双胚苗水稻中单倍体与二倍体基因表达芯片分析

本研究运用基因芯片技术,对双胚苗水稻SARⅡ-628品系中的单倍体及其对应二倍体的RNA水平的表达变化情况进行对比分析,发现单倍化后基因表达迅速发生了不同程度的变异,主要结果如下：（i）单倍化后,表达变化的序列占水稻总探针序列的2．47％,随机分布在水稻的12条染色体上,激活的序列数量多于沉默的：（ii）有33个序列在染色体上的分布具有成簇分布的特点;（iii）对单倍化中已经预测功能的575条序列进行功能分类,发现都涉及了生物过程、细胞成份和分子功能这3个方面．SNF2家族中控制转录调节的位点表达下降居多;与DNA重组修复相关的RAD54家族中只有2个位点分别发生了表达上升和降低的变化;（iv）采用RT．PCR验证基因芯片的结果,总体上83．78％序列表达结果与芯片结果相符;任选7个极端（激活和沉默）表达方式的序列,其表达结果与芯片相符率达到91．86％．     

不同倍性黄瓜遗传差异的AFLP分析

采用AFLP(Amplified fragment length polymorphism)技术对黄瓜(Cucumis sativusL.)品种‘津绿4号’的单倍体、二倍体和四倍体进行基因组DNA多态性比较。结果表明:(1)从35对引物扩增获得2 188条60~500 bp的条带,多态性位点仅有20个,占0.92%,其中22对引物组合扩增的不同倍性材料的AFLP指纹没有明显差异;(2)在多态性位点表现中,以二倍体的条带存在,单倍体和(或)四倍体的条带丢失为主,在四倍体中扩增出1条特异带;(3)与相应的二倍体相比,单倍体和四倍体有特异片段的消失和增加。     

金鱼etv2基因的克隆及在雌核发育单倍体和自交二倍体胚胎中的差异表达

为研究鱼类雌核发育单倍体循环系统发育异常的原因, 从金鱼(Carassius auratus)中克隆了血管发生主调控基因etv2(Ets variant 2)的全长cDNA序列, 并比较分析了该基因在雌核发育单倍体和自交二倍体中的表达。金鱼etv2 cDNA全长1531 bp, 其开放阅读框为1116 bp, 编码371个氨基酸。序列对比分析表明, 金鱼ETV2蛋白的C端含有ETS(E26 transformation-specific)转录因子家族所共有的ETS DNA结合结构域, 该结构域氨基酸序列与其他脊椎动物ETV2的同源性超过60%。RT-PCR和荧光实时定量PCR分析结果表明, etv2在自交二倍体金鱼成体的肝脏、心脏、肌肉、肾脏、精巢、脑和脾脏等多种器官组织中表达, 但在卵巢和成熟卵子中不表达; 在金鱼胚胎发育过程中, etv2从尾芽期开始表达, 在体节形成后, etv2表达水平随胚胎发育而升高, 在20体节期达到峰值, 随后其表达水平降低。整胚原位杂交显示etv2特异性表达于自交二倍体金鱼胚胎的侧板中胚层、成血管细胞以及血管内皮细胞。在14体节期和20体节期, 雌核发育单倍体胚胎中etv2在躯干及尾部的部分区域表达减弱或缺失, 特别是在胚胎中线表达消失, 并且其整体表达水平显著低于自交二倍体。上述结果说明在金鱼雌核发育单倍体胚胎中成血管细胞数量减少并存在向中线迁移的障碍, 可能是导致雌核发育单倍体血管发生异常的重要原因。     

单倍体高度不育的原因?

