华南植物园锥栗F-MSAP采样策略及遗传多样性分析

基因组甲基化修饰受环境因素的影响。在以甲基化为代表的表观遗传学研究中,如何减少保存环境对异地采后样品的影响,提高整个实验的准确性和科学性,目前尚未有系统的认知。该研究选取5种常用的采后样品保存方式(液氮冷冻、-20℃冷冻、变色硅胶干燥、密封袋密封、75%酒精浸泡),分别用Wilcoxon signed ranks tests统计分析和UPGMA聚类分析方法,对华南植物园锥栗进行F-MSAP研究,以期找出最佳保存方式。同时,利用正交试验法对F-MSAP体系进行优化,筛选出9对引物(E3-H/M2;E5-H/M2;E6-H/M1;E6-H/M5;E8-H/M1;E8-H/M5;E9-H/M2;E11-H/M5;E14-H/M1),并对不同发育时期的锥栗甲基化水平及遗传多样性进行了论述。结果表明:在锥栗F-MSAP的研究中,Willcoxon signed ranks tests统计分析和UPGMA聚类分析结论一致,密封袋保存效果最佳;成熟叶半甲基化率(27.83%)和总甲基化率(51.13%)高于幼叶(21.35%,45.90%),全甲基化率(23.30%)低于幼叶(24.55%),平均多态位点百分数39.60%,香农信息指数0.207±0.002,表现出较高的甲基化水平和遗传多样性。     

东乡野生稻耐冷渐渗系DNA甲基化水平和模式变化研究

根据水稻全基因组和特定位点的CpG岛序列设计引物,采用McrBC酶酶切DNA,以东乡野生稻耐冷渐渗系(IL5335和IL5423)及其双亲为试材,研究其基因组和特定位点的DNA甲基化水平和模式变化特征,探讨甲基化变异对野生优异基因渐渗的影响。结果显示:(1)在覆盖全基因组的83个CpG岛中,IL5335和IL5423的基因组甲基化频率分别为46.6%和53.8%,低于受体亲本协青早B的62.6%;大部分(75.9%~80.7%)受体亲本的甲基化模式在两耐冷渐渗中能稳定遗传,另外一些位点发生了甲基化模式的改变,主要表现为脱甲基化(13.3%~18.1%)和过甲基化(4.4%~6.0%)。(2)在耐冷QTLs区间,两耐冷渐渗系的甲基化水平为13.3%~26.7%,远低于受体亲本的61.4%;它们在该区域的甲基化模式变异主要为脱甲基化(33.3%~40.0%),高于全基因组的平均变异率。(3)分析逆转座子Houba和Osr14区域的51个CpG岛发现,耐冷渐渗系在该区域具有较高频率的甲基化修饰和较低的甲基化模式变异。研究表明,种间杂交渐渗诱发了受体亲本广泛的甲基化水平和模式变异,为野生优异基因的有效利用提供了新的机遇和挑战。     

高黎贡山中山湿性常绿阔叶林树种的分布格局及其地形影响因子

基因组甲基化修饰受环境因素的影响。在以甲基化为代表的表观遗传学研究中,如何减少保存环境对异地采后样品的影响,提高整个实验的准确性和科学性,目前尚未有系统的认知。该研究选取5种常用的采后样品保存方式（液氮冷冻、-20 ℃冷冻、变色硅胶干燥、密封袋密封、75%酒精浸泡）,分别用Wilcoxon signed ranks tests统计分析和UPGMA聚类分析方法,对华南植物园锥栗进行F MSAP研究,以期找出最佳保存方式。同时,利用正交试验法对F MSAP体系进行优化,筛选出9对引物（E3 H/M2;E5 H/M2;E6 H/M1;E6 H/M5;E8 H/M1;E8 H/M5;E9 H/M2;E11 H/M5;E14 H/M1）,并对不同发育时期的锥栗甲基化水平及遗传多样性进行了论述。结果表明：在锥栗F MSAP的研究中,Willcoxon signed ranks tests统计分析和UPGMA聚类分析结论一致,密封袋保存效果最佳;成熟叶半甲基化率（27.83%）和总甲基化率（51.13%）高于幼叶（21.35%,45.90%）,全甲基化率（23.30%）低于幼叶（24.55%）,平均多态位点百分数39.60%,香农信息指数0.207±0.002,表现出较高的甲基化水平和遗传多样性。     

