Genome-Wide Mapping of Cytosine Methylation Revealed Dynamic DNA Methylation Patterns Associated with Sporophyte Development of Saccharina japonica.

Cytosine methylation plays vital roles in regulating gene expression and plant development. However, the function of DNA methylation in the development of macroalgae remains unclear. Through the genome-wide bisulfite sequencing of cytosine methylation in holdfast, stipe and blade, we obtained the complete 5-mC methylation landscape of Saccharina japonica sporophyte. Our results revealed that the total DNA methylation level of sporophyte was less than 0.9%, and the content of CHH contexts was dominant. Moreover, the distribution of CHH methylation within the genes exhibited exon-enriched characteristics. Profiling of DNA methylation in three parts revealed the diverse methylation pattern of sporophyte development. These pivotal DMRs were involved in cell motility, cell cycle and cell wall/membrane biogenesis. In comparison with stipe and blade, hypermethylation of mannuronate C5-epimerase in holdfast decreased the transcript abundance, which affected the synthesis of alginate, the key component of cell walls. Additionally, 5-mC modification participated in the regulation of blade and holdfast development by the glutamate content respectively via glutamine synthetase and amidophosphoribosyl transferase, which may act as the epigenetic regulation signal. Overall, our study revealed the global methylation characteristics of the well-defined holdfast, stipe and blade, and provided evidence for epigenetic regulation of sporophyte development in brown macroalgae.

Non-Coding RNAs Participate in the Regulation of CRY-DASH in the Growth and Early Development of Saccharina japonica (Laminariales, Phaeophyceae).

CRY-DASH, a new cryptochrome blue light receptor, can repair damaged DNA and regulate secondary metabolism and development of fungus. However, its role in regulation during the growth of Saccharina japonica is still unclear. After cloning the full-length of CRY-DASH from S. japonica (sjCRY-DASH), we deduced that its open reading frame was 1779 bp long and encoded 592 amino acids. sjCRY-DASH transcription was rapidly upregulated within 30 min in response to blue light and exhibited 24 h periodicity with different photoperiods. Moreover, sjCRY-DASH maintained the same periodicity in suitable growth temperature, suggesting a close relationship between this periodicity and circadian rhythm regulation. Novel-m3234-5p, which was targeted to sjCRY-DASH, decreased with increasing sjCRY-DASH transcription, acting as a negative modulator of sjCRY-DASH. Six long non-coding RNAs classified as long intergenic non-coding RNAs (lincRNAs) exhibited co-expression with sjCRY-DASH. A miRNA sjCRY DASH lincRNA network was consequently identified. By predicting the endogenous competing mRNAs of novel-m3234-5p, we found that sjCRY-DASH indirectly participated in the regulation of DNA damage repair, protein synthesis and processing, and actin transport. In conclusion, our results revealed that non-coding RNAs participate in the regulation of sjCRY-DASH, which played vital roles in the growth and early development of S. japonica.

Transcriptome sequencing of Saccharina japonica sporophytes during whole developmental periods reveals regulatory networks underlying alginate and mannitol biosynthesis.

