桔梗SRAP反应体系的优化

目的：建立适合桔梗的比较稳定的SRAP反应体系,用于桔梗遗传多样性分析。方法：用改进的CTAB法提取桔梗叶片的总DNA,通过对不同镁离子浓度、dNTP浓度、模板DNA含量、引物浓度、DNA聚合酶量条件下的SRAP扩增反应的效果。结果：桔梗SRAP扩增反应的最佳体系：模板DNA 20ng,引物0.8μmol/L,dNTP150μmol/L,MgCl22.0mmol/L,TaqDNA聚合酶1unit,10×Buffer2.0μL;反应程序为94℃预变性5min;94℃变性1min,35℃退火1min,72℃延伸1min,5个循环;94℃变性1min,50℃退火1min,72℃延伸1min,35个循环;最后72℃延伸5min,4℃保存。结论：按此优化的SRAP条件进行实验,重现性良好,可用于桔梗遗传多样性分析。     

高山嵩草种群在放牧干扰下遗传多样性的变化

利用SRAP (Sequence-related amplified polymorphism)分子标记, 对放牧干扰下的高山嵩草(Kobresia pygmaea)种群进行了遗传多样性研究, 获得了下述结果: 1) 20对SRAP引物组合共检测出448条清晰条带, 其中376条条带具有多态性, 多态位点百分率为83.93%, 随着放牧强度的增加, 高山嵩草种群多态位点百分数、Nei’s遗传多样性指数、Shannon信息指数均下降。2)高山嵩草种群具有较高的遗传多样性和较低的遗传分化(总的遗传多样性H_t为0.276 6, 种群内遗传多样性H_s为0.243 6, 遗传分化系数G_st为0.119 4, 基于G_st估计的基因流N_m^*为1.843 4), 但随着放牧强度的增加, G_st增加, N_m^*降低, 说明放牧限制了种群间的基因交流, 使种群发生遗传分化。3)不同放牧梯度的高山嵩草种群间的遗传距离很小, 但是随着放牧强度的增加, 种群间的遗传距离逐渐增加, 遗传一致度降低。根据遗传距离所构建的UPGMA聚类图中高山嵩草4个种群随着牧压的增加, 逐级聚在一起。     

草鱼种质相关SRAP及SCAR的分子标记

采用相关序列扩增多态性(Sequence-realted Amplified Polymorphism, SRAP)技术分析野生草鱼和家养草鱼,筛选与草鱼种质退化相关的分子遗传标记.共进行88对引物组合的检测, 产生标记数目共计905个.依据标记在群体中出现的频率和变化规律,共筛选出2 1个可能与种质相关的特异性标记,对这些特异性标记进行测序并将测序结果进行BLAST分析 .发现测得片段中有8个片段在GenBank中找到同源性较高的序列,而其他片段与数据库中序列的相似性较低.根据序列信息分别设计了3对引物.用这3对引物分别对草鱼三个群体进行 PCR扩增,分别产生了SCAR1(308 bp)、SCAR2(66 bp)、SCAR3(114 bp)3个扩增带.采用大样本对这3个标记进行验证,发现其中SCAR1在家养群体中呈现阳性,在野生群体中为阴性,可区分出这两种群体.以SCAR3为引物在174条家养群体中得到目的片段,在26个家养群体没有扩增出条带,分布频率为87%;在100个野生群体中有6个个体检测到该条带,分布频率为6%.以SCAR2为引物在野生群体中完全扩增出目的条带,淡水中心群体中有7条扩增到条带,前洲群体中没有扩增出条带,标记在家养种群中的分布频率为96.50%.因此SCAR1可作为草鱼家养群体的一个重要的分子遗传特征指标,为进一步进行分子标记辅助育种奠定了基础 [动物学报 54(3):475-481,2008].     

新疆石榴种质资源遗传多样性的SRAP分析

采用SRAP分子标记技术,对21份新疆石榴品种的遗传多样性与亲缘关系进行分析。从36对引物组合中筛选出16对扩增条带清晰、多态性高的引物组合分析供试材料,共检测出271个位点,其中194个为多态性位点,多态性比率达71.59%,平均每对引物组合产生16.94个位点和12.13个多态性位点。21份石榴样品间的相似性系数为0.63~0.89,平均为0.77。UPGMA聚类结果显示,在相似性系数为0.776时,21份材料可被分为5个类群。新疆石榴品种间平均观察等位基因数(Na)、有效等位基因数(Ne)、Neis基因多样性指数(H)及Shannons信息指数(I)分别为1.712 2、1.352 8、0.199 9及0.310 5。研究表明,新疆喀什地区石榴品种的遗传多样性最为丰富;SRAP聚类结果与石榴的表型特征存在一致性,且SRAP分子标记技术是研究石榴遗传多样性简单而有效的手段。     

番木瓜基因型遗传关系的SRAP分析

利用SRAP标记对中国22个番木瓜主要栽培品种（系）进行亲缘关系和遗传多样性研究。结果表明:（1）筛选出的20对SRAP引物共扩增249条谱带,其中多态性条带110条,多态性比率为43.20%,平均每对引物扩增的条带数和多态性条带数分别为12.45条和5.50条。（2）引物多态性信息含量（PIC）值变化范围为0.15～0.79,平均值为0.49。（3）聚类分析和主坐标分析显示,供试材料间的遗传相似系数为0.72～0.96,遗传多样性水平较低,在相似性系数为0.87时,可将所有试材分为3个类群。该研究结果有效地揭示了中国番木瓜主要栽培品种（系）资源的遗传背景和亲缘关系,可为番木瓜种质资源的分类、保护和有效利用以及新品种选育提供理论依据。     

利用SRAP标记研究四个暗纹东方鲀群体的遗传多样性

利用相关序列扩增多态性SRAP(Sequence-related amplified polymorphism)分子标记首次对4个暗纹东方鲀(Takifugu obscurus)群体(1个长江捕捞群体、1个放流群体和2个养殖群体)进行了遗传多样性分析。从49对引物组合中筛选出18对扩增条带清晰、稳定的引物组合对4个群体进行扩增,共获得231个位点,其中多态位点数为156个,总多态位点百分率为67.53%。4个群体内多态位点比例为54.98%—58.87%,群体的Nei’s基因多样性指数(H)为0.1992—0.2005,群体Shannon多样性指数(I)为0.2953—0.3016,群体间基因流(Nm)为4.1291。长江捕捞群体的多态位点比例、基因多样性指数、Shannon多样性指数均略高于其他三个群体。采用UPGMA法对4个群体进行聚类分析显示,上海养殖群体单独聚为一类,其余3个群体聚为另一类。AMOVA结果表明遗传变异主要来源于群体内,占总遗传变异的87.40%。以上结果表明,4个暗纹东方鲀群体具有较高的遗传多样性,群体间相似性较大,并且存在一定的基因交流。     

Genetic diversity and population structure of Opisthopappus longilobus and Opisthopappus taihangensis (Asteraceae) in China determined using sequence related amplified polymorphism markers

The genetic diversity and structure of four Opisthopappus longilobus and nine Opisthopappus taihangensis populations from Mts. Taihang were analyzed using SRAP markers. The results showed that O. longilobus (PPB = 95.16%, H = 0.349, I = 0.517) and O. taihangensis populations (PPB = 94.58%, H = 0.332, I = 0.504) had high genetic diversity. The genetic diversity within populations varied according to two species. Low genetic differentiation was detected among O. longilobus (Φ_ST = 0.105) and O. taihangensis (Φ_ST = 0.188) populations, respectively. Significantly genetic variation was found between O. longilobus and O. taihangensis (Φ_ST = 0.232). The dendrogram generated from pairwise genetic distance among all populations showed two distinct clusters, each corresponding to one species. A Mantel test revealed insignificant positive correlation between genetic distances and geographic distances (r_{O. longilobus} = 0.3021; r_{O. taihangensis} = 0.3658; P > 0.05). Those results indicated that self-compatibility, effective gene flow, biological traits, and historical factors may be the main factors causing geographical differentiation in the structure of O. longilobus and O. taihangensis populations.     

Genetic diversity analysis of Pinellia ternata based on SRAP and TRAP markers

An assessment of the genetic diversity and variation of Pinellia ternata collected from 43 populations in China was undertaken using SRAP + TRAP markers. A total of 13 SRAP primers in addition to 3 TRAP primer combinations yielded 292 bands in total of which 286 were polymorphic (98.0%), with an average of 16 for each. The PIC value ranged from 0.88 to 0.95, with a mean polymorphic information content (PIC) of 0.92 over all the primers. A cluster analysis was conducted based on the unweighted pair group method with arithmetic average (UPGMA), and a principle coordinate analysis (PCA) was performed relying on Nei's genetic distance, both of which showed similar outcomes in that the results of clustering did not correlate to the geography locations but showed an association with morphology. An analysis of molecular variance (AMOVA) was performed to detect the source of variation; the results indicated that the main variation existed within populations (67.2%), whereas there was still 32.8% differentiation existing among the populations.     

基于SRAP分子标记新疆野核桃的遗传多样性分析

利用SRAP分子标记技术对新疆野核桃遗传多样性进行分析。通过筛选出的15对具有多态性的SRAP引物组合进行PCR扩增,得到新疆野核桃遗传分化系数Gst为0.1152,说明新疆野核桃的遗传变异绝大部分存在于区域内部,占总变异的88.48%;多态位点百分率为94.07%,Shannon's信息指数I=0.4954,等位基因平均数Na=1.9454,表明新疆野核桃具有较高的遗传多样性;各区域间遗传相似系数在0.8981~0.9496之间,遗传距离在0.0553~0.1075之间,说明新疆野核桃资源间存在着丰富的遗传变异。通过聚类分析可聚为2类,进一步明确了新疆野核桃各区域之间的亲缘关系。     

Genetic diversity of Hypericum perforatum collected from the Qinling Mountains of China

The genetic diversity among samples of Hypericum perforatum L. collected from the Qinling Mountains in China were studied using SRAP molecular markers and 12 SRAP primer combinations. A total of 183 bands and 153 polymorphic bands were obtained that accounted for 83.6% of the polymorphism. Analysis by the UPGMA method grouped the samples into three main clusters, with coefficients of similarity ranging from 0.57 to 0.97. Morphological traits were correlated with four of the contents of phenolic compounds, chlorogenic acid, rutin, hyperoside and quercitroside. Correlation analysis revealed that the hyperoside concentration was significantly and positively correlated with the density of black glands found along the vertical edges of the stem. The information generated by the SRAP markers was closely associated with some morphological variability. However, there was only a partial correlation between the chemistry and genetic data.