Duan Delin

Assessing the germplasm ofLaminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

Eighteen gametophytes includingL. japonica, L. ochotensis andL. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains ofLaminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species ofLaminaria.

Genetic study of Kelp “901” strain

Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901” strain, genetic study on variation was conducted to its parents and offsprings of F6, F7, F8, and F9 generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp were obtained in the average of 9.9 loci for each primer. This indicated a high polymorphic rate with RAPD detection. UPGMA (unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.3212–0.4767, and the maximum was between F7 and F8 (0.4767). Identity analysis showed that F6 generation was more close to the female parent (0.6593), and F7 generation was more close to the male parent (0.578 8). To the sporophytes study in 24 selected primers for RAPD amplification, 191 loci ranging from 230–2800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations were male parent (0.2239), female parent (0.1072), F6 (0.2164), F7(0.2286), F8(0.2296) and F9(0.3172). The nearest genetic distance was 0.083 5(F8, F9). Total heterozygosity (HT) of F6, F7, F8 and F9 generations was 0.3186, the average heterozygosity (HS) for F6, F7, F8 and F9 generations was 0.2480, and deduced coefficient of population differentiation (Gst) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed to be verified in detail as they are significant for molecular marker assistance in breeding and selectingLaminaria.

Use of rapd for detecting and identifyingPorphyra (Bangiales, Rhodophyta)

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variations and phylogenetic relationships in 4 species ofPorphyra. The samples were collected from the coast of Canada, Vietnam, Zhoushan, Fujian and Qingdao in China. Amplifications with 20 primers were carried out under predetermined optimal reaction conditions (samples were first heated at 94°C for 5 min. and followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C, then held at 72°C for 10 min). The amplified products were scored as present (1) or absent (0) for each DNA sample and an index of genetic similarity (F) was calculated by using Nei & Li’s matching coefficient method (1979). The value of (1−F) was used to quantify the genetic distances between species and construct a phylogenetic tree. The relationship indicated by the UPGMA and NJ cluster analysis on the values of the genetic distance is in good overall agreement with classical taxonomy. The obvious differences between natural and cultivated population ofP. haitanensi suggest that variation or hybridization with other species occurred during the culture.

Effects of Blue Light on Gametophyte Development of Laminaria japonica (Laminariales, Phaeophyta)

Laminaria gametophyte was greatly influenced by light in its growth and development. Using light-emitting diodes (LED) as blue and red light sources, we analyzed the light effect on gametophytes development ofLaminaria japonica Aresch. The gametophytes were obtained from zoospores collected in April, May, July, 2003 and September, 2004. We found that the growth of gametophytes was stimulated by increasing intensity of blue light (BL) and red light (RL) illumination, of which BL was obviously stronger than that of RL. The fertilization of gametophytes depended largely on BL, and only sufficient BL illumination could take the reproductive effect. In addition, we noticed that there was a significant difference in light responses for gametophytes developed from zoospore collected in different times. For zoospores released in April, under BL1 (73.90 μmol photons/m·s), the unicellular female gametophytes and multi-cellular male gametophytes produced eggs and sperms respectively, and further developed towards sporophytes. However, for gametophytes developed in May, July or September, they became multi-cellular and never formed oogonia or antheridia. It is believed that theLaminaria sporangium maturation stage could affect the gametophytes reaction to BL under laboratory culture conditions. Therefore, cryptochrome- or phototropin-like BL photoreceptors is probably involved in BL-induced development ofLaminaria gametophytes.

Early development ofChondrus ocellatus holm (Gigartinaceae, Rhodophyta)

Chondrus is an economically important red algae widely used for food and biochemical purpose. It early development is crucial for the culture and seedling propagation. We chose tetraspores and carpospores ofChondrus ocellatus as examples for experiment of the culture, induction and release in laboratory condition, aiming to understand early development ofC. ocellatus and to apply in seedling production. MatureC. ocellatus were collected in Qingdao, China, from Nov. to Dec. 2004. After the gametophyte and tetrasporophyte were brushed and washed with sterilized seawater, the algal materials were treated in 1.5% KI for 20 min, then were dried for 1h to stimulate the releasing of spores. After the spores released overnight, it were cultured in PES medium, incubated at 18 °C, 10±2 μmol/(m2·s1) in 12∶12h (light: dark). The observation and recording under microscope were carried out. Continuous observation of the early development showed that both tetraspore and carpospore are similar to each other. In general, three stages of the early development were shown being division, discoid crust and seedling stages. To the division stage, the most obvious feature was the increasing of cell number; during the discoid crust stage, the discoid crust had a three-dimensional axis, and it began to differentiate into two types of cells: the basal cells and the apical cells; and to the seedling stage, several protuberances-like appeared on the discoid crusts and formed juvenile seedlings. Carpospores and tetraspores exhibited a similar development process that included division stage, discoid crust stage and seedling stage.

