Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish

Shandong Key Laboratory of Greenhouse Vegetable Biology, Shandong Branch of National Vegetable Improvement Center, Institute of Vegetables, Shandong Academy of Agricultural Sciences, Jinan 250100, China.
Plant Cell Reports (Impact Factor: 3.07). 04/2012; 31(8):1437-47. DOI: 10.1007/s00299-012-1259-3
Source: PubMed


The tuberous root of radish is an important vegetable, but insufficient transcriptomic and genomic data are currently available to understand the molecular mechanisms underlying tuberous root formation and development. High-throughput transcriptomic sequencing is essential to generate a large transcript sequence data set for gene discovery and molecular marker development. In this study, a total of 107.3 million clean reads were generated using Illumina paired-end sequencing technology. De novo assembly generated 61,554 unigenes with an average length of 820 bp. Based on a sequence similarity search with known proteins or nucleotides, 85.51 % (52,634), 90.18 % (55,507) and 54 % (33,242) consensus sequences showed homology with sequences in the Nr, Nt and Swiss-Prot databases, respectively. Of these annotated unigenes, 21,109 and 17,343 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. A total of 27,809 unigenes were assigned to 123 pathways in the Kyoto Encyclopedia of Genes and Genomes database. Analysis of transcript differences between libraries from the early and late seedling developmental stages demonstrated that starch and sucrose metabolism and phenylpropanoid biosynthesis may be the dominant metabolic events during tuberous root formation and plant hormones probably play critical roles in regulation of this developmental process. In total, 14,641 potential EST-SSRs were identified among the unigenes, and 12,733 primer pairs for 2,511 SSR were obtained. Summarily, this study gave us a clue to understand the radish tuberous root formation and development, and also provided us with a valuable sequence resource for novel gene discovery and marker-assisted selective breeding in radish. KEY MESSAGE: De novo assembled and characterized the radish tuberous root transcriptome; explored the mechanism of radish tuberous root formation; development of EST-SSR markers in radish.

