Marine plankton support global biological and geochemical processes. Surveys of their biodiversity have hitherto been geographically restricted and have not accounted for the full range of plankton size. We assessed eukaryotic diversity from 334 size-fractionated photic-zone plankton communities collected across tropical and temperate oceans during the circumglobal Tara Oceans expedition. We analyzed 18S ribosomal DNA sequences across the intermediate plankton-size spectrum from the smallest unicellular eukaryotes (protists, >0.8 micrometers) to small animals of a few millimeters. Eukaryotic ribosomal diversity saturated at ~150,000 operational taxonomic units, about one-third of which could not be assigned to known eukaryotic groups. Diversity emerged at all taxonomic levels, both within the groups comprising the ~11,200 cataloged morphospecies of eukaryotic plankton and among twice as many other deep-branching lineages of unappreciated importance in plankton ecology studies. Most eukaryotic plankton biodiversity belonged to heterotrophic protistan groups, particularly those known to be parasites or symbiotic hosts.

/pdf/eukaryotic-plankton-diversity-in-the-sunlit-ocean-pxea35iar7.pdf

Eukaryotic plankton diversity in the sunlit ocean

Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. We use this approach to sequence two SSU rDNA diversity markers-the variable V4 and V9 regions-from 10 L of anoxic Norwegian fjord water. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-294X.2009.04480.x

Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water

The green lineage is reportedly 1,500 million years old, evolving shortly after the endosymbiosis event that gave rise to early photosynthetic eukaryotes. In this study, we unveil the complete genome sequence of an ancient member of this lineage, the unicellular green alga Ostreococcus tauri (Prasinophyceae). This cosmopolitan marine primary producer is the world's smallest free-living eukaryote known to date. Features likely reflecting optimization of environmentally relevant pathways, including resource acquisition, unusual photosynthesis apparatus, and genes potentially involved in C(4) photosynthesis, were observed, as was downsizing of many gene families. Overall, the 12.56-Mb nuclear genome has an extremely high gene density, in part because of extensive reduction of intergenic regions and other forms of compaction such as gene fusion. However, the genome is structurally complex. It exhibits previously unobserved levels of heterogeneity for a eukaryote. Two chromosomes differ structurally from the other eighteen. Both have a significantly biased G+C content, and, remarkably, they contain the majority of transposable elements. Many chromosome 2 genes also have unique codon usage and splicing, but phylogenetic analysis and composition do not support alien gene origin. In contrast, most chromosome 19 genes show no similarity to green lineage genes and a large number of them are specialized in cell surface processes. Taken together, the complete genome sequence, unusual features, and downsized gene families, make O. tauri an ideal model system for research on eukaryotic genome evolution, including chromosome specialization and green lineage ancestry.

/pdf/genome-analysis-of-the-smallest-free-living-eukaryote-3cbmwsox9v.pdf

Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features

Background 
Massively parallel pyrosequencing of amplicons from the V6 hypervariable regions of small-subunit (SSU) ribosomal RNA (rRNA) genes is commonly used to assess diversity and richness in bacterial and archaeal populations. Recent advances in pyrosequencing technology provide read lengths of up to 240 nucleotides. Amplicon pyrosequencing can now be applied to longer variable regions of the SSU rRNA gene including the V9 region in eukaryotes.
Methodology/Principal Findings 
We present a protocol for the amplicon pyrosequencing of V9 regions for eukaryotic environmental samples for biodiversity inventories and species richness estimation. The International Census of Marine Microbes (ICoMM) and the Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites (MIRADA-LTERs) projects are already employing this protocol for tag sequencing of eukaryotic samples in a wide diversity of both marine and freshwater environments.
Conclusions/Significance 
Massively parallel pyrosequencing of eukaryotic V9 hypervariable regions of SSU rRNA genes provides a means of estimating species richness from deeply-sampled populations and for discovering novel species from the environment.

/pdf/a-method-for-studying-protistan-diversity-using-massively-1u2hpx3461.pdf

A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes.

The green lineage (Viridiplantae) comprises the green algae and their descendants the land plants, and is one of the major groups of oxygenic photosynthetic eukaryotes. Current hypotheses posit the early divergence of two discrete clades from an ancestral green flagellate. One clade, the Chlorophyta, comprises the early diverging prasinophytes, which gave rise to the core chlorophytes. The other clade, the Streptophyta, includes the charophyte green algae from which the land plants evolved. Multi-marker and genome scale phylogenetic studies have greatly improved our understanding of broad-scale relationships of the green lineage, yet many questions persist, including the branching orders of the prasinophyte lineages, the relationships among core chlorophyte clades (Chlorodendrophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae), and the relationships among the streptophytes. Current phylogenetic hypotheses provide an evolutionary framework for molecular evolutionary studies and comparative genomics. This review summarizes our current understanding of organelle genome evolution in the green algae, genomic insights into the ecology of oceanic picoplanktonic prasinophytes, molecular mechanisms underlying the evolution of complexity in volvocine green algae, and the evolution of genetic codes and the translational apparatus in green seaweeds. Finally, we discuss molecular evolution in the streptophyte lineage, emphasizing the genetic facilitation of land plant origins.

/pdf/phylogeny-and-molecular-evolution-of-the-green-algae-4244e09mtu.pdf

Phylogeny and Molecular Evolution of the Green Algae

Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene.

Eukaryotic picoplankton communities of the Mediterranean Sea in summer assessed by molecular approaches (DGGE, TTGE, QPCR)

Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.

https://www.frontiersin.org/articles/10.3389/fpls.2022.1075838/pdf

Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

Suitable pollen source for the improvement of fruit and seed traits in Xanthoceras sorbifolium

Variability in seed and seedling traits of Xanthoceras sorbifolium Bunge was evaluated at the population level. Seed samples were collected from four provenances in China and examined for variations in morphometric traits, chemical components, and seedling growth in the nursery stage. There were significant differences in the seed length, width, dry weight, 1000-seed weight, oil concentration, Mg and Cu concentrations, root biomass, and root–stem biomass ratio. The largest seed in terms of size and weight was from Ongniud Banner, Inner Mongolia (OB), but these seeds also had the lowest seed oil concentration. At the end of the first growing season in the nursery, seedlings stopped growing one month earlier in height than in diameter. The provenance difference in height was significant at the first 2 months after sowing but disappeared later. Genotypic variance (Vg) was found to be higher than corresponding environmental (Ve) variance for seed length, seed width, seed dry weight, 1000-seed weight, diameter, and root biomass, indicating that these parameters were strongly inherited and there was ample scope for improvement. Moreover, correlations between seed and seedling traits and climatic and geographical factors were assessed. Some significant intercharacter correlations were found, such as between seed length, width and seed weight, between oil concentration and seed size, and between seedling height, diameter, and root biomass. Combining the seed- and seedling-related parameters, our results indicated that Mulei, Xinjiang (ML) may be used as an ideal material for a further improvement program.

/pdf/phenotypic-variation-in-seed-morphochemical-and-seedling-2652lk8x.pdf

Fei Zhu

Papers

Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene.

Eukaryotic picoplankton communities of the Mediterranean Sea in summer assessed by molecular approaches (DGGE, TTGE, QPCR)

Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

Suitable pollen source for the improvement of fruit and seed traits in Xanthoceras sorbifolium

Phenotypic Variation in Seed Morphochemical and Seedling Traits in Four Chinese Provenances of Xanthoceras sorbifolium