BACKGROUND: Alginate is an important cell wall component and mannitol is a soluble storage carbon substance in the brown seaweed Saccharina japonica. Their contents vary with kelp developmental periods and harvesting time. Alginate and mannitol regulatory networks and molecular mechanisms are largely unknown. RESULTS: With WGCNA and trend analysis of 20,940 known genes and 4264 new genes produced from transcriptome sequencing of 30 kelp samples from different stages and tissues, we deduced that ribosomal proteins, light harvesting complex proteins and "imm upregulated 3" gene family are closely associated with the meristematic growth and kelp maturity. Moreover, 134 and 6 genes directly involved in the alginate and mannitol metabolism were identified, respectively. Mannose-6-phosphate isomerase (MPI2), phosphomannomutase (PMM1), GDP-mannose 6-dehydrogenase (GMD3) and mannuronate C5-epimerase (MC5E70 and MC5E122) are closely related with the high content of alginate in the distal blade. Mannitol accumulation in the basal blade might be ascribed to high expression of mannitol-1-phosphate dehydrogenase (M1PDH1) and mannitol-1-phosphatase (M1Pase) (in biosynthesis direction) and low expression of mannitol-2-dehydrogenase (M2DH) and Fructokinase (FK) (in degradation direction). Oxidative phosphorylation and photosynthesis provide ATP and NADH for mannitol metabolism whereas glycosylated cycle and tricarboxylic acid (TCA) cycle produce GTP for alginate biosynthesis. RNA/protein synthesis and transportation might affect alginate complex polymerization and secretion processes. Cryptochrome (CRY-DASH), xanthophyll cycle, photosynthesis and carbon fixation influence the production of intermediate metabolite of fructose-6-phosphate, contributing to high content of mannitol in the basal blade. CONCLUSIONS: The network of co-responsive DNA synthesis, repair and proteolysis are presumed to be involved in alginate polymerization and secretion, while upstream light-responsive reactions are important for mannitol accumulation in meristem of kelp. Our transcriptome analysis provides new insights into the transcriptional regulatory networks underlying the biosynthesis of alginate and mannitol during S. japonica developments.

Verification of the Saccharina japonica Translocon Tic20 and its Localization in the Chloroplast Membrane in Diatoms.

Tic20 is an important translocon protein that plays a role in protein transport in the chloroplast. The sequence of Tic20 was determined in the lower brown alga Saccharina japonica. Structural analysis of SjTic20 revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain (a helix-loop-helix structural domain) and a C-terminal cyanobacterium-originated Tic20 domain. Subcellular localization and transmembrane analysis indicated that SjTic20 featured an "M"-type Nin-Cin-terminal orientation, with four transmembrane domains in the innermost membrane of the chloroplast in the microalga Phaeodactylum tricornutum, and the EF-hand domain was entirely extruded into the chloroplast stroma. Our study provides information on the structure, localization, and topological features of SjTic20, and further functional analysis of SjTic20 in S. japonica is needed.

Acclimation of Antarctic Chlamydomonas to the sea-ice environment: a transcriptomic analysis.

The Antarctic green alga Chlamydomonas sp. ICE-L was isolated from sea ice. As a psychrophilic microalga, it can tolerate the environmental stress in the sea-ice brine, such as freezing temperature and high salinity. We performed a transcriptome analysis to identify freezing stress responding genes and explore the extreme environmental acclimation-related strategies. Here, we show that many genes in ICE-L transcriptome that encoding PUFA synthesis enzymes, molecular chaperon proteins, and cell membrane transport proteins have high similarity to the gens from Antarctic bacteria. These ICE-L genes are supposed to be acquired through horizontal gene transfer from its symbiotic microbes in the sea-ice brine. The presence of these genes in both sea-ice microalgae and bacteria indicated the biological processes they involved in are possibly contributing to ICE-L success in sea ice. In addition, the biological pathways were compared between ICE-L and its closely related sister species, Chlamydomonas reinhardtii and Volvox carteri. In ICE-L transcripome, many sequences homologous to the plant or bacteria proteins in the post-transcriptional, post-translational modification, and signal-transduction KEGG pathways, are absent in the nonpsychrophilic green algae. These complex structural components might imply enhanced stress adaptation capacity. At last, differential gene expression analysis at the transcriptome level of ICE-L indicated that genes that associated with post-translational modification, lipid metabolism, and nitrogen metabolism are responding to the freezing treatment. In conclusion, the transcriptome of Chlamydomonas sp. ICE-L is very useful for exploring the mutualistic interaction between microalgae and bacteria in sea ice; and discovering the specific genes and metabolism pathways responding to the freezing acclimation in psychrophilic microalgae.

A trehalose-6-phosphate synthase gene from Saccharina japonica (Laminariales, Phaeophyceae).

