Microsatellite DNA Variation of the Gametophyte Clones Isolated from Introduced Laminaria japonica （Phaeophyta） and L. Iongissima of China and Varieties Derived from them

The variation of 90 Laminaria gametophyte clones representing the introduced Laminaria japonica （Group 1） and Laminaria Iongissima （Group 2）, the varieties of L. japonica （Group 3） and the varieties derived from interspecific hybrids （Group 4） was determined with 18 microsatellite markers. The allelic diversity and Nei＇s gene diversity of Group 1 were significantly higher than those of Group 2 （2.9 vs. 1.8 and 0.414 vs. 0.161, respectively）, demonstrating that the variation of the introduced L. japonica is richer than that of L. Iongissima. Both allelic diversity and Nei＇s gene diversity of Group 3 were lower than those of Group 1, indicating that only a portion of variation of L. japonica was incorporated into the varieties of L. japonica. Significant genetic differentiation was detected between four groups and between female （Population 1 ） and male （Population 2） gametophyte clones in each group. The variation among groups accounted for 39.95%, while that among populations accounted for 21.65% of the total. The genetic distance between Group 1 and Group 4 was obviously longer than that between Group 2 and Group 4 （0.686 vs. 0.291）, indicating that maternal gametophyte clone contributed more variation to the hybrids than the paternal gametophyte clone did.     

海带(Laminaria japonica)丝状体遗传多样性的比较研究

采用垂直板式聚丙烯酰胺凝胶电泳技术对分别来自中国青岛与日本北海道海带(Laminaria japonica)丝状体的6种酶系统（乙醇脱氢酶、谷氨酸脱氢酶、异柠檬酸脱氢酶、乳酸脱氢酶、山梨醇脱氢酶和超氧化物歧化酶）进行了检测,发现存在22个基因位点,分析了不同来源海带丝状体的遗传多样性与遗传分化程度。结果表明：日本北海道海带丝状体的预期杂合度、每个位点有效等位基因数等遗传参数均大于中国青岛海带丝状体。对日本北海道海带丝状体与中国青岛海带丝状体的遗传相似系数和遗传距离进行了计算分析,显示两者发生了遗传分化。日本北海道海带丝状体的遗传多样性高于中国青岛海带丝状体。研究结果还发现在中国青岛海带丝状体的Idh、Ldh 和Sdh中,以及日本北海道海带丝状体的Adh、Gdh、Idh、Ldh、Sdh和Sod中存在着不同迁移率的不等距双带酶,可能由特殊基因控制,并集中在第3个基因位点上。     

Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.     

Sensitivity of Laminariales zoospores from Helgoland (North Sea) to ultraviolet and photosynthetically active radiation: implications for depth distribution and seasonal reproduction

Depth distribution of kelp species in Helgoland (North Sea) is characterized by occurrence of Laminaria digitata in the upper sublittoral, whereas L. saccharina and L. hyperborea dominate the mid and lower sublittoral region. Laminaria digitata is fertile in summer whereas both other species are fertile in autumn/winter. To determine the light sensitivity of the propagules, zoospores of L. digitata, L. saccharina and L. hyperborea were exposed in the laboratory to different exposure times of photosynthetically active radiation (PAR; 400–700 nm), PAR + UVA radiation (UVAR; 320–400 nm) and PAR + UVAR + UVB radiation (UVBR; 280–320 nm). Optimum quantum yield of PSII and DNA damage were measured after exposure. Subsequently, recovery of photosynthetic efficiency and DNA damage repair, as well as germination rate were measured after 2 and 3 d cultivation in dim white light. Photosynthetic efficiency of all species was photoinhibited already at 20 µmol photons m⁻² s⁻¹ PAR, whereas UV radiation (UVR) had a significant additional effect on photoinhibition. Recovery of the PSII function was observed in all species but not in spores exposed to irradiation longer than 4 h of PAR + UVA + UVB and 8 h of PAR + UVA. The amount of UVB-induced DNA damage measured as cyclobutane–pyrimidine dimers (CPDs) increased with exposure time and highest damage was detected in the spores of lower subtidal L. hyperborea relative to the other two species. Significant removal of CPDs indicating repair of DNA damage was observed in all species after 2 d in low white light especially in the spores of upper subtidal L. digitata. Therefore, efficient DNA damage repair and recovery of PSII damage contributed to the germination success but not in spores exposed to 16 h of UVBR. UV absorption of zoospore suspension in L. digitata is based both on the absorption by the zoospores itself as well as by exudates in the medium. In contrast, the absorption of the zoospore suspension in L. saccharina and L. hyperborea is based predominantly on the absorption by the exudates in the medium. This study indicates that UVR sensitivity of zoospores is related to the seasonal zoospore production as well as the vertical distribution pattern of the large sporophytes.     

