SSR Markers Research Articles

Development of a robust SNP marker set for genotyping diverse gene bank collections of polyploid roses.

Due to genetic depletion in nature, gene banks play a critical role in the long-term conservation of plant genetic resources and the provision of a wide range of plant genetic diversity for research and breeding programs. Genetic information on accessions facilitates gene bank management and can help to conserve limited resources and to identify taxonomic misclassifications or mislabelling. Here, we developed SNP markers for genotyping 4,187 mostly polyploid rose accessions from large rose collections, including the German Genebank for Roses. We filtered SNP marker information from the RhWag68k Axiom SNP array using call rates, uniformity of the four allelic dosage groups and chromosomal position to improve genotyping efficiency. After conversion to individual PACE® markers and further filtering, we selected markers with high discriminatory power. These markers were used to analyse 4,187 accessions with a mean call rate of 91.4%. By combining two evaluation methods, the mean call rate was increased to 95.2%. Additionally, the robustness against the genotypic groups used for calling was evaluated, resulting in a final set of 18 markers. Analyses of 94 pairs of assumed duplicate accessions included as controls revealed unexpected differences for eight pairs, which were confirmed using SSR markers. After removing the duplicates and filtering for accessions that were robustly called with all 18 markers, 141 out of the 1,957 accessions showed unexpected identical marker profiles with at least one other accession in our PACE® and SSR analysis. Given the attractiveness of NGS technologies, 13 SNPs from the marker set were also analysed using amplicon sequencing, with 76% agreement observed between PACE® and amplicon markers. Although sampling error cannot be completely excluded, this is an indication that mislabelling occurs in rose collections and that molecular markers may be able to detect these cases. In future applications, our marker set could be used to develop a core reference set of representative accessions, and thus optimise the selection of gene bank accessions.

Identification and genetic diversity analysis of specific walnut F1 progeny based on SSR molecular markers: taking heart-shaped walnuts and Jinghong 1 as examples.

To gain a deeper understanding of the genetic diversity and authenticity of natural pollinated F1 progeny populations of specific walnuts (heart-shaped walnuts and Jinghong 1), as well as the genetic relationship between F1 offspring and their maternal parent. 32 heart-shaped walnut F1 offspring, 45 Jinghong 1 F1 offspring, and their maternal parent were selected as materials, and14 pairs of SSR markers were used for genetic diversity and parent-child relationship analysis. The results showed that Jr05, Jr11, Jr29, Jr44, Jr45, Jr52, Jr53, Jr55, and Jr56 could be used as SSR markers for the identification of hybrid offspring in heart-shaped walnuts. Jr35, Jr40, Jr45, Jr50, Jr52, and Jr53 could be used as SSR markers for the identification of hybrid offspring of Jinghong 1. XX21 had the closer genetic relationship with heart-shaped walnuts, and HR20 had the closer genetic relationship with Jinghong 1. Besides, the classification results of F1 offspring based on UPGMA clustering and STRUCTURE were different. This study not only laid the foundation for parental identification of specific walnut F1 progeny, but also provided theoretical reference for fixing hybrid advantages, germplasm innovation, and new variety breeding.

Identification of rice samples with rice blast resistance genes Pi-2 and Pi-b

Since rice blast is a disease that causes significant damage to rice all over the world, the intensive development of disease-resistant genotypes is of great urgency. The use of molecular biology technologies, new biotechnological approaches and molecular genetic methods allows creating disease-resistant varieties while reducing the time of their development, which plays an important role in growing competitive products. The current paper has highlighted the results of estimating rice samples developed at the FSBSI “ARC “Donskoy” for blast resistance. The objects of the study were new samples developed in the laboratory of rice breeding and seed production. In 2022–2023, there were screened one hundred eighty-seven rice samples. They were identified on a presence of the blast resistance gene Pi-2. One hundred fifteen samples were estimated for the blast resistance gene Pi-b. Genomic DNA was isolated from young rice leaves. After estimating the quantity and quality of the isolated DNA there was conducted a polymerase chain reaction for the target genes Pi-2 and Pi-b. In order to identify the alleles of the desired genes there were used specific intragenic molecular SSR markers of dominant (for Pi-2) and codominant (for Pi-b) types. Electrophoresis of the obtained amplicons was performed on 2 % agarose gels during an hour. The results of the analysis were recorded in ultraviolet light and processed in ImageLab 5.1 software. As a result of the conducted study, there were identified new rice breeding samples that carry blast resistance genes Pi-2 and Pi-b. These samples are recommended for use in the further breeding process for blast resistance.

