Capsicum chinense germplasms. The study observed significant variability in carotenoid and capsaicinoid content among the germplasms. Correlation analysis revealed a strong positive correlation between violaxanthin and antheraxanthin. In contrast, capsaicin and dihydrocapsaicin displayed negative correlations with individual carotenoids but exhibited a strong positive correlation between the two compounds (r = 0.90 ***). Genotyping-by-sequencing (GBS) was performed on 160 genotypes of pepper germplasm, which identified 47,810 high-quality SNPs. A comprehensive genome-wide association analysis was performed using these SNPs to identify SNPs associated with carotenoids and capsaicinoids, revealing 193 SNPs that exhibited significant associations. Specifically, 4 SNPs were associated with violaxanthin, 2 with antheraxanthin, 86 with capsorubin, 5 with capsanthin, 63 with zeaxanthin, 3 with β-cryptoxanthin, and 2 with α-carotene. With further studies, the significantly associated SNPs identified in this study have the potential to be utilized for selecting pepper accessions with high carotenoid and capsaicinoid contents. Additionally, the genes associated with these significant SNPs will be used to understand their roles and involvement in the biosynthesis pathway of carotenoids and capsaicinoids. Understanding the function of these genes can provide insights into the molecular mechanisms underlying the production of these bioactive compounds in pepper. The findings of this study hold valuable implications for selecting pepper varieties with desirable traits and developing breeding programs aimed at enhancing the nutritional and medicinal properties of pepper.Pepper is a highly important vegetable globally, both economically and nutritionally. However, to efficiently select and identify genetic resources for pepper breeding programs, it is crucial to understand the association between important traits and genetic factors. In this study, we investigated the genetic basis of carotenoid and capsaicinoid content in 160  Solanaceae) family and the genus Capsicum. The genus Capsicum has approximately 35 species [Capsicum annum L., Capsicum chinese Jacq., Capsicum frutescens L., Capsicum baccatum L., and Capsicum pubescens Ruiz and Pav. [Pepper is a member of the nightshade  have become the preferred choice and are extensively utilized in agricultural breeding programs . To idenCapsicum chinense, a highly genetically diverse pepper species. The results of this study have the potential to significantly contribute to the breeding of improved pepper varieties with enhanced nutritional and medicinal properties. The use of SNP markers can accelerate the breeding process and facilitate the selection of plants with desirable traits, while the underlying genes can provide insights into the biochemical pathways involved in the biosynthesis of bioactive compounds in pepper. Further research can be conducted to explore the potential applications of these findings in pepper genomics and breeding.This study aimed to identify SNPs associated with carotenoid and capsaicinoid contents in the pepper germplasm of The results of the descriptive analysis for ten bioactive traits  in a sample of 160 germplasms are summarized in The present study used correlation analysis to investigate the pairwise associations among 10 variables labeled “A” through “J” . The corC. chinense accessions are provided in C. chinense accessions. A window size of 1 Mb was used for this analysis. The heatmap colors in the figure depict the density of SNPs, providing a visual representation of their distribution patterns. These SNPs were used for genetic association studies to explore the genetic basis of important pepper bioactive compounds (carotenoids and capsaicinoids).GBS was performed on 160 pepper germplasms using the Illumina HiSeq X Ten platform, resulting in a substantial dataset comprising approximately 1.2 billion total trimmed reads. The average mapping depth for a single accession was determined to be 17.61×, indicating a robust and thorough coverage of the pepper genome. After the variant calling process, a comprehensive set of 1,859,683 single nucleotide polymorphisms (SNPs) was identified, encompassing all 12 pepper chromosomes. The sequencing statistics for 160 The genome-wide association analysis conducted using 47,810 SNPs aimed to identify SNPs associated with carotenoids and capsaicinoids. The results of the analysis were visualized in Manhattan plots  and quan10 = 6.0. Out of these, 57 SNPs also exceeded a higher threshold of −log10 = 6.7. The SNPs that exhibited significant association with capsorubin were distributed across various chromosomes. Notably, the highest number of associations was observed on chromosomes 8, 9, and 2, with 24, 16, and 14 SNPs, respectively.Of the SNPs associated with violaxanthin, two were identified on chromosome 12. The first SNP  was found in an intergenic region, while the second SNP  was located within the gene encoding 6,7-dimethyl-8-ribityllumazine synthase. Another significant association was observed with a SNP on chromosome 3 , which resides within the gene encoding 4-hydroxycinnamoyl-CoA ligase 2. Additionally, one SNP on chromosome 07  was also found to be significantly associated with violaxanthin. Furthermore, two SNPs located on chromosome 12 demonstrated significant associations with both violaxanthin and antheraxanthin in relation to capsorubin, a total of 86 SNPs showed significant association, surpassing a Benferroni-corrected threshold of −logC. chinense. The characteristics of the gene  in which significant SNPs were identified can be found in In relation to capsanthin, β-cryptoxanthin, and α-carotene, a total of 10 SNPs were identified, with eight SNPs located in genic regions and three SNPs located in intergenic regions. Among these SNPs, five were associated with capsanthin, with three situated within genic regions and two found in intergenic regions. The genic SNP was specifically located within the CA12g19380 gene, which encodes for 6,7-dimethyl-8-ribityllumazine synthase. For β-cryptoxanthin, three SNPs were identified, with one SNP located in an intergenic region. In the case of α-carotene, two SNPs were found, both residing within genic regions. One genic SNP was located within the CA00g41080 gene, which encodes for RAB1X, a protein involved in intracellular transport. The other genic SNP was located within the CA12g19190 gene, which encodes for ubiquitin carboxyl-terminal hydrolase 19-like. A significant association with zeaxanthin was observed for a total of 63 SNPs. Among these, the major SNPs were distributed across chromosomes 3, 8, 12, and 1, with 12, 9, 7, and 7 SNPs, respectively. Among the identified SNPs associated with zeaxanthin, a total of 21 were selected and presented in 10 = 6.0 for capsaicin. Out of the significant SNPs linked to dihydrocapsaicin, 15 SNPs exceeded the highly significant threshold of −log10 = 6.7. These SNPs associated with dihydrocapsaicin were distributed across different chromosomes, including Chr08 , Chr01 , Chr01 , Chr01 , Chr01 , Chr01 , Chr01 , Chr01 , Chr06 , and Chr07 . Several of these SNPs were located within genes encoding various proteins, such as ATP binding protein, late embryogenesis abundant protein (LEA) family protein, steroleosin-B, and small MutS-related (Smr). Additionally, SNPs were also identified in intergenic regions, which are regions that do not correspond to known genes.For capsaicinoids, a total of 28 significant SNPs were found to be associated with dihydrocapsaicin, while no SNP reached the significant threshold of −logThe common SNPs associated with multiple traits are presented in Capsicum species and cultivars [Peppers’ widespread popularity stems from their visually appealing colors, diverse flavors, culinary versatility, and nutritional benefits. Peppers contain a variety of nutrients and bioactive compounds, including vitamins, carotenoids, capsaicinoids, anthocyanins, phenolic acids, and flavonoids ,37. Caroultivars .Capsaicinoids, a category of alkaloids responsible for the hot or spicy flavor, are synthesized and accumulated by the plant and are found largely in placental tissue near the seeds . Their cCucurbita maxima Duchesne, SNP markers showing pleiotropic effects among the different analyzed carotenoids were reported [The impact of genetic variation on the biochemical components of plant species has been explored using a number of molecular biology approaches. GWAS analysis plays a critical role in identifying genetic markers associated with traits of interest. A marker-trait association study was carried out utilizing 47,810 high-quality SNPs derived from 160 pepper accessions. A total of 193 SNPs were identified as significantly associated with individual carotenoids (165 SNPs) and capsaicinoids (28 SNPs). The SNPs discovered in this study were located in both genic and intergenic regions. SNPs can occur at different frequencies in different regions of chromosomes, including coding sequences of genes, non-coding regions of genes, and intergenic