DNA NUCLEOSIDE COMPOSITION AND METHYLATION IN SEVERAL SPECIES OF MICROALGAE1

Total DNA was isolated from 10 species of microalgae, including representatives of the Chlorophyceae (Chlorella ellipsoidea, Chlamydomonas reinhardtii, and Monoraphidium minutum), Bacillariophyceae (Cyclotella cryptica, Navicula saprophila, Nitzschia pusilla, and Phaeodactylum tricornutum), Charophyceae (Stichococcus sp.), Dinophyceae (Crypthecodinium cohnii), and Prasinophyceae (Tetraselmis suecica). Control samples of Escherichia coli and calf thymus DNA were also analyzed. The nucleoside base composition of each DNA sample was determined by reversed-phase high performance liquid chromatography. All samples contained 5-methyldeoxycytidine, although at widely varying levels. In M. minutum, about one-third of the cytidine residues were methylated. Restriction analysis supported this high degree of methylation in M. minutum and suggested that methylation is biased toward 5′-CG dinucleotides. The guanosine + cytosine (GC) contents of the green algae were, with the exception of Stichococcus sp., consistently higher than those of the diatoms. Monoraphidium minutum exhibited an extremely high GC content of 71%. Such a value is rare among eukaryotic organisms and might indicate an unusual codon usage. This work is important for developing strategies for transformation and gene cloning in these algae.     

Growth Rate of the Microalga Tetraselmis suecica Changes the Biochemical Composition of Artemia Species

The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved. Received October 15, 2000; accepted December 29, 2000.     

A unique recent origin of the allotetraploid species Arabidopsis suecica: Evidence from nuclear DNA markers

A coalescent-based method was used to investigate the origins of the allotetraploid Arabidopsis suecica, using 52 nuclear microsatellite loci typed in eight individuals of A. suecica and 14 individuals of its maternal parent Arabidopsis thaliana, and four short fragments of genomic DNA sequenced in a sample of four individuals of A. suecica and in both its parental species A. thaliana and Arabidopsis arenosa. All loci were variable in A. thaliana but only 24 of the 52 microsatellite loci and none of the four sequence fragments were variable in A. suecica. We explore a number of possible evolutionary scenarios for A. suecica and conclude that it is likely that A. suecica has a recent, unique origin between 12,000 and 300,000 years ago. The time estimates depend strongly on what is assumed about population growth and rates of mutation. When combined with what is known about the history of glaciations, our results suggest that A. suecica originated south of its present distribution in Sweden and Finland and then migrated north, perhaps in the wake of the retreating ice.     

Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations

Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.     

Chloroplast DNA indicates a single origin of the allotetraploid Arabidopsis suecica

DNA sequencing was performed on up to 12 chloroplast DNA regions [giving a total of 4288 base pairs (bp) in length] from the allopolyploid Arabidopsis suecica (48 accessions) and its two parental species, A. thaliana (25 accessions) and A. arenosa (seven accessions). Arabidopsis suecica was identical to A. thaliana at all 93 sites where A. thaliana and A. arenosa differed, thus showing that A. thaliana is the maternal parent of A. suecica. Under the assumption that A. thaliana and A. arenosa separated 5 million years ago, we estimated a substitution rate of 2.9 x 10(-9) per site per year in noncoding single copy sequence. Within A. thaliana we found 12 substitution (single bp) and eight insertion/deletion (indel) polymorphisms, separating the 25 accessions into 15 haplotypes. Eight of the A. thaliana accessions from central Sweden formed one cluster, which was separated from a cluster consisting of central European and extreme southern Swedish accessions. This latter cluster also included the A. suecica accessions, which were all identical except for one 5 bp indel. We interpret this low level of variation as a strong indication that A. suecica effectively has a single origin, which we dated at 20 000 years ago or more.     

Analysis of microbiota in cultures of the green microalga Tetraselmis suecica

ABSTRACT

Bacteria associated with microalgae strongly affect algal biomass and derived product yield and quality. Nevertheless, only a few studies have addressed the detailed phylogenetic characterization of bacterial communities associated with microalgae. In this study, the phycospheric bacterial communities associated with different Tetraselmis suecica F&M-M33 cultures, a green marine microalga with several industrial applications, were analysed using a metagenomic approach. The T. suecica F&M-M33 cultures used originated from the same ancestral microalgal non-axenic culture but were physically and geographically separated for years and maintained under different growing conditions. Despite the different history of the cultures, a ‘core’ bacterial community was identified, accounting for 70% of the total bacterial community and formed by at least 13 families. Among the ‘core’ operational taxonomic units (OTUs), 24 different genera were identified. Nevertheless, there was a high variability in the relative proportions of the taxa forming the ‘core’ community, indicating that the growing conditions and/or external contamination influence the relative abundance of these microorganisms. Our study allowed the identification of persistent taxa that may be used to deepen the knowledge of the complex relationship between T. suecica and its associated bacteria.     