Genetic diversity and population structure of local avocado (Persea americana mill.) from northern Tanzania assessed using SSR markers

Genetic diversity and population structure of local avocado (Persea americana mill.) from northern Tanzania assessed using SSR markers

Assessment of genetic biodiversity and association of micronutrients and agronomic traits using microsatellites and staining methods which accelerates high-micronutrients variety selections within different wheat groups

Evaluation of genetic biodiversity for micronutrients is crucial for breeding high-quality crops and addressing the negative impacts of mineral deficiencies. The objectives of this research were to assess genetic variation and the relationship between grain Fe and Zn levels and agronomic traits in a diverse collection of wheat varieties. Additionally, the study aimed to determine the correlation between microsatellite markers (SSR) and micronutrient quantities. A total of 42 genotypes (Iranian commercial cultivars, landraces, and Afghan and Swiss varieties) were evaluated over a two-year period. Fe and Zn levels were measured using two semi quantitative staining assays and atomic absorption spectrophotometry (AAS) facility. Semi-quantitative staining methods and AAS showed high correlations for micronutrient contents. Landraces exhibited higher Fe (63.79 mg/kg) and Zn (44.76 mg/kg) but lower grain yield compared with commercial cultivars. Heritability estimates ranged 53%-79.43%, suggesting that genetic variance played a higher contribution in the phenotypic variation of traits than environmental factors. Notably, Fe content displayed significant correlations with days to maturity. Canonical correlation analysis (CCA) revealed that Zn content was correlated with four agronomic traits. Evaluation of genetic diversity using SSR markers demonstrated high genetic variation among the genotypes tested. The analysis of polymorphism information content (PIC) indicated that SSR primers had an average PIC of 0.75, with the Xgwm192 primer exhibiting higher PIC than others. Several SSR markers revealed association with micronutrient content that can be used in marker-assisted selection (MAS) programs aimed at selection of high micronutrient genotypes. In conclusion, the findings underscored the substantial genetic diversity present in micronutrient levels among global wheat genotypes, the potential of landraces for micronutrients biofortification of wheat cultivars through cross hybridization, the utility of staining methods for screening high/low micronutrient genotypes, and use of microsatellite markers for marker-assisted breeding aiming to micronutrient improvement in breeding programs.

Development of Microsatellite Markers from Transcriptome of Eriocheir sinensis and Their Application in Multiplex PCR Panels

Background: The development of genetic markers is crucial for aquaculture and population genetics. Polyallelic microsatellite markers are widely used for breeding and family management of Eriocheir sinensis (E. sinensis), contributing to the sustainable development of the industry. Methods: Through transcriptome analysis, 100 highly polymorphic SSR markers were identified and developed. From these, four multiplex PCR panels, each containing 16 markers, were constructed and applied to parentage assignment in six E. sinensis families. Results: The number of alleles (Na) ranged from 7 to 21, the observed heterozygosity (Ho) varied from 0.356 to 0.951, and the expected heterozygosity (He) varied from 0.656 to 0.909. The polymorphism information content (PIC) ranged from 0.633 to 0.900. The CERVUS analysis showed that using only the two most informative multiplex PCR panels, assignment success rates exceeded 90.34%, with 100% accuracy in assigning offspring to parents when three or four panels were used. Conclusions: This method significantly enhances the efficiency of microsatellite marker analysis, which provides a convenient and cost-effective method for breeding programs and family management of E. sinensis.

