Fanna Kong

Complete Sequence and Analysis of Plastid Genomes of Two Economically Important Red Algae: Pyropia haitanensis and Pyropia yezoensis

Background Pyropia haitanensis and P. yezoensis are two economically important marine crops that are also considered to be research models to study the physiological ecology of intertidal seaweed communities, evolutionary biology of plastids, and the origins of sexual reproduction. This plastid genome information will facilitate study of breeding, population genetics and phylogenetics. Principal Findings We have fully sequenced using next-generation sequencing the circular plastid genomes of P. hatanensis (195,597 bp) and P. yezoensis (191,975 bp), the largest of all the plastid genomes of the red lineage sequenced to date. Organization and gene contents of the two plastids were similar, with 211–213 protein-coding genes (including 29–31 unknown-function ORFs), 37 tRNA genes, and 6 ribosomal RNA genes, suggesting a largest coding capacity in the red lineage. In each genome, 14 protein genes overlapped and no interrupted genes were found, indicating a high degree of genomic condensation. Pyropia maintain an ancient gene content and conserved gene clusters in their plastid genomes, containing nearly complete repertoires of the plastid genes known in photosynthetic eukaryotes. Similarity analysis based on the whole plastid genome sequences showed the distance between P. haitanensis and P. yezoensis (0.146) was much smaller than that of Porphyra purpurea and P. haitanensis (0.250), and P. yezoensis (0.251); this supports re-grouping the two species in a resurrected genus Pyropia while maintaining P. purpurea in genus Porphyra. Phylogenetic analysis supports a sister relationship between Bangiophyceae and Florideophyceae, though precise phylogenetic relationships between multicellular red alage and chromists were not fully resolved. Conclusions These results indicate that Pyropia have compact plastid genomes. Large coding capacity and long intergenic regions contribute to the size of the largest plastid genomes reported for the red lineage. Possessing the largest coding capacity and ancient gene content yet found reveal that Pyropia are more primitive multicellular red algae.

R-ISSR as a new tool for genomic fingerprinting, mapping, and gene tagging

In the present study we propose and test the concept of R-ISSR, a new tool for genomic fingerprinting, mapping, and gene tagging. The concept is based on the fact that primers for inter-simple sequence repeat (ISSR) and random-amplified polymorphic DNA (RAPD) analysis elicit different genomic information, and the combined use of these 2 kinds of primers in the same polymerase chain reaction (PCR) reactions might reveal new genomic loci that could not be detected with either technique alone. The feasibility of this tool was first electronically simulated with sequence analysis software andArabidopsis chromosome sequence. Next, different combinations of ISSR and RAPD primers were applied in real PCR reactions to detect new genomic loci in 2 maize lines (Q319 and 1145). Sequencing gels were used to separate PCR products and showed good resolving ability in comparison with agarose gels. RAPD primers could be successfully used with ISSR primers for the detection of new genomic loci and applied in a new way for genomic mapping, fingerprinting, and gene tagging.

Transcriptome de novo assembly sequencing and analysis of the toxic dinoflagellate Alexandrium catenella using the Illumina platform

In this article, high-throughput de novo transcriptomic sequencing was performed in Alexandrium catenella, which provided the first view of the gene repertoire in this dinoflagellate based on next-generation sequencing (NGS) technologies. A total of 118,304 unigenes were identified with an average length of 673bp (base pair). Of these unigenes, 77,936 (65.9%) were annotated with known proteins based on sequence similarities, among which 24,149 and 22,956 unigenes were assigned to gene ontology categories (GO) and clusters of orthologous groups (COGs), respectively. Furthermore, 16,467 unigenes were mapped onto 322 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). We also detected 1143 simple sequence repeats (SSRs), in which the tri-nucleotide repeat motif (69.3%) was the most abundant. The genetic facts and significance derived from the transcriptome dataset were suggested and discussed. All four core nucleosomal histones and linker histones were detected, in addition to the unigenes involved in histone modifications.190 unigenes were identified as being involved in the endocytosis pathway, and clathrin-dependent endocytosis was suggested to play a role in the heterotrophy of A. catenella. A conserved 22-nt spliced leader (SL) was identified in 21 unigenes which suggested the existence of trans-splicing processing of mRNA in A. catenella.

Comparative transcriptome profiling of Pyropia yezoensis (Ueda) M.S. Hwang & H.G. Choi in response to temperature stresses.

