转基因白桦的遗传变异分析

应用细胞学方法分析了由农杆菌介导法获得的转基因白桦的细胞学变异情况,结果表明转基因白桦的染色体变异频率为78.5％,远远高于非转基因白桦的变异频率(15.3％),且变异以非整倍体占多数。同时用RAPD标记方法研究了转基因白桦在DNA水平的变异情况,结果显示DNA多态性指数为31.67,并与其它转基因植物的变异情况作了比较研究。最后分析、讨论了产生变异的原因:(1)组织培养过程中产生突变;(2)外源基因的整合及重排时宿主基因组的插入位点及相邻基因转录表达的干扰;(3)应用抗生素和除草剂等筛选转基因植株时促进了转基因植株的变异程度。并提出减少转基因植物体细胞克隆变异的建议。     

转基因植物的表型变异、分子检测与遗传分析

本讨论了转化方法、体细胞克隆和选育过程等影响转基因植物表型变异的因素,并对转基因植物不同群体的表型变异组成和效应进行了比较分析,提出了转基因植物分子检测和遗传分析的技术策略。多数情况下,分析转基因植物回交后代(BClF1)比分析T1代能获得更可靠和有价值的结论。     

不同种源桃金娘表型性状多样性研究

为解析桃金娘表型性状多样性及其种源间关系，该文以20个不同来源的桃金娘为研究对象，在同质园栽培条件下，对其营养器官和花器官表型性状进行观测，采用方差分析、变异分析、Shannon-Wiener多样性指数分析和聚类分析等方法，探讨不同种源桃金娘表型性状多样性。结果表明：(1)不同种源桃金娘表型性状存在显著差异(P<0.05),Shannon-Wiener多样性指数均值在1.35以上，表型性状多样性丰富。(2)种源内表型性状变异系数均值在10.81%～63.75%之间，种源间的变异系数均值在13.08%～74.04%之间，种源间变异(23.33%)高于种源内变异(19.79%),营养器官变异(29.52%)高于花器官变异(14.06%)。(3)部分性状存在极显著或显著相关性，株高与分枝数呈极显著负相关，而与叶长、叶宽和叶面积等却呈显著正相关。(4)在欧式距离10处，20个种源桃金娘可分为A、B、C三类，A类包含8个种源，该类种源表现为植株高大、分枝少、叶大和花大等特征；B类包含11个种源，该类种源表现为株高中等、叶较大和花中等等特征；C类仅包含1个种源，表现为植株低矮、分枝多、叶小和...     

抗冻蛋白(afp)基因表达载体的构建及对香蕉胚性细胞悬浮系的转化

通过PCR从‘京都七寸人参'胡萝卜基因组DNA中扩增抗冻蛋白基因,测序结果表明该基因的核苷酸序列与从宁夏‘吴忠'胡萝卜中克隆的完全一致。先后将获得的胡萝卜afp基因克隆和亚克隆至pMD18-T和pBI121,构建植物表达载体pBI121-afp。通过冻融法将pBI121-afp导入根癌农杆菌EHA105中。以香蕉栽培品种‘北大矮蕉'的胚性细胞悬浮系为受体,采用农杆菌介导法将胡萝卜afp基因导入其中,然后在Kanamycin的选择压力下通过体细胞胚发生途径进行植株再生。共获得抗性再生植株9株,其中两株经PCR检测呈阳性,可初步确定目的基因已经整合到这两株转基因香蕉植株的基因组中。     

云南含笑天然居群的表型多样性分析

为揭示云南含笑天然居群表型变异程度和变异规律,以云南昆明地区天然分布的云南含笑为研究对象,调查了6个居群180个单株的14个表型性状,采用巢式方差分析、变异系数、相关分析、Shannon-Weaver多样性指数分析和聚类分析等方法,分析了居群间和居群内表型多样性.结果表明:(1)云南含笑表型性状在居群间和居群内存在极其丰富的多样性,14个表型性状平均表型分化系数(24.38％)小于居群内变异(75.62％),居群内变异是表型变异的主要来源;14个表型性状的变异系数(CV)在16.20％～60.11％之间,表明云南含笑居群内表型性状离散程度较高.(2)对云南含笑各居群的Shannon-Weaver指数分析表明,云南含笑各居群具有丰富的多样性,总体表型多样性指数为1.772.(3)利用居群间欧氏距离进行的UPGMA聚类分析结果表明,云南含笑6个天然居群可以聚为3类,而且表型性状并没有严格依地理距离而聚类.     