Comparison of the chloroplast genomics of nine endangered Habenaria species and phylogenetic analysis

BackgroundHabenaria, a genus in the family Orchidaceae, are the nearly cosmopolitan orchids, and most species have significant medicinal and ornamental values. Despite the morphological and molecular data that have been studied in recent years, the phylogenetic relationship is still unclear.ResultsWe sequenced, assembled, and annotated the chloroplast (cp) genomes of two species (Habenaria aitchisonii Rchb.f. and Habenaria tibetica Schltr.ex Limpricht) of Habenaria grown on the Qinghai-Tibetan Plateau (QTP), and compared them with seven previously published cp genomes which may aid in the genomic profiling of these species. The two genomes ranged from 155,259–155,269 bp in length and both included 132 genes, encoding 86 proteins, 38 tRNAs and 8 rRNAs. In the cp genomes, the tandem repeats (797), SSRs (2195) and diverse loci (3214) were identified. Comparative analyses of codon usage, amino frequency, microsatellite, oligo repeats and transition and transversion substitutions revealed similarities between the species. Moreover, we identified 16 highly polymorphic regions with a nucleotide diversity above 0.02, which may be suitable for robust authentic barcoding and inferring in the phylogeny of Habenaria species. Among the polymorphic regions, positive selection was significantly exerted on several genes, such as cemA, petA, and ycf1. This finding may suggest an important adaptation strategy for the two Habenaria species on the QTP. The phylogenetic relationship revealed that H. aitchisonii and H. tibetica were more closely related to each other than to the other species, and the other seven species were clustered in three groups. In addition, the estimated divergence time suggested that the two species separated from the others approximately 0.39 Mya in the Neogene period. Our findings also suggest that Habenaria can be divided into different sections.ConclusionsThe results of this study enriched the genomics resources of Habenaria, and SSR marker may aid in the conservation management of two endangered species.

Thousands of trait-specific KASP markers designed for diverse breeding applications in rice (Oryza sativa).

This study aimed to broaden applicability of KASP for Oryza sativa across diverse genotypes through incorporation of ambiguous (degenerate) bases into their primer designs and to validate 4000 of them for genotyping applications. A bioinformatics pipeline was used to compare 129 rice genomes from 89 countries with the indica reference genome R498 and generate ∼1.6 million KASP designs for the more common variants between R498 and the other genomes. Of the designs, 98,238 were for predicted functional markers. Up to five KASP each for 1024 breeder-selected loci were assayed in a panel of 178 diverse rice varieties, generating 3366 validated KASP. The 84% success rate was within the normal range for KASP demonstrating that the ambiguous bases do not compromise efficacy. The 3366-trait-specific marker panel was applied for population structure analysis in the diversity panel and resolved them into four expected groups. Target variations in thirteen of the genome sequences used for designs were compared with the corresponding KASP genotypes of other accessions of the same thirteen varieties in the diversity panel. There was agreement across 12 varieties for 79% of markers. Ten varieties had high agreement (>88%) but a variety selected from a landrace had only 46.5% agreement. Breeders can now search for the validated KASP and >1 million so-far untested designs across three alternative reference genomes (including Niponbare MSU7), search for designs proximal to previously published SSR markers and retrieve the target variations in 129 rice genomes plus their genomic locations with +/-25 bp flanking sequences.

Species Differentiation of Prunus serrulata and Prunus xueluoensis Based on Combined Analysis of SSR and cpDNA Markers

Species Differentiation of Prunus serrulata and Prunus xueluoensis Based on Combined Analysis of SSR and cpDNA Markers

Genetic Diversity and Population Structure Analysis of Pinus elliottii Germplasm Resources in Jiangxi Province

This study aimed to compare and assess the genetic diversity and trends among the introduced family provenance, first-cycle superior trees breeding provenance, and improved-generation superior trees breeding provenance of Pinus elliottii using EST-SSR markers. The goal was to provide a foundation for advanced genetic improvement and sustainable utilization of P. elliottii in Jiangxi Province. A total of 417 individuals were analyzed for their genetic diversity and population structure using 19 pairs of SSR markers. The analysis identified 103 alleles across all the samples, with an average of 5.421 alleles per locus. Compared to other coniferous species, P. elliottii exhibited a moderate to high level of genetic diversity (I = 0.862, He = 0.457). Analysis of the molecular variance (AMOVA) revealed that 97.90% of the genetic variation occurred within provenances, consistent with a low genetic differentiation coefficient (Fst = 0.016 < 0.05) and high gene flow (Nm = 15.715) among provenances. In addition, analysis using STRUCTURE v. 2.3.4 software divided the 417 germplasm samples into two distinct groups, corroborating the results of the principal coordinates analysis (PCoA) and the unweighted pair group method with arithmetic (UPGMA) clustering analysis. Overall, the germplasm resources of P. elliottii exhibited rich genetic diversity, with the majority of the genetic variation occurring within provenances. For the genetic improvement of high-resin-yielding slash pines, breeding programs should prioritize populations with high genetic diversity while carefully selecting superior individuals from within those populations. These findings provide a solid foundation for breeding high-resin-yielding varieties and for future research on the sustainable utilization of these valuable resources.

