Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili
© Futamura et al; licensee BioMed Central Ltd. 2008
Received: 28 January 2008
Accepted: 11 August 2008
Published: 11 August 2008
Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages.
We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes.
Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species.
Molecular studies of angiosperm model plants have identified large numbers of genes that are expressed during stamen and pollen development. Genetic analyses have revealed the roles of some of these genes in the specification of stamen identity, the regulation of anther cell division and the differentiation of anthers, the control of male meiosis, the development of pollen, and anther dehiscence . Pollen development in gymnosperms and angiosperms involves several similar developmental and physiological processes [2, 3]. Homologs to floral transcription factors in angiosperms have been isolated from gymnosperm strobili and characterized. For example, the B-class MADS-box genes, which in angiosperms determine petal and stamen identities, and C-class genes that control the identities of stamens and carpels have been identified in conifers [4–7]. The Norway spruce gene DAL1, belongs to the AGL6 subfamily and DAL10, belongs to gymnosperm-specific subgroup were identified as other MADS-box genes in conifers [8, 9]. The flowering time gene, SOC1 and LEAFY, and A class gene APETALA2 have also been identified [10–13]. However, available information about transcripts that are expressed in the male reproductive structures of the gymnosperms is still limited .
The gymnosperm Cryptomeria is a monoecious plant that is distributed throughout Japan and in some parts of China. Cryptomeria japonica D. Don is widely grown in Japan because of its high productivity and utility. This species comprises 18% of the forests and covers 12% of the landmass of Japan. However, allergic reactions to its pollen have become a severe public health problem in Japan. A recent nationwide epidemiological survey found that at least 13% of the Japanese population suffers from pollinosis due to pollen of C. japonica .
The male strobili of C. japonica develop in axils of small branches near the tips of these branches. The primordia of male strobili are initiated from June to August and first become visible from July to September under natural conditions [16, 17]. The development of strobili can also be initiated by treatment with gibberellic acid (GA3). The promotion of flower formation by exogenous GA3 occurs even in one-year-old seedlings in spite of the fact that formation of strobili usually requires around 20 years after germination under natural conditions . Meiosis of microsporocytes begins in the middle of October, and microspores are formed from late October to late November. Differentiation of generative cells and tube cells occurs in December. The staminate strobili then remain in an arrested state of development until the following March when pollen grains are released. Each male strobilus is oval, pale yellow, and close to 5 mm in length and 2 mm in diameter, and each consists of microsporophylls attached to a main axis. Three to five rounded microsporangia develop on the lower surface of each microsporophyll. Each microsporangium contains 3,000 or more pollen grains, and as many as 400,000 pollen grains may be produced in a single strobilus .
Large-scale analysis of expressed sequence tags (ESTs) should help us to clarify the overall expression of genes in the male strobili of C. japonica. Some ESTs have already been derived from male strobili and from the pollen of C. japonica [18, 19]. However, the numbers of ESTs were relatively small, namely, 739 from male strobili and 3,655 from pollen, and most isolated cDNAs were not full-length. In the present study, we constructed a full-length enriched cDNA library using RNA derived from male strobili at various developmental stages, and we obtained more than 30,000 ESTs. We performed sequence-similarity searches using TBLASTX and BLASTX to compare the ESTs from C. japonica male strobili to sequences in UniProt, amino acid sequences from Arabidopsis and rice (Oryza sativa L.), and EST sequences from poplar, spruce and pine. We also identified ESTs that encoded MADS-box genes, which play a variety of important developmental roles in plants. In this report, we discuss both the utility of full-length cDNAs for gene discovery and the characterization of MADS-box genes in C. japonica.
Construction and quality check of a full-length enriched cDNA library
Summary of characteristics of the full-length cDNA library from male strobili of C. japonica
Number of 5'sequences
Number of 3' sequences
Number of cDNA clones
Number of contigsa
Number of singletsa
Number of unique transcriptsb
Number of unique transcripts corresponded to
Base composition at each position in the 5'-end sequences
To confirm the addition of G at the 5' end of full-length cDNA clones, we compared the 5'-terminal sequences of cDNA clones that encoded Cry j 2 with genomic sequences, including the promoter and coding region of Cry j 2, that we determined in a previous study . We found seven clones that encoded Cry j 2 among the 5'-end sequences of 18,843 clones. All seven of them were 5'-extended cDNAs, as compared with three Cry j 2 cDNAs that we generated previously . Five of the seven had one G, one clone had two Gs and one clone had TG at its 5' terminus; these nucleotides were not present in the corresponding region of Cry j 2 genes (data not shown). The comparison between these seven cDNAs and Cry j 2 genes indicated that full-length cDNAs had one or two additional nucleotides at their 5' termini, with most clones having a single G.
