An overview of the Phalaenopsis orchid genome by BAC end sequence analysis Chia-Chi Hsu1, Yu-Lin Chung1, Yu-Ling Lee1, Tien-Chih Chen1, Yi-Tzu Kuo1, Wen-Chieh Tsai2,3, Yu-Yun Hsiao1, Yun-Wen Chen1, Wen-Luan Wu1,2,3, and Hong-Hwa Chen1,2,3 1Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan 2Institute of Tropical Plant Sciences, National Cheng Kung University, Tainan 701, Taiwan 3Orchid Research Center, National Cheng Kung University, Tainan 701, Taiwan. Abstract Phalaenopsis orchid is one of the most popular floral crops and is an economically important floriculture plant worldwide. Bacterial artificial chromosome (BAC) end sequences (BESs) can provide the first glimpse of the sequence composition of an unsequenced genome and yield molecular markers useful for genetic mapping and breeding. We used two BAC libraries (BamH I and Hind III) constructed from Phalaenopsis equestris. Pair-end sequences were generated from 2,920 BAC clones with a success rate of 95.7%. A total of 5,535 BESs generated represented 4.5 Mb, about 0.3% of the Phalaenopsis genome. The trimmed sequences ranged from 123 to 1,397 bp, with an average edited read length of 821 bp. These BESs underwent sequence homology searches. In total, 641 BESs (11.6%) were predicted to be protein-coding regions. In addition, 1,272 BESs (23.0%) were identified to contain repetitive DNA. Most of these repetitive DNA sequences were gypsy- and copia-like retrotransposons, with ratios of 41.9% and 12.8%, respectively, and only 10.8% were DNA transposons. Furthermore, 950 potential simple sequence repeats (SSRs) were discovered. Microsynteny analysis revealed that higher numbers of BESs mapped to the whole-genome sequences of poplar, then grape and Arabidopsis, and less to that of rice. Overall, BES analysis can give an overview of the Phalaenopsis genome in terms of the abundance of genes, repetitive DNA, SSR markers, and microsynteny with other plant species. BAC libraries are useful for constructing the physical map of the Phalaenopsis genome, thus improving the whole-genome sequencing of Phalaenopsis. 1 Background The family of Orchidaceae is one of the largest of the flowering plants, and the number of species may exceed 25,000 [1]. Like all other living organisms, present-day orchids have evolved from ancestral forms as a result of selection pressure and adaptation. They show a wide diversity of epiphytic and terrestrial growth forms and have successfully colonized almost every habitat on earth. The factors promoting the richness of orchid species may be specific interactions between orchid flowers and pollinators [2], sequential and rapid interplay between drift and natural selection [3], obligate orchid–mycorrhizal interactions [4], and epiphytism, the growth form of more than 70% of all orchids, which probably make up two-thirds of the epiphytic flora of the world. The expanding diversity of the orchid family may have taken place in a shorter time that that of most flowering plant families, which had already started to diversify in the Mid-Cretaceous [5]. The time of origin of orchids is in dispute, although they have been suggested to originate 80 to 40 million years ago (late Cretaceous to late Eocene) [6]. Perhaps the only general statement that can be made about the origin of orchids is that most extant groups are probably very young. Recently, the Orchidaceae were dated by a fossil record of an orchid pollinia on the back of its pollinator, a stingless bee, in amber. The most recent common ancestor of extant orchids was deduced to live in the late Cretaceous (76-84 Mya) [7]. Orchids are known for their diversity of specialized reproductive and ecological strategies. For successful reproduction, the formation of labellum and gynostemium (a fused structure of androecium and gynoecium) to facilitate pollination is well documented, and the co-evolution of orchid flowers and their pollinators is also well known [8-9]. In addition, the successful evolutionary progress of orchids may be 2 explained by mature pollen grains packaged as pollinia, pollination-regulated ovary/ovule development, synchronized timing of micro- and mega-gametogenesis for effective fertilization, and the release of thousands or millions of immature embryos (seeds without endosperm) in a mature capsule [10]. However, despite their unique developmental reproductive biology, specialized pollination and ecological strategies, molecular studies of orchids remain underrepresented relative to that of other species-rich plant families [11]. The genomic sequence resources for orchids are limited. A number of studies have used Sanger sequencing to develop expressed sequence tag (EST) resources for orchids [12-14]. These studies have highlighted the usefulness of cDNA sequencing for discovery of candidate genes for orchid floral development [15-16], floral scent production [13, 17] and flowering time [18] in the absence of a genomic sequence. However, a comprehensive description of the full complement