Systemic Markers Research Articles

Optimization of high-throughput marker systems for genomic prediction in alfalfa family bulks.

Alfalfa (Medicago sativa L.) is a perennial forage legume esteemed for its exceptional quality and dry matter yield (DMY); however, alfalfa has historically exhibited low genetic gain for DMY. Advances in genotyping platforms paved the way for a cost-effective application of genomic prediction in alfalfa family bulks. In this context, the optimization of marker density holds potential to reallocate resources within genomic prediction pipelines. This study aimed to (i) test two genotyping platforms for population structure discrimination and predictive ability (PA) of genomic prediction models (G-BLUP) for DMY, and (ii) explore optimal levels of marker density to predict DMY in family bulks. For this, 160 nondormant alfalfa families were phenotyped for DMY across 11 harvests and genotyped via targeted sequencing using Capture-seq with 17K probes and the DArTag 3K panel. Both platforms discriminated similarly against the population structure and resulted in comparable PA for DMY. For genotyping optimization, different levels of marker density were randomly extracted from each platform. In both cases, a plateau was achieved around 500 markers, yielding similar PA as the full set of markers. For phenotyping optimization, models with 500 markers built with data from five harvests resulted in similar PA compared to the full set of 11 harvests and full set of markers. Altogether, genotyping and phenotyping efforts were optimized in terms of number of markers and harvests. Capture-seq and DArTag yielded similar results and have the flexibility to adjust their panels to meet breeders' needs in terms of marker density.

A prospective multicentre open label study of Magseed Pro marker and Sentimag Gen3 system to localise breast cancer and axillary lymph nodes: initial results

A prospective multicentre open label study of Magseed Pro marker and Sentimag Gen3 system to localise breast cancer and axillary lymph nodes: initial results

Оценка профессиональной педагогической подготовки в контексте принципа инклюзии в Беларуси

<p>The results of an empirical study of a comprehensive assessment of the professional training of future teachers of various specialties (preschool educators, primary school teachers, subject teachers, psychology teachers, social educators, speech pathologists) at higher education institutions in the Republic of Belarus are presented. The complexity of the assessment is ensured by the study of all components of professional teacher training (educational conditions, educational process, educational results) and the representativeness of the multi-subject groups of respondents (vice-rectors for academic affairs of institutions of higher education, teaching staff, graduate students and undergraduates). In order to obtain the research results, there were used author's checklists, 4 types of questionnaires correlated with the marker system (marker 1 – "educational conditions", marker 2 – "educational process", marker 3 – "educational results"), criteria, indicators. The study involved 8242 respondents, including the authority (10 vice-rectors for academic affairs), the teaching staff (1131 people), future teacher students of various specialties (7101 people). Mathematical and statistical data processing included the usee of Pearson's χ2 criterion (STATISTICA statistical package version 10.0). The study allowed us to see an important problem – "deficits" in the quality of university teaching staff training (especially its methodological component) that enables to solve problems of forming professional competencies in future teachers. The assessment of professional teacher training made it possible to determine the vectors of improvement of organizational and pedagogical conditions, content and methodological support of higher teacher-training education in the context of the implementation of the principle of inclusion, new methodological approaches to improving the quality of differentiated professional teacher training and to create a basis for making a number of managerial decisions.</p>

Overview on Current Selectable Marker Systems and Novel Marker Free Approaches in Fruit Tree Genetic Engineering.

Selectable marker genes are useful for recognizing which cells have integrated specific sequences in their genome after genetic transformation processes. They are especially important for fruit trees genetic transformation to individuate putatively genetically modified events, because most of the protocols used to genetic engineer these species are often unsuccessful or with low efficiency. Traditional selectable marker genes, mainly of bacterial origin, confer antibiotics/herbicides-resistance or metabolic advantages to transformed cells. Genes that allow the visual recognition of engineered tissues without using any selective agent, such as morphogenic regulators and reporter genes, are also used as selection tools to in vitro identify genetically modified regenerated lines. As final step, genetic engineered plants should be tested in field conditions, where selectable marker genes are no longer necessary, and strongly unpopular especially for the commercial development of the new products. Thus, different approaches, mainly based on the use of site-specific recombinases and/or editing nucleases, are being now used to recover marker-free fruit crops. This review describes and comments the most used and suitable selection tools of interest, particularly for fruit tree genetic engineering. Lastly, a spotlight highlights the biosafety aspects related to the use of selectable marker genes exploited for fruit species genetic engineering.

