Analysis of genetic diversity for wild and captive green peafowl populations by random amplified polymorphic DNA technique

The genetic diversity of the populations for 14 wild green peafowls (Pavo muticus) and 18 captive green pea-fowls was investigated by using the technology of random amplified polymorphic DNA (RAPD). Totally 161 and 166 ampli-fied bands were obtained by using 23 arbitrary primers to amplify the genomic DNA of wild and captive green peafowls re-spectively. The results showed that the average relative genetic distance of the wild and captive green peafowls popula-tions was 0.0555 and 0.1355, respectively, and difference of the average relative genetic distances between the two popu-lations was 0.1635. The Shannon diversity index for the wild and captive green peafowl populations was 0.4348 and 1.0163, respectively, which means that there exists significant difference in genetic diversity between the two populations, and the genetic diversity of wild green peafowl was low. The two populations originated from two different families according to analysis by the UPGMA method. This research can provide the theoretical basis for supervising genealogies management of peafowl populations.

Use of random amplified polymorphic DNA （RAPD） analysis to detect jujube witches＇ broom phytoplasmas

Jujube witches＇ broom is a devastating disease of Ziziphusjujube that occurs in various jujube regions of China. Nucleic acid extracted from midribs of samples collected from three jujube varieties （＂Suanzao＂, ＂Lajiaozao＂ and ＂Langzao＂） from symptomatic and asymptomatic shoots were tested by random amplified polymorphic DNA analyses. Using 13 different 10 and 11-bp random primers the amplification of jujube DNA was achieved from all the samples; AMI4 primer provided amplification of specific DNA fragments of about 400 bp, only from samples collected from symptomatic plants. No genetic variations in these varieties were identified using the other 11 arbitrary primers; only with primer AL07 it was possible to differentiate ＂Langzao＂ from the other two varieties tested. All the experiments were repeated 2 times and the results were consistent. Compared with PCR analyses with phytoplasma-specific primers, RAPD techniques resulted to be an alternative rapid and sensitive method for detecting jujube phytoplasmas presence in different jujube varieties.

Authentication and Genetic Origin of Medicinal <i>Angelica acutiloba</i>Using Random Amplified Polymorphic DNA Analysis

Some Angelica species are used for medicinal purposes. In particular, the roots of Angelica acutiloba var. acutiloba and A. acutiloba var. sugiyamae, known as “Toki” and “Hokkai Toki”, respectively, are used as important medicinal materials in traditional Japanese medicine. However, since these varieties have recently outcrossed with each other, it is difficult to determine whether the Japanese Angelica Root material used as a crude drug is the “pure” variety. In this study, we developed an efficient method to authenticate A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae from each other and from other Angelica species/varieties. The random amplified polymorphic DNA (RAPD) method efficiently discriminated each Angelica variety. A. acutiloba var. sugiyamae was identified via a characteristic fragment amplified by the decamer primer OPD-15. This fragment showed polymorphisms among Angelica species/varieties. The unique fragment derived from A. acutiloba var. sugiyamae was also found in one strain of A. acutiloba var. acutiloba, implying that this strain arose from outcrossing between A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae. This RAPD marker technique will be useful for practical and accurate authentication among A. acutiloba var. acutiloba, A. acutiloba var. sugiyamae, and their adulterants.

Rapid detection of self-biting disease of mink by specific sequence-characterized amplified regions

Self-biting disease occurred in most farmed fur animals in the world. The mechanism and rapid detection method of this disease has not been reported. We applied bulked sergeant analysis （BSA） in combination with RAPD method to analyze a molecular genetic marker linked with self-biting trait in mink group. The molecular marker was converted into sequence-characterized amplified regions （SCAR） marker for rapid detection of this disease. A single RAPD marker A8 amplified a specific band of 263bp in self-biting minks, which was designated as SRA8-250, and non-specific band of 315bp in both self-biting and healthy minks. The sequences of the bands exhibited 75% and 88% similarity to Canis familiarizes major histocompatibility complex （MHC） class II region and Macaca mulatta MHC class I region, respectively. A SCAR marker SCAR-A8 was designed for the specific fragment SRA8-250 and validated in 30 self-biting minks and 30 healthy minks. Positive amplification of SCAR-A8 was detected in 24 self-biting minks and 12 healthy minks. χ2 test showed significant difference （p〈0.01） in the detection rate between the two groups. This indicated that SRA8-250 can be used as a positive marker to detect self-biting disease in minks. Furthermore, the finding that self-biting disease links with MHC genes has significant implications for the mechanism of the disease.

Panax ginseng-specific sequence characterized amplified region (SCAR) marker for testing medicinal products

To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA （RAPD） was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced （Genl3ank access number KU553472）. Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples.