1 Follower
32 Reads
  • Source
    • "Fortunately , with the advent of next-generation sequencing (NGS) technology in recent years, genomic information in previously uncharacterized systems can be generated (Grabherr et al. 2011). Currently, high-throughput RNA sequencing (RNA-Seq) has emerged as a powerful and cost-efficient tool for transcriptome analysis, and it has also been used for transcript profiling in various nonmodel plant species, including potato (Solanum tuberosum L.) (Zhang et al. 2014), pennycress (Thlaspi arvense) (Dorn et al. 2013), sweet potato (Ipomoea batatas) (Firon et al. 2013) and radish (Raphanus sativus L.) (Wang et al. 2012a). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ipomoea nil is widely used as an ornamental plant due to its abundance of flower color, but the limited transcriptome and genomic data hinder research on it. Using illumina platform, transcriptome profiling of I. nil was performed through high-throughput sequencing, which was proven to be a rapid and cost-effective means to characterize gene content. Our goal is to use the resulting information to facilitate the relevant research on flowering and flower color formation in I. nil. In total, 268 million unique illumina RNA-Seq reads were produced and used in the transcriptome assembly. These reads were assembled into 220,117 contigs, of which 137,307 contigs were annotated using the GO and KEGG database. Based on the result of functional annotations, a total of 89,781 contigs were assigned 455,335 GO term annotations. Meanwhile, 17,418 contigs were identified with pathway annotation and they were functionally assigned to 144 KEGG pathways. Our transcriptome revealed at least 55 contigs as probably flowering-related genes in I. nil, and we also identified 25 contigs that encode key enzymes in the phenylpropanoid biosynthesis pathway. Based on the analysis relating to gene expression profiles, in the phenylpropanoid biosynthesis pathway of I. nil, the repression of lignin biosynthesis might lead to the redirection of the metabolic flux into anthocyanin biosynthesis. This may be the most likely reason that I. nil has high anthocyanins content, especially in its flowers. Additionally, 15,537 simple sequence repeats (SSRs) were detected using the MISA software, and these SSRs will undoubtedly benefit future breeding work. Moreover, the information uncovered in this study will also serve as a valuable resource for understanding the flowering and flower color formation mechanisms in I. nil.
    MGG Molecular & General Genetics 04/2015; 290(5). DOI:10.1007/s00438-015-1034-6 · 2.73 Impact Factor
  • Source
    • "After removing the reads with adaptors, reads with unknown nucleotides larger than 5% and low quality reads, 66,110,340 clean PE reads consisting of 5,949,930,600 nucleotides (nt) were obtained with an average GC content of 47.34% (Table 1). The output was similar to a previous study on radish transcriptome from two root cDNA libraries, which generated a total of 53.6 million and 53.7 million clean reads, respectively [13]. All high-quality clean reads were assembled into 150,455 contigs with an average length of 299 bp, and the length distribution of the assembled contigs was as shown in Additional file 1A. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Radish (Raphanus sativus L.), is an important root vegetable crop worldwide. Glucosinolates in the fleshy taproot significantly affect the flavor and nutritional quality of radish. However, little is known about the molecular mechanisms underlying glucosinolate metabolism in radish taproots. The limited availability of radish genomic information has greatly hindered functional genomic analysis and molecular breeding in radish. In this study, a high-throughput, large-scale RNA sequencing technology was employed to characterize the de novo transcriptome of radish roots at different stages of development. Approximately 66.11 million paired-end reads representing 73,084 unigenes with a N50 length of 1,095 bp, and a total length of 55.73 Mb were obtained. Comparison with the publicly available protein database indicates that a total of 67,305 (about 92.09% of the assembled unigenes) unigenes exhibit similarity (e --value <= 1.0e-5) to known proteins. The functional annotation and classification including Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the main activated genes in radish taproots are predominately involved in basic physiological and metabolic processes, biosynthesis of secondary metabolite pathways, signal transduction mechanisms and other cellular components and molecular function related terms. The majority of the genes encoding enzymes involved in glucosinolate (GS) metabolism and regulation pathways were identified in the unigene dataset by targeted searches of their annotations. A number of candidate radish genes in the glucosinolate metabolism related pathways were also discovered, from which, eight genes were validated by T-A cloning and sequencing while four were validated by quantitative RT-PCR expression profiling. The ensuing transcriptome dataset provides a comprehensive sequence resource for molecular genetics research in radish. It will serve as an important public information platform to further understanding of the molecular mechanisms involved in biosynthesis and metabolism of the related nutritional and flavor components during taproot formation in radish.
    BMC Genomics 11/2013; 14(1):836. DOI:10.1186/1471-2164-14-836 · 3.99 Impact Factor
  • Source
    • "Based on MISA results, Primer3 v2.23 ( was used to design the primer pairs following the criteria described in Wang et al. [14]. In total, 30 pairs of primers were randomly selected and validated by PCR reactions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The pine tip moth, Rhyacionia leptotubula (Lepidoptera: Tortricidae) is one of the most destructive forestry pests in Yunnan Province, China. Despite its importance, less is known regarding all aspects of this pest. Understanding the genetic information of it is essential for exploring the specific traits at the molecular level. Thus, we here sequenced the transcriptome of R. leptotubula with high-throughput Illumina sequencing. In a single run, more than 60 million sequencing reads were generated. De novo assembling was performed to generate a collection of 46,910 unigenes with mean length of 642 bp. Based on Blastx search with an E-value cut-off of 10(-5), 22,581 unigenes showed significant similarities to known proteins from National Center for Biotechnology Information (NCBI) non-redundant (Nr) protein database. Of these annotated unigenes, 10,360, 6,937 and 13,894 were assigned to Gene Ontology (GO), Clusters of Orthologous Group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. A total of 5,926 unigenes were annotated with domain similarity derived functional information, of which 55 and 39 unigenes respectively encoding the insecticide resistance related enzymes, cytochrome P450 and carboxylesterase. Using the transcriptome data, 47 unigenes belonging to the typical "stress" genes of heat shock protein (Hsp) family were retrieved. Furthermore, 1,450 simple sequence repeats (SSRs) were detected; 3.09% of the unigenes contained SSRs. Large numbers of SSR primer pairs were designed and out of randomly verified primer pairs 80% were successfully yielded amplicons. A large of putative R. leptotubula transcript sequences has been obtained from the deep sequencing, which extensively increases the comprehensive and integrated genomic resources of this pest. This large-scale transcriptome dataset will be an important information platform for promoting our investigation of the molecular mechanisms from various aspects in this species.
    PLoS ONE 11/2013; 8(11):e81096. DOI:10.1371/journal.pone.0081096 · 3.23 Impact Factor
Show more