Molecular Detection And Phylogenetic Analysis Of The Bloom-forming Alga In China

From 2007 to 2010 year, massive green tides have occurred every year in the Yellow Sea. Especially in 2008, Qingdao experienced the largest Ulva bloom in the world, which has caused ecological disaster and attracted wide attention. The taxonomy identification and the source of bloom algae, as well as the mechanism, prevention and comprehensive utilizationhas of green tide have become the key and difficult issues. In this paper, the taxonomy identification and the algal source have been focused, and the DNA sequence of Ulva spp. in GenBank and the Ulva samples of the south Yellow Sea from 2008 to 2010 were analyzed. This study laid the significant foundations for the species identification and source tracing of the Ulva bloom in the Yellow Sea.The DNA sequences of Ulva spp. in GenBank were searched and collected thoroughly. Ulva intestinalis represented the most abundant and then Ulva prolifera, Ulva 1inza, Ulva compressa were followed. There were no sequences of Ulva tubulosa. The rDNA internal transcribed spacer (ITS) region and the plastid encoded large subunit ribulose 1,5-bisphosphate carboxylase gene (rbcL) were most frequently submitted. Based on the ITS and rbcL sequences, the phylogenetic analysis on usual green seaweeds such as Ulva prolifera, Ulva 1inza, Ulva compressa has showed that genetic distances of Ulva compressa and Ulva flexuosa were 0-0.006 and 0-0.053 respectively, which were very close, however, the genetic distances of Ulva prolifera and Ulva 1inza were larger, 0-0.171 and 0-0.135 respectively. It is necessary to establish the unified molecular classification standard.Based on the pre-investigation of Ulva spp. in the coastal base line of our country, Ulva samples were collected from the offshore in Fujian Province, Guangdong Province, Guangxi Province and Hainan Province. The genomic DNA of samples were extracted. The ITS and rbcL genes were amplified and sequenced. The phylogenetic analysis of Ulva samples showed that the 21 samples from the south sea in China have far relationship with the dominant free-floating Ulva in Yellow Sea in 2008. However, the molecular phylogeny of Ulva species from Qingdao coast, Lianyungang coast and Rudong coast in 2008 to 2010 revealed that they presented highly concordance. The similarity of the dominant free-floating Ulva was 100%.In 2009, the survey and monitoring of green seaweeds were conducted in the Rudong sea area of Jiangsu Province. The nuclear encoded internal transcribed spacer (ITS) and associated 5.8S rDNA regions as well as the chloroplast rbcL genes were sequenced and analysed for the green algae collected from Rudong coasts, including 11 attached samples and 15 free-floating samples. The investigation showed that a large amount of sessile Ulva existed in Rudong coasts, in addition, the floating green algae clusters in Rudong appeared earliest and drifted northward gradually. Furthermore, the phylogenetic analysis of ITS sequence revealed that the samples fell into five distinct clades: the Ulva compressa clade (6 samples), the Ulva linza-procera-prolifera (LPP) complex clade (12 samples), the Ulva flexuosa clade (3 samples), the Blidingia sp. clade (3 samples)and the Urospora spp. clade (2 samples). However, 26 samples just formed four clades in the phylogenetic tree of rbcL sequences, which indicated the higher conservation of rbcL. DNA sequencing analysis implied there were Ulva compressa, Ulva flexuosa and LPP complex in both the free-floating Ulva and the attached species from Rudong coasts and the final free-floating strain was the same as the dominant Ulva species (U. prolifera) of the 2008 bloom in the Yellow Sea.A series of field collections of both Ulva samples and water samples in various sites along Jiangsu coasts were conducted in April to July of 2010. Density of microscopic Ulva germlings in the waters sampled from different sites ranged from 3 to 233 individuals L-1, indicating the wide spreading and long term existence of the algae in the investigated region. ITS nrDNA and rbcL gene comparisons revealed that the algae collected from the Yellow Sea in 2010 was identical to the dominant bloom forming alga in 2008 and 2009. The principal and original source of Ulva bloom in the Yellow Sea in 2010 was probably from the Rudong coasts, then reached to Dafeng, Sheyang and Lianyungang, finally drifted to the Qingdao sea area.