Analysis of expressed sequence tags from the Ulva prolifera (Chlorophyta)

In 2008, a green tide broke out before the sailing competition of the 29th Olympic Games in Qingdao. The causative species was determined to be Enteromorpha prolifera (Ulva prolifera O. F. Müller), a familiar green macroalga along the coastline of China. Rapid accumulation of a large biomass of floating U. prolifera prompted research on different aspects of this species. In this study, we constructed a nonnormalized cDNA library from the thalli of U. prolifera and acquired 10 072 high-quality expressed sequence tags (ESTs). These ESTs were assembled into 3 519 nonredundant gene groups, including 1 446 clusters and 2 073 singletons. After annotation with the nr database, a large number of genes were found to be related with chloroplast and ribosomal protein, GO functional classification showed 1 418 ESTs participated in photosynthesis and 1 359 ESTs were responsible for the generation of precursor metabolites and energy. In addition, rather comprehensive carbon fixation pathways were found in U. prolifera using KEGG. Some stress-related and signal transduction-related genes were also found in this study. All the evidences displayed that U. prolifera had substance and energy foundation for the intense photosynthesis and the rapid proliferation. Phylogenetic analysis of cytochrome c oxidase subunit I revealed that this green-tide causative species is most closely affiliated to Pseudendoclonium akinetum (Ulvophyceae).     

High efficiency of protoplast preparation for artificially cultured Ulva prolifera (Ulvophyceae,Chlorophyta)

Journal of Oceanology and Limnology - Protoplast isolation was relevant for gene manipulation in Ulva, and universal protocols have been proposed based on evaluation for various wildly collected...     

Isolation,Expression and Characterization of rbcL Gene from Ulva prolifera J.Agardh (Ulvophyceae,Chlorophyta)

Ulva prolifera is a typical green alga in subtidal areas and can grow tremendously fast. A highly efficient Rubisco enzyme which is encoded by Up Rbc L gene may contribute to the rapid growth. In this study, the full-length Up Rbc L open reading frame(ORF) was identified, which encoded a protein of 474 amino acids. Phylogenetic analysis of Up Rbc L sequences revealed that Chlorophyta had a closer genetic relationship with higher plants than with Rhodophyta and Phaeophyta. The two distinct residues(aa11 and aa91) were presumed to be unique for Rubisco catalytic activity. The predicted three-dimensional structure showed that one α/β-barrel existed in the C-terminal region, and the sites for Mg~(2+)coordination and CO_2 fixation were also located in this region. Gene expression profile indicated that Up Rbc L was expressed at a higher level under light exposure than in darkness. When the culture temperature reached 35℃, the expression level of Up Rbc L was 2.5-fold lower than at 15℃, and the carboxylase activity exhibited 13.8-fold decrease. Up Rbc L was heterologously expressed in E. coli and was purified by Ni~(2+) affinity chromatography. The physiological and biochemical characterization of recombinant Rubisco will be explored in the future.     

Combined effects of light intensity and NH^＋4-enrichment on growth,pigmentation, and photosynthetic performance of Ulva prolifera （Chlorophyta）

The aim of this study was to investigate the effects of light intensity and enhanced nitrogen supply on the growth and photosynthesis of the green-tide macroalga, Ulva prolifera. Thalli of U. prolifera were grown in natural or NH 4 +-enriched seawater under two different light intensities for 7 days, and then the growth rate, pigmentation, and photosynthetic performance of the thalli were evaluated. The results show that the relative growth rate(RGR) was markedly higher under the high light level than under the low light level. Enrichment with NH 4 + enhanced the RGR under high light intensity, but did not affect RGR under low light intensity. In low light conditions, NH 4 +-enrichment resulted in a marked decrease in the maximal photosynthetic rate( P m) and the maximum carbon fixation rate( V max), but it did not affect the half saturation constant for carbon( K 0.5) or the ratio of V max to K 0.5, which reflects the carbon acquisition efficiency. In high light conditions, P m, K 0.5, and the dark respiration rate( R d) increased under NH 4 + enrichment, but V max and the V max / K 0.5 ratio decreased. Regardless of the light intensity, NH 4 +-enrichment did not affect the apparent photosynthetic efficiency( α), which refl ects the ability of the alga to use light energy at low light levels. Under both low and high light intensities, the chlorophyll a(Chl a), chlorophyll b(Chl b), and carotenoids(Car) contents in thalli were higher in NH 4 +-enriched than in natural seawater, except that there was a decrease in the Chl b content of thalli in NH 4 +-enriched seawater under low light intensity. Therefore, NH 4 + enrichment improved the growth and photosynthetic performance of U. prolifera under high light intensity, but not under low light intensity. We discuss the possible mechanisms underlying these physiological responses.     

