Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family.

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.

Alleviative Effect of Iodine Pretreatment on the Stress of Saccharina japonica (Phaeophyceae, Laminariales) Caused by Cadmium and Its Molecular Basis Revealed by Comparative Transcriptomic Analysis.

Iodide is accumulated by the brown alga Saccharina japonica at a high concentration and has been proven to be an inorganic antioxidant that plays an important role in oxidative metabolism. Vanadium-dependent bromoperoxidases (vBPOs) and iodoperoxidases (vIPOs), which catalyze the oxidation of iodide, are essential for iodine accumulation and metabolism. Heavy metal pollutant cadmium (Cd) from anthropogenic activities can cause damage to algae mainly by producing oxidative stress. Here, the effects of iodine pretreatment on the stress of S. japonica caused by cadmium were analyzed. The growth experiment showed that iodine pretreatment could reduce the damage of low concentration cadmium on S. japonica young thalli. At the transcriptomic level, gene ontology (GO) enrichment analysis confirmed that cadmium stress could cause a peroxidation reaction in S. japonica. However, the most significant GO term was "photosystem I" in the series with iodine pretreatment. Weighted gene co-expression network analysis (WGCNA) indicated that iodine pretreatment alleviated cadmium stress responses of S. japonica by affecting the photosynthesis process. Analysis of the differentially expressed genes (DEGs) showed that five enzymes from the vBPO family and 13 enzymes from the vIPO family might play crucial roles in this process.

The Inheritable Characteristics of Monoecy and Parthenogenesis Provide A Means for Establishing A Doubled Haploid Population in the Economically Important Brown Alga Undaria pinnatifida (Laminariales, Alariaceae).

Monoecy and parthenogenesis exist in certain male and female gametophytes of the brown alga Undaria pinnatifida. The inheritance of these traits is not known. In this study, we made a cross between a male and a female gametophyte clone which could exhibit monoecy and parthenogenesis phenotypes, respectively, and obtained their next-generation gametophyte offspring. We found that under conditions suitable for gametogenesis, all of the male offspring (n = 100) exhibited monoecy phenotype and all of the female offspring (n = 100) only formed oogonia and underwent parthenogenesis, suggesting that monoecy and parthenogenesis phenotypes are inheritable. Then, we established a doubled haploid (DH) population through monoecious selfing and parthenogenesis, and evaluated the young sporophyte growth and the maximum quantum yield (Fv /Fm ) of 10 "male" and 10 "female" DH lines. On day 60, the average length of the "male" DH lines was significantly larger than that of the "female" DH lines, while their average Fv /Fm values were not significantly different. Monoecious selfing seemed superior to parthenogenesis as the sporophyte formation efficiency, and the young sporophyte growth was better in the former than in the latter. We also crossed two monoecious gametophytes with another male gametophyte, and a parentage analysis showed success of obtaining hybrid sporophytes, indicating that the female gametes released by the monoecious gametophyte can actually be fertilized by sperm. The approach of establishing a DH population proposed here will be useful in genetic breeding and quantitative trait loci mapping in U. pinnatifida and may be applicable to other kelp species.

A method to establish an "immortalized F2 " sporophyte population in the economic brown alga Undaria pinnatifida (Laminariales: Alariaceae).

Studies of quantitative trait loci based on genetic linkage maps require the establishment of a mapping population. Permanent mapping populations are more ideal than temporary ones because they can be used repeatedly. However, there has been no reported permanent sporophyte population of economically important kelp species. Based on the characteristics of the kelp life cycle, we proposed a method to establish, and then constructed experimentally, an "immortalized F2 " (IF2 ) population of Undaria pinnatifida. Doubled-haploid "female" and "male" sporophytes were obtained through the parthenogenesis of a female gametophyte clone and the selfing of a "monoicous" gametophyte clone (originally male), respectively, and they were used as the parents. The F1 hybrid line was generated by crossing the female and male gametophytes derived from the respective female and male parents. Full-sibling F2 gametophyte clones, consisting of 260 females and 260 males, were established from an F1 hybrid sporophyte. Thirty-five females and 35 males were randomly selected and paired to give rise to an IF2 population containing 35 crossing lines. A parentage analysis using 10 microsatellite markers confirmed the accuracy of the IF2 population and indicated the feasibility of this method. This proposed method may be adapted for use in other kelp species, and thus, it will be useful for genetic studies of kelp.

Effects of Cobalt on Spore Germination, Gametophyte Growth and Gametogenesis of Undaria pinnatifida (Phaeophyceae).