答:单倍体是指细胞中含有本物种配子染色体数目(n)的个体。在高等植物中,单倍体植株与相应的二倍体植株比较,一般表现为根、茎、叶、花等器官都较小,有时还会出现白化苗。在减数分裂过程中,由于染色体之间没有相互联会的同源染色体,染色体将无规律地分配到配子中,结果是极大多数不能育成有效的配子,从而表现出高度不育。     

应用微卫星分型方法进行大熊猫父亲鉴定

1979年我们利用水稻花粉单倍体植株的 茎节、幼穗、叶鞘和叶片等组织进行离体培养, 诱导出二倍体植株。     

实时荧光定量PCR检测三七SS基因表达的初步实践

运用含有SYBR Green I的Real Time RT-PCR法分析SS基因在一年生三七根、茎、芦头3个部位中转录水平的相对表达差异。统计分析表明SS基因在根中的表达量最高。本研究取得了特异性高、重复性好的结果,标准曲线斜率均在-3.33～-4范围内,扩增效率均在95%～100%之间,熔解曲线分析显示产物特异性的单一峰,为Real Time RT-PCR技术用于三七植物基因的差异表达分析建立了相应的技术平台。     

菘蓝二倍体及其同源四倍体遗传差异的ISSR分析

以1个菘蓝二倍体及其10个同源四倍体株系为材料,利用19个随机扩增ISSR引物分析其遗传差异性,为菘蓝多倍体诱变的基因表达调控以及遗传改良提供依据。结果显示:菘蓝二倍体与其同源四倍体及四倍体之间的ISSR多态性有明显差异,除主要遗传位点相同外,有些四倍体株系扩增条带数多于二倍体,有些四倍体株系扩增条带数少于二倍体,四倍体株系间亦有多态位点;11个株系共扩增111条多态性条带,多态性达55.22%。聚类分析显示,不同四倍体株系与二倍体的遗传差异大小亦不同。研究表明,菘蓝二倍体与其同源四倍体具有中等偏高的遗传差异性。     

表达乙肝病毒融合表面抗原基因SA-28的二倍体酵母工程菌的选育

将乙肝病毒融合表面抗原基因SA-28的单倍体酵母工程菌Y19/YFD158和与其不同接合型的单倍体酵母菌Y95接合,筛选到二倍体酵母工程菌Y95xY19/YFD158。对两种工程菌的研究表明：二倍体工程菌发酵密度为单倍体工程菌的3倍;表达质粒在二倍体酵母中的稳定性明显高于单倍体工程菌;二倍体工程菌对融合抗原的表达量为单倍体的3倍以上;表达质粒在二倍体细胞中的平均拷贝数略低于单倍体工程菌。     

单套染色体组在泽蛙雌核单倍体发育中的作用

本文比较了泽蛙(Rana limnocharis Boie)单倍体和二倍体的胚胎发育。结果表明:泽蛙单倍体的生活力低下,器官发生异常,自原肠胚起发育速度减慢。据此,作者讨论了单套染色体组在个体发育中的作用。     

表达乙肝病毒融合表面抗原基因SA-28的二倍体酵母工程菌的选育

将乙肝病毒融合表达抗原基因ＳＡ－２８的单倍体酵母工程菌Ｙ１９／ＹＦＤ１５８和与其不同接合型的单倍体酵母菌Ｙ９５接合,筛以二倍体酵母工程菌Ｙ９５ｘＹ１９／ＹＦＤ１５８。对两种工程菌的研究表明：三倍体工程菌发酵密度为单倍休工程菌的３倍;表达质粒在二倍体酵母中的稳定性明显高于单倍体工程菌;二倍体工程菌对融合抗原的表达量为单倍体的３倍以上,表达质粒在二倍体细菌中的平均拷贝数略低于单倍体工程菌。     

The microarray analysis for gene expression in haploids and diploids derived from twin-seedling rice

In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Different degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 sequences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the biological process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.     

The microarray analysis for gene expression in haploids and diploids derived from twin-seedling rice

In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Differ- ent degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 se- quences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the bio- logical process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.     

Mating system and gene flow in the red seaweed Gracilaria gracilis: effect of haploid-diploid life history and intertidal rocky shore landscape on fine-scale genetic structure

