The effect of developmental transitions on small RNAs in Arabidopsis thaliana shoots

We were interested in changes in small RNA abundance changes in response to developmental transitions in Arabidopsis thaliana shoots, with special focus on vegetative phase change. We specifically wanted to separate the temporal changes in gene expression that result from vegetative phase change and those from flowering. Because of the close timing between the juvenile-to-adult and adult-to-reproductive developmental transitions in Arabidopsis grown under long day conditions, we used the late-flowering genotype FRI;FLC developed by the lab of Richard Amasino by introgressing the FRI allele from Sf-2 into the Col-0 genetic background, which is fri;FLC. For the early flowering genotype, we used FRI;flc-3, also developed by the Amasino lab by EMS-mutagenizing FRI;FLC, identifying early flowering mutants, and backcrossing multiple times to eliminate other EMS-induced mutations. The onset of vegetative phase change in FRI;FLC and FRI;flc-3 under our growth conditions was identical, but the progression was slower in FRI;FLC. By sequencing small RNAs from shoot apices at different time points and fully-expanded leaves at different positions on the shoot and comparing the results between the two genotypes, we were able to obtain a clear picture of changes in small RNA abundance in response to vegetative phase change and flowering in Arabidopsis. For the small RNA samples, we performed two replicates using two different indices in the 5'-adapter and ran each replicate pair on the same sequencing lane. For the cotyledon and leaf samples we only performed one replicate using the same index for all samples because we obtained significantly different results with the two adapters used for the shoot apices, preventing us from using them as true replicates. Genome-wide small RNA profiling was done by a home assembled version of Illumina's 2007 small RNA library sample preparation followed by high-throughput sequencing with Illumina's Genome Analyzer II platform.

本研究旨在探究拟南芥（Arabidopsis thaliana）地上组织中小RNA丰度响应发育转变的变化特征，重点关注营养生长期相变（vegetative phase change），核心目标为区分由营养生长期相变与开花过程各自介导的基因表达时序变化。鉴于长日照培养条件下，拟南芥的幼年期-成年期转变与成年期-生殖期转变的发生时序高度重合，我们选用了理查德·阿玛西诺（Richard Amasino）实验室构建的晚开花基因型FRI;FLC：该基因型通过将Sf-2的FRI等位基因回交导入至背景为fri;FLC的Col-0生态型中获得。针对早开花基因型，我们采用了同样由阿玛西诺实验室构建的FRI;flc-3：通过对FRI;FLC进行EMS诱变筛选早开花突变体，并经多次回交以清除EMS诱导的其他随机突变。在本实验的培养条件下，FRI;FLC与FRI;flc-3的营养生长期相变起始时间完全一致，但FRI;FLC的相变进程更为缓慢。通过对不同时间点的茎尖以及地上组织不同部位的完全展开叶片中的小RNA进行测序，并比较两种基因型的测序结果，我们得以清晰解析拟南芥中小RNA丰度响应营养生长期相变与开花过程的变化规律。针对小RNA样本，我们采用5'接头带有两种不同索引（index）的方法完成了两次生物学重复，并将每对重复样本置于同一测序泳道中进行测序。而对于子叶与叶片样本，我们仅完成了一次生物学重复，且所有样本均使用同一索引：由于茎尖样本使用的两种接头得到的结果差异显著，无法将其作为有效重复样本。全基因组小RNA表达谱分析采用了本实验室自主构建的Illumina 2007版小RNA文库制备方案，并通过Illumina Genome Analyzer II平台完成高通量测序。