Sequencing fragments of cryptophyte plastomes from 16S rRNA to rbcL genes and phylogenetic analyses based on the protein-encoding genes located in these fragments
In recent phylogenetic analyses combining nuclear and nucleomorph RNA genes of the ribosomal operons, three different colourless lineages were found in the genus Cryptomonas. This raised questions about the evolutionary history of these interesting objects and their relatives as well as the role of plastid genomes such as whether these three lineages resulted from similar or from different evolutionary events or what are the mutual relationship or/and roles of photosynthetic genes in the absence of photosynthetic activities, etc. To answer the interesting questions, the biological information has to been collected systematically from their plastid genomes. At the first stage of the thesis, the cryptophyte plastid rbcL gene (1,5- biphosphate carboxylase/oxygenase [RuBisCO] large subunit) was chosen to amplify by BioTherm� Taq DNA Polymerase and read their DNA compositions by SequiTherm EXCEL� II DNA Sequencing Kit-LC and Li-Cor 4200L bidirectional sequencer. Eighteen newly rbcL sequences of Cryptomonas strains were obtained. Of these, five sequences were from heterotrophic (colorless) strains and the remaining was from photosynthetic (pigmented) strains. The results of rbcL phylogeny analyses showed that the colorless C. paramecium and their closely relative photosynthetic Cryptomonas had increased their evolutionary rates significantly. These were congruent with those of nuclear rDNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA) and nucleomorph SSU rDNA that had been examined in previously. They were combined with other result done by Dr. Kerstin Hoef-Emden such as analyzing the shift from NNC to NNU in two-fold degenerate NNY codon in rbcL gene in Cryptomonas to discuss some hypotheses of the loss of photosynthetic activities in the colorless C. paramaecium strains. Detail results and discussion were published in BMC Evolutionary Biology 2005; 5:56. In the second part of the thesis, the goals were to amplify the cryptophyte plastome 16S rRNA-rbcL fragments by MasterAmpTM Extra-long PCR kit and read their DNA sequences by BigDye Terminator v1.1 Cycle sequencing kit and automated ABI3730 sequencer, then exploited the sequencing information for further understanding the evolutionary history of cryptophyte plastomes. The task also attempted to find new evidence to explain the relationship between the changing from autotrophic to heterotrophic lifestyle in colorless Cryptomonas lineages and the elevation of evolutionary rates of photosynthetic genes that were located in the plastome 16S rRNA-rbcL fragment. Twenty-two cryptophyte strains (four of them were colorless) were participated in this part. Most of the fragments (15) were read completely as planned while several fragments (7) were not, due to lack of time. The colorless strains possessed the smallest fragments; their plastomes, thus, were predicted to be the smallest among those of Cryptomonas. Strain C. erosa CCAC 0018 and C. obovoidea CCAC 0031 seemed to have the largest plastomes as their 16S-rbcL fragments contained an additional gene � ycf26 � that was not found in other Cryptomonas strains. Advantages and disadvantages of long-range PCR and primer-walking sequencing combination were discussed. Based on the conserved domain analyses, all ycf26 from secondary plastids seems to be inactive and on the way to become pseudogene than alter its function. Another additional gene � ORF403 encoding Tic22 protein � also was examined the conserved domains and done a phylogenetic analysis. Some specific characteristics of ORF403 in rhodoplasts and cryptophyte plastome were found. Three protein-coding genes � chlI, rps4 and rbcL � were used as separated phylogenetic markers or in combined. The results confirmed that one colorless lineage (presented by CCAC 0056, CCAP 977/2a, M2452, M2180) had accelerated evolutionary rates in all gene or/and protein trees. The observations also suggested that chlI gene increased its substitution rate earlier than rps4 and rbcL genes as well as the elevated evolutionary rates could be ordered by chlI > rps4 > rbcL. Although having moderate size (609 bp), rps4 had an evolution rate neither as high as in chlI gene nor as low as in rbcL gene, producing acceptable phylogenetic trees for both nucleotide and protein levels. Therefore, rps4 gene seems to be more suitable protein-enoding plastid gene maker for phylogenies than its sisters, chlI and rbcL genes. The ratio of NNC to NNU in two-fold degenerate NNY codons was calculated for each gene and discussed. It is possible that the shift in codon usage from NNC to NNU did not correlate to the relaxation of functional constraints and/or reduction of gene expression levels. Furthermore, the usage of NNU codons over the NNC in two-fold degenerate NNY codon seemed to be controlled by neutral mutation pressure rather than by selection followed by the gradually acceleration of evolutionary rate. A hypothetical scenario for the relations among the loss of photosynthesis, increasing of substitution rate of interring genes and time of diverging in colorless lineages was discussed
