Plant Physiology Preview. Published on March 17, 2011, as DOI:10.1104/pp.110.171579
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Running head: Analysis of Barley FLcDNAs
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Corresponding author: Takashi Matsumoto
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National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki
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305-8602, Japan
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Tel: +81-29-838-7441
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e-mail: [email protected]
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Research area: Genome Analysis
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Comprehensive Sequence Analysis of 24,783 Barley Full-length cDNAs derived
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from Twelve Clone Libraries
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Takashi Matsumoto*#, Tsuyoshi Tanaka*, Hiroaki Sakai, Naoki Amano, Hiroyuki
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Kanamori, Kanako Kurita, Ari Kikuta, Kozue Kamiya, Mayu Yamamoto, Hiroshi Ikawa,
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Nobuyuki Fujii, Kiyosumi Hori, Takeshi Itoh and Kazuhiro Sato
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National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki
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305-8602, Japan (T. M., T. T., H. S., N. A., K. H., T. I.), Institute of Society for
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Techno-Innovation of Agriculture, Forestry and Fisheries, 446-1, Kamiyokoba, Tsukuba,
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Ibaraki 305-0854, Japan (H. K., K. Ku., A. K., K. Ka., M. Y., H. I.), Hitachi
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Government & Public Corporation System Engineering, Ltd., 2-4-8, Toyo-cyo, Koto,
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Tokyo 135-8633, Japan (N. F.), 4Institute of Plant Science and Resources, Okayama
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University, 2-20-1, Chuo, Kurashiki, Okayama, 710-0046, Japan (K. H., K. S.)
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Financial sources: This work was supported by grants to T. M. and T. I. from the
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Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural
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Innovation, TRC-1008, GIR-1001; and Integrated Research Project for Plant, Insect and
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Animal using Genome Technology, GD-1001, GD-1002).
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* These authors contributed equally to this work.
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# Corresponding author.
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Takashi Matsumoto
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e-mail: [email protected]
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Abstract
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Full-length cDNA (FLcDNAs) libraries consisting of 172,000 clones were constructed
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from a two-row malting barley cultivar, Hordeum vulgare L. cv. Haruna Nijo, under
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normal and stressed conditions. After sequencing the clones from both ends and
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clustering the sequences, a total of 24,783 complete sequences were produced. By
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removing duplicates between these and publicly available sequences, 22,651
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representative sequences were obtained; 17,773 were novel barley FLcDNAs, and 1,699
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were barley specific. Highly conserved genes were found in the barley FLcDNA
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sequences for 721 of 881 rice trait genes with 50% or greater identity. These FLcDNA
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resources from our Haruna Nijo cDNA libraries and the full-length sequences of
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representative clones, will improve our understanding of the biological functions of
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genes in barley, which is the cereal crop with the fourth-highest production in the world,
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and will provide a powerful tool for annotating the barley genome sequences that will
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become available in the near future.
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Introduction
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In 2009, approximately 54 million hectares of barley (Hordeum vulgare) were
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cultivated, and harvests of this species produced approximately 150 million tons of
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grain worldwide, according to the Food and Agriculture Organization of the United
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Nations (FAOSTAT; http://faostat.fao.org/). Barley belongs to the Poaceae family (i.e.,
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grasses), which also includes rice (Oryza sativa), maize (Zea mays), wheat (Triticum
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spp.), and rye (Secale sereade; cross-pollinated). These members of the Poaceae,
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including barley, are the major cereal crops cultivated throughout the world. Barley is
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utilized for animal feed, malting, and as a human food source. Because barley is
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self-pollinated and has a diploid (2n = 14) genome, it is recognized as a genetic model
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of the Triticeae tribe within the Poaceae. Studies of the barley genome, transcriptome,
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and proteome are currently advancing our understanding of the molecular functions of
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agriculturally important barley genes (Sreenivasulu and Wobus 2008; Sreenivasulu et al.
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2008).
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Although barley has been extensively studied in terms of its genetics and breeding,
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molecular biology and genomics studies have been limited until recently because of the
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large genome size of this species (>5 Gb; ca. 12 times that of rice) (Varshney et al.
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2007). To further promote barley genomics, a multi-national collaboration, the
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International Barley Sequencing Consortium (IBSC), has been developed with the
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objective of obtaining the whole genome sequence of barley (http://barleygenome.org;
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Schulte et al. 2009).
