Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China.
National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China.
BMC Plant Biol. 2015 Apr 18;15:105. doi: 10.1186/s12870-015-0480-y.
Radish (Raphanus sativus L., 2n = 2x = 18) is a major root vegetable crop especially in eastern Asia. Radish root contains various nutritions which play an important role in strengthening immunity. Repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. To date, studies about repetitive elements of radish are still limited. To better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish.
We conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. Results showed that about 31% of the genome was composed of repetitive sequences. Satellite repeats were the most dominating elements of the genome. The distribution pattern of three satellite repeat sequences (CL1, CL25, and CL43) on radish chromosomes was characterized using fluorescence in situ hybridization (FISH). CL1 was predominantly located at the centromeric region of all chromosomes, CL25 located at the subtelomeric region, and CL43 was a telomeric satellite. FISH signals of two satellite repeats, CL1 and CL25, together with 5S rDNA and 45S rDNA, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. The centromere-specific histone H3 (CENH3) has been used as a marker to identify centromere DNA sequences. One putative CENH3 (RsCENH3) was characterized and cloned from radish. Its deduced amino acid sequence shares high similarities to those of the CENH3s in Brassica species. An antibody against B. rapa CENH3, specifically stained radish centromeres. Immunostaining and chromatin immunoprecipitation (ChIP) tests with anti-BrCENH3 antibody demonstrated that both the centromere-specific retrotransposon (CR-Radish) and satellite repeat (CL1) are directly associated with RsCENH3 in radish.
Proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. Fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. Our study provides a valuable basis for future genomic studies in radish.
萝卜（Raphanus sativus L.，2n=2x=18）是一种主要的根茎类蔬菜作物，尤其在东亚地区广泛种植。萝卜根中含有多种营养物质，对增强免疫力具有重要作用。重复元件是基因组序列的主要组成部分，也是高等真核生物基因组大小变异的最重要因素。迄今为止，关于萝卜重复元件的研究仍然有限。为了更好地了解萝卜的基因组结构，我们进行了一项研究，以评估萝卜中重复元件的比例及其分布。
我们通过低覆盖度基因组测序进行了萝卜全基因组重复元件的特征分析，并进行了基于相似性的聚类分析。结果表明，约 31%的基因组由重复序列组成。卫星重复是基因组中最主要的重复元件。利用荧光原位杂交（FISH）技术对萝卜染色体上的三种卫星重复序列（CL1、CL25 和 CL43）的分布模式进行了研究。CL1 主要位于所有染色体的着丝粒区域，CL25 位于端粒附近，CL43 是端粒卫星。FISH 信号显示，两种卫星重复序列 CL1 和 CL25 与 5S rDNA 和 45S rDNA 一起，为鉴定每个体细胞中期染色体提供了有用的细胞遗传学标记。着丝粒特异性组蛋白 H3（CENH3）已被用作鉴定着丝粒 DNA 序列的标记。我们从萝卜中克隆并鉴定了一个假定的 CENH3（RsCENH3）。其推导的氨基酸序列与芸薹属物种的 CENH3 具有高度相似性。针对 B. rapa CENH3 的抗体特异性地标记了萝卜着丝粒。免疫染色和染色质免疫沉淀（ChIP）试验用抗 BrCENH3 抗体表明，萝卜中的着丝粒特异性逆转座子（CR-Radish）和卫星重复（CL1）都与 RsCENH3 直接相关。
萝卜中重复元件的比例进行了估计，卫星重复是最主要的重复元件。建立了精细的核型分析方法，可以方便地鉴定每个体细胞中期染色体。免疫荧光和 ChIP 基于测定的方法证明了卫星和着丝粒特异性逆转座子在着丝粒处的功能意义。我们的研究为萝卜的未来基因组研究提供了有价值的基础。
