Exploring the potential of nuclear and mitochondrial sequencing data generated through genome‐skimming for plant phylogenetics: A case study from a clade of neotropical lianas

Few botanical studies have explored the potential of nuclear ribosomal DNA (nrDNA) and mitochondrial DNA (mtDNA) data obtained through genome skimming for phylogeny reconstruction. Here, we analyzed the phylogenetic information included in the nrDNA and mtDNA of 44 species of the “Adenocalymma‐Neojobertia” clade (Bignoniaceae). To deal with intraindividual polymorphisms within the nrDNA, different coding schemes were explored through the analyses of four datasets: (i) “nrDNA contig,” with base call following the majority rule; (ii) “nrDNA ambiguous,” with ambiguous base calls; (iii) “nrDNA informative,” with ambiguities converted to multistate characters; and, (iv) “mitochondrial,” with 39 mitochondrial genes. Combined analyses using the nrDNA and mtDNA data and previously published “plastid” datasets were also conducted. Trees were obtained using Maximum Likelihood and Bayesian criteria. The congruence among genomes was assessed. The nrDNA datasets were shown to be highly polymorphic within individuals, while the “mitochondrial” dataset was the least informative, with 0.36% of informative bases within the ingroup. The topologies inferred using the nrDNA and mtDNA datasets were broadly congruent with the tree derived from the analyses of the “plastid” dataset. The topological differences recovered were generally poorly supported. The topology that resulted from the analyses of the “combined” dataset largely resembles the “plastid” tree. These results highlight limitations of nuclear ribosomal DNA and mitochondrial genes for phylogeny reconstruction obtained through genome skimming and the need to include more data from both genomes. The different topologies observed among genomes also highlight the importance of exploring data from various genomes in plant phylogenetics.     

Evidence for new nuclear and mitochondrial genome organizations among high-frequency somatic embryogenesis-derived plants of allotetraploid Coffea arabica L. (Rubiaceae)

 The most important commercial species of coffee, Coffea arabica, which produces 73% of the world's coffee crop and almost all of the coffee in Latin America, is the only tetraploid (allotetraploid, 2n=4x=44) species known in the genus. High-frequency somatic embryogenesis, plant regeneration and plant recovery were achieved from leaf explants of a mature, elite plant of C. arabica cv. Cauvery (S-4347) using a two-step culture method. To assess the genetic integrity of the nuclear, mitochondrial and chloroplast genomes among the hardened regenerants, we employed multiple DNA markers (RFLP, RAPD, ISSR) for sampling various regions of the genome. Although the nuclear and mitochondrial genomes of the mother plant and five ramets derived from the mother ortet were similar in organization, this was not so in the somatic embryo-derived plants where both nuclear and mitochondrial genomes changed in different, characteristic ways and produced novel genome organizations. A total of 480 genetic loci, based on the data obtained from a total of 16 nuclear, mitochondrial and chloroplast gene probes, in combination with nine restriction enzyme digests, 38 RAPD and 17 SSR primers, were scored in 27 somatic embryo-derived plants and the single control. Among these, 44 loci were observed to be polymorphic. A relatively low level of polymorphism (4.36%) was found in the nuclear genome, while polymorphism in the mitochondrial genome (41%) was much higher. No polymorphism was detected in the chloroplast genome. The polymorphism in the mitochondrial genome was found in only 4 plants. Such selective polymorphism was not true for the nuclear genome. Thus, this in-depth and comprehensive study demonstrates, for the first time, the presence of subtle genetic variability and novel genome organizations in the commercially well-established somatic embryogenesis-derived plants of this important coffee species. Received: 2 July 1999 / Revision received: 1 February 2000 / Accepted: 17 February 2000     

Assembly-free quantification of vagrant DNA inserts

Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called “vagrantDNA” and code to simulate test data sets.     

植绥螨科线粒体基因组研究进展

植绥螨科属于囊螨总科,大部分植绥螨为害螨、害虫的重要天敌,在农业生产上具有重要的应用价值。其线粒体基因组具有独特特征,引起了生物学家的广泛关注。本文就植绥螨科线粒体基因组的结构、非编码区、碱基组成、基因重排和tRNA的特征进行综述,其特征有：（1）植绥螨科中发现了螯肢动物最大的线粒体基因组;（2）植绥螨科线粒体基因组非编码区的AT含量差异大,编码区的AT含量差异小;（3）植绥螨科线粒体基因组均发生了不同程度的的基因重排;（4）已测定的植绥螨科部分物种tRNA基因二级结构出现了截短和碱基错配的现象,部分物种的线粒体基因组出现反密码子突变的情况。在今后的研究中应进一步测定植绥螨科关键类群的线粒体基因组,深入分析植绥螨科出现大量基因重排的原因,以期能反映植绥螨科的真实进化历程。     

