Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

The chloroplast genome of Phacus orbicularis (Euglenophyceae): an initial datum point for the phacaceae

The Euglenophyceae chloroplast was acquired when a heterotrophic euglenoid engulfed a green alga and subsequently retained the algal chloroplast, in a process known as secondary endosymbiosis. Since this event, Euglenophyceae have diverged widely and their chloroplast genomes (cpGenomes) have as well. Changes to the cpGenome include extensive gene rearrangement and the proliferation of introns, the analyses of which have proven to be useful in examining cpGenome changes throughout the Euglenophyceae. The Euglenales fall into two families, Euglenaceae and Phacaceae. Euglenaceae contains eight genera and at least one cpGenome has been published for each genus. Phacaceae, on the other hand, contains three genera, none of which have had a representative chloroplast genome sequenced. Members of this family have many small disk‐shaped chloroplasts that lack pyrenoids. We sequenced and annotated the cpGenome of Phacus orbicularis in order to fill in the large gap in our understanding of Euglenophyceae cpGenome evolution, especially in regard to intron number and gene order. We compared this cpGenome to those of species from both the Euglenaceae and Eutreptiales of the Euglenophyceae phylogenetic tree. The cpGenome showed characteristics that were more derived than that of the basal species Eutreptia viridis, with extensive gene rearrangements and nearly three times as many introns. In contrast, it contained fewer introns than all but one of the previously reported Euglenaceae cpGenomes, had a smaller estimated genome size, and shared greater synteny with two main branches of that family.     

Identification of chloroplast genome loci suitable for high‐resolution phylogeographic studies of Colocasia esculenta (L.) Schott (Araceae) and closely related taxa

Recently, we reported the chloroplast genome‐wide association of oligonucleotide repeats, indels and nucleotide substitutions in aroid chloroplast genomes. We hypothesized that the distribution of oligonucleotide repeat sequences in a single representative genome can be used to identify mutational hotspots and loci suitable for population genetic, phylogenetic and phylogeographic studies. Using information on the location of oligonucleotide repeats in the chloroplast genome of taro (Colocasia esculenta), we designed 30 primer pairs to amplify and sequence polymorphic loci. The primers have been tested in a range of intra‐specific to intergeneric comparisons, including ten taro samples (Colocasia esculenta) from diverse geographical locations, four other Colocasia species (C. affinis, C. fallax, C. formosana, C. gigantea) and three other aroid genera (represented by Remusatia vivipara, Alocasia brisbanensis and Amorphophallus konjac). Multiple sequence alignments for the intra‐specific comparison revealed nucleotide substitutions (point mutations) at all 30 loci and microsatellite polymorphisms at 14 loci. The primer pairs reported here reveal levels of genetic variation suitable for high‐resolution phylogeographic and evolutionary studies of taro and other closely related aroids. Our results confirm that information on repeat distribution can be used to identify loci suitable for such studies, and we expect that this approach can be used in other plant groups.     

Genomic differentiation and patterns of gene flow between two long‐tailed tit species (Aegithalos)

Patterns of heterogeneous genomic differentiation have been well documented between closely related species, with some highly differentiated genomic regions (“genomic differentiation islands”) spread throughout the genome. Differential levels of gene flow are proposed to account for this pattern, as genomic differentiation islands are suggested to be resistant to gene flow. Recent studies have also suggested that genomic differentiation islands could be explained by linked selection acting on genomic regions with low recombination rates. Here, we investigate genomic differentiation and gene‐flow patterns for autosomes using RAD‐seq data between two closely related species of long‐tailed tits (Aegithalos bonvaloti and A. fuliginosus) in both allopatric and contact zone populations. The results confirm recent or ongoing gene flow between these two species. However, there is little evidence that the genomic regions that were found to be highly differentiated between the contact zone populations are resistant to gene flow, suggesting that differential levels of gene flow is not the cause of the heterogeneous genomic differentiation. Linked selection may be the cause of genomic differentiation islands between the allopatric populations with no or very limited gene flow, but this could not account for the heterogeneous genomic differentiation between the contact zone populations, which show evidence of recent or ongoing gene flow.     