An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava.

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene (HbAN1, LOC110667474) from a rubber tree (Hevea brasiliensis Müll. Arg.) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Analysis of Inter-Primer Binding Site Retrotransposon Length Polymorphism in Selected Group of Vaccinium corymbosum

Abstract The genetic variability in 13 selected genotypes of Vaccinium corymbosum using re-trotransposon-based iPBS markers system was characterized in this study. The four selected iPBS primers amplified 232 fragments, average 58 fragments per primer, approximately 53.16% of which were polymorphic. The polymorphism information content of iPBS marker was 0.238. The hierarchical cluster using unweighted pair group method with arithmetic mean analysis divided selected genotypes into two main groups and several subgroups. The values of the Jaccard coefficient of genetic relatedness were between 0.036–0.243. The similarity between two sample sets was 3.6%–24.3%. This study confirmed inter-PBS amplification technique as a reproducible and trustable marker system, but has proved to be an effective DNA fingerprinting technology to generate polymorphism in Vaccinium corymbosum.

Assessment of Genomic Integrity of Vitex negundoL., An Important Indian Medicinal Plant, Using RAPD Markers

Vitex negundo is an Indian medicinal plant containing steroids, flavonoids, lignans and terpenoids that can be used as a precursor for commercial production. An efficient marker system such as Random Amplified Polymorphic DNA (RAPD) was used to assess the genetic integrity of V. negundo. The straightforward method RAPD can be used to evaluate genomic integrity because it uses a small amount of DNA for PCR amplification. Six out of thirteen RAPD primers generated 150 distinct bands, of which 31 were polymorphic, with an average of 5.16 polymorphic bands per primer. A maximum of up to 32 fragments were amplified, and an average of 25 per primer, and the amplicons varied in size between 100 and 2000bp. The percentage of polymorphism ranges from 12.9 to 22.5, with an average of 16.6. The PIC values ranged from 0.11 to 0.63 for RAPD primers. The study pointed out that RAPD markers evaluate the genetic fidelity in Vitex negundo. The UPGMA cluster analysis grouped all in vitro raised plantlets treated with different growth regulators such as BAP, DPU, TDZ, and mT. The principal component analysis also substantiates this clustering pattern. Thus, the phylogenetic relationship and a high genetic variation revealed in the present study could provide baseline data for the conservation and improvement of this plant in future. Also, the molecular marker identified in this study will be helpful in the authentication of this species to prevent adulteration in herbal medicine.

Implications of newly developed SNPs for conservation programmes for the threatened Nubian ibex (Capra nubiana) in Oman

Conservation of elusive species affected by habitat degradation, population fragmentation and poaching is challenging. The remaining wild populations of a desert-adapted ungulate, Nubian ibex (Capra nubiana), within Oman are small and fragmented. The appropriateness of captive insurance populations for reinforcing existing, or establishing new, wild populations remains uncertain for Oman due to ambiguity regarding their genetic provenance. For effective management of this threatened species, it is essential to assess the genetic relationships between the wild and captive populations, and to investigate hybridisation with domestic goats (Capra hircus). We identified 5,775 high-quality SNPs using double digest restriction-site associated DNA (ddRAD), to assess genetic structure, gene flow and divergence between Oman’s wild populations of Nubian ibex and in captivity, which are likely of North African provenance. We detected hybridisation with goats in captivity and recommend that genetic assessments of captive individuals are routinely used to evaluate their suitability for conservation programs. Building on previous mitochondrial evidence, substantial nuclear divergence (FST = 0.540) was found between wild Oman and captive populations, providing further evidence that Nubian ibex may be composed of multiple species and urgently needs a taxonomic review. Additionally, an appropriate insurance population should be established for Oman’s threatened wild population. The data provided here will be invaluable for developing marker systems to assess wild populations using low-quality DNA from non-invasive sampling. Consequently, it will support further research into Nubian ibex throughout their range and highlights the need to integrate genetic information for effective conservation management of Nubian ibex.