We performed a BLASTX comparison of amino acids encoded by 5'-terminal sequences of 18,843 cDNA clones and proteins from Arabidopsis. Our analysis revealed that 9,208 ESTs (48.7%) exhibited strong homology to Arabidopsis proteins (E-value < 1e-20). Among these ESTs, the starting positions in the alignment of 7,480 clones (81.2%) were upstream of the initiation codon of the corresponding protein. We also performed a BLASTN comparison with nucleotide sequences of protein-coding regions in Arabidopsis. We found that 2,131 ESTs (11.5%) derived from 5'-terminal sequences of cDNAs exhibited strong homology (E-value < 1e-10) to protein-coding regions in the Arabidopsis genome. The starting positions in the alignment of 1,755 ESTs (82.4%) were upstream of the corresponding coding region of Arabidopsis. These results suggest that most of our cDNA clones are full-length cDNAs.
Classification of ESTs
Sequence comparisons with other species
Occurrence of the 25 most common Pfam domains in the predicted proteins of unique transcripts from male strobili of C. japonica
Description of Pfam domain
Number of C. japonica transcriptsa
Number of genes in A. thaliana genomeb
Protein kinase domain
RNA recognition motif.
(a.k.a. RRM, RBD, or RNP domain)
NAD-dependent epimerase/dehydratase family
Zinc finger; C3HC4 type (RING finger)
Myb-like DNA-binding domain
WD domain; G-beta repeat
UDP-glucuronosyl and UDP-glycosyl transferase
Alpha/beta hydrolase fold
Sugar (and other) transporter
2OG-Fe(II) oxygenase superfamily
Core histone H2A/H2B/H3/H4
Eukaryotic aspartyl protease
Aldo/keto reductase family
Alcohol dehydrogenase, GroES-like domain
Mitochondrial carrier protein
Similarity of products of ESTs to stamen- or male gametophyte-specific proteins of Arabidopsis
Pfam domains found in transcripts of both A. thaliana stamen- or male gametophyte-specific genes and in transcripts from male strobili of C. japonica
Description of Pfam domain
Pfam accession number
Number of transcripts in C. japonica male strobili
Number of A. thaliana stamen- specific transcriptsa
Number of A. thaliana male gametophyte-specific transcriptsb
Protein kinase domain
Plant invertase/pectin methylesterase inhibitor
Protein tyrosine kinase
Glycosyl hydrolase family 28
Sodium/hydrogen exchanger family
ABC-2 type transporter
No apical meristem (NAM) protein
RNA recognition motif.
(a.k.a. RRM, RBD, or RNP domain)
Sugar (and other) transporter
Haloacid dehalogenase-like hydrolase
Glycosyl hydrolase family 1
Galactose-binding lectin domain
Similarity of the deduced proteins to pollen allergens
Products of ESTs that resemble pollen allergens
Cry j 1
Cryptomeria japonica (Sugi)
Cry j 2
Cryptomeria japonica (Sugi)
Cry j 3.8
Cryptomeria japonica (Sugi)
Cryptomeria japonica (Sugi)
Class IV chitinase
Cryptomeria japonica (Sugi)
Isoflavone reductase family
Jun o 4
Juniperus oxycedrus L. (Prickly juniper)
Amb a 3
Ambrosia artemisiifolia L. (Ragweed)
Cat r 1
Catharanthus roseus (L.) G. Don (Madagascar periwinkle)
Che a 1
Chenopodium album L. (Lamb's-quarters)
Cor a 1.04
Corylus avellana L. (Hazel)
Cor a 10
Corylus avellana L. (Hazel)
Cro s 1
Crocus sativus L. (Saffron)
Cyn d 22
Cynodon dactylon (L.) Pers. (Bermuda grass)
Cyn d 24
Cynodon dactylon (L.) Pers. (Bermuda grass)
Hum j Profilin
Humulus japonicus Siebold & Zucc. (Japanese hop)
Hum j 1
Humulus japonicus Siebold & Zucc. (Japanese hop)
Lol p 1
Lolium perenne L. (perennial ryegrass)
Ole e 5
Olea europaea L. (Olive)
Ole e 9
Olea europaea L. (Olive)
Ole e 10
Olea europaea L. (Olive)
Sal k 1.03
Salsola kali L. (Russian-thistle)
Sal k 2
Salsola kali L. (Russian-thistle)
Families of putative transcription factors
Identification of transcripts encoding putative transcription factors in male strobilus of C. japonica
Description of Pfam domains
Number of C. japonica male strobilus transcripts
Zinc finger, C3HC4 type (RING finger)
Myb-like DNA-binding domain
No apical meristem (NAM) protein; NAC domain
SRF-type transcription factor; MADS box
Histone-like transcription factor (CBF/NF-Y) and archaeal histone