Assessing Genetic Variability and Population Structure of Alnus glutinosa (Black Alder) in Kazakhstan Using SSR Markers.

Alnus glutinosa plays a crucial role in flood control, riverbank stabilization, and water purification. Recognized for its ecological significance, it is listed in the Red Book of Kazakhstan. This study investigated the genetic variability of A. glutinosa populations in Kazakhstan, analyzing 78 trees from seven populations in the Bayanaul mountain forest massif and the northern Turgay regions using 12 SSR markers. The study identified an average of 6.3 alleles and 2.783 effective alleles, as well as observed and expected heterozygosities of 0.570 and 0.562, respectively, reflecting genetic diversity. Among the populations, KS1 (northern Turgay) and PVL3 (Bayanaul) displayed the highest diversity, while PVL5 (Bayanaul) showed slightly lower diversity. The analysis of molecular variance results indicated that 86% of the genetic diversity occurred within populations, with 14% attributed to differences between populations. A UPGMA tree based on Nei's genetic distance revealed three distinct clusters, suggesting geographically structured genetic variability in A. glutinosa populations.

Diversity of Northwestern and North Caucasian Populations of <i>Puccinia striiformis</i> f. sp. <i>tritici</i> by Virulence and Microsatellite Loci in 2022

Yellow rust (Puccinia striiformis f. sp. tritici, Pst) is a potentially dangerous disease of wheat. Genetic protection of wheat is an environmentally safe method of control. For its successful application, information on the structure of regional populations of the pathogen is needed. The purpose of these studies is to characterize the virulence and molecular polymorphism of Russian Pst populations in 2022. Wheat leaves with Pst urediniopustules were obtained from the North Caucasus (Krasnodar Territory, Dagestan, Kalmykia) and Northwestern (Leningrad region) regions. Virulence analysis was performed on 14 isogenic lines (AvocetNIL) and 15 differentiator varieties. The polymorphism of 20 microsatellite loci was evaluated in molecular analysis. We used SSR markers recommended by the Global Rust Reference Center. Virulence was studied in 74 monopustular isolates: 29 Dagestan, 10 Krasnodar, 5 Kalmyk, 30 northwestern. Resistance to all isolates was shown for lines with genes Yr5, Yr10, Yr15, Yr24, Yr26 and varieties Moro (Yr10, YrMor) and Nord Desprez (Yr3, YrND, Yr+). Isolates virulent to the AvYr17 line were detected for the first time in Dagestan and Krasnodar populations. They were moderately represented in the northwestern population (13%). Significant variation was observed in lines and varieties with the Yr1 and Yr3 genes. There was a decrease in virulence to Yr7 and YrSp compared to 2019–2021. 28 phenotypes (races) were determined in the virulence analysis (15 in Dagestan, 11 in North West, 3 in Krasnodar and 2 in Kalmyk). A common phenotype was noted for three North Caucasian Pst samples. The genetic distances between the identified phenotypes were estimated. In the multidimensional diagram, most of them combined together in a common group, with the exception of three Dagestan phenotypes with the fewest virulence alleles. According to the Fst index, Dagestan and Kalmyk Pst collections were characterized by high similarity; others differed moderately from them. The long-term virulence dynamics (2019–2022) of Pst populations in the Northwestern and North Caucasian regions was assessed. High similarity was determined between all regional population samples in 2019 and 2020. The northwestern and Dagestan populations differed slightly from them in 2021 and 2022. In 2022, the Krasnodar and Kalmyk populations were divided into separate groups, which differed from each other and from the main group. Long-term results of virulence analysis indicate a high dynamic of the structure of Pst populations in Russia. All North Caucasian isolates and 23 Northwestern isolates were used in the SSR analysis. Six loci (RYN3, RYN9, RYN12, WU6, RJO21, RJO24) were monomorphic. Three polymorphic alleles were identified in the RYN13 and RJO27 loci and two in the remaining studied ones. Significant deviations from the Hardy-Weinberg equilibrium are noted for most loci. The observed heterozygosity exceeded the expected one, which indicates an excess of heterozygotes and the clonal origin of the population. The studied collection of isolates was represented by 20 multilocus genotypes (MGs) (Dagestan and Northwestern – 11 each, Krasnodar – 3, Kalmyk – 1). Common genotypes were detected in Dagestan, Krasnodar and Northwestern populations (MG_1); Dagestan, Kalmyk and Northwestern (MG_2); Dagestan and Krasnodar (MG_3, MG_4). The genetic distances between MGs were estimated. In the multidimensional diagram, they are divided into 4 groups. The main group included 80% of MGs. One Dagestan MG, two Northwestern MG and MG_3 common for Dagestan and Krasnodar collections, significantly differentiated from the main group and among themselves. According to the Fst index, most regional Pst collections were moderately differentiated among themselves, with the exception of Dagestan and Kalmyk, which is consistent with the virulence analysis. According to the Mantel test, a moderate correlation was found between the results of virulence analysis and SSR (r = 0.6). This indicates that both analyses can be used in assessing the genetic polymorphism of Pst. The high variability of Russian populations based on virulence and microsatellite loci determines the need for annual monitoring of regional Pst populations in Russia.