regions between genes . The prereported .The present study identified a SNP on chromosome 09 associated with the carotenoid capsorubin within a gene encoding an auxin response factor (ARF). This finding aligns with the existing literature that emphasizes the involvement of ARFs in plant growth and development, as well as their potential roles in carotenoid biosynthesis and responses to abiotic stresses . ResearcCapsicum and the expression profiles of specific bHLH transcription factors, such as CabHLH009, CabHLH032, CabHLH048, CabHLH095, and CabHLH100 from clusters C1, C2, C3, and C4, showed a correlation with the accumulation of carotenoids, including zeaxanthin, in the pericarp [bHLH66, PIF4, LOB13, NAC92, and APL were found to be enriched in multiple categories related to chlorophyll biosynthesis, regulation of chlorophyll biosynthesis, and carotenoid biosynthetic process [Another SNP was found on chromosome 04, which showed a significant association with zeaxanthin, one of the individual carotenoids. This SNP is located within a gene that encodes a member of the helix–loop–helix (bHLH) transcription factor (TFs) family. The bHLH is one of the largest TFs and plays a crucial role in regulating plant growth and development by interacting with other TFs in various biological processes ,65. Exispericarp . Additiopericarp . In anotpericarp . Moreove process . These fThe present study identified 28 SNPs associated with dihydrocapsaicin, among which one SNP was found in the gene encoding an ATP-binding protein. ATP-binding proteins are recognized for their critical roles in various cellular processes in plants, encompassing energy metabolism, signaling pathways, transport processes, and enzymatic activities . AdditioIn conclusion, the development of improved plant varieties with enhanced bioactive compounds and nutritional values is a crucial goal in plant breeding. Marker-assisted plant breeding approaches offer a valuable tool by enabling the study of genetic variants and their association with important traits of interest. In this study, we have identified a large number of significantly associated SNPs with carotenoids and capsaicinoids. These findings can assist future studies aiming to identify potential markers for the selection of pepper germplasm with high carotenoid and capsaicinoid content. Furthermore, the genes associated with the identified SNPs will provide valuable insights into their functions and their involvement in the biosynthesis pathways of these bioactive compounds for future studies.In this study, analytical grade reagents, extraction solvents, and carotenoid and capsaicinoid standards were used. The chemicals used were sourced from Sigma-Aldrich  and included carotenoid standards such as capsanthin, capsorubin, antheraxanthin, violaxanthin, zeaxanthin, beta-cryptoxanthin, alpha-carotene, and beta-carotene, as well as capsaicinoid standards including capsaicin and dihydrocapsaicin. Additional chemicals used in the study were potassium hydroxide, dichloromethane, methanol, sodium chloride, ascorbic acid, ammonium acetate, and methyl tert-butyl ether.Capsicum chinense genetic materials used in the study consisted of 160 accessions sourced from the National Agrobiodiversity Center’s (NAC) gene bank under the Rural Development Administration (RDA) in Jeonju, Republic of Korea. Each accession was represented by ten to twelve pepper plants, with three replications, which were cultivated in NAC greenhouses following the RDA’s pepper growing methods. Fully matured pepper fruits were collected, freeze-dried, powdered, and stored in a deep freezer at −70 °C for further analysis. Detailed information about the IT (introduction) numbers and origins of the 160 C. chinense pepper materials can be found in The w/v). The mixture was vortexed for 20 s and then placed in an 85 °C water bath for 5 min. Subsequently, saponification of the extract was carried out for 10 min in an 85 °C water bath using potassium hydroxide . After saponification, the samples were immediately cooled on ice, and 1.5 mL of cold deionized water was added. The extraction process was repeated twice using 1.5 mL of hexane. The resulting extracts were then centrifuged at 12,009× g, and the supernatant was filtered through a 0.2 µm syringe filter to obtain the final carotenoid extract for analysis.The pepper samples used in this study were all freeze-dried and powdered. The extraction, separation, and measurement of carotenoids by High-Performance Liquid Chromatography (HPLC) were conducted with minor modifications, following the procedure outlined by Kim et al. . To extrv/v) containing 10 mM ammonium acetate, and Solvent B, which was 100% methyl tert-butyl ether. The carotenoids were separated using HPLC  on a C30 YMC column  and detected at 450 nm. Solvent A was a mixture of methanol and water (92:8 v/v), containing 10 mM ammonium acetate. Solvent B was made of 100% methyl tert-butyl ether. The following gradient elution conditions were employed: 0 min (83% A and 17% B), 23 min (70% A and 30% B), 29 min (59% A and 41% B), 35 min (30% A and 70% B), 40 min (30% A and 70% B), 44 min (83% A and 17% B), and 55 min (83% A and 17% B) at a 1 mL/min flow rate. Calibration curves were created for quantification purposes by graphing four distinct concentrations of carotenoid standards based on the peak area ratios of the standards. The analysis was executed in triplicate.The separation of carotenoids was carried out using HPLC on an Agilent 1260/90 Infinity II system  equipped with a C30 YMC column . The detection of carotenoids was performed at a wavelength of 450 nm. The mobile phase consisted of two solvents: Solvent A, a mixture of methanol and water  0.2 μm syringe filter to remove any remaining particulate matter and prepared for analysis.The capsaicinoid analysis was conducted using Ultra-Performance Liquid Chromatography (UPLC) with an AQUITY UPLC H-Class instrument  and an ACQUITY UPLC HSS T3 1.8 µm 2.1 × 50 mm column. The column temperature was maintained at 30 °C, and a flow rate of 0.4 mL/min was used for the mobile phase. The sample temperature was set at 20 °C. The total run time for the analysis was 7 min, with a delay time of 5 min. An injection volume of 2 µL was used. The mobile phase consisted of a 45% acetonitrile isocratic condition. The seal solvent, purge solvent, and needle solvent were all 45% acetonitrile. The Photo Diode Array (PDA) detector was set to measure the absorbance at a wavelength of 280 nm, which is suitable for detecting capsaicinoids. To prepare the capsaicin and dihydrocapsaicin standards for analysis, precise measurements were taken. Initially, 5 mg of each standard was accurately weighed and dissolved in 5 mL of acetonitrile, resulting in a working solution with a concentration of 1000 mg/L. Working solutions for capsaicin and dihydrocapsaicin were prepared in a concentration range from 1.95 mg/L to 1000 mg/L and 1.95 mg/L to 250 mg/L, respectively. These prepared standards were then utilized in the subsequent analysis to determine the concentration of capsaicinoids present in the sample. The analysis was executed in triplicate.For the extraction of genomic DNA from the young leaves of each accession, we followed the cetyltrimethylammonium bromide (CTAB) protocol . The DNAC. chinense reference genome v1.2 .Demultiplexing was performed using barcode sequences, followed by removal of adapter sequences and sequence quality trimming. Adapter and barcode sequences were removed using the software Cutadapt (version 1.8.3) . Low-quaDuring the mapping process, a SAM file was generated with default parameter values, except for specific options set as follows: a seed length (−l) of 30, maximum differences allowed in the seed (−k) of 1, number of threads (−t) used for processing of 16, mismatch penalty (−M) of 6, gap opening penalty (−O) of 15, and gap extension penalty (−E) of 8. The resulting SAM files were used for raw SNP discovery using SAMtools (version 0.1.16) . From thPrior to SNP detection, SNP validation was carried out using the SEEDERS  in-houseThe association analysis was conducted using the 47,810 SNPs obtained from 160 pepper individuals. Imputation of missing genotypes was performed using the BEAGLE algorithm . For theCapsicum genome database  CDS), tomato and potato genomes at https://solgenomics.net/ and the NCBI  database were utilized in this analysis. The search focused on a 200-kb region surrounding the SNP, including 100 kb on each side. The flanking sequences of the SNP were obtained from the C. chinense genome database. These sequences were then compared against the Capsicum genome database and the NCBI database to identify genes or gene regions that exhibited similarity or alignment.A Basic Local Alignment Search Tool (BLAST) was employed to search for candidate genes adjacent to the SNP of interest. The The Microsoft Excel program was utilized for data summary and descriptive statistics of the carotenoids and capsaicinoids. Furthermore, the R software (version 4.2.1) was employed to perform correlation analysis (“pheatmap” package).