Helix-loop-helix DNA-binding domain
B-box zinc finger
Dof domain, zinc finger
WRKY DNA-binding domain
Response regulator receiver domain
bZIP transcription factor
GATA zinc finger
B3 DNA binding domain; ABI3/VP1 transcription factor
GRAS family transcription factor
ZF-HD protein dimerisation region
CCT motif; CO-like protein
Auxin response factor
ARID/BRIGHT DNA binding domain
CCAAT-binding transcription factor (CBF-B/NF-YA) subunit B
TCP family transcription factor
CG-1 domain; CAMTA protein
Plant protein of unknown function; BZR1/LAT61 family
Whirly transcription factor
Identification and phylogenetic analysis of MADS-box genes
The genomes of gymnosperm species are usually large and replete with highly repetitive sequences. The haploid DNA content of C. japonica was estimated to correspond to 11 pg of DNA . This value is about half of the mean value for Pinaceae species , but about eighty times that of the Arabidopsis genome. EST analysis is a low-cost approach to characterization of the coding component of the genome. ESTs derived from several tissues of C. japonica were analyzed previously [18, 19, 35]. However, most of the previously analyzed ESTs were not full-length. Full-length cDNAs are essential for the determination of sites of initiation of transcription and the functional analysis of genes . In the present study, we constructed a full-length enriched cDNA library from C. japonica male strobili at various stages of development. To our knowledge, this is the first report of a full-length enriched cDNA library from a gymnosperm. In fact, the information of 6,464 Sitka spruce full-length cDNA sequences were registered in GenBank (accession numbers EF081469 to EF087932), but it has not been published. These full-length cDNAs should be a valuable tool for gene discovery and analysis in gymnosperms. We found that 90% of our cDNA clones included G at the 5' ends (Table 2). The G at the 5' end of full-length cDNA can be explained by the addition of C to the 3' end of full-length first-strand cDNA in a non-template-directed manner by the reverse transcriptase . Our observation of bias towards G at the 5' end of cDNAs is consistent with the addition of C to the 3' end of full-length cDNAs. The sequences of 5' ends imply that our cDNA library had been enriched for full-length cDNA clones. We also found that the second nucleotide of most cDNAs at their 5' termini was a purine nucleotide, which is common at sites of initiation of transcription in Arabidopsis and rice . This result suggests that a purine nucleotide is common at sites of initiation of transcription in C. japonica as well as Arabidopsis and rice. However, further studies about the comparison between genome sequences and the full-length cDNAs are needed to identify the sequence characteristics around the transcription start site in C. japonica.
We isolated 36,011 ESTs from either one or both ends of 19,437 cDNA clones. These ESTs were clustered into 23,658 consensus sequences (7,686 tentative contigs and 15,972 singletons) by PHRAP. The redundancy is extremely low compared with assemblies of ESTs derived from other conifers. In previous studies, the assembly of 49,101 ESTs derived from 16 cDNA libraries from different tissues of white spruce resulted in 16,578 consensus sequences , and that of 59,797 ESTs from wood-forming tissues of loblolly pine represented 20,377 consensus sequences . The normalization step in the generation of our cDNA library contributed the high rate of gene discovery. We obtained 10,463 transcripts after a round-robin BLASTN search and clustering of 5'- and 3'-end sequences derived from the same respective clones. We found that 1,317 transcripts (12.6%) did not exhibit any similarity in terms of predicted amino acid sequences to those encoded by Arabidopsis and rice genomes or to those derived from Pinus, Picea and Populus ESTs using BLASTX and TBLASTX (E- value > 1e-5). These results indicate that the ESTs generated in the present study have added to the known complement of gymnosperm transcripts.