Kinetics of non-photosynthetic callus production from the brown alga Laminaria setchellii (Laminariales)

White, filamentous, callus-like tissue was produced from the temperate marine brown alga Laminaria setchellii Silva under non-photosynthetic conditions. Explant disks from the medullary core of the stipe section were plated on solid medium (1.5 wt% agar in seawater base, pH 8) at 8 ± 2°C in the dark, and the formation of callus-like tissue was followed as a function of time up to 8 weeks. Although 30% of the total explants plated formed filamentous, callus-like growths, only 3 to 6% of the total explants produced relatively large amounts of filamentous and rounded callus-like cells. The fresh weight yield of this callus-like tissue was 3.74 ± 0.93 mg per 10 mm explant disk (0.053 g callus per g explant). Attempts at establishing a growing, cell suspension of the dispersed callus tissue in PES liquid medium at 24 μmol photon m^?2 s^?1 were not successful.     

ON THE TAXONOMY OF CALIFORNIA LAMINARIA (PHAEOPHYTA)1

It is suggested that Laminaria setchellii Silva be retained as a species separate from L. dentigera Kjellman as there is no evidence suggesting they are conspecific. Further, L. saccharina (L.) Lamouroux and L. groenlandica Rosenvinge should also continue to be recognized as valid components of the California flora.     

Guluronate content of alginate in Laminaria japonica and Laminaria angustata fronds in laboratory culture

The guluronate (G) content of alginate in the fronds of Laminaria japonica and Laminaria angustata, cultured in the laboratory from zoospore via gametophyte using PESI medium, was determined by the ¹H‐nuclear magnetic resonance spectroscopic method. The G content of alginates in young fronds cultured in various conditions were shown to exceed 55%. These values were remarkably higher than those in field kelp. The G content increased with extending culture period and at low temperature.     

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES,PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS1

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.     

Effect of temperature and photon fluence rate on gametophytes and young sporophytes of Laminaria ochroleuca Pylaie

We analysed the effects of temperature and photon fluence rate on meiospore germination, growth and fertility of gametophytes, and growth of young sporophytes of Laminaria ochroleuca. Maximum percentages of germination (91–98%) were obtained at 15°C and 18°C, independent of photon fluence rate. Optimal development of female gametophyte and maximum fecundity and reproductive success of gametophytes occurred at 15°C and 18°C and at 20 and 40 μmol m^–2 s^–1. Maximum relative growth rate of young sporophytes after 2 weeks of culture was achieved under the same conditions. L. ochroleuca gametophytes cannot reproduce and growth of its sporophytes is not competitive at temperatures close to 10°C. Received in revised form: 31 August 2001 Electronic Publication     

Assessment of Genetic Diversities of Selected Laminaria （Laminariales, Phaeophyta） Gametophytes by Inter-Simple Sequence Repeat Analysis

Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10 pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10 selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility.of applying ISSR markers to evaluate Laminaria germplasm diversities.     

Assessment of Genetic Diversities of Selected Laminaria (Laminariales,Phaeophyta) Gametophytes by Inter-Simple Sequence Repeat Analysis

Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility of applying ISSR markers to evaluate Laminaria germplasm diversities.     

Research note: Salinity tolerance of Arctic kelps from Spitsbergen

The effect of hypo‐ and hypersaline treatments on the effective quantum yield of photosystem II was comparatively studied with a pulse amplitude modulated fluorometer (PAM) in the brown algal species Alaria esculenta, Fucus distichus, Laminaria digitata, Laminaria solidungula, Saccharina latissima (formerly Laminaria saccharina) and Saccorhiza dermatodea collected in the Arctic Kongsfjorden (Spitsbergen). While the euryhaline F. distichus was not affected at all by salinities ranging from 5 to 60 psu, A. esculenta, S. latissima and L. solidungula exhibited under hyposaline conditions strong loss of pigments (bleaching) or even high mortality reflecting stenohaline features. In contrast to the latter species, L. digitata and S. dermatodea survived all salinities, but showed reduced photosynthetic activities at the lowest and highest salt treatments and hence, can be characterized as stenohaline‐euryhaline organisms. The data are discussed in terms of vertical zonation (eulittoral versus sublittoral habitat), in terms of interactive effects with other abiotic factors such as temperature and in terms of the species‐specific acclimation potential.     

