The use of retrotransposon-based molecular markers to analyze genetic diversity

Molecular markers play an essential role in all aspects of genetics, modern plant breeding, in human forensics, for map-based cloning of genes, ranging from the identification of genes responsible for desired traits to the management of backcrossing programs. Retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their “copy and paste” life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Following the demonstration that retrotransposons are ubiquitous, active, and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of genetic diversity in plants and the way for the rapid isolation of retrotransposon termini.Peer reviewe

Comparison Between O and OH Intermediates of Cytochrome c Oxidase Studied by FTIR Spectroscopy

Cytochrome c oxidase is terminal enzyme in the respiratory chain of mitochondria and many aerobic bacteria. It catalyzes reduction of oxygen to water. During its catalysis, CcO proceeds through several quite stable intermediates (R, A, PR/M, O/OH, E/EH). This work is concentrated on the elucidation of the differences between structures of oxidized intermediates O and OH in different CcO variants and at different pH values. Oxidized intermediates of wild type and mutated CcO from Paracoccus denitrificans were studied by means of static and time-resolved Fourier-transform infrared spectroscopy in acidic and alkaline conditions in the infrared region 1800–1000 cm−1. No reasonable differences were found between all variants in these conditions, and in this spectral region. This finding means that the binuclear center of oxygen reduction keeps a very similar structure and holds the same ligands in the studied conditions. The further investigation in search of differences should be performed in the 4000–2000 cm−1 IR region where water ligands absorb.Peer reviewe

Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants

Species specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm’ region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.Non Peer reviewe

In Silico Estimation of the Abundance and Phylogenetic Significance of the Composite Oct4-Sox2 Binding Motifs within a Wide Range of Species

High-throughput sequencing technologies have greatly accelerated the progress of genomics, transcriptomics, and metagenomics. Currently, a large amount of genomic data from various organisms is being generated, the volume of which is increasing every year. Therefore, the development of methods that allow the rapid search and analysis of DNA sequences is urgent. Here, we present a novel motif-based high-throughput sequence scoring method that generates genome information. We found and identified Utf1-like, Fgf4-like, and Hoxb1-like motifs, which are cis-regulatory elements for the pluripotency transcription factors Sox2 and Oct4 within the genomes of different eukaryotic organisms. The genome-wide analysis of these motifs was performed to understand the impact of their diversification on mammalian genome evolution. Utf1-like, Fgf4-like, and Hoxb1-like motif diversity was evaluated across genomes from multiple species.Peer reviewe

A major gene for grain cadmium accumulation in oat

A population of 150 F2 plants was derived from a cross between two spring oat individuals, one from cv. Aslak (Boreal Plant Breeding LTd., Finland) and the other from cv. Salo (Svalöf-Weibull AB, Sweden). Cadmium was tested by inductively coupled plasma mass spectrometry (ICP-MS) method

Editorial: mobile elements and plant genome evolution, comparative analyzes and computational tools