of genes expressed in orchids remains unavailable. P. equestris is a diploid plant with an estimated haploid genome size of 1,600 Mb (3.37 pg/diploid genome) and 2n = 2x = 38 chromosomes, which are small and uniform in size (< 2 μm) [19]. Public databases of floral bud ESTs of P. equestris and P. bellina have been analyzed [13, 16]. These collections provide valuable resources for direct access to the genes of interest [15-17] and for the development of molecular markers for marker-assisted breeding programs or cultivar identification (unpublished data). However, basic genomic information about the genome organization and structure of Phalaenopsis orchids is lacking. To understand the sequence content and complexity of the Phalaenopsis genome, an efficient and viable strategy is to construct bacterial artificial chromosome (BAC) libraries and end-sequence randomly selected BAC clones from the BAC libraries. 3 The BAC end sequences (BESs) can be used as a primary scaffold for genome shotgun-sequence assembly and to generate comparative physical maps [20]. Moreover, analysis of BES data can provide an overview of the sequence composition, including gene density, presence of potential transposable elements (TEs) and microsatellites of an unsequenced genome [21-24]. In addition, BESs can yield molecular markers for genomic mapping, cloning of genes and phylogenetic analysis. Even for the sequenced rice genome, the Oryza Map Alignment Project (OMAP) constructed deep-coverage large-insert BAC libraries from 11 wild and 1 cultivated African Oryza species (O. glaberrima); the project fingerprinted and end-sequenced the clones from all 12 BAC libraries to construct physical maps of these Oryza species for study of evolution, genome organization, domestication, and gene regulatory networks and to contribute to crop improvement [25-26]. In this study, we analyzed 5,535 BESs from 2 genomic BAC libraries of Phalaenopsis equestris and focused on simple sequence repeat (SSR) or microsatellite content, repeat element composition, GC content and protein-coding regions. These BESs offer the first detailed insight into the sequence composition of the P. equestris genome, and the annotated BESs can be useful resources for molecular marker development with P. equestris. Results and Discussion BAC end sequencing DNA samples extracted from 2,920 BAC clones were sequenced from both ends, with a success rate of 95.7%. Of these, 71.4% and 28.6% BACs were from BamH I and Hind III libraries, respectively. After trimming ambiguous sequences, removing of vector sequence and mitochondrial DNA, we generated a collection of 5,535 4 high-quality BESs that included 5,360 paired-end reads (Table 1). The size of these BESs ranged from 123 to 1,397 bp, with an average of 821 bp, which corresponded to a total length of 4,544,250 bp equivalent to 0.3% of the P. equestris genome (Table 1). The 5535 BESs can be assembled into 340 contigs (with the average coverage of 2.99) and 4518 singletons (data not shown). For the read-length distribution of the BESs, both 800-899 bp and 900-999 bp were the most abundant for 1,567 BESs (28%) and 1,684 BESs (30%), respectively (Fig. 1). The GC content was 35.95%, close to that of the genomic DNA of P. amabilis BLUME (Sarcanthinae, Vandeae) and 34.09% as estimated by using buoyant density [34], which indicates that the Phalaenopsis genome is AT-rich. Buoyant density was also used to analyze Brassica maculate R. BR. (Oncidiinae, Vandeae), Cattleya schombocattleya LINDL. (Epidendrinae, Epidendrae), and Cymbidium pumilum SWARTZ cv. “Gareth Latangor” (Cymbidiinae, Vandeae) and revealed their GC contents to be 32.05%, 34.09%, and 32.05%, respectively [34], which suggests that most orchids have AT-rich genomes. All BES sequences have been submitted to GenBank, with the accession numbers HN176659-HN182163. Database sequence searches The P. equestris BESs underwent sequence homology analysis in RepBase and TIGR plant repeat databases by using RepeatMasker and BLAST for prediction of repeat sequences and potential TEs. A total of 1,272 BESs were homologous to transposable elements and repeats, for 23.0% of the total 5,535 BESs analyzed. These BESs were RepeatMasked and compared to the NCBI non-redundant protein databases. In total, 641 sequences (11.6%) contained the genic region of protein coding sequences. In addition, 29 BESs (0.5%) matched chloroplasts (Table 1). 5 Analysis of repetitive DNA in BES The large genome size of P. equestris (1,600 kb) implies a highly repetitive DNA content (i.e., more similar to that of maize than rice). After removal of 29 BESs containing chloroplast and mitochondria sequences, 5,506 BESs were used to identify the repetitive DNA sequences with RepeatMasker and the TIGR plant repeat database. Similar to other eukaryotic genomes, that of Phalaenopsis contains a significant portion of the repeat sequences and potential TEs: 1,272 BESs contain the potential TEs, for 23% of the total number of BESs (Table 1).