Triticum aestivum L. plants subjected to dehydration  was investigated. SNP pretreatment stimulated germination and seedling growth in normal conditions and protected them under dehydration. These effects were confirmed by percentage of seed germination, changes in fresh and dry weight of 5–6-day-old seedlings, as well as by seedlings’ linear dimensions, visual appearance, and mitotic index of the root apical meristem. Assessment of the transpiration intensity (TI) and relative water content (RWC) showed that SNP pretreatment helped to maintain the water status of seedlings subjected to dehydration stress. The data obtained by enzyme-linked immunosorbent assay (ELISA) suggested that the positive effects of SNP may be due to its influence on the phytohormonal system. SNP pretreatment induced an increase in the level of indolylacetic acid (IAA) and especially cytokinins (CK), while essential changes in ABA content were not detected. Water deficiency caused a substantial increase in ABA content and a decrease in the levels of CK and IAA. Pre-sowing SNP treatment decreased stress-induced fluctuations in the content of all studied phytohormones. Using reverse-transcription PCR (RT-PCR), we obtained data on the increase in expression of the TADHN dehydrin gene in SNP-pretreated seedlings under normal and, especially, under dehydration conditions. These findings may indicate the participation of dehydrins in NO-induced defense reactions in wheat plants under water stress. Furthermore, exogenous NO had a stabilizing effect on membrane cellular structures, as evidenced by the reduction of electrolyte leakage (EL) levels and malondialdehyde (MDA) content in dehydrated wheat seedlings under the influence of pre-sowing SNP treatment.Nitric oxide (NO) is a universal signaling molecule with important regulatory functions in the plant’s life cycle and adaptation to a wide spectrum of environmental stresses including drought. The effect of pre-sowing seed treatment with the donor of NO sodium nitroprusside  on wheat  Drought is one of the most widespread abiotic stress factors, with detrimental effects on metabolic reactions and physiological processes throughout the plant life cycle ,2,3. TheData on drought mitigation in different plant species under exogenous NO treatment suggest that it can be practically applied to improve the stress tolerance and productivity of crops ,21,22,23Among the most numerous dehydration-inducible genes are dehydrins, encoding LEA (Late Embryogenesis Abundant) proteins of group II, which accumulate in the seeds at late stages of their maturation during natural desiccation. These proteins are characterized by high hydrophilicity and structural plasticity and are essential for maintaining the embryo structures’ viability. Dehydrins also accumulate in vegetative plant tissues under water deficit conditions and exert various protective functions related to their chaperone, ion-binding, and antioxidant properties ,28. The Triticum aestivum L. with the SNP at the concentration of 200 μM on germination, subsequent growth, transpiration intensity, relative water content (RWC), the state of the hormonal system, and the integrity of membrane cell structures of wheat seedlings under dehydration conditions simulated by treatment with PEG-8000. Considering the fact that dehydrins contribute significantly to the plant tolerance to water deficit conditions, a comparative analysis of expression of the TADHN dehydrin gene in wheat seedlings, pretreated with SNP and subjected to PEG-induced dehydration, was performed.The present work is devoted to the investigation of the effect of pre-sowing seed treatment of wheat The influence of pre-sowing SNP treatment on wheat growth intensity under normal and dehydration conditions was assessed by the following indicators: seed germination , fresh wSoaking seeds prior to sowing in the 200 µM SNP solution for 3 h stimulated their germination under normal conditions, increasing seedling emergence by about 15% relative to the control level . GerminaPre-sowing SNP treatment had a stimulating and protective effect on the subsequent vegetative growth of wheat seedlings under normal and dehydration conditions, respectively . The SNPAn important component of plant growth is the process of cell division in the apical root meristem. Pre-sowing SNP treatment promoted the root growth of wheat seedlings, as indicated by the increase of the mitotic index (MI) by more than 20% in comparison with SNP-untreated plants a, as welSNP treatment contributed to the maintenance of the MI value and cell division process in roots of wheat seedlings under the influence of water deficit, which could be judged by the recovery of cell size and the character of their staining a,b. The SNP seed treatment had no significant effect on the transpiration process under normal growing conditions in wheat seedlings, in which values of transpiration intensity (TI) were close to the control levels a. DehydrPre-sowing SNP seed treatment had a significant effect on the hormonal system of wheat seedlings as judged by changes in the abscisic acid (ABA), indolylacetic acid (IAA), and cytokinin (CK) content. In 5–6-day-old SNP-pretreated seedlings under normal growing conditions, in the absence of any significant shifts in ABA content, an increase in the concentration of IAA and CK by approximately 20% and more than 50%, respectively, was revealed. Their enhanced levels were kept in the seedlings throughout the experiment a–c. UndeTADHN dehydrin gene expression in 5–6-day-old seedlings, which was manifested by an increase in the level of its transcript accumulation by about 25–30% compared to that of the control SNP-untreated samples (TADHN gene transcript content. SNP pretreatment further increased the dehydration-induced expression of the TADHN dehydrin gene by about 50% so that at the end of the experiment (24 h), the level of dehydrin gene transcript accumulation reached more than 3.5-fold values relative to those of the control  and electrolyte leakage (EL) levels in 5–6-day-old seedlings . DehydraTriticum aestivum L.) seeds with 200 µM SNP for 3 h on germination, subsequent seedling growth, their transpiration rate, relative water content, hormone balance, and expression of the TADHN dehydrin gene under normal conditions and under exposure to PEG-induced dehydration were analyzed.Drought is one of the most widespread and unpredictable stress factors, negatively affecting all aspects of plant metabolism and development and significantly limiting the productivity of different crops, including wheat . The regA. thaliana, barley, lettuce, and other plant species [It was found that SNP pretreatment had a stimulating and protective effect on the wheat seeds’ germination under normal and water deficit conditions , which i species ,33,34. U species . Supplem species . The ger species ,37. Evid species . The pra species ,19.Atnoa1 mutant lines of A. thaliana with reduced endogenous NO production are characterized by severe growth inhibition and defective root development [Glycine max) with 100 μM SNP improved their growth under normal conditions and under water stress, as evidenced by the data of their biomass increasing [Physalis angulata plants with SNP at concentrations of 50–100 μM had a positive effect on their growth and photosynthetic activity under soil drought [Lycopersicon esculentum) and cucumber (Cucumis sativus) plants, increasing their branching by enhancing the growth of lateral and adventitious roots [CYCD 3;1 and CYCA 2;1 was observed under the influence of SNP treatment, indicating the involvement of NO in the regulation of the cell cycle [We obtained data on the effectiveness of pre-sowing SNP seed treatment on the growth stimulation of wheat seedlings in normal conditions and their protection under water stress . SNP preelopment . Sprayincreasing . Pretrea drought . Incubat drought . SNP treus roots ,43. In tll cycle . This isBrassica juncea plants incubated in the presence of 100 µM SNP for 4 days maintained their indices of RWC, transpiration rate, and stomatal conductance under water stress  [Lactuca sativa L.) improved photosynthesis by increasing chlorophyll synthesis; activated antioxidant enzymes, protecting lettuce plants against oxidative damage; and increased irrigation water consumption, thus increasing the yield under soil drought [The establishment of drought tolerance in plants is highly dependent on their ability to maintain water status and limit water loss. In our experiments, it was shown that stress treatment 12% PEG) caused serious disturbances in the water balance of wheat, which were manifested in a significant decrease in the TI indices of leaves and RWC of roots and shoots of 5-day-old seedlings . SNP seeEG, 10%) . SNP treEG, 10%) . Exogeno drought . Thus, o% PEG cau drought ,45,46,47The fact that various physiological processes, in particular, germination, vegetative growth, and water metabolism, are regulated by phytohormones under normal or stress conditions ,49 suggeTADHN dehydrin gene [TADHN gene transcripts  plants was observed under the treatment with spermidine belonging to the polyamines, which are one of the sources of oxidative NO production in photosynthetic organisms [nNOS gene with increased endogenous NO production, an enhancement in the expression activity of the transcription factors OsDREB2A and OsDREB2B, which are involved in the regulation of expression of many drought-related genes, including dehydrins, was observed, as well as an increase in the gene expression of the OsLEA3 protein, which belongs to the family of late embryogenesis abundant proteins [Haberlea rhodopensis under drought conditions was accompanied by the accumulation of dehydrin proteins, which was detected by Western blot analysis [The stress-protective effect of NO at the molecular level is achieved by inducing transcription factors followed by activating the gene expression of defensive proteins ,3,4,10. rin gene  in SNP-pnscripts . Exposur control . There arganisms ,55. In tproteins . The incanalysis . Thus, t2●−), peroxynitrite (ONOO−) is formed, which belongs to the class of active nitrogen forms and is characterized by high reactivity. However, the level of its toxicity is much lower than that of O2●− or H2O2, so its formation is considered as a direct NO-induced mitigation of oxidative burst during stress impact [It is well-known that the rapid nonspecific plant response to environmental stresses is a sharp increase in the generation of reactive oxygen species (ROS), which induce oxidative damage of biopolymers and membrane lipids, affecting the configuration and functions of cellular structures. NO can reduce the level of oxidative stress by interacting with ROS or by activating the antioxidant system (AOS) of plants ,10,13,20s impact ,57. An iTriticum aestivum L., variety Ekada-70, were collected from the Chishminsky Breeding station . Seeds were sterilized in 96% ethanol for 1 min, washed thoroughly with running water, drained of excess moisture with filter paper, and soaked for 3 h in a solution of NO donor sodium nitroprusside (SNP) at a concentration of 200 μM. Samples soaked in distilled water served as control.Seeds of spring soft wheat To evaluate the protective effect of NO on wheat germination under drought conditions, SNP-treated and untreated seeds were sown in Petri dishes containing a disk of filter paper moistened with 10 mL of water (control) or PEG-8000 solutions at concentrations of 3%, 6%, 9%, and 12%. Samples were placed in the thermostat TS-1/80 SPU  with a temperature of 21 °C, and seeds were germinated in the dark for 3 days. Germination was determined by the percentage ratio of the number of germinated seeds to the total number of sown seeds.