六倍体小黑麦品种资源的抗旱性聚类分析

本研究选用10个六倍体小黑麦品种(系)为材料,利用20%PEG-6000模拟干旱,检测各品种芽期的发芽势、发芽率、胚芽鞘长、主根长以及苗期的POD和SOD活性、相对含水量、MDA含量,并计算各性状的抗旱系数（T/CK）,根据抗旱系数的欧氏距离将其聚为两大类,一类为抗旱性强的S1、S3、S5、S8、 S10,另一类为抗旱性弱的S2、S4、S6、S7、S9。同时,利用RAPD分子标记分析其遗传多样性,以0.34为阈值将其分为三类,其中S4、S5为一类;S1、S2、S3、S6、S7、S8、S9为一类, S10自成一类。对比分析发现抗旱性强的一类品种分散分布在分子标记的三大类群里,表明这些分子标记与品种的抗旱性不相关。但反过来可以说明这些抗旱性强的品种其遗传差异较大,遗传变异丰富,为在小麦或小黑麦的抗旱育种中更好地利用这些品种资源提供了依据。     

与黄瓜抗黑星病相关基因紧密连锁的SSR标记

本研究以黄瓜抗黑星病母本Q6和感黑星病父本F51及其F2代分离群体为试材,采用BSA法和SSR技术建立了对黑星病的抗病组和感病组,SSR引物CSWCTT02D在抗感组间表现多态性,且呈共显性.经162个F2单株验证,在高抗单株和高感单株中分别仅扩增出246 bp和256 bp的特异片段,而在中间类型个体中同时扩增出了两个特异片段.连锁分析结果表明,该标记与黄瓜黑星病抗病相关基因紧密连锁,距离为3.1 cM.测序结果显示,两个片段的差异在于10个碱基的插入或缺失.     

利用Cre/loxP系统和rd29A启动子构建诱导型植物标记基因删除载体

出于对转基因作物中标记基因的安全性考虑,植物转基因育种中标记基因的删除将成为未来研究的热点之一。位点特异性重组系统之一Cre／loxP系统是目前在植物遗传转化中应用较多,较成熟的一个标记基因删除系统。为了利用Cre／loxP系统构建一种可调控的标记基因删除系统,本研究首先从拟南芥中克隆了逆境诱导型的启动子rd29A,同时从质粒pCre上克隆了Cre基因,构建了含有rd29A：Cre：Tnos表达元件的中间载体,将这一表达元件插入到另一植物双元表达载体pKsb中,最终构建了含有loxP-Pnos—nptⅡ-Toes—rd29A—Cre-Tnos-loxP的可诱导型删除标记基因nptⅡ的植物双元表达载体。     

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Torr. & Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction (PCR) assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045. Foundation project: This study was supported by the Deutsche Forschungsgemeinschaft DFG (grant number SCHN 1080/1-1)     

两种微卫星多样PCR系统应用于胡杨高效生产基因型

对已在橡树岭国家实验室建立起来的美洲山杨(Populus tremuloides Michx.)和毛果杨(Populus trichocarpa Torr.＆ Gray)18个微卫星引物进行筛选,用于胡杨相关基因的扩增.发现有13个基因位点可以表达基因多态性,范围是2～17个等位基因.选择了8个最易变的基因位点,来构建和优化2种多样PCR法.用来自3个种群的436株树木表征选择的位点,确定他们在亲缘关系分析、基因型研究的适用性.通过克隆基因型鉴定树木性别的交叉检验,我们估计并入基因型最大误差率小于O.045.     

Tryptophan synthesis block-associated sugar response, a physiological marker for rapid selection of Arabidopsis thaliana transformants, marked with feedback-inhibition-insensitive anthranilate synthase gene

The feedback-insensitive anthranilate synthase (AS) gene was used as a selection marker for transformants of Arabidopsis thaliana . The mutant gene ( mAS1-2 ) was constructed by substituting nucleotide at the effector-binding site of the intrinsic AS gene via PCR-mediated site-directed mutagenesis and flanked with the myrosinase promoter pyk10 to drive its expression during initial root elongation. This inducible gene cassette was first introduced into Agrobacterium tumefaciens and then delivered into A. thaliana by floral-dip inoculation. 5-methyltryptophan (5-MT) inhibited AS and suppressed seedling growth of wild type plants as a result of tryptophan starvation. With the addition of sucrose (10 mg/ml), 5-MT inhibited cotyledon opening and caused anthocyanin to accumulate in juvenile seedlings. The present mutant reversed the tryptophan starvation caused by 5-MT and blocked subsequent sugar responses. The sugar responses were detected in non-transformed plants grown on a selection medium containing 10 mg/ml of sucrose and 10 μg/ml of 5-MT after 3 days of incubation. Thus, true transformants could be selected after a short incubation, compared to the conventional kanamycin-selection method that did not eliminate all non-transformed plants.     

