Pulmonary tuberculosis epidemiology and genetics in Kazakhstan.

Background: Tuberculosis (TB) is a major public health emergency in many countries, including Kazakhstan. Despite the decline in the incidence rate and having one of the highest treatment effectiveness in the world, the incidence rate of TB remains high in Kazakhstan. Social and environmental factors along with host genetics contribute to pulmonary tuberculosis (PTB) incidence. Due to the high incidence rate of TB in Kazakhstan, our research aimed to study the epidemiology and genetics of PTB in Kazakhstan. Materials and methods: 1,555 participants were recruited to the case-control study. The epidemiology data was taken during an interview. Polymorphisms of selected genes were determined by real-time PCR using pre-designed TaqMan probes. Results: Epidemiological risk factors like diabetes (χ2 = 57.71, p < 0.001), unemployment (χ2 = 81.1, p < 0.001), and underweight-ranged BMI (<18.49, χ2 = 206.39, p < 0.001) were significantly associated with PTB. VDR FokI (rs2228570) and VDR BsmI (rs1544410) polymorphisms were associated with an increased risk of PTB. A/A genotype of the TLR8 gene (rs3764880) showed a significant association with an increased risk of PTB in Asians and Asian males. The G allele of the rs2278589 polymorphism of the MARCO gene increases PTB susceptibility in Asians and Asian females. VDR BsmI (rs1544410) polymorphism was significantly associated with PTB in Asian females. A significant association between VDR ApaI polymorphism and PTB susceptibility in the Caucasian population of Kazakhstan was found. Conclusion: This is the first study that evaluated the epidemiology and genetics of PTB in Kazakhstan on a relatively large cohort. Social and environmental risk factors play a crucial role in TB incidence in Kazakhstan. Underweight BMI (<18.49 kg/m2), diabetes, and unemployment showed a statistically significant association with PTB in our study group. FokI (rs2228570) and BsmI (rs1544410) polymorphisms of the VDR gene can be used as possible biomarkers of PTB in Asian males. rs2278589 polymorphism of the MARCO gene may act as a potential biomarker of PTB in Kazakhs. BsmI polymorphism of the VDR gene and rs2278589 polymorphism of the MARCO gene can be used as possible biomarkers of PTB risk in Asian females as well as VDR ApaI polymorphism in Caucasians.

Vitamin D Status, VDR, and TLR Polymorphisms and Pulmonary Tuberculosis Epidemiology in Kazakhstan.

BACKGROUND: Tuberculosis (TB) and vitamin D deficiency remain major public health problems in Kazakhstan. Due to the high incidence of pulmonary tuberculosis in the country and based on the importance of vitamin D in the modulation of the immune response and the association of its deficiency with many health conditions, the aim of our research was to study the vitamin D status, VDR and TLR gene polymorphisms, and pulmonary tuberculosis epidemiology in Kazakhstan. METHODS: A case-control study included 411 individuals diagnosed with pulmonary TB and 686 controls with no family history of pulmonary tuberculosis. Concentrations of serum vitamin D (25-(OH)D) levels were measured by electrochemiluminescence immunoassay. The gene polymorphisms were determined by real-time polymerase chain reaction (PCR) allelic discrimination assay using TaqMan probes. The association between the risk of pulmonary TB and polymorphisms was evaluated using multimodal logistic regression and assessed with the ORs, corresponding to 95% Cis, and the significance level was determined as p < 0.05. RESULTS: 1097 individuals were recruited from 3 different regions of Kazakhstan. Biochemical data showed vitamin D deficiency (25-(OH)D < 20 ng/mL) was present in both groups, with the case group accounting for almost 95% and 43.7% in controls. Epidemiological data revealed that socioeconomic factors such as BMI < 25 kg/m2 (p < 0.001), employment (p < 0.001), diabetes (p < 0.001), and vitamin D deficiency (p < 0.001) were statistically different between case and control groups. Logistic regression analysis, adjusted by sex, age, BMI, residence, employment, smoking, alcohol consumption, and diabetes, showed that T/T polymorphism of the VDR gene (rs1544410, OR = 1.97, 95% CI: 1.04-3.72, p = 0.03) and A/A polymorphism of the TLR8 gene (rs3764880, OR = 2.44, 95% CI: 1.20-4.98, p = 0.01) were associated with a high risk of developing pulmonary tuberculosis. CONCLUSIONS: Vitamin D deficiency remains prevalent in our study cohort and is associated with TB progression. Socioeconomic determinants such as unemployment, BMI under 25 kg/m2, and diabetes are the main risk factors for the development of pulmonary TB in our study. A/A polymorphism of TLR8 (rs3764880) and T/T polymorphism (BsmI, rs1544410) of VDR genes may act as biomarkers for pulmonary tuberculosis in the Kazakh population.

Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap.

Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA's electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA's negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability.

Overcoming genetic paucity of Camelina sativa: possibilities for interspecific hybridization conditioned by the genus evolution pathway.

Camelina or false flax (Camelina sativa) is an emerging oilseed crop and a feedstock for biofuel production. This species is believed to originate from Western Asian and Eastern European regions, where the center of diversity of the Camelina genus is located. Cultivated Camelina species arose via a series of polyploidization events, serving as bottlenecks narrowing genetic diversity of the species. The genetic paucity of C. sativa is foreseen as the most crucial limitation for successful breeding and improvement of this crop. A potential solution to this challenge could be gene introgression from Camelina wild species or from resynthesized allohexaploid C. sativa. However, both approaches would require a complete comprehension of the evolutionary trajectories that led to the C. sativa origin. Although there are some studies discussing the origin and evolution of Camelina hexaploid species, final conclusions have not been made yet. Here, we propose the most complete integrated evolutionary model for the Camelina genus based on the most recently described findings, which enables efficient improvement of C. sativa via the interspecific hybridization with its wild relatives. We also discuss issues of interspecific and intergeneric hybridization, aimed on improving C. sativa and overcoming the genetic paucity of this crop. The proposed comprehensive evolutionary model of Camelina species indicates that a newly described species Camelina neglecta has a key role in origin of tetra- and hexaploids, all of which have two C. neglecta-based subgenomes. Understanding of species evolution within the Camelina genus provides insights into further research on C. sativa improvements via gene introgression from wild species, and a potential resynthesis of this emerging oilseed crop.

Telomere sequence variability in genotypes from natural plant populations: unusual block-organized double-monomer terminal telomeric arrays.

BACKGROUND: Telomeres are the nucleoprotein complexes that physically cap the ends of eukaryotic chromosomes. Most plants possess Arabidopsis-type telomere sequences (TSs). In addition to terminal TSs, more diverse interstitial TSs exists in plants. Although telomeres have been sufficiently studied, the actual diversity of TSs in land plants is underestimated. RESULTS: We investigate genotypes from seven natural populations with contrasting environments of four Chenopodium species to reveal the variability in TSs by analyzing Oxford Nanopore reads. Fluorescent in situ hybridization was used to localize telomeric repeats on chromosomes. We identified a number of derivative monomers that arise in part of both terminal and interstitial telomeric arrays of a single genotype. The former presents a case of block-organized double-monomer telomers, where blocks of Arabidopsis-type TTTAGGG motifs were interspersed with blocks of derivative TTTAAAA motifs. The latter is an integral part of the satellitome with transformations specific to the inactive genome fraction. CONCLUSIONS: We suggested two alternative models for the possible formation of derivative monomers from telomeric heptamer motifs of Arabidopsis-type. It was assumed that derivatization of TSs is a ubiquitous process in the plant genome but occurrence and frequencies of derivatives may be genotype-specific. We also propose that the formation of non-canonical arrays of TSs, especially at chromosomal termini, may be a source for genomic variability in nature.

Regulation of plant epigenetic memory in response to cold and heat stress: towards climate resilient agriculture.

Plants have evolved to adapt and grow in hot and cold climatic conditions. Some also adapt to daily and seasonal temperature changes. Epigenetic modifications play an important role in regulating plant tolerance under such conditions. DNA methylation and post-translational modifications of histone proteins influence gene expression during plant developmental stages and under stress conditions, including cold and heat stress. While short-term modifications are common, some modifications may persist and result in stress memory that can be inherited by subsequent generations. Understanding the mechanisms of epigenomes responding to stress and the factors that trigger stress memory is crucial for developing climate-resilient agriculture, but such an integrated view is currently limited. This review focuses on the plant epigenetic stress memory during cold and heat stress. It also discusses the potential of machine learning to modify stress memory through epigenetics to develop climate-resilient crops.