−2 s−1. Then, 4-day-old samples were transferred into glasses of up to 1 L, 30 seedlings in each glass containing 40 mL of 10% Hoagland-Arnon solution, and incubated for 24 h to stabilize the growth process. After that, 5-day-old seedlings were subjected to dehydration by using PEG-8000 at a concentration of 12% for 3, 9, and 24 h. Growth intensity was judged by changes in fresh and dry weight of 5–6-day-old seedlings; linear sizes of roots, shoots, and whole seedlings; their visual appearance; and mitotic index of root apical meristem, which were evaluated after 24 h of stress treatment. For subsequent analysis of phytohormone content, MDA production, and accumulation of TADHN dehydrin gene transcripts, plant material was fixed in liquid nitrogen and stored at −70 °C.SNP-pretreated and untreated seeds were sown in Petri dishes with filter paper moistened with 10 mL of water and germinated at 20-21°C, 16 h photoperiod, and illumination of 180 µmol mhttps://imagej.net/Fiji (accessed on 20 October 2020)).To determine the MI, the apical part of seedling roots together with the root cap was fixed for 1.5 h in a 1:3 mixture of acetic acid and ethanol. After fixation, plant material was washed for 30 min in running tap water and then treated for 1 h with a mixture of 5% pectinase (EC 3.2.1.15)  and 5% cellulase (EC 3.2.1.4) ; enzyme treatment was carried out at 37 °C. The pressed preparations were stained with acetocarmine made in 45% acetic acid solution. MI of apical root meristem cells was counted as the sum of cells in prophase, metaphase, anaphase, and telophase stages, expressed as the percent of the total number of counted cells . In eachTo assess the intensity of transpiration, which was carried out using the gravimetric method, 10 seedlings were used per experimental variant. The 5-day-old seedlings were placed into the small glasses containing 10 mL of 10% Hoagland–Arnon solution or a mixture of 10% Hoagland–Arnon solution with 12% PEG-8000. Then, the glasses were covered with aluminum foil to prevent evaporation. The glasses were weighed every hour from 1 h to 5 h of stress treatment. The transpiration rate was calculated by subtracting the value of the final weight of the sample from the value of the initial weight, followed by division by the number of seedlings .RWC analysis was performed using the same samples in which TI was evaluated. For calculation of the RWC, it was necessary to determine the fresh weight (FW), turgor weight (TW), and dry weight (DW) of roots and shoots . For thig, and the supernatant was evaporated in a stream of air to an aqueous residue. An aliquot of the aqueous residue was used for the subsequent determination of the total content of zeatin derivatives  immunopositive to serum obtained against zeatin riboside. ABA and IAA were extracted with sulfuric ether from the remaining aqueous extract and subjected to immunoassay using polyclonal rabbit antibodies to ABA and IAA.The content of free IAA, ABA, and cytokinins in one plant sample was determined by enzyme-linked immunosorbent assay (ELISA) using rabbit antibodies specific to each of these hormones and peroxidase-labeled anti-rabbit antibodies. The extraction of phytohormones  from one plant sample was carried out according to the method described in detail earlier . SamplesTADHN dehydrin gene was investigated using a semi-quantitative RT-PCR analysis, as was described in detail previously [2, 75 mM KCl). The obtained cDNA samples were amplified using gene-specific primers . To determine the electrolyte leakage, the samples of plant material (1 g) were cut into equal sections of about 1 mm and placed into the small glasses with 20 mL distilled water. Samples were stirred on a shaker  at 140 rpm, 35 °C for 1.5 h, filtered, and used for electrical conductivity measuring using a conductometer/TDS meter HI 8633 . Electrolyte leakage levels were judged from the obtained conductivity values expressed in µS g−1 FW [Changes in MDA content and electrolyte leakage levels served as indicators of lipid peroxidation and membrane permeability, respectively . To detep < 0.05.All experiments were performed in at least three or four biological and analytical replicates. Statistical analysis of the obtained data was carried out by ANOVA, SPSS version 19.0 for Windows . The significance of differences was evaluated using the LSD test at TADHN dehydrin gene in seedlings under the influence of SNP pretreatment under normal and especially under stress conditions may indicate the dehydrins’ involvement in the protective effects of NO on wheat plants under dehydration. The data on the decline in MDA production and electrolyte leakage levels in dehydrated wheat seedlings under the influence of SNP pretreatment support the ability of NO to participate in plant defense against oxidative stress under drought.The data obtained in this work indicate that SNP, which is a donor of NO, displayed the properties of a growth stimulator and inductor of wheat plants’ resistance to dehydration when used at a concentration of 200 μM for a 3 h seed treatment. This was clearly demonstrated in a comparative analysis of seed germination as well as when assessing the fresh and dry weights, linear dimensions, and cell division rates of SNP-pretreated wheat seedlings subjected to PEG-induced dehydration. The beneficial effect of NO donor on wheat plants under normal and water deficit conditions may be attributed to its participation in the regulation of water regime, as indicated by RWC and TI data, and achieved through its interaction with the phytohormonal system, judging from the changes in the content of ABA, IAA and CK. SNP treatment of seeds caused the accumulation of auxins and cytokinins in the seedlings, both of them being known activators of plant metabolic activity, which can explain the pronounced growth-stimulating effect of NO. At the same time, these hormones are also involved in the regulation of stress responses in plants. In addition, SNP-pretreated plants were characterized by a decrease in the amplitude of stress-induced fluctuations in the content of all studied phytohormones. The data obtained in this work on the increase in the expression of the


Aspergillus fumigatus is a ubiquitous, critical priority human fungal pathogen. Despite its clinical importance, there is limited knowledge regarding the variations of the genome within mitochondria, the powerhouse organelle within eukaryotic cells. In this study, we leveraged publicly available, raw, whole genome sequence data isolates from 1939 to investigate the variations in the mitochondrial genomes of A. fumigatus. These isolates were isolated from 22 countries on six continents, as well as from outer space and from within the International Space Station. In total, our analysis revealed 39 mitochondrial single nucleotide polymorphisms (mtSNPs) within this global sample, and, together, these 39 mtSNPs grouped the 1939 isolates into 79 mitochondrial multilocus genotypes (MLGs). Among the 79 MLGs, 39 were each distributed in at least two countries and 30 were each shared by at least two continents. The two most frequent MLGs were also broadly distributed: MLG11 represented 420 isolates from 11 countries and four continents and while MLG79 represented 418 isolates from 18 countries and five continents, consistent with long-distance dispersals of mitogenomes. Our population genetic analyses of the mtSNPs revealed limited differentiation among continental populations, but highly variable genetic differences among national populations, largely due to localized clonal expansions of different MLGs. Phylogenetic analysis and Discriminant Analysis of Principal Components of mtSNPs suggested the presence of at least three mitogenome clusters. Linkage disequilibrium, Index of Association, and phylogenetic incompatibility analyses collectively suggested evidence for mitogenome recombination in natural populations of A. fumigatus. In addition, sequence read depth analyses revealed an average ratio of ~20 mitogenomes per nuclear genome in this global population, but the ratios varied among strains within and between certain geographic populations. Together, our results suggest evidence for organelle dynamics, genetic differentiation, recombination, and both widespread and localized clonal expansion of the mitogenomes in the global A. fumigatus population. Aspergillus fumigatus is a ubiquitous airborne saprophytic fungus, and a leading cause of human fungal infections [A. fumigatus’ ecological niche is primarily in organic debris and soil [A. fumigatus spreads via sporulation of minute conidia, roughly 2–3 µm in diameter, light enough for airborne dissemination and small enough to penetrate lung alveoli [fections . Playingand soil . Howeverand soil ,4. Repro alveoli . The com alveoli .A. fumigatus spores often develop into invasive aspergillosis (IA), with mortality rates reaching as high as 90% [A. fumigatus a Critical Priority fungal pathogen [Despite the near constant inhalation of spores, a healthy host’s innate immune system is usually able to eliminate the foreign cells, such that clinical symptoms are mild and rarely seen . Howeverh as 90% . Consequpathogen . Antifunpathogen . Howeverpathogen ,9.A. fumigatus requires energy generated by mitochondria through oxidative respiration. In addition, aside from energy generation, fungal mitochondria have also been implicated in various cellular processes, including temperature and oxidative stress responses, pathogenicity, antifungal resistance, iron homeostasis, and fungal dormancy [A. fumigatus and potential variations among strains in their mitogenomes of this fungal pathogen are unknown.For effective growth and reproduction both in vitro and in vivo, like most other eukaryotes, dormancy ,13,14,15dormancy ,17. Typidormancy . Howeverdormancy ,20. Amondormancy . HoweverSince mitogenomes are small, relatively abundant, and well conserved yet often distinct among species, mitogenome analysis is a popular method for population genetics, physiological, and evolutionary studies ,22. IndeAspergillus species, is not well represented in the literature. Furthermore, investigations into the intraspecies structure or whole-genome level analysis of mitogenomic single nucleotide polymorphisms (mtSNPs) in A. fumigatus are currently nonexistent. Previous studies examining interspecies population dynamics and phylogenetics in the Aspergillus genus have predominantly relied on a single gene, such as cytochrome b or concatenated alignments of multiple mitochondrial genes, most of which were conducted more than a decade ago. Notably, Kozlowski and Stepien [Aspergillus genus. Subsequent studies by Wang et al. [A. fumigatus and other pathogenic Aspergillus species using cytochrome b gene sequences. More recently, Joardar et al. [Aspergillus and Penicillium species, including employing 14 concatenated mitochondrial proteins for inter-species and inter-genus phylogenetic analyses. Although relatively less common than single gene focused assays, genome-wide mtSNPs for phylogenetic and population analyses has been successfully demonstrated in studies on crops, cattle, humans, and selected fungi [In recent years, there has been a notable increase in the number of studies focusing on mitochondrial genomics. However, the application of mitochondria-based analyses to investigate fungal population structure, particularly within the  Stepien  pioneereg et al. ,27 succer et al.  publisheed fungi ,29,30,31A. fumigatus, we conducted a large-scale intraspecific analysis of mtSNPs in a global A. fumigatus population. Specifically, raw sequencing reads of about 2000 isolates of A. fumigatus were available (as of 2 May 2023) in the National Centre for Biology Information’s (NCBI) Sequence