Multiple changes that occur constantly in the plant genome allow an organism to develop from a single-celled embryo to a multicellular organism. A significant part of these changes is associated with the recombination activity of numerous classes of interspersed repeats. These numerous families of interspersed repeats were often called "junk DNA" as they were not associated with vital protein-coding processes (1). Transposable elements (TEs), such as DNA transposons and retrotransposons, are the main part of these interspersed repeats (2). DNA transposons can rightfully be called true mobile elements, the activity of which can occur at any stage of cell development and manifest itself at any moment and stage of the organism's development. The diverse families of retrotransposons are highly abundant genetic elements that are related to retroviruses (3). Although retrotransposons are not true mobile elements like DNA transposons, retrotransposable elements (RTEs) form a variety of chromosomal structures, such as centromeric and telomeric regions (4), and are the main intergenic part of the genome (5). Retrotransposons move to new chromosomal locations via an RNA intermediate that is converted into extrachromosomal DNA by the encoded reverse transcriptase/RNaseH enzymes prior to reinsertion into the genome. This replicative mode of transposition can rapidly increase the copy number of elements and can thereby greatly increase plant genome size. RTEs can be clustered into distinct families each traceable to a single ancestral sequence or a closely related group of ancestral sequences. In contrast to multigene families, which are defined based on their biological role, repetitive families are usually defined based on their active ancestors (called master or source genes) and on their generation mechanisms. Over time, individual elements from repetitive families may acquire diverse biological roles. Some RTEs can provide evolutionary advantages to the host and increase their chances of survival (6). While the view that RTEs are beneficial to the host is not new, recent progress in the field has placed RTEs squarely in the center of the ongoing debate on eukaryotic evolution. To advance this important research field, in the Research Topic "Mobile Elements and Plant Genome Evolution, Comparative Analyses, and Computational Tools" we focus on the role of mobile elements with host genome evolution, discovery, and comparative and genome-wide profiling analysis of transposable elements. Different retrotransposon families, each with its own lineage and structure, may have been active at distinct phases in the evolution of a species. Retrotransposon sequences bear the promoters that bind the nuclear factors of transcription initialization and initiate RNA synthesis by polymerases II or III. In the article entitled "Additional ORFs in Plant LTR-Retrotransposons" by Vicient C.M. and Casacuberta J.M., LTR-retrotransposons that carry additional, not retrotransposon-specific open reading frames (aORF), were discovered and analyzed. This discovery expands on the unique potential of LTR-retrotransposons as evolutionary tools, as LTR-retrotransposons can be used to deliver new gene variants within a genome. The presence of a unique aORF in some characterized LTR-retrotransposon families like maize Grande, rice RIRE2, or Silene Retand, are just as typical as retrovirus gene transduction. As dispersed and ubiquitous mobile elements, the life cycle of replicative transposition leads to genome rearrangements that affect cellular function (7). Transposable elements are important drivers of species diversity and exhibit great variety in structure, size, and mechanisms of transposition, making them important putative actors in genome evolution. The research group led by Kashkush K., reported the potential impact of miniature transposable element insertions on the expression of wheat genes in different wheat species in the articles entitled "The Evolutionary Dynamics of a Novel Miniature Transposable Element in the Wheat Genome" and "Where the Wild Things Are: Transposable Elements as Drivers of Structural and Functional Variations in the Wheat Genome". The induced genetic rearrangements and insertions of mobile genetic elements in regions of active euchromatin contribute to genome alteration, which leads to "genomic stress" (8). TEmediated epigenetic modifications lead to phenotypic diversity, genetic variation, and environmental stress tolerance. TEs also contribute to genome plasticity and have a dramatic impact on the genetic diversity and evolution of the wheat genome. Using transposon display (9) and genome-wide profiling analysis of insertional polymorphisms of transposable elements (10), the authors discovered large genomic rearrangement events, such as deletions and introgressions in the wheat genome. High-throughput bioinformatics with next-generation sequencing (NGS) were key tools in these studies (11). Chromosomal rearrangements, gene duplications, and transposable element content may have a large impact on genomic structure, which could generate new phenotypic traits (7). In the article entitled "Genome Size Variation and Comparative Genomics Reveal Intraspecific Diversity in Brassica rapa", de Carvalho J.F. et al investigated structural variants and repetitive content between two accessions of Brassica rapa genomes and genome-size variation among a core collection using comparative genomics and cytogenetic approaches. Large genomic variants with a chromosome length difference of 17.6% between the A06 chromosomes of 'Z1' compared to 'Chiifu' belonging to different cultigroups of B. rapa highlighted the potential impact of differential insertion of repeat elements and inversions of large genomic regions in genome size intraspecific variability. Transposable elements are also the driving force in the evolution of epigenetic regulation and have a long-term impact on genomic instability and evolution. Remnants of RTEs appear to be overrepresented in transcription regulatory modules and other regions conserved among distantly related species, which may have implications for our understanding of their impact on speciation. RTEs are dynamic and play a role in chromosome crossing over recognition and in DNA recombination between homologous chromosomes. In the article entitled "Sequencing Multiple Cotton Genomes Reveals Complex Structures and Lays Foundation for Breeding", Wang X. et al revealed that post-polyploidization of cotton genome instability resulted in numerous genomic structural changes, DNA inversion and translocation, illegitimate recombinations, accumulation of repetitive sequences, and functional innovation accompanied by elevated evolutionary rates of genes. This genome study also revealed the evolutionary past of cotton plants, which were recursively affected by polyploidization, with a decaploidization contributing to the formation of the genus Gossypium, and a neo-tetraploidization contributing to the formation of the currently widely cultivated cotton plants. The centromere is a unique part of the chromosome that combines a conserved function with extreme variability in its DNA sequence. In the article entitled "Functional Allium fistulosum centromeres comprise arrays of a long satellite repeat, insertions of retrotransposons and chloroplast DNA" Kirov G.I., et al studied the largest plant genomic organization of the functional centromere in large-sized chromosomes in Allium fistulosum and A. cepa. Long, high-copy repeats are associated with insertions of retrotransposons and plastidial DNA, and the landscape of the centromeric regions of these species possess insertions of plastidial DNA. Among evolutionary factors, repetitive sequences play multiple roles in sex chromosome evolution. As such, the Spinacia genus serves as an ideal model to investigate the evolutionary mechanisms underlying the transition from homomorphic to heteromorphic sex chromosomes. This was studied in the article entitled "Genome-Wide Analysis of Transposable Elements and Satellite DNAs in Spinacia Species to Shed Light on Their Roles in Sex Chromosome Evolution" by Li N., et al. Major repetitive sequence classes in male and female genomes of Spinacia species and their ancestral relative, sugar beet, were elucidated in the evolutionary processes of sex chromosome evolution using NGS data. The differences of repetitive DNA sequences correlate with the formation of sex chromosomes and the transition from homomorphic sex chromosomes to heteromorphic sex chromosomes, as heteromorphic sex chromosomes existed exclusively in Spinacia tetrandra.Non peer reviewe