Phylogenetic position of the coral symbiont Ostreobium (Ulvophyceae) inferred from chloroplast genome data

The green algal genus Ostreobium is an important symbiont of corals, playing roles in reef decalcification and providing photosynthates to the coral during bleaching events. A chloroplast genome of a cultured strain of Ostreobium was available, but low taxon sampling and Ostreobium's early‐branching nature left doubt about its phylogenetic position. Here, we generate and describe chloroplast genomes from four Ostreobium strains as well as Avrainvillea mazei and Neomeris sp., strategically sampled early‐branching lineages in the Bryopsidales and Dasycladales respectively. At 80,584 bp, the chloroplast genome of Ostreobium sp. HV05042 is the most compact yet found in the Ulvophyceae. The Avrainvillea chloroplast genome is ~94 kbp and contains introns in infA and cysT that have nearly complete sequence identity except for an open reading frame (ORF) in infA that is not present in cysT. In line with other bryopsidalean species, it also contains regions with possibly bacteria‐derived ORFs. The Neomeris data did not assemble into a canonical circular chloroplast genome but a large number of contigs containing fragments of chloroplast genes and showing evidence of long introns and intergenic regions, and the Neomeris chloroplast genome size was estimated to exceed 1.87 Mb. Chloroplast phylogenomics and 18S nrDNA data showed strong support for the Ostreobium lineage being sister to the remaining Bryopsidales. There were differences in branch support when outgroups were varied, but the overall support for the placement of Ostreobium was strong. These results permitted us to validate two suborders and introduce a third, the Ostreobineae.     

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein‐coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein‐coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Optimizing the widely used nuclear protein‐coding gene primers in beetle phylogenies and their application in the genus Sasajiscymnus Vandenberg (Coleoptera: Coccinellidae)

Advances in genomic biology and the increasing availability of genomic resources allow developing hundreds of nuclear protein‐coding (NPC) markers, which can be used in phylogenetic research. However, for low taxonomic levels, it may be more practical to select a handful of suitable molecular loci for phylogenetic inference. Unfortunately, the presence of degenerate primers of NPC markers can be a major impediment, as the amplification success rate is low and they tend to amplify nontargeted regions. In this study, we optimized five NPC fragments widely used in beetle phylogenetics (i.e., two parts of carbamoyl‐phosphate synthetase: CADXM and CADMC, Topoisomerase, Wingless and Pepck) by reducing the degenerate site of primers and the length of target genes slightly. These five NPC fragments and 6 other molecular loci were amplified to test the monophyly of the coccinellid genus Sasajiscymnus Vandenberg. The analysis of our molecular data set clearly supported the genus Sasajiscymnus may be monophyletic but confirmation with an extended sampling is required. A fossil‐calibrated chronogram was generated by BEAST, indicating an origin of the genus at the end of the Cretaceous (77.87 Myr). Furthermore, a phylogenetic informativeness profile was generated to compare the phylogenetic properties of each gene more explicitly. The results showed that COI provides the strongest phylogenetic signal among all the genes, but Pepck, Topoisomerase, CADXM and CADMC are also relatively informative. Our results provide insight into the evolution of the genus Sasajiscymnus, and also enrich the molecular data resources for further study.     

The ANGULATA7 gene encodes a DnaJ‐like zinc finger‐domain protein involved in chloroplast function and leaf development in Arabidopsis

The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid‐localized proteins that perform essential functions in leaf growth and development. A large‐scale screen previously allowed us to isolate ethyl methanesulfonate‐induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7‐1 (anu7‐1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic‐lethal mutations. ANU7 encodes a plant‐specific protein that contains a domain similar to the central cysteine‐rich domain of DnaJ proteins. The observed genetic interaction of anu7‐1 with a loss‐of‐function allele of GENOMES UNCOUPLED1 suggests that the anu7‐1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7‐1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid‐encoded genes, we found that anu7‐1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed.     

A novel 111‐nucleotide duplication in the G gene of human metapneumovirus

In 2017, novel human metapneumovirus (HMPV) A2b subgroup strains with a 111‐nucleotide duplication in the G gene was detected by the present team. These strains were related to previously identified HMPV A2b strains with a 180‐nucleotide duplication; however, they appeared to be different strains, produced by an independent duplication event. The recent evolution of HMPV suggests that careful monitoring of this virus is required.