Genetic diversity of the breeding collection of tomato varieties in Kazakhstan assessed using SSR, SCAR and CAPS markers.

Tomato is one of the most prominent crops in global horticulture and an important vegetable crop in Kazakhstan. The lack of data on the genetic background of local varieties limits the development of tomato breeding in the country. This study aimed to perform an initial evaluation of the breeding collection of tomato varieties from the point of view of their genetic structure and pathogen resistance using a set of PCR based molecular markers, including 13 SSR markers for genetic structure analysis, and 14 SCAR and CAPS markers associated with resistance to five pathogens: three viruses, fungus Fusarium oxysporum, and oomycete P hytophthora infestans. Nine SSR markers were with a PIC value varying from 0.0562 (low information content) to 0.629 (high information content). A weak genetic structure was revealed in the samples of varieties including local cultivars and, predominantly, varieties from Russia and other ex-USSR countries. The local varieties were closely related to several groups of cultivars of Russian origin. Screening for a set of resistance markers revealed the common occurrence of the resistance locus I against Fusarium oxysporum and only the occasional presence of resistance alleles of other markers. No markers of resistance to the three considered viruses were revealed in local tomato varieties. Only two local cultivars had markers of resistance to P. infestans, and only the 'Meruert' cultivar had a combination of resistance markers against P. infestans and F. oxysporum. The obtained results have demonstrated the need for further studies of local tomato varieties with a wider range of molecular markers and source germplasm to lay a foundation for the development of tomato breeding in Kazakhstan.

Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. This primer set is designed to set up any variants of the primers pool for whole-genome sequencing of SARS-CoV-2 using single- or double-tiled amplicons from 1.2 to 4.8 kb with the Oxford Nanopore. This multiplexed set of primers is also applicable for tasks like targeted SARS-CoV-2 genome sequencing. We proposed here an optimized protocol to synthesize cDNA using Maxima H Minus Reverse Transcriptase with a set of SARS-CoV-2 specific primers, which has high yields of cDNA template for RNA and is capable of long-length cDNA synthesis from a wide range of RNA amounts and quality. The proposed protocol allows whole-genome sequencing of the SARS-CoV-2 virus with tiled amplicons up to 4.8 kb on low-titer virus samples and even where RNA degradation has occurred. This protocol reduces the time and cost from RNA to genome sequence compared to the Midnight multiplex PCR method for SARS-CoV-2 genome sequencing using the Oxford Nanopore.

The power of retrotransposons in high-throughput genotyping and sequencing.

The use of molecular markers has become an essential part of molecular genetics through their application in numerous fields, which includes identification of genes associated with targeted traits, operation of backcrossing programs, modern plant breeding, genetic characterization, and marker-assisted selection. Transposable elements are a core component of all eukaryotic genomes, making them suitable as molecular markers. Most of the large plant genomes consist primarily of transposable elements; variations in their abundance contribute to most of the variation in genome size. Retrotransposons are widely present throughout plant genomes, and replicative transposition enables them to insert into the genome without removing the original elements. Various applications of molecular markers have been developed that exploit the fact that these genetic elements are present everywhere and their ability to stably integrate into dispersed chromosomal localities that are polymorphic within a species. The ongoing development of molecular marker technologies is directly related to the deployment of high-throughput genotype sequencing platforms, and this research is of considerable significance. In this review, the practical application to molecular markers, which is a use of technology of interspersed repeats in the plant genome were examined using genomic sources from the past to the present. Prospects and possibilities are also presented.

Genetic Diversity in Natural Populations of Rhodiola Species of Different Adaptation Strategies.

