雷公藤ISSR反应体系的建立与优化

[目的]建立适合雷公藤的ISSR-PCR反应体系,为雷公藤种质资源的亲缘关系鉴定和遗传多样性分析提供技术支持.[方法]采用单因素试验设计优化影响雷公藤ISSR-PCR扩增效果的Taq DNA聚合酶、dNTPs、Mg2+、引物浓度和DNA模板含量及退火温度,并以32份雷公藤样品对优化的ISSR-PCR反应体系进行验证.[结果]优化的雷公藤种质资源ISSR-PCR反应体系为:20.00μL反应液中含1.50 U Taq DNA聚合酶、0.30 mmol/L dNTPs、2.00 mmol/L Mg2+、0.40μmol/L引物、20 ng DNA模板和2.00μL 10×Buffer.ISSR-PCR扩增程序中最佳退火温度为54.7℃.[结论]建立优化的雷公藤ISSR-PCR反应体系具有较高的稳定性、重现性和适用性,可应用于雷公藤不同地理种源间的遗传多样性鉴定.     

杨树腐烂病菌表观特征及遗传多样性分析

为探究杨树腐烂病菌的表观特征、遗传多样性以及它们与不同寄主杨树派别和病原菌地理来源的关系,本文收集来自我国14个省份的106份杨树腐烂病标本,纯培养观察记录菌落生长状况,分析菌株培养形态多样性,并从中选取背景信息差异较大的菌株34份,采用SRAP和ISSR两种标记方法分析其遗传多样性。结果显示:杨树腐烂病菌同一菌株在不同条件下的培养性状稳定;不同菌株的培养形态差异明显,且各类别间菌株的培养性状与寄主植物、地理来源没有明显相关性。利用获得多态性良好的12对SRAP引物和20条ISSR引物分析得出群体的Nei's多样性指数为0.31(SRAP)、0.28(ISSR),各菌株间存在较大的遗传差异;并从寄主角度分析其群体遗传结构,得出群体总的遗传多样性为0.27,群体内的遗传多样性为0.23,可以解释总体多样性的87%,遗传分化系数(G_ST)为0.13,遗传变异主要存在于群体内。从地理角度分析得出,总的群体遗传多样性为0.28,群体内的遗传多样性为0.13,可以解释总体多样性的48%,G_ST为0.52,来自群体内和群体间的多样性差异相当,而地理群体间的迁移对总体的遗传分化产生了较大的影响,各群体的遗传多样性水平表现为从西到东依次降低的趋势。      

白花泡桐11个种源间亲缘关系的ISSR分析

[目的]对白花泡桐11个种源间亲缘关系进行ISSR分析。[方法]选取10个不同地理种源的白花泡桐个体及"绿树"(又名新桐)无性系组培苗为研究对象,采用ISSR分子标记进行分析。[结果]共筛选出22对有效引物,扩增出112条带,其中有67条多态带,多态性比例为59.82%。根据ISSR聚类分析结果,在遗传距离为0.280时,11份白花泡桐材料可分为6类,第1类为江西、福建种源个体;第2类为"绿树"无性系与湖南、湖北、河南、浙江种源个体;第3类为江苏种源个体;第4类为河北种源个体;第5类为广西种源个体;第6类为广东种源个体。[结论]筛选得到的引物可以有效地将不同种源加以区分,并且能准确地将"绿树"无性系从众多种源中鉴定出来,该研究结果一方面为白花泡桐的品种选育提供分子辅助育种的技术支持,另一方面也有助于"绿树"无性系的鉴定及知识产权的维护。     

Genetic diversity of the complex Paspalum notatum Flügge (Paniceae: Panicoideae)

The genus Paspalum L. comprises approximately 400 species worldwide and about 220 in Brazil. Paspalum is ecologically and economically important, and has been very useful as pasture and P. notatum Flügge (bahiagrass) is a valuable forage grass in the subtropics. This species consists of several sexual (diploid) and apomictic (tetraploid, ocasionally tri and pentaploids) biotypes. In this work, inter simple sequence repeats (ISSR) markers were used to assess the genetic variability of a bahiagrass (P. notatum) collection. Vegetative tissues of 95 bahiagrass accessions were obtained from various locations in South America (Brazil, Argentina and Uruguay). A total of 91 reproducible ISSR fragments were observed and 89 fragments (97.5% of the total observed) were polymorphic. Cluster analyses (UPGMA) were performed from the ISSR data set and the results illustrate the genetic relationships among the 95 accessions of P. notatum. A comparison among molecular, morphological and ploidy levels data were done. ISSR markers were effective in distinguishing the genotypes analyzed, and a wide variability was observed for this species. These results add new information regarding the genetic diversity in P. notatum, thus contributing toward the biological knowledge of this species, and providing with subsides for future plant breeding and conservation programs.     

