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Genetic variation among the Azerbaijani isolates of anamorphs of Cochliobolus spp., the causal agents of common root rot of wheat, was evaluated using pathogenicity assessments, sequence analyses of the internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, as well as inter-simple sequence repeat (ISSR) and inter-primer binding site (iPBS) markers. Twenty-eight isolates used in this study were obtained from diseased wheat plants in cereal growing regions of Azerbaijan in 2017. Bipolaris sorokiniana, Curvularia spicifera, and Curvularia inaequalis were identified. Bipolaris sorokiniana isolates were the most virulent on wheat seedlings, followed by isolates of C. spicifera and C. inaequalis. Phylogenetic analyses of a combined dataset of the ITS and GPDH regions grouped the isolates into three clusters, each of which contained isolates of one species. The dendrogram derived from the unweighted pair-grouped method by arithmetic average (UPGMA) cluster analyses based on the data of ISSR and iPBS markers divided the isolates into three clusters in concordance to their taxonomic grouping at species level, but without correlation to their geographic origins. Population structure of isolates was estimated based on Bayesian modelling, and this showed three populations (K = 3) supporting the separation of isolates in the dendrogram with the greatest mean value of Ln likelihood (-893,8). Utilization of the markers either separately or together produced a high level of polymorphism at interspecies level, which allowed for the separation of species. Although both marker systems had similar discrimination power to reveal genetic differences among the species, ISSR markers were more informative for eliciting intraspecies polymorphisms within B. sorokiniana and C. spicifera isolates. This is the first study on genetic diversity and population structure of anamorphic stages of Cochliobolus associated with common root rot of wheat using iPBS markers.

Photoautotrophic micropropagation (PAM), which employs sugar-free media and ventilated culture vessels to enhance plantlet photoautotrophic capacity and overall quality, has substantial potential to increase propagation efficiency when integrated with an appropriate propagation protocol. In this study, we established a PAM propagation protocol for medicinal cannabis (Cannabis sativa L.) using two genotypically different cultivars, ‘Charlotte’ and ‘Auto Charlotte’, and systematically evaluated the genetic similarity of plantlets across all successive batches via inter-simple sequence repeat (ISSR) markers as well as their performance after transplanting. Furthermore, a cultivar-specific logistic growth model was used to estimate annual yield and optimize the mother plant culture cycle for maximizing production. Results showed that under PAM conditions, mother plants sustained 11 batches of shoot tip harvest within a 98-d cycle. The propagation coefficients of ‘Charlotte’ and ‘Auto Charlotte’ reached 12.0 (95%CI: 11.0-13.0) and 11.3 (95%CI: 10.5-12.2), respectively. After 35 days of culture, plantlets from all batches exhibited uniform morphological and physiological traits, with rooting rates exceeding 70%. These plantlets could be directly transplanted without acclimatization, and minor growth differences between batches were eliminated by extending the culture cycle by one week. ISSR markers detected no obvious polymorphism between regenerated plantlets and their respective mother plants, with all similarity coefficients exceeding 0.90. Model simulations suggested that a 70-day culture cycle could maximize annual production, reaching a yield of 50,417–54,034 plants m−2. In conclusion, this study established an efficient PAM propagation protocol with high genetic similarity for the two tested medicinal cannabis cultivars, thereby providing technical support for cannabis plantlet production.

To identify the powdery mildew (PM) resistance gene in mungbean, inter-simple sequence repeat (ISSR) markers and newly developed ISSR-anchored resistance gene analog (ISSR-RGA) markers were evaluated. When F 2:7 and F 2:8 recombinant inbred line populations derived from a cross between CN72 (susceptible cultivar in Thailand) and V4718 (resistant line from Asian Vegetable Research and Development Center) were evaluated for PM resistance under field conditions, the PM resistance gene from V4718 was found to be inherited as a single major gene. Fifteen out of 75 ISSR primers produced 27 DNA bands putatively associated with PM resistance in bulk segregant analysis (BSA). Ten ISSR primers were combined with four RGA primers homologous to the nucleotide-binding site and kinase domains of resistance ( R ) genes to generate 40 ISSR-RGA primer combinations. When these 40 ISSR-RGA primer combinations and 10 corresponding ISSR primers were used in BSA, 873 ISSR and 756 ISSR-RGA loci were amplified. Fifty-two of 756 ISSR-RGA loci were new, and 11 of these 23 ISSR-RGA loci were putatively associated with the PM resistance. Simple linear regression confirmed that 5 of the 27 ISSR markers and 3 of the 11 ISSR-RGA markers were significantly associated with the PM resistance gene. When these eight ISSR and ISSR-RGA markers were used for quantitative trait loci (QTL) analysis, multiple interval mapping identified a major QTL, qPMC72V18-1 , explaining up to 92.4% of the phenotypic variation, flanked by I42PL229 and I85420 markers at the distance of 4 and 9 cM, respectively. These results suggest that ISSR and ISSR-RGA markers are highly efficient tools for mapping PM resistance gene in mungbean. The markers closely linked to the PM resistance gene will be useful for future marker-assisted selection to develop mungbean varieties resistant to PM.