The full-length cDNA sequence of a trehalose-6-phosphate synthase gene from Saccharina japonica (designated as SjaTPS) (Accession: KC578568) was isolated based on homologous cloning and RACE-PCR. It was 4,127 bp, with 320 bp 5'-UTR, 21 bp 3'-UTR, and open reading frame (ORF) of 3,786 bp. The deduced 1,261 amino acids characterized with predicted molecular weight of 137.84 kDa and theoretical isoelectric point of 7.12. The SjaTPS had one N-terminal CBM20 (family 20 carbohydrate-binding module) domain, one TPS domain (trehalose-6-phosphate synthase) in the middle region and a single TPP (trehalose-6-phosphate phosphatase) domain near the C-terminus. Structural analysis suggested that the SjaTPS putatively functioned as trehalose-6-phosphate synthase, and might be related to laminaran metabolism in S. japonica. Homology analysis indicated that the SjaTPS shared 49-70 % similarities with the 13 known TPS sequences of other algae; only 55 % amino acid similarities were detected between SjaTPS and the previously reported TPS sequence of S. japonica (Accession: DQ666325). Phylogenetic analysis revealed close affinity between SjaTPS and TPS of brown alga Ectocarpus siliculosus (Accession: CBJ29609). Transcriptional analysis showed that desiccation greatly enhanced SjaTPS expression and the maximum appeared at 3 h, which was about 300-fold compared to that of the start, implied that SjaTPS was involved with drought adaption in kelp. In vitro expression of SjaTPS showed that one distinct band existed at ~115 kDa, and western blot detection proved that it was positive to the anti-His antibody with high specificity. Our results increased the knowledge of trehalose-6-phosphate synthase properties in S. japonica and also important for better understanding the role trehalose plays in kelp abiotic tolerance for adaption to the sublittoral habitats.

Expression of the novel all-trans retinoic acid-related resistance gene HA117 in pediatric solid tumors.

This study aimed to detect the protein expression of HA117 in pediatric solid tumors. Immunohistochemistry was performed to detect the expression of HA117 and P-gp in pediatric solid tumors. In Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), nephroblastoma (WT), and neuroblastoma (NB), the positive expression rate of HA117 was 65.4%, 58.3%, 81.3%, and 74.1%, and that of P-gp was 57.7%, 70.8%, 65.6%, and 66.7%, respectively. HA117 expression was closely related to the clinical stage of HL (P=0.004) and to the International Prognostic Index score, mediastinal lesions, and clinical stages of NHL (P=0.01, 0.03, and 0.01). The expression of HA117 in WT was higher than in adjacent normal tissues, but there was no statistical significance (P=0.21). The positive expression of HA117 in NB was markedly higher than that in normal tissues (P=0.002), which closely associated with histologic type and lymph node metastasis (P=0.03 and 0.001). Spearman correlation analysis revealed that HA117 expression was not correlated with P-gp in these 4 tumors. This suggests that HA117 might be an important resistance gene in pediatric solid tumors. The mechanism underlying the resistance to all-trans retinoic acid conferred by HA117 is different from that of P-gp.

Predicting the medium-temperature thermal stability of impact-strengthened medium-thick plate aluminum alloy using a back propagation artificial neural network.

A normalized medium-thick plate of aluminum alloy (4038) was impact-strengthened using a free-fall method at room temperature (approximately 20 °C). Specimens were then aged at 450 °C, 550 °C and 650 °C for 10, 20, 30 and 40 min respectively. Micro-hardness of each sample was tested. Micro-structure of samples annealed at 650 °C for different durations was characterized. A three-layer back propagation artificial neural network (BPANN) was trained using actual state parameters of the prepared samples. Results reveal that medium-temperature thermal stability of the prepared plate can be predicted through the BPANN model. Deviation of predicted values from the experimental ones is within 6 %, with a prediction accuracy exceeding 94 %. Variation trend of the predicted and the experimental thermal stability is consistent, but the predicted values are all higher than the measurements. Prediction accuracy of BPANN can be improved by increasing convergence rate of the error function. By adding relevant parameters of the micro-structure from samples aged at 650 °C to the input layer, BPANN model further improve its output and approach the real state of samples. The findings of this study can help researchers reduce the number and cost of experiments. The aim of this work was to predict the medium-temperature thermal stability of impact-strengthened normalized medium-thick plate of aluminum alloy annealed at different temperatures, and it also can be used as reference for other similar experiments.

