Development Of EST-based Microsatellite Markers Of Green Algae Of Ulva (Ulvaceae, Chlorophyta) And Complete Sequencing Of The Mitochondrial DNA Of Gracilariopsis Lemaneiformis (Gracilariaceae, Rhodophyta)

Ulva species can grow rapidly in nutrient-rich habitats causing green tides andmarine fouling. A more complete understanding of the reasons behind these outbreaksis urgently required. Accordingly, this study attempts to use microsatellite (simplesequence repeat, SSR) markers based expressed sequence tag (EST) to analyze thegenetic relationships between several Ulva populations in the south Yellow Sea ofChina. Two-hundred and thirty eight SSRs were identified from8,179unique ESTs(6,203were newly sequenced and1,976downloaded from NCBI database) and37pairs of primers were successfully designed according to the ESTs;11pairs wereselected to detect the genetic diversity and relationships of69attached Ulva proliferasamples and13free-floating samples collected from coastal and off-coast areas ofsouth Yellow Sea. The result of cross-species transferability showed that6of11EST-SSR amplifiable primers from U. prolifera could produce amplicons in otherfive Ulva species and the average allele number was4.67. The percentage ofpolymorphic fragments was92.96%. The genetic distance coefficients betweenpopulations ranged from0.0167to0.2177with an average of0.1136. To compare thelevel of genetic diversity among different sampling sites, Shannon’s informationindex (I) and Nei’s gene diversity (H) were calculated. The overall level of geneticdiversity was moderate at I=0.2742and H=0.1610. Results of all U. prolifera analysesindicated that82individuals were clearly divided; most samples collected from thesame place clustered together as a group in the dendrogram produced by unweightedpair-group mean analysis (UPGMA), the results showed some consistency with theirgeographical origins. All thirteen floating samples (except HT-001-2) were clusteredas a single group separated from the attached samples. According to the genetic diversity of U. prolifera samples and considering the relevant collecting location andsampling time, we guess the free-floating U. prolifera experienced a process ofcoalescence and redispersion, and we conclude the dispersal and migratory routes ofthe free-floating U. prolifera on the sea based on the analyses of the genetic similaritybetween different samples.Gracilariopsis is one of the most important economic marine red seaweed and iswidely used in food industry, agar extraction etc. However, few data are available onthe mtDNAs of Rhodophyta algae. Therefore, more genetic background informationabout it will be assistant for further study about molecular phylogenetic analysis ofred algae and germplasm improvement for the economic species. In this research, thecomplete mitochondrial DNA of Gracilariopsis lemaneiformis was sequenced(25883bp) and mapped to a circular model based on our ability to amplify thegenomes in entirely overlapping fragments. The A+T composition was72.5%,consistent with previously reports of red algal mitochondrial genomes. Forty six genesincluding24protein-coding genes,2rRNA genes and20tRNA genes were identified.No intron was inserted in the any of these genes. Compared to the six known red algalmitochondrial genomes, the protein-coding gene content and order in all sevengenomes are virtually identical. Besides, there is considerable sequence syntenyacross the five mitochondreal genomes which fall in Florideophyceae including Gr.lemaneiformis in this study and previously sequenced: Gracilariopsis andersonii,Gracilariophila oryzoides, Chondrus crispus and Plocamiocolax pulvinata.In the mtDNA of Gr. lemaneiformis, one unknown ORF (orf60) was presentbetween nad6and sdhB gene. This potential functional ORF was unique in Gr.lemaneiformis and showed weak sequence similarity with ORFs in other obtainedalgal mt genomes. In addition, firstly found in Gracilariaceae, at the demarcationpoint of the two opposite transcriptional units, a long and stable stem-loop (63nt) wasidentified in intergenic regions of Gr. lemaneiformis. Nearly diametrally opposite onthe circular mt genome, another transcriptional demarcation point, a second hairpinstructure (21nt) was found. Which were believed to be involved with transcriptionand replication. The stem-loop structure is likely to cause the termination of transcription, which also displays a high A+T content, could be involved in DNAreplication. Phylogenetic analyses of the red algal species were conducted with a setof19protein-coding genes and two rRNA genes, which are commonly present in allseven mt genomes publicly available in the GenBank database. Phylogenetic treesusing maximum likelihood (ML) and maximum parsimony (MP) methods wereperformed using PAUP version4.0. Both MP and ML trees had the same topologies,in which Porphyra purpurea and Cyanidioschyzon merolae formed single clade asthey belong to the Bangiophyceae and Cyanidiophyceae respectively; the remainingfive species falling into the same class (Florideophyceae) were clustered together.Phylogenetic analyses based on mitochondrial protein-coding genes and rRNA genesdemonstrated Gracilariopsis andersonii had closer phylogenetic relationship with itsparasite Gracilariophila oryzoides than Gracilariopsis lemaneiformis which was fromthe same genus of Gracilariopsis.