BackgroundPyropia yezoensis is a model organism often used to investigate the mechanisms underlying stress tolerance in intertidal zones. The digital gene expression (DGE) approach was used to characterize a genome-wide comparative analysis of differentially expressed genes (DEGs) that influence the physiological, developmental or biochemical processes in samples subjected to 4 treatments: high-temperature stress (HT), chilling stress (CS), freezing stress (FS) and normal temperature (NT).ResultsEqual amounts of total RNAs collected from 8 samples (two biological replicates per treatment) were sequenced using the Illumina/Solexa platform. Compared with NT, a total of 2202, 1334 and 592 differentially expressed unigenes were detected in HT, CS and FS respectively. Clustering analysis suggested P. yezoensis acclimates to low and high-temperature stress condition using different mechanisms: In heat stress, the unigenes related to replication and repair of DNA and protein processing in endoplasmic reticulum were active; however at low temperature stresses, unigenes related to carbohydrate metabolism and energy metabolism were active. Analysis of gene differential expression showed that four categories of DEGs functioning as temperature sensors were found, including heat shock proteins, H2A, histone deacetylase complex and transcription factors. Heat stress caused chloroplast genes down-regulated and unigenes encoding metacaspases up-regulated, which is an important regulator of PCD. Cold stress caused an increase in the expression of FAD to improve the proportion of polyunsaturated fatty acids. An up-regulated unigene encoding farnesyl pyrophosphate synthase was found in cold stress, indicating that the plant hormone ABA also played an important role in responding to temperature stress in P. yezoensis.ConclusionThe variation of amount of unigenes and different gene expression pattern under different temperature stresses indicated the complicated and diverse regulation mechanism in response to temperature stress in P. yezoensis. Several common metabolism pathways were found both in P. yezoensis and in higher plants, such as FAD in low-temperature stress and HSP in heat stress. Meanwhile, many chloroplast genes and unigene related to the synthesis of abscisic acid were detected, revealing its unique temperature-regulation mechanism in this intertidal species. This sequencing dataset and analysis may serve as a valuable resource to study the mechanisms involved in abiotic stress tolerance in intertidal seaweeds.

The complete mitochondrial genome of Pyropia haitanensis Chang et Zheng

The complete mitochondrial genome (mitogenome) of a red alga, Pyropia haitanensis Chang et Zheng, a circular-mapping molecule, was determined to be 37,023 bp. The overall AT content of the mitogenome is 69.32%. It contains 54 genes, including 49 conserved genes, 4 intronic reading frames, and an additional free-standing open reading frame. The rnl gene and cox1 gene are the only two interrupted genes in the mitogenome. All the protein-coding genes and Open Reading Frames (ORF) have typical ATG start codon, except for cox1 and cox2, which contain the unusual GTG and CTG as an initiator codon, respectively. Relative differences are found by comparing the mitogenome of P. haitanensis with that of Porphyra purpurea, reinforcing the viewpoint that the two species belong to different genus.

De novo assembly and characterization of the transcriptome of seagrass Zostera marina using Illumina paired-end sequencing.

Background The seagrass Zostera marina is a monocotyledonous angiosperm belonging to a polyphyletic group of plants that can live submerged in marine habitats. Zostera marina L. is one of the most common seagrasses and is considered a cornerstone of marine plant molecular ecology research and comparative studies. However, the mechanisms underlying its adaptation to the marine environment still remain poorly understood due to limited transcriptomic and genomic data. Principal Findings Here we explored the transcriptome of Z. marina leaves under different environmental conditions using Illumina paired-end sequencing. Approximately 55 million sequencing reads were obtained, representing 58,457 transcripts that correspond to 24,216 unigenes. A total of 14,389 (59.41%) unigenes were annotated by blast searches against the NCBI non-redundant protein database. 45.18% and 46.91% of the unigenes had significant similarity with proteins in the Swiss-Prot database and Pfam database, respectively. Among these, 13,897 unigenes were assigned to 57 Gene Ontology (GO) terms and 4,745 unigenes were identified and mapped to 233 pathways via functional annotation against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). We compared the orthologous gene family of the Z. marina transcriptome to Oryza sativa and Pyropia yezoensis and 11,667 orthologous gene families are specific to Z. marina. Furthermore, we identified the photoreceptors sensing red/far-red light and blue light. Also, we identified a large number of genes that are involved in ion transporters and channels including Na+ efflux, K+ uptake, Cl− channels, and H+ pumping. Conclusions Our study contains an extensive sequencing and gene-annotation analysis of Z. marina. This information represents a genetic resource for the discovery of genes related to light sensing and salt tolerance in this species. Our transcriptome can be further utilized in future studies on molecular adaptation to abiotic stress in Z. marina.