Editorial: Innovative Applications of Sequencing Technologies in Plant Science

Sequencing and sequencing technologies are amongst the techniques in life sciences that have brought about a revolution, opening up many possibilities to explore the hidden secrets of life in DNA. Sequencing and sequencing technologies have also advanced to make for more accurate and time-efficient discoveries that can be employed on a large scale. This is particularly useful in plant science research when screening and selecting for particular traits of interest. Despite this advancement in sequencing technologies, the full potential this power to explore the genetic code gives is yet to be fully explored. New studies showing innovative ways sequencing has been employed to understand plants better are emerging. It is the goal of this Research topic to highlight these innovative applications of sequence technologies in Plant Science – such as in genetic mapping, the identification and characterization of candidate genes, innovative use of DNA metabarcoding, expansion of opportunities and solvable problems by PCR technologies, and the novel combination of sequence-based technologies to answer plant science questions aligned with making for a healthy planet.Non peer reviewe

Palindromic sequence-targeted (PST) PCR, version 2: an advanced method for high-throughput targeted gene characterization and transposon display

Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that exist in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, which offer the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as Palindromic Sequence-Targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5’ tail attached to the sequence-specific primer and the other anneals to a different 5’ tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2–3 hours to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific Random Amplified Polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display, which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the corn (Zea mays) genome was used as a sequence tag during the transposon display procedure to characterize the Ac integration sites.Peer reviewe

Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy

Crystal structure analyses at atomic resolution and FTIR spectroscopic studies of cytochrome c oxidase have yet not revealed protonation or deprotonation of key sites of proton transfer in a time-resolved mode. Here, a sensitive technique to detect protolytic transitions is employed. In this work, probing a proton-loading site of cytochrome c oxidase from Paracoccus denitrificans with time-resolved Fourier transform infrared spectroscopy is presented for the first time. For this purpose, variants with single-site mutations of N131V, D124N, and E278Q, the key residues in the D-channel, were studied. The reaction of mutated CcO enzymes with oxygen was monitored and analyzed. Seven infrared bands in the “fast” kinetic spectra were found based on the following three requirements: (1) they are present in the “fast” phases of N131V and D124N mutants, (2) they have reciprocal counterparts in the “slow” kinetic spectra in these mutants, and (3) they are absent in “fast” kinetic spectra of the E278Q mutant. Moreover, the double-difference spectra between the first two mutants and E278Q revealed more IR bands that may belong to the proton-loading site protolytic transitions. From these results, it is assumed that several polar residues and/or water molecule cluster(s) share a proton as a proton-loading site. This site can be propionate itself (holding only a fraction of H+), His403, and/or water cluster(s)