Formation of primary pit connection during conchocelis phase ofPorphyra yezoensis (Bangiophyceae, Rhodophyta)

The formation of pit connection during conchocelis phase ofPorphyra yezoensis Ueda was observed and examined with transmission electron microscope (TEM) and epifluorence microscope. It is indicated that the pit connection was formed in late stage of conchocelis phase and the early stages of conchosporangial cell development, and disappeared in bispore stage. The pit connection contained a thin membrane layer at outer pit plug. Stained with 4′, 6′-diamidino-2-phenylidole dihydrochloride hydrate (DAPI), transferring of DNA or RNA between adjacent cells were observed in late stage of conchocelis development, it was deduced that pit connection might serve as a channel for signal transduction and genetic substance transportation in conchocelis phase.

New phenomenon in early development of sporelings inGracilaria asiatica Chang et Xia (Gracilariaceae, Rhodophyta)

Study on the early development of sporelings from carpospores ofGracilaria asiatica Chang et Xia was conducted indoors under controlled culture conditions. Besides normal development of sporelings, a new developmental phenomenon of filamentous frond was observed. It was composed of one or two rows of cells, and took place from the outmost brim of the basal disc. During the early disc stage of germinated carpospores, one or two filamentous fronds formed on about 10% basal discs. Simultaneously, young fronds began to arch slightly from the centers of single and coalescent discs; lately more filamentous fronds up to 80% appeared on the brims of basal discs. Meanwhile one or more upright fronds protuberated on the basal discs. Generally, filamentous fronds exhibited in self-existence or co-existence forms with normal young sporelings on the same basal disc, and single cell detached from filamentous fronds developed into a new filamentous frond. This new phenomenon exhibited a unique differentiation pathway during the early development ofG. asiatica, which would be potential for the application in artificial sporelings nursery.

Identification of cytokinin in a green algae extract

Isopentenyladenosine (i6Ado) was identified, and trans-zeatin (trans-Z) and trans-zeatin riboside (trans-ZR) were detected by high pressure liquid chromatography (HPLC) but not verified with chromatography—mass spectrometry (GC-MS) analysis of cytokinin from the extracts of green algae (Ulva pertusa (Kjellm),Enteromopha compressa andMonostroma sp.). This indicated that the green algae mixture contained cytokinin—like substances.

Phenological study of Sargassum thunbergii (Fucales, Phaeophyta) in Lidao Bay, Rongcheng, China

We conducted a phenological evaluation of Sargassum thunbergii, a common algal species, along the coast of Lidao Bay, Rongcheng, China. The local S. thunbergii population entered a maturation period from early June to mid-August, when seawater temperature was 14.4 to 25.1°C. Our results suggest an increase in temperature and day length during this period initiated thalli maturation. Inhabitants of the low tidal zone had higher thalli length and biomass compared with those from the mid-and high tidal zones. We observed a switch in the length frequency distribution and contribution of length classes to biomass between the small (<20 cm) and long (20–79.9 cm) length classes at the time of thallus maturity. This suggests there is a trade-offbetween sexual reproduction and vegetative regeneration.

Observation of female gamete (Carpogonia) germination ofPorphyra yezoensis andP. oligospermatangia

Several papers have reported that part or whole leafy thallus seemingly consisting of zygotospores can give rise to both blades and conchocelis in the same culture ofPorphyra. Study on samples of wild and cultivatedPorphyra yezoensis andP. oligospermatangia were conducted to clarify the origination of the young blades in the culture. It is confirmed that single cells on the blade of both species, which normally intermixed with zygotospores, germinated into young blades. TEM and SEM observation has shown that the single cells ofPorphyra yezoensis had typical features of female gamete (carpogonia) but archeospore. Therefore, the female gametes are responsible in developing leafy thalli.