Case Report: Anal canal duplication associated with anorectal stenosis-A rare presentation.

Anal canal duplication is a rare gastrointestinal malformation characterized by extra anal orifices at 6 o'clock in the lithotomy position. To date, there have been only 110 reported cases. The purpose of this study is to contribute two infant cases, one of which is associated with anorectal stenosis, which has never been described.

MiR8181 is involved in the cell growth regulation of Saccharina japonica.

Aureochrome, a blue-light receptor specifically found in photosynthetic stramenopiles, plays an important role in algal growth and development. It holds a reversed effector-sensor topology for the reception of blue light, acting as a candidate of optogenetic tool in transcriptional regulation. However, the inner regulatory mechanism of aureochrome is still unclear. In this study, we explored the potential regulatory relationship between microRNAs (miRNAs) and mRNAs by small RNA, transcriptome and degradome sequencing in Saccharina japonica. Through screening miRNA-mRNA interaction networks at the whole-genome level, we found that 18 miRNAs perfectly paired with aureochrome. Among these screened miRNAs, miR8181 was negatively correlated with aureochrome5 with high credibility, exhibiting tissue-specific expression in sporophyte of S. japonica. Degradome analysis further revealed the exact cleavage site of miR8181 on aureochrome5, confirming their targeting relationship. For the 54 target genes of miR8181, nine genes that exhibited similar expression to that of aureochrome5 competed for the same binding site, thus establishing a competing endogenous RNA network. Functional enrichment of the target genes revealed that miR8181 was involved in the regulation of cell differentiation and development in S. japonica. Moreover, overexpression of miR8181 resulted in significant decreases in the cell growth rates of Phaeodactylum tricornutum, suggesting negative roles of miR8181 in regulating cell growth. Our study revealed that miR8181, the targeting miRNA of aureochrome5, played negative roles in cell growth and development.

High-density SNP-based QTL mapping and candidate gene screening for yield-related blade length and width in Saccharina japonica (Laminariales, Phaeophyta).

Saccharina japonica is one of the most important marine crops in China, Japan and Korea. Candidate genes associated with blade length and blade width have not yet been reported. Here, based on SLAF-seq, the 7627 resulting SNP loci were selected for genetic linkage mapping to 31 linkage groups with an average spacing of 0.69 cM, and QTL analyses were performed to map the blade length and blade width phenotypes of S. japonica. In total, 12 QTLs contributing to blade length and 10 to width were detected. Some QTL intervals were detected for both blade length and width. Additive alleles for increasing blade length and width in S. japonica came from both parents. After the QTL interval regions were comparatively mapped to the current reference genome of S. japonica (MEHQ00000000), 14 Tic20 (translocon on the inner envelope membrane of chloroplast) genes and three peptidase genes were identified. RT-qPCR analysis showed that the transcription levels of four Tic20 genes were different not only in the two parent sporophytes but also at different cultivation times within one parent. The SNP markers closely associated with blade length and width could be used to improve the selection efficiency of S. japonica breeding.

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.

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.

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.

The clinical significance of pathological studies of congenital intestinal atresia.

