Laboratorio de Citogenética y Evolución, Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Instituto de Ecología, Genética y Evolución de Buenos Aires (IEGEBA, CONICET-UBA), Universidad de Buenos Aires, Intendente Güiraldes 2160, Pabellón II, Ciudad Universitaria, C1428EHA, Ciudad Autónoma de Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, Ciudad Autónoma de Buenos Aires, C1425FQB, Argentina.
J Plant Res. 2021 May;134(3):559-575. doi: 10.1007/s10265-021-01279-3. Epub 2021 Mar 23.
Differential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and vegetative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual differentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a first step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of differential fragments. A significant positive correlation was found between epigenetic and indirect 'genetic' information for both species, indicating influence of the genetic background on the epigenetic variation. Higher epigenetic than genetic diversities were estimated. Our outcomes showed up to 1.86 times more representation of mCG subepiloci than mCCG in all organs sampled. Along the maturing stages of floral buds, the frequency of mCG evidenced an incremental trend, whereas mCCG and unmethylated conditions showed opposite tendencies. Reproductive and vegetative samples tended to cluster apart based on epigenetic patterns; at gene expression level, organs exhibited clear-cut distinctive patterns, nonetheless profiles of young leaves and floral primordia resemble. Epigenetic and expression data allowed discrimination of I. dumosa´s samples according to the gender of the donor; more elusive patterns were observed for I. paraguariensis. In total, 102 differentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, finding statistical differences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indicative of gender in both species. Thirty-three organ-specific and 34 gender-specific methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application.
我们研究了在不同发育阶段的巴拉圭冬青和多毛冬青的生殖器官和营养器官中的差异表观遗传（DNA 胞嘧啶甲基化）和基因表达模式。我们的目的是为阐明这些雌雄异株物种中完全未知的性分化过程的主要分子变化做出贡献。同时，作为开发早期性别鉴定系统的第一步，我们寻找了有希望的分子标记。通过甲基化敏感扩增多态性（MSAP）和 cDNA 扩增片段长度多态性（cDNA-AFLP）技术，并应用判别多元分析和差异片段的生物信息学特征来评估。发现两种物种的表观遗传和间接“遗传”信息之间存在显著正相关，表明遗传背景对表观遗传变异的影响。估计表观遗传多样性高于遗传多样性。我们的研究结果表明，在所采样的所有器官中，mCG 亚基因座的代表率比 mCCG 高 1.86 倍。在花芽成熟过程中，mCG 的频率呈递增趋势，而 mCCG 和非甲基化条件则呈相反趋势。基于表观遗传模式，生殖和营养器官趋于聚类分开；在基因表达水平上，器官表现出明显独特的模式，然而幼叶和花原基的模式相似。表观遗传和表达数据允许根据供体的性别来区分 I.dumosa 的样本；对于 I. paraguariensis 观察到更难以捉摸的模式。总共对 102 个差异甲基化和表达片段进行了生物信息学分析。在各种功能类别中注释了 43 个片段；通过 qPCR 验证了 4 个候选标记，发现器官之间存在统计学差异，但性别之间没有差异。epilocus C13m33 的甲基化条件在两种物种中似乎都表明了性别。区分了 33 个器官特异性和 34 个性别特异性甲基化标记，值得进一步研究，特别是在叶子中表达的那些标记。我们的研究提供了具有实际应用潜力的具体候选标记。