Agricultural sustainability is increasingly challenged by climate variability, habitat loss, and erosion of genetic diversity by narrowing the genetic base of cultivated crops. Wild relatives of crop species harbor valuable adaptive traits for climate resilience, yet many remain underexplored and endangered. The present study aimed to develop an integrated conservation framework combining biotechnological propagation, molecular characterization, and ecological reintroduction of endangered wild Brassica species. Field surveys were conducted in arid and semi-arid regions of northwestern India to collect ten wild and allied species. In vitro micropropagation protocols were optimized using Murashige and Skoog medium with different hormonal combinations, followed by acclimatization and field reintroduction. Molecular characterization was performed using DNA barcoding (matK, rbcL, and cox2) along with ISSR and SCoT markers to assess genetic identity and diversity. Brassica tournefortii exhibited a maximum shoot regeneration rate of 81.2% on medium supplemented with 2.0 mg L⁻¹ BAP (6-benzylaminopurine) and 0.5 mg L⁻¹ NAA (α-naphthaleneacetic acid), while B. insularis showed the highest callus induction (77.9%) at 2.0 mg L⁻¹ 2,4-D. Reintroduced plantlets demonstrated a field survival rate of 72%. Molecular analyses revealed 89% interspecific polymorphism and clear ecological clustering among species. This study demonstrates the effectiveness of integrating micropropagation with molecular diagnostics and ecological restoration, highlighting endangered wild Brassica species as valuable genetic resources for climate-resilient agriculture.  

Lentil (Lens culinaris Medik. subsp. culinaris) constitutes one of the most important legumes globally, considering a major food source of protein, carbohydrates, and essential micronutrients. Nowadays, its cultivation is under increasing threat due to adverse climatic conditions. Knowledge of the genetic diversity and structure of the lentil germplasm will lead to a better optimization of its genetic resources in breeding programs in order to adapt to climate change. This study aimed to assess the genetic diversity and population structure of 31 lentil accessions consisting of 22 landraces and 9 commercial cultivars of small- and large-seeded types from Greece using ISSR markers. The ISSR-based characterization of the evaluated lentil populations revealed moderate genetic diversity, with polymorphism ranging from 14.29% to 45.45% and corresponding variation in Nei’s gene diversity (GD = 0.063-0.202) and Shannon’s information index (I = 0.091-0.289). Results from AMOVA indicated that 75% of the total variation fell within populations and so revealed strong intra-population heterogeneity, despite the species being highly self-pollinating. UPGMA and PCoA analyses revealed a central cluster of closely related populations and a set of genetically distinct lineages (DIG, KOR, PEL), while geographic structuring was not detected, suggesting the shaping impact of farmer-mediated seed exchange. Thus, these genetically diverse populations are considered important resources for widening the lentil gene pool for breeding, whereas those presenting low diversity may maintain locally adapted or rare alleles. In conclusion, the obtained results provide an important molecular basis for the conservation and further exploitation of Greek lentil landraces in future breeding programs.