Read Archive (SRA), each including both the nuclear and mitochondrial sequence reads. While the nuclear genomes of these strains have been analyzed and published in a diversity of papers and journals [With the increasing abundance and availability of whole genome sequencing (WGS) reads in journals ,34,35,36journals . Our stuAspergillus fumigatus” and Whole Genome Sequencing (WGS) and Whole Genome Amplicon (WGA) assay types. The raw sequence data for each accession was obtained using the sra-toolkit (v3.0.0), and subsequently subjected to quality checking and trimming using Fastp (v0.23.1) [NCBI’s SRA, which includes raw data from published experiments, the European Bioinformatics Institute (EBI), and the DNA Database of Japan (DDBJ), served as the data source and repository for this study. A query was performed on 2 May 2023 on the SRA to identify all accessions associated with “v0.23.1) .A. fumigatus mitochondrial genome  using Burrows–Wheeler Aligner (BWA-MEM v0.7.17-r1188) [To ensure data integrity, reads were filtered based on quality (requiring at least 60% of bases with a phred score of 30 or higher), length (minimum of 30 base pairs), and complexity. The filtered reads were aligned to a reference 7-r1188) . The alignments were further processed using samtools (v1.17)  to ensurCountry and continent of origin for each strain was initially determined from associated metadata collected from NCBI. In cases where metadata was incomplete, a manual literature search was conducted to confirm the origin information. Accessions with unconfirmed or artificial origins were labelled as having “Unknown” geographic origins. All isolates retrieved and analyzed, including their relevant metadata, and are shown in A. fischeri isolate as the outgroup. The Interactive Tree Of Life (iTOL) web viewer (v5) [To investigate the relationships among strains and mitogenomes, we conduct phylogenetic analysis using concatenated SNPs. The SNPs were aligned using the program vcf2phylip (v2.0) , and thewer (v5)  was usedA. fischeri root, (2) a clone-corrected (CC) full pooled dataset, (3) a dataset excluding countries with fewer than nine samples and samples with unknown or non-geographic origins, and (4) a country wise CC dataset where only one representative for each unique MLG type from each country was included.To investigate the potential influence of geographic isolation on mitogenomic population structure, population genetic analyses were performed using the R statistical software and the R package poppr (v.2.9.4) . The firPopulation structure was explored by performing a Discriminant Analysis of Principal Components (DAPC) on both the full and country wise clone corrected datasets. For both datasets, Kernel (K) values were chosen from where the Bayesian Information Criterion (BIC) began to decrease minimally, based on 10 repetitions using the find.clusters function from the adegenet R package (v2.1.10) . DAPC waThe impact of geography on genetic distribution was further explored through an Analysis of MOlecular VAriance (AMOVA). The country-filtered and country-wise CC datasets were subjected to AMOVA at both the country and continental levels. Additionally, pairwise Nei’s Fst values were calculated between pairs of countries to assess the genetic differentiation among them. To evaluate the correlation between geographic and genetic distances, a Mantel test was performed using the non-CC filtered dataset. This analysis involved comparing the geographic distances (as measured by latitude and longitude) with the corresponding genetic distances (measured as pairwise Nei’s Fst in this implementation) between countries. Significance testing was conducted using 999 permutation bootstrap iterations, with the null hypothesis assuming no genetic differentiation among the geographic populations.A. fumigatus. The first test employed the common multilocus Index of Association (IA) [A, we generated 999 artificially recombined datasets through random allele permutation while maintaining allelic proportions at each locus, and evaluated each for IA. A significantly higher observed IA compared to the randomly recombined permutations would indicate a lack of random recombination in the dataset, leading to the rejection of the null hypothesis of panmixia . To facilitate comparisons between studies, we standardized IA by dividing it by the number of loci analyzed, yielding the measure rD. IA and rD calculations were performed on the clone-corrected dataset, where only one representative of each unique MLG was included. These calculations were conducted using the R package poppr [Three tests were conducted to assess the presence of recombination in the mitogenome of ion (IA) ,48 to inge poppr .2) were computed, with statistical significance assessed through a χ2 test against the null hypothesis of panmixia . Considering the extensive number of locus pairs, p-values from the pairwise χ2 tests were adjusted using the Bonforroni method. Both LD calculations and χ2 analyses were performed in R.To further investigate the potential recombination patterns in the mitogenome, the second test investigated allelic associations between all pairs of SNP loci across the mitogenome, using the clone-corrected pooled dataset. For each loci pair, both the coefficient (D) of linkage disequilibrium (LD) and the correlation coefficient  or recombination . For biacyp51A (GeneID: 3509526), to represent the nuclear genome and compared it with the average read depth of the mitogenome. The cyp51A read depth for each strain was obtained by aligning the adapter-trimmed and quality-filtered reads from each sample to the reference genome Af293 using the same methodology as described earlier. The entire mitogenome sequence was used to obtain the average read depth of the mitogenome for each strain. The read depth for each nucleotide position for both the cyp51A gene and the mitogenome in each strain was determined using samtools depth (v1.17) [cyp51A read depth.To assess the relative abundance of the mitogenome and the nuclear genome within a cell, we used two methods. In the first, we obtained the average read depth of a single-copy gene,  (v1.17) , incorpoA. fumigatus reference strain, Af293 (GenBank: GCA_000002655.1), minus that of the mitogenome. The obtained coverage was then compared with the mitogenome coverage described in method #1 above. The obtained ratios were then analyzed for their potential phylogenetic and geographic patterns.In the second method, we estimated the nuclear genome read depth based on whole-genome sequencing coverage. To estimate coverage, the total count of bases in each sequence file that remained post quality filtering and trimming was divided by the aggregate genome size of the A. fumigatus that had their whole-genome sequences deposited in the SRA by 2 May 2023, and one Aspergillus fischeri sample used as an outgroup for later phylogenetic analysis. The retrieved 1939 strains came from 22 countries representing six continents, as well as from outer space (2 isolates) and from within the International Space Station (2 isolates)  . At the solates) . At the solates) .Using the mitogenome sequence of Af293 as reference and the retrieved sequence reads from the 1939 isolates, we identified a total of 39 SNP sites in this global collection of isolates. Of these 39 SNPs, 15 (38.5%) were intergenic while 24 (61.5%) were in protein-coding regions. Among the 24 SNPs in protein-coding regions, 3 (7.69%) were synonymous and 21 (53.85%) were non-synonymous. All nonsynonymous mutations were predicted to have low-to-moderate effects on protein structure and functions . These 3Among the 79 MLGs, 39 were shared by at least two countries and 30 were shared by at least two continents. The remaining 40 were each found in only one country so far and 35 of these were only represented by one isolate each . The twoThe relationships among strains and MLGs based on mitogenome SNPs are shown in We analyzed whether the 79 MLGs could be grouped into distinct genetic clusters. To accomplish this, we used the Discriminant Analysis of Principal Components (DAPC) of pairwise distances among MLGs. The DAPC analysis showed a clustering pattern very similar to that observed in the cladograms, with three groups appearing as the most parsimonious. However, there were four cases of incongruence between the DAPC grouping of three genetic clusters and the NJ tree, highlighted by different colors shown in The Bayesian Information Criterion (BIC) plot exhibited a sharp drop from a k value of one to two, followed by a less steep decline from two to three, and three to four A. SubseqTo investigate the geographic structure of mitogenome SNPs and estimate the potential quantitative relationships between geographic distance and genetic distance among our samples, we conducted AMOVA at the continental and country levels and the Mantel analyses at the country level.AMOVA was conducted using three different datasets: (1) the non-clone-corrected dataset including all countries with sample sizes greater than eight, (2) the same dataset after clone correction on a country-wise basis, and (3) the country-wise clone corrected dataset but included only countries with more than nine MLGs. The AMOVA test of the first dataset revealed a statistically significant genetic differentiation among countries, but the differentiation was statistically insignificant among continents. However, after clone correction, no statistically significant genetic differentiations were observed at either the country or the continental levels for both the second and third dataset. Taken together, the results suggested that localized clonal expansion of mitogenome MLGs were responsible for the observed statistically significant genetic differentiations in the total samples among countries.A. fumigatus samples were genetically differentiated, we obtained Fst values between pairs of national samples using both the non-clone-corrected data and the clone-corrected data. Similar to the analyses above, only countries with an initial sample size exceeding eight for the original dataset  and exceeding eight MLGs for clone-corrected data  were analyzed. The preliminary pairwise comparison revealed a considerable and diverse distribution of Fst values between countries  differentiation. The two pairings that resulted in the highest Fst values both included Cote d’Ivoire. The two highest Fst values, 0.7538 and 0.7308, were observed between Cote d’Ivore and the Netherlands and New Zealand, respectively. However, after clone correction, there was a substantial reduction in variability among country pairs. In addition, six of the initial 14 countries assessed failed to meet our sample size criteria for comparison, and thus only eight countries were compared .To further identify which pairs of national ountries . Among tcompared . As showA. fumigatus. Here, only the global sample of 79 clone-corrected MLGs were included in the analyses. In the first analysis of multilocus index of association (IA) among the 39 SNP loci, our sample showed significant deviations from random mating and recombination, with the overall IA and standardized rD values of 2.50 and 0.07, respectively. Both values were significantly higher than what would be expected in randomly recombining populations (p < 0.01). In the second test, phylogenetic compatibility analysis of the 39 SNP loci also demonstrated a significant departure from random mating. Approximately 80% of the loci pairs displayed phylogenetic compatibility, which is significantly higher than what would be observed under the hypothesis of random recombination (p < 0.01). In addition, out of the 741 SNP loci pairs, 73 were found to exhibit significant allelic associations, indicative of