镉胁迫下萝卜基因组DNA甲基化敏感扩增多态性分析

应用甲基化敏感扩增多态性(MSAP)技术分析了重金属镉(cd)胁迫处理后萝卜基因组DNA甲基化程度的变化。结果表明,经50、250和500mg/L CdCl_2处理后,MSAP比率分别为37%、43%和51%,均高于对照(34%);全甲基化率(双链C~mCGG)分别为23%、25%和27%,而其对照为22%,表明重金属CdCl_2胁迫后,某些位点发生了重新甲基化。萝卜叶片DNA中总甲基化水平的增加与CdCl_2处理浓度呈显著正相关。甲基化变异可分为重新甲基化、去甲基化、不定类型以及与对照相同的甲基化模式等类型,Cd胁迫处理引起的植株基因组DNA甲基化程度的提高主要是重新甲基化。     

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。     

橡胶树MSAP反应体系优化及其不同开割高度单株DNA甲基化分析

以‘热研7-33-97’橡胶树无性系幼嫩叶片为材料,通过利用单因素和正交试验相结合的方法,对酶切、预扩增和选择性扩增3个影响甲基化敏感扩增多态性(MSAP)分析的关键步骤的反应体系中关键影响因素进行了优化,建立橡胶树MSAP反应最佳体系,并用于高低割线橡胶树基因组DNA甲基化差异分析。结果表明:在50μL反应体系中,750 ng基因组DNA用EcoRⅠ20 U,HpaⅡ20 U或MspⅠ10 U于37℃恒温同步酶切10 h,酶切完全。最佳预扩增体系(20μL)为:连接产物4μL,Mg Cl_2(25 mmol·L~(-1))0.15μL,d NTPs(2.5 mmol·L~(-1))0.1μL,上下游引物E-00/HM-00(10μmol·L~(-1))各0.3μL,Taq酶(5 U·μL~(-1))0.1μL,10×PCR Buffer 2μL。最佳选择性扩增反应体系(20μL)为:稀释20倍的预扩增产物2μL,Mg Cl_2(25 mmol·L~(-1))0.1μL,d NTPs(2.5 mmol·L~(-1))0.125μL,上下游引物E+3/HM+3(10μmol·L~(-1))各0.4μL,Taq酶(5 U·μL~(-1))0.1μL,10×PCR Buffer 2μL。高低割线树DNA的甲基化比例分别为37.22%和36.43%,2种开割胶树基因组CCGG位点胞嘧啶全甲基化率明显高于半甲基化率,推测橡胶树基因组甲基化主要模式可能是Cp G型。综上表明,建立的MSAP反应体系稳定可靠且重复性好,为后续橡胶树不同胁迫(割胶)程度DNA甲基化的研究奠定了基础。     

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。     

孝顺竹开花过程中DNA甲基化水平动态研究

以孝顺竹为材料,利用HPLC和MSAP技术在其未开花至开花的生长过程中进行甲基化水平的检测,分析竹子开花过程中基因组DNA甲基化的动态,以揭示DNA甲基化水平与竹子开花现象的相关性。结果显示:(1)孝顺竹基因组DNA甲基化率在不同的时间处于动态变化中,进入开花状态的孝顺竹植株其甲基化水平极显著低于开花竹丛中未开花植株和未开花竹丛;开花和未开花植株的总甲基化率分别为9.00%和12.42%,全甲基化率分别为5.06%和7.53%。(2)MSAP位点中有66.83%的位点在开花和未开花材料中甲基化状态保持一致,33.17%的位点在开花和未开花植株中发生甲基化变化:其中22.28%的位点在开花植株中发生完全的去甲基化,1.98%的谱带在开花植株中发生甲基化,8.91%的位点在开花和未开花材料中甲基化水平呈现上升或降低的趋势。研究表明,开花的孝顺竹同时发生甲基化和去甲基化,但发生去甲基化的概率明显大于发生甲基化的概率,最终导致其甲基化水平极显著降低。     

生长因子颗粒蛋白前体和肿瘤坏死因子受体基因启动子区改变与阿尔茨 海默病的相关性研究

目的:探讨生长因子颗粒蛋白前体(PGRN)、肿瘤坏死因子受体(TNFR)基因启动子区改变以及全基因组DNA甲基化与阿尔茨海默病的相关性。方法:收集阿尔茨海默病患者血液样本80例以及健康对照血液样本80例,PCR扩增PGRN和TNFR基因启动子区并进行测序,观察两组间的单核苷酸多态性位点是否有差异。同时,用甲基化特异性PCR法检测启动子区DNA甲基化情况以及用ELISA法检测全基因组DNA甲基化水平。结果:在TNFR基因启动子区域发现阿尔茨海默病和对照组之间在多态性位点rs4149570和rs4149569有显著性差异(P0.001和P=0.033)。阿尔茨海默病患者全基因组甲基化水平为(0.79±0.29)%,显著低于对照组的(1.00±0.36)%(P0.001)。结论:TNFR基因多态性位点rs4149570和rs4149569的变异可能与阿尔茨海默病相关,全基因组甲基化水平降低可能与阿尔茨海默病相关。     

不同倍性西瓜基因组DNA甲基化水平与模式的MSAP分析

DNA甲基化是表观遗传修饰的主要方式之一,在基因表达调控中发挥重要作用。本研究以不同倍性（2x、3x、4x）西瓜为试材,采用基于DNA甲基化敏感酶的扩增多态性分析（Methylation-Sensitive Ampliftcation Polymorphism,MSAP）方法,在全基因组水平上探究西瓜同源多倍化过程中DNA序列中CCGG位点的甲基化水平及模式变化特征。研究中选用23对选扩引物,共检测到1883个基因位点。二倍体、三倍体、四倍体中检测到的位点数分别为647、655和581;其中发生甲基化的位点数分别为181、150和159。相应的扩增总甲基化率分别为28．0％、22．9％和27．4％：全甲基化位点数分别为121、80和82,相应的全甲基化率分别为18．7％、12．2％和14．1％。进一步对不同倍性西瓜DNA甲基化模式的变化特征进行分析,结果显示：四倍体西瓜与二倍体西瓜相比有超过半数的位点（54．4％）DNA甲基化模式发生了变化,其与三倍体西瓜相比也有近一半的位点（45．4％）DNA甲基化模式发生了变化,并且变化趋势都以四倍体西瓜甲基化程度升高为主：而三倍体西瓜与二倍体西瓜相比．虽然也有41．6％的位点DNA甲基化模式发生了改变,但变化趋势以三倍体西瓜甲基化程度降低略占优势：与之相似,三倍体西瓜与四倍体相比较。甲基化的变化趋势也是以三倍体西瓜甲基化程度降低为主。以上结果表明：不同倍性西瓜中DNA甲基化事件虽均有发生．但不论是从总甲基化率还是全甲基化率来看,DNA甲基化水平与倍性高低关系不大．三倍体西瓜表现出较为显著的低甲基化水平特征。DNA甲基化模式的分析也表明。与二倍体及四倍体西瓜相比．三倍体西瓜DNA甲基化模式的调整主要以去甲基化为主。显示出三倍体西瓜基因组独特的DNA甲基化特征。本研究为进一步从表观遗传学的角度探讨西瓜的三倍体优?     

五月季竹开花及复壮过程中DNA甲基化的MSAP分析

以五月季竹为材料,采用MSAP技术对其开花及花后无性复壮过程中的DNA甲基化状况进行检测,分析其开花前后的甲基化动态,以揭示竹子开花及复壮过程中的表观遗传变化规律。结果显示:（1）五月季竹开花时其叶片甲基化水平降低,而在无性复壮产生不再开花新竹的过程中其叶片甲基化水平又逐渐回升;（2）与未开花竹株相比,五月季竹开花时有29.09%的甲基化位点发生了变异,其中有17.88%的位点在开花植株中发生了完全的去甲基化,远高于发生甲基化位点的比率;（3）复壮竹株与未开花竹株之间发生变异的位点数和所占比率,尤其是发生去甲基化的位点数和比率,低于开花竹株;（4）开花五月季竹花器官的甲基化水平低于叶片,同时有28.58%的位点发生了甲基化状态的改变,且同样以去甲基化为主。     

Analysis of DNA methylation during the germination of wheat seeds

DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. Here, we focused on the DNA methylation dynamics during the germination of wheat seeds using the adapted AFLP technique so called methylation-sensitive amplified polymorphism (MSAP). The MSAP profiles of genomic DNA in embryo and endosperm tissues of germinating seeds, as well as dry seeds were characterized and notable changes of cytosine methylation were detected. Comparisons of MSAP profiles in different tissues tested showed that the methylation level in dry seeds is the highest. The alteration analysis of cytosine methylation displayed that the number of demethylation events were three times higher than that of de novo methylation, which indicated that the demethylation was predominant in germinating wheat seeds, though the methylation events occurred as well. Sixteen differentially displayed DNA fragments in MSAP profiles were cloned and the sequencing analysis confirmed that nine of them contained CCGG sites. The further BLAST search showed that four of the cloned sequences were located in coding regions. Interestingly, three of the sixteen candidates were homologous to retrotransposons, which indicated that switches between DNA methylation and demethylation occurred in retrotransposon elements along with the germination of wheat seeds.     