Morpho-biochemical and molecular characterization of new mung bean [Vigna radiata (L.) Wilczek] landraces for Cercospora leaf spot (CLS) disease resistance

Mung bean production is significantly lowered by Cercospora leaf spot (CLS) in various parts of the globe. The most effective way to increase mung bean resilience to this stress is to identify new CLS resistance sources. Therefore, a panel population of 13 landraces along with 2 susceptible check cultivar, and 11 SSR markers were taken for the present experiment. Correlation analysis suggested significant relationships between the quantitative traits, retained resistance (Banapur local, and Kalampur local), and moderately resistance (Dikhitapada local, and Fakirpur local 1) to CLS. However, total chlorophyll content (TCL) showed strong positive correlations with phenolic content (PHE) (0.984), peroxidase activity (POX) (0.931), and polyphenol oxidase activity (PPO) (0.969). Moreover, PHE (−0.769) and POX (−0.867) were also found negatively correlated with CLS disease reaction. The eigenvalues from the principal component analysis (PCA) indicated the first three principal components collectively explained 84.88 % of the total variance. Molecular analysis of the panel population suggested a PIC value range from 0.30 to 0.88, with an average 0.696, and markers such as cp01713 (0.84), cp02181 (0.87), and CEDG147 (0.88) had higher PIC values. Principal coordinate analysis (PCoA) suggested capturing of 69.06 % of the total variance by the first three axes, and the landraces were categorizing into three clusters (cluster A, B and C). The genetic distances among the landraces ranged from 0 to 0.608, and phylogenetic tree differentiated them into three major clusters at respective Jaccard's similarity coefficient i.e., Cluster I (0.68), II (0.75) and III (0.83). Meanwhile, the resistance and moderately resistance landraces were observed in cluster A and cluster B, and in cluster II and cluster III respectively. The resistance landraces obtained from the present study could be used as potential donor for breeding process towards CLS resistance in mung bean.

Genetic Diversity of Yellow and Red Berries Arabica Coffee Populations Grown in a Mix Populations in Garut, West Java, Indonesia, Based on SSR Markers

Farmers in Garut, West Java, grow mixed varieties of Arabica coffee (Coffea arabica L.). Subsequently, they use harvested beans as seeds. Intercrossing among varieties may result in hybrid progenies and harvesting hybrid progenies as seed results in genetic variations. This research aims to evaluate the genetic diversity of Arabica coffee grown in a mixed population. Ninety-one Arabica coffees comprised 37 Arabica cv. “Ahernt GRT KN” (yellow-), 45 “Sigararutang” (red-), and nine “S795” (red berries) were sampled. Twenty SSR primer pairs were validated using 15 samples representing three varieties; six were polymorphic and used to genotype 91 Arabica accessions. Genetic data were analyzed using PowerMaker 3.25 and Darwin version 6 software. The results showed that the six SSR loci generated from 2 – 3 alleles, with an average of 2.17 alleles per locus. Genetic analysis of Arabica coffee from Garut, West Java, generated SSR markers with an average PIC of 0,33 across loci and varieties. The PIC within Arabica coffee cv. “Ahernt GRT KN” and “Sigararutang” were low, and within “S795” was moderate. Those PICs indicate the presence of more genetic diversity within “S795” than the other two cultivars. The Ho across Arabica coffee cv. “Sigararutang” and “S795” were lower than the He values, confirming their self-pollination nature. However, the Ho values of Arabica coffee cv. “Ahernt GRT KN” was larger than the others, indicating the presence of residual heterozygosity and a low percentage of recent outcrossing. The low Ho values of “Sigararutang” suggest that Arabica coffee is homozygous. Arabica coffee cv. “S795” also showed a low Ho value, but its moderate He value indicates the presence of more genetic diversity than the othercultivars.

