markers

Forty-seven new microsatellite markers were generated and applied, together with the AFLP (Amplified Fragment Length Polymorphism) technique using two different enzyme combinations, to the genetic analysis of two carp species, Cyprinus carpio L. and Ctenopharyngodon idella. The extent of polymorphism and the genetic relationships between nine carp populations were studied. The incidence of microsatellites containing CA and CT motifs was estimated to be one every 17.4 and one every 126.3 kb, respectively, and their average allele numbers were four and five, respectively. Across populations, the average proportion of individuals that were heterozygous for microsatellite markers was 44.2% and the average allele number was 4.02. The EcoRI/TaqI combination generated more analyzable AFLP bands than the EcoRI/MseI pair, making the former preferable for the analysis of carp populations. The proportion of polymorphic AFLP bands within populations ranged from 6.7% in grass carp to 59.9% in Kohaku strain (Koi) of the ornamental carp. The fixation index (FST) for microsatellites in these populations was estimated to be 0.37, and for AFLP markers the value was 0.39. Genetic distance matrices derived from microsatellites and from two AFLP analyses were positively correlated. Grass carp showed fewer AFLP bands than other populations and was genotyped by only half of the microsatellite markers. These findings agree with genetic distance estimates in suggesting that the grass carp is phylogenetically quite remote from all the other populations examined.     

转基因抗虫棉SSR和AFLP遗传变异研究

利用SSR和AFLP两种分子标记技术,分析了52份转基因抗虫棉品种（系）的遗传多样性。结果表明：在61对SSR引物中,有4对引物在供试材料中表现出多态性,共扩增出102个标记,其中多态性标记25个,多态性百分率为24．51％,每对引物的扩增带数变化在17～30之间;在100对AFLP引物中,有9对引物在供试材料中产生多态性,共扩增出618个标记,多态性标记33个,占总数的5．34％,每对引物组合扩增的标记数分布于47～81之间。成对品种的欧式距离变化在2．00～5．57之间,平均值为4．21,单一品种欧氏距离的平均值分布在3．73～4．75之间,表明不同品种之间遗传差异不大。基于SSRs和AFLPs多态性数据的聚类分析,可以将供试材料划分为3个类群（SAGs）,但类群划分与品种地理来源不十分吻合。     

Genetic diversity in wild populations of Paulownia fortunei

The genetic diversities of 16 Paulownia fortunei populations involving 143 individuals collected from 6 provinces in China were analyzed using amplified fragment length polymorphism (AFLP). A total of 9 primer pairs with 1169 polymorphic loci were screened out, and each pair possessed 132 bands on average. The percentage of polymorphic bands (98.57%), the effective number of alleles (1.2138–1.2726), Nei’s genetic diversity (0.1566–0.1887), and Shannon’s information index (0.2692–0.3117) indicated a plentiful genetic diversity and different among Paulownia fortune populations. The genetic differentiation coefficient between populations was 0.2386, while the gene flow was 1.0954, and the low gene exchange promoted genetic differentiation. Analysis of variance indicated that genetic variation mainly occurred within populations (81.62% of total variation) rather than among populations (18.38%). The 16 populations were divided by unweighted pair-group method with arithmetic means (UPGMA) into 4 groups with obvious regionalism, in which the populations with close geographical locations (latitude) were clustered together.