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The study of an organism's transcriptome (i.e., all transcribed sequences) is one of
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the most effective ways to investigate the structure and function of its active genes. In
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many plant species, transcript contigs have been constructed by assembling all the
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expressed sequence tag (EST) data available in PlantGDB, with the aim of identifying a
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dataset of unique mRNA sequences and maximizing the information obtained for both
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protein-coding and non-coding (nc) regions in these sequences (Duvick et al. 2008). A
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large set of ESTs (501,620 from the vulgare subspecies and 24,161 from the
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spontaneum
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www.ncbi.nlm.nih.gov/dbEST/;
subspecies
in
the
and
NCBI-dbEST
522,561
sequences
release-100110:
in
PlantGDB:
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www.plantgdb.org/) has been accumulated in the public domain. These ESTs have been
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assembled
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(http://www.ncbi.nlm.nih.gov/unigene) and into 134,482 Plant GDB-assembled unique
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transcripts
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estimated that approximately 75% of the genes in the barley genome have been captured
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(Sreenivasulu et al. 2008). Although these assemblies provide researchers with a
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tremendous amount of information for understanding partial barley gene structures, the
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assembly of sequences from different barley varieties might result in erroneous contigs
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or lead to the fusion of unrelated transcripts via common domains or motif sequences.
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Moreover, these data might lead to an overestimation of the number of genes in barley
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because these assemblies do not consider virtual connections between 5 and 3 end
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sequences, where both sequences are derived from the same cDNA clone.
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into
(PUTs)
23,595
clusters
in
NCBI’s
UniGene
(http://www.plantgdb.org/prj/ESTCluster/progress.php).
′
dataset
It
is
′
Capturing the transcripts of all active genes under defined (temporal, spatial, and
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stressed) conditions can provide a “snapshot” of living cells. The resultant data can be
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used to obtain quantitative (expression frequency) and qualitative (predicted protein
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function) information about these genes. For barley transcriptome analysis, a 22K
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Barley1 GeneChip probe array based on an EST database containing 350,000 sequences
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from 84 different RNA sources has been established (Close et al. 2004). The results of
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high-throughput transcript profiling of barley using this chip can be retrieved from
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PLEXdb
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(http://www.ebi.ac.uk/microarray-as/ae/). Using several platforms for transcript
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profiling, researchers have described the responses of barley to conditions such as high
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salinity (Ueda et al. 2006; Walia et al. 2006), low temperature or freezing (Koo et al.
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2008), and drought (Talame et al. 2007; Tommasini et al. 2008), as well as during
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various stages, such as grain development (Sreenivasulu et al. 2006), germination
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(Sreenivasulu et al. 2008), and growth (Druka et al. 2006), by means of coordinated up
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or down regulation of sets of genes.
(http://www.plexdb.org/)
and
ArrayExpress
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Moreover, the construction of a comprehensive gene set for barley by the genome
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sequencing is underway. However, following sequencing, genome annotation in the
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absence of information on exon-intron junctions is not an easy task, even for organisms
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for which complete genome sequences have been revealed. Although several gene
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prediction programs have been developed, the predicted gene structures might not
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always be correct because these programs may select and connect incorrectly predicted
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exons (Bennetzen et al. 2004; Cruveiller et al. 2004; Jabbari et al. 2004). Hence, most
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gene annotation projects refer to EST or mRNA sequences for the accurate structural
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annotation of genes (Imanishi et al. 2004; Itoh et al. 2007).
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Full-length cDNA (FLcDNA) is defined as the DNA complementary to an mRNA
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sequence that extends from the region near the 5' cap structure to the poly(A) tail, which
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are structures specific to eukaryotic mRNAs (Maruyama and Sugano 1994; Suzuki et al.
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1997; Carninci et al. 2000). FLcDNA technology has been applied to the genomes of
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humans (Ota et al. 2004), mice (Kawai et al. 2001), and several plant species, including
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Arabidopsis thaliana (Seki et al. 2002), O. sativa (Kikuchi et al. 2003), T. aestivum
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(Ogihara et al. 2004; Kawaura et al. 2009), soybean (Glycine max) (Umezawa et al.
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2008), Z. mays (Soderlund et al. 2009) and tomato (Solanum lycopersicum) (Aoki et al.
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2010). Recently, an FLcDNA library has been constructed for the Japanese malting
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barley variety Haruna Nijo (Sato et al. 2009). From more than 45,000 cDNA clones,
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5,006 non-overlapping sequences were obtained. These sequences and clones have been
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made publicly available through the Barley DB (http://www.shigen.nig.ac.jp/barley/),
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and they have proven effective in the identification of functional genes. However, this
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information is insufficient because the number of obtained FLcDNAs is much lower
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than all of the expressed genes in the barley genome.
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In the present study, we constructed twelve FLcDNA libraries for Haruna Nijo
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from various organs or under different conditions to produce a comprehensive dataset
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for this barley variety. After 5 and 3 end-sequencing and clustering of approximately
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172,000 cDNA clones, 24,783 complete FLcDNAs sequences were retrieved. In
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conjunction with publicly available barley FLcDNAs, a total of 22,651 non-redundant
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(nr) barley FLcDNAs were obtained. These sequenced clones should provide
′
′
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comprehensive information about the barley gene repertory.
