大花蕙兰子房授粉前后基因组DNA胞嘧啶甲基化状态的MSAP分析

应用甲基化敏感扩增多态性(Metyhfion sensitive amplified polymorphism,MSAP)技术分析了大花蕙兰(Cymbidium hybridium)授粉前后子房DNA甲基化状态的变化(甲基化水平和甲基化差异模式).采用72对引物进行选择性扩增,共得到5892条带,其中748条带为甲基化多态性带.结果显示DNA甲基化在大花蕙兰子房发育过程中发牛频繁,从授粉前后子房的总扩增位点甲基化水平(14%和11.4%)和全甲基化率(9.5%和7.8%)来看,授粉后都略低于未授粉子房,表明子房在授粉后的发育过程中在某些位点发生了去甲基化.除甲基化水平有变化外,大花蕙兰子房授粉前后的DNA甲幕化模式也存在较大差异,共榆测到14种带型,分为两大类(Ⅰ和Ⅱ型).其中,授粉前后DNA甲基化状态保持不变的位点较少,只占25.6%,归为Ⅰ型;大部分榆测位点(占74.4%,归为Ⅱ型)的DNA甲基化模式在授粉前后存在显著差异.上述结果表明,大化蕙兰子房发育过程中以DNA甲基化为代表的表观遗传调控起重要作用.本研究的开展将促进对与大花蕙兰子房发育相关的甲基化差异片段及受DNA甲基化调控的关键基因的克隆,进而为从表观遗传学这一新角度揭示大花蕙兰子房发育的分子机制奠定基础.     

二斑叶螨不同发育时期基因组DNA甲基化的F-MSAP分析

DNA甲基化是表观遗传调控的重要机制,在真核生物基因表达调控中发挥重要作用。本研究通过荧光标记的甲基化敏感扩增多态性技术(F-MSAP,fluorescence-labeled methylation-sensitive amplified polymorphism)对二斑叶螨Tetranychus urticae Koch 4个龄期(卵、幼螨、若螨、成螨)基因组DNA中CCGG位点的胞嘧啶甲基化水平和模式进行分析。研究结果显示3种甲基化模式:无甲基化(TypeⅠ),半甲基化(TypeⅡ),全甲基化(TypeⅢ)在4个龄期均有出现,扩增的总甲基化位点共有641个,其中半甲基化率(TypeⅡ)均高于全甲基化率(TypeⅢ),各个龄期的平均总甲基化率(TypeⅡ+TypeⅢ)为16.01%,平均半甲基化率为10.24%,平均全甲基化率为5.77%。F-MSAP分析结果表明不同发育时期的二斑叶螨基因组DNA的甲基化水平和模式存在差异。     

运用MSAP技术测定谷子基因组DNA胞嘧啶甲基化水平

DNA甲基化是真核生物一种重要的表观修饰形式。为了探讨谷子基因组DNA胞嘧啶甲基化的水平和模式,以谷子Setaria italica的两个品种朝谷58号和豫谷1号为实验材料,利用Eco RⅠ和HpaⅡ/MspⅠ双酶切建立适合于谷子基因组的甲基化敏感扩增多态性(MSAP)分析体系。结果表明,从100对MSAP选扩引物中,筛选出32对MSAP引物组合,在朝谷58号和豫谷1号中分别扩增产生1 615、1 482条清晰可辨且可重复的DNA条带,其中包括3种类型的甲基化条带,朝谷58号和豫谷1号的基因组中CCGG序列胞嘧啶甲基化水平分别为6.93%和8.77%。这种谷子不同品种间甲基化水平和分布位点的差异为从表观遗传学的角度培育新品种提供了初步的理论依据和参考。     

叶锈菌胁迫下的小麦基因组MSAP分析

内源DNA甲基化是真核生物表观遗传调控的重要组成部分, 在真核生物的基因表达调控中具有重要的作用。生物胁迫为植物提供一种内在的表观遗传进化动力。研究生物胁迫下DNA甲基化的变异模式, 有助于全面理解DNA甲基化的表观调控生物学功能。小麦近等基因系TcLr19、TcLr41及其感病亲本Thatcher在苗期对叶锈菌生理小种THTT、TKTJ分别表现为小种特异性抗病反应和感病反应。文章利用甲基化敏感扩增多态性(Methylation-sensitive amplified polymorphism, MSAP)技术分析了小麦的甲基化水平, 同时比较了苗期在生物胁迫前后基因组DNA胞嘧啶甲基化模式。用60对MSAP引物对接种前后的小麦DNA进行全基因组筛选, 没有直接分离得到接菌前后的甲基化模式的差异, 结果初步表明, 叶锈菌并没有诱导稳定且特异的植物基因组DNA胞嘧啶位点的甲基化模式变化, 但发现TcLr41及其感病亲本Thatcher之间存在表观遗传学差异。     