Free amino acid analysis of five microalgae

The HPLC separation of fluorescent o-phtaldialdehyde (OPA) derivatives has been applied to the assay of free amino acids from five microalgae commonly used in aquaculture: Tetraselmis suecica, Skeletonema costatum, Chaetoceros calcitrans, Thalassiosira sp. and Isochrysis galbana, as part an assessment of their potential use in cosmetic products. Thirteen free amino acids were analyzed using High Performance Liquid Chromatography. There were considerable differences between species. However, four amino acids were responsible for more than 60% total concentration in all species: ASP, GLU, ARG and TYR; the next most important (accounting for less than 30%) were: ALA, VAL, PHE and LYS. This revised version was published online in June 2006 with corrections to the Cover Date.     

SYSTEMATIC SIGNIFICANCE OF THE ABSENCE OF PIT-PLUG CAP MEMBRANES IN THE BATRACHOSPERMALES (RHODOPHYTA)1

Four species of the Batrachospermales were examined by transmission electron microscopy to determine whether or not cap membranes, a typical structural component of pit plugs in several orders of red algae, were present. Routine specimen preservation methods used in past studies led to contradictory reports, so Batrachospermum keratophytum Bory, B. sirodotii Skuja ex Reis, Sirodotia suecica Kylin, and S. tenuissima (Collins) Skuja ex Flint were prepared by secondary fixation in potassium permanganate or a combination of potassium ferrocyanide-osmium tetroxide to enhance membrane contrast. These fixation procedures produced clear, well-contrasted images in which cap membranes were absent. The absence of cap membranes in S. suecica and S. tenuissima and the presence of cap membranes in two members of the Nemaliales was confirmed by freeze-substitution methods. Absence of cap membranes in representatives of the Batrachospermales further distinguishes the Batrachospermales from the Nemaliales, the order in which they long resided, and demonstrates the value of this character in elucidating ordinal alliances among the Rhodophyta.     

基于短序列测序数据的四倍体拟南芥转录组研究

为了促进对四倍体拟南芥(A.suecica)的研究,阐明多倍体植物在染色体加倍过程中遗传物质的变化,从而在分子层面上解释多倍体植物的环境适应和进化机制,描述了一套基于第二代测序技术的转录组短序列组装和生物信息学分析方法.通过对23 000 000条来至于Illumina测序平台的序列数据进行SOAPdenovo组装,以...     

Phenotypic responses to light,water, and nutrient conditions in the allopolyploid Arabidopsis suecica and its parent species A. thaliana and A. arenosa: Does the allopolyploid outrange its parents?

Polyploid species possess more than two sets of chromosomes and may show high gene redundancy, hybrid vigor, and masking of deleterious alleles compared to their parent species. Following this, it is hypothesized that this makes them better at adapting to novel environments than their parent species, possibly due to phenotypic plasticity. The allopolyploid Arabidopsis suecica and its parent species A. arenosa and A. thaliana were chosen as a model system to investigate relationships between phenotypic plasticity, fitness, and genetic variation. Particularly, we test if A. suecica is more plastic, show higher genetic diversity, and/or have higher fitness than its parent species. Wild Norwegian populations of each species were analyzed for phenotypic responses to differences in availability of nutrient, water, and light, while genetic diversity was assessed through analysis of AFLP markers. Arabidopsis arenosa showed a higher level of phenotypic plasticity and higher levels of genetic diversity than the two other species, probably related to its outbreeding reproduction strategy. Furthermore, a general positive relationship between genetic diversity and phenotypic plasticity was found. Low genetic diversity was found in the inbreeding A. thaliana. Geographic spacing of populations might explain the clear genetic structure in A. arenosa, while the lack of structure in A. suecica could be due to coherent populations. Fitness measured as allocation of resources to reproduction, pointed toward A. arenosa having lower fitness under poor environmental conditions. Arabidopsis suecica, on the other hand, showed tendencies toward keeping up fitness under different environmental conditions.