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Results
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Barley FLcDNA dataset
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A total of 24,783 FLcDNAs were completely sequenced from the EST library (see
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Supplemental Information) (Fig.1). After eliminating contaminated sequences and
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possible fusions of unrelated transcripts, we obtained 23,614 barley FLcDNA sequences
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(henceforth, the "National Institute of Agrobiological Sciences FLcDNAs, or NIAS
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FLcDNAs"). These FLcDNAs were derived from the 12 cDNA libraries distinguished
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by library-specific tag sequences and an unknown group because of lacking the tag
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sequences (Supplemental Table S1). The number of FLcDNAs in the various libraries
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ranged from 857 in the library derived from flowers in the juvenile stage to 2,643 in the
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library derived from the plants treated with jasmonic acid (JA). To characterize the
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dataset, we compared the sequences with 4,999 Haruna Nijo FLcDNA sequences that
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had been published previously (henceforth, "Public FLcDNAs") (Sato et al. 2009). We
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found that the NIAS FLcDNA inserts were clearly longer (1,701 ± 846 bp) than the
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Public FLcDNA inserts (1,455 ± 742 bp) (Supplemental Figure S1). For comparison,
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the average lengths of rice FLcDNAs in sets of 37,139 O. sativa var. japonica
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FLcDNAs and 10,083 O. sativa var. indica FLcDNAs (DDBJ/EMBL/GenBank) were
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1,746 ± 945 bp and 1,042 ± 535 bp, respectively. Moreover, the estimated proportion of
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clones that might contain complete predicted open reading frames (ORFs; from Met to
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the stop codon) was similar between the NIAS and Public FLcDNAs (84.9% and 85.6%,
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respectively; Table I). These results suggest that the qualities of the two FLcDNA
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datasets are quite similar in terms of capturing complete ORFs.
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To evaluate the novel barley FLcDNAs revealed by our data, we compared the
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sequence similarity between the NIAS and Public FLcDNAs. We found that 21,586 of
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the 23,614 NIAS FLcDNAs showed no significant hits for any of the Public FLcDNAs.
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After redundant clones were removed, the remaining 17,773 FLcDNAs represented
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novel barley sequences (Fig. 1). Combined with the 4,878 nonredundant Public
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FLcDNAs, 22,651 representative barley FLcDNAs were constructed. We have
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designated these FLcDNAs “Uni-FLcDNAs” in this study and used this set for further
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comparative analyses. The average length of the Uni-FLcDNAs was 1,711 ± 863 bp.
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Recently, many paired sense/antisense transcripts referred to as natural antisense
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transcripts (NATs) (Osato et al. 2003; Wang et al. 2005) have been identified. In
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Arabidopsis, 958 NAT pairs have been confirmed (Alexandrov et al. 2006), and in rice,
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687 bidirectional transcription units have been discovered by means of FLcDNA
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mapping onto genome sequences (Osato et al. 2003). To test for the existence of NATs
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in the obtained FLcDNAs, we used blastn to screen for paired sense/antisense
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transcripts among all of the FLcDNAs, with a positive match criterion of greater than
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95% identity and an E-value of less than 1 × 10−5. We determined that 2,051 FLcDNAs
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could form sense/antisense pairs. An example of a sense/antisense pair presented by
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ClustalX alignment software is shown in Supplemental Figure S2. NIASHv1002F20
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and NIASHv1022F01 exhibit complete reverse homology, despite the fact that they
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both contain predicted ORFs. This suggests that our dataset contains NATs, even for
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sequences that encode predicted proteins.
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Protein-coding genes
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Based on homology searches and predictions of the longest ORF, 22,623 of
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22,651 representative ORFs were identified; 19,212 of these ORFs (84.9%) were
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homologous to known functional genes according to the results of blastx searches
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against the RefSeq (Pruitt et al. 2009) and UniProtKB (The UniProt Consortium 2009)
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databases. The proportion of representative FLcDNAs that were deemed to contain
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complete ORFs was 85.4% (Table I), indicating that most of the FLcDNAs were
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associated with protein-coding genes.
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To identify sequences in the Uni-FLcDNAs that were homologous to the
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previously cloned, rice trait genes, we employed 881 genes from Oryzabase
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(http://www.shigen.nig.ac.jp/rice/oryzabase/, Kurata and Yamazaki 2006). Barley
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homologs of these genes could play important roles in barley traits (phenotypes). We
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searched for homologs of these sequences among the Uni-FLcDNAs using blastp
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(Supplemental Table S2). We found that 721 of the 881 genes (81.8%) had barley
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homologs with a high similarity (≥50% identity). Although we currently do not know
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whether these homologs are orthologs, this result indicates that these trait genes are
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highly conserved between barley and rice. The detection of homologous sequences
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among the Uni-FLcDNAs could accelerate the comparative mapping of barley genes
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and functional prediction of these homologous cDNAs to promote future barley
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genomics research.
