Comparative analysis of plastid genomes in the non-photosynthetic genus Thismia reveals ongoing gene set reduction

Heterotrophic plants provide intriguing examples of reductive evolution. This is especially evident in the reduction of their plastid genomes, which can potentially proceed toward complete genome loss. Several milestones at the beginning of this path of degradation have been described; however, little is known about the latest stages of plastome reduction. Here we analyze a diversity of plastid genomes in a set of closely related non-photosynthetic plants. We demonstrate how a gradual loss of genes shapes the miniaturized plastomes of these plants. The subject of our study, the genus Thismia, represents the mycoheterotrophic monocot family Thismiaceae, a group that may have experienced a very ancient (60–80 mya) transition to heterotrophy. In all 18 species examined, the plastome is reduced to 14–18 kb and is highly AT-biased. The most complete observed gene set includes accD, seven ribosomal protein genes, three rRNA, and two tRNA genes. Different clades of Thismia have undergone further gene loss (complete absence or pseudogenization) compared to this set: in particular, we report two independent losses of rps2 and rps18

Sequencing and Analysis of Plastid Genome in Mycoheterotrophic Orchid Neottia nidus-avis

Plastids are the semiautonomous organelles that possess their own genome inherited from the cyanobacterial ancestor. The primary function of plastids is photosynthesis so the structure and evolution of plastid genomes are extensively studied in photosynthetic plants. In contrast, little is known about the plastomes of nonphotosynthetic species. In higher plants, plastid genome sequences are available for only three strictly nonphotosynthetic species, the liverwort Aneura mirabilis and two flowering plants, Epifagus virginiana and Rhizanthella gardneri. We report here the complete sequence of a plastid genome of nonphotosynthetic mycoheterotrophic orchid Neottia nidus-avis, determined using 454 pyrosequencing technology. It was found to be reduced in both genome size and gene content; this reduction is however not as drastic as in the other nonphotosynthetic orchid, R. gardneri. Neottia plastome lacks all genes encoding photosynthetic proteins, RNA polymerase subunits but retains most genes of translational apparatus. Those genes that are retained have an increased rate of both synonymous and nonsynonymous substitutions but do not exhibit relaxation of purifying selection either in Neottia or in Rhizanthella

Mabs, a suite of tools for gene-informed genome assembly

Abstract Background Despite constantly improving genome sequencing methods, error-free eukaryotic genome assembly has not yet been achieved. Among other kinds of problems of eukaryotic genome assembly are so-called "haplotypic duplications", which may manifest themselves as cases of alleles being mistakenly assembled as paralogues. Haplotypic duplications are dangerous because they create illusions of gene family expansions and, thus, may lead scientists to incorrect conclusions about genome evolution and functioning. Results Here, I present Mabs, a suite of tools that serve as parameter optimizers of the popular genome assemblers Hifiasm and Flye. By optimizing the parameters of Hifiasm and Flye, Mabs tries to create genome assemblies with the genes assembled as accurately as possible. Tests on 6 eukaryotic genomes showed that in 6 out of 6 cases, Mabs created assemblies with more accurately assembled genes than those generated by Hifiasm and Flye when they were run with default parameters. When assemblies of Mabs, Hifiasm and Flye were postprocessed by a popular tool for haplotypic duplication removal, Purge_dups, genes were better assembled by Mabs in 5 out of 6 cases. Conclusions Mabs is useful for making high-quality genome assemblies. It is available at https://github.com/shelkmike/Mab

RNA-seq highlights parallel and contrasting patterns in the evolution of the nuclear genome of fully mycoheterotrophic plants

Abstract Background While photosynthesis is the most notable trait of plants, several lineages of plants (so-called full heterotrophs) have adapted to obtain organic compounds from other sources. The switch to heterotrophy leads to profound changes at the morphological, physiological and genomic levels. Results Here, we characterize the transcriptomes of three species representing two lineages of mycoheterotrophic plants: orchids (Epipogium aphyllum and Epipogium roseum) and Ericaceae (Hypopitys monotropa). Comparative analysis is used to highlight the parallelism between distantly related fully heterotrophic plants. In both lineages, we observed genome-wide elimination of nuclear genes that encode proteins related to photosynthesis, while systems associated with protein import to plastids as well as plastid transcription and translation remain active. Genes encoding components of plastid ribosomes that have been lost from the plastid genomes have not been transferred to the nuclear genomes; instead, some of the encoded proteins have been substituted by homologs. The nuclear genes of both Epipogium species accumulated nucleotide substitutions twice as rapidly as their photosynthetic relatives; in contrast, no increase in the substitution rate was observed in H. monotropa. Conclusions Full heterotrophy leads to profound changes in nuclear gene content. The observed increase in the rate of nucleotide substitutions is lineage specific, rather than a universal phenomenon among non-photosynthetic plants

Mitochondrial Genome of Fagopyrum esculentum and the Genetic Diversity of Extranuclear Genomes in Buckwheat

Fagopyrum esculentum (common buckwheat) is an important agricultural non-cereal grain plant. Despite extensive genetic studies, the information on its mitochondrial genome is still lacking. Using long reads generated by single-molecule real-time technology coupled with circular consensus sequencing (CCS) protocol, we assembled the buckwheat mitochondrial genome and detected that its prevalent form consists of 10 circular chromosomes with a total length of 404 Kb. In order to confirm the presence of a multipartite structure, we developed a new targeted assembly tool capable of processing long reads. The mitogenome contains all genes typical for plant mitochondrial genomes and long inserts of plastid origin (~6.4% of the total mitogenome length). Using this new information, we characterized the genetic diversity of mitochondrial and plastid genomes in 11 buckwheat cultivars compared with the ancestral subspecies, F. esculentum ssp. ancestrale. We found it to be surprisingly low within cultivars: Only three to six variations in the mitogenome and one to two in the plastid genome. In contrast, the divergence with F. esculentum ssp. ancestrale is much higher: 220 positions differ in the mitochondrial genome and 159 in the plastid genome. The SNPs in the plastid genome are enriched in non-synonymous substitutions, in particular in the genes involved in photosynthesis: psbA, psbC, and psbH. This presumably reflects the selection for the increased photosynthesis efficiency as a part of the buckwheat breeding program

Assembly and Analysis of the Complete Mitochondrial Genome of Capsella bursa-pastoris

Shepherd’s purse (Capsella bursa-pastoris) is a cosmopolitan annual weed and a promising model plant for studying allopolyploidization in the evolution of angiosperms. Though plant mitochondrial genomes are a valuable source of genetic information, they are hard to assemble. At present, only the complete mitogenome of C. rubella is available out of all species of the genus Capsella. In this work, we have assembled the complete mitogenome of C. bursa-pastoris using high-precision PacBio SMRT third-generation sequencing technology. It is 287,799 bp long and contains 32 protein-coding genes, 3 rRNAs, 25 tRNAs corresponding to 15 amino acids, and 8 open reading frames (ORFs) supported by RNAseq data. Though many repeat regions have been found, none of them is longer than 1 kbp, and the most frequent structural variant originated from these repeats is present in only 4% of the mitogenome copies. The mitochondrial DNA sequence of C. bursa-pastoris differs from C. rubella, but not from C. orientalis, by two long inversions, suggesting that C. orientalis could be its maternal progenitor species. In total, 377 C to U RNA editing sites have been detected. All genes except cox1 and atp8 contain RNA editing sites, and most of them lead to non-synonymous changes of amino acids. Most of the identified RNA editing sites are identical to corresponding RNA editing sites in A. thaliana

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid Δ1-dehydrogenase and 3-ketosteroid 9α-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology