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。     

北京油鸡MSAP毛细管电泳荧光检测技术的建立

采用毛细管电泳荧光检测技术, 对北京油鸡肌肉组织基因组进行甲基化敏感扩增片段多态性(Methylation sensitive amplified polymorphism, MSAP)检测。通过对基因组DNA用量、预扩产物稀释倍数、选择性引物浓度、Mg2+浓度、dNTPs浓度和电泳内标量等6个主要参数进行分析和优化, 建立了适合北京油鸡基因组DNA甲基化分析的MSAP毛细管电泳荧光检测技术。重复实验表明, 建立的毛细管电泳荧光标记的MSAP检测技术能够自动地、高通量地分析北京油鸡基因组的DNA甲基化状态, 也适用于其他动植物基因组的DNA甲基化研究。     

欧石楠体细胞胚发生过程中的DNA甲基化

利用甲基化敏感扩增多态性（MSAP）方法,对欧石楠大田苗、胚性愈伤组织和再生苗的DNA甲基化进行了研究。从64对选扩增引物中筛选出19对,共扩增得到506条带,统计显示,大田苗、胚性愈伤组织和再生苗的全基因组DNA甲基化水平分别为31．42％、27．86％和29．05％,3种试材发生甲基化变异的有175条带,变异率为34．58％。体细胞胚诱导形成胚性愈伤组织过程中,甲基化水平降低,而在再生苗中有所恢复,与大田苗接近。在外侧胞嘧啶甲基化水平上,胚性愈伤组织的甲基化水平有所增加,且在再生苗中可部分维持。另外,在175条变异带中,再生苗恢复到大田苗DNA甲基化模式的有62条,占总变异条带的35．43％,而与胚性愈伤组织维持相同DNA甲基化模式的有59条,占33．71％。回收部分甲基化变异条带,最终得到8条有效的基因组DNA序列。BLASTnI：对分析表明,在欧石楠基因组中,包括抗性基因、蛋白激酶、质体基因等在内的多种DNA序列均存在DNA基化修饰现象。     

利用 MSAP 分析18个芥蓝齐口期的表观遗传多样性

本研究利用MSAP检测18个芥蓝齐口期DNA甲基化水平,分析了表观遗传多样性,探讨DNA甲基化模式对齐口期的影响。结果表明,18个芥蓝齐口期平均为50d,叶片数平均为10片,齐口期和叶片数不相关(相关系数为0.296);变异系数分别为21%和18%;遗传距离分布在0~40,平均值为12.2276,在10.62处分为3类。MSAP分析表明,5对引物组合扩增得到432条多态性条带,201条片段表现出多态性,多态性比率为47%;Nei遗传距离分布在0.004~0.467,平均值为0.0958,表明遗传多样性水平较低;在0.04处分为3类。Mantel测验表明两种分析的遗传距离相关系数为-0.1366,显示齐口期、叶片数与DNA甲基化多态性没有相关性。DNA甲基化模式分析表明,非甲基化片段为110条,甲基化多态性片段为322条,分为3种带型,类型一为非甲基化带型(110条),类型二为甲基化带型(110条),类型三为半甲基化带型(152条),非甲基化片段和半甲基化片段在不同品种之间呈现多态性,甲基化片段在不同品种之间呈现多态性与单态性相差不大,显示MSAP多态性主要来源于非甲基化和半甲基化片段,芥蓝甲基化模式以半甲基化为主。本文推测DNA甲基化水平降低参与芥蓝齐口期调控,MSAP分析既可用于基因组结构研究,又可用于基因组水平上性状的功能研究。     

长白猪×蓝塘猪及其杂种基因组甲基化片段克隆与分析

应用甲基化敏感扩增多态性(MSAP)技术对6头长白公猪和50头蓝塘母猪及5头长白×蓝塘杂交F1代3个群体基因组DNA胞嘧啶甲基化位点进行检测,旨在克隆和分析父母代猪及其杂种F1代之间基因组DNA共同甲基化片段和差异片段,并找到其同源基因.结果显示,从MSAP条带中分离、克隆得到18条3个群体共同的甲基化片段、10条母代独有的甲基化片段和9条杂交一代独有的甲基化片段,其中有1条3个群体共有的甲基化片段通过EST拼接和电子延伸后在NCBI数据库中找到同源基因,即猪类酪氨酸蛋白激酶Lyn基因(GeneID:LOC100152890,序列号:XM_001926250).结果表明,长蓝杂交F1代与其父母代之间的基因组甲基化存在异同,为通过MSAP技术克隆猪基因组DNA甲基化片段及寻找其对应的甲基化基因提供可能,也会为将来研究这些甲基化基因表达调控机制奠定基础.     