     

Non‐invasive,whole‐plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants

Leaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high‐sensitivity, non‐invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image‐based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less‐mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1‐5 phot2‐1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual‐imaging platform also allowed us to develop a straightforward approach to correct non‐photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy‐dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the overall photosynthetic performance of higher plants.     

Plastid phylogenetics of Oceania yams (Dioscorea spp., Dioscoreaceae) reveals natural interspecific hybridization of the greater yam (D. alata)

Phylogenetic relationships of Oceanian staple yams (species of Dioscorea section Enantiophyllum) were investigated using plastid trnL‐F and rpl32‐trnL^(UAG) sequences and nine nuclear co‐dominant microsatellites. Analysis of herbarium specimens, used as taxonomic references, allowed the comparison with samples collected in the field. It appears that D. alata, D. transversa and D. hastifolia are closely related species. This study does not support a direct ancestry from D. nummularia to D. alata as previously hypothesized. The dichotomy in D. nummularia previously described by farmers in semi‐perennial and annual types was reflected by molecular markers, but the genetic structure of D. nummularia appears more complex. Dioscorea nummularia displayed two haplotypes, each corresponding to a different genetic group. One, including a D. nummularia voucher from New Guinea, is closer to D. tranversa, D. alata and D. hastifolia and encompasses only semi‐perennial types. The second group is composed of semi‐perennial and annual yams. However, some of these annual yams also displayed D. alata haplotypes. Nuclear markers revealed that some annual yams shared alleles with D. alata and semi‐perennial D. nummularia, suggesting a hybrid origin, which may explain their intermediate morphotypes and the difficulty met in classifying them.     

Chloroplast Genome Evolution in the Euglenaceae

Over the last few years multiple studies have been published outlining chloroplast genomes that represent many of the photosynthetic euglenid genera. However, these genomes were scattered throughout the euglenophyceaean phylogenetic tree, and focused on comparisons with Euglena gracilis. Here, we present a study exclusively on taxa within the Euglenaceae. Six new chloroplast genomes were characterized, those of Cryptoglena skujai, E. gracilis var. bacillaris, Euglena viridis, Euglenaria anabaena, Monomorphina parapyrum, and Trachelomonas volvocina, and added to six previously published chloroplast genomes to determine if trends existed within the family. With this study: at least one genome has now been characterized for each genus, the genomes of different strains from two taxa were characterized to explore intraspecific variability, and a second taxon has been characterized for the genus Monomorphina to examine intrageneric variability. Overall results showed a large amount of variability among the genomes, though a few trends could be identified both within Euglenaceae and within Euglenophyta. In addition, the intraspecific analysis indicated that the similarity of a genome sequence between strains was taxon dependent, and the intrageneric analysis indicated that the majority of the evolutionary changes within the Euglenaceae occurred intergenerically.     

Haplotype‐based genotyping‐by‐sequencing in oat genome research

In a de novo genotyping‐by‐sequencing (GBS) analysis of short, 64‐base tag‐level haplotypes in 4657 accessions of cultivated oat, we discovered 164741 tag‐level (TL) genetic variants containing 241224 SNPs. From this, the marker density of an oat consensus map was increased by the addition of more than 70000 loci. The mapped TL genotypes of a 635‐line diversity panel were used to infer chromosome‐level (CL) haplotype maps. These maps revealed differences in the number and size of haplotype blocks, as well as differences in haplotype diversity between chromosomes and subsets of the diversity panel. We then explored potential benefits of SNP vs. TL vs. CL GBS variants for mapping, high‐resolution genome analysis and genomic selection in oats. A combined genome‐wide association study (GWAS) of heading date from multiple locations using both TL haplotypes and individual SNP markers identified 184 significant associations. A comparative GWAS using TL haplotypes, CL haplotype blocks and their combinations demonstrated the superiority of using TL haplotype markers. Using a principal component‐based genome‐wide scan, genomic regions containing signatures of selection were identified. These regions may contain genes that are responsible for the local adaptation of oats to Northern American conditions. Genomic selection for heading date using TL haplotypes or SNP markers gave comparable and promising prediction accuracies of up to r = 0.74. Genomic selection carried out in an independent calibration and test population for heading date gave promising prediction accuracies that ranged between r = 0.42 and 0.67. In conclusion, TL haplotype GBS‐derived markers facilitate genome analysis and genomic selection in oat.