Studies On The Molecular Markers Of Taxonomic Status And Production Traits In Arthrospira

1. Studies on phylogenetic relationship of the genus Arthrospira and SpirulinaThe 16S rRNA sequences of ten Arthrospira strains were sequenced and compared with the sequences of related cyanobacteria strains which were downloaded from the Nucleotide sequences database (GenBank). Based on 16S rRNA sequences, the molecular phylogenetic tree of order Oscillatoriales was constructed. And the results showed that: (1) The Spirulina strains formed a separate cluster distinct from the Arthrospira cluster, and it was consistent with the point of view that separating these two genus in Bergey's Manual of Systematic Barteriology. (2) The tight cluster of Arthrospira had a close relationship with the cluster of Oscillatoria and Planktothrix. And the sequence similarity values of 16S rRNA gene between different strains of Arthrospira were more than 99%, indicating that these Arthrospira strains belonged to the same genus. (3) The seven Spirulina strains were distributed dispersedly in the phylogenetic tree, and divided into two genetically clustered lineages. One sub-cluster was formed by the strains FACHB351 and AB2002/06 which the sequence similarity value of 16S rRNA gene was 98.1 %, and closely related to strains CCCBaja-95C1.3 and MPI S3 of Halospirulina. The other five strains formed another sub-cluster, and the 16S rRNA sequence similarity values were more than 96%. The similarity values between these two sub-clusters strains were 92% which were at the same level as ones of most of other different genus in Oscillatoriales. (4) Although belonged to the Arthrospira cluster, Sp-12 located most peripherally alone in the cluster which was very distinct from others. The main reasons were that none but its sequence from 321 to 1512 bp of 16S rRNA gene was comparatively homologous with that of other Arthrospira , whereas the similarity values of their sequences from 1 to 320 bp were only 66% and reached 90% and 89% with Flectobacillus rubber and Bellia baltica BA1 (Flexibacteraceae), respectively. It is probable that the specific 16S rRNA sequence of Sp-12 was a result of horizontal gene transfer between strains Arthrospira and Flexibacteraceae. Thus the 16S rRNA gene sequence was not a sufficient criterion to guarantee species identity. 2. Studies on phylogenetic relationship of Arthrospira platensisThe genetic diversity of sixteen Arthrospira platensis strains is investigated by supertree and supermatrix approaches based on the sequences of 16S rRNA gene, nuclear ribosomal DNA internal transcribed spacer, 23S rRNA gene and the cpcHID operon. (1) With the supertree approach, the optimal phylogentic tree was constructed step by step. Firstly, the sixteen strains of Arthrospira platensis were divided into three sub-clusters by the sequences of 16S rRNA gene supported by a bootstrap value of 100%. Secondly, based on the sequences of nuclear ribosomal DNA internal transcribed spacer, strain Sp-14 was separated from the other strains of the cluster II. Thirdly, the phylogenetic trees of the cluster II and III were constructed based on the sequences of the cpcHID operon, respectively. (2) With the supermatrix approach, a concatenated 4084 bp sequence was composed by the sequences of 16S rRNA gene, nuclear ribosomal DNA internal transcribed spacer, 23S rRNA gene and the cpcHID operon in turn. These sequences were also used to calculate the sequence similarity values and construct a phylogenetic tree of sixteen strains of Arthrospira platensis. (3) The two phylogenetic trees constructed with those two approaches discussed above were basically similar. The sixteen strains of Arthrospira platensis were divided into three genetically clustered lineages definitely: Sp-11 formed the cluster I, Sp-1, Sp-2, Sp-6, Sp-9, Sp-10, Sp-14 and Sp-18 formed the cluster II, and Sp-3, Sp-4, Sp-5, Sp-7, Sp-8, Sp-15, Sp-16 and Sp-17 formed the cluster III. The main difference between thses two phylogentic trees was the cluster II. This cluster was subdivided into two lineages that one sub-cluster contained strains Sp-1, Sp-2, Sp-9 and Sp-10, and the other contained strains Sp-6, Sp-14 and Sp-18 with the supermatrix approach while there was no lineage in the cluster II with the supertree approach.3. Molecular markers to evaluate the production traits in Arthrospira platensisFour molecular marker techniques were established which could evaluate production traits of Arthrospira platensis conveniently, fleetly and efficiently for the first time, described as below: (1) In the 16S-23S rRNA ITS, the sequences from 1 to 50 bp (ITS-Marker1) and 281 to 400 bp (ITS-Marker2) were distinct markedly, consequently they could be used to evaluate the production traits. (2) The points of 7^th, 20^th, 30^th, 56^th and 72^nd in amino acid sequences of CpcI in 1 to 80 bp were diverse, thus they could become a molecular marker. (3) Another molecular marker was special bands of RAPD electrophoretic map amplified by S35 and S99 primers. Namely, it was considered as a superior production trait group when the bands were at 680 bp and 900 bp, amplified by S35 and S99 respectively, and an inferior one at 900 bp and 650 bp. (4) The production traits could also be evaluated by the exDNA in total DNA, two bands was represented superior production traits, and one band inferior.