马哈利樱桃矮化砧的DNA甲基化水平及模式分析

该研究利用MSAP技术,对25株矮化马哈利樱桃和25株半矮化马哈利樱桃进行甲基化水平和模式分析,以探讨其矮化的表观性状与其基因组甲基化修饰的关系。结果表明:(1)从64对引物中筛选出15对引物,在半矮化组中共扩增4 577个条带,其中半甲基化336个,全甲基化1 274个;在矮化组中共扩增4 444个条带,其中半甲基化349个,全甲基化1 383个;t检验和方差分析表明,矮化组与半矮化组在总甲基化水平和全甲基化水平上差异极显著,在半甲基化水平上差异显著,矮化组甲基化水平高于半矮化组。(2)半矮化组单态性位点23个,多态性位点136个;矮化组单态性位点17个,多态性位点142个,表明矮化组多态性高于半矮化组。(3)多态性类型分析表明,矮化组出现A4类型的频率较半矮化组高,A2类型的频率较半矮化组低,即矮化组中发生超甲基化的位点多于半矮化组,且‘马哈利’基因组甲基化多态性位点主要发生在双链内侧甲基化位点以及超甲基化位点上。研究认为,马哈利樱桃矮化和半矮化的基因组甲基化水平及模式存在差异,马哈利砧木的矮化性状与其基因组甲基化修饰有关。     

光暗条件下大蒜DNA甲基化差异的初步研究

用甲基敏感扩增多态性(MSAP)技术分析光暗条件下生长的大蒜DNA甲基化水平差异。结果表明,用8对引物组合扩增出343条带,完全一致的带型240条,不一致的103条。在不一致的条带中,光照条件下相同的47条带与黑暗条件下相同的带型有差异,与黑暗相比光照下出现的去甲基化带28条,有56条差异带在2个处理内个体间也有差异。总的趋势是光照引起大蒜DNA去甲基化。     

常压下强电场辐射对国稻6号DNA甲基化的影响

目的:研究常压下强电场辐射对国稻6号DNA甲基化的影响.方法:常压下在相同时间下,以不同强度(和在相同强度下,以不同时间)辐射国稻6号种子,应用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism,MSAP)技术,研究在不同辐射条件下,水稻基因组DNA甲基化的水平及差异.结果:所有强电场辐射的试验组甲基化水平都比未辐射的对照组有所降低,并与辐射的时间和强度有关.细胞DNA全甲基化、半甲基化扩增位点占总扩增位点的比例为:12.89％～13.62％,3.36％～4.63％,随着辐射强度和时间的增加有所降低.结论:说明经强电场辐射的国稻6号水稻基因组DNA甲基化较亲本发生了明显的变异,强电场能引起表观遗传变异,为研究其规律奠定了基础,也为探讨水稻甲基化对水稻生长调控的分子机理提供了帮助.     

橡胶树体细胞胚发生过程中DNA甲基化分析

为探讨巴西橡胶树(Hevea brasiliensis)自根幼态无性系与供体间差异产生的原因,应用甲基化敏感扩增多态性扩增技术,对巴西橡胶树体细胞胚发生过程中基因组DNA 胞嘧啶甲基化程度和模式进行了分析。结果表明,在巴西橡胶树体细胞胚发生过程中不同阶段的DNA 甲基化程度不同,以花药的DNA 甲基化程度最高,体细胞胚的DNA 甲基化水平最低。在体细胞胚发生过程中出现了I、Ⅱ和Ⅲ 3 种类型的甲基化多态性带型的改变,包括他们的出现与消失。因此,橡胶树体细胞胚发生过程中可能通过DNA 甲基化甲基化和去甲基化来调控基因的表达。     

利用F-MSAP分析菜心表观遗传多样性

菜心杂交除了引起DNA序列变化,还可能引起不依赖于DNA序列的表观遗传变化,为揭示菜心表观遗传多样性形成机理,该文利用F-MSAP检测49份菜心的DNA甲基化水平和模式变化。结果表明：(1)F-MSAP检测效率较高,菜心DNA甲基化多态性较高,杂交可以提高DNA甲基化多态性。(2)菜心表观遗传多样性较低,均质化严重,大部分遗传变异来源于种内,自交增加自交系的表观遗传差异,杂交增加杂种的表观遗传差异。(3)49份菜心的DNA甲基化水平较高,以全甲基化模式为主,自交降低DNA甲基化水平,杂交通过DNA甲基化模式变化增加自交系杂种的DNA甲基化水平。(4)49份菜心分成五类,聚类分析和主成分分析结果基本一致,杂种倾向于按照母本亲缘关系分类。该研究利用F-MSAP检测菜心表观遗传多样性,提高了菜心的鉴定效率和准确性,为进一步开展杂交育种提供了理论基础和技术支持。     