linkage disequilibrium . Taken together, these results indicated no evidence of random recombination in the mitogenome of the global A. fumigatus population.Three analyses were performed to investigate the possibility of recombination in the mitogenome in A. fumigatus. However, signatures of recombination were found in the mitogenomes of this global sample. Specifically, 145 of the 741 pairs of SNP loci showed phylogenetic incompatibility , consistcyp51A gene varied widely among the 1939 isolates, from a few reads to over 400 reads , 1.79 and 2.83, respectively  or very low (<1) ratios were either transcriptome data or synthetic metagenome data but were deposited as whole-genome sequence data. These ten strains were removed from subsequent analyses of mitogenome to nuclear genome ratios. Interestingly, though the raw ratios had a significantly skewed distribution (γ = 1.10), the square-rooted ratios had a normal distribution (γ = 0.18), with a mean ratio of 20.70 among the 1929 isolates ; p < 0.05, p < 0.01). Subsequent post hoc pairwise Wilcoxon rank sum testing uncovered significant (p < 0.05) differences between Group #3 and both Group #1 and Group #2. Specifically, the Group #3 strains had an overall higher mitogenome to nuclear genome ratio than those in Groups #1 and #2, 29 compared to 21 and 18, respectively. In contrast, no significant differences were observed between Groups #1 and #2. To investigate the potential patterns of mitogenome to nuclear genome ratios among strains and among genetic and geographic populations, we compared the ratio patterns across both the DAPC grouping and among countries with sample size greater than 8. Kruskal–Wallis testing revealed statistically significant heterogeneity in ratio distribution within both the DAPC groups and the countries examined  differences in their mitogenome to nuclear genome ratios (p < 0.05) (Among the 14 compared countries (each country with sample size greater than 8 isolates), 38 pairs  showed statistically significant (e ratios A. Canadae ratios . Converse ratios . Canada e ratios . Interes < 0.05) B.A. fumigatus, a critical priority human fungal pathogen. Our analyses identified 39 SNPs and 79 multi-locus genotypes among the analyzed isolates. In addition, we found a range of mitogenome to nuclear genome copy number ratios, from about 11 to over 100, with a mean of ~20. Our examination of concatenated SNP sequences separated the strains into at least three genetic groups. However, though multiple genotypes are often overrepresented in certain geographic regions, there was limited geography-based clustering at either the country or the continent level for the 79 mitochondrial genotypes. Interestingly, evidence for mitogenome recombination was found between several pairs of SNP loci. Below, we discuss the relevance and potential implications of our results.In this study, we conducted a broad analysis of mitogenome SNPs in a global collection of 1939 isolates of In this study, 39 SNPs were identified among the 1939 isolates from around the world. Among the 24 SNPs in protein-coding genes, 3 were synonymous while 21 were nonsynonymous. This high ratio of nonsynonymous to synonymous SNPs suggested potential positive selection might have acted on the mitogenome during the evolution of A. fumigatus. However, all nonsynonymous mutations were predicted to have low to moderate effects on protein structure and functions. Indeed, the two most frequently and most broadly distributed mitogenome MLGs, MLG11 and MLG79, were found to differ by six nonsynonymous mutations. At present, the functional impacts of these mutations are not known. Additional functional comparisons are needed in order to determine their potential roles in nature. Despite the uncertainty of functional importance of the observed mitogenome SNPs, mitogenome sequence variations provided valuable information for understanding natural populations of A. fumigatus.A. fumigatus isolates representing 22 countries in 6 continents, including two strains from the International Space Station. Among the 79 MLGs from these 1939 isolates, 19 were each distributed in at least three countries, indicative of continental and global clonal expansion. Indeed, all the sequenced samples from Africa, Oceana, South America, and the International Space Station shared MLGs with samples from other geographic regions and none had private mitochondrial MLGs. Interestingly, the two samples from the International Space Station were genetically divergent from each other, similar to the extensive differences found in their nuclear genomes [The analyzed global population included 1939  genomes .Our phylogenetic and DAPC analyses showed our samples can be optimally clustered into three major clades. This finding differed from previous studies conducted by Pringle et al. , KlassenA. fumigatus to be genetically highly distinct from other national populations. It is likely that the mitogenomes from the Cameroonian populations will be genetically distinct as well. However, there is currently no genome sequence information from Cameroonian isolates of A. fumigatus. In addition, among the 22 sampled countries, the distributions of isolates were also highly skewed. Specifically, over half of the isolates (1036) originated from western Europe, and an additional 30% (563) were from the United States. Furthermore, we were unable to find the geographic origins for 114 of the isolates. Together, we believe that better data submission and including more strains from under-sampled regions and strains from not-yet sampled regions will reveal additional novel mitogenome diversity in this species.We note that despite the large sample size (1939 isolates) and broad geographic representation (22 countries from across six continents), the analyzed samples were still limited in their geographic distributions. Specifically, the 22 countries represented only about 12% of the total countries in the world. A previous study by Ashu et al.  revealedA. fumigatus. In A. fumigatus, the mitochondrial inheritance has not been critically examined in sexual crosses in the lab. However, sexual mating through hyphal fusion between strains of different mating types should result in significant cytoplasmic mixing, including the potential for mitochondrial fusion and mitogenome recombination. The observation of cleistothecia development along junction zones between mating partners is consistent with hyphal fusion being a dominant mode of mating in A. fumigatus [A. fumigatus contrasts with those observed in the majority of eukaryotic organisms, where mitogenomes are inherited uniparentally in sexual crosses without recombination [Aspergillus genus have provided potential evidence of intra- and inter-species mitochondrial recombination. For instance, investigations on Aspergillus nidulans using genetic crosses, gel electrophoresis, Southern hybridization, and electron microscopy suggested genetic exchange between mitogenomes [Aspergillus niger, a related opportunistic human pathogen, caused by the homing of mobile mitochondrial group I introns [A. fumigatus natural populations, evidence for recombination was observed between SNPs located across the mitogenome.We observed signatures of recombination in the mitogenome of this global population of umigatus . Our obsbination . Howeverbination ,55,56. Iogenomes ,58. Simi introns . In bothA. fumigatus. Wide variations of the ratio, from about 11 to over 100, were found among strains and among geographic populations. Based on the redundancy in their assembled sequence, a previous study estimated a clinical isolate of A. fumigatus Af293 having a mitochondrial to nuclear DNA ratio of ~12:1 [Aside from mitogenome SNPs, we also investigated the relative copy number differences between mitochondrial and nuclear genomes among the 1939 strains of of ~12:1 . In a moof ~12:1  estimateA. fumigatus, a critical human fungal pathogen. Our analyses revealed evidence for both global and local clonal expansions of mitogenomes. The over-representation of relatively few mitochondrial genotypes in the global sample suggested potential selective advantages of these mitochondrial MLGs and/or their associated nuclear genomes in both natural environments and patients. Due to their biased clinical prevalence, whenever possible, the overrepresented mitogenome MLGs should be used for virulence and drug development studies. The identified mitogenome SNPs and MLGs could be used as molecular markers to track future epidemiological patterns of A. fumigatus at both local and global scales. In addition, evidence of mitogenome recombination was found among several mtSNP loci, indicating a departure from typical uniparental mitochondrial inheritance pattern in eukaryotes. Similarly, the investigation of relative copy number differences between mitochondria and nuclear genomes revealed a wide range of ratios. At present, the potential biological significance of the observed variations in mitogenome to nuclear genome ratios is not known. However, it’s tempting to speculate that the variable ratios likely reflect the dynamic nature of mitochondrial genome replication and maintenance, which are regulated by various environmental and metabolic cues. Our study underscores the need for further investigation into the functional implications of the observed mitogenome variations in A. fumigatus. Collectively, these findings help lay the foundation for future studies into the genetic mechanisms underlying the diversity and evolution of this critical fungal pathogen.Our study provides novel insights into mitogenome variations in a large global collection isolates of


Lonicera japonica Thunb. are widely used in Chinese medicine for their anti-inflammatory properties, and they have played an important role in the fight against SARS COVID-19 and other major epidemics. However, due to the lack of scientific and accurate variety identification methods and national unified standards, scattered and non-standardized management in flower bud production has led to mixed varieties that have caused significant difficulties in the cataloging and preservation of germplasm resources and the identification, promotion, and application of new L. japonica varieties.The flower buds of Lonicera in China using simplified genome sequencing technology.In this study, we evaluated the population structure, genetic relationships, and genetic fingerprints of 39 germplasm resources of Lonicera were divided into four subgroups, and the population structure and genetic relationships among existing Lonicera germplasm resources were determined using principal component analysis, population structure analysis, and phylogenetic tree analysis. Through several stringent selection criteria, 15 additional streamlined, high-quality DNA fingerprints were filtered out of the validated 50 SNP loci and verified as being able to effectively identify the 39 Lonicera varieties.A total of 13,143,268 single nucleotide polymorphisms (SNPs) were identified. Thirty-nine samples of Lonicera varieties in China. These results have greatly broadened our understanding of the diversity, phylogeny, and population structure of Lonicera. The results may enhance the future analysis of genetic diversity, species identification, property rights disputes, and molecular breeding by providing a scientific basis and reference data for these efforts.To our knowledge, this is the first comprehensive study measuring the diversity and population structure of a large collection of  Lonicera japonica Thunb. a perennial semi-evergreen twining shrub of the Caprifoliaceae family, is native to China and is primarily distributed in temperate regions of the northern hemisphere, with smaller numbers in Japan and Korea. The honeysuckle flower is a dry flower bud or open flower of L. japonica and is rich in organic acids, flavonoids    and Foury (HPLC)  have beeNicotiana tabacum L.  