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

ABSTRACT: 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.

SUBMITTER: He Q

PROVIDER: S-EPMC4417506 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

He Qunyan Q Cai Zexi Z Hu Tianhua T Liu Huijun H Bao Chonglai C Mao Weihai W Jin Weiwei W

BMC plant biology 20150418

<h4>Background</h4>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 s ...[more]

PMID: 25928652

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Project description:BackgroundRadish (Raphanus sativus L.) is an economically important root vegetable crop, and the taproot-thickening process is the most critical period for the final productivity and quality formation. MicroRNAs (miRNAs) are a family of non-coding small RNAs that play an important regulatory function in plant growth and development. However, the characterization of miRNAs and their roles in regulating radish taproot growth and thickening remain largely unexplored. A Solexa high-throughput sequencing technology was used to identify key miRNAs involved in taproot thickening in radish.ResultsThree small RNA libraries from 'NAU-YH' taproot collected at pre-cortex splitting stage, cortex splitting stage and expanding stage were constructed. In all, 175 known and 107 potential novel miRNAs were discovered, from which 85 known and 13 novel miRNAs were found to be significantly differentially expressed during taproot thickening. Furthermore, totally 191 target genes were identified for the differentially expressed miRNAs. These target genes were annotated as transcription factors and other functional proteins, which were involved in various biological functions including plant growth and development, metabolism, cell organization and biogenesis, signal sensing and transduction, and plant defense response. RT-qPCR analysis validated miRNA expression patterns for five miRNAs and their corresponding target genes.ConclusionsThe small RNA populations of radish taproot at different thickening stages were firstly identified by Solexa sequencing. Totally 98 differentially expressed miRNAs identified from three taproot libraries might play important regulatory roles in taproot thickening. Their targets encoding transcription factors and other functional proteins including NF-YA2, ILR1, bHLH74, XTH16, CEL41 and EXPA9 were involved in radish taproot thickening. These results could provide new insights into the regulatory roles of miRNAs during the taproot thickening and facilitate genetic improvement of taproot in radish.

Project description:Radish (Raphanus sativus L.) is one of the most important vegetable crops worldwide. Taproot thickening represents a critical developmental period that determines yield and quality in radish life cycle. To isolate differentially expressed genes (DGEs) involved in radish taproot thickening process and explore the molecular mechanism underlying taproot development, three cDNA libraries from radish taproot collected at pre-cortex splitting stage (L1), cortex splitting stage (L2), and expanding stage (L3) were constructed and sequenced by RNA-Seq technology. More than seven million clean reads were obtained from the three libraries, from which 4,717,617 (L1, 65.35%), 4,809,588 (L2, 68.24%) and 4,973,745 (L3, 69.45%) reads were matched to the radish reference genes, respectively. A total of 85,939 transcripts were generated from three libraries, from which 10,450, 12,325, and 7392 differentially expressed transcripts (DETs) were detected in L1 vs. L2, L1 vs. L3, and L2 vs. L3 comparisons, respectively. Gene Ontology and pathway analysis showed that many DEGs, including EXPA9, Cyclin, CaM, Syntaxin, MADS-box, SAUR, and CalS were involved in cell events, cell wall modification, regulation of plant hormone levels, signal transduction and metabolisms, which may relate to taproot thickening. Furthermore, the integrated analysis of mRNA-miRNA revealed that 43 miRNAs and 92 genes formed 114 miRNA-target mRNA pairs were co-expressed, and three miRNA-target regulatory networks of taproot were constructed from different libraries. Finally, the expression patterns of 16 selected genes were confirmed using RT-qPCR analysis. A hypothetical model of genetic regulatory network associated with taproot thickening in radish was put forward. The taproot formation of radish is mainly attributed to cell differentiation, division and expansion, which are regulated and promoted by certain specific signal transduction pathways and metabolism processes. These results could provide new insights into the complex molecular mechanism underlying taproot thickening and facilitate genetic improvement of taproot in radish.

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

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

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