With rapid development of the construction of nuclear power plants along the coast, the concern of negative effects of potentially unexpected release of nuclides on marine organisms has increased. Cobalt (Co) is one of the crucial nuclides in nuclear polluted seawater. The effect of its presence in seawater on life cycle of macroalgae has seldom been studied. In this investigation, a series of Co concentrations including 1, 10, 100 µg L-1, and 1, 10 mg L-1 (the background concentration of Co in culture seawater was determined to be at the level of 0.75 ± 0.11 µg L-1) were used to test the effects of their presence on spore germination, gametophyte growth and gametogenesis of the important brown macroalga Undaria pinnatifida. It was found that the spore germination rate of 10 mg L-1 group was significantly lower than that of the control group after 1- and 2 days exposure. The gametophyte sizes of 1 and 10 mg L-1 groups were much smaller than that of the control group after 6- and 12-days exposure. Oogonia and juvenile sporophytes were observed to appear in 1, 10 µg L-1 and the control groups after 12 and 15 days, respectively, but not in the higher concentration groups. In the recovery test, sporophytes appeared in the 100 µg L-1 group on the 5th day, but not in 1 and 10 mg L-1 groups. These results demonstrate that presence of Co at high concentrations in seawater disturbs the life cycle by suppressing both the gametophyte growth and gametogenesis in U. pinnatifida.

Phytoremediation Potential of Saccharina japonica and Sargassum horneri (Phaeophyceae): Biosorption Study of Strontium.

Strontium (Sr) is an important nuclide in nuclear polluted seawater. Algal biosorption is regarded as an efficient and cost-effective way to scavenge detrimental metal ions of low concentration from polluted waters. In this study, we selected two algal species (Saccharina japonica and Sargassum horneri) through comparing background Sr contents in six algal species and explored their potential to scavenge Sr of low concentration from seawater in culture conditions. It was found that biosorption of Sr in S. japonica and S. horneri increased in response to increased Sr concentrations in the seawater and S. horneri adsorbed more Sr than S. japonica. The equilibrium established in their biosorption process follows both Langmiur and Freundlich equations well. The Sr biosorption process of S. horneri fits the pseudo-second-order kinetic model well, implying that adsorption was the rate-limiting step of the biosorption process. Results suggest S. japonica and S. horneri could become efficient biosorbents for in situ scavenging Sr from nuclear polluted seawater.

Construction of a high-density genetic map and mapping of a sex-linked locus for the brown alga Undaria pinnatifida (Phaeophyceae) based on large scale marker development by specific length amplified fragment (SLAF) sequencing.

BACKGROUND: Undaria pinnatifida is an important economic brown alga in East Asian countries. However, its genetic and genomic information is very scarce, which hinders further research in this species. A high-density genetic map is a basic tool for fundamental and applied research such as discovery of functional genes and mapping of quantitative trait loci (QTL). In this study the recently developed specific length amplified fragment sequencing (SLAF-seq) technology was employed to construct a high-density genetic linkage map and locate a sex determining locus for U. pinnatifida. RESULTS: A total of 28.06 Gb data including 140.31 M pair-end reads was obtained. After linkage analysis 4626 SLAF markers were mapped onto the genetic map. After adding the sex linked simple sequence repeat (SSR) marker [GenBank:AY738602.1], the final genetic map was 1816.28 cM long, consisting of 30 linkage groups with an average distance of 0.39 cM between adjacent markers. The length of LGs ranged from 20.12 to 106.95 cM. A major sex associated QTL was mapped to LG22 within a window starting at 29.01 cM and ending at 68.81 cM with a total of 68 SLAF markers. The SSR marker and five SLAF markers (Marker6556, 19020, 43089, 60771 and 26359) were identified as tightly sex-linked markers, as indicated by the absence of recombination between them and the sex phenotype. These markers were located at the position of 59.50 cM, which was supposed to be the sex determining region. CONCLUSIONS: A high-density genetic linkage map was constructed using SLAF-seq technique and F1 gametophyte population for the first time in the economically important brown alga U. pinnatifida. For the first time, a major sex associated QTL suggesting a sex determining region was mapped to a single LG. This map will facilitate the further fundamental and applied research such as QTL mapping and map-based gene clone in U. pinnatifida and provide a reference for studies in other kelp species.

Breeding in the Economically Important Brown Alga Undaria pinnatifida: A Concise Review and Future Prospects.

