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Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes.

ABSTRACT: Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT-, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale-V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.

SUBMITTER: Hamaji T

PROVIDER: S-EPMC4856071 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes.

Hamaji Takashi T Mogi Yuko Y Ferris Patrick J PJ Mori Toshiyuki T Miyagishima Shinya S Kabeya Yukihiro Y Nishimura Yoshiki Y Toyoda Atsushi A Noguchi Hideki H Fujiyama Asao A Olson Bradley J S C BJ Marriage Tara N TN Nishii Ichiro I Umen James G JG Nozaki Hisayoshi H

G3 (Bethesda, Md.) 20160503 5

Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to ...[more]

PMID: 26921294

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Project description:BackgroundColonial and multicellular volvocine green algae have been extensively studied recently in various fields of the biological sciences. However, only one species (Pandorina morum) has been cryopreserved in public culture collections.ResultsHere, we investigated conditions for cryopreservation of the multicellular volvocine alga Gonium pectorale using vegetative colonies or cells and zygotes. Rates of vegetative cell survival in a G. pectorale strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3% and 6%) of the cryoprotectant N,N-dimethylformamide (DMF) and two types of tubes (0.2-mL polymerase chain reaction tubes and 2-mL cryotubes) used for cryopreservation. Among the four conditions investigated, the highest rate of survival [2.7 ± 3.6% (0.54-10%) by the most probable number (MPN) method] was obtained when 2.0-mL cryotubes containing 1.0 mL of culture samples with 6% DMF were subjected to cryogenic treatment. Using these optimized cryopreservation conditions, survival rates after freezing in liquid nitrogen were examined for twelve other strains of G. pectorale and twelve strains of five other Gonium species. We obtained ≥ 0.1% MPN survival in nine of the twelve G. pectorale strains tested. However, < 0.1% MPN survival was detected in eleven of twelve strains of five other Gonium species. In total, ten cryopreserved strains of G. pectorale were newly established in the Microbial Culture Collection at the National Institute for Environmental Studies. Although the cryopreservation of zygotes of volvocine algae has not been previously reported, high rates (approximately 60%) of G. pectorale zygote germination were observed after thawing zygotes that had been cryopreserved with 5% or 10% methanol as the cryoprotectant during two-step cooling and freezing in liquid nitrogen.ConclusionsThe present study demonstrated that cryopreservation of G. pectorale is possible with 6% DMF as a cryoprotectant and 1.0-mL culture samples in 2.0-mL cryotubes subjected to two-step cooling in a programmable freezer.

Project description:BackgroundGreen algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of approximately 16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri.ResultsStable nuclear transformation of G. pectorale was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (aphVIII) of Streptomyces rimosus, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from Chlamydomonas reinhardtii or from Volvox carteri. After particle gun bombardment of wild type Gonium cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of approximately 30-50% when cells were co-bombarded with aphVIII-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod Gaussia princeps, which was previously engineered to match the codon usage in C. reinhardtii, Gonium transformants show luciferase activity through light emission in bioluminescence assays.ConclusionFlanking sequences that include promoters from C. reinhardtii and from V. carteri work in G. pectorale and allow the functional expression of heterologous genes, such as the selectable marker gene aphVIII of S. rimosus or the co-transformed, codon-optimized G. princeps luciferase gene, which turned out to be a suitable reporter gene in Gonium. The availability of a method for transformation of Gonium makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular Chlamydomonas, the 16-celled Gonium, and the multicellular Volvox.

Project description:BackgroundAstrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections.ResultsTo establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36-33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest "MPN survival" [5.5 ± 5.9% (0.12-12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES.ConclusionsThe optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed.

Dataset Information

Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes.

Publications

Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes.

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