Repetitive sequence analysis and karyotyping reveals centromere-associated DNA sequences in radish (Raphanus sativus L.).
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. xiaoyuhqy@163.com.|National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China. xiaoyuhqy@163.com.
Qunyan
Q
He
National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China. caizexi123@cau.edu.cn.
Zexi
Z
Cai
Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. hutianh@hotmail.com.
Tianhua
T
Hu
National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China. huijunliu1022@gmail.com.
Huijun
H
Liu
Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. baocl@126.com.
Chonglai
C
Bao
Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. maowh@126.com.
Weihai
W
Mao
National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China. weiweijin@cau.edu.cn.
Weiwei
W
Jin
D002503:Centromere / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D059785:Karyotype; D008677:Metaphase; D016366:Open Reading Frames; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D031224:Raphanus / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D018744:DNA, Plant; D006657:Histones; D010940:Plant Proteins