Undaria pinnatifida is the commercially second most important brown alga in the world. Its global annual yield has been more than two million tonnes since 2012. It is extensively cultivated in East Asia, mainly consumed as food but also used as feed for aquacultural animals and raw materials for extraction of chemicals applicable in pharmaceutics and cosmetics. Cultivar breeding, which is conducted on the basis of characteristics of the life history, plays a pivotal role in seaweed farming industry. The common basic life history shared by kelps determines that their cultivar breeding strategies are similar. Cultivar breeding and cultivation methods of U. pinnatifida have usually been learned or directly transferred from those of Saccharina japonica. However, recent studies have revealed certain peculiarity in the life history of U. pinnatifida. In this article, we review the studies relevant to cultivar breeding in this alga, including the peculiar component of the life history, and the genetics, transcriptomics and genomics tools available, as well as the main cultivar breeding methods. Then we discuss the prospects of cultivar breeding based on our understanding of this kelp and what we can learn from the model brown alga and land crops.

Transcriptome profiling of the meristem tissue of Saccharina japonica (Phaeophyceae, Laminariales) under severe stress of copper.

Copper (Cu) is an essential metal involved in many physiological processes of living organisms. However, beyond a certain threshold, Cu can become highly toxic. For instance, in the summer sporeling production of the economic kelp Saccharina japonica, the excess Cu accidently released from the low-quality alloys of the refrigerating machine was deadly to the seedlings and led to the failure of hatchery operations. However, the molecular basis underlying high toxicity of Cu remains unclear. In this study, juvenile sporophytes were cultured in seawater containing different concentrations of Cu2+ (10, 100, and 200 µg L-1). Bleaching was observed in the meristem of individuals in the 100 and 200 µg L-1 treatment groups on the third day, indicating that Cu has caused severe harm at these concentrations. RNA-Seq was used to profile transcriptomic changes under different Cu2+ concentrations. Compared with the control, the number of differentially expressed genes (DEGs) was 11,350 (4944 up- and 6406 down-regulated) in the 200 µg L-1 treatment group and 2868 (1075 up- and 1793 down-regulated) in the 100 µg L-1 treatment group, whereas much fewer DEGs were detected in the 10 µg L-1 treatment group. Genes coding for glutathione-S-transferase and vanadium-dependent bromoperoxidase and iodoperoxidase were found to be remarkably regulated, especially in the 200 µg L-1 treatment group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that only down-regulated DEGs were enriched. There were 45 enriched GO terms and four enriched KEGG pathways common to the 100 and 200 µg L-1 treatment groups, which were associated with diverse essential biological processes such as photosynthesis, protein synthesis, redox activity, and metabolism and biosynthesis of functional biomolecules, among others. Suppression of these biological processes at the transcriptional level likely contributes to the observed high toxicity of Cu2+ in S. japonica.

The structure-activity relationship between polysaccharides from Sargassum thunbergii and anti-tumor activity.

Polysaccharides derived from Sargassum thunbergii were prepared to investigate the structure-activity relationship between polysaccharides and anti-tumor activity in vitro. Many factors were examined. Overall, STW (polysaccharide extracted by hot water) had the best activity, followed by STJ (polysaccharide extracted by dilute alkali), and then STA (polysaccharide extracted by dilute acid). Location of algae had no effect at 500µg/mL and 1000µg/mL, while STW-QD (algae collected from Qingdao, China) had the best activity, followed by STW-WZ (algae collected from Wenzhou, China) and STW-LJ (algae collected from Lianjiang, China) and then STW-DL (algae collected from Dalian, China) and STW-RC (algae collected from Rongcheng, China) at 250µg/mL. Moreover, molecular weight had no effect at 1000µg/mL, while higher molecular weights were associated with better activities at 250µg/mL and 500µg/mL. Sulfate content had no effect at 1000µg/mL, while anti-tumor activities decreased accompanying with the changes of sulfate content. Uronic acid content was an important factor influencing activity. The fractions of STW showed little anti-tumor activity; however, the mixture of the fractions of STW showed approximately 60% inhibition. Overall, these findings suggested that the anti-tumor activity of polysaccharides required multilateral cooperation and that some of the effective components were lost.

Understanding the recurrent large-scale green tide in the Yellow Sea: temporal and spatial correlations between multiple geographical, aquacultural and biological factors.

The coast of Jiangsu Province in China - where Ulva prolifera has always been firstly spotted before developing into green tides - is uniquely characterized by a huge intertidal radial mudflat. Results showed that: (1) propagules of U. prolifera have been consistently present in seawater and sediments of this mudflat and varied with locations and seasons; (2) over 50,000 tons of fermented chicken manure have been applied annually from March to May in coastal animal aquaculture ponds and thereafter the waste water has been discharged into the radial mudflat intensifying eutrophication; and (3) free-floating U. prolifera could be stranded in any floating infrastructures in coastal waters including large scale Porphyra farming rafts. For a truly integrated management of the coastal zone, reduction in nutrient inputs, and control of the effluents of the coastal pond systems, are needed to control eutrophication and prevent green tides in the future.