Assessing the population structure and genetic diversity of wheat germplasm with the iPBS-retrotransposons marker system

Context Wheat (Triticum aestivum L.) is an important crop that provides food to millions of people all over the world. Currently, wheat production is limited due to various biotic and abiotic stresses resulting from uneven patterns of climate change. Therefore, it is very important to develop climate-resilient wheat cultivars. Crop genetic diversity allows the scientific community to identify genetic variations that can be utilised in the development of improved cultivars. Aims This study planned to characterise the wheat germplasm with the iPBS-retrotransposons marker system. Methods A total of 30 iPBS-retrotransposons markers were screened and among these, the 12 most polymorphic markers were selected for further analysis. Key results Molecular characterisation yielded a total of 170 bands, of which 143 were polymorphic. A substantial level of genetic diversity was observed (mean effective number of alleles: 1.37, Shannon’s information index: 0.23, gene diversity: 0.35). Maximum genetic distance was observed in G9 and G60 genotypes. Analysis of molecular variance revealed that most genetic variation (95%) occurred within the populations. The model-based structure algorithm divided the studied germplasm into three populations based on their collection regions. Similarly, the neighbour-joining analysis also divided 70 tested wheat genotypes into three populations, whereas principal coordinate analysis divided the evaluated germplasm into four populations. Conclusions This study confirms the iPBS-retrotransposons as an ideal marker for the genetic diversity assessment studies for any crop, especially for wheat. Implications The results presented here will be helpful for the scientific community in the marker-assisted breeding of wheat.

Gender differences in postoperative pain, sleep quality, and recovery outcomes in patients undergoing visual thoracoscopic surgery

ObjectiveThe purpose of our study was to investigate the effect of gender on postoperative pain, sleep quality, and recovery outcomes in patients undergoing VATS surgery under general anesthesia. MethodPerioperative peripheral blood inflammatory markers system inflammation Index (SII) was recorded for perioperative inflammatory response. The visual analog scale (VAS) was used to evaluate pain level. And the Athens Insomnia Scale (AIS) was evaluated on the night before surgery (sleep preop 1), the first night after surgery (sleep POD 1), and the third night after surgery (sleep POD 3) for postoperative sleep. ResultIn this prospective cohort study, 79 males and 79 females were consecutively included. Females had significantly higher pain score (both rest and cough pain) compared to the males at 3 h after the surgery (3.85 ± 1.2 vs. 3.16 ± 1.1) (rest) (p < 0.001) and 5.10 ± 1.3 vs. 4.46 ± 1.6 (coughing) (p = 0.006)). Patients in the male group had significantly lower AIS scores than those in the female group at Sleep POD 1 and Sleep POD 3 (p = 0.024 and p = 0.045). And in both groups, postoperative SII was increased and statistically significant compared to preoperative SII (p < 0.001, respectively). Women presented higher levels of SII on the first day after surgery, and the increase of postoperative SII in females groups was significantly higher than that in male group when compared to preoperative SII (1806.33 ± 1314.8 vs 1430.55 ± 958.4) (p = 0.042). ConclusionThese findings highlight the complex multidimensional nature of postoperative pain, nausea and vomiting, sleep quality and the potential contributory role of sex in shaping these outcomes. Women had worse sleep quality, higher postoperative inflammatory response level and pain level than men.

Mosquitoes on a chip-environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR.