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We found 75 representative FLcDNAs that were longer than 5,000 bp in length,
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and some of these had the capacity to encode relatively long ORFs. For example, four
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of the ten longest FLcDNAs encoded ORFs longer than 1,000 amino acids. In contrast,
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three of their ORFs were shorter than 100 amino acids (Table II).
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We estimated that there were 28 nc FLcDNAs among the Uni-FLcDNAs (see
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Materials and Methods). Although these nc FLcDNAs could have been derived from
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miRNAs, a blastn search against rice miRNAs revealed no significant homologies.
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Moreover, as 26 of the 28 nc FLcDNAs exhibited no homologies with four fully
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sequenced grass genomes (O. sativa, Z. mays, Sorghum bicolor and Brachypodium
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distachyon), they might represent poorly conserved nc RNAs.
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An analysis using InterProScan revealed that 16,859 of the 22,623 representative
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ORFs (74.5%) contained conserved domains, and 12,595 ORFs (55.7%) were assigned
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GO terms(Barrell et al. 2009). Using GO2slim, we found that “binding” (GO:0005488)
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and “catalytic activity” (GO:0003824) were the most common second-level GO terms
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found in the data (Supplemental Fig. S3). The distribution of GO terms related to the
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barley Uni-FLcDNAs was quite similar to that seen in representative rice RAP data
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(Tanaka et al. 2008).
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Comparison of barley FLcDNAs with the Triticeae transcriptome
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We described 17,773 novel barley nr FLcDNAs (Fig. 1). To evaluate the amount
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of novel gene information in these sequences, homology searches of the FLcDNAs were
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conducted against sequences from publicly available transcripts. For this purpose, 6,625
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mRNAs and 525,559 ESTs from barley and 3,433 mRNAs and 1,107,168 ESTs from
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wheat were downloaded from DDBJ/EMBL/GenBank.
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A blastn search showed that 3,278 of the representative FLcDNAs had no
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homologous sequences. The average insert length of these novel FLcDNAs (1,602 ±
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921 bp) was slightly shorter than that of the known FLcDNAs (1,729 ± 851 bp) (Fig. 2).
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Characterization of protein functions using the second-level GO terms showed a similar
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distribution of terms among the novel and known FLcDNAs, except for “structural
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molecule activity” (GO:0005198) (Fig. 3) (2.4% in the “known” gene set and 5.1% in
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the “novel” gene set), indicating that the molecular functions of the novel FLcDNAs
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were distributed similarly to those of the known FLcDNAs. The novel transcripts were
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predicted to code for many different kinds of essential proteins, such as proteins
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involved in protein synthesis, including ribosomal proteins and elongation factors,
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transcription factors, cytochromes and stress-related proteins, including JA-induced
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protein, CBFII-5.1, heat shock protein, and NBS-LRR disease resistance protein
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homologue (Supplemental Table S3). Hence, we anticipate that these novel transcripts
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will support the construction of an essential gene network for barley.
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Of the 3,278 novel FLcDNAs, 1,974 were conserved in all four sequenced grass
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genomes (i.e., O. sativa, Z. mays, S. bicolor, and B. distachyon (International Rice
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Genome Sequencing Project 2005; Paterson et al. 2009; Schnable et al. 2009; The
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International Brachypodium Initiative 2010)) according to the results of blastn searches,
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but 942 had no grass homologs (Supplemental Table S4). We conducted blastp
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searches of the amino acid sequences derived from the novel ORFs against predicted
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protein sequences from the four species, and found that 1,731 ORFs were homologous
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to genes annotated in at least one species. These results suggest that the molecular
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functions of approximately two-thirds of the novel barley FLcDNAs could be predicted
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by comparison with annotated genes in major grass species.
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5
Detection of barley-specific FLcDNAs
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To elucidate whether the Uni-FLcDNAs contained gene structures that are
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conserved among crop plants, blastn searches were conducted against the four
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completed genome sequences. The results showed that 1,699 FLcDNAs (Specific
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FLcDNAs) were not associated with homologous regions in the four genomes, whereas
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20,952 FLcDNAs (Common FLcDNAs) had a homolog in at least one of the four plant
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species (Supplemental Table S5). Most FLcDNAs (19,778) were conserved in all four
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species, but more barley ORFs were homologous to ORFs in O. sativa than in B.
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distachyon, which shares a more recent common ancestor with barley than rice does.