We classified and annotated the transcripts isolated in this study (Fig. 1; Table 3; Additional file 2). In our functional classification, categories with no concrete assignment, such as "prediction of general function only", "function unknown" and "unassigned", accounted for a large fraction of transcripts (Fig. 1). Among assigned pollen transcripts of C. japonica, close to 31% of the predicted proteins are involved in the synthesis and modification of proteins, which are categorized as J and O . However, the percentage in those categories was only approximately 14% in the present study. The difference might be due to the large population of transcripts in the present study and/or to the specific characteristics of the male strobili of C. japonica. Table 3 shows the functional annotation of the most abundant families identified in this study. Protein kinase, cytochrome P450, and the RNA recognition motif were the most frequent domains. These domains are also present at relatively high levels in deduced products of the genome of Arabidopsis. We also found that the numbers of transcripts that encoded certain protein families or domains, such as NAD-dependent epimerase/dehydratase, the C3HC4 type zinc finger, the WD domain, aspartyl proteases and aldo/keto reductases, were larger than those that encoded the corresponding protein families or domains in the Arabidopsis genome. In general, the degree of complexity of multigene families seems to be correlated with the size of the plant genome. Southern hybridization patterns and comparative sequence analysis of conserved ortholog sets suggested that genomes of gymnosperms include complex families of genes [39, 40]. Our results suggest the increased complexity of gene families in C. japonica as compared to Arabidopsis. Since the number of cDNA clones in the present study was insufficient to allow us to prove this hypothesis and since the complete sequence of each clone has not been determined, further studies are needed to clarify the complexity of gene families in C. japonica.
We also searched potential candidates for novel allergens in C. japonica. In previous study, we analyzed similarity of the deduced proteins encoded by ESTs derived from C. japonica pollen and known plant allergens, and found cDNA clones that encoded proteins similar to six types of pollen allergens, eight types of food allergens and three types of latex allergens . In the present study, we found 180 transcripts that encoded proteins similar to 22 pollen allergens including all of five known allergens in C. japonica (Table 5). The major allergens in C. japonica are known to have similarity to pollen allergens of other plants . The existence of numerous allergens in C. japonica pollen has been suggested but only a few antigens have been identified. These newly deduced proteins in C. japonica are potential candidates for novel plant allergens.
In this study, we identified 207 transcripts that encoded Pfam domains of transcription factors, including those encoded by MADS-box genes. In plants, MADS-box genes are involved in various aspects of vegetative and reproductive growth, including the morphogenesis of flowers. Plant MADS-box genes are classified into types I and II . Most of plant type II genes have three more domains than type I, namely, an intervening (I) domain, a keratin-like (K) domain, and a C-terminal (C) domain. The plant type II genes are divided into two types based on the intron structure, namely, MIKCC- and MIKC*-type genes . The MIKCC-type genes can be subdivided into several well-defined gene clades, known as 'subfamilies' . We found twelve MADS-box genes derived from transcripts from male strobili of C. japonica, one of which was a type I gene while the other eleven were MIKCC-type genes. The MIKCC-type genes in C. japonica could be subdivided into five subfamilies: DEF/GLO/GGM13-, TM8-, AG-, AGL6-, and TM3-like genes. To our knowledge, this is the first report to identify type I genes and TM8-like genes in a gymnosperm. Functional characterization of type I genes in plants has been very limited to date. The type I gene in C. japonica (CMFL_009_N22) was most similar to AGL80, which is required for development of the central cell of the female gametophyte . The functions of type I genes in reproductive organs in C. japonica remain to be determined.
The MADS-box gene family has been subjected to a model of birth-and death evolution . TM8-like genes have been found in rosids (Cucurbitaceae) and asterids (Solanaceae) but not in Arabidopsis . A recent review noted that TM8-like genes exist in the basalmost angiosperms (Amborellaceae) and in magnoliids (Lauraceae) . We found evidence for at least one TM8-like gene in Cryptomeria (Cupressaceae sensu lato). None has been reported in Pinaceae to date, to our knowledge. These results suggest that TM8-like genes were established in the common ancestor of angiosperms and gymnosperms and that they have been lost relatively recently in some lineages. It seems likely that the rate of birth-and-death evolution of TM8-like genes has been high.