Fucoidan Isolated from Laminaria angustata var. longissima Induced Macrophage Activation

We investigated macrophage activation by fucoidan from Laminaria angustata var. longissima in a murine macrophage cell line, RAW 264.7. The ratio of the chemical composition of the fucoidan was L-fucose:D-galactose:D-glucose:D-xylose:uronic acid:sulfate = 1.00:0.54:0.08:0.08:0.64:0.87. The fucoidan induced production of nitric oxide, tumor necrosis factor-α, and interleukin-6 in RAW 264.7 cells. These results indicate that the fucoidan induced macrophage activation.     

海带内生芽胞杆菌DNN7和HSN2胞外蛋白抗肿瘤活性初步研究

为检测海带内生芽胞杆菌DNN7和HSN2胞外蛋白的抗肿瘤活性,用0.22μm滤膜过滤将海带内生芽胞杆菌DNN7和HSN2的菌体和发酵液分开,用硫酸铵沉淀发酵液的蛋白组分并用SDS-PAGE检测,用海虾毒性法和MTT法检测胞外蛋白的抗肿瘤活性。结果表明,海带内生芽胞杆菌DNN7的粗蛋白主要由分子量约为66×103、52×103和34×103的3条带组成,HSN2的粗蛋白主要由分子量约为44×103和52×103的2条带组成。DNN7胞外蛋白对体外慢性粒细胞白血病癌细胞K562、肝肿瘤细胞HepG2和乳腺癌细胞MDA-MB-231均具有较好的抑制活性,其抑制率分别高达96.46%、42.99%和85.70%;HSN2胞外蛋白对体外慢性粒细胞白血病癌细胞K562、肝肿瘤细胞HepG2和乳腺癌细胞MDA-MB-231也具有较好的抑制活性,其抑制率分别为92.65%、66.80%、88.48%。     

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997     

Identification and Assessing the Cultivars of Laminaria Lamx.(Phaeophyceae) with Molecular Markers

Molecular markers were used to identify and assess cultivars ofLaminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956.Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars of Laminaria japonica Aresch. used for breed ing in China fell into one cluster. L. japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm.formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including ITS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.     

Ethanol production from seaweed extract

Extracts from Laminaria hyperborea could possibly be fermented to ethanol commercially. In particular, seaweed harvested in the autumn contains high levels of easily extractable laminaran and mannitol. Four microorganisms were tested to carry out this fermentation, one bacterium and three yeasts. Only Pichia angophorae was able to utilise both laminaran and mannitol for ethanol production, and its substrate preferences were investigated in batch and continuous cultures. Laminaran and mannitol were consumed simultaneously, but with different relative rates. In batch fermentations, mannitol was the preferred substrate. Its share of the total laminaran and mannitol consumption rate increased with oxygen transfer rate (OTR) and pH. In continuous fermentations, laminaran was the preferred substrate at low OTR, whereas at higher OTR, laminaran and mannitol were consumed at similar rates. Optimisation of ethanol yield required a low OTR, and the best yield of 0.43 g ethanol (g substrate)⁻¹ was achieved in batch culture at pH 4.5 and 5.8 mmol O₂ l⁻¹ h⁻¹. However, industrial production of ethanol from seaweed would require an optimisation of the extraction process to yield a higher ethanol concentration. Journal of Industrial Microbiology & Biotechnology (2000) 25, 249–254. Received 25 February 2000/ Accepted in revised form 05 August 2000     

Growth of the Brown Alga <Emphasis Type="Italic">Laminaria japonica</Emphasis> in a Laboratory Environment

The growth of an individual thallus of the brown alga Laminaria japonica was studied in a laboratory environment. It was found that daily accretion of the Laminaria thallus can remain constant for a long time. The blade grew in length at a distance from 0 to 15–25 cm from the stipe-blade border, which makes up 60% of the blade length. In width, the blade grew both in the central and in the lateral parts of the blade, 0 to 7–10 cm from the stipe-blade border, which makes up 30% of the frond length. The transposition of a perforation hole mark from the stipe-blade border toward the blade tip evidenced the formation of new tissue in the growth zone even when the thallus diminished in size due to destruction of the blade tip. Based on the results of this observation, it was concluded that both restraint of accumulation and even reduction of the algal thallus in biomass and size cannot be taken as an indication of algal growth cessation.Original Russian Text Copyright ¢ 2005 by Biologiya Morya, Skriptsova, Leletkin.