Construction of a genetic linkage map in Pyropia yezoensis (Bangiales, Rhodophyta) and QTL analysis of several economic traits of blades.

Pyropia yezoensis is an economically important seaweed but its molecular genetics is poorly understood. In the present study, we used a doubled haploid (DH) population that was established in our previous work to construct a genetic linkage map of P. yezoensis and analyze the quantitative trait loci (QTLs) of blades. The DH population was genotyped with fluorescent sequence-related amplified polymorphism (SRAP) markers. A chi-square test identified 301 loci with normal segregation (P ≥ 0.01) and 96 loci (24.18%) with low-level skewed segregation (0.001 ≤ P < 0.01). The genetic map was constructed after a total of 92 loci were assembled into three linkage groups (LGs). The map spanned 557.36 cM covering 93.71% of the estimated genome, with a mean interlocus space of 6.23 cM. Kolmogorov-Smirnov test (α = 5%) showed a uniform distribution of the markers along each LG. On the genetic map, 10 QTLs associated with five economic traits of blades were detected. One QTL was for length, one for width, two for fresh weight, two for specific growth rate of length and four for specific growth rate of fresh weight. These QTLs could explain 2.29-7.87% of the trait variations, indicating that their effects were all minor. The results may serve as a framework for future marker-assisted breeding in P. yezoensis.

realDB: a genome and transcriptome resource for the red algae (phylum Rhodophyta).

With over 6000 species in seven classes, red algae (Rhodophyta) have diverse economic, ecological, experimental and evolutionary values. However, red algae are usually absent or rare in comparative analyses because genomic information of this phylum is often under-represented in various comprehensive genome databases. To improve the accessibility to the ome data and omics tools for red algae, we provided 10 genomes and 27 transcriptomes representing all seven classes of Rhodophyta. Three genomes and 18 transcriptomes were de novo assembled and annotated in this project. User-friendly BLAST suit, Jbrowse tools and search system were developed for online analyses. Detailed introductions to red algae taxonomy and the sequencing status are also provided. In conclusion, realDB (realDB.algaegenome.org) provides a platform covering the most genome and transcriptome data for red algae and a suite of tools for online analyses, and will attract both red algal biologists and those working on plant ecology, evolution and development.Database URL: http://realdb.algaegenome.org/.

Construction of a dense genetic linkage map and mapping quantitative trait loci for economic traits of a doubled haploid population of Pyropia haitanensis (Bangiales, Rhodophyta).

BACKGROUND: Pyropia haitanensis is one of the most economically important mariculture crops in China. A high-density genetic map has not been published yet and quantitative trait locus (QTL) mapping has not been undertaken for P. haitanensis because of a lack of sufficient molecular markers. Specific length amplified fragment sequencing (SLAF-seq) was developed recently for large-scale, high resolution de novo marker discovery and genotyping. In this study, SLAF-seq was used to obtain mass length polymorphic markers to construct a high-density genetic map for P. haitanensis. RESULTS: In total, 120.33 Gb of data containing 75.21 M pair-end reads was obtained after sequencing. The average coverage for each SLAF marker was 75.50-fold in the male parent, 74.02-fold in the female parent, and 6.14-fold average in each double haploid individual. In total, 188,982 SLAFs were detected, of which 6731 were length polymorphic SLAFs that could be used to construct a genetic map. The final map included 4550 length polymorphic markers that were combined into 740 bins on five linkage groups, with a length of 874.33 cM and an average distance of 1.18 cM between adjacent bins. This map was used for QTL mapping to identify chromosomal regions associated with six economically important traits: frond length, width, thickness, fresh weight, growth rates of frond length and growth rates of fresh weight. Fifteen QTLs were identified for these traits. The value of phenotypic variance explained by an individual QTL ranged from 9.59 to 16.61 %, and the confidence interval of each QTL ranged from 0.97 cM to 16.51 cM. CONCLUSIONS: The first high-density genetic linkage map for P. haitanensis was constructed, and fifteen QTLs associated with six economically important traits were identified. The results of this study not only provide a platform for gene and QTL fine mapping, map-based gene isolation, and molecular breeding for P. haitanensis, but will also serve as a reference for positioning sequence scaffolds on a physical map and will assist in the process of assembling the P. haitanensis genome sequence. This will have a positive impact on breeding programs that aim to increase the production and quality of P. haitanensis in the future.

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.

Assessment of four molecular markers as potential DNA barcodes for red algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta).

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.

An assessment of vertical inheritance versus endosymbiont transfer of nucleus-encoded genes for mitochondrial proteins following tertiary endosymbiosis in Karlodinium micrum.