We identified six DEF/GLO/GGM13- like genes in this study. DEF- and GLO-like genes are known as B-class MADS genes, which are key regulators of petal and stamen specification in model species of angiosperms, such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. GGM13- like genes are the sister lineage of the B-class genes and are, hence, also known as Bsister (Bs) genes . Bs genes are expressed mainly in female reproductive organs and a member of the Bs gene family is involved in development of the seed coat . B-class and Bs genes were treated as one clade in the present study. Three genes (CMFL_009_E12, CMFL_023_G10, CMFL_052_L02) seem to be ancestral DEF- and GLO-like genes, while the other three genes (CMFL_007_I11, CMFL_018_D08, CMFL_046_H20) seem to constitute a sister clade of B-class and Bs genes. Several gene duplications appear to have occurred in the lineage to Cryptomeria after it diverged from other conifers.
The phylogenetic analysis of MADS-box genes in the present study has implications for the evolution of gymnosperms. The phylogenetic tree of TM3-like genes indicates that the Gnetales and the Pinaceae are nested as sister groups and that Cryptomeria is a sister of the Gnetales plus Pinaceae clade. A similar result was obtained for AG-like genes, but the bootstrap support was not very strong. Our data support those in prior multigene phylogenetic studies that suggested that all conifers, with the exception of Pinaceae, are a sister clade of the Gnetales plus Pinaceae clade [48, 49]. The evolution and divergence of the MADS-box family of genes has been studied extensively. However, most of the studied MADS-box genes in gymnosperms were derived from Gnetum, Pinus, and Picea [50, 51]. The identification and characterization of MADS-box genes in Cryptomeria and other gymnosperms should help us to understand the evolution of the structure of MADS-box genes and their roles in reproductive development.
We established a full-length enriched cDNA library using RNA derived from male strobili at various developmental stages, and we obtained 36,011 ESTs that was grouped into 10,463 clusters as unique transcripts. These full-length cDNAs provide information on expression of genes during the development of male reproductive organs. Our full-length enriched cDNA library will be useful for large-scale gene discovery for studies of gymnosperm species.
Male strobili of C. japonica were collected at seven-days intervals from mid August to mid November of 2003 and 2004. All the samples were immediately frozen in liquid nitrogen and stored at -80°C until use. RNA was extracted from each set of strobili, and a total RNA mixture derived from all the samples was used for construction of a full-length enriched cDNA library.
Total RNA was isolated by a previously described method  with slight modifications, using the SV Total RNA Isolating System (Promega, Madison WI). Frozen male strobili were powdered with Multi-beads shocker® (Yasui Kikai, Osaka, Japan). Then we added 7.5 ml of a solution of 100 mM Tris-HCl (pH 9.5), 20 mM EDTA, 1.4 M NaCl, 2% (w/v) polyvinylpyrrolidinone, 5% (w/v) β-mercaptoethanol and 0.5 mg/ml spermidine per gram of powdered male strobili with through mixing. After incubation of the mixture at 65°C for 5 min, RNA was extracted twice with an equal volume of a mixture of chloroform and isoamyl alcohol (24:1, v/v) and centrifugation at 15,000 × g for 20 min. One-fourth volume of 10 M LiCl was added to the final aqueous phase. Total RNA was allowed to precipitate for 2 h and collected by centrifugation at 15,000 × g for 30 min. The pelleted RNA was dissolved in SV RNA Lysis Buffer (Promega) and purified with the SV Total RNA Isolating System according to the protocol provided by the manufacturer. Poly(A)+ RNA was prepared with a μMACS™ mRNA Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the protocol provided by the manufacturer.
Construction of a full-length enriched cDNA library and DNA sequencing
A full-length enriched cDNA library was generated from poly(A)+ RNA by the biotinylated CAP trapper method using trehalose-thermoactivated reverse transcriptase as described previously [36, 53]. The resultant cDNAs were normalized, inserted into λ-FLC-III vectors and packaged [54, 55]. The packaged DNA produced approximately 1.1 × 106 plaques and the original phage library was amplified once. Part of the one-round-amplified phage library was excised in vivo and resultant plasmids were used to transform E. coli DH10B. Colonies of bacteria were picked with picking machines (Q-bot; Genetix, New Milton, UK) and transferred to 384-microwell plates. DNA templates corresponding to cDNA inserts were prepared from glycerol stocks by the rolling circle amplification (RCA) method with a TempliPhi™ DNA Amplification Kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). Products of RCA were sequenced with BigDye® Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA) and automatic sequencers (ABI 3700; Applied Biosystems). The M13-21 primer (5'-TGT AAA ACG ACG GCC AGT-3') was used for forward sequencing and the 1233 primer (5'-AGC GGA TAA CAA TTT CAC ACA GGA-3') was used for reverse sequencing. Sequences were quality-trimmed by reference to the high-quality (PHRED 20 or better) contiguous region, as determined with PHRED software  and then vector and poly(A) regions were removed. Sequences of less than 100 nucleotides after trimming were discarded. Contaminating regions of E. coli genome were identified with BLASTN , and ESTs with an E- value < 1e-10 were excluded. The remaining sequences were used for further analysis.