Introgression of Herbicide-Resistant Gene from Genetically Modified Brassica napus L. to Brassica rapa through Backcrossing.

Interspecific hybridization between two different Brassicaceae species, namely Brassica rapa ssp. pekinensis (♀) (AA, 2n = 2x = 20) and genetically modified Brassica napus (♂) (AACC, 2n = 4x = 38), was performed to study the transmission of a herbicide resistance gene from a tetraploid to a diploid Brassica species. Initially, four different GM B. napus lines were used for hybridization with B. rapa via hand pollination. Among the F1 hybrids, the cross involving the B. rapa (♀) × GM B. napus (♂) TG#39 line exhibited the highest recorded crossability index of 14.7 ± 5.7. However, subsequent backcross progenies (BC1, BC2, and BC3) displayed notably lower crossability indices. The F1 plants displayed morphological characteristics more aligned with the male parent B. napus, with significant segregation observed in the BC1 generation upon backcrossing with the recurrent parent B. rapa. By the BC2 and BC3 generations, the progeny stabilized, manifesting traits from both parents to varying degrees. Cytogenetic analysis revealed a substantial reduction in chromosome numbers, particularly in backcrossing progenies. BC1 plants typically exhibited 21-25 chromosomes, while BC2 progenies showed 21-22 chromosomes, and by the BC3 generation, stability was achieved with an average of 20 chromosomes. SSR marker analysis confirmed the progressive reduction of C-genome regions, retaining minimal C-genome-specific bands throughout successive backcrossing. Despite the extensive elimination of C-genome-specific genomic regions, the glyphosate resistance gene from the male parent B. napus was introgressed into BC3 progenies, suggesting that the glyphosate resistance gene located and introgressed in A-chromosome/genome regions of the Brassica plants.

R2R3-MYB transcription factor GmMYB68 is involved in the accumulation of soybean isoflavones

We aimed to investigate the regulatory function of the soybean transcription factor R2R3-MYB (GmMYB68) in isoflavone biosynthesis. Through comprehensive subcellular and chromosomal localization analyses, we found that GmMYB68 was predominantly localized to the nucleus and mapped to chromosome Gm04. Notably, SSR markers near this gene significantly correlated with seed isoflavone content. GmMYB68 overexpression markedly increased isoflavone contents, confirming its positive role in regulating isoflavone synthesis. GmMYB68 also played a crucial role in the response of soybean to abiotic stress. Using RNA-seq and yeast one-hybrid techniques, we discovered an intricate interaction between GmMYB68 and key isoflavone biosynthesis genes GmCHS7 and GmCHS8. These findings provide novel insights into the mechanisms underlying isoflavone biosynthesis. Furthermore, using yeast two-hybrid experiments, we identified proteins interacting with GmMYB68, suggesting roles in the synthesis of physiologically active compounds and abiotic stress response. We not only elucidated the regulatory mechanisms of GmMYB68 in isoflavone biosynthesis and abiotic stress response but also constructed a molecular network encompassing GmMYB68, GmCHS7, and GmCHS8. This network provides a theoretical basis for a better understanding of and strategies for improving soybean isoflavone biosynthesis.

Diversity of Northwestern and North Caucasian Populations of Puccinia striiformis f. sp. tritici by Virulence and Microsatellite Loci in 2022.