Analysis of Cytosine Methylation Pattern in Response to Water Deficit in Pea Root Tips

Abstract: The correlation between environmental stress and DNA methylation has been studied by following the methylation status of cytosine residues in the DNA of pea root tips exposed to water deficit. DNA methylation was evaluated by two complementary approaches: (i) immunolabelling by means of a monoclonal antibody against 5-methylcytosine; (ii) MSAP (Methylation-Sensitive Amplified Polymorphism) to verify if methylation and de-methylation in response to water deficit may be related to specific DNA sequences. Immunolabelling showed that water stress induces cytosine hypermethylation in the pea genome. Regarding the CCGG target sequence, an increase in methylation specifically in the second cytosine (about 40 % of total site investigated) was revealed by MSAP analyses. In addition, MSAP band profile detected in three independent repetitions was highly reproducible suggesting that, at least for the CCGG target sequence, methylation was addressed to specific DNA sequences.     

MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild‐growing species

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation‐sensitive amplification polymorphism (MS‐AFLP or MSAP) have been often used to assess methyl‐cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome‐wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome‐wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl‐cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context‐specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation‐based epigenetic processes in nonmodel plants.     

MSAP技术及其在植物上的应用

DNA甲基化在植物的很多生命过程中具有重要作用,检测DNA甲基化的技术应运而生。依据对DNA甲基化敏感程度不同的同裂酶,在AFLP技术的基础上发展而来的MSAP技术可以方便的检测全基因组范围内胞嘧啶甲基化模式及程度。该文对MSAP技术的原理、特点、基本程序及应用进行了阐述。     

The influence of Al<Superscript>3+</Superscript> on DNA methylation and sequence changes in the triticale (× <Emphasis Type="Italic">Triticosecale</Emphasis> Wittmack) genome

Abiotic stressors such as drought, salinity, and exposure to heavy metals can induce epigenetic changes in plants. In this study, liquid chromatography (RP-HPLC), methylation amplified fragment length polymorphisms (metAFLP), and methylation-sensitive amplification polymorphisms (MSAP) analysis was used to investigate the effects of aluminum (Al) stress on DNA methylation levels in the crop species triticale. RP-HPLC, but not metAFLP or MSAP, revealed significant differences in methylation between Al-tolerant (T) and non-tolerant (NT) triticale lines. The direction of methylation change was dependent on phenotype and organ. Al treatment increased the level of global DNA methylation in roots of T lines by approximately 0.6%, whereas demethylation of approximately 1.0% was observed in NT lines. DNA methylation in leaves was not affected by Al stress. The metAFLP and MSAP approaches identified DNA alterations induced by Al³⁺ treatment. The metAFLP technique revealed sequence changes in roots of all analyzed triticale lines and few mutations in leaves. MSAP showed that demethylation of CCGG sites reached approximately 3.97% and 3.75% for T and NT lines, respectively, and was more abundant than de novo methylation, which was observed only in two tolerant lines affected by Al stress. Three of the MSAP fragments showed similarity to genes involved in abiotic stress.     

Musa methylated DNA sequences associated with tolerance toMycosphaerella fijiensis toxins

DNA methylation is an epigenetic phenomenon associated with gene silencing in transgenic plants, retrotransposons and virus infection. Expression analysis of specific genes in Arabidopsis methylation mutants showed an association between DNA methylation and gene expression. To determine whether DNA methylation is associated with resistance to black Sigatoka (BS) andMycosphaerella fijiensis (MF), we used anin vitro assay of mesophyll cell suspensions of reference cultivars with known resistance to BS. Methylation of CC^mGG sequences was evaluated by methylation-sensitive amplification polymorphism (MSAP) markers of reference cultivars and somaclonal variants to identify molecular markers associated with resistance to MF toxins and BS. Four MSAP markers were associated with resistance (MAR) to MF toxins. The MSAP markers show a high degree of sequence similarity with resistance gene analog and with retrotransposon sequences. The MSAP markers are useful as molecular indicators of tolerance to MF toxins and resistance to BS.