The impact of haploid-diploidy and the intertidal landscape on a fine-scale genetic structure was explored in a red seaweed Gracilaria gracilis. The pattern of genetic structure was compared in haploid and diploid stages at a microgeographic scale (< 5 km): a total of 280 haploid and 296 diploid individuals located in six discrete, scattered rock pools were genotyped using seven microsatellite loci. Contrary to the theoretical expectation of predominantly endogamous mating systems in haploid-diploid organisms, G. gracilis showed a clearly allogamous mating system. Although within-population allele frequencies were similar between haploids and diploids, genetic differentiation among haploids was more than twice that of diploids, suggesting that there may be a lag between migration and (local) breeding due to the long generation times in G. gracilis. Weak, but significant, population differentiation was detected in both haploids and diploids and varied with landscape features, and not with geographic distance. Using an assignment test, we establish that effective migration rates varied according to height on the shore. In this intertidal species, biased spore dispersal may occur during the transport of spores and gametes at low tide when small streams flow from high- to lower-shore pools. The longevity of both haploid and diploid free-living stages and the long generation times typical of G. gracilis populations may promote the observed pattern of high genetic diversity within populations relative to that among populations.     

Morphometric and cytogenetic characteristics of haploid tomato plants

Stable differences have been found between haploid and diploid Mikado tomato plants with respect to quantitative characteristics of their vegetative and generative organs, whereas these plants were phenotypically similar with respect to qualitative characteristics, such as the bush habitus and the shapes of leaves, flowers, and fruit. A simple indirect method based on counting the chloroplasts in guard cells has been suggested to diagnose haploids. Analysis of meiosis during microspore formation in haploids has revealed considerable anomalies leading to heterogeneity and a high sterility rate of pollen, which has been confirmed by differences in DNA content. The data obtained suggest that formation of fertile gametes is accounted for by the absence of reduction division. It has been hypothesized that meiosis in haploids induces the genotypic variation that is subject to selection.     

Unpredictable fitness transitions between haploid and diploid strains of the genetically loaded yeast Saccharomyces cerevisiae.

Mutator strains of yeast were used to accumulate random point mutations. Most of the observed changes in fitness were negative and relatively small, although major decreases and increases were also present. The average fitness of haploid strains was lowered by approximately 25% due to the accumulated genetic load. The impact of the load remained basically unchanged when a homozygous diploid was compared with the haploid from which it was derived. In other experiments a heterozygous diploid was compared with the two different loaded haploids from which it was obtained. The fitness of such a loaded diploid was much less reduced and did not correlate with the average fitness of the two haploids. There was a fitness correlation, however, when genetically related heterozygous diploids were compared, indicating that the fitness effects of the new alleles were not entirely lost in the heterozygotes. It is argued here that to explain the observed pattern of fitness transitions it is necessary to invoke nonadditive genetic interactions that go beyond the uniform masking effect of wild-type alleles. Thus, the results gathered with haploids and homozygotes should be extrapolated to heterozygotes with caution when multiple loci contribute to the genetic load.     

Effects of ploidy and sex-locus genotype on gene expression patterns in the fire ant Solenopsis invicta

Males in many animal species differ greatly from females in morphology, physiology and behaviour. Ants, bees and wasps have a haplodiploid mechanism of sex determination whereby unfertilized eggs become males while fertilized eggs become females. However, many species also have a low frequency of diploid males, which are thought to develop from diploid eggs when individuals are homozygous at one or more sex determination loci. Diploid males are morphologically similar to haploids, though often larger and typically sterile. To determine how ploidy level and sex-locus genotype affect gene expression during development, we compared expression patterns between diploid males, haploid males and females (queens) at three developmental timepoints in Solenopsis invicta. In pupae, gene expression profiles of diploid males were very different from those of haploid males but nearly identical to those of queens. An unexpected shift in expression patterns emerged soon after adult eclosion, with diploid male patterns diverging from those of queens to resemble those of haploid males, a pattern retained in older adults. The finding that ploidy level effects on early gene expression override sex effects (including genes implicated in sperm production and pheromone production/perception) may explain diploid male sterility and lack of worker discrimination against them during development.     

