应用RAMP分子标记分析小豆栽培型种质资源遗传多样性

利用42对引物组合，对93份小豆栽培型种质资源的基因组DNA进行了RAMP分析，得到308条扩增谱带，其中297条（96.43%）具有多态性，每对引物组合可扩增出3～17条多态性谱带，平均7.1条，总遗传多样性指数或平均期望杂合度（Ht）达0.693，表明小豆栽培型种质资源内存在较丰富的遗传多样性；小豆种质间遗传距离变异幅度为0.066～0.494，平均值为0.281，表明来源于不同生态区的小豆栽培型种质间的亲缘关系较近；利用RAMP扩增谱带数据进行聚类分析，可将93份小豆栽培型种质材料中的91份区分开，并划分为5个类群；其RAMP分子标记表现出明显的生育特性和生长习性趋同性，及一定的地域相关性。     

RAMP与REMAP分子标记技术及其应用

引物设计是RAMP和REMAP标记分析的关键，二者都使用锚定的微卫星序列引物。目前已开发出多个RAMP锚定微卫星序列引物，与不同的RAPD引物进行扩增，得到更多的引物组合。REMAP使用反转录转座子的外向LTR引物和锚定微卫星序列引物。而RAMP分析采用不对称PCR循环，在PCR扩增的后15个循环中，退火温度设为2个；REMAP则采用常规PCR循环。扩增产物可在聚丙烯酰胺凝胶、变性聚丙烯酰胺凝胶、琼脂糖凝胶上进行电泳分离，检测时可使用同位素、银染或溴化乙锭染色等技术。目前，这两种标记技术已在作物和动物种质资源的遗传多样性分析、遗传连锁图谱构建、转基因植株基因稳定性分析等方面都有成功的应用。     

山羊基因组DNA RAMP-PCR反应体系的建立

以山羊基因组DNA为材料,研究了MgCl2浓度、引物浓度、dNTP浓度、模板DNA用量、TaqDNA聚合酶用量对RAMP-PCR反应的影响.建立了一套适合山羊的最佳RAMP反应体系.反应总体积为25μL,MgCl2浓度2.0 mmol/L、引物为0.4 mmol/L、dNTP浓度为0.2 mmol/L、模板DNA为25 ng,TaqDNA聚合酶为1U.PCR反应程序为:94℃ 5 min;94℃ 30 s,40℃ 45s,72℃ 90s,30 Cycles;72℃ 10 min.     

利用RAMP和ISSR标记分析大麦种质资源的遗传多样性

 利用RAMP和ISSR两种标记分别对60份大麦种质资源遗传多样性进行了检测。结果发现，两种标记都能揭示材料间较高的遗传多样性，其中ISSR标记多态性又高于RAMP标记。在RAMP分析中，每个引物可扩增出1～10条DNA片段，平均为5.27条；22个RAMP引物共扩增出116条DNA片段，其中98条具有多态性，多态性比率（PPB）为84.48%；平均多态信息量（PIC）为0.277；每个位点有效等位基因数（Ne）为1.602；材料间遗传相似系数GS变化范围为0.551～0.965，平均值为0.830。在ISSR分析中，每个引物可获得1～10条DNA片段，平均为5.95条；19个ISSR引物共扩增出113条DNA片段，其中111条具有多态性，多态性比率（PPB）为98.23%；平均多态信息量（PIC）为0.427；每个位点有效等位基因数（Ne）为2.033；材料间遗传相似系数GS变幅为0.203～0.931，平均达0.676。聚类分析表明，两种标记都能将供试材料完全区分开，聚类结果具有一定的相似性，但也存在明显差异。Mantel检测表明，这两种标记极显著相关（r = 0.346, t = 2.808）。     

应用RAMP标记分析长白山地区中国林蛙的遗传多样性

应用随机扩增徽卫星多态性DNA（RAMP）标记技术对长白山地区中国林蛙60个个体间遗传多样性进行了分析。以便为中国林蛙种质资源的保护和利用以及物种分化研究提供基础资料。结果表明：120个RAMP引物中。有14个引物组合可扩增出清晰且具多态性的条带。这14个引物组合共扩增出107条带,其中105条具有多态性,多态条带比率为98．13％。每个引物可扩增出3～10条多态性条带,平均7．5条。多态条带比率、Shannon多样性指数（I）以及Nei氏基因多样度（h）均显示长白山地区中国林蛙具有较为丰富的遗传多样性。RAMP标记遗传相似性系数（岱）变异范围为0．4860～0．8505,平均值为0．6634,以GS平均值0．6634为闻值,可将所有供试材料划分为10类,其中大部分个体归为同一类,群体内并没有形成较明显的遗传分化。     