空间搭载水稻种子后代基因组多态性及其与空间重离子辐射关系的探讨

神舟三号飞船搭载带核径迹辐射探测器的水稻种子装置,回收后应用随机扩增多态性DNA(randomamplified polymorphic DNA,RAPD)技术,分析了201粒升空种子长出植株的基因组多态性。在检测的189个基因座位范围内,30.2%的植株中发现与地面对照不同的扩增带,单株的多态性座位数为1￣25。特异扩增带的测序及单核苷酸多态性(single-nucleotide polymorphism,SNP)分析进一步证明了空间搭载水稻种子确实可导致当代植株基因组发生变异。同一技术分析个别种子连续世代的基因组多态性,结果显示,当代的部分多态性可遗传至后代。7粒受空间高原子序数、高能粒子轰击的种子,在当代植株均显示不同程度的基因组多态性,从胚受粒子击中的3粒种子后代中,筛选出农艺性状明显变异的突变株系,初步暗示了空间高能重离子辐射对诱导基因组的多态性,乃至遗传性表型变异的有效性。     

根癌农杆菌介导的水稻高效转化系统的建立

比较了影响根癌农杆菌转化水稻的各种因素后,建立了农杆菌介导的水稻高效转基因实验体系。按该体系,水稻品种中花11号预培养4d的幼胚经农杆菌EHA105／pCAMBIA1301感染后,具有GUS基因瞬间表达的幼胚比例在50％以上,最高可达90％;按产生潮霉素抗性愈伤和转基因植株的比例计算,转化率分别达到87．6％和64．6％。转基因植株总DNA的Southern杂交分析表明T－DNA上的外源基因已整合进了水稻基因组,且在大多数转基因植株中表现为单拷贝插入;遗传分析证明T1代的表型分离符合孟德尔法则。此转化系统的建立为高效地将有用的外源DNA导入水稻植株奠定了基础。     

利用改进的MutMap方法克隆水稻雄性不育基因

通过对籼稻黄华占EMS(甲磺酸乙酯)诱变, 筛选得到一隐性核不育的水稻雄性不育突变体osms55, 遗传分析表明该突变体为单基因控制的隐性核不育, 采用高通量的Illumina Infinium iSelect SNP(50 K)芯片检测技术鉴定该突变体的遗传背景, 确认该突变体的遗传背景与黄华占一致。文章利用改进的MutMap方法成功克隆该雄性不育基因, 突变位点与突变表型的共分离分析表明LOC_Os02g40450(MER3)是控制osms55突变体雄性不育的基因, 该基因的剪切识别位点发生变异后导致剪切异常, 造成第5外显子缺失15个碱基, 从而产生雄性不育。改进的MutMap方法无需精确组装的野生型基因组序列作对照, 而是通过将定位群体中有突变表型植株的DNA pool和野生型植株DNA的重测序结果分别与日本晴参考基因组进行比对, 然后再比较突变体和野生型的差异SNP来确定候选基因, 该方法大大降低了野生型基因组测序和组装成本, 进一步扩大了MutMap方法的应用范围。     

灯盏花种质资源群体表型多样性研究

以采集自云南和贵州的21份灯盏花种质资源为材料,观测了它们的群体表型性状特征,并分析了这些表型性状的遗传多样性.结果表明:灯盏花种质资源的表型质量性状在居群内外均有丰富变异,不同质量性状的频率不同,并以多叶型的植株、绿色的茎、疏的茎毛、倒披针型的基生叶、全缘叶、锐尖的叶尖、花色淡紫和黄色的管状花冠口等为代表性表型性状.居群内各数量性状间以单株基生叶数和单株分枝数变异系数较高(均超过50%),而花序直径变异系数最低(18.14%);居群间各表型性状以株高和单序管状花数变异系数较高(52.98%和41.98%),而单株基生叶数和基生叶长变异系数较低(<20%).地理类群间表型分化系数为26.58%,地理类群内表型分化系数为73.42%.灯盏花性状表型多样性指数以株高最高(2.243),以单株分枝数和单株基生叶数较低(1.723和1.874),总体平均表型多样性指数为2.028;不同地理类群的表型多样性指数为1.589~1.890,并以楚雄地区最高,曲靖地区最低.研究发现,灯盏花种质资源具有丰富的质量和数量性状变异,多数性状的地理类群内变异大于地理类群间,且表型多样性指数相对较高,对其地理类群内变异的保护和利用对灯盏花育种具有重要的意义.     