Binucleation to breed new plant species adaptable to their environments

Classical plant breeding approaches may fall short to breed new plant species of high environmental and ecological interests. Biotechnological and genetic manipulations, on the other hand, may hold more effective capabilities to circumvent the limitations of sexual incompatibility and conventional breeding programs. Given that plant cells encompass multiple copies of organellar genomes (mitochondrial and plastidial genomes), an important question could be raised about whether an artificial attempt to duplicate the nuclear genome might also be conceivable through a binucleation approach (generating plant cells with 2 nuclei from 2 different plant species) for potential production of new polyploidies that would characterize new plant species. Since the complexities of plant genomes are the result of multiple genome duplications, an artificial binucleation approach would thus be of some interest to eventually varying plant genomes and producing new polyploidy from related or distal plant species. Here, I discuss the potentiality of such an approach to engineer binucleated plant cells as a germ of new plant species to fulfill some environmental applications such as increasing the biodiversity and breeding new species adaptable to harsh environmental stresses and increasing green surfaces to reduce atmospheric pollutions in arid lands with poor vegetation.     

All-male asexuality: origin and maintenance of androgenesis in the Asian clam Corbicula

Androgenesis is a rare form of asexual male reproduction found in disparate taxa across the Tree of Life. Phylogenetic analyses of mitochondrial genes suggest that androgenesis has arisen repeatedly in the Asian clam genus Corbicula. Two of these androgenetic species have been introduced to North America. Multiple lines of genetic evidence suggest that although nuclear recombination between these two species is rare, mitochondrial genome capture is a frequent consequence of androgenetic parasitism of heterospecific eggs. Egg parasitism may also rarely result in partial nuclear genome capture between closely related species of Corbicula, which provides a mechanism for the otherwise clonal species to avoid the deleterious effects of asexuality. Egg parasitism among congeners may explain why androgenesis has been maintained in Corbicula after fixation and has not yet led to population extinction. This mechanism also provides an explanation for the apparent multiple origins of androgenesis in Corbicula as seen on the mitochondrial DNA phylogeny. We suggest that a single androgenetic lineage may have repeatedly captured mitochondrial genomes (as well as portions of nuclear genomes) from various sexual species, resulting in several distinct androgenetic species with distantly related mtDNA genomes and divergent morphologies.     

Pest species diversity enhances control of spider mites and whiteflies by a generalist phytoseiid predator

To test the hypothesis that pest species diversity enhances biological pest control with generalist predators, we studied the dynamics of three major pest species on greenhouse cucumber: Western flower thrips, Frankliniella occidentalis (Pergande), greenhouse whitefly, Trialeurodes vaporariorum (Westwood), and two-spotted spider mites, Tetranychus urticae Koch in combination with the predator species Amblyseius swirskii Athias-Henriot. When spider mites infested plants prior to predator release, predatory mites were not capable of controlling spider mite populations in the absence of other pest species. A laboratory experiment showed that predators were hindered by the webbing of spider mites. In a greenhouse experiment, spider mite leaf damage was lower in the presence of thrips and predators than in the presence of whiteflies and predators, but damage was lowest in the presence of thrips, whiteflies and predators. Whitefly control was also improved in the presence of thrips. The lower levels of spider mite leaf damage probably resulted from (1) a strong numerical response of the predator (up to 50 times higher densities) when a second and third pest species were present in addition to spider mites, and (2) from A. swirskii attacking mobile spider mite stages outside or near the edges of the spider mite webbing. Interactions of spider mites with thrips and whiteflies might also result in suppression of spider mites. However, when predators were released prior to spider mite infestations in the absence of other pest species, but with pollen as food for the predators, we found increased suppression of spider mites with increased numbers of predators released, confirming the role of predators in spider mite control. Thus, our study provides evidence that diversity of pest species can enhance biological control through increased predator densities.     

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.     