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This result might have been caused by differences in the methodology used to sequence
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O. sativa and B. distachyon and the resulting quality of the reference genome sequences.
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The number of conserved genes detected in Z. mays was less than half the number
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detected in S. bicolor, even though the evolutionary distances of barley from Z. mays
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and S. bicolor are similar. The length of the inserts in the barley-specific FLcDNAs was
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shorter (1,301 ± 897 bp) than in the other FLcDNAs (1,745 ± 851 bp).
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Of the specific FLcDNAs, 263 contained known functional domains based on
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InterProScan analysis, and 25 were nc transcripts; thus, 85% of the specific FLcDNAs
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were not assigned to any putative function. GO data suggested that the relative
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1
proportions of the genes related to “signal transducer activity” (GO:0004871) and
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“enzyme regulator activity” (GO:0030234) were greater in the specific FLcDNAs than
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in the total representative FLcDNAs (data not shown). This suggests that barley exhibits
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species-specific regulatory networks involved in signal transduction, transcription, and
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metabolism.
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Comparison of FLcDNA sequences with published BAC sequences of barley
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To evaluate the barley FLcDNAs dataset in terms of its ability to capture a large
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number of active FLcDNAs, we mapped all of the FLcDNAs on publicly available
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barley bacterial artificial chromosome (BAC) sequences. Taketa and colleagues reported
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(Taketa et al. 2008) a Haruna BAC contig (AP009567) in which two genes were
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predicted. BAG12385.1 encodes a putative iron-deficiency specific 4 protein, and
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BAG12386.1 is a putative ethylene-responsive transcription factor responsible for the
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nud (hull-less) phenotype. Even though the public FLcDNAs could not be mapped to
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the predicted loci, multiple FLcDNAs determined in this study could be mapped at each
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locus, which suggests that the structures of these genes are accurate (Supplemental Fig.
17
S4). We also mapped the FLcDNAs on BACs of a different cultivar, Morex. Haruna
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Nijo is an established Japanese two-row malting cultivar, whereas Morex is a six-row
19
cultivar that is used as the American malting industry's standard cultivar. Because
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Morex will provide the first reference sequence for the barley genome, there have been
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increasing numbers of submissions of Morex sequences in the DNA Data Bank of Japan
22
(DDBJ; www.ddbj.nig.ac.jp/). To estimate the conservation of genes sequences between
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1
these cultivars, we mapped the FLcDNAs to publicly available Morex BAC sequences
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using EST2genome (Mott 1997). We found that 373 FLcDNAs mapped to genomic
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sequences from 113 of 181 BACs. Forty-five FLcDNAs (31 representatives) mapped to
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more than one locus. For example, NIASHv1123K08 was mapped to four loci in three
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BACs (one locus each of AY758233.1 and DQ249273.1 and two loci of AC239053.1).
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These genes were highly conserved (99.4% identity on average, Fig. 4), and 61 of them
7
were completely identical. The results of this analysis indicate that the Haruna Nijo
8
FLcDNAs can be utilized by IBSC to annotate the Morex genome.
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Discussion
2
3
Scientists have recently recognized that FLcDNAs are indispensable resources for
4
gene identification and annotation. As the barley genome sequencing project progresses,
5
obtaining information about barley FLcDNAs has become increasingly important.
6
There is a considerable amount of Triticeae (barley and wheat) transcript data in the
7
public domain; however, the number of barley FLcDNAs is insufficient compared with
8
the number available for other crops, such as O. sativa, Z. mays, and G. max (Kikuchi et
9
al. 2003; Umezawa et al. 2008; Soderlund et al.2009).
10
The sequences developed in this study represent the largest collection of Triticeae
11
FLcDNA data produced to date. To comprehensively cover the entire barley
12
transcriptome with FLcDNA clones, we constructed novel libraries from Haruna Nijo
13
from 12 different developmental stages, organs and/or stressed conditions and isolated
14
more than 170,000 clones (see Supplemental information). To identify the source of
15
clones in the pooled library constructions, tagged sub-libraries were used.
16
The ESTs we identified were clustered, and representative cDNA clones were
17
selected for full sequencing. The quality of the FLcDNAs obtained in this study was
18
evaluated by comparing their average sequence length (1,701bp) with that of publicly
19
available barley and rice FLcDNAs. A recent report in which the average length of
20
27,455 maize FLcDNAs was found to be 1,442 bp (Soderlund et al.2009) also supports
21
the length quality of the FLcDNAs in the present study. The method of FLcDNA
22
construction used here was the CAP-trapper method (Carninci et al. 1996), which has
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1
been used in FLcDNA projects in mice, Arabidopsis (Seki et al. 2002; Seki et al. 2004),
2
rice (Kikuchi et al. 2003), and soybean (Umezawa et al. 2008). Because this
3
methodology is based on the selection of mRNAs with an existing cap structure, it
4
provides high coverage of mRNA structures.