Standl. (Triticum aestivum L. (Camellia sinensis L. (Beta vulgaris L. (Ipomoea batatas (L.) Lam.  and single nucleotide polymorphism (SNP) markers have been used to construct fingerprint databases, identify genetic relationships, construct genetic maps, and isolate and clone diverse genes of bacum L. , Brassic italica , Lagenar Standl. , Triticutivum L. , Camelliensis L. , Beta vugaris L. , and IpoL.) Lam. , and othL.) Lam.  construcL.) Lam. .via high-throughput automated detection  library construction of qualified sample DNA. First, 500 ng of genomic DNA was reacted with 0.6U EcoRI (NEB), T4 DNA ligase (NEB), ATP (NEB), and EcoRI connectors (including index sequences of differentiated samples) at 37°C for 3 h and annealed at 65°C for 1 h. The restriction enzyme NlaIII (NEB) and the NlaIII connector were then added and allowed to react for 3 h at 37 °C. After the reaction, the endonuclease was inactivated at 65°C for 30 min in a polymerase chain reaction (PCR) amplifier. Digested products of 400–600 bp were recovered via agarose gel electrophoresis and quantified using Qubit 3.0 (Life Technology). After mixing 39 samples in equal quantities, an Illumina TruSeq kit was used to construct a DNA library of the mixed products , and the quality and concentration of DNA were measured using a NanoDrop2000 UV spectrophotometer . Then, a paired-end library with a length range of 300–500 bp was constructed products .L. Lonicera) (download website: https://ngdc.cncb.ac.cn/search/?dbId=gwh&q=SAMC097356), and the parameters were set as -M -R. The insert size and coverage depth of each sample were counted, and variation was detected by comparing the positions of clean reads on the reference genome  HaplotypeCaller function was used to call variable outliers. The FilterVCF function was used to filter low-quality mutation sites to obtain the final SNP site set and obtain SNP statistics   was used to perform principal component analysis (PCA)   with theis (PCA) . The max samples . Finally samples  . The PCR reaction program involved pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing for 30 s to 63°C, and extension at 72°C for a total of 30 s; this was repeated for 30 cycles, after which there was a final extension at 72°C for 10 min before storage at 4°C. After PCR amplification, 5 μl of PCR products were taken and subjected to 1% agarose gel electrophoresis at 150 V and 100 mA. After 10–20 min of observation, the target PCR band was cut from the gel, recovered, and then sequenced using 3730XL. The sequencing data were searched in the results group, and sequence analysis software SnapGene was used for analysis. Finally, SeqMan software was used to observe the peak map to test the reproducibility, genetic stability, and specificity of 15 SNPs.Primers for the 15 SNP loci were designed using Primer Premier 5 software Table 2,Lonicera samples on the Illumina NovaSeq 6000 PE150 yielded a total of 84.88 Gb of clean data, and Q30 reached more than 93.25% per sample , “Yincuilei” (SPL019), and “Jincuilei” (SPL020) could only be distinguished by Marker 7; “Yujin 2” (SPL010) and “Yate 1” (SPL004) could only be distinguished by Marker 6. “Yujin 5” (SPL014) and “Yujin 5 1-2” (SPL028) could only be distinguished by Marker 11. These results also illustrate that it is difficult to develop fingerprints against the background of complex and chaotic genetic relationships in L. japonica germplasm.Some We randomly selected four samples, “Yujin 1,” “Yujin 2,” “Mixian Damaohua,” and “Telei 1,” from a set of 39 samples to verify the reproducibility of SNPs. Fifteen SNPs had the same site specificity as the previous test results Table 5,Lonicera, China has three main production areas: Fengqiu in Henan Province, Julu in Hebei Province, and Pingyi in Shandong Province  that provided a basis for the quality control analysis of L. japonica and L. macranthoides. L. japonica varieties in Henan, Hebei, Shandong, and other areas and concluded that the index components of Shandong L. japonica were the highest. This work provided a scientific basis for the selection of raw materials for honeysuckle decoctions. L. japonica using HPLC fingerprints and found that wild L. japonica contained more chemical components, while tree-shaped L. japonica was more consistent. L. japonica and L. macranthoides, providing data support for their application in traditional Chinese medicine. The related studies focused on Lonicera in the early stages; most of the studies on the germplasm focused on morphological analysis, index component differences, and quality evaluation among varieties, while there was almost no research on the genetic relationships and population structure of Lonicera species and varieties.The 2015 Edition of the Chinese Pharmacopoeia details the differences between Lonicera germplasm resources in China were collected and divided into four subgroups via simplified sequencing, PCA, population structure analysis, and evolutionary tree construction. Subgroup I contains the red variety L. acuminata of L. japonica. Subgroup II contained L. macranthoides. Subgroup III comprises the main, wild, and hybrid varieties of L. japonica from Shandong and Henan. Subgroup IV contained cultivars of L. japonica that comprised varieties with high yields, strong resistance to pests and diseases, and good characteristics that have been recognized on the current market, for example, SPL009, SPL002, SPL003, and other varieties. Based on these results, it should be clear that the lineages of wild Lonicera species were extremely complex, while the lineages of systematically bred L. japonica were relatively simple. For example, “Mixian xianhua” (SPL006), “Mixian wild” (SPL007), and “Mixian Damaohua” (SPL039) are wild varieties of L. japonica with extremely complex genetic relationships. In the population structure analysis, they all had ancestral lines from the four subgroups. However, the genetic relationship between “Yujin 1” (SPL009) and “Yate liangzhong” (SPL035) was simple and derived from one pedigree. After further analysis, this study also clarified the controversial genetic relationship of “Longyao” (SPL023), a variety produced in Hunan, as belonging to L. macranthoides. However, our results confirmed that “Longyao” (SPL023) was a cross-species hybrid of a female parent L. japonica and a male parent L. macranthoides, which explained why it simultaneously had the pedigree of two subgroups in the PCA and population structure analysis. The genetic relationship of “Yujin 6” (SPL027) was further clarified, and the results showed that it was crossbred from the female parent “Telei 1” (SPL011) and the male parent “Yujin 1” (SPL009). This finding well explained its position in Group IV of the evolutionary tree. In addition, four other hybrids were used in this study. Population structure analysis showed that all hybrids have genetic genes from their parents, which was largely consistent with expectations. At present, this research group is conducting a more in-depth analysis of hybrid characteristics.In this study, 39 existing L. japonica, excavating the differences among varieties of L. japonica, and using SNP to distinguish varieties of L. japonica. This work can not only explain the population relationships of existing Lonicera germplasm resources but can also further clarify the genetic relationships between Lonicera varieties. These results provide a favorable reference and a basis for the management of Lonicera germplasm resources and have important significance for the breeding of new Lonicera varieties in the future.Different from most previous studies focusing on honeysuckle morphology and quality evaluation , the preLonicera; 50 core SNPs with a strong ability to identify Lonicera germplasm were screened; and DNA fingerprints were constructed. A fingerprint map constructed with 50 core SNP markers effectively identified and distinguished differences among different species and varieties of Lonicera. In addition, this study further filtered out 15 SNPs as the most compact site combination. The DNA fingerprints constructed based on the 15 SNP loci were used to detect different Lonicera samples, and ≥1 different locus was found in each Lonicera variety that could be used to distinguish different species and varieties rapidly, accurately, and at a low cost.In this study, SNP molecular marker technology and DNA fingerprinting technology were used; 3,374,929 SNPs were obtained from 39 samples of Lonicera germplasm, this study conducted further research on varieties that had several different loci ≤1 in the fingerprints of 15 SNP loci. After further analysis, it was found that these varieties not only had high repeatability in DNA fingerprints but also had high similarity in origin, species, and characteristics. For example, “Mihua 1” (SPL017) and “Fengjin 1” (SPL012) were introduced and domesticated from “Mixian Damaohua” (SPL008). The varieties “Longhua” (SPL022), “Jincuilei” (SPL020), and “Yincuilei” (SPL019) originated from Hunan Province and were L. macranthoides “Yujin 2” (SPL010) and “Yate 1” (SPL004) had purplish red flower buds and were red varieties of L. japonica. “Yujin 5” (SPL014) and “Yujin 5 1-2” (SPL028) were both hybrids, and their parents were the same varieties (Considering the extremely complex genetic background of arieties Table 1.via PCR and Sanger sequencing. The 15 SNP verification results were completely consistent with the previous test results. Subsequently, genetic analysis was conducted on 10 honeysuckle samples of the same variety from different years. The results showed that the genetic stability of these honeysuckle samples was extremely strong, reaching 97.33%, a level that was consistent with the characteristics of honeysuckle as a vegetatively propagated plant. However, it is worth noting that the genotypes of the two-year-old samples “J01,” “YJ02,” and “YJ03” and the five-year-old sample “YJ07” changed from A/A to A/G in Marker 10, a result that may have been related to the second allele of the SNP. However, as a vegetatively propagated plant, the genetic traits of honeysuckle are very stable, and the probability of genetic mutation between the same varieties is very low; thus, this phenomenon was most likely caused by the duality of SNP sites. Finally, three new varieties verified the specificity of SNPs, further demonstrating the reliability of the SNPs and DNA fingerprints developed in this study.To verify the reliability of the DNA fingerprints comprising 15 SNP loci, four samples were randomly selected from the original 39 samples, and 15 SNP loci were analyzed In general, the verification results of the 15 SNP loci and the genetic stability of honeysuckle were in line with expectations; the analysis not only confirmed that the 15 SNP loci constituted the authenticity and reliability of DNA fingerprints but also verified the high genetic stability of honeysuckle, providing a data reference and theoretical support for the identification of honeysuckle germplasm and the SNP fingerprint identification method.The original contributions presented in the study are publicly available. This data can be found here: NCBI, PRJNA895242.JL conceived of the project. JL and XC designed the experiments. XC performed most of the experiments. QH assisted in preparing seedlings for experiments. PL and XZ helped overcome technical difficulties in SNP site validation. CC, XZ, FL, and YW assisted in analyzing the data. JL, XC, and CC interpreted the data generated in the figures and wrote the manuscript. All authors contributed to the article and approved the submitted version.