Penstemon bicolor is a relatively rare, short-lived perennial herb of conservation concern and is found in the Mojave Desert and surrounding areas of southern Nevada, southeastern California, and northwestern Arizona. Two varieties of P. bicolor are named according to the color of their corolla: variety bicolor, the yellow or pink-colored form, and variety roseus, the magenta-colored form. The rarity of P. bicolor var. bicolor, coupled with its limited distribution, raises concerns about the genetic diversity present in the species, which is critical for the survival of species in the face of environmental stressors, such as habitat loss. Conservation management strategies for P. bicolor rely partially on the taxonomic status of variety bicolor and variety roseus; if these varieties are in fact distinct genetic lineages more appropriately defined as different species, then the implementation and urgency of management strategies would require updating accordingly. We analyzed amplified fragment length polymorphisms and inter-simple sequence repeats for 13 populations of P. bicolor from southern Nevada and northwestern Arizona to assess genetic diversity within the species and to identify the genetic distinctiveness, if any, between varieties. Our findings reveal surprisingly high levels of genetic diversity, in contrast to expectations for rare, perennial, outcrossing plants. Penstemon bicolor does, however, face ongoing threats of habitat destruction and potential local extirpation due to urbanization of the greater Las Vegas area, and we recommend that the current protective status of the species be maintained. Additionally, our analyses of differentiation failed to reveal genetic distinctiveness between varieties; we thus do not recommend that varieties of P. bicolor be elevated to species level.

As a new crop in Malaysia, forty-four Bambara groundnut ( Vigna subterranea L. verdc.) genotypes were sampled from eleven distinct populations of different origins to explore the genetic structure, genetic inconsistency, and fixation index. The Bambara groundnut, an African underutilized legume, has the capacity to boost food and nutrition security while simultaneously addressing environmental sustainability, food availability, and economic inequalities. A set of 32 ISSRs were screened out of 96 primers based on very sharp, clear, and reproducible bands which detected a total of 510 loci with an average of 97.64% polymorphism. The average calculated value of PIC  = 0.243, RP  = 5.30, H  = 0.285, and MI  = 0.675 representing the efficiency of primer set for genetic differentiation among the genotypes. The ISSR primers revealed the number of alleles ( Na  = 1.97), the effective number of alleles ( Ne  = 1.38), Nei's genetic diversity ( h  = 0.248), and a moderate level of gene flow ( Nm  = 2.26) across the genotypes studied. The estimated Shannon’s information index ( I  = 0.395) indicates a high level of genetic variation exists among the accessions. Based on Nei’s genetic dissimilarity a UPMGA phylogenetic tree was constructed and grouped the entire genotypes into 3 major clusters and 6 subclusters. PCA analysis revealed that first principal component extracted maximum variation (PC1 = 13.92%) than second principal component (PC2 = 12.59%). Bayesian model-based STRUCTURE analysis assembled the genotypes into 3 (best ΔK = 3) genetic groups. The fixation-index (F st ) analysis narrated a very great genetic diversity (F st  = 0.19 to 0.40) exists within the accessions of these 3 clusters. This investigation specifies the effectiveness of the ISSR primers system for the molecular portrayal of V. subterranea genotypes that could be used for genetic diversity valuation, detection, and tagging of potential genotypes with quick, precise, and authentic measures for this crop improvement through effective breeding schemes.