Cytotype variation and allopolyploidy in North American species of the Sphagnum subsecundum complex (Sphagnaceae)

Allopolyploid speciation is likely the predominant mode of sympatric speciation in plants. The Sphagnum subsecundum complex includes six species in North America. Three have haploid gametophytes, and three are thought to have diploid gametophytes. Microsatellite analyses indicated that some plants of S. inundatum and S. lescurii are heterozygous at most loci, but others have only one allele at each locus. Flow cytometry and Feulgen staining showed that heterozygous plants have twice the genome size as plants with one allele per locus; thus, microsatellite patterns can be used to survey the distribution and abundance of haploid and diploid gametophytes. Microsatellite analyses also revealed that S. carolinianum is consistently diploid, but S. lescurii and S. inundatum include both haploid and diploid populations. The frequency of diploid plants in S. lescurii increases with latitude. In an analysis of one population of S. lescurii, both cytotypes co-occurred but were genetically differentiated with no evidence of interbreeding. The degree of genetic differentiation showed that the diploids were not derived from simple genome duplication of the local haploids. Heterozygosity appears to be fixed or nearly so in diploids, strongly suggesting that although morphologically indistinguishable from the haploids, they are derived by allopolyploidy.     

Natural and Sexual Selection on Many Loci

A method is developed that describes the effects on an arbitrary number of autosomal loci of selection on haploid and diploid stages, of nonrandom mating between haploid individuals, and of recombination. We provide exact recursions for the dynamics of allele frequencies and linkage disequilibria (nonrandom associations of alleles across loci). When selection is weak relative to recombination, our recursions provide simple approximations for the linkage disequilibria among arbitrary combinations of loci. We show how previous models of sex-independent natural selection on diploids, assortative mating between haploids, and sexual selection on haploids can be analyzed in this framework. Using our weak-selection approximations, we derive new results concerning the coevolution of male traits and female preferences under natural and sexual selection. In particular, we provide general expressions for the intensity of linkage-disequilibrium induced selection experienced by loci that contribute to female preferences for specific male traits. Our general results support the previous observation that these indirect selection forces are so weak that they are unlikely to dominate the evolution of preference-producing loci.     

Genetic structure and genealogy in the Sphagnum subsecundum complex (Sphagnaceae: Bryophyta)

Allopolyploidy is probably the most extensively studied mode of plant speciation and allopolyploid species appear to be common in the mosses (Bryophyta). The Sphagnum subsecundum complex includes species known to be gametophytically haploid or diploid, and it has been proposed that the diploids (i.e., with tetraploid sporophytes) are allopolyploids. Nucleotide sequence and microsatellite variation among haploids and diploids from Newfoundland and Scandinavia indicate that (1) the diploids exhibit fixed or nearly fixed heterozygosity at the majority of loci sampled, and are clearly allopolyploids, (2) diploids originated independently in North America and Europe, (3) the European diploids appear to have the haploid species, S. subsecundum, as the maternal parent based on shared chloroplast DNA haplotypes, (4) the North American diploids do not have the chloroplast DNA of any sampled haploid, (5) both North American and European diploids share nucleotide and microsatellite similarities with S. subsecundum, (6) the diploids harbor more nucleotide and microsatellite diversity than the haploids, and (7) diploids exhibit higher levels of linkage disequilibrium among microsatellite loci. An experiment demonstrates significant artifactual recombination between interspecific DNAs coamplified by PCR, which may be a complicating factor in the interpretation of sequence-based analyses of allopolyploids.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth

The AtNRT1.1 (CHL1) transporter provides a primary mechanism for nitrate uptake in Arabidopsis and is expected to localize to the epidermis and cortex of the mature root, where the bulk of nitrate uptake occurs. Using fusions to GFP/GUS marker genes, we found CHL1 expression concentrated in the tips of primary and lateral roots, with very low signals in the epidermis and cortex. A time-course study showed that CHL1 is activated in the primary root tip early in seedling development and at the earliest stages of lateral root formation. Strong CHL1 expression also was found in shoots, concentrated in young leaves and developing flower buds but not in the shoot meristem. These expression patterns were confirmed by immunolocalization and led us to examine CHL1 function specifically in the growth of developing organs. chl1 mutants showed a reduction in the growth of nascent roots, stems, leaves, and flower buds. The growth of nascent primary roots was inhibited in the mutants even in the absence of added nitrate, whereas elongation of lateral root primordia was inhibited specifically at low nitrate and acidic pH. Interestingly, chl1 mutants also displayed a late-flowering phenotype. These results indicate that CHL1 is activated and functions in the growth of nascent organs in both shoots and roots during vegetative and reproductive growth.