5
The 22,651 representative barley FLcDNAs described here were constructed
6
using the clones produced in this study and publicly available barley FLcDNAs.
7
Functional categorization of the Haruna Nijo FLcDNAs based on ORF prediction and
8
GO assignments produced results similar to those found for rice FLcDNAs. This could
9
indicate conservation of the entire gene sets between barley and rice. We also detected
10
28 nc genes. Only 4 out of the 28 nc transcripts were included in the NAT pairs,
11
suggesting that NATs are not the sole reason underlying the observed the nc transcripts.
12
Comparative analysis of the Uni-FLcDNAs against public data indicated the
13
importance of this dataset. First, 17,773 FLcDNAs were found to be novel in barley,
14
and 3,278 cDNAs from the Uni-FLcDNAs exhibited no homology to public EST or
15
mRNA data from barley or wheat. We consider it unlikely that this paucity of
16
homologous sequences resulted from natural variation among Haruna Nijo and other
17
barley varieties, because 1,974 of the 3,278 novel FLcDNAs had common homologs in
18
all four grass species examined (i.e., O. sativa, Z. mays, S. bicolor, and B. distachyon).
19
As full-length sequences are available for these four grass species, these 1,974 genes
20
might be structurally conserved and functionally active in grasses in general. These
21
findings should help us to assign gene functions to the novel barley FLcDNAs.
22
Second, we detected 1,699 FLcDNAs that showed no homology to any of the four
19
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
1
grass genomes. Even though the mean insert length of these FLcDNAs was shorter than
2
that of the other cDNAs, these 1,699 FLcDNAs still have the capacity to encode
3
functional proteins. We therefore concluded that these genes are Triticeae-specific (or at
4
least Hordeum-specific) sequences. Unfortunately, only 263 specific FLcDNAs could
5
be assigned putative functions based on InterPro domains, and the gene functions of the
6
other genes are unknown. The complete gene structure information and the FLcDNA
7
clones of the barley-specific genes can be used in future experimental studies, such as
8
for overexpression of recombinant proteins or microarray analyses, to reveal the
9
functions of these genes. We note that the 20,952 FLcDNAs with homologs in all four
10
grass genomes might still include some barley-specific genes, because the coding
11
potential of their mapped regions has not been verified; additionally, there were cases
12
where FLcDNAs mapped to non-genic regions in the latest annotated genome data.
13
The barley cultivar Haruna Nijo, which was used here as the source of the cDNA
14
libraries generated, was released as a malting barley variety in 1981 and has been
15
intensively used in the pedigree of Japanese malting barleys because of its excellent
16
quality profiles for brewing. Additionally, several genetic/genomic resources have been
17
established for this cultivar. More than 140,000 ESTs have been sequenced, and the
18
positions of more than 2,890 of these have been located in genetic maps (Sato et al.
19
2009). A BAC library has been constructed (Saisho et al. 2007), and some of these BAC
20
clones have been beneficial for map-based cloning of trait genes in barley (Taketa et al.
21
2008). These resources have made Haruna Nijo a useful variety for investigating barley
22
genetics and genomics (see http://www.shigen.nig.ac.jp/barley/index.html).
20
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
1
The IBSC is currently sequencing the complete genome of the Morex cultivar.
2
Mapping of all of the Haruna Nijo FLcDNAs to genome sequences from Morex BACs
3
clearly showed that the Haruna Nijo FLcDNAs can serve as good resources for the
4
genome annotation of Morex. Based on the density of the mapped FLcDNAs (i.e., 48.8
5
kb per gene; 277 loci mapped to 181 BAC sequences, covering 13.52 Mb), we
6
estimated that the total gene number in the barley genome is approximately 100,000.
7
This estimation is much larger than an estimation presented in a previous report (Mayer
8
et al. 2009) of 38,000 to 48,000 genes in the barley genome (i.e., 104 to 132 kb per gene
9
on average). It is likely that we have overestimated the number of genes, because the
10
BAC clones used for genome sequencing come from gene-rich regions targeted for the
11
purpose of map-based cloning or the investigation of genic regions. Based on the
12
previously estimated gene number (Mayer et al. 2009), our Hruna Nijo dataset of
13
22,651 nr FLcDNAs could represent 47% to 59% of the total number of genes present
14
in barley. The fact that 54% to 70% of the rice genes predicted by RAP have been
15
validated by rice FLcDNAs indicates that our dataset is similarly comprehensive. Not
16
all genes present in barley are expected to have been expressed in the 12 conditions
17
examined in the present study, so we suggest that the proportion of active genes
18
captured could be much greater than 47% to 59%.