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai).Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences,after redundancy elimination.Seventeen polymorphic EST-SSRs were developed.The number of alleles per locus varied from 2-17,with an average of 6.8 alleles per locus.The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922,respective...     

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai). Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences, after redundancy elimination. Seventeen polymorphic EST-SSRs were developed. The number of alleles per locus varied from 2–17, with an average of 6.8 alleles per locus. The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922, respectively. Twelve of the 17 loci (70.6%) were successfully amplified in H. diversicolor. Seventeen loci segregated in three families, with three showing the presence of null alleles (17.6%). The adequate level of variability and low frequency of null alleles observed in H. discus hannai, together with the high rate of transportability across Haliotis species, make this set of EST-SSR markers an important tool for comparative mapping, marker-assisted selection, and evolutionary studies, not only in the Pacific abalone, but also in related species.     

Identification and characterization of 43 microsatellite markers derived from expressed sequence tags of the sea cucumber (Apostichopus japonicus)

The sea cucumber Apostichopus japonicus is a commercially and ecologically important species in China. A total of 3056 potential unigenes were generated after assembling 7597 A. japonicus expressed sequence tags (ESTs) downloaded from Gen-Bank. Two hundred and fifty microsatellite-containing ESTs (8.18%) and 299 simple sequence repeats (SSRs) were detected. The average density of SSRs was 1 per 7.403 kb of EST after redundancy elimination. Di-nucleotide repeat motifs appeared to be the most abundant type with a percentage of 69.90%. Of the 126 primer pairs designed, 90 amplified the expected products and 43 showed polymorphism in 30 individuals tested. The number of alleles per locus ranged from 2 to 26 with an average of 7.0 alleles, and the observed and expected heterozygosities varied from 0.067 to 1.000 and from 0.066 to 0.959, respectively. These new EST-derived microsatellite markers would provide sufficient polymorphism for population genetic studies and genome mapping of this sea cucumber species.     

Ulva and Enteromorpha (Ulvaceae, Chlorophyta) from two sides of the Yellow Sea: analysis of nuclear rDNA ITS and plastid rbcL sequence data

Ulvacean green seaweeds are common worldwide; they formed massive green tides in the Yellow Sea in recent years, which caused marine ecological problems as well as a social issue. We investigated two major genera of the Ulvaceae, Ulva and Enteromorpha, and collected the plastid rbcL and nuclear ITS sequences of specimens of the genera in two sides of the Yellow Sea and analyzed them. Phylogenetic trees of rbcL data show the occurrence of five species of Enteromorpha (E. compressa, E. flexuosa, E. intestinalis, E. linza and E. prolifera) and three species of Ulva (U. pertusa, U. rigida and U. ohnoi). However, we found U. ohnoi, which is known as a subtropical to tropical species, at two sites on Jeju Island, Korea. Four ribotypes in partial sequences of 5.8S rDNA and ITS2 from E. compressa were also found. Ribotype network analysis revealed that the common ribotype, occurring in China, Korea and Europe, is connected with ribotypes from Europe and China/Japan. Although samples of the same species were collected from both sides of the Yellow Sea, intraspecific genetic polymorphism of each species was low among samples collected worldwide.     

The Ulva prolifera genome reveals the mechanism of green tides

The genome of green microalgae has rarely been reported.Ulva prolifera is a green microalga that has received much attention.Despite research articles about U.prolifera in recent years,we know very little about its genome.Therefore,the 87.9-Mb haploid genome(containing 10 311 protein-coding genes)of U.prolifera was studied,and the genome was compared with that of U.mutabilis,which is the only published Ulva species.Results showed that the two species are closely related.A phylogenetic tree was constructed among U.prolifera and other green algae available in GenBank,revealing the evolutionary status of U.prolifera in Chlorophyta.To understand why U.prolifer a could grow rapidly,we identified some genes related to growth,such as those involved in cell division,phosphorylation,and cell proliferation.In addition,genes related to stress resistance were found,which supports the notion that U.prolifera grows vigorously in nature.These results help to characterize green tides from a new perspective and reveal some important insight into the biology of U.prolifera.     