The differentiation of Cinchona spp. hybrids genotypes are challenging due to their shared origins and morphological similarities. This study utilized ISSR (Inter Simple Sequence Repeat) markers to distinguish between genotypes of Cinchona spp.: LF40, LC29, LF74, LF74GB, and LF211 maintained in vitro and ex vitro in Colombia. Genomic DNA was extracted from the plant materials, and eight ISSR primers were used for PCR amplification. In total 61 loci were amplified, of which 37 (60.92%) were polymorphic. The total number of loci per primer ranged from 5 to 12, with an average of 7.62, and polymorphic loci varied from 3 to 6, averaging 4.62 per primer. Cluster analysis based on the unweighted pair group method with arithmetic mean (UPGMA) grouped the genotypes into distinct clusters, showing genetic differences. Principal coordinate analysis (PCA) confirmed the clustering patterns, further distinguishing the genotypes despite their shared origins. The primer ISSR4 was the most effective, with the highest polymorphism rate (75%) and PIC value (0.473), followed by ISSR6 which had a polymorphism rate of 71% and a PIC value of 0.426, both primers allowed the identification of a group of plants under field conditions from in vitro cultures, with unknown genotype origin. As a result, it was possible to confirm a cluster of plants belonging to the LF40 genotype using only two primers. The results demonstrate the genetic distinctiveness of the selected Cinchona genotypes and underscore the utility of ISSR markers as a reliable tool for identifying genetic differences of in vitro and ex vitro Cinchona plant selections. La diferenciación de genotipos híbridos de Cinchona spp.  es un desafío debido a sus orígenes compartidos y similitudes morfológicas. En este estudio se utilizaron marcadores ISSR para distinguir entre los genotipos de Cinchona spp.: LF40, LC29, LF74, LF74GB y LF211, mantenidos en condiciones in vitro y ex vitro en Colombia. Se extrajo ADN genómico de los materiales vegetales y se utilizaron ocho cebadores ISSR para la amplificación por PCR. En total, se amplificaron 61 loci, de los cuales 37 (60,92%) fueron polimórficos. El número total de loci por cebafdor varió de 5 a 12, con un promedio de 7,62, y los loci polimórficos variaron de 3 a 6, con un promedio de 4,62 por cebador. El análisis de conglomerados basado en el método de grupos de pares no ponderados con media aritmética (UPGMA) agrupó los genotipos en distintos clusters, mostrando diferencias genéticas. El análisis de coordenadas principales (PCA) confirmó los patrones de agrupamiento, distinguiendo aún más los genotipos a pesar de sus orígenes compartidos. El cebador ISSR4 fue el más efectivo, con la mayor tasa de polimorfismo (75%) y valor PIC (0.473), seguido del ISSR6 que tuvo una tasa de polimorfismo del 71% y un valor PIC de 0.426, ambos cebadores permitieron la identificación de un grupo de plantas en condiciones de campo a partir de cultivos in vitro, con origen genotípico desconocido. Como resultado, fue posible confirmar un grupo de plantas pertenecientes al genotipo LF40 utilizando sólo dos cebadores. Los resultados demuestran la distinción genética de los genotipos de Cinchona seleccionados y subrayan la utilidad de los marcadores ISSR como una herramienta confiable para identificar diferencias genéticas de selecciones de plantas de Cinchona in vitro y ex vitro.

The species belonging to the Rhododendron genus are well-known for their colorful corolla. Molecular marker systems have the potential to elucidate genetic diversity as well as to assess genetic fidelity in rhododendrons. In the present study, the reverse transcription domains of long terminal repeat retrotransposons were cloned from rhododendrons and used to develop an inter-retrotransposon amplified polymorphism (IRAP) marker system. Subsequently, 198 polymorphic loci were generated from the IRAP and inter-simple sequence repeat (ISSR) markers, of which 119 were derived from the IRAP markers. It was shown that in rhododendrons, IRAP markers were superior to the ISSRs in some polymorphic parameters, such as the average number of polymorphic loci (14.88 versus 13.17). The combination of the IRAP and ISSR systems was more discriminative for detecting 46 rhododendron accessions than each of the systems on their own. Furthermore, IRAP markers demonstrated more efficiency in genetic fidelity detection of in-vitro-grown R. bailiense Y.P.Ma, C.Q.Zhang and D.F.Chamb, an endangered species recently recorded in Guizhzhou Province, China. The available evidence revealed the distinct properties of IRAP and ISSR markers in the rhododendron-associated applications, and highlighted the availability of highly informative ISSR and IRAP markers in the evaluation of genetic diversity and genetic fidelity of rhododendrons, which may facilitate preservation and genetic breeding of rhododendron plants.

The growing extractive activity in forest areas, the inefficient use of management practices combined with the intensification of socioeconomic activities, are the main factors in the loss of biodiversity. In order to reduce environmental impacts, genetic characterization of populations allows to infer about their real situation. Thus, the present study aimed to perform an analysis of the genetic diversity of populations of Syagrus cearensis using ISSR markers. Populations AQU (Aquicultura), MTB (Mata do Bebo) and MOD (Mata Olho d´água) were sampled, totaling 53 individuals. ISSR markers generated a total of 61 loci. The AQU population had the highest polymorphism index (71%), followed by MTB (57%) and MOD (53%). AQU showed the highest index of genetic diversity, compared to the MTB and MOD populations. There was a high and significant genetic differentiation between populations. Bayesian analysis identified the existence of two groups (K = 2). The genetic bottleneck test was significant for the AQU and MOD populations, according to the SMM model. Thus, the populations of genetic diversity index close to the averages found for tropical species with a similar life history. The presence of a genetic bottleneck was detected in populations. The AQU population presented low sharing of genotypes with the others and should be prioritized in conservation activities.