Xiuliang Wang

Transcriptome Sequencing and Comparative Analysis of Saccharina japonica (Laminariales, Phaeophyceae) under Blue Light Induction

Background Light has significant effect on the growth and development of Saccharina japonica, but there are limited reports on blue light mediated physiological responses and molecular mechanism. In this study, high-throughput paired-end RNA-sequencing (RNA-Seq) technology was applied to transcriptomes of S. japonica exposed to blue light and darkness, respectively. Comparative analysis of gene expression was designed to correlate the effect of blue light and physiological mechanisms on the molecular level. Principal Findings RNA-seq analysis yielded 70,497 non-redundant unigenes with an average length of 538 bp. 28,358 (40.2%) functional transcripts encoding regions were identified. Annotation through Swissprot, Nr, GO, KEGG, and COG databases showed 25,924 unigenes compared well (E-value <10−5) with known gene sequences, and 43 unigenes were putative BL photoreceptor. 10,440 unigenes were classified into Gene Ontology, and 8,476 unigenes were involved in 114 known pathways. Based on RPKM values, 11,660 (16.5%) differentially expressed unigenes were detected between blue light and dark exposed treatments, including 7,808 upregulated and 3,852 downregulated unigenes, suggesting S. japonica had undergone extensive transcriptome re-orchestration during BL exposure. The BL-specific responsive genes were indentified to function in processes of circadian rhythm, flavonoid biosynthesis, photoreactivation and photomorphogenesis. Significance Transcriptome profiling of S. japonica provides clues to potential genes identification and future functional genomics study. The global survey of expression changes under blue light will enhance our understanding of molecular mechanisms underlying blue light induced responses in lower plants as well as facilitate future blue light photoreceptor identification and specific responsive pathways analysis.

Development of expressed sequence tag-derived microsatellite markers for Saccharina (Laminaria) japonica

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.

Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers

Genetic variation of four populations of Sargassum thunbergii (Mert.) O. Kuntze and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China was studied with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. A total of 28 RAPD primers and 19 ISSR primers were amplified, showing 174 loci and 125 loci, respectively. Calculation of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon’s information index) revealed low or moderate levels of genetic variations within each S. thunbergii population. High genetic differentiations were determined with pairwise Nei’s unbiased genetic distance (D) and fixation index (FST) between the populations. The Mantel test showed that two types of matrices of D and FST were highly correlated, whether from RAPD or ISSR data, r = 0.9310 (P  = 0.008) and 0.9313 (P = 0.009) respectively. Analysis of molecular variance (AMOVA) was used to apportion the variations between and within the S. thunbergii populations. It indicated that the variations among populations were higher than those within populations, being 57.57% versus 42.43% by RAPD and 59.52% versus 40.08% by ISSR, respectively. Furthermore, the Mantel test suggested that the genetic differentiations between the four populations were related to the geographical distances (r > 0.5), i.e., they conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. As a whole, the high genetic structuring between the four S. thunbergii populations along distant locations was clearly indicated in the RAPD and ISSR analyses (r > 0.8) in our study.

The genetic analysis and germplasm identification of the gametophytes of Undaria pinnatifida (Phaeophyceae) with RAPD method

Eleven pairs of Undaria pinnatifida (Harv.) Suringar gametophytes were identified with random amplified polymorphic DNA (RAPD) technique. After screening 100 primers, 20 ten-base primers were determined for the RAPD analysis. A total of 312 polymorphic loci were obtained, of which 97.7% were polymorphic. The primer S198 was found to distinguish all the selected Undariapinnatifida gametophytes. The genetic distances between each two of the twenty-two U.pinnatifida gametophytes ranged from 0.080 to 0.428, while the distances to the Laminaria was 0.497 on average. After reexamination, two sequences characterized amplification region (SCAR) markers were successfully converted, which could be applied to U.pinnatifida germplasm identification. All these results demonstrated the feasibility of applying RAPD markers to germplasm characterization and identification of U. pinnatifida gametophytes, and to provide a molecular basis for Undaria breeding.

Chloroplast genome of one brown seaweed, Saccharina japonica (Laminariales, Phaeophyta): Its structural features and phylogenetic analyses with other photosynthetic plastids

The chloroplast genome sequence of one brown seaweed, Saccharina japonica, was fully determined. It is characterized by 130,584 base pairs (bp) with a large and a small single-copy region (LSC and SSC), separated by two copies of inverted repeats (IR1 and IR2). The inverted repeat is 5015 bp long, and the sizes of SSC and LSC are 43,174 bp and 77,378 bp, respectively. The chloroplast genome of S. japonica consists of 139 protein-coding genes, 29 tRNA genes, and 3 ribosomal RNA genes. One intron was found in one tRNA-Leu gene in the chloroplast genome of S. japonica. Four types of overlapping genes were identified, ycf24 overlapped with ycf16 by 4 nucleotides (nt), ftrB overlapped with ycf12 by 6 nt, rpl4 and rpl23 overlapped by 8 nt, finally, psbC overlapped with psbD by 53 nt. With two sets of concatenated plastid protein data, 40-protein dataset and 26-protein dataset, the chloroplast phylogenetic relationship among S. japonica and the other photosynthetic species was evaluated. We found that the chloroplast genomes of haptophyte, cryptophyte and heterokont were not resolved into one cluster by the 40-protein dataset with amino acid composition bias, although it was recovered with strong support by the 26-protein dataset.