Yellow rust (Puccinia striiformis f. sp. tritici (Pst)) is a potentially dangerous disease of wheat. Genetic protection of wheat is an environmentally safe method to control the disease, but its successful application requires information on the structure of regional populations of the pathogen. Virulence and molecular polymorphism were characterized in two Russian Pst populations in 2022. Wheat leaves with Pst urediniopustules were collected from the North Caucasus (Krasnodar Krai, Dagestan, and Kalmykia) and Northwestern Russia (Leningrad Oblast). Virulence was tested on 14 isogenic lines (AvocetNIL) and 15 differentiator cultivars. Polymorphism at 20 microsatellite loci was evaluated in molecular analyses. The SSR markers were as recommended by the Global Rust Reference Center. Virulence was assessed in 74 monopustular isolates, including 29 Dagestan, 10 Krasnodar, 5 Kalmyk, and 30 Northwestern ones. Resistance to all isolates was observed in lines with the genes Yr5, Yr10, Yr15, Yr24, and Yr26 and the cultivars Moro (Yr10, YrMor) and Nord Desprez (Yr3, YrND, Yr+). Isolates virulent to the AvYr17 line were detected for the first time in the Dagestan and Krasnodar populations. Their occurrence was moderate (13%) in the Northwestern population. A significant variation was observed in lines and cultivars with the Yr1 and Yr3 genes. Virulence to Yr7 and YrSp was found to be lower than in 2019 to 2021. In total, 28 phenotypes (races) were determined in the virulence analysis (15 in Dagestan, 11 in the Northwestern region, 3 in Krasnodar, and 2 in Kalmykia). A common phenotype was detected in three North Caucasian Pst samples. Genetic distances between the phenotypes were estimated. Most phenotypes grouped together in a multidimensional diagram, with the exception of three Dagestan phenotypes with the fewest virulence alleles. Based on the Fst index, the Dagestan and Kalmyk Pst collections were high similar to each other and differed moderately from the other collections. Long-term trends in virulence (from 2019 to 2022) were assessed in the Pst populations. A high similarity was observed between all regional population samples in 2019 and 2020. The Northwestern and Dagestan populations slightly differed from the other populations in 2021 and 2022. In 2022, the Krasnodar and Kalmyk populations formed separate groups, which differed from each other and from the main group. The long-term virulence analysis indicated that the structure of Pst populations is highly dynamic in Russia. All North Caucasian isolates and 23 Northwestern isolates were used in the SSR analysis. Six loci (RYN3, RYN9, RYN12, WU6, RJO21, and RJO24) were monomorphic. Three polymorphic alleles were identified in each of the RYN13 and RJO27 loci; two alleles, in each of the remaining loci examined. Significant deviations from Hardy-Weinberg equilibrium were observed for most loci. The observed heterozygosity exceeded the expected one, suggesting a clonal origin of the Pst population. Twenty multilocus genotypes (MGs) were found in the total isolate collection (11 in Dagestan, 11 in the Northwestern region, 3 in Krasnodar, and 1 in Kalmykia). Common MGs were detected in the Dagestan, Krasnodar, and Northwestern populations (MG_1); Dagestan, Kalmyk, and Northwestern populations (MG_2); and Dagestan and Krasnodar populations (MG_3 and MG_4). Genetic distances between MGs were estimated. MGs formed four groups in a multidimensional diagram. A major group included 80% of MGs. One Dagestan MG, two Northwestern MGs, and MG_3 common for the Dagestan and Krasnodar collections were significantly differentiated from the major group and differed from each other. Based on Fst, most regional Pst collections were moderately differentiated from each other, with the exception of the Dagestan and Kalmyk collections. The finding was consistent with the virulence analysis results. The Mantel test detected a moderate correlation between the virulence and SSR data (r = 0.6). This indicates that both analyses can be used to assess genetic polymorphism in Pst. The high variability of the virulence and microsatellite loci warrants annual monitoring of regional Pst populations in Russia.

Integrated Transcriptomics and Metabolomics Studies Reveal Steroid Biosynthesis Pathway and BCL2 Inhibitory Diazo-Progesterone of Drimia indica for Conservation and Sustainable Utilization.

This study is the first report on the sequence of the transcriptome of Drimia indica, a non-model plant with medicinal properties found in a forest tribal belt, using the Illumina NovaSeq platform. The primary objectives of this study were to elucidate the gene expression profiles in different tissues, identify key regulatory genes and pathways involved in secondary metabolite biosynthesis, and explore the plant's potential pharmacological properties. The study generated 670087 unigenes from both leaves and roots and identified putative homologs of annotated sequences against UniProt/Swiss-Prot and KEGG databases. The functional annotation of the identified unigenes revealed the secondary metabolite biosynthetic process as the most prominent pathway, with gene enrichment analysis predominantly accounting for secondary metabolite pathways, such as terpenoid, steroid, flavonoid, alkaloid, selenocompound, and cortisol synthesis. The study also identified regulatory genes NAC, Bhlh, WRKY, and C2H2 on the transcriptome dataset. The functionally annotated unigenes suggested phytocompounds in Drimia indica to have multi-potent properties, such as anti-cancer, anti-inflammatory, and anti-diabetic activities, which has been further validated by GC-MS-based metabolite profiling. Notably, we have identified two novel molecules, di-azo progesterone and 4H-pyran-4-one 2,3-dihydro-3,5-dihydroxy- 6-methyl, with potential BCL2 inhibitory anticancer properties, supported by stable binding interactions observed in molecular docking and dynamics simulations. Additionally, an abundance of mono-nucleotide SSR markers has been identified, useful for genetic diversity studies. This study provides a foundational understanding of the molecular mechanisms in Drimia indica, highlighting its potential as a source for novel therapeutic agents and contributing valuable insights for future pharmacological and agricultural applications. However, further in vivo studies are warranted to confirm these findings and validate their pharmacological efficacy and therapeutic potential. The SSR markers identified also offer valuable tools for molecular genetics, plant breeding, and sustainable drug development.

Phenotypic, genetic, variation, and molecular function of CaMYB113 in pepper (Capsicum annuum L.).

Pepper (Capsicum annuum L.) is widely consumed vegetables worldwide, and F1 hybrids are highly sought after in the pepper seed industry. However, studies on gene mutations affecting the color of cotyledon are rare, and the same is true for peppers. In this study, a segregating population was developed by crossing the pepper accession 21C1344 with purple cotyledon and accession 21C912 with green cotyledon. Initially, a target genomic region was identified by screening polymorphic SSR markers distributed across 12 chromosomes. Subsequently, polymorphic markers were developed based on resequencing data from the two parental lines, and genetic linkage analysis was performed. This approach ultimately identified Capana10g001433 (CaMYB113) as the candidate gene responsible for the purple cotyledons. The gene mutation type in 21C912 represents a new mutation type distinct from the reported missense mutation types, and this mutation affects the biosynthesis of anthocyanins. Virus-induced gene silencing (VIGS) of CaMYB113 substantially decreased anthocyanin accumulation in the cotyledons. Subsequent overexpression of CaMYB113 resulted in purple callus and leaves of pepper, and changed the expression levels of downstream genes involved in anthocyanin synthesis. Yeast one-hybrid and dual-luciferase transient expression assays demonstrated the binding of CaMYB113 to anthocyanin biosynthesis-related genes, thereby regulating anthocyanin accumulation in pepper cotyledons.

A Study on the Morphology and Molecular Biology of Tropical Strawberries

The studies on the morpho-molecular characteristics of different strawberry genotypes were conducted at Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur, Bangladesh. BARI Strawberry 1, BARI Strawberry 2, BARI Strawberry 3, FA 005, and Festival were used for this experiment. This study was undertaken to assess five tropical strawberry genotypes at morphological and molecular level. BARI Strawberry 2 was recognised as the preeminent genotype among five strawberry cultivars based on fruit production per plant (594.73 grammes), yield per hectare (19.39 tonnes), and fruit count per plant (32.42). The SSR technique was utilised to assess genetic variation and connections among five strawberry cultivars. Initially, ten primers were evaluated, and finally, three primers—EMFv104, ARSFL-10, and ARSFL-15—were selected for analysis. ARSFL-10 produced the fewest polymorphic alleles (3), while the markers EMFv104 and ARSFL-15 generated the most polymorphism alleles (4). The allele frequency for each location ranged from 0.4 (EMFv104) to 0.6 (ARSFL-10). The PIC values ranged from 0.672 on EMFv104 to 0.4992 on ARSFL-10. EMFv104 and ARSFL-15 demonstrated gene diversity scores of 0.72, whereas ARSFL-10 achieved a value of 0.56. The genotypes demonstrating the minimal genetic dissimilarity were BARI Strawberry 1 and Festival, with a value of 0.16667. Primer EMFv104 and ARSFL-15 are regarded as the most efficacious SSR markers for strawberry genotypes.