OBJECTIVE: The purpose of this study was to explore the mechanisms of postoperative intestinal motility disorders in intestinal atresia patients by investigating the expression profiles of proteins, including calretinin (CR), glial-derived neurotrophic factor (GDNF), bone morphogenetic protein 2 (BMP-2), c-kit, α-smooth muscle actin (α-SMA), and S-100 protein; to decipher the correlation between the area of the pathological segment and the alteration of the above 6 proteins; and thereby to provide a clinical specific reference values to determine the removal length for intestinal tract resection. METHODS: Immunohistochemistry technique was applied to detect the CR, c-kit, GDNF, BMP-2, α-SMA, and S-100 protein in specimens of atretic, proximal, and distal intestine from 25 cases of intestinal atresia and samples of intestinal walls from 10 non-atresia control specimens. The alteration of the enteric nervous system, nerve growth and its regulatory factors, the interstitial cells of Cajal (ICCs), and the enteric muscle system were examined, with particular attention being paid to pathological changes and the lesion area. RESULTS: The expression of all of the abovementioned 6 proteins in the proximal side of the atresia was significantly lower than in control group. The expression of the abovementioned proteins tended to be higher farther away from the atresia site. The expressions of both GDNF and BMP-2 had returned to normal level at 10 cm proximal to the atresia site, whereas the expressions of CR, c-kit, α-SMA, and S-100 protein only returned to normal at 15 cm proximal to the atresia site. On the distal side, the expression of all 6 markers at 3 cm distal to the atresia site was normal. CONCLUSION: Pathological deterioration of the myenteric ganglia, nerve growth factor, and ICCs are the causes of intestinal motility disorders after the surgical repair of intestinal atresia. Our data support resecting an intestinal segment extending from 15 cm proximal to 3 cm distal to the atretic segment. In proximal jejunal atresia, when it is not possible to resect 15 cm, we suggest resecting as much of the hypertrophic proximal intestine as possible. Based on our data, we believe this surgical practice could improve postoperative dysmotility in these patients.

Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice.

The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T1 to T4 generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice.

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.

QTL mapping for frond length and width in Laminaria japonica aresch (Laminarales, Phaeophyta) using AFLP and SSR markers.

In Laminaria japonica Aresch breeding practice, two quantitative traits, frond length (FL) and frond width (FW), are the most important phenotypic selection index. In order to increase the breeding efficiency by integrating phenotypic selection and marker-assisted selection, the first set of QTL controlling the two traits were determined in F(2) family using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Two prominent L. japonicas inbred lines, one with "broad and thin blade" characteristics and another with "long and narrow blade" characteristics, were applied in the hybridization to yield the F(2) mapping population with 92 individuals. A total of 287 AFLP markers and 11 SSR markers were used to construct a L. japonica genetic map. The yielded map was consisted of 28 linkage groups (LG) named LG1 to LG28, spanning 1,811.1 cM with an average interval of 6.7 cM and covering the 82.8% of the estimated genome 2,186.7 cM. While three genome-wide significant QTL were detected on LG1 (two QTL) and LG4 for "FL," explaining in total 42.36% of the phenotypic variance, two QTL were identified on LG3 and LG5 for the trait "FW," accounting for the total of 36.39% of the phenotypic variance. The gene action of these QTL was additive and partially dominant. The yielded linkage map and the detected QTL can provide a tool for further genetic analysis of two traits and be potential for maker-assisted selection in L. japonica breeding.

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Laminaria japonica (Laminariaceae, Phaeophyta).

In this study, a full-length cytosolic heat shock protein 70 complementary DNA (cDNA) of Laminaria japonica (designated as LJHsp70) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends. The full length of LJHsp70 cDNA was 2,918 bp, with a 5' untranslated region of 248 bp, a 3' untranslated region of 696 bp, and an open reading frame of 1,974 bp encoding a polypeptide of 657 amino acids with an estimated molecular mass of 72.03 kDa and an estimated isoelectric point of 4.97. There was highly repeated sequence of CAA in 5' untranslated region of LJHsp70. The result of phylogenetic tree of Hsp70s, the BLAST program, analysis and cytosolic Hsp70-specific motif of LJHsp70 verified that the cloned LJHsp70 belonged to cytosolic Hsp70 family. Three typical Hsp70 signature motifs were detected in LJHsp70 by InterPro analysis. Under different stress conditions, messenger RNA (mRNA) expression levels of LJHsp70 were quantified by quantitative RT-PCR. To L. japonica sporophytes kept in different temperatures for 1 h, the expression level of LJHsp70 at 30 degrees C was highest and twofold higher than that at 10 degrees C. To L. japonica sporophytes kept at 25 degrees C for different times, the mRNA expression level of LJHsp70 reached a maximum level after 7 h and then dropped progressively. The expression level of LJHsp70 at 0 or 5 per thousand salt concentration for 2 h was twofold higher than that at 30 per thousand salt concentration for 2 h. The results showed that LJHsp70 may be a kind of potential biomarker used to monitor environment conditions.

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES, PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS(1).

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.