Application of SSR and AFLP to the analysis of genetic diversity in Gracilariopsis lemaneiformis (Rhodophyta)

The agarophyte Gracilariopsis lemaneiformis is both important for biological research and of significant economic value. However, the genetic diversity of wild populations of the alga has not been studied. We used amplified fragment length polymorphism (AFLP) PCR and simple sequence repeat (SSR) analysis to investigate diversity in four field populations, three from the coast of Qingdao and one from Weihai, China. Forty G. lemaneiformis isolates collected from the four different geographical groups were analyzed using 16 pairs of SSR primers for PCR amplification. However, no polymorphisms were detected, indicative of a degree of genetic homogeneity. A total of 347 reproducible bands were then amplified using eight AFLP primer pairs, and genetic indices of diversity within and between populations were calculated. This analysis revealed only low levels of genetic diversity both within and between the four geographical groups of G. lemaneiformis. The Weihai population showed a higher within-population genetic diversity than any of the Qingdao populations. The data suggest that there is only limited gene flow between populations. An UPGMA dendrogram revealed two main clusters, and one of these included all the Qingdao isolates. The order of clustering was in accordance with their geographical distribution. These results suggest that the wild G. lemaneiformis populations are closely related and that there is little genetic diversity within wild germplasm in the regions sampled.

Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR

Quantitative real-time PCR (qRT-PCR) is frequently used for gene expression analysis. The selection of reference genes is required for normalization of the variation to avoid misinterpretation of experimental results and erroneous analyses. Pyropia yezoensis, growing in the intertidal zone, is an economically important seaweed. Although this intertidal seaweed has been an experimental system for understanding stress tolerance and developmental mechanisms, reference genes suitable for the normalization of qRT-PCR data have not previously been identified. In this study, expression stability of six candidate reference genes, including ACT, eIF4A, EF1α, GAPDH, TUA, and UBQ (traditional housekeeping genes), has been validated in a diverse set of samples representing different developmental stages and stress conditions of P. yezoensis. Three qRT-PCR analysis methods, geNorm, NormFinder, and BestKeeper, were evaluated systematically. The results indicated that ACT3, eIF4A, and EF1α were the optimal reference genes for P. yezoensis under stress conditions; UBQ, EF1α, and eIF4A were suitable for studying the expression of genes related to P. yezoensis development. TUA showed the lowest expression stability both under stress conditions and over developmental stages. Our results have provided a reference gene application guideline for P. yezoensis gene expression characterization using the qRT-PCR system.

Complete mitochondrial genome of Pyropia yezoensis: reasserting the revision of genus Porphyra.

Abstract Pyropia yezoensis Ueda, belonging to the Bangiales order Rhodophyte, is economically important seaweed distributed along the coastal region of the northwest Pacific. In this study, we report the complete mitochondrial DNA sequences of P. yezoensis. The entire genome comprised 41,688 bp with circular organization, which is the longest among the red algae sequenced to date. The overall AT composition is 62.22% and the average AT content of PCG, 16S rRNA, tRNA and noncoding region are 67.51%, 65.23%, 61.23% and 72.67%, respectively The mitochondrial DNA of P. yezoensis encoded 23 proteins, 4 open reading frames, 3 ribosomal RNAs and 27 transfer RNA genes. The comparison analyses showed that obvious variation occurred on the gene content and gene structure among P. yezoensis. P. haitanensis and Porphyra purpurea. Phylogenetic analysis revealed that P. yezoensis is more closely related to P. haitanensis than to P. purpurea, and P. purpurea was phylogenetically distinct from the two species, which confirmed the reassignment of Pyropia and Porphyra in Bangiaceae.

Generation and analysis of expressed sequence tags fromthe salt-tolerant eelgrass species, Zostera marina

Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To investigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was constructed. Single-pass sequencing of the 5′ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags (ESTs), which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100–1 500 bp. Basic Local Alignment Search Tool (BLASTX) analysis revealed that 1 664 unigenes had significant homology to known genes in the National Center for Biotechnology Information (NCBI) non-redundant (nr) database (E-value⩽5–10). Among them, the two most abundant genes encoded metallothionein (157 ESTs) and chlorophyll a/b-binding protein (38 ESTs), accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes (KEGG), 1 462 unigenes were assigned to 1 161 pathways (E-value⩽5–10). A total of 938 unigenes were assigned Gene Ontology (GO) terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.