The appearance of Ulva laetevirens (Ulvophyceae,Chlorophyta) in the northeast coast of the United States of America

Introduced species may outcompete or hybridize with native species, resulting in the loss of native biodiversity or even alteration of ecosystem processes. In this study, we reported an alien distromatic Ulva species, which was found in an embayment (Holly Pond) connected with Long Island Sound, USA. The morphological and anatomical observations in combination with molecular data were used for its identification to species. Anatomy of collected specimens showed that the cell shape in rhizoidal and basal regions was round and the marginal teeth along the basal and median region were not found. These characteristics were primarily identical to the diagnostic characteristics of Ulva laetevirens Areschoug (Chlorophyta). The plastid-encoding tufA and nucleusencoding ITS1 were used for its molecular identification. Phylogenetic analysis for the tufA gene placed the specimens from Holly Pond in a well-supported clade along with published sequences of U. laetevirens identified early without any sequence divergence. In ITS tree, the sample also formed well-supported clades with the sequences of U. laetevirens with an estimated sequence divergence among the taxa in these clades as low as 1%. These findings confirmed the morpho-anatomical conclusion. Native to Australia, this species was reported in several countries along the Mediterranean coast after the late of 1990s. This is the first time that U. laetevirens is found in the northeast coast of United States and the second record for Atlantic North America.     

Acute toxicity of live and decomposing green alga Ulva (Enteromorpha) prolifera to abalone Haliotis discus hannai

From 2007 to 2009, large-scale blooms of green algae (the so-called “green tides”) occurred every summer in the Yellow Sea, China. In June 2008, huge amounts of floating green algae accumulated along the coast of Qingdao and led to mass mortality of cultured abalone and sea cucumber. However, the mechanism for the mass mortality of cultured animals remains undetermined. This study examined the toxic effects of Ulva (Enteromorpha) prolifera, the causative species of green tides in the Yellow Sea during the last three years. The acute toxicity of fresh culture medium and decomposing algal effluent of U. prolifera to the cultured abalone Haliotis discus hannai were tested. It was found that both fresh culture medium and decomposing algal effluent had toxic effects to abalone, and decomposing algal effluent was more toxic than fresh culture medium. The acute toxicity of decomposing algal effluent could be attributed to the ammonia and sulfide presented in the effluent, as well as the hypoxia caused by the decomposition process.     

Comparative chloroplast genomes of Ulva prolifera and U. linza (Ulvophyceae) provide genetic resources for the development of interspecific markers

Journal of Oceanology and Limnology - The green seaweeds Ulva linza and U. prolifera are closely related species. They usually co-occur widely and have important ecological significance as primary...     

Existence of Microsatellites in Expressed Sequence Tags of Common Carp （Cyprinus carpio L. ） Available in GenBank dbEST Database

Common carp expressed sequence tags （ESTs） were analyzed for the existence of microsatellites, or simple sequence repeats （SSRs）. In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2-6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were independent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a vari- ety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies （98/179） of hits on zebrafish sequences.     

Purification and Characterization of a Novel Hydrolase That Can Specifically Degrade the Polysaccharide Isolated from Green Seaweed Ulva prolifera

The extracellular polysaccharide hydrolase-producing strain EP-1 was isolated from seawater and identified as Paenibacillus pabuli. Furthermore, a homogeneous extracellular polysaccharide hydrolase from Paenibacillus pabuli EP-1 was purified by combining ion-exchange chromatography and size exclusion chromatography with a purification fold of 90.69 and recovery of 16.23%. Characterization of the purified polysaccharide hydrolase revealed a molecular mass of 38 k Da and optimum activity at 45℃ and pH 6.0. The polysaccharide hydrolase maintained its stability within a wide range of pH(3.0–12.0) and thermal stability when the temperature was below 50℃. The presence of Hg²⁺, Fe²⁺, Mn²⁺, Co²⁺ and SDS notably decreased hydrolase activity, and organic solvents such as formaldehyde, acetone, DMF and acetonitrile completely inhibited hydrolase activity. The purified hydrolase had no activity on agar, carrageenan, gellan gum, sodium alginate, or starch, but effectively hydrolyzed the polysaccharide from Ulva prolifera. The Km and Vmax values of this hydrolase were 43.84 mg m L^-1 and 4.33 mg m L^-1 min^-1, respectively. The sequence analysis with quantitative time-of-flight mass spectrometry indicated that the hydrolase was an endoglucanase.     

The Overwintering Capability of Ulva prolifera Spores and Gametes in the Yellow Sea,China

Green tides caused by the bloom of Ulva prolifera have affected the Yellow Sea annually since 2007.To explore the intrinsic causes of successive blooms,field investigations and indoor simulation experiments were conducted using micro-propagules of U.prolifera.From January to February 2014,micro-propagules of various green algae,including U.prolifera,Ulva flexuosa,and Ulva linza,were prevalent in the coastal waters of Rudong,Lianyungang,and Qingdao.However,in March 2014,micro-propagules of U.prol...     

Investigation of the decline of Ulva prolifera in the Subei Shoal and Qingdao based on physiological changes

It has been a scientific consensus that Ulva prolifera green tide that break out annually between May and August in the recent decade off eastern coast of Yellow Sea. To investigate the ecological mechanism of the decline of U. prolifera green tide, we compared the physiological characteristics of U. prolifera samples collected in three stages of the green tide in Subei Shoal north Jiangsu, the initiation point, on May 10(early, the initiation), July 10(middle, the outbreak), and in Taipingjiao, Qingdao on August 10(late, the decline). Compared to the early samples, the middle, and late samples showed a lower chlorophyll content and photosynthetic e fficiency at a higher lipid peroxidation level. The middle and later samples had fewer chloroplasts in the cells of U. prolifera than those in the early samples. In addition, the cells of U. prolifera sampled in the late stage showed ruptured and deformed cell walls. Differences in physiological function were reflected in the growth rate. Both the middle and late U. prolifera samples presented negative growth rates. Correlation analysis results show that the rising temperature was mainly responsible for the local decline of U. prolifera green tide. The high light-intensity that could induce the photoinhibition was also an important factor for the decline. Therefore, U. prolifera population that remained in the Subei Shoal and those that northward drifted to Qingdao experienced the same decline processes: rising temperature and continuous high light conditions in the late phase of green tide reduced the photosynthetic capacity and destroyed the antioxidant system of the thallus, which lead to decrease of biomass. The only difference is that the temperature in the Subei Shoal rose earlier than that in Qingdao; thus, U. prolifera remaining in the Subei Shoal went disappeared first.     

Influence of Sea Surface Temperature on Outbreak of Ulva prolifera in the Southern Yellow Sea,China

In this study, using Moderate Resolution Imaging Spectroradiometer(MODIS) satellite images and environmental satellite CCD images, the spatio-temporal distribution of Ulva prolifera in the southern Yellow Sea during the period of 2011–2018 was extracted and combined with MODIS Level3 Photosynthetically Active Radiation(PAR) product data and Earth System Research Laboratory(ESRL) Sea Surface Temperature(SST) data to analyze their influences on the growth and outbreak of Ulva prolifera. The following conclusions were drawn: 1) comprehensive analysis of Ulva prolifera distribution during the eight-year period revealed that the coverage area of Ulva prolifera typically exhibited a gradually increasing trend. The coverage area of Ulva prolifera reached a maximum of approximately 1714.21 km~2 during the eight-year period in late June 2015. The area affected by Ulva prolifera fluctuated. In mid-July 2014, the area affected by Ulva prolifera reached a maximum of approximately 39 020.63 km~2. 2) The average growth rate of Ulva prolifera was positive in May and June but negative in July. During the outbreak of Ulva prolifera, the SST in the southern Yellow Sea tended to increase each month. The SST anomaly and average growth rate of Ulva prolifera were positively correlated in May(R~2 = 0.62), but not significantly correlated in June or July. 3) The variation trends of PAR and SST were approximately the same, and the PAR during this time period maintained a range of 40–50 mol/(m~2·d), providing sufficient illumination for the growth and outbreak of Ulva prolifera. In addition, the abundant nutrients and suitable temperature in the sea area near northern Jiangsu shoal resulted in a high growth rate of Ulva prolifera in May. In summary, the outbreak of Ulva prolifera was closely related to the environmental factors including SST, nutrients, and PAR. Sufficient nutrients and suitable temperatures resulted in a fast growth rate of Ulva prolifera. However, under poor nutrient conditions, even more suitable temperatures were not sufficient to trigger an outbreak of Ulva prolifera.     

Microscopic observation of pyrenoids in Order Ulvales (Chlorophyta) collected from Qingdao coast

Pyrenoids found in green algae Order Ulvales have taxonomical significance.Their morphology,microstructure,and the location and number in the cells have been widely used as identification criteria. As these traits can be affected by environmental conditions,it is necessary to look for other different characteristics.Here pyrenoids in several algae species collected along the Qingdao coast were studied for such purpose. The morphology and distribution of pyrenoids in the cells,as well as their change in diff...