Zea mays Transcriptome or Gene expression. Zea mays

Small RNAs (sRNAs) are hypothesized to contribute to hybrid vigor because they maintain genome integrity, contribute to genetic diversity, and control gene expression. We used Illumina sequencing to assess how sRNA populations vary between two maize inbred lines (B73, Mo17) and their hybrid. We sampled sRNAs from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids. We found that parental differences in siRNAs primarily originate from repeat regions. Although the maize genome contains greater number and complexity of repeats compared to Arabidopsis or rice, we confirmed that like these simpler plant genomes, 24-nt siRNAs whose abundance differs between maize parents also show a trend of downregulation following hybridization. Surprisingly, hybrid vigor is fully maintained when 24-nt siRNAs are globally reduced by mutation of the RNA-dependent RNA polymerase2 (RDR2) encoded by modifier of paramutation1 (mop1). We also discovered that 21-22nt siRNAs derived from a number of distinct retrotransposon families differentially accumulate between B73 and Mo17 as well as their hybrid. Thus, maize possesses a novel source of genetic variation for regulating both transposons and genes at a genomic scale, which may contribute to its high degree of observed heterosis. Overall design: sRNA libraries were derived from RNA isolated from the seedling shoot apex and developing ear tissues from B73, Mo17, B73xMo17 and Mo17xB73. The shoot apex was chosen because it is enriched for meristematic tissue where cell proliferation occurs, rates of organ initiation are determined, and organ size is specified. The developing ear was examined because it is enriched in meristematic tissue and is undergoing rapid growth, and also because the mature ear shows the highest degree of heterosis. Total RNA was isolated and separated on a 15% TBE-Urea polyacrylamide gel. Using a 10-bp ladder, the sRNA fraction representing 10-40-bp was excised. sRNA libraries were prepared according to Lu et al. (2007) or manufacturer's instructitions (Illumina). A combination of Perl scripts and FASTX toolkit scripts were used to remove adapters, collapse identical sequences and count reads per sequence. Supplementary processed data text files contain the distinct sRNA sequences for all of the genotypes analyzed in that experiment. Abundance (reads per million) was calculated for each distinct sequence by dividing the number of reads of distinct sRNA in a library by the total number of sRNA reads for that library and multiplying this by 1 million. Genome builds: B73 genome, maizesequence.org release 4a.53 (October, 2009); Mo17 whole genome shotgun clones.

小RNA（small RNAs，sRNAs）被假设可参与杂种优势（hybrid vigor）形成，因其能够维持基因组完整性、贡献遗传多样性并调控基因表达。我们采用Illumina测序（Illumina sequencing）技术，评估了两个玉米自交系（maize inbred lines，B73、Mo17）及其杂交种之间的sRNA群体差异。我们从幼苗茎尖（seedling shoot apex）与发育中的雌穗（developing ear）这两种快速生长的组织中取材，这两类组织正是决定玉米杂交种更强生长势的关键部位。我们发现，亲本间的小干扰RNA（small interfering RNAs，siRNAs）差异主要源自重复序列区域（repeat regions）。尽管与拟南芥（Arabidopsis）或水稻（rice）相比，玉米基因组拥有更多且更复杂的重复序列，但我们证实，与这些结构更简单的植物基因组一致，玉米亲本间丰度存在差异的24-nt小干扰RNA（24-nucleotide siRNAs，24-nt siRNAs）在杂交后同样呈现出下调（downregulation）趋势。令人意外的是，当通过编码副突变修饰因子1（modifier of paramutation1，mop1）的RNA依赖的RNA聚合酶2（RNA-dependent RNA polymerase2，RDR2）的突变，全局降低24-nt siRNA的水平时，杂种优势完全得以维持。我们还发现，源自多个不同反转录转座子（retrotransposons）家族的21-22nt siRNA在B73、Mo17及其杂交种之间的积累模式存在显著差异。由此可见，玉米存在一种可在基因组规模上调控转座子（transposons）与基因表达的新型遗传变异来源，这或许与其观测到的高度杂种优势密切相关。总体实验设计（Overall design）：sRNA文库的构建原料为从B73、Mo17、B73×Mo17及Mo17×B73的幼苗茎尖与发育中的雌穗组织中分离得到的RNA。选择幼苗茎尖作为取材部位，是因其富含分生组织（meristematic tissue）——该组织存在细胞增殖过程，决定器官起始速率并确定器官大小。选择发育中的雌穗，则是因为其同样富含分生组织且处于快速生长阶段，同时成熟雌穗表现出最高程度的杂种优势。提取总RNA（Total RNA）后，将其在15% TBE-尿素聚丙烯酰胺凝胶（TBE-Urea polyacrylamide gel）上进行电泳分离。以10-bp分子量标准（10-bp ladder）为参照，切取10-40 bp区间的sRNA组分。参照Lu等人（2007）的方法或Illumina制造商的说明书构建sRNA文库。使用Perl脚本（Perl scripts）与FASTX工具包（FASTX toolkit）移除接头序列（adapters）、合并相同序列并统计每条序列的读段数。补充处理后的文本数据文件包含本实验中分析的所有基因型的独特sRNA序列。我们对每条独特序列计算其丰度（每百万读段数，reads per million）：将该文库中该独特sRNA的读段数除以该文库的总sRNA读段数，再乘以100万，即可得到其丰度值。基因组组装版本（genome builds）：B73基因组采用maizesequence.org于2009年10月发布的4a.53版本；Mo17基因组采用全基因组鸟枪法克隆文库。