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES, PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS

To establish a molecular‐marker‐assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F2 cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P ≤ 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular‐marker‐assisted breeding for Laminaria.

Identification of SCAR marker linking to longer frond length of Saccharina japonica (Laminariales, Phaeophyta) using bulked-segregant analysis

To construct a molecular-marker-assisted selection (MAS) system, research was done on identifying molecular markers linking to longer frond length, a crucial selection index in the breeding of the commercially important seaweed Saccharina japonica. An F2-segregant population of 92 individuals was obtained by crossing two prominent S. japonica strains. Genomic DNA from ten individuals with the longest frond and ten individuals with the shortest frond in the F2-segregant population were mixed to create two DNA pools for screening polymorphic markers. In bulked-segregant analysis (BSA), out of 100 random amplified polymorphic DNA (RAPD) primers only two produced three polymorphic RAPD markers between the two DNA pools. In conversion of the three RAPD markers into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a SCAR marker FL-569 linking to the trait of longer frond. Test of the marker FL-569 showed that 80% of the individuals with longest fronds in a wild population and 87.5% of individuals with the longest fronds in an inbred line “Zhongke No. 2” could be detected by FL-569. Additionally, genetic linkage analysis showed that the SCAR marker could be integrated into the reported genetic map and QTL mapping showed that FL-569 linking to qL1-1. The obtained marker FL-569 will be beneficial to MAS in S. japonica breeding.

Characterization of Mannitol-2-Dehydrogenase in Saccharina japonica: Evidence for a New Polyol-Specific Long-Chain Dehydrogenases/Reductase

Mannitol plays a crucial role in brown algae, acting as carbon storage, organic osmolytes and antioxidant. Transcriptomic analysis of Saccharina japonica revealed that the relative genes involved in the mannitol cycle are existent. Full-length sequence of mannitol-2-dehydrogenase (M2DH) gene was obtained, with one open reading frame of 2,007 bp which encodes 668 amino acids. Cis-regulatory elements for response to methyl jasmonic acid, light and drought existed in the 5′-upstream region. Phylogenetic analysis indicated that SjM2DH has an ancient prokaryotic origin, and is probably acquired by horizontal gene transfer event. Multiple alignment and spatial structure prediction displayed a series of conserved functional residues, motifs and domains, which favored that SjM2DH belongs to the polyol-specific long-chain dehydrogenases/reductase (PSLDR) family. Expressional profiles of SjM2DH in the juvenile sporophytes showed that it was influenced by saline, oxidative and desiccative factors. SjM2DH was over-expressed in Escherichia coli, and the cell-free extracts with recombinant SjM2DH displayed high activity on D-fructose reduction reaction. The analysis on SjM2DH gene structure and biochemical parameters reached a consensus that activity of SjM2DH is NADH-dependent and metal ion-independent. The characterization of SjM2DH showed that M2DH is a new member of PSLDR family and play an important role in mannitol metabolism in S. japonica.

Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)

A high quality cDNA library was constructed from the brown alga Laminaria japonica, with the titer of 1.2×105 pfu/ml. The average insert size of the cDNA library is about 1.6 kb. From the cDNA library, 591 cDNA clones were randomly selected and sequenced. As a result, 574 EST (expressed sequence tag) sequences were generated. All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198. The cDNA library was screened with a α-32p labeled 453 bp TPS gene probe, which is a partial sequence yielded from Porphyra yezoensis. Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L. japonica TPS cDNA sequence. After PCR amplification, sequencing and assembling, the entire ORF (open reading frame) sequence of the TPS gene was obtained, which was named LjTPS. LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons. The LjTPS gene was successfully expressed in E. coli and rice. The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the TPS gene function and mechanism to stress tolerance.

Effect of domestication on the genetic diversity and structure of Saccharina japonica populations in China

Saccharina japonica is a commercially and ecologically important seaweed and is an excellent system for understanding the effects of domestication on marine crops. In this study, we used 19 selected simple sequence repeat (SSR) markers to investigate the influence of domestication on the genetic diversity and structure of S. japonica populations. Wild kelp populations exhibited higher genetic diversity than cultivated populations based on total NA, HE, HO, NP and AR. Discriminant analysis of principal components (DAPC), a neighbour-joining (NJ) tree and STRUCTURE analyses indicated that S. japonica populations could be divided into two groups (a cultivated/introduced group and a wild indigenous group) with significant genetic differentiation (P < 0.0001). Divergent selection, continuous inbreeding and inter-specific hybridization have caused the divergence of these two genetically separate gene pools. The significant genetic differentiation between northern and southern cultivated populations appears to be due to inter-specific hybridization and wild germplasm introduction during the domestication process. In addition, the cultivation of S. japonica has not resulted in any serious genetic disturbance of wild introduced S. japonica populations. An understanding of the genetic diversity and genetic structure of domesticated S. japonica will be necessary for further genetic improvement and effective use of germplasm.