2014 Vol. 33, No. 2

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2014, Vol. 33, No. 2 Content
2014, 33(2): .
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Reviews
Transcriptome sequencing of essential marine brown and red algal species in China and its significance in algal biology and phylogeny
WU Shuangxiu, SUN Jing, CHI Shan, WANG Liang, WANG Xumin, LIU Cui, LI Xingang, YIN Jinlong, LIU Tao, YU Jun
2014, 33(2): 1-12. doi: 10.1007/s13131-014-0435-4
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Most phaeophytes (brown algae) and rhodophytes (red algae) dwell exclusively in marine habitats and play important roles in marine ecology and biodiversity. Many of these brown and red algae are also important resources for industries such as food, medicine and materials due to their unique metabolisms and metabolites. However, many fundamental questions surrounding their origins, early diversification, taxonomy, and special metabolisms remain unsolved because of poor molecular bases in brown and red algal study. As part of the 1 000 Plant Project, the marine macroalgal transcriptomes of 19 Phaeophyceae species and 21 Rhodophyta species from China's coast were sequenced, covering a total of 2 phyla, 3 classes, 11 orders, and 19 families. An average of 2 Gb per sample and a total 87.3 Gb of RNA-seq raw data were generated. Approximately 15 000 to 25 000 unigenes for each brown algal sample and 5 000 to 10 000 unigenes for each red algal sample were annotated and analyzed. The annotation results showed obvious differences in gene expression and genome characteristics between red algae and brown algae; these differences could even be seen between multicellular and unicellular red algae. The results elucidate some fundamental questions about the phylogenetic taxonomy within phaeophytes and rhodophytes, and also reveal many novel metabolic pathways. These pathways include algal CO2 fixation and particular carbohydrate metabolisms, and related gene/gene family characteristics and evolution in brown and red algae. These findings build on known algal genetic information and significantly improve our understanding of algal biology, biodiversity, evolution, and potential utilization of these marine algae.
Articles
Transcriptome-wide evolutionary analysis on essential brown algae (Phaeophyceae) in China
SUN Jing, WANG Liang, WU Shuangxiu, WANG Xumin, XIAO Jingfa, CHI Shan, LIU Cui, REN Lufeng, ZHAO Yuhui, LIU Tao, YU Jun
2014, 33(2): 13-19. doi: 10.1007/s13131-014-0436-3
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Brown algae (Chromista, Ochrophyta, Phaeophyceae) are a large group of multicellular algae that play important 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 phylogenetic 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-Desmarestiale-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 phylogenetic taxonomy of brown algae.
Transcriptome characterization of Ishige okamurae (Phaeophyceae) shows strong environmental acclimation
QU Jieqiong, WANG Xumin, CHI Shan, WU Shuangxiu, SUN Jing, LIU Cui, CHEN Shengping, YU Jun, LIU Tao
2014, 33(2): 20-26. doi: 10.1007/s13131-014-0437-2
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Ishige okamurae, with leathery branched narrow fronds consisting of cylindrical hairs, is the typical species of the genus Ishige, which is considered as one of the most basal genera in the phylogeny of the Phaeophyceae. Apart from great public interest from the evolutionary respect, more attention has been brought on the abundant bioactive compounds in I. okamurae for therapeutic or economic considerations, such as diphlorethohydroxycarmalol and ishigoside. Yet little is known about related key genes or metabolic pathways involved in I. okamurae, which calls upon us to carry out global analyses of transcriptome by next generation sequencing. Altogether, we obtained 78 583 assembled scaffolds with N50 of 1 709 nucleotides, and 25 357 unigenes with significant BLAST matches (E-value cutoff of 10?5). In terms of characterization of the transcriptome of I. okamurae, we focused on anti-stress metabolic pathways and synthetic routes of bioactive compounds in an attempt to obtain a better understanding of the interactive organism-environment regulatory networks. Pathway-based analysis helped us to deepen our comprehension of the interaction between I. okamurae and its surroundings, with MAPK signal pathway as an example. Furthermore, we discovered a wide range of novel putative functional proteins that could be of wide application, such as Rab family, using sequence-based transcriptome. In conclusion, transcriptome characterization of I. okamurae (Phaeophyceae) shows strong environmental acclimation.
Analysis of Saccharina japonica transcriptome using the high-throughput DNA sequencing technique and its vanadium-dependent haloperoxidase gene
LIANG Xiayuan, WANG Xumin, CHI Shan, WU Shuangxiu, SUN Jing, LIU Cui, CHEN Shengping, YU Jun, LIU Tao
2014, 33(2): 27-36. doi: 10.1007/s13131-014-0438-1
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Saccharina is one of the most important cold-water living marine brown algal genera. In this study we analyzed the transcriptome of S. japonica, which belongs to the 1 000 Plants (OneKP) Project, by using a nextgeneration high-throughput DNA sequencing technique. About 5.16 GB of raw data were generated, and 65 536 scaffolds with an average length of 454 bp were assembled with SOAP de novo assembly method. In total, 19 040 unigenes were identified by BLAST; 25 734 scaffolds were clustered into 37 Gene ontology functional groups; 6 760 scaffolds were classified into 25 COG categories, as well as 2 665 scaffolds that were assigned to 306 KEGG pathways. Majority of the unigenes exhibited more similarities to algae including brown algae and diatom than other cyanobacteria, marine diatom, and plant. Saccharina japonica has the outstanding capability to accumulate halogen such as Br and I via halogenation processes from seawater. We acquired 42 different vanadium-dependent haloperoxidases (vHPO) in S. japonica transcriptome data, including 5 segments of vanadium-dependent iodoperoxidase (vIPO) and 37 segments of vanadium-dependent bromoperoxidase (vBPO). Complicated analyses of identified fulllength S. japonica vBPO1 and S. japonica vBPO2 revealed the importance of vBPO among species of brown algae and the strong relationship between marine algal vBPOs and vIPOs. This study will enhance our understanding of the biological characteristics and economic values of S. japonica species.
Comparative analysis on transcriptome sequencings of six Sargassum species in China
WANG Guoliang, SUN Jing, LIU Guiming, WANG Liang, YU Jun, LIU Tao, CHI Shan, LIU Cui, GUO Haiyan, WANG Xumin, WU Shuangxiu
2014, 33(2): 37-44. doi: 10.1007/s13131-014-0439-0
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Species of Sargassum are distributed worldwide, and are of great ecological and economic importance in marine ecosystems and bioresources. In this study, transcriptome sequencings of six Sargassum species were performed for the first time using an Illumina platform. For each sample, a total of 2.1-2.5 Gb of nucleotides are collected and assembled into 69 871-116 790 scaffolds, with an average length of 410-550 bp and N50 length of 756-1 462 bp. A total of 20 512-28 684 unigenes of each sample were annotated and compared well with known gene sequences from nr database. Clusters of Orthologous Groups (COG), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also performed for further understanding of gene functions and regulation pathways. Gene expression levels were calculated based on RPKM values and compared among these species, especially for those genes related to carbohydrate metabolism. Cluster analyses indicated that the differences of global gene expression between S. fusiforme, which was nominated as Hizikia fusiformis before, and other five species were not significant. Further phylogenetic analysis of 108 orthologous genes confirmed that S. fusiforme had closer relationship with S. hemiphyllum rather than S. horneri. These transcriptome data provided valuable information for better understanding of genome and gene characteristics of Sargassum algae and benefiting comparative and phylogenetic studies of Phaeophyceae species in future studies.
De novo sequencing and comparative analysis of three red algal species of Family Solieriaceae to discover putative genes associated with carrageenan biosysthesis
SONG Lipu, WU Shuangxiu, SUN Jing, WANG Liang, LIU Tao, CHI Shan, LIU Cui, LI Xingang, YIN Jinlong, WANG Xumin, YU Jun
2014, 33(2): 45-53. doi: 10.1007/s13131-014-0440-7
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Betaphycus gelatinus, Kappaphycus alvarezii and Eucheuma denticulatum of Family Solieriaceae, Order Gigartinales, Class Rhodophyceae are three important carrageenan-producing red algal species, which produce different types of carrageenans, beta (β)-carrageenan, kappa (κ)-carrageenan and iota (ι)-carrageenan. So far the carrageenan biosynthesis pathway is not fully understood and few information is about the Solieriaceae genome and transcriptome sequence. Here, we performed the de novo transcriptome sequencing, assembly, functional annotation and comparative analysis of these three commercial-valuable species using an Illumina short-sequencing platform Hiseq 2000 and bioinformatic software. Furthermore, we compared the different expression of some unigenes involved in some pathways relevant to carrageenan biosynthesis. We finally found 861 different expressed KEGG orthologs which contained a glycolysis/gluconeogenesis pathway (21 orthologs), carbon fixation in photosynthetic organisms (16 orthologs), galactose metabolism (5 orthologs), and fructose and mannose metabolism (9 orthologs) which are parts of the carbohydrate metabolism. We also found 8 different expressed KEGG orthologs for sulfur metabolism which might be importantly related to biosynthesis of different types of carrageenans. The results presented in this study provided valuable resources for functional genomics annotation and investigation of mechanisms underlying the biosynthesis of carrageenan in Family Solieriaceae.