拷贝数变异: 基因组多样性的新形式

基因拷贝数变异是指DNA片段大小范围从kb到Mb的亚微观突变, 是一可能具有致病性、良性或未知临床意义的基因组改变。Fosmid末端配对序列比较策略、比较基因组杂交芯片是当前较多使用的检测手段。染色体非等位的同源重排、非同源突变和非b DNA结构是造成基因组拷贝数变异的重要原因。拷贝数变异可导致不同程度的基因表达差异, 对正常表型的构成及疾病的发生发展具有一定作用。文章在总结基因拷贝数变异的认识过程和研究策略的基础上, 分析了拷贝数变异的形成和作用机制, 介绍了第一代人类基因组拷贝数变异图谱, 阐述了拷贝数变异研究的临床意义, 提示在探索疾病相关的遗传变异时不能错失拷贝数变异这一基因组多样性的新形式。     

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verfied with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram.The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and i their self-pollination progenies.While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points toin vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.     

Genomic stability in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transgenic plants obtained by floral dip

The occurrence of DNA modification is an undesired phenomenon accompanying plant cell transformation. The event has been correlated with the stress imposed by the presently utilised transformation procedures, all depending on plant differentiation from in vitro cell culture, but other causes have not been excluded. In this work, transgenic Arabidopsis thaliana plants have been produced by an approach that does not require cell dedifferentiation, being based on in planta Agrobacterium-mediated gene transfer by flower infiltration, which is followed by recovery and selection of transgenic progeny. Genomic DNA changes in transgenic and control plants have been investigated by AFLP and RAMP analysis. Results show no statistically relevant genomic modifications in transgenic plants, as compared with control untreated plants. Variations were observed in callus-derived A. thaliana plants, thus supporting the conclusion that somaclonal variation is essentially correlated with the stress imposed by the in vitro cell culture, rather than with the integration of a foreign gene.     

Somaclonal variation at the nucleotide sequence level in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) as revealed by RAPD and ISSR markers,and by pairwise sequence analysis

The nature of somaclonal variation at the nucleotide sequence level was studied in rice cv. Nipponbare. First, we investigated genomic variations by using 2 molecular marker systems: RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This was followed by sequencing of selected bands that represented genomic variations, and pairwise sequence analysis taking advantage of the whole genome sequence of rice. In addition, transpositional activity of the active MITE, mPing, was analysed by locus-specific PCR amplifications. The 2-year-old calli and their regenerated plants, analysed with 24 RAPD and 20 ISSR primers, showed moderate levels of genomic variation (20.83% and 17.04%, respectively). To test whether DNA methylation plays a role in somaclonal variation, the calli were treated with 5-azacytidine, a chemical agent that reduces cytosine methylation by blocking the activity of DNA methyltransferase. Though dwarfism occurred in regenerants from treated calli (a hallmark of the drug treatment), there was only a slight increase in the frequency of somaclonal variation detected in the treated calli and their regenerated plants relative to untreated controls. The transposon mPing also remained immobile in both treated and untreated calli. Nevertheless, dendrograms constructed according to the Jaccard coefficient calculated by UPGMA of the ISSR bands revealed that the 5-azacytidine-treated and untreated somaclones were grouped into 2 distinct clusters, suggesting a possible indirect effect of the treatment on the genomic changes, depending on the marker used. Sequence analysis indicated a low level of variation (0.31%), with single-base-pair substitutions predominating.     

Microsatellite DNA somaclonal variation in micropropagated trembling aspen (Populus tremuloides)

Microsatellite DNA markers of ten simple sequence repeat (SSR) loci were used to examine somaclonal variation in randomly selected micropropagated plantlets derived from three different Populus tremuloides donor trees (genotypes). The plantlets were obtained from tissue cultures of dormant vegetative buds, and those derived from the same donor tree, grown in the greenhouse, did not exhibit any sign of visible morphological variation. No microsatellite DNA variation was observed among 13 somaclones of one tree and 4 somaclones of another tree at eight of the ten SSR loci. However, despite the small number of micropropagated progeny per tree sampled, microsatellite DNA variation was detected among the plantlets derived from the same donor trees at two SSR loci. The primer pair for the SSR locus PTR5 revealed somaclonal variation in 1 out of the 13 plantlets obtained from one genotype, while the primer pair for the PTR2 SSR locus revealed somaclonal variation in one out of the four plantlets obtained from another genotype. The variation at the PTR2 locus resulted in the appearance of a new allele of increased size, possibly due to an addition of the repeat units, while the variation at the PTR5 locus resulted in the appearance of third allele, presumably due to the presence of a single extra chromosome or duplication of a chromosomal segment. These results demonstrate that the genetic fidelity of micropropagated plants of P. tremuloides cannot always be assured and somaclonal variation can occur even when tissues of well organized vegetative buds are used for tissue cultures; that somaclonal variation cannot always be detected at the gross morphological level; and that microsatellite DNA markers provide useful and sensitive markers for determining the clonal fidelity and somaclonal variation in P. tremuloides.     

