Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19 DOI: 10.1007/s13131-014-0436-3 http://www.hyxb.org.cn E-mail: [email protected] Transcriptome-wide evolutionary analysis on essential brown algae (Phaeophyceae) in China SUN Jing1,3,4†, WANG Liang1,3,4†, WU Shuangxiu1,3†, WANG Xumin1,3, XIAO Jingfa1,3, CHI Shan2, LIU Cui2, REN Lufeng1,3, ZHAO Yuhui1,4, LIU Tao2*, YU Jun1,3* 1 CAS Key Laboratory of Genome Sciences and Information, Beijing Key Laboratory of Genome and Precision Medicine Technologies, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China 2 College of Marine Life Science, Ocean University of China, Qingdao 266003, China 3 Beijing Key Laboratory of Functional Genomics for Dao-di Herbs, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China 4 University of Chinese Academy of Sciences, Beijing 100049, China Received 1 April 2013; accepted 18 July 2013 ©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2014 Abstract Brown algae (Chromista, Ochrophyta, Phaeophyceae) are a large group of multicellular algae that play im- portant roles in the ocean's ecosystem and biodiversity. However, poor molecular bases for studying their phylogenetic evolutions and novel metabolic characteristics have hampered progress in the field. In this study, we sequenced the de novo transcriptome of 18 major species of brown algae in China, covering six orders and seven families, using the high-throughput sequencing platform Illumina HiSeq 2000. From the transcriptome data of these 18 species and publicly available genome data of Ectocarpus siliculosus and Phaeodactylum tricornutum, we identified 108 nuclear-generated orthologous genes and clarified the phy- logenetic relationships among these brown algae based on a multigene method. These brown algae could be separated into two clades: Clade Ishigeales-Dictyotales and Clade Ectocarpales-Laminariales-Desmares- tiale-Fucales. The former was at the base of the phylogenetic tree, indicating its early divergence, while the latter was divided into two branches, with Order Fucales diverging from Orders Ectocarpales, Laminariales, and Desmarestiale. In our analysis of taxonomy-contentious species, Sargassum fusiforme and Saccharina sculpera were found to be closely related to genera Sargassum and Saccharina, respectively, while Petalonia fascia showed possible relation to genus Scytosiphon. The study provided molecular evidence for the phylo- genetic taxonomy of brown algae. Key words: Phaeophyceae, transcriptome sequencing, multigene, phylogeny Citation: Sun Jing, Wang Liang, Wu Shuangxiu, Wang Xumin, Xiao Jingfa, Chi Shan, Liu Cui, Ren Lufeng, Zhao Yuhui, Liu Tao, Yu Jun. 2014. Transcriptome-wide evolutionary analysis on essential brown algae (Phaeophyceae) in China. Acta Oceanologica Sinica, 33(2): 13–19, doi: 10.1007/s13131-014-0436-3 1 Introduction these questions are issues of taxonomy and phylogeny in Pha- Brown algae are a large group of multicellular algae of Class eophyceae. For example, Sargassum fisiforme has a debated Phaeophyceae, Phylum Ochrophyta, Kingdom Chromista. membership in genus Sargassum or Hizikia; Orders Ishigeales They usually live in cold and temperate oceans (Charrier et al., and Dictyotales were considered to have diverged early in Pha- 2012; Zambounis et al., 2012) and distribute very broadly in the eophyceae, despite a lack of evolutionary evidence. Moreover, Northern Hemisphere, ranging from the North Pacific to North among Phaeophyceae, only one species, Ectocarpus siliculo- Atlantic regions (Coyer et al., 2011). Desmarestiales, Dictyotales, sus, has a completely sequenced genome (Cock et al., 2010), Ishigeales, Laminariales, Ectocarpales and Fucales are major or- and only three species, E. siliculosus, Saccharina japonica and ders of Phaeophyceae found in China and are dominant sea- S. latissima, have transcriptome data, showing poor molecular weeds along the western Pacific shore. They play important bases for the study of phylogenetic relationship within Phaeo- roles in marine ecosystems both as the predominant primary phyceae (Deng et al., 2012; Dittami et al., 2009; Heinrich et al., producers in the food chain and as underwater canopies for 2012). Fortunately, with the development of the next generation marine organisms (Eom et al., 2012). sequencing technology with the improvement of high through- From Linnaeus' first descriptions in his 1 753 book Species put and large decreasing of cost, such as 454 GS Junior (Roche) Plantarum to the present day, brown algae have continued to be and Hiseq 2000 (Illumina), de novo transcriptome sequencing a source for research. But despite the wealth of existing brown on a broad range of non-model species could realize to provide algae research, a number of unanswered evolutionary ques- more gene information to meet the demands for phylogenetic tions still persist (Dorrell and Smith, 2011; Green, 2011). Among analysis. Foundation item: The National Natural Science Foundation of China under contract Nos 31140070, 31271397 and 41206116; the algal transcrip- tome sequencing was supported by 1KP Project (www.onekp.com). *Corresponding author, E-mail: [email protected], [email protected] †Contributed equally. 