Identification and analysis of differentially expressed genes in differentiating xylem of Chinese fir (Cunninghamia lanceolata) by suppression subtractive hybridization

Abstract

Wood is an important raw material for global industries with rapidly increasing demand. To isolate the genes differentially expressed during xylogenesis of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), we used a novel system. Forward and reverse subtracted cDNA libraries were constructed using the suppression subtractive hybridization method; for the forward library we used cDNA from the mutant Dugansha as the tester and cDNA from the wild-type clone Jurong 0 as the driver, and for the reverse library we used Jurong 0 cDNA as the tester and Dugansha cDNA as the driver. Transcriptional profiling was performed using a macroarray with 4 digoxigenin-labeled probes. We obtained 618 and 409 clones from the forward and the reverse subtracted library, respectively. A total of 405 unique expressed sequence tags (ESTs) were obtained. Forty percent of the ESTs exhibited homologies with proteins of known function and fell into 4 major classes: metabolism, cell wall biogenesis and remodeling, signal transduction, and stress. Real-time PCR was performed to confirm the results. The expression levels of 11 selected ESTs were consistent with both macroarray and real-time PCR results. The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation and is an important resource for forest research that can be directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Transcriptome Characterization of the Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook.) and Expression Analysis of Candidate Phosphate Transporter Genes

“…However, the length distribution of the unigenes was similar to that of the transcripts, and 75.18% unigenes were less than 500 bp, indicating that the length distribution of the transcripts and unigenes was mainly represented by short sequences with relatively little redundancy. These findings are roughly consistent with the results of previous studies [23,24], suggesting that the quality of our data is comparable to similar data of Chinese fir transcriptomic research. The Trinity software we used is a popular assembly tool, but different assembly strategies have an effect on the accuracy of data, and the comparative study of different assembly software such as AbySS and SOAPdenovo can help to improve assembly accuracy [46].…”

“…However, the quality of the data may be affected by several factors, including the contamination of adapter sequences, poor quality reads, and assembly errors [23]. In this study, a total of 236,529,278 clean reads and 35.47 Gbs of databases were obtained from Chinese fir transcriptomes, which is much more than any previously published transcriptome research on Chinese fir [23][24][25]43]. Due to a lack of homologous unigenes, the average length of unigenes for non-model plants is usually less than 500 bp [26,44,45].…”

“…As a non-model plant species with a very large genome size that is typical of conifers (20 to 30 Gb), the limited molecular research of Chinese fir has focused primarily on transcript profile investigations and patterns of gene expression [24,25]. However, the development of next-generation DNA sequencing technologies provides a revolutionary tool for transcriptomics to detect information about a given sample's RNA content [23].…”

Identification and Analysis of Differentially Expressed Genes During Seed Development Using Suppression Subtractive Hybridization (SSH) in Phaseolus vulgaris

“…The efficiency and reproducibility of SSH are very useful in studies of tissue-specific, developmental, or induced differentially expressed genes (Von Stein et al 1997;Basyuni et al 2011;Prabu et al 2011;Yang et al 2011b). In addition, in some plant species, SSH has proved useful for identifying genes differentially expressed during zygotic and somatic embryogenesis (Bishop-Hurley et al 2003;Namasivayam and Hanke 2006;Legrand et al 2007;Tsuwamoto et al 2007;Wang et al 2007;Geng et al 2009). Despite its utility and efficiency in isolating differentially expressed genes, SSH has not yet been widely applied in Phaseolus embryogenesis.…”

Comparison of Different Methods for RNA Extraction from Floral Buds of Tree Peony (<i>Paeonia suffruticosa</i> Andr.)

“…The CTAB method has been widely used for RNA isolation from polyphenols and polysaccharides-rich plants, including pine needles (Chang et al, 1993), a variety of tissues and organs of Chinese fir (Wang et al, 2007(Wang et al, , 2011, cotton (Zhao et al, 2012), Spurge (Xu et al, 2010), and tea (Muoki et al, 2012). Unexpectedly, it failed to extract high quality RNA from floral buds of tree peony.…”