Somaclonal variation in insect‐resistant transgenic sugarcane (Saccharum hybrid) plants produced by cell electroporation

A population of 42 transgenic sugarcane ( hybrid, cv. Ja605) clones expressing a truncated cryIA(b) gene from Bacillus thuringiensis was evaluated in field trials under artificial borer (Diatraea saccharalis Fab.) infection. Five clones displaying the highest borer tolerance were selected and analysed with molecular tools (RAPD, AFLP and RAMP) to verify genomic changes. Results of field trials provided evidence both for the expression of the resistance trait and for the occurrence of limited but consistent morphological, physiological and phytopathological variation, as compared with control plants regenerated from dedifferentiated culture without transformation (C1control) or with plants that were clonally propagated in the field (C2control). The five elite transgenic clones, selected for consistent borerresistance and good agronomic traits, were further evaluated in a large scale field trial. It was found that the majority of agronomic and industrial traits were those of the original cv. Ja605, but that a small number of qualitative traits was different. DNA changes were verified in the five selected clones. A total of 51 polymorphic DNA bands (out of the 1237 analysed bands) was identified by extensive AFLP and RAMP analysis, thus showing rare but consistent genomic changes in the transgenic plants, as compared with C1 and C2control plants. It is proposed that the increased variability verified in transgenic plants by field trials and DNA analysis is essentially correlated with cell growth in the dedifferentiated state during the transformation procedure. The results, which are consistent with those published in the case of other transgenic plant populations, are discussed in the context of selecting approaches to gene transfer that minimize somaclonal variation. This is important especially in cases, such as that of sugarcane, where success of backcrosses to restore the original genotype is made difficult by the complex ploidy state of the plant.     

Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency.

Summary An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15–20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4–5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and -glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7–8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.     

Morphological and molecular analysis of genetic stability in micropropagated Fragaria×Ananassa cv. pocahontas

Summary Micropropagated strawberry plants (Fragaria×ananassa L.) grown on 5 μM and 15 μM BA medium or cold-stored were grown in the field to examine morphological variation. Except for plant height, morphological characteristics did not differ for field-grown plants micropropagated on 5 μM and 15 μM BA medium. Cold-stored plants were less vigorous, both vegetatively and reproductively, than BA-treated plants. Random amplified polymorphic DNA (RAPD) markers were used to determine if cold storage or supraoptimal levels of N⁶-benzyladenine (BA) in the culture medium caused genetic changes leading to somaclonal variation. No mutations were observed in 246 loci amplified by the 29 primers tested. Possible changes in methylation patterns of ribosomal DNA genes of strawberries were also examined. Changes in methylation patterns were observed in only one DNA sample from plants grown on 15 μM BA medium and in one of the cold-stored plants. Length polymorphism was observed in two samples from plantlets derived from one explant. The low levels of RAPD variation and methylation observed, and the apparently epigenetic changes in morphological characteristics in plants used in this study, indicated that mutations had not occurred. Part of a thesis submitted by M. B. K. in partial fulfillment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

General features of chromosome substitutions in Triticum aestivum x T. timopheevii hybrids

Summary Tissue culture of the Zea mays inbred line A188 resulted in the regeneration of plants having a high level of phenotypic variation compared to seed-grown control plants. To determine how such variation was induced and whether this could be related to specific in vitro culture methods, callus cultures were established and maintained on different, commonly used culture media. Plants were regenerated and the genomic DNA of callus cultures and regenerants analysed for RFLP differences. The results show that regardless of the gene probe used, callus formation resulted in significant deviations from the DNA pattern normally found in seed-grown control plants. Alterations in gene copy number also occurred. As differentiation and organogenesis began, the level of DNA variation fell, and most of the regenerated plants showed a genetic similarity to the controls; those with RFLP differences were the somaclonal variants.