19
21
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
1
Conclusion
2
We cloned more than 170,000 Haruna Nijo barley FLcDNAs, and identified more
3
than 24,000 complete FLcDNAs. The final set of 22,651 representative FLcDNAs
4
obtained will be a very useful resource for future studies of barley, as well as for the
5
annotation of barley genomic sequences in the ongoing IBSC genome sequencing
6
project. These data will also support the future wheat genome sequencing project
7
currently being organized by the International Wheat Genome Sequencing Consortium
8
(IWGSC, http://www.wheatgenome.org/).
9
10
All FLcDNA data obtained in this study are available from our full length Barley
cDNA Database (http://barleyflc.dna.affrc.go.jp/hvdb/index.html).
11
22
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
1
Materials and Methods
2
3
Full-sequencing of representative clones
4
For each representative clone determined after clustering the end sequences of
5
clones, both the 5 and the 3 ends were resequenced using the Big Dye Terminator v3.1
6
Cycle Sequencing Kit and then analyzed with an ABI 3730xl DNA sequencer (Applied
7
Biosystems Inc., Foster City, CA, USA) to confirm the clone-sequence identity. Next,
8
the internal regions were sequenced using a primer-walking method until the sequences
9
from opposite directions overlapped. We designed the sequencing strategy so that at
10
least two reads covered every part of the insert region of each cDNA clone to assure
11
sequence quality. Finally, we assembled these reads to create a consensus sequence for
12
each clone.
′
′
13
14
Removing redundancy
15
All-against-all blastn searches (Altschul et al. 1990) were conducted among the
16
barley FLcDNA sequences to detect redundant sequences. When an FLcDNA was
17
similar to another FLcDNA with 90% identity and 95% coverage, the FLcDNAs
18
were clustered. If two FLcDNAs that did not overlap were in the same cluster, we
19
considered that a fused transcript that bridged the two FLcDNAs was contained in this
20
cluster. In this case, the cDNA was discarded and the cluster was separated into two
21
clusters.
≥
≥
22
23
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
1
Prediction of open reading frames and functional annotation
2
Representative ORFs were predicted by blastx searches against the RefSeq and
3
UniProtKB databases with a positive match cutoff set at an E-value less than 1 × 10−20.
4
If there were no homologous proteins in the databases, the longest ORFs (>70 amino
5
acids in length) were assigned as predicted ORFs. If no ORFs were predicted, for an
6
FLcDNA, it was defined as nc FLcDNA. nc FLcDNAs were compared with rice
7
microRNAs
8
Griffiths-Jones et al. 2008) using blastn. To assign a gene function on the basis of
9
conserved domains or motifs, InterProScan searches (Zdobnov and Apweiler 2001;
10
Hunter et al. 2009) were conducted using the predicted ORFs. Based on the results of
11
the InterProScan searches, GOs categories were assigned to each predicted ORF
12
(Barrell et al. 2009). The GOs assignments were categorized using map2slim software
13
(http://www.geneontology.org/GO.slims.shtml#whatIs).
(miRNAs)
downloaded
from
miRBase
(http://www.mirbase.org/;
14
15
Comparison of FLcDNA sequences with complete genome sequences from other
16
crop species
17
To conduct genome-wide comparisons of the FLcDNA sequences, genome
18
sequences and annotation data from four completely sequenced crop plants were
19
obtained at the following sites: the Rice Annotation Project Database (RAP-DB;
20
http://rapdb.dna.affrc.go.jp/)
21
(http://www.maizesequence.org/index.html)
22
(http://genome.jgi-psf.org/Sorbi1/Sorbi1.home.html) for S. bicolor, and BrachyBase
for
O.
sativa,
for
the
MaizeSequence
Z.
mays,
24
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
the
database
JGI
1
(http://www.brachybase.org/) for B. distachyon. The FLcDNA sequences were mapped
2
onto the genome sequences using blastn search at an E-value of less than 1 × 10-5.
3
4
Detection of novel barley FLcDNAs
5
mRNAs and ESTs from barley and wheat were downloaded from DDBJ. The
6
poly(A) sequences were then trimmed, and repetitive regions were masked using
7
RepeatMasker software (http://repeatmasker.org). We conducted blastn searches of the
8
sequences against all barley FLcDNAs with the threshold of a positive match set at an
9
identity 90% and a coverage 90%. Because the dataset of barley FLcDNAs contained
10
redundant sequences, multiple hits for the query mRNA or EST sequences were
11
accepted.