Identification of Random Amplified Polymorphic DNA Markers Linked to Sex Determination in Calamus simplicifolius C. F. Wci

The random amplified polymorphic DNA （RAPD） molecular marker technique was used to determine the sex of Calamus simplicifolius C. F. Wei In the present study, DNA samples were extracted individually from 10 male and 10 female plants. After a total of 1 040 decamer primers had been tested, an approximate 500-bp male-specific DNA fragment was generated with the S 1443 primer. It is feasible to identify sex at the early stages of plant life, which is beneficial for improving breeding programs of this dioecious species. In addition, we have obtained a proper RAPD protocol that is useful for other species of rattan.

栽培稻和普通野生稻基因组的随机扩增多态性DNA（RAPD）初步分析

用３５个随机引物对５个籼稻品种、５个粳稻品种和２７份中国普通野生稻进行了ＲＡＰＤ分析。６０％以上的引物能在籼稻和粳稻基因组间显示差异；中国普通野生稻与栽培稻的差异主要表现在与籼稻的不同，绝大部分供试野生稻的ＲＡＰＤ带型与粳稻的相同。这说明多数中国普通野生稻偏粳，但也存在偏籼的普通野生稻。

我国杂交水稻主要恢复系的DNA多态性研究

利用 RAPD引物对我国杂交稻主要的 31个恢复系进行了 DNA多态性分析。从 15 2个随机引物中筛选出 48个引物扩增恢复系的基因组 DNA,选取 9个引物的扩增结果进行分析 ,共获得 78条带 ,其中 6 1条带为多态性标记 ,每个引物平均提供 8.7个标记信息。由 UPMGA方法得到的聚类分析结果表明了 31个恢复系间的亲缘关系 ,聚类结果与它们的系谱关系基本吻合。

三系杂交稻亲本随机扩增多态性DNA(RAPD)分析

选用９个随机引物对３１份杂交水稻亲本材料进行了ＲＡＰＤ分析，共检测到６０条多态性带。聚类分析结果表明，所有供试材料可以被明确地区分。在９个随机引物中，有８个具有较高的多态性检测能力。以这８个引物为基础，选用任两个引物即可在任一对材料中检测出多态性的频率在９６１３％以上，而选用任３个引物则该频率在９９２１％以上。这显示了运用ＲＡＰＤ鉴定稻种具有简便、灵敏、高效的优点，在鉴定杂交稻种的实践中有着良好的应用前景。

植物中药材总DNA提取方法的比较

随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术已经应用于中药材的鉴别，如何从多种中药材提取到总ＤＮＡ来进行相关性研究是一个关键的课题。从植物中提取基因组ＤＮＡ的方案众多，其中较为流行的方案有三类：（１）用苯酚－氯仿（变性蛋白）作为提取介质。（２）用ＣＴＡＢ（既能裂解细胞壁，又能去除多糖）作为提取介质。（３）用高盐低ｐＨ介质作为提取介质（有效的蛋白沉淀剂）。经过比较，我们认为第（３）套方案快捷、经济，比较适于基层单位应用。

密穗高粱总DNA导入水稻的研究

为了选育高产、优质、多抗的水稻新品种，采用花粉管通道法，将密穗高粱总ＤＮＡ导入水稻晚粳品种鄂宜１０５，从Ｄ１至Ｄ５代变异材料中选育出ＤＨ３，ＤＨ４，ＤＨ５等多个具有高光效、高产和优质的新品系．经随机扩增多态ＤＮＡ技术对供体、受体、后代进行分子验证，后代中出现了在供体中存在而在受体中不存在的泳带．解剖学观察发现了后代剑叶、穗颈及穗基的输导组织比受体发达，维管束数目比受体增加１０％～３０％．后代ＤＨ３，ＤＨ４的光合速率亦显著高于受体．在形态解剖、光合特性和分子水平上进一步证明了高粱ＤＮＡ片段在受体中的表达和稳定遗传．对ＤＨ３，ＤＨ４，ＤＨ５等品系进行生产示范，增产效果显著，单产高达７５００ｋｇ／ｈｍ２．ＤＨ５已于１９９９年２月通过长沙市品种审定，定名为长晚粳１号．米质分析结果表明，整精米率、蛋白质含量。

芸薹类蔬菜基因组DNA遗传多样性的RAPD分析

采用随机引物扩增多态性 DNA( RAPD)技术 ,对芸薹类 ( 2 n=2 0 )蔬菜作物的 3 3个品种进行了遗传多样性检测 .从 70个引物中筛选出 3 7个引物 ,共扩增出 3 53带 .其中 ,多态带有 2 60条 ,扩增片段长度大多数集中在 0 .9～ 1 .6kb之间 .不同引物的检测效率相差很大 .运用 5个引物扩增的 1 0条RAPD特征带 ,可以作为一组 DNA指纹 ,区分所有供试

随机扩增多态性DNA分型法用于酵母菌基因分型

目的研究酵母菌随机扩增多态性DNA分型法(RAPD)的最佳实验条件。方法在改变引物、Mg2+、模板浓度、Taq多聚酶用量等不同条件下,采用RAPD法对酵母菌进行基因分型。结果经优化后的RAPD方法能清晰地对酵母菌进行基因分型。结论RAPD是从分子水平对酵母菌感染进行病原学、流行病学研究的一种快速、可靠的分子生物学分型方法。

随机扩增多态性DNA技术在中药红曲基原菌系统分类中的应用

目的 :建立中药红曲基原菌的随机扩增多态性 DNA( RAPD)分析方法。 方法 :以 CTAB法提取紫色红曲霉等 7种红曲霉和 1株土曲霉的基因组 DNA;溴化乙啶荧光强度和分光光度双重测定法确定 DNA浓度 ;琼脂糖凝胶电泳法检测 PCR扩增产物 ,PHYL IP 3.5 c进行聚类分析。 结果 :从 6 0个随机引物中筛选出 S2 82 等 10个引物的扩增产物谱带多 ,特征好 ;红曲霉属种间指纹图谱差异较大 ,呈现出明显的 DNA扩增产物多态性。 结论 :RAPD分析技术可作为真菌系统分类的客观而有效的手段之一。

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增，共获扩增片段１０３ 条，分子量在０．３ － ３ｋｂ 之间，其中５ 种引物ＯＰＪ－ １ ，７ ，９，１１ ，１２ 扩增出差异片段１２ 条。结果表明，低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变；ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响，以及氮离子注入诱变效应的可能机制。

广东野百合DNA提取和RAPD条件的优化

以野百合(Lilium brownii)新鲜叶片、硅胶干燥叶片及鳞片为材料,研究了DNA的提取方法,并对影响随机扩增多态DNA(RAPD)反应的各因素进行了优化。建立了野百合RAPD的优化反应体系及程序,即在20μl反应体系中,含20 ng模板DNA,2.0 mmol/L Mg2+、0.2 mmol/L dNTPs、1.5 U Taq DNA聚合酶、0.3μmol/L随机引物S1519;扩增程序为:94℃预变性5 min,然后94℃30 s,38℃50 s,72℃1 min,35个循环,最后72℃延伸10 min,4℃保存。

野生与笼养绿孔雀种群的随机扩增多态DNA研究

利用随机扩增多态DNA(RAPD)技术对野生 14只和笼养 18只绿孔雀 (Pavomuticus)个体进行了种群遗传多样性分析。用 2 3个随机引物 ,野生与笼养绿孔雀分别获得 16 1和 16 6个扩增片段 ,计算发现野生与笼养绿孔雀的种群内平均相对遗传距离分别是 0 .0 5 5 5和 0 .135 5 ,两种群间的为 0 .16 35 ;两种群的Shannon多样性指数平均分别是 0 .434 8和 1.0 16 3,有显著性差异。以上分析都显示野生绿孔雀的遗传多样性很低。用UPGMA法聚类显示两个种群都是分别来源于两个家系 ,可据此进行繁育管理。

杂交一代(尼罗罗非鱼♀×奥利亚罗非鱼♂)及其亲本基因组DNA的比较

采用RAPD技术 ,用 2 0个随机引物对尼罗罗非鱼、奥利亚罗非鱼及其杂交一代的精巢DNA进行扩增反应 ,发现引物OPZ - 14和OPZ - 18在尼罗罗非鱼和奥利亚罗非鱼之间扩增出有差异的DNA片段 ,作为鉴定两种鱼的分子标记有较高可信度。对这两种鱼的杂交一代基因组DNA的RAPD扩增能获得足够的多态性 ,2 0个引物中有 8个引物扩增出差异性产物 ,表明杂交一代基因杂合性增强 ,这是杂种优势得以形成的重要遗传物质基础之一。

西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。

用随机扩增多态DNA(RAPD)技术鉴别中药材天花粉及其类似品

目的：对来源于１３个种３个变种的天花粉及其类似品进行鉴别研究，并进行方法学的探讨。方法：应用随机扩增多态ＤＮＡ（ＲＡＰＤ）技术进行鉴别，采用聚类分析方法分析结果，并对药材贮存时间及产地对实验结果的影响进行探讨。结果：把天花粉正品与类似品有效地分成三大类。结论：认为在实验时采取设对照组，对结果采用聚类分析等方法，ＲＡＰＤ技术鉴别药材具有一定的可靠性和实用价值，这为解决粉末及破碎药材的鉴别提供了新的方法。