14 SUN Jing et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 13–19 In this study, a total of 18 major species of brown algae in ligase. Fragment size selection was performed using agarose China, covering six orders and seven families, were collected in gel, and fragments of 200–250 bp were extracted from agarose the Bohai Bay. Except S. japonica, most brown algal transcrip- gel. The selected cDNA fragments were amplified by PCR. The tomes were for the first time sequenced (RNA-seq) using high- constructed cDNA library was sequenced by Illumina HiSeq throughput sequencing technology on an Illumina HiSeq 2000 2000. platform. A multi-gene evolutionary analysis was applied based on these transcriptome sequencing data to clarify the phylo- 2.3 De novo assembly genetic relationship among these brown algae. The research Strict reads filtering was performed before the assembly. not only provided molecular evidence for species taxonomy in Pair-end reads with primer or adaptor sequences were removed. brown algae but also provided valuable gene and methodology Reads with more than 10% of bases below Q20 quality or more information for further molecular research of brown algae. than 5% unknown nucleotides (Ns) were filtered from the total reads. De novo assembly was carried out using SOAPdenovo- Trans with the following parameters: -p 1, -K 25, -e 2, -r, -F, -L 2 Material and methods 100, -t 5 (http://soap.genomics.org.cn/SOAPdenovo-Trans. 2.1 Algal samples and total RNA extraction html) (Li et al., 2008). Gapcloser was then used for gap filling of All the brown algal samples (Table 1) were provided by the the scaffolds (Luo et al., 2012). laboratory of the Culture Collection of Seaweed in the Ocean 2.4 Predicting protein sequence and coding sequence (cds) of University of China. Total RNA was extracted using an improved CTAB method (Ghangal et al., 2009; Li and Ren, 2012; Xu et al., transciptome 2010; Yao et al., 2009). The quality and quantity of extracted RNA A protein sequence for each transcript was predicted by were assessed using Nanodrop ND 1000 spectrophotometer FASTX search (Pearson, 1996; Pearson and Lipman, 1988; Pear- (Labtech International Ltd, Lewes, UK) and Agilent 2100 bio- son et al., 1997) against the proteome of E. siliculosus from Eu- analyzer (Agilent Biotechnologies, Santa Clara, USA). ropean Nucleotide Archive (ENA) (http://www.ebi.ac.uk/ena/) with E-value less than 10−5 (Deng et al., 2012; Lu et al., 2012). 2.2 Transcriptome sequencing Then, coding sequences (cds) were obtained by getting the DNA cDNA library construction and sequencing were performed sequence between the start and end position of the alignment by the BGI (Shenzhen, China) on Illumina (San Diego, USA) and replacing insertions and deletions in the alignment; the in- HiSeq instruments according to the manufacturer's instruc- sertion of cds was signified by “N” while the insertion of protein tions. Briefly, mRNA was isolated from total RNA by Sera-mag sequence was signified by “X”. The predicted protein sequences Magnetic Oligo (dT) Beads. The mRNA with fragment buffer were filtered by length and identity to improve protein identi- was sheared into short fragments of about 200 bp. Using these fication accuracy and maintain consistent sequencing quality mRNA fragments as templates, the first-strand cDNA was syn- between each species. Protein sequences with length over 90% thesized by random hexamers-primers and reverse transcrip- of the aligned protein and identity over 30% were retained. tase. The second-strand cDNA was synthesized using DNA polymerase I, together with RNase H and dNTPs, and was pu- 2.5 Getting orthologs between 20 species rified by QiaQuick PCR purification kit (Qiagen). The double- We searched for gene orthologs from the 18 brown algal stranded cDNA was end-repaired and phosphorylated using transcriptome sequencing data of this study and two genome T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK. PE data of E. siliculosus and Phaeodactylum tricornutum CCMP adapter was added to the repaired cDNA fragments by T4 DNA 1055 (http://www.jgi.doe.gov/) according to the following pro- Table 1. Brown algal species selected for the transcriptome sequencing analysis Class Order Family Species Phaeophyceae Dictyotales Dictyotaceae Dictyopteris undulata Ishigeales