We searched for the nucleotide and amino acid sequences in TAIR (The Arabidopsis Information Resources; genome release version 6.0)  homologous to nucleotide and predicted amino acid sequences encoded by the 5'-end sequences of ESTs using the BLASTN and BLASTX program. We also searched for sequence homologies between the 5'-end sequences of ESTs and the cDNA and promoter region of Cry j 2 using the BLASTN program. Assessment of possible contamination by chloroplast and mitochondrial genomes and by ribosomal RNAs was performed with the BLASTN program (E-value < 1e-10). We used the chloroplast and mitochondrial genomes of Arabidopsis thaliana and genes for ribosomal RNAs from A. thaliana, Populus species, Platanus occidentalis and Zea mays as query sequences.
Grouping of ESTs
Related cDNA sequences from both 5' and 3' ends were grouped as contig sequences with the PHRAP program according to the following criteria: -penalty -5; -minmatch 300; -minscore 300; and -trim_qual 20 . After grouping ESTs as contigs, we used the BLASTN program to compare contig sequences. When contig sequences overlapped by more than 200 nucleotides and were more than 99% homologous in the overlapping regions and, in addition, when the overlap began within 5 bp from the ends of the contig sequences, we grouped the contig sequences together. We performed BLASTN searches separately for the EST sequences of between 100 and 200 bp and the resultant contig sequences. When an EST sequence and a contig were more than 98% homologous and the length of non-overlapping sequence in the EST sequence was less than 10 bp and, in addition, the overlap began within 5 bp from the end of the EST sequence or the contig, we grouped the EST sequence and the contig together. Finally both 5'- and 3'-end sequences derived from the same clone were grouped together.
Functional classification and annotation of ESTs
We categorized ESTs on the basis of the putative functions of encoded products using a database of clusters of orthologous groups from seven eukaryotic genomes (COG) . We used databases of proteins from more than three species (KOGs), proteins from two species (TWOGs) and lineage-specific expansion groups (LSEs). We compared the results of BLASTX analysis of each sequence in each cluster and adopted the functional category with the highest score.
We performed similarity searches with the BLASTX and TBLASTX programs. We used RefSeq of Arabidopsis from the National Center for Biotechnology Information , the database of the Knowledge-based Oryza Molecular Biological Encyclopedia  and UniProtKB/TrEMBL Release 33.3  as protein databases, and we used Pinus Gene Index Release 6.0, Spruce Gene Index Release 2.0, and Poplar Gene Index Release 3.0 from the TIGR Gene Indices  as EST databases. Sequences of stamen- and pollen-specific transcripts of Arabidopsis were obtained from previous studies [29, 30]. Similarities to ESTs that had previously been derived from C. japonica were determined with the BLASTN program. Similarities to known plant allergens were determined by BLASTX comparison with proteins in Allergome database http://www.allergome.org. We annotated protein families using a list of Pfam domain sequence (Pfam-A.fasta; release 21.0)  using the BLASTX program and an E- value < 1e-10. We obtained domain annotations of Arabidopsis thaliana proteins from the TAIR website  and selected Pfam families with an E- value < 1e-10.
To reconstruct a phylogenetic tree of MADS-box genes, we obtained amino acid sequences from EMBL/DDBJ/GenBank DNA databases and aligned them using the Clustal W program . The alignments of sequences of MADS, intervening (I-) and keratin-like (K-) domains were edited manually using the MacClade program . Maximum likelihood distances were calculated with the NJdist and ProtML programs in MOLPHY . A neighbor-joining (NJ) tree was obtained with NJdist, and was based on ML distances in the JTT model . This tree was used as the starting tree for a local rearrangement search with the ProtML program . The local bootstrap probability of each branch was estimated by the resampling-of-estimated-log-likelihood (RELL) method .
The authors are grateful to the members of Sequence Technology Team, RIKEN genomic Sciences Center for technical support. This research was supported in part by a Grant-in-Aid "Research Project for Utilizing Advanced Technologies in Agriculture, Forestry and Fisheries" and in part by a Grant-in-Aid for Scientific Research (No. 18780123) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and in part by a research grant (No. 200607) from the Forestry and Forest Products Research Institute.
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