Guoliang Wang

Genome sequence and genetic diversity of the common carp, Cyprinus carpio

The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

Complete Plastid Genome of the Brown Alga Costaria costata (Laminariales, Phaeophyceae)

Costaria costata is a commercially and industrially important brown alga. In this study, we used next-generation sequencing to determine the complete plastid genome of C. costata. The genome consists of a 129,947 bp circular DNA molecule with an A+T content of 69.13% encoding a standard set of six ribosomal RNA genes, 27 transfer RNA genes, and 137 protein-coding genes with two conserved open reading frames (ORFs). The overall genome structure of C. costata is nearly the same as those of Saccharina japonica and Undaria pinnatifida. The plastid genomes of these three algal species retain a strong conservation of the GTG start codon while infrequently using TGA as a stop codon. In this regard, they differ substantially from the plastid genomes of Ectocarpus siliculosus and Fucus vesiculosus. Analysis of the nucleic acid substitution rates of the Laminariales plastid genes revealed that the petF gene has the highest substitution rate and the petN gene contains no substitution over its complete length. The variation in plastid genes between C. costata and S. japonica is lower than that between C. costata and U. pinnatifida as well as that between U. pinnatifida and S. japonica. Phylogenetic analyses demonstrated that C. costata and U. pinnatifida have a closer genetic relationship. We also identified two gene length mutations caused by the insertion or deletion of repeated sequences, which suggest a mechanism of gene length mutation that may be one of the key explanations for the genetic variation in plastid genomes.

Complete Plastid Genome Sequence of the Brown Alga Undaria pinnatifida

In this study, we fully sequenced the circular plastid genome of a brown alga, Undaria pinnatifida. The genome is 130,383 base pairs (bp) in size; it contains a large single-copy (LSC, 76,598 bp) and a small single-copy region (SSC, 42,977 bp), separated by two inverted repeats (IRa and IRb: 5,404 bp). The genome contains 139 protein-coding, 28 tRNA, and 6 rRNA genes; none of these genes contains introns. Organization and gene contents of the U. pinnatifida plastid genome were similar to those of Saccharina japonica. There is a co-linear relationship between the plastid genome of U. pinnatifida and that of three previously sequenced large brown algal species. Phylogenetic analyses of 43 taxa based on 23 plastid protein-coding genes grouped all plastids into a red or green lineage. In the large brown algae branch, U. pinnatifida and S. japonica formed a sister clade with much closer relationship to Ectocarpus siliculosus than to Fucus vesiculosus. For the first time, the start codon ATT was identified in the plastid genome of large brown algae, in the atpA gene of U. pinnatifida. In addition, we found a gene-length change induced by a 3-bp repetitive DNA in ycf35 and ilvB genes of the U. pinnatifida plastid genome.

Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.

Validation of Suitable Reference Genes for Assessing Gene Expression of MicroRNAs in Lonicera japonica

MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as 21 cultivated varieties collected from 16 production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica.

Development and utility of EST-SSR markers in Ulva prolifera of the South Yellow Sea

Ulva species can grow rapidly in nutrient-rich habitats causing green tides and marine fouling. A more complete understanding of the reasons behind these outbreaks is urgently required. Accordingly, this study attempts to use microsatellite markers based expressed sequence tag (EST) to analyze the genetic variation of several Ulva prolifera populations in the South Yellow Sea of China. Two hundred and thirty-eight SSRs were identified from 8 179 unique ESTs (6 203 newly sequenced and 1 976 downloaded from NCBI database) and 37 primer pairs were successfully designed according to the ESTs; 11 pairs were selected to detect the genetic diversity and relationship of 69 attached U. prolifera samples and 13 free-floating samples collected from coastal and off-coast areas of the South Yellow Sea. The results of cross-species transferability showed that six of the 11 EST-SSR primers could give good amplification in other five Ulva species and the average allele number was 4.67. Genetic variation analysis indicated that all 82 U. prolifera samples were clearly divided and most samples collected from the same site clustered together as a group in the dendrogram tree produced by unweighted pair-group mean analysis (UPGMA) method and the cluster results showed some consistency with the geographical origins. In addition, 13 free-floating samples (except HT-001-2) were grouped as a single clade separated from the attached samples.

Comparative analysis on transcriptome sequencings of six Sargassum species in China

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.

Comparative genomics and systematics of Betaphycus , Eucheuma , and Kappaphycus (Solieriaceae: Rhodophyta) based on mitochondrial genome

Betaphycus Doty, Eucheuma J. Agardh, and Kappaphycus Doty (Solieriaceae, Gigartinales) are the three most commercially important seaweed genera that produce carrageenan. In the present study we provide mitogenomes of Betaphycus gelatinus, Eucheuma denticulatum and Kappaphycus alvarezii. The mitogenomes of these three species contain a set of 50 genes, including 24 protein-coding genes, 2 rRNA genes, and 24 tRNA genes. The mitogenome length ranges from 25,198xa0bp (Kappaphycus alvarezii) to 25,327xa0bp (Eucheuma denticulatum). As compared with the previous published mitogenomes of Florideophyceae species, only the species in Gelidiaceae and Pterocladiaceae have smaller mitochondrial genome size than these reported here. At the junction of two transcription units, we identified a stem-loop structure in six representative Gigartinales species, which is presumed to play an important role in the replication and transcription of mitochondrial genes. In Gigartinales the difference in gene order among the four Solieriaceae (B. gelatinus, E. denticulatum, K. alvarezii, K. striatus) and other two Gigartinales species (Chondrus crispus and Mastocarpus papillatus) can be explained by inversion of two tRNA genes. Collinearity analysis of the 12 mitochondrial genomes of Florideophyceae showed considerable sequence synteny across all the species compared, with the exception of a highly variable region between atp6 and rpl20 genes. Phylogenetic analyses based on 21 shared mitochondrial genes showed that the four Solieriaceae species form one clade (Solieriaceae clade). Within this clade, B. gelatinae is basal relative to the other three species. The genus Kappaphycus is more closely related to Eucheuma than Betaphycus.

Functional genomics analysis reveals the biosynthesis pathways of important cellular components (alginate and fucoidan) of Saccharina

Although alginate and fucoidan are unique cellular components and have important biological significance in brown algae, and many possible involved genes are present in brown algal genomes, their functions and regulatory mechanisms have not been fully revealed. Both polysaccharides may play important roles in the evolution of multicellular brown algae, but specific and in-depth studies are still limited. In this study, a functional genomics analysis of alginate and fucoidan biosynthesis routes was conducted in Saccharina, and the key events in these pathways in brown algae were identified. First, genes from different sources, including eukaryotic hosts via endosymbiotic gene transfer and bacteria via horizontal gene transfer, were combined to build a complete pathway framework. Then, a critical event occurred to drive these pathways to have real function: one of the mannose-6-phosphate isomerase homologs that arose by gene duplication subsequently adopted the function of the mannose-1-phosphate guanylyltransferase (MGP) gene, which was absent in algal genomes. Further, downstream pathway genes proceeded with gene expansions and complex transcriptional mechanisms, which may be conducive to the synthesis of alginate and fucoidan with diverse structures and contents depending on the developmental stage, tissue structure, and environmental conditions. This study revealed the alginate and fucoidan synthesis pathways and all included genes from separate phylogenetic sources in brown algae. Enzyme assays confirmed the function of key genes and led to the determination of a substitute for the missing MPG. All gene families had constitutively expressed member(s) to maintain the basic synthesis; and the gene function differentiation, enzyme characterization and gene expression regulation differences separated brown algae from other algae lineages and were considered to be the major driving forces for sophisticated system evolution of brown algae.

Phylogenetic analyses of the genes involved in carotenoid biosynthesis in algae

Carotenoids play a crucial role in absorbing light energy for photosynthesis, as well as in protecting chlorophyll from photodamage. In contrast to the Streptophyta, few studies have examined carotenoid biosynthetic pathways in algae, owing to a shortage of datasets. As part of the 1000 Plants Project, we sequenced and assembled the transcriptomes of 41 marine macroalgal species, including 22 rhodophytes and 19 phaeophytes, and then combined the datasets with publicly available data from GenBank (National Center for Biotechnology Information) and the U.S. Department of Energy Joint Genome Institute. As a result, we identified 68 and 79 fulllength homologs in the Rhodophyta and Phaeophyceae, respectively, of seven inferred carotenoid biosynthetic genes, including the genes for phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), ζ-carotene isomerase (Z-ISO), prolycopene isomerase (crtISO), lycopene β-cyclase (LCYB), and lycopene ε-cyclase (LCYE). We found that the evolutionary history of the algal carotenoid biosynthetic pathway was more complex than that of the same pathway in the Streptophyta and, more specifically, that the evolutionary history involved endosymbiotic gene transfer, gene duplication, and gene loss. Almost all of the eukaryotic algae that we examined had inherited the seven carotenoid biosynthesis genes via endosymbiotic gene transfer. Moreover, PSY, crtISO, and the ancestral lycopene cyclase gene (LCY) underwent duplication events that resulted in multiple gene copies, and the duplication and subsequent divergence of LCYB and LCYE specialized and complicated the cyclization of lycopene. Our findings also verify that the loss of LCYE in both the microphytic rhodophytes and phaeophytes explains the differences in their carotenoid patterns, when compared to the macrophytic rhodophytes. These analyses provide a molecular basis for further biochemical and physiological validation in additional algal species and should help elucidate the origin and evolution of carotenoid biosynthetic pathways.