Comparative analysis of four essential Gracilariaceae species in China based on whole transcriptomic sequencing
XU Jiayue, SUN Jing, YIN Jinlong, WANG Liang, WANG Xumin, LIU Tao, CHI Shan, LIU Cui, REN Lufeng, WU Shuangxiu, YU Jun
2014, 33(2): 54-62. doi: 10.1007/s13131-014-0441-6
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Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilariopsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous species which are important resources for the production of special proteins, phycobilisomes, special carbohydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lemaneiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.
Tryptophan synthase of Phaeophyceae originated from the secondary host nucleus
ZHANG Yalan, CHI Shan, WU Shuangxiu, LIU Cui, YU Jun, WANG Xumin, CHEN Shengping, LIU Tao
2014, 33(2): 63-72. doi: 10.1007/s13131-014-0442-5
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Tryptophan synthase (TS, EC 4.2.1.20) catalyzes the last two steps of L-tryptophan biosynthesis. In prokaryotes, tryptophan synthase is a multi-enzyme complex, and it consists of α and β subunit which forms an α-ββ-α complex. In fungi and diatoms, TS is a bifunctional enzyme. Because of the limited genomic and transcriptomic data of algae, there are few studies on TS evolution of algae. Here we analyzed the data of the 1000 Plants Project (1KP), and focused on red algae and brown algae. We found out that the TS of Phaeophyceae were fusion genes, which probably originated from the secondary host nucleus, and that the TS of Rhodophyta contained two genes, TSA and TSB, which both display a possible cyanobacterial origin at the time of primary endosymbiosis. In addition, there were two types of TSB genes (TSB1 and TSB2). Through the multiple sequence alignment of TSB proteins, we found several residues conserved in TSB1 but variable in TSB2 which connect with α subunit. The phenomenon may suggest that the TSB2 sequences of Rhodophyta cannot form stable complex with TSA.
Origin and evolution of alginate-c5-mannuronan-epimerase gene based on transcriptomic analysis of brown algae
WANG Ren, WANG Xumin, ZHANG Yalan, YU Jun, LIU Tao, CHEN Shengping, CHI Shan
2014, 33(2): 73-85. doi: 10.1007/s13131-014-0443-4
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The coding product of alginate-c5-mannuronan-epimerase gene (algG gene) can catalyze the conversion of mannuronate to guluronate and determine the M/G ratio of alginate. Most of the current knowledge about genes involved in the alginate biosynthesis comes from bacterial systems. In this article, based on some algal and bacterial algG genes registered on GenBank and EMBL databases, we predicted 94 algG genes open reading frame (ORF) sequences of brown algae from the 1 000 Plant Transcriptome Sequencing Project (OneKP). By method of transcriptomic sequence analysis, gene structure and gene localization analysis, multiple sequence alignment and phylogenetic tree construction, we studied the algal algG gene family characteristics, the structure modeling and conserved motifs of algG protein, the origin of alginate biosynthesis and the variation incidents that might have happened during evolution in algae. Although there are different members in the algal algG gene family, almost all of them harbor the conserved epimerase region. Based on the phylogenetic analysis of algG genes, we proposed that brown algae acquired the alginate biosynthesis pathway from an ancient bacterium by horizontal gene transfer (HGT). Afterwards, followed by duplications, chromosome disorder, mutation or recombination during evolution, brown algal algG genes were divided into different types.
Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts for marine red algae (Rhodophyta) in China
JIA Shangang, WANG Xumin, QIAN Hao, LI Tianyong, SUN Jing, WANG Liang, YU Jun, LI Xingang, YIN Jinlong, LIU Tao, WU Shuangxiu
2014, 33(2): 86-93. doi: 10.1007/s13131-014-0444-3
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The chloroplast and mitochondrion of red algae (Phylum Rhodophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using red algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Rhodophyta. We have found that red algae were divided into three clades of orders, Florideophyceae, Bangiophyceae and Cyanidiophyceae. Taxonomy resolution for Class Florideophyceae showed that Order Gigartinales was close to Order Halymeniales, while Order Gracilariales was in a clade of Order Ceramials. We confirmed Prionitis divaricata (Family Halymeniaceae) was closely related to the clade of Order Gracilariales, rather than to genus Grateloupia of Order Halymeniales as reported before. Furthermore, we found both mitochondrial and chloroplastic genes in Rhodophyta under negative selection (Ka/Ks < 1), suggesting that red algae, as one primitive group of eukaryotic algae, might share joint evolutionary history with these two organelles for a long time, although we identified some differences in their phylogenetic trees. Our analysis provided the basic phylogenetic relationships of red algae, and demonstrated their potential ability to study endosymbiotic events.
Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown algae (Phaeophyceae) in China
JIA Shangang, WANG Xumin, LI Tianyong, QIAN Hao, SUN Jing, WANG Liang, YU Jun, REN Lufeng, YIN Jinlong, LIU Tao, WU Shuangxiu
2014, 33(2): 94-101. doi: 10.1007/s13131-014-0445-2
Abstract:
The chloroplast and mitochondrion of brown algae (Class Phaeophyceae of Phylum Ochrophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others (Phylum Cryptophyta and Haptophyta) in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassifolia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.
Endogenous viral elements in algal genomes
WANG Liang, WU Shuangxiu, LIU Tao, SUN Jing, CHI Shan, LIU Cui, LI Xingang, YIN Jinlong, WANG Xumin, YU Jun
2014, 33(2): 102-107. doi: 10.1007/s13131-014-0446-1
Abstract:
Endogenous viral elements (EVEs) are host-genomic fragments originated from viral genomes. They have been found universally in animal and plant genomes. Here we carried out a systematic screening and analysis of EVEs in algal genomes and found that EVEs commonly exist in algal genomes. We classified the EVE fragments into three categories according to the length of EVE fragments. Due to the probability of sequence similarity by chance, we ignored the potential function of medium-length EVE fragments. However, longlength EVE fragments probably had capability to encode protein domains or even entire proteins, and some short-length EVE fragments had high similarity with host's siRNA sequences and possibly served functions of small RNAs. Therefore, short and long EVE fragments might provide regulomic and proteomic novelty to the host's metabolism and adaptation. We also found several EVE fragments shared by more than 3 algal genomes. By phylogenetic analysis of the shared EVEs and their corresponding species, we found that the integration of viral fragments into host genomes was an ancient event, possibly before the divergence of Chlorophytes and Ochrophytes. Our findings show that there is a frequent genetic flow from viruses to algal genomes. Moreover, study on algal EVEs shed light on the virus-host interaction in large timescale and could also help us understand the balance of marine ecosystems.
The discovery of archaea origin phosphomannomutase in algae based on the algal transcriptome
FENG Yanjing, CHI Shan, LIU Cui, CHEN Shengping, YU Jun, WANG Xumin, LIU Tao
2014, 33(2): 108-113. doi: 10.1007/s13131-014-0447-0
Abstract:
phosphomannomutase (PMM; EC 5.4.2.8) is an enzyme that catalyzes the interconversion reaction between mannose-6-phosphate and mannose-1-phosphate. However, its systematic molecular and functional investigations in algae have not hitherto been reported. In this work, with the accomplishment of the 1 000 Plant Project (OneKP) in which more than 218 species of Chromista, including 19 marine phaeophytes, 22 marine rhodophytes, 171 chlorophytes, 5 cryptophytes, 4 haptophytes, and 5 glaucophytes were sequenced, we used a gene analysis method to analyze the PMM gene sequences in algae and confirm the existence of the PMM gene in the transcriptomic sequencing data of Rhodophyta and Ochrophyta. Our results showed that only one type of PMM with four conserved motifs exists in Chromista which is similar to human PMM. Moreover, the phylogenetic tree revealed that algae PMM possibly originated from archaea.