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Tissue culture is an essential tool for the regeneration of uniform plant material. However, tissue culture conditions can be a source of abiotic stress for plants, leading to changes in the DNA sequence and methylation patterns. Despite the growing evidence on biochemical processes affected by abiotic stresses, how these altered biochemical processes affect DNA sequence and methylation patterns remains largely unknown. In this study, the methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach was used to investigate de novo methylation, demethylation, and sequence variation in barley regenerants derived by anther culture. Additionally, we used Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy to identify the spectral features of regenerants, which were then analyzed by mediation analysis. The infrared spectrum ranges (710&ndash, 690 and 1010&ndash, 940 cm&minus, 1) identified as significant in the mediation analysis were most likely related to &beta, glucans, cellulose, and S-adenosyl-L-methionine (SAM). Additionally, the identified compounds participated as predictors in moderated mediation analysis, explaining the role of demethylation of CHG sites (CHG_DMV) in in vitro tissue culture-induced sequence variation, depending on the duration of tissue culture. The data demonstrate that ATR-FTIR spectroscopy is a useful tool for studying the biochemical compounds that may affect DNA methylation patterns and sequence variation, if combined with quantitative characteristics determined using metAFLP molecular markers and mediation analysis. The role of &beta, glucans, cellulose, and SAM in DNA methylation, and in cell wall, mitochondria, and signaling, are discussed to highlight the putative cellular mechanisms involved in sequence variation.

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations (&lsquo, Cu*Ag&rsquo, ) acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of &lsquo, and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis

Fungi and oomycetes encompass many pathogens affecting crops worldwide. Their effective control requires screening pathogens across the local and international trade networks along with the monitoring of pathogen inocula in the field. Fundamentals to all of these concerns are their efficient detection, identification, and quantification. The use of molecular markers showed the best promise in the field of plant pathogen diagnostics. However, despite the unquestionable benefits of DNA-based methods, two significant limitations are associated with their use. The first limitation concerns the insufficient level of sensitivity due to the very low and uneven distribution of pathogens in plant material. The second limitation pertains to the inability of widely used diagnostic assays to detect cryptic species. Targeting mtDNA appears to provide a solution to these challenges. Its high copy number in microbial cells makes mtDNA an attractive target for developing highly sensitive assays. In addition, previous studies on different pathogen taxa indicated that mitogenome sequence variation could improve cryptic species delimitation accuracy. This review sheds light on the potential application of mtDNA for pathogen diagnostics. This paper covers a brief description of qPCR and DNA barcoding as two major strategies enabling the diagnostics of plant pathogenic fungi and oomycetes. Both strategies are discussed along with the potential use of mtDNA, including their strengths and weaknesses.

Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY). The nuclear ribosomal DNA (rDNA) sequence of P. brassicae, comprising 6932 base pairs (bp), was cloned and sequenced and found to include the small subunits (SSUs) and a large subunit (LSU), internal transcribed spacers (ITS1 and ITS2), and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs), oligonucleotide polymorphisms, and insertions/deletions (InDels). A combination of three markers was able to distinguish the geographical isolates into two groups.