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。     

草鱼全同胞鱼苗不同个体甲基化位点的差异

本研究通过甲基化敏感扩增多态性(Methylation sensitive amplification polymorphism)对一对草鱼亲本的20个子代甲基化位点进行了研究。从20对引物组合中扩增出311个位点,其中甲基化位点236个,占总扩增位点的75.9%,表明草鱼水花期基因组甲基化水平已经很高,说明它们大部分组织分化基本完成;其中甲基化多态位点65个,占甲基化位点的27.5%,说明这些子代草鱼甲基化位点已经有相当的差异。对其他两对亲本的后代用六个引物组合扩增的结果表明,同一亲本的子代在甲基化模式上有差异可能是普遍现象。本研究结果说明,即使来自同一对草鱼亲本的不同子代个体在基因表达上也有较大的差异,因此很多性状在草鱼后代的分离和一些基因表达的改变有一定的关系。     

病毒侵染对西伯利亚百合DNA甲基化的影响

采用基于AFLP的甲基化敏感扩增多态性(MSAP)技术,用10对引物对侵染百合花叶病毒和丛簇病毒的西伯利亚百合植株和无毒植株进行DNA甲基化水平和模式分析.结果发现,西伯利亚百合无毒植株和病毒侵染植株的平均甲基化水平分别为40.1%和31.5%;平均全甲基化率分别为13.0%和9.7%;半甲基化率分别为27.1%和21.8%.研究表明,百合DNA甲基化多以半甲基化的形式存在;病毒侵染导致百合植株DNA甲基化水平降低,且对整体甲基化水平、全甲基化水平和半甲基化水平均产生了影响;说明病毒侵染百合后植株出现的症状在一定程度上与DNA甲基化存在关联.     

红豆杉种质资源遗传多样性的AFLP分析

目的：通过对5份红豆杉种质资源的AFLP分析,探求各种质间的遗传多样性。方法：采用扩增片段长度多态性（AFLP）标记,在DNA水平上进行遗传多样性研究,筛选了32对选择性扩增引物,将扩增出的条带作为原始矩阵,用NTSYS-PC软件计算并分析了红豆杉种质间的相似度,构建了遗传系统进化树。结果：（1）SDS法提取的红豆杉基因组DNA质量较佳,能够满足AFLP分析的要求;（2）从32对选择性扩增引物中,筛选出10对多态性较强、带型较好、分辨率较高的组合;（3）构建了红豆杉AFLP指纹图谱,将5个红豆杉种质全部区分开来;（4）通过构建进化树,把5个种质分成3类。结论：红豆杉种质资源有丰富的遗传多样性。     

单链DNA纯化对变性PAGE凝胶银染片段回收效率的提高

为了提高变性聚丙烯酰胺凝胶电泳（PAGE）银染后片段回收的效率,本实验在传统的煮沸法的基础上加入单链DNA纯化的步骤对水稻基因组CCGG为点甲基化敏感限制性酶切多态性（MSAP）分析片段进行回收,并对加入该步骤后的再扩增的效率与传统煮沸法进行了比较。实验结果显示,加入单链DNA纯化步骤后的回收效率比传统煮沸法提高了约6倍。改进后的方法可以有效地用于扩增片段长度多态性（AFLP）以及MSAP等差异显示PAGE凝胶银染DNA片段的回收。     

Epigenetic instability in genetically stable micropropagated plants of <Emphasis Type="Italic">Gardenia jasminoides</Emphasis> Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.     

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.     

马铃薯（Solanum tuberosum L.）茎尖的超低温保存及其遗传变异的初步观察

研究马铃薯茎尖超低温保存技术的结果表明,4℃低温下锻炼6d,在添加二甲基亚砜（DMSO）和乙酰胺的培养基中预培养5d,60％PVS2于室温下装载30min,0℃下PVS2脱水40min时,茎尖成活率最高（71．6％）,再生植株生长分化正常。进一步对再生植株进行AFLP分析,6对引物组合共扩增出385条带,超低温保存前后的材料之间未见到明显差的异带,但用MSAP技术分析超低温保存前后植株甲基化的结果显示：超低温保存后的材料均有不同程度的甲基化。在扩增的624条带中,处理与否之间完全一致的带型为584条;有变化的带型为40条,处理2（茎尖经过完整的超低温保存过程,区别于处理1,增加了冷冻、解冻和洗涤后恢复培养）有13个位点的甲基化增加,21个位点去甲基化。     

Genomic Change,Retrotransposon Mobilization and Extensive Cytosine Methylation Alteration in Brassica napus Introgressions from Two Intertribal Hybridizations

Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4–39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.     

猕猴桃倍性混合居群基因组遗传和表观遗传变异

植物倍性混合居群的形成和维系常伴随着明显的基因组遗传及表观遗传变异。利用AFLP和MSAP两种分子标记探讨了中华猕猴桃复合体(Actinidia chinensis)倍性混合居群的遗传变异和结构及其基因组甲基化变异方式。结果表明, 该倍性混合居群具有较高的遗传和表观遗传多样性, 但两者之间没有明显的相关性。种群的遗传多样性与海拔呈显著的负相关(P<0.05), 但表观遗传多样性与海拔不具显著相关性。AMOVA分析显示, 主要的遗传和表观遗传分化出现在倍性小种内部(97.65% vs 99.84%, P<0.05); 同时, AFLP邻接聚类分析显示二者存在一定程度的倍性相关性, MSAP分析则未显示有明显的倍性相关性。进一步研究发现, 中华猕猴桃居群的总甲基化程度为24.86%, 且多倍体具有更多的甲基化位点变异。该研究结果为深入探讨猕猴桃倍性混合居群的形成和维系机制奠定了基础。