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Selection of marker systems for determining the allelic composition of high molecular glutenin loci in winter wheat

Aim. Selection of marker systems and conducting multiplex polymerase chain reactions to determine the allelic composition of the Glu-A1, Glu-B1 and Glu-D1 loci, which determine the high quality of flour in common wheat samples. Methods. Polymerase chain reaction (PCR) using specific primers for the Glu-A1, Glu-B1 and Glu-D1 loci of wheat. Results. PCR analysis was performed to detect the insertion at 43 b. p. in the MAR region, in varieties with the Glu-B1al allele, the presence of an amplicon 563 b. p. long was observed, for varieties that carry other alleles of the locus – an amplicon 520 b. p. long. The results on the Glu-D1 locus showed that for № 2, № 3, № 4, № 7, № 8, № 10, № 11, № 19, № 20, № 21 subunits (5 + 10) were identified. Conclusions. With the help of allele-specific primers, the allelic composition of loci of high molecular weight glutenins was determined in 22 lines wheat. Three alleles Glu-A1a, Glu-A1b, Glu-A1c were found in the Glu-A1 locus; in the Glu-B1 locus, only the Glu-B1al allele was detected, there are two alleles at the Glu-D1 locus: Glu-D1a and Glu-D1d (the Glu-D1a allele predominated). As a result of our research, samples of soft wheat (lines № 2, № 7, № 21) were selected, which can be attributed to genotypes with high baking quality of the flour.

Utility of a breast biopsy clip and a point marker system in tailored axillary surgery for patients with breast cancer after neoadjuvant chemotherapy.

Tailored axillary surgery (TAS) is a new approach for selective removal of metastatic lymph nodes. This study evaluated the safety and utility of TAS using a breast biopsy clip inserted into a metastatic lymph node and a point marker consisting of a short hook wire and nylon thread to remove the clipped lymph node. Patients with breast cancer and clinically confirmed metastases to one-to-three axillary lymph nodes were included in this study. A breast biopsy clip was inserted into the metastatic lymph nodes before neoadjuvant chemotherapy. TAS was performed in patients with ycN0 disease after neoadjuvant chemotherapy. The lymph nodes containing the clips were removed using a point marker. The success criteria for TAS were the removal of the lymph node into which the clip was inserted using a point marker and the identification of the sentinel lymph node. The false-negative rate was calculated for cases in which TAS and axillary lymph node dissection were performed. Thirty individuals from two institutions were enrolled between May 2021 and November 2022, of whom 20 underwent TAS. Ten patients had clinically positive axillary lymph nodes and underwent axillary lymph node dissection. No adverse events were observed in any patient using the clips or point markers. TAS was successful in 18 of the 20 patients (90%). Seven patients underwent TAS and axillary lymph node dissection with a false-negative rate of 0%. The use of clips and point markers to perform TAS is clinically feasible.

Streamlining of Simple Sequence Repeat Data Mining Methodologies and Pipelines for Crop Scanning.

Genetic markers are powerful tools for understanding genetic diversity and the molecular basis of traits, ushering in a new era of molecular breeding in crops. Over the past 50 years, DNA markers have rapidly changed, moving from hybridization-based and second-generation-based to sequence-based markers. Simple sequence repeats (SSRs) are the ideal markers in plant breeding, and they have numerous desirable properties, including their repeatability, codominance, multi-allelic nature, and locus specificity. They can be generated from any species, which requires prior sequence knowledge. SSRs may serve as evolutionary tuning knobs, allowing for rapid identification and adaptation to new circumstances. The evaluations published thus far have mostly ignored SSR polymorphism and gene evolution due to a lack of data regarding the precise placements of SSRs on chromosomes. However, NGS technologies have made it possible to produce high-throughput SSRs for any species using massive volumes of genomic sequence data that can be generated fast and at a minimal cost. Though SNP markers are gradually replacing the erstwhile DNA marker systems, SSRs remain the markers of choice in orphan crops due to the lack of genomic resources at the reference level and their adaptability to resource-limited labor. Several bioinformatic approaches and tools have evolved to handle genomic sequences to identify SSRs and generate primers for genotyping applications in plant breeding projects. This paper includes the currently available methodologies for producing SSR markers, genomic resource databases, and computational tools/pipelines for SSR data mining and primer generation. This review aims to provide a 'one-stop shop' of information to help each new user carefully select tools for identifying and utilizing SSRs in genetic research and breeding programs.

A cyclical marker system enables indefinite series of oligonucleotide-directed gene editing in Chlamydomonas reinhardtii.

CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for non-selectable target mutations. This becomes challenging when many sequential modifications are required in a single cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, non-selectable genes. We demonstrate that this 'Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)' technology enables an essentially limitless series of genetic modifications to be introduced into a single cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.