Genetic erosion and in situ conservation of Lima bean (Phaseolus lunatus L.) landraces in its Mesoamerican diversity center

Lima bean (Phaseolus lunatus L.) is an important crop in traditional Mayan agriculture of the Yucatan Peninsula, Mexico, its Mesoamerican center of diversity. Genetic erosion in this species is currently a threat in this region out of 3 of 21 landraces dominate 71.24% of the cultivated area, and 12 are rare landraces grown only in 6.29%. Using 90 ISSR loci, we estimated the diversity and genetic relationships for 21 landraces to analyzing their risk of genetic erosion, and generate data for their in situ conservation. Total genetic diversity was high (h = 0.29), however it was lower than wild gene pool reported (h = 0.69). The abundant landraces had genetic diversity values lower (h = 0.13, I = 0.17) than the common (h = 0.26, I = 0.33) and rare landraces (h = 0.24, I = 0.27). However, the rare landraces are in a higher risk of genetic erosion due to local extinction. The cluster analysis showed no groups corresponding to morpho-phenological characteristics, geographic origin or traditional classification, which resulted from high inter-landraces gene flow levels. The molecular data confirmed that the domesticated Lima bean pool of the Yucatan Peninsula has a high risk of genetic erosion. If current tendencies in landrace cultivation continue, many will no longer be planted within two to three generations, with a consequent loss of their alleles. Programs urgently need to be established for in situ conservation of Lima bean landraces in this region.     

九节茶ISSR反应体系的建立及优化

以九节茶叶片提取的基因组DNA为材料,对影响ISSR-PCR扩增效果的一些因素,诸如dNTPs浓度、TaqDNA聚合酶用量、引物用量、模板DNA用量、退火温度以及循环次数等指标进行筛选和优化.结果表明:20μL ISSR反应体系含10×PCR Buffer、0.4 mmol.L-1dNTPs、2 UTaqDNA聚合酶、0.6μmol.μL-1引物、5 ng模板DNA;PCR扩增程序为:94℃预变性2 min,94℃变性30 s,44.8℃退火30 s,72℃延伸1 min,35个循环,最后于72℃延伸7 min,置4℃保存.应用该ISSR体系对10份九节茶种质进行了扩增,证实了该体系的适用性和稳定性.     

基于ISSR标记的伏山楂起源及分类地位研究

利用ISSR标记对59份山楂材料进行了遗传多样性分析.从51条引物中筛选出15条,用于山楂的ISSR扩增,共扩增出122条带,其中多态性条带113条,多态性为92.62%.根据ISSR扩增结果,利用DPS软件进行分析,通过UPGMA对59份材料进行聚类分析,认为伏山楂为山楂属的一个独立种,与山楂亲缘关系较近.同时,探讨了我国北方分布的山楂属植物的亲缘关系.     

丁(鱼岁)养殖群体遗传多样性的ISSR分析

[目的]分析丁[鱼][岁]养殖群体的遗传多样性水平,为其种质资源的评价、保护和合理利用提供依据。[方法]利用简单重复序列区间扩增多态性（ISSR）分子标记技术。[结果]选用77个随机引物对其20个个体的基因组DNA进行PCR扩增,有18个引物可以扩增出稳定且清晰的条带,共扩增出115条稳定的DNA位点,片段大小在100-2000bp,其中多态性位点14个,多态位点比例为12.17％。个体间遗传距离为0-0.1376,平均为0.0329±0.0056。[结论]丁[鱼][岁]群体的遗传多样性水平较低。     

基于ISSR分子标记的桉树遗传分析

采用ISSR分子标记技术对5个桉树原种和3个推广的杂交桉树品种进行基因组DNA多态性分析。从100条引物中筛选出10条用于ISSR-PCR扩增,共扩增出155条DNA条带,其中多态性条带148条,占总条带数的95.5%。通过NTSY Spc 2.10e软件分析ISSR扩增结果,结果表明,桉树资源具有较丰富的遗传多样性,8份桉树材料的遗传距离(GD)值范围为0.419 5～1.003 4,遗传相似性(SM)值范围为0.420 0～0.693 3。应用UPGMA法进行聚类分析,将8份桉树材料划分为3个大类,邓恩桉和赤桉为一类;巨尾桉、尾巨桉、尾细桉、韦塔桉、粗皮桉为一类;托里桉则独成一类。     

绿茶DNA提取方法及分子鉴定的初步研究

以绿茶为原料,通过改进的CTAB方法来提取分离绿茶总DNA,实验表明,所采用改进的CTAB 微量提取DNA法,可以得到高质量的绿茶总DNA,1.0 g茶样DNA含量在101～498 μg/g之间,平均249 μg/g。采用ISSR分子标记对10个绿茶产品进行鉴定,结果表明ISSR标记能有效地区别不同的绿茶产品,为进一步研究绿茶分子鉴定技术提取了参考依据。