红豆杉脱分化过程中的遗传和表观遗传变异

采用扩增片段长度多态性（AFLP）和甲基化敏感扩增多态性（MSAP）技术分析红豆杉脱分化前后基因组DNA和DNA甲基化状态的变化。选用32个AFLP引物组合从红豆杉植株及其愈伤组织分别扩增出1834个片段,无多态性片段产生。这说明红豆杉植株在诱导形成愈伤组织的过程中基因组DNA保持高度的遗传稳定性。另用32个MSAP引物组合从红豆杉植株及其愈伤组织分别扩增出1197个片段,总扩增位点的甲基化水平由脱分化前的12．4％上升为16．2％,表明红豆杉在脱分化过程中的某些位点发生了甲基化。红豆杉脱分化前后的DNA甲基化模式也存在较大差异,说明DNA甲基化对愈伤组织形成有调控作用。     

Analysis of DNA methylation patterns of PLBs derived from <Emphasis Type="Italic">Cymbidium hybridium</Emphasis> based on MSAP

The best known and most thoroughly studied epigenetic phenomenon is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, the methylation-sensitive amplified polymorphism (MSAP) technique was carried out to determine differences in methylation profiles between two forms of protocorm-like bodies (PLBs), continuously proliferating PLBs (cPLBs) and spontaneously-differenting PLBs (sdPLBs), derived from cultures of Cymbidium hybridium. A total of 72 selective primer combinations were used to assess the status of cytosine methylation of DNA in these tissues. Of 4,440 fragments obtained 911 fragments, each representing a recognition site cleaved by one or both of the isoschizomers (Hpa II and Msp I), were amplified and were significantly different between the two forms of PLBs. Frequency of total and full-methylation of cPLBs and sdPLBs were 26.7/12.2%, 24.1/11.1%, respectively. In addition, 14 types of MSAP patterns detected in the two forms of PLBs belonged to two classes, type I and II. Sequencing of 14 differentially methylated fragments and their subsequent blast search revealed that cytosine methylated 5′-CCGG-3′ sequences were equally distributed in the coding and non-coding regions. Southern blotting was conducted to verify the methylation polymorphism.     

大花蕙兰与墨兰种间杂交子房培养研究初报

大花蕙兰与墨兰种间远缘杂交是培育具香味、易开花、早开花、抗性强的大花蕙兰品种的有效途径之一。本研究以大花蕙兰为母本,墨兰为父本进行种间杂交,共设计三个杂交组合。结果显示,同一组合子房不同离体时间培养萌发率差异明显,且不同组合间最适宜子房培养的离体时间有所差异。三个杂交组合最适宜的子房离体时间分别为授粉后90 d、120 d及100 d,萌发率分别为80.0%、80.0%及60.0%。     

Comparative analysis of DNA methylation changes in two contrasting wheat genotypes under water deficit

DNA methylation is one of the epigenetic mechanisms regulating gene expression in plants in response to environmental conditions. In this study, analysis of methylation patterns was carried out in order to assess the effect of water stress in two contrasting wheat genotypes using methylation-sensitive amplified polymorphism (MSAP). The results revealed that demethylation was higher in drought-tolerant genotype (C306) as compared to drought-sensitive genotype (HUW468) after experiencing drought stress. Comparisons of different MSAP patterns showed a high percentage of polymorphic bands between tolerant and susceptible wheat genotypes (from 74.79 % at anthesis to 88.89 % at tillering). Furthermore, differential DNA methylation in roots and leaves also revealed tissue-specific methylation of genomic DNA. Interestingly, 54 developmental stage-specific bands and 23 bands that were found contrasting between these two wheat genotypes were detected. Furthermore, a few sites with stable DNA methylation differences were identified between drought-tolerant and drought-sensitive cultivars, thus providing genotype-specific epigenetic markers. These results not only provide data on differences in DNA methylation changes but also contribute to dissection of molecular mechanisms of drought response and tolerance in wheat.     

Utility of the methylation-sensitive amplified polymorphism (MSAP) marker for detection of DNA methylation polymorphism and epigenetic population structure in a wild barley species (<Emphasis Type="Italic">Hordeum brevisubulatum</Emphasis>)

We report here that by using a modified scoring criterion, the methylation-sensitive amplified polymorphism or MSAP marker can be used effectively to detect polymorphism in DNA methylation patterns within and among populations of a perennial wild barley species, Hordeum brevisubulatum. Twenty-four selected individual genotypes representing four natural populations of H. brevisubulatum distributed in the Songnen Prairie in northeastern China were studied. The utility of MSAP was evidenced by its detection of high levels of polymorphism in DNA methylation patterns between individuals within a given population, and the clear inter-population differentiation in methylation patterns (methylation-based epigenetic population structure) revealed among the four populations. The resolving power of MSAP to detect DNA methylation polymorphism was found to be comparable with that of a retrotransposon-based sequence-specific amplified polymorphism marker, or SSAP, to detect genetic polymorphism in the same set of plants, suggesting that MSAP with a modified scoring criterion can be used efficiently to detect DNA methylation polymorphism and assess epigenetic population structure in natural plant populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.