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

Abstract

BackgroundRadish (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.ResultsWe 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.ConclusionsProportions 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0480-y) contains supplementary material, which is available to authorized users.

Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat

“…Usually, centromeric DNA sequences in plant species consist of species-specific tandem repeats and retrotransposons, and these sequences are located on all centromeric regions (Zhong et al, 2002 ; Nagaki et al, 2003 , 2009 , 2011 ; Nagaki and Murata, 2005 ; Houben et al, 2007 ; Tek et al, 2010 , 2011 ; Wang et al, 2011 ; Neumann et al, 2012 ; He et al, 2015 ). In this study of sunflower, we identified two types of centromeric DNA sequences (Supplementary Table 2 and Figure 4 ).…”

“…Among the constitutive proteins of kinetochores, centromere-specific histone H3 (CENH3) acts as a base for assembling other kinetochore proteins, and its presence epigenetically determines the kinetochore position (Perpelescu and Fukagawa, 2011 ). The first identified CENH3 was CENP-A in humans (Earnshaw and Rothfield, 1985 ), and its orthologs have been isolated from 11 of 63 APG III orders, including Poales, Asparagales, Rosales, Fabales, Malpighiales, Malvales, Brassicales, Myrtales, Solanales, Asterales, and Apiales, in this decade (Talbert et al, 2002 ; Zhong et al, 2002 ; Nagaki et al, 2004 , 2005 , 2012b ; Nagaki and Murata, 2005 ; Sanei et al, 2011 ; Wang et al, 2011 ; Neumann et al, 2012 , 2015 ; Dunemann et al, 2014 ; Masonbrink et al, 2014 ; He et al, 2015 ; Maheshawari, 2015 ). However, no CENH3 has been isolated from Asterales species.…”

“…Additionally, CENH3 localizes to functional centromeres (Warburton et al, 1997 ; Nasuda et al, 2005 ; Han et al, 2006 ). Therefore, centromeric DNA sequences have been identified in plant species by chromatin immunoprecipitation (ChIP) using anti-CENH3 antibodies (Zhong et al, 2002 ; Nagaki et al, 2003 , 2004 , 2009 , 2011 , 2012a , b ; Nagaki and Murata, 2005 ; Houben et al, 2007 ; Tek et al, 2010 , 2011 ; Wang et al, 2011 ; Gong et al, 2012 ; Neumann et al, 2012 ; He et al, 2015 ). In most cases, species-specific microsatellites, minisatellites, macrosatellites, and retrotransposons have been identified as the centromeric sequences, and these sequences are located on all centromeric regions in the species (Zhong et al, 2002 ; Nagaki et al, 2003 , 2009 , 2011 ; Nagaki and Murata, 2005 ; Houben et al, 2007 ; Tek et al, 2010 , 2011 ; Wang et al, 2011 ; Neumann et al, 2012 ; He et al, 2015 ).…”

Efficient anchoring of Erianthus arundinaceus chromatin introgressed into sugarcane by specific molecular markers

“…6C). Many studies have shown that satellite DNAs comprising head-to-tail tandem repeats are believed to be the most dynamic components (Biscotti et al 2015;Cai et al 2014;He et al 2015), undergoing the most rapid changes in the number and position of sites within a short evolutionary period (Mehrotra and Goyal 2014). It may also explain the largest number of E. arundinaceus-speci c sequences from the SSH library.…”

The Organization of Repetitive DNA in the Genomes of Amazonian Lizard Species in the Family Teiidae

“…Thus, these sequences are probably involved in the structural and functional organization of the centromere [Padeganeh et al, 2013;He et al, 2015;Padeken et al, 2015;Steiner and Henikoff, 2015].…”