Representatives of the Crassulaceae family's genus Rhodiola are succulents, making them distinctive in a changing environment. One of the most significant tools for analyzing plant resources, including numerous genetic processes in wild populations, is the analysis of molecular genetic polymorphism. This work aimed to look at the polymorphisms of allelic variations of the superoxide dismutase (SOD) and auxin response factor (ARF) gene families, as well as the genetic diversity of five Rhodiola species, using the retrotransposons-based fingerprinting approach. The multi-locus exon-primed intron-crossing (EPIC-PCR) profiling approach was used to examine allelic variations in the SOD and ARF gene families. We implemented the inter-primer binding site (iPBS) PCR amplification technique for genome profiling, which demonstrated a significant level of polymorphism in the Rhodiola samples studied. Natural populations of Rhodiola species have a great capacity for adaptation to unfavorable environmental influences. The genetic variety of wild populations of Rhodiola species leads to their improved tolerance of opposing environmental circumstances and species evolutionary divergence based on the diversity of reproductive systems.

Investigation of mutation load and rate in androgenic mutant lines of rapeseed in early generations evaluated by high-density SNP genotyping.

Oilseed rape (Brassica napus) is an important oil crop distributed worldwide with a broad adaptation to different climate zones. The cultivation of rapeseed is one of the most commercially viable areas in crop production. Altogether 269,093 ha of rapeseed are cultivated in Kazakhstan. However, all rapeseed cultivars and lines cultivated in Kazakhstan on an industrial scale predominantly belong to the foreign breeding system. Therefore, the formation of a diverse genetic pool for breeding new, highly productive cultivars adopted to the environmental conditions of Kazakhstan is the most important goal in country selection programs. In this work, we have developed ethyl methanesulfonate (EMS) doubled haploid mutant lines from plant material of cultivars 'Galant' and 'Kris' to broad diversity of rapeseed in Kazakhstan. The development of mutant lines was performed via embryo callusogenesis or embryo secondary callusogenesis. Mutants were investigated by Brassica90k SNP array, and we were able to locate 24,657 SNPs from 26,256 SNPs filtered by quality control on the genome assembly (Bra_napus_v2.0). Only 18,831 SNPs were assigned to the available annotated genomic features. The most frequent combination of mutations according to reference controls was adenine with guanine (70%), followed by adenine with cytosine (28.8%), and only minor fractions were cytosine with guanine (0.54%) and adenine with thymine (0.59%). We revealed 5606.27 markers for 'Kris' and 4893.01 markers for 'Galant' by mutation occurrence. Most mutation occurrences were occupied by double mutations where progenitors and offspring were homozygous by different alleles, enabling the selection of appropriate genotypes in a short period of time. Regarding the biological impact of mutations, 861 variants were reported as having a low predicted impact, with 1042 as moderate and 121 as high; all others were reported as belonging to non-coding sequences, intergenic regions, and other features with the effect of modifiers. Protein encoding genes, such as wall-associated receptor kinase-like protein 5, TAO1-like disease resistance protein, receptor-like protein 12, and At5g42460-like F-box protein, contained more than two variable positions, with an impact on their biological activities. Nevertheless, the obtained mutant lines were able to survive and reproduce. Mutant lines, which include moderate and high impact mutations in encoding genes, are a perfect pool not only for MAS but also for the investigation of the fundamental basis of protein functions. For the first time, a collection of mutant lines was developed in our country to improve the selection of local rapeseed cultivars.

The role of LTR retrotransposons in plant genetic engineering: how to control their transposition in the genome.

KEY MESSAGE: We briefly discuss that the similarity of LTR retrotransposons to retroviruses is a great opportunity for the development of a genetic engineering tool that exploits intragenic elements in the plant genome for plant genetic improvement. Long terminal repeat (LTR) retrotransposons are very similar to retroviruses but do not have the property of being infectious. While spreading between its host cells, a retrovirus inserts a DNA copy of its genome into the cells. The ability of retroviruses to cause infection with genome integration allows genes to be delivered to cells and tissues. Retrovirus vectors are, however, only specific to animals and insects, and, thus, are not relevant to plant genetic engineering. However, the similarity of LTR retrotransposons to retroviruses is an opportunity to explore the former as a tool for genetic engineering. Although recent long-read sequencing technologies have advanced the knowledge about transposable elements (TEs), the integration of TEs is still unable either to control them or to direct them to specific genomic locations. The use of existing intragenic elements to achieve the desired genome composition is better than using artificial constructs like vectors, but it is not yet clear how to control the process. Moreover, most LTR retrotransposons are inactive and unable to produce complete proteins. They are also highly mutable. In addition, it is impossible to find a full active copy of a LTR retrotransposon out of thousands of its own copies. Theoretically, if these elements were directly controlled and turned on or off using certain epigenetic mechanisms (inducing by stress or infection), LTR retrotransposons could be a great opportunity to develop a genetic engineering tool using intragenic elements in the plant genome. In this review, the recent developments in uncovering the nature of LTR retrotransposons and the possibility of using these intragenic elements as a tool for plant genetic engineering are briefly discussed.

Epigenetic stress memory: A new approach to study cold and heat stress responses in plants.

Understanding plant stress memory under extreme temperatures such as cold and heat could contribute to plant development. Plants employ different types of stress memories, such as somatic, intergenerational and transgenerational, regulated by epigenetic changes such as DNA and histone modifications and microRNAs (miRNA), playing a key role in gene regulation from early development to maturity. In most cases, cold and heat stresses result in short-term epigenetic modifications that can return to baseline modification levels after stress cessation. Nevertheless, some of the modifications may be stable and passed on as stress memory, potentially allowing them to be inherited across generations, whereas some of the modifications are reactivated during sexual reproduction or embryogenesis. Several stress-related genes are involved in stress memory inheritance by turning on and off transcription profiles and epigenetic changes. Vernalization is the best example of somatic stress memory. Changes in the chromatin structure of the Flowering Locus C (FLC) gene, a MADS-box transcription factor (TF), maintain cold stress memory during mitosis. FLC expression suppresses flowering at high levels during winter; and during vernalization, B3 TFs, cold memory cis-acting element and polycomb repressive complex 1 and 2 (PRC1 and 2) silence FLC activation. In contrast, the repression of SQUAMOSA promoter-binding protein-like (SPL) TF and the activation of Heat Shock TF (HSFA2) are required for heat stress memory. However, it is still unclear how stress memory is inherited by offspring, and the integrated view of the regulatory mechanisms of stress memory and mitotic and meiotic heritable changes in plants is still scarce. Thus, in this review, we focus on the epigenetic regulation of stress memory and discuss the application of new technologies in developing epigenetic modifications to improve stress memory.

Retrotransposons: How the continuous evolutionary front shapes plant genomes for response to heat stress.

Long terminal repeat retrotransposons (LTR retrotransposons) are the most abundant group of mobile genetic elements in eukaryotic genomes and are essential in organizing genomic architecture and phenotypic variations. The diverse families of retrotransposons are related to retroviruses. As retrotransposable elements are dispersed and ubiquitous, their "copy-out and paste-in" life cycle of replicative transposition leads to new genome insertions without the excision of the original element. The overall structure of retrotransposons and the domains responsible for the various phases of their replication is highly conserved in all eukaryotes. The two major superfamilies of LTR retrotransposons, Ty1/Copia and Ty3/Gypsy, are distinguished and dispersed across the chromosomes of higher plants. Members of these superfamilies can increase in copy number and are often activated by various biotic and abiotic stresses due to retrotransposition bursts. LTR retrotransposons 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. Additionally, LTR retrotransposons influence the gene expression patterns of adjacent genes by modulating potential small interfering RNA (siRNA) and RNA-directed DNA methylation (RdDM) pathways. Furthermore, comparative and evolutionary analysis of the most important crop genome sequences and advanced technologies have elucidated the epigenetics and structural and functional modifications driven by LTR retrotransposon during speciation. However, mechanistic insights into LTR retrotransposons remain obscure in plant development due to a lack of advancement in high throughput technologies. In this review, we focus on the key role of LTR retrotransposons response in plants during heat stress, the role of centromeric LTR retrotransposons, and the role of LTR retrotransposon markers in genome expression and evolution.

Correction: Ramakrishnan et al. The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation. Int. J. Mol. Sci. 2021, 22, 11387.

The authors would like to make the following corrections to the original publication [...].