Gene recruitment--a common mechanism in the evolution of transfer RNA gene families

The evolution of alloacceptor transfer RNAs (tRNAs) has been traditionally thought to occur vertically and reflect the evolution of the genetic code. Yet there have been several indications that a tRNA gene could evolve horizontally, from a copy of an alloacceptor tRNA gene in the same genome. Earlier, we provided the first unambiguous evidence for the occurrence of such "tRNA gene recruitment" in nature--in the mitochondrial (mt) genome of the demosponge Axinella corrugata. Yet the extent and the pattern of this process in the evolution of tRNA gene families remained unclear. Here we analyzed tRNA genes from 21 mt genomes of demosponges as well as nuclear genomes of rhesus macaque, chimpanzee and human. We found four new cases of alloacceptor tRNA gene recruitment in mt genomes and eleven cases in the nuclear genomes. In most of these cases we observed a single nucleotide substitution at the middle position of the anticodon, which resulted in the change of not only the tRNA's amino-acid identity but also the class of the amino-acyl tRNA synthetases (aaRSs) involved in amino-acylation. We hypothesize that the switch to a different class of aaRSs may have prevented the conflict between anticodon and amino-acid identities of recruited tRNAs. Overall our results suggest that gene recruitment is a common phenomenon in tRNA multigene family evolution and should be taken into consideration when tRNA evolutionary history is reconstructed.     

石松类和蕨类植物质体基因组结构演化研究进展

近年来, 随着测序技术的发展, 石松类和蕨类植物的核基因组、质体基因组以及线粒体基因组研究发展迅速, 质体基因组研究工作更是呈爆发式增长。截至2019年3月1日, GenBank公布的石松类和蕨类植物的175个质体基因组中, 约3/4为最近两年新增。研究内容从早期对个别质体基因组结构和序列特征的简单报道, 逐渐发展到综合性的比较基因组学和系统发育基因组学研究。目前已发表的质体基因组覆盖了石松类和蕨类植物的所有目和大部分科, 这两大类群的质体基因组结构变异和系统发育的基本框架已逐渐清晰。这些研究为我们理解维管植物的早期演化提供了重要参考。本文对石松类和蕨类植物的质体基因组结构特征进行了系统梳理, 发现其结构变异主要包括大片段倒位、IR区边界变动、基因或内含子丢失等, 其中一些结构变异可作为较高分类阶元的共衍征。RNA编辑和长片段非编码序列插入普遍存在于石松类和蕨类植物的质体基因组中, 但其起源、演化机制和功能等仍不清楚。我们对质体基因组的应用、系统发育研究中质体和核基因组的优劣性, 以及系统发育基因组学的前景进行了评述。     

A Comparative Genomics Analysis of Codon Reassignments Reveals a Link with Mitochondrial Proteome Size and a Mechanism of Genetic Code Change Via Suppressor tRNAs

Using a comparative genomics approach we demonstrate a negative correlation between the number of codon reassignments undergone by 222 mitochondrial genomes and the mitochondrial genome size, the number of mitochondrial ORFs, and the sizes of the large and small subunit mitochondrial rRNAs. In addition, we show that the TGA-to-tryptophan codon reassignment, which has occurred 11 times in mitochondrial genomes, is found in mitochondrial genomes smaller than those which have not undergone the reassignment. We therefore propose that mitochondrial codon reassignments occur in a wide range of phyla, particularly in Metazoa, due to a reduced “proteomic constraint” on the mitochondrial genetic code, compared to the nuclear genetic code. The reduced proteomic constraint reflects the small size of the mitochondrial-encoded proteome and allows codon reassignments to occur with less likelihood of lethality. In addition, we demonstrate a striking link between nonsense codon reassignments and the decoding properties of naturally occurring nonsense suppressor tRNAs. This suggests that natural preexisting nonsense suppression facilitated nonsense codon reassignments and constitutes a novel mechanism of genetic code change. These findings explain for the first time the identity of the stop codons and amino acids reassigned in mitochondrial and nuclear genomes. Nonsense suppressor tRNAs provided the raw material for nonsense codon reassignments, implying that the properties of the tRNA anticodon have dictated the identity of nonsense codon reassignments. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Laura Landweber]     

High-quality genomes reveal significant genetic divergence and cryptic speciation in the model organism Folsomia candida (collembola)