Most photosynthetic dinoflagellates harbour a red alga-derived secondary plastid. In the dinoflagellate Karlodinium micrum, this plastid was replaced by a subsequent endosymbiosis, resulting in a tertiary plastid derived from a haptophyte. Evolution of endosymbionts entails substantial relocation of endosymbiont genes to the host nucleus: a process called endosymbiotic gene transfer (EGT). In K. micrum, numerous plastid genes from the haptophyte nucleus are found in the host nucleus, providing evidence for EGT in this system. In other cases of endosymbiosis, notably ancient primary endosymbiotic events, EGT has been inferred to contribute to remodeling of other cell functions by expression of proteins in compartments other than the endosymbiont from which they derived. K. micrum provides a more recently derived endosymbiotic system to test for evidence of EGT and gain of function in non-plastid compartments. In this study, we test for gain of haptophyte-derived proteins for mitochondrial function in K. micrum. Using molecular phylogenies we have analysed whether nucleus-encoded mitochondrial proteins were inherited by EGT from the haptophyte endosymbiont, or vertically inherited from the dinoflagellate host lineage. From this dataset we found no evidence of haptophyte-derived mitochondrial genes, and the only cases of non-vertical inheritance were genes derived from lateral gene transfer events.

An improved and cost-effective cDNA-AFLP method to investigate transcription-derived products when high throughput sequencing is not available.

Although the cost of high throughput sequencing is decreasing, the cost is still often too high for individual projects targeted at, e.g., genome-wide transcription profiling in non-model organisms. Then, a low-cost alternative is cDNA-AFLP, which we have now considerably modified in order to develop a faster and simpler method to identify and analyze genes involved in specific, possibly adaptive characteristics. Particularly, we wanted to exclude repetitive PCR amplifications, extensive cloning and the presence of overlapping transcripts, which all lower the efficiency of the method.

Production of phycocyanin--a pigment with applications in biology, biotechnology, foods and medicine.

C-phycocyanin (C-PC) is a blue pigment in cyanobacteria, rhodophytes and cryptophytes with fluorescent and antioxidative properties. C-PC is presently extracted from open pond cultures of the cyanobacterium Arthrospira platensis although these cultures are not very productive and open for contaminating organisms. C-PC is considered a healthy ingredient in cyanobacterial-based foods and health foods while its colouring, fluorescent or antioxidant properties are utilised only to a minor extent. However, recent research and developments in C-PC synthesis and functionality have expanded the potential applications of C-PC in biotechnology, diagnostics, foods and medicine: The productivity of C-PC has been increased in heterotrophic, high cell density cultures of the rhodophyte Galdieria sulphuraria that are grown under well-controlled and axenic conditions. C-PC purification protocols based on various chromatographic principles or novel two-phase aqueous extraction methods have expanded in numbers and improved in performance. The functionality of C-PC as a fluorescent dye has been improved by chemical stabilisation of C-PC complexes, while protein engineering has also introduced increased stability and novel biospecific binding sites into C-PC fusion proteins. Finally, our understanding of the physiological functions of C-PC in humans has been improved by a mechanistic hypothesis that links the chemical properties of the phycocyanobilin chromophores of C-PC to the natural antioxidant, bilirubin, and may explain the observed health benefits of C-PC intake. This review outlines how C-PC is produced and utilised and discusses the novel C-PC synthesis procedures and applications.

Evidence of a chimeric genome in the cyanobacterial ancestor of plastids.

BACKGROUND: Horizontal gene transfer (HGT) is a vexing fact of life for microbial phylogeneticists. Given the substantial rates of HGT observed in modern-day bacterial chromosomes, it is envisaged that ancient prokaryotic genomes must have been similarly chimeric. But where can one find an ancient prokaryotic genome that has maintained its ancestral condition to address this issue? An excellent candidate is the cyanobacterial endosymbiont that was harnessed over a billion years ago by a heterotrophic protist, giving rise to the plastid. Genetic remnants of the endosymbiont are still preserved in plastids as a highly reduced chromosome encoding 54 - 264 genes. These data provide an ideal target to assess genome chimericism in an ancient cyanobacterial lineage. RESULTS: Here we demonstrate that the origin of the plastid-encoded gene cluster for menaquinone/phylloquinone biosynthesis in the extremophilic red algae Cyanidiales contradicts a cyanobacterial genealogy. These genes are relics of an ancestral cluster related to homologs in Chlorobi/Gammaproteobacteria that we hypothesize was established by HGT in the progenitor of plastids, thus providing a 'footprint' of genome chimericism in ancient cyanobacteria. In addition to menB, four components of the original gene cluster (menF, menD, menC, and menH) are now encoded in the nuclear genome of the majority of non-Cyanidiales algae and plants as the unique tetra-gene fusion named PHYLLO. These genes are monophyletic in Plantae and chromalveolates, indicating that loci introduced by HGT into the ancestral cyanobacterium were moved over time into the host nucleus. CONCLUSION: Our study provides unambiguous evidence for the existence of genome chimericism in ancient cyanobacteria. In addition we show genes that originated via HGT in the cyanobacterial ancestor of the plastid made their way to the host nucleus via endosymbiotic gene transfer (EGT).