Validation of SNP Markers for Diversity Analysis, Quality Control, and Trait Selection in a Biofortified Cassava Population.

A validated marker system is crucial to running an effective genomics-assisted breeding program. We used 36 Kompetitive Allele-Specific PCR (KASP) markers to genotype 376 clones from the biofortified cassava pipeline, and fingerprinted 93 of these clones with DArTseq markers to characterize breeding materials and evaluate their relationships. The discriminating ability of the 36-quality control (QC) KASP and 6602 DArTseq markers was assessed using 92 clones genotyped in both assays. In addition, trait-specific markers were used to determine the presence or absence of target genomic regions. Hierarchical clustering identified two major groups, and the clusters were consistent with the breeding program origins. There was moderate genetic differentiation and a low degree of variation between the identified groups. The general structure of the population was similar using both assays. Nevertheless, KASP markers had poor resolution when it came to differentiating the genotypes by seed sources and overestimated the prevalence of duplicates. The trait-linked markers did not achieve optimal performance as all markers displayed variable levels of false positive and/or false negative. These findings represent the initial step in the application of genomics-assisted breeding for the biofortified cassava pipeline, and will guide the use of genomic selection in the future.

Nontechnical Skills Assessment in Acute Care Trauma Simulations: A Mixed Methods Approach Using Eye Tracking and Behavioral Marker Systems.

The condition of trauma patients and the urgent need for timely resuscitation present unique challenges to trauma teams. These difficulties are exacerbated for military trauma teams in combat environments. Consequently, there is a need for continued improvement of nontechnical skills (NTS) training for trauma teams. However, current approaches to NTS assessment rely on subjective ratings, which can introduce bias. Accordingly, objective methods of NTS evaluation are needed. Eye-tracking (ET) methods have been applied to studying communication, situation awareness, and leadership in many health care settings, and could be applied to studying physicians' NTS during trauma situations. In this study, we aimed to assess the relationship between trauma team leaders' objective gaze patterns and subjective expert NTS ratings during patient care simulations. After Institutional Review Board approval, 9 trauma teams from first-year post-graduate general surgery and emergency medicine residents were recruited to participate in 1 of 2 trauma simulations (a difficult airway case and a multi-patient trauma). Each scenario lasted approximately 15 minutes. All team leaders wore a mobile ET system to evaluate gaze metrics-time to first fixation (TTFF), average fixation duration (AFD), and total percentage of the scenario (TPS) focused on Areas of Interest (AOI), which included patient, care team, diagnostic equipment, and patient care equipment. Trained faculty raters completed the Non-Technical Skills for Surgeons (NOTSS) assessment tool and the Trauma Non-Technical Skills (T-NOTECHS) scale. One-way analysis of variance, Kruskal-Wallis, and appropriate post-hoc pairwise comparison tests were run to assess differences between ET metrics across AOI groups. Spearman's Rho tests were used to assess correlations between ET and subjective NTS ratings. Compared to other NTS domains, trauma teams scored relatively poorly on communication across both T-NOTECHS (3.29$ \pm $0.61, maximum = 5) and NOTSS (2.87$ \pm $0.66, maximum = 4). We found significant differences in trauma team leaders' TTFF between teammates and the patient (Team: 1.56 vs Patient: 29.82 seconds, P < .001). TTFF on the diagnostic equipment was negatively correlated (P < .05) to multiple measures of subjective NTS assessments. There were no significant differences in AFD between AOIs, and AFD on teammates was positively correlated (P < .05) to communication and teamwork. There were significant differences in TPS across most AOI pairs (P < .05), and the average TPS fixated was highest on the patient (32%). Finally, there were several significant correlations between additional ET and NTS metrics. This study utilized a mixed methods approach to assess trauma team leaders' NTS in simulated acute care trauma simulations. Our results provide several objective insights into trauma team leaders' NTS behaviors during patient care simulations. Such objective insights provide a more comprehensive understanding of NTS behaviors and can be leveraged to guide NTS training of trauma physicians in the future. More studies are needed to apply these methods to capture NTS from a larger sample of teams in both simulated and real trauma environments.