Leishmania aethiopica is a zoonotic Old World parasite transmitted by Phlebotomine sand flies and causing cutaneous leishmaniasis in Ethiopia and Kenya. Despite a range of clinical manifestations and a high prevalence of treatment failure, L. aethiopica is one of the most neglected species of the Leishmania genus in terms of scientific attention. Here, we explored the genome diversity of L. aethiopica by analyzing the genomes of twenty isolates from Ethiopia. Phylogenomic analyses identified two strains as interspecific hybrids involving L. aethiopica as one parent and L. donovani and L. tropica respectively as the other parent. High levels of genome-wide heterozygosity suggest that these two hybrids are equivalent to F1 progeny that propagated mitotically since the initial hybridization event. Analyses of allelic read depths further revealed that the L. aethiopica - L. tropica hybrid was diploid and the L. aethiopica - L. donovani hybrid was triploid, as has been described for other interspecific Leishmania hybrids. When focusing on L. aethiopica, we show that this species is genetically highly diverse and consists of both asexually evolving strains and groups of recombining parasites. A remarkable observation is that some L. aethiopica strains showed an extensive loss of heterozygosity across large regions of the nuclear genome, which likely arose from gene conversion/mitotic recombination. Hence, our prospection of L. aethiopica genomics revealed new insights into the genomic consequences of both meiotic and mitotic recombination in Leishmania. Depending on the Leishmania species, the disease can present itself in various clinical representations, ranging from cutaneous to visceral leishmaniasis. Leishmania aethiopica is an Old World species transmitted by sand flies of the Phlebotomus genus, mainly P. longipes  that mostly reside in rocky cliffs   . Such fiL. aethiopica to gain a better understanding of the evolutionary history and population biology of this species. We generated a total of 28 Leishmania genomes and complemented our dataset with publicly available genomes from L. donovani, L. infantum, L. tropica and L. major for comparative purposes. Population genomic and phylogenomic analyses provide genomic confirmation of interspecific hybridization including L. aethiopica as one of the two parental species and confirm that L. aethiopica is genetically highly diverse. Our prospection of L. aethiopica genomics may promote future studies on the genomic basis of treatment failure and clinical outcome.Here, we present the first genome diversity study of 22.1Throughout this manuscript, we use the term ‘strain’ when referring to an isolate that has been characterized (here through whole genome sequencing), ‘clone’ when referring to a strain that has been cloned and ‘genome’ when referring to strains/clones that have been sequenced .Leishmania genomes from Ethiopia within the context of this study  or the WHO International Leishmania Reference Center . For two strains (GERE and KASSAYE), only the derived clone was sequenced. For one strain (L100), both the strain and a derived clone were sequenced. The L100 strain is the WHO reference strain that represents the same strain as the one used to create the reference genome (https://tritrypdb.org/tritrypdb/app/record/dataset/NCBITAXON_1206056#description). (ii) One genome of the L. donovani strain HUSSEN. (iii) One genome of the putative L. aethiopica/L. tropica strain L86. (iv) Six genomes of the putative L. aethiopica/L. donovani strain ABAUY (hereafter referred to as LEM3469) and its derived clones . All genomes were sequenced (150bp paired-end) on the Illumina sequencing platform of GenomeScan, Leiden, The Netherlands.We have sequenced a total of 28 in vitro culturing done within the context of this study. The numbers below reflect the minimum number of passages, as we don’t know how many passages occurred between isolation from patients and shipment from the reference center to ITM. The L100 strain has undergone 29 passages; its derived clone L100cl1 was generated at passage 27 and has undergone a total of 31 passages. Hence, L100 and L100cl1 differ by only 2 passages. The LEM3469 strain has undergone 3 passages; its derived clones were generated after 26  or 28 (clone 1 and 9) passages, and have undergone a total of 30  or 34 (clone 1 and 9) passages. Hence, LEM3469 and derived clones differ by about 30 passages, and the clones themselves by either 0 or 4 passages.For strains L100 and LEM3469, for which we sequenced both the strain and derived clone(s), we here provide information about the number of passages that occurred at ITM before the Leishmania species (L. donovani from Ethiopia (n=5), L. infantum (n=1), L. major (n=1) and L. tropica (n=1)  . We onlyAltogether, this provided us with a total of 36 genomes for downstream analyses.2.2L. aethiopica’s reference genome L147 (available on tritrypdb.org as TriTrypDB-54_LaethiopicaL14710) using BWA (Leishmania genomes and once including 20 L. aethiopica genomes.All sequences were mapped against the sing BWA . Resultising BWA . All dupsing BWA . BAM-filLeishmania genomes) or larger than 40 (when genotyping was done on the 20 L. aethiopica genomes). SNPs were annotated using the L. braziliensis M2904 annotation file with SNPEFF v4.5  . In addiEFF v4.5 . SNPs we2.3L. aethiopica genomes and the two interspecific hybrids L86 and LEM3469 based on the per-site read depth as obtained with SAMTools depth. To obtain haploid copy numbers (HCN) for each chromosome, the median chromosomal read depths were divided by the median genome-wide read depth. Somy variation was then obtained by multiplying HCN by two  or three . To obtain local HCN, the median read depth in non-overlapping 2kb windows was divided by the median genome-wide read depth. These calculations were done using the depth2window.py script (available at github.com/FreBio/mytools/blob/master/depth2window.py).Chromosomal and local copy number variations were studied for the 20 L. aethiopica genomes from the HCN as estimated per Leishmania genome. This results in a distribution of HCN centered around zero, allowing us to identify 2kb windows with increased (z-score > 5) or decreased (z-score < -5) HCN in each of the 20 L. aethiopica genomes and the two interspecific hybrids (LEM3469 and L86). Consecutive 2kb windows showing a significant increase/decrease in HCN were joined by averaging HCN across the consecutive windows, allowing us to detect larger copy number variants. Small deletions/amplifications (i.e. <= 6kb) that do not cover protein coding genes were ignored.Windows of reduced or increased HCN were identified by deducting the median HCN across 20 2.4The quality-filtered SNP VCF files were converted to the fasta format using the vcf2fasta.py script (available at github.com/FreBio/mytools/blob/master/vcf2fasta.py). To get an initial idea on the phylogenetic relationships between the genomes, a phylogenetic network was reconstructed with SplitsTree version 4.17.2  based onL. aethiopica was investigated after excluding putative near-identical genomes (see results), i.e. genomes showing fixed differences at <3 sites. In addition, sites exhibiting high linkage disequilibrium (LD) were removed in a pairwise manner (–indep-pairwise) with plink v1.9 . ADMIXTURE v1.3 was run for K equals 1 to 5 along with a five-fold cross validation [23]. Principal component analysis (PCA) was done using the glPCA function within the Adegenet R-package [25] (ST (mean and weighted), were calculated in non-overlapping windows of 50kb for the populations as inferred by ADMIXTURE using VCFtools v0.1.13 (The population structure of ink v1.9  within 5age [25] . Nucleot v0.1.13 .Loss-of-Heterozygosity (LOH) regions were identified by analyzing the distribution of heterozygous sites in non-overlapping 10kb windows as described elsewhere , using tL. aethiopica was investigated by calculating the LD decay with PopLDdecay v3.41  belonging to the same genetic population as inferred by ADMIXTURE and sampled over a period of four years. FIS was calculated as 1-Ho/He, with Ho the observed heterozygosity and He the expected heterozygosity.The recombination history of ay v3.41  and the 33.1L. aethiopica L147 reference genome, resulting in a median coverage of 27x to 70x for the publicly available sequence data, and 44x to 119x for the L. aethiopica genomes , L. donovani species complex including L. donovani (11 genomes) and L. infantum (1 genome), L. major (1 genome) and L. tropica (1 genome). Two strains did not cluster with any of these species: LEM3469 and its derived clones are positioned in between L. aethiopica and the L. donovani species complex, and L86 is positioned in between L. aethiopica and L. tropica , L. major  and L. tropica  genomes contain a large amount of homozygous SNPs, and are thus genetically distant from L. aethiopica L147   and heterozygous SNPs (where one haplotype is similar to the L147 reference and the other different). This showed that the When investigating the distribution of SNPs across the 35 chromosomes in LEM3469 and L86, we found that the majority of chromosomes consists almost entirely of heterozygous SNPs , L. donovani species complex  and L. major . This revealed that 85% of the fixed SNPs specific to L. tropica were heterozygous in L86, compared to 0.3% of the L. donovani species complex or L. major-specific SNPs. Similarly, we found that 91% of the fixed SNPs specific to L. donovani species complex were heterozygous in LEM3469, compared to 0.01% of the L. major and L. tropica-specific SNPs. We next focused on the fixed SNPs that were specific to the six L. donovani strains  and the L. infantum strain . This revealed that 1% of the L. infantum-specific SNPs and 88% of the L. donovani-specific SNPs were heterozygous in LEM3469, suggesting L. donovani rather than L. infantum was the parental species.In order to identify and confirm the other parental species of the hybrid parasites, we identified fixed SNPs specific to L. aethiopica and L. tropica (in case of L86) or between L. aethiopica and L. donovani (in case of LEM3469).Altogether, our results demonstrate that L86 and LEM3469 are the result of hybridization, rather than the result of a mixed infection, between 3.2L