≥
≥
12
13
Comparison with rice trait genes
14
A
15
(http://www.shigen.nig.ac.jp/rice/oryzabase/genes/genesTop.jsp). Of 4,124 trait genes,
16
881 genes had RAP-DB transcript information. The amino acid sequences of these
17
genes were used as queries for blastp analyses with the predicted ORFs of the
18
Uni-FLcDNAs.
list
of
rice
trait
genes
was
downloaded
from
Oryzabase
19
20
Comparison with barley BAC sequences
To compare the FLcDNAs with BAC sequences, one Haruna Nijo (Taketa et al.
21
22
2008)
and
181
Morex
BAC
sequences
were
downloaded
25
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
from
the
1
DDBJ/EMBL/GenBank. The Morex BACs were downloaded using the keywords
2
“Morex” and “BAC”. After masking repeat sequences using RepeatMasker and the
3
libraries
4
(http://mips.helmholtz-muenchen.de/plant/genomes.jsp) (Spannagl et al. 2007) and
5
Triticeae
6
http://wheat.pw.usda.gov/ITMI/Repeats/), the FLcDNAs were mapped onto the BACs
7
using the same method as was used for RAP annotation (Tanaka et al. 2008). The
8
FLcDNAs were first mapped using blastn to determine the possible mapping regions in
9
the genomic sequences and further mapped using EST2genome (Mott 1997) with
10
thresholds of 95% identity and 90% coverage. When two or more FLcDNAs were
11
mapped to the same locus, the longest FLcDNA was used for further analysis.
of
Repeat
≥
MIPS
Repeat
Sequence
Element
Database
release
Database
10
4.3
(TREP,
≥
12
13
14
15
Sequence publication in the public domain
All of the sequence data for the representative FLcDNAs have been submitted to
the DDBJ (AK353559-AK377172).
16
26
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
1
Acknowledgments
2
3
The authors are grateful to Sapporo Breweries Co. Ltd for providing the
4
germinating barley seeds and to Dr. J. F. Ma from Okayama University for experimental
5
assistance. Published barley FLcDNA information on was provided by the National
6
Bio-Resource Project of the MEXT, Japan.
7
27
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2011 American Society of Plant Biologists. All rights reserved.
1
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identification of cis-natural antisense transcripts in Arabidopsis thaliana. Genome Biol
12
6:R30
13
Wang J, Shi ZY, Wan XS, Sheu GZ and Zhang JL (2008) The expression pattern of
14
a rice proteinase inhibitor gene OsP18-1 implies its role in plant development. J Plant
15
Physiol 165:1519-1529
16
Zdobnov E M and Apweiler R (2001) InterProScan--an integration platform for the
17
signature-recognition methods in InterPro. Bioinformatics 17:847-848
18
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1
Figure Legends
2
Figure 1.
3
study
Determination of the representative barley FLcDNAs obtained in this
4
5
Figure 2.
Distribution of nucleotide lengths of known and novel FLcDNAs
6
Size distributions of FLcDNAs with a high homology to known grass genes (white bar)
7
and with no homology to known gene s (black bars).
8
9
Figure 3.
GO categorization of known and novel FLcDNAs
10
Functional motif/domains were detected and categorized using GO criteria. Results for
11
the representative FLcDNAs with a high homology to known grass genes (Known) and
12
with no homology to known genes (Novel).
13
14
Figure 4.
Distribution of sequence identities of mapped Haruna Nijo FLcDNAs to
15
published Morex BAC sequences
16
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1
Tables
2
Table I. Comparison of the completeness of the ORFs in the barley FLcDNAs
3
NIAS
Complete ORF
Public
Representative
20,055
4,279
19,335
5'-truncated ORF
3,404
685
3,181
3'-truncated ORF
100
11
79
5’-, 3’-truncated ORFa
29
4
28
Non-protein codingb
26
20
28
23,614
4,999
22,651
Total
4
a
5
proteins or longest ORFs whose lengths were ≥70 amino acids.
ORF had neither start nor stop codon.
b
FLcDNAs had no ORFs that were similar to known
6
37
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1
Table II. The ten longest FLcDNAs in the Uni-FLcDNA library
2
Nucleotide Amino acid length
Clone name
Gene function
length (bp) (AA)
NIASHv2010H17
7,384
460
Mitogen-activated protein kinase
NIASHv3115C23
7,155
2,230
Acetyl-coenzyme A carboxylase
NIASHv2090B16
7,012
90
Cycloeucalenol cycloisomerase
FLbaf76j08
6,765
98
Hypothetical protein
NIASHv2019K03
6,637
1,731
Chromatin remodeling complex subunit
NIASHv2035I12
6,311
227
Conserved hypothetical protein
NIASHv2017F18
6,183
152
KN1-type homeobox transcription factor
NIASHv1031N03
6,072
33
Hypothetical protein
NIASHv2125A18
6,037
1,859
DNA-directed RNA polymerase
NIASHv3085A02
6,036
1,792
Callose synthase 1 catalytic subunit
3
38
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