RNA-Seq data of R. agastum and R. irroratum

R. agastum and R. irroratum from experimental field at National Forest Garden of GuiZhou province were employed in this paper. Flower samples during the four developmental stages (S1-S4) were sheared and harvested. According to instructions of RNA pure Plant Kit (CWBIO, China), total RNA was extracted from flowers of R. agastum and R. irroratum. RNA concentration and integrity was identified through Nanodrop spectrophotometer (Life Technologies, USA) and bioanalyzer 2100 (Agilent, USA) respectively. cDNA libraries preparation were finished via Biomarker Technology (Beijing, China) and then subjected to sequence using Illumina HiSeq X™ Ten platform. The high-quality clean reads/data were generated by removing the low-quality, adapter- and N-base reads, and assembled basing on the genome of Rhododendron delavayi assessed by Rhododendron plant Genome Database (RPGD, http://bioinfor.kib.ac.cn/RPGD/index.html). Function of the assembled unigene was annotated against the following database: KEGG (Kyoto Encyclopedia of Genes and Genomes); PFAM (protein family); Nt (non-redundant nucleotide sequences, NCBI); Swiss-Prot (a protein sequence database); Nr (non-redundant protein sequences, NCBI); KO (Kyoto Encyclopedia of Genes and Genomes Ortholog database); GO (Gene Ontology); and COG (Clusters of Orthologous Groups of proteins). To normalize the gene expression level, FPKM (Fragments Per Kilobase of transcriptome per Million mapped reads) method was selected. Differential expression analysis of two groups (Ra and Ri) was conducted using DESeq2 package with the screening parameters: fold change ≥2 and FDR (false discovery rate) < 0.01 to identify the genes expressed significantly different. Meanwhile, GO and KEGG enrichment analysis were applied for analyzing the biological function of DEGs through using TopGO and KOBAS.

本研究以采自贵州省国家森林公园试验田的杜鹃属植物R. agastum（Rhododendron agastum）与R. irroratum（Rhododendron irroratum）为实验材料，采集其四个发育阶段（S1-S4）的花组织样品。参照植物总RNA纯净提取试剂盒（CWBIO，中国）的操作说明书，分别从两种杜鹃的花组织中提取总RNA；分别采用Nanodrop分光光度计（Life Technologies，美国）与Agilent 2100生物分析仪（Agilent，美国）对RNA的浓度及完整性进行检测。由北京百迈客生物科技有限公司（Biomarker Technology，中国北京）完成cDNA文库的构建，随后采用Illumina HiSeq X™ Ten测序平台进行高通量测序。通过去除低质量序列、接头序列与含N碱基的reads，获得高质量clean reads/clean data；随后以杜鹃属植物基因组数据库（RPGD，http://bioinfor.kib.ac.cn/RPGD/index.html）中收录的马缨杜鹃（Rhododendron delavayi）基因组为参考进行序列组装。将组装得到的Unigene与以下数据库进行功能注释：京都基因与基因组百科全书（KEGG，Kyoto Encyclopedia of Genes and Genomes）、蛋白质家族数据库（PFAM，protein family）、NCBI非冗余核苷酸序列数据库（Nt，non-redundant nucleotide sequences）、Swiss-Prot蛋白质序列数据库、NCBI非冗余蛋白质序列数据库（Nr，non-redundant protein sequences）、KEGG直系同源数据库（KO，Kyoto Encyclopedia of Genes and Genomes Ortholog database）、基因本体论数据库（GO，Gene Ontology）以及蛋白质直系同源簇数据库（COG，Clusters of Orthologous Groups of proteins）。为对基因表达水平进行标准化处理，本研究采用每百万比对片段中每千碱基转录本的片段数（FPKM，Fragments Per Kilobase of transcriptome per Million mapped reads）法。采用DESeq2软件包对两组样品（Ra对应R. agastum，Ri对应R. irroratum）进行差异表达分析，筛选标准为倍数变化≥2且错误发现率（FDR，false discovery rate）<0.01，以鉴定显著差异表达基因（DEGs，Differentially Expressed Genes）；同时分别通过TopGO与KOBAS工具，对DEGs进行GO富集分析与KEGG富集分析，以解析其生物学功能。