The collembolan Folsomia candida Willem, 1902, is widely distributed throughout the world and has been frequently used as a test organism in soil ecology and ecotoxicology studies. However, it is questioned as an ideal “standard” because of differences in reproductive modes and cryptic genetic diversity between strains from various geographical origins. In this study, we obtained two high-quality chromosome-level genomes of F. candida, for a parthenogenetic strain (named FCDK, 219.08 Mb, 25,139 protein-coding genes) and a sexual strain (named FCSH, 153.09 Mb, 21,609 protein-coding genes), reannotated the genome of the parthenogenetic strain reported by Faddeeva-Vakhrusheva et al. in 2017 (named FCBL, 221.7 Mb, 25,980 protein-coding genes) and conducted comparative genomic analyses of the three strains. High genome similarities between FCDK and FCBL based on synteny, genome architecture, mitochondrial and nuclear gene sequences suggest that they are conspecific. The seven chromosomes of FCDK are each 25%–54% larger than the corresponding chromosomes of FCSH, showing obvious repetitive element expansions and large-scale inversions and translocations but no whole-genome duplication. The strain-specific genes, expanded gene families and genes in nonsyntenic chromosomal regions identified in FCDK are highly related to the broader environmental adaptation of parthenogenetic strains. In addition, FCDK has fewer strain-specific microRNAs than FCSH, and their mitochondrial and nuclear genes have diverged greatly. In conclusion, FCDK/FCBL and FCSH have accumulated independent genetic changes and evolved into distinct species after 10 million years ago. Our work provides important genomic resources for studying the mechanisms of rapidly cryptic speciation and soil arthropod adaptation to soil ecosystems.     

Complete Mitochondrial Genome of Haplorchis taichui and Comparative Analysis with Other Trematodes

Mitochondrial genomes have been extensively studied for phylogenetic purposes and to investigate intra- and interspecific genetic variations. In recent years, numerous groups have undertaken sequencing of platyhelminth mitochondrial genomes. Haplorchis taichui (family Heterophyidae) is a trematode that infects humans and animals mainly in Asia, including the Mekong River basin. We sequenced and determined the organization of the complete mitochondrial genome of H. taichui. The mitochondrial genome is 15,130 bp long, containing 12 protein-coding genes, 2 ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). Like other trematodes, it does not encode the atp8 gene. All genes are transcribed from the same strand. The ATG initiation codon is used for 9 protein-coding genes, and GTG for the remaining 3 (nad1, nad4, and nad5). The mitochondrial genome of H. taichui has a single long non-coding region between trnE and trnG. H. taichui has evolved as being more closely related to Opisthorchiidae than other trematode groups with maximal support in the phylogenetic analysis. Our results could provide a resource for the comparative mitochondrial genome analysis of trematodes, and may yield genetic markers for molecular epidemiological investigations into intestinal flukes.     

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).     

Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus

We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time.The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.     

Complete mitochondrial genomes offer insights into the evolutionary relationships and comparative genetic diversity of New Zealand’s iconic kiwi (Apteryx spp.)

ABSTRACT

Kiwis (Apterygidae) are an enigmatic family of flightless birds endemic to New Zealand. Apterygidae is made up of a single genus, Apteryx with five species, four of which are characterised as at risk of greater by the New Zealand Department of Conservation. These five species are further separated into two morphologically and genetically distinguishable clades, containing A.haastii, and A.owenii in one and A.rowi, A.mantelli, and A.australis in the other. We reconstructed 17 kiwi mitochondrial genomes from previously published genomic data, nine from A.rowi and eight from A.owenii. Mitochondrial diversity analyses uncovered low levels of genetic diversity consistent with their reduced ranges and conservation concern. We further used one of the assembled A.rowi mitochondrial genomes together with mitochondrial genomes from A.haastii, A.owenii, A.mantelli, and several other individuals from Palaeognathae to estimate the within and between clade divergence times of kiwis. Our study exemplifies how available published data can be used in novel ways to provide new and complementary evolutionary insights to previous studies.     

虱目裂化线粒体基因组研究进展

虱目是哺乳类和鸟类体表的专性寄生虫。在虱科、阴虱科、长角鸟虱科和兽羽虱科的某些寄生虱种中发现了线粒体基因组裂化现象, 其线粒体基因组裂化成了多个环状的线粒体染色体, 如体虱(Pediculus humanus)、头虱(pediculus capitis)和阴虱(Pthirus pubis)的线粒体基因组分别裂化形成20个、20个和14个微环染色体。微环染色体可能是基因删除和同源重组的结果, 关于线粒体基因组裂化的具体原因和机制, 目前并不清楚, 推测可能是进化选择或随机遗传漂变的结果或与线粒体单链DNA结合蛋白的缺失有关。鉴于线粒体基因组裂化研究对于深入理解线粒体的起源和进化方面具有重要意义, 文章以虱目裂化线粒体基因组为主线, 列举了动物裂化线粒体基因组和裂化特征, 阐述了虱目裂化线粒体基因组的研究现状, 分析了虱目线粒体基因组裂化的类型、原因和机制, 并对该领域未来的研究方向进行了展望。