The Genetic Diversity and Similarities Among <Emphasis Type="Italic">Kengyilia</Emphasis> Species Based on Random Amplified Microsatellite Polymorphism (RAMP)

The genetic diversity and similarities among 32 Kengyilia accessions, distributed to 14 species and one variety were analyzed by using random amplified microsatellite polymorphism (RAMP) markers. Of the 160 RAMP primer combinations tested, 40 (25%) produced polymorphic and clear bands. A total of 264 bands were produced by 40 primer combinations, among which 231 out of 264 bands (87.5%) were polymorphic. Two to 11 polymorphic bands could be amplified from each primer combination, with an average of 5.8 bands. The data of 264 bands were used for RAMP assay. By NTSYS-pc program, genetic similarity coefficients were generated and dendrogram was constructed using UPGMA. The genetic similarity coefficients ranged from 0.477 to 0.965 with the mean of 0.714. The results showed as follows: (1) distinct genetic differences were present among the different species; (2) the different accessions in a species were clustered together, respectively, which had larger genetic similarities and closer relations; (3) the species with similar morphological characters and the species from the same areas or neighboring geographical regions were clustered together; (4) the lowest genetic similarity was found between K. hirsuta (PI531618) and K. laxiflora (PI531631), while the highest genetic similarity was observed between K. hirsuta (Y2364) and K. hirsuta (Y2368); (5) RAMP results are basically comparable with those obtained from studies on morphology and cytology. It is a useful method for analysis of the genetic diversity and similarities in Kengyilia.     

杨树RAMP体系的建立与优化

以杨树基因组DNA为模板,通过正交试验设计,从Mg2+、Taq酶、dNTPs、引物4种因素3个水平对杨树RAMP反应体系进行优化,建立了适合于杨树的RAMP-PCR优化反应体系,该体系为25μL:Mg2+浓度为1.5 mmol/L,dNTPs为0.2 mmol/L,Taq酶1.0 U,引物各为0.4μmol/L。PCR反应程序为:94℃预变性5 min,94℃变性1 min,45℃复性1 min,72℃延伸2 min,45个循环,72℃延伸10 min。     

应用RAMP标记分析水貂与彩貂种属间的遗传多样性

对标准黑褐色水貂、蓝宝石貂、咖啡色貂3个品种的89个个体进行了RAMP分析。结果表明,品种间遗传差异明显,多态性高。14个引物组合扩增出的81条DNA片段中,75条具有多态性,多态比例为92.59%,每个引物组合可扩增4～8条多态性条带,平均5.78条。多态条带比率、Shannon多样性指数以及Nei氏基因多样度（h）均显示水貂与彩貂品种较〖JP2〗为丰富的遗传多样性。Nei遗传距离、Gst基因分化系数显示水貂与彩貂品种间遗传分化程度不高,遗传变异主要来自种群内。     

葡萄品种遗传关系的RAMP分析

对目前南方大力推广的 18个葡萄品种进行了RAMP分析。结果表明 ,品种间遗传差异明显 ,多态性高。 33个引物组合产生的 14 8条DNA扩增片段中 ,12 0条具有多态性 ,占 81 1% ,每个引物组合可扩增 1～ 8条多态性带 ,平均 4 5条。遗传相似系数变化范围 0 75 9～ 0 94 5 ,平均值为 0 84 4。聚类分析显示 ,RAMP标记能将 18个品种完全分开 ,鉴定出引入栽培的京秀和信浓笑两个品种遗传关系很近。同时 ,RAMP标记的聚类结果与葡萄品种抗感霜霉病类型基本一致。     

The Genetic Diversity Among Leymus Species Based on Random Amplified Microsatellite Polymorphism (RAMP)

The genetic diversity and similarities among 40 accessions of Leymus Hochst., distributed in 19 species and 1 subspecies, were analyzed by using random amplified microsatellite polymorphism (RAMP) markers. Of the 120 RAMP primer combinations tested, 24 (20%) produced polymorphic and clear bands. A total of 192 bands were amplified by 24 primer combinations, among which 179 (93.23%) bands were found to be polymorphic. Three to thirteen polymorphic bands were amplified by each primer combination, with an average of 7.64 bands. The data of 192 RAMP bands were used to generate Jaccard's similarity coefficients and to construct a dendrogram by means of UPGMA in the NTSYS-pc computer program. The genetic similarity coefficients ranged from 0.10 to 0.73 with the mean of 0.34. The results showed as follows: (1) Distinct genetic differences were present among the different species; (2) The different accessions in a species were clustered together, respectively, which had larger genetic similarities and closer relations; (3) The species with similar morphological characters and the species from the same areas or neighboring geographical regions were clustered together; (4) RAMP results are basically comparable with those obtained from studies on morphology. It is a useful method for analysis of the genetic diversity and similarities in Leymus.