A high-throughput, low cost gel-based SNP assay for positional cloning and marker assisted breeding of useful genes in cereals

Several SNP (single nucleotide polymorphism) genotyping methods have been developed in the past most of which require sophisticated instrumentation and large initial investments. We describe here a high-throughput SSCP (single strand conformation polymorphism) system on our HEGS (high efficiency genome scanning) platform, which is simple, accurate, cost effective and requires neither restriction digestion of the amplification products nor elaborate post-PCR processing detection. Several parameters critical to SSCP analysis were optimized viz., gel matrix and concentration, gel running temperature, buffer composition, running conditions and PCR primer design so as to identify SNPs in amplicons ranging from 100 to 750 bp in size. A simple post-PCR processing system was developed using fluorescent dye for quick and easy detection of SNP polymorphism. HEGS-SSCP was also found to be useful in uncovering simple sequence repeat differences between different genotypes that differ by one or few di/tri nucleotide repeats. The practical utility of this system is illustrated with two successful efforts towards construction of high resolution linkage maps of a lesion mimic locus on chromosome 7 and a major quantitative trait locus conditioning field blast resistance on chromosome 4 in rice.     

用SSR标记研究谷子品种的遗传多样性

用37对SSR标记对40个谷子品种(来自我国的37个谷子品种及从印度、俄罗斯引进的3个谷子品种)进行遗传多样性研究,37对引物共检测出228个等位变异,引物检测出的等位变异数在3～12之间,平均每个位点检测出的等位变异数为6.16,37个位点的平均多态信息量(PIC)为0.697.研究这37个SSR标记重复单元出现次数与多态信息量及等位变异数之间的相关性,发现多态信息量与等位变异数呈极显著相关,相关系数达到0.802(P<0.01),而多态信息量与重复单元出现次数之间相关性也达到显著水平.相关系数达到0.429(P<0.01).对40个谷子品种进行聚类,在遗传相似系数为0.79时聚为5组,聚类结果表明除山西外几乎所有来自我国不同地区的谷子育成品种都被聚成1组,多数农家品种聚类群与生态类型存在一定的一致性.     

月季SSR-PCR反应体系的建立和优化

采用L16(54)正交设计和二因素完全随机试验,分析了月季SSR-PCR反应体系的主要成分Taq DNA聚合酶、样本浓度、Mg2+浓度、dNTP浓度以及引物浓度对扩增结果的影响,建立了适合月季的SSR反应体系.研究结果表明:在20μL反应体系中,Taq DNA聚合酶宜加入1.5U,样本最适宜浓度为30～40 ng,Mg2+的最适浓度为1～1.5 mmol/L,dNTP最适浓度为0.2 mmol/L,单引物的最适浓度均为1μmol/L.用建成的反应体系对19对引物筛选,对12个月季品种扩增,3%的琼脂糖凝胶电泳检测,品种间DNA谱带多态性丰富,证明该体系稳定可靠.     

新麦草种质遗传多样性的ISSR分析

利用ISSR标记对15份新麦草(Psathyrostachys juncea)种质资源进行遗传多样性检测.为获得稳定的新麦草ISSR反应体系,对反应条件中的dNTP、Taq酶浓度、引物浓度、Mg_(2+)浓度等因子进行了优化,建立起的最佳反应体系为:25 μL的反应体系中含有dNTP 2.5 μL、Taq酶0.2 μL、Primers 1 μL、Mg~(2+)1.5 μL、2.5 μL 10×Buffer、2 μL模板DNA和ddH2O 15.3 μL.ISSR标记结果显示,筛选出的重复性好、谱带清晰的ISSR引物8条,共获得84个扩增位点,其中多态性位点72个,多态性比率(PPB)为85.7%.通过遗传相似系数(GS)的计算,供试种质材料间的GS值在0.440 5～0.904 8之间,表现出丰富的遗传多样性.通过聚类分析,将15份新麦草种质材料划分为四大类,且呈现出一定的地域性分布规律.