Development of a CRISPR/Cas9-mediated gene knockout method for functional genomics of the barley spot blotch pathogen Bipolaris sorokiniana

Bipolaris sorokiniana (=Cochliobolus sativus) is an important fungal pathogen that causes spot blotch, common root rot and kernel blight in barley and wheat. In this study, we aimed to develop a CRISPR/Cas9-mediated approach for efficiently knocking out genes of B. sorokiniana. We first assessed the efficiency of Cas9/sgRNA combined with the split marker system for gene replacement. We designed sgRNAs to target a polyketide synthase gene (PKS1) that is required for melanin biosynthesis of the fungus. When the preassembled Cas9/gRNA ribonucleoproteins (RNPs) and the split hygromycin B resistance gene (HygB) fragments each harboring 449 bp upstream and 595 bp downstream flanking sequences of PKS1 were co-transformed into the protoplasts of B. sorokiniana, the number of transformants with the PKS1 gene replaced by the selection marker were significantly increased compared to the control without RNPs. We then used the RNPs with PCR-amplified HygB gene cassette carrying 40 bp or 60 bp arms homologous to the flanking sequences of the PKS1 target site for fungal transformation and showed that the RNPs significantly enhanced gene disruption efficiency through the short-homology recombination, and more gene knockout mutants were obtained with longer (60 bp) homologous arms than the shorter (40 bp) ones. Finally, we disrupted an uncharacterized non-ribosomal peptide synthetase (NRPS) gene (NPSx) in B. sorokiniana strain ND90Pr using the RNP-mediated gene knockout approach and showed the mutants lost virulence on barley cv. Bowman. The Cas9/sgRNA-mediated gene knockout method developed will facilitate large-scale functional genomics studies of B. sorokiniana.

Plant Genetic Diversity Studies: Insights from DNA Marker Analyses

The plant adaptation response to a changing environment depends on the genetic diversity level it possesses. Genetic diversity and a thorough understanding of population indices are pivotal for decoding plant adaptation to dynamic environmental stressors. The development of polymerase chain reaction (PCR)-based molecular markers enables comprehensive population analyses and the precise detection of individuals and groups with unique genetic variations. Various molecular markers have been employed to assess genetic diversity, examine population structure, and delineate cluster patterns within and among populations. DNA markers revolutionize plant diversity studies by allowing detailed analyses of genetic variations, including economically significant trait-influencing genes. Despite their simplicity, they offer high reproducibility, ensuring accurate estimations of plant variation. Integrating multiple marker systems with advanced high-throughput sequencing techniques is poised to enhance the understanding and management of depleting plant genetic resources by providing a comprehensive picture of diversity at the genome-wide level. This review explores diverse molecular markers, elucidating their advantages and limitations, and highlights their impact on evaluating the genetic diversity and population structure of plants.

Diversity of genome size, endopolyploidy and SCoT markers in 20 Trigonella (Fabaceae) species

The Trigonella species possess medicinal, nutraceutical and pharmaceutical properties due to the presence of many bioactive compounds. Its therapeutic effects are mostly valuable in medicine, cosmetics and the functional food industry. Correct genetic characterisation of plant material is needed to increase the potential of Trigonella species by breeding and conservation programs. The aim of this study was to develop a reliable marker system to support the morphological and phytochemical analysis in Trigonella taxonomic research, species identification and characterization as well as determination of the interspecific variation within this genus along with relationships between species. For this purpose, flow cytometry and SCoT molecular markers were combined. Flow cytometric analyses revealed that Trigonella species possess very small and small genomes. The range of genome sizes was from 1.10 to 5.76 pg/2C, with most species possessing very small genomes (< 2.8 pg/2C). In seeds of 14 species endopolyploid nuclei were detected. Flow cytometric analysis of genome size enabled quick identification of four out of 20 species, while combined with endopolyploidy detection in seeds, facilitated distinction of the next seven species. ScoT molecular markers helped to identify closely related species with similar genome size and cell cycle activity. Therefore, flow cytometry was proposed as the first-choice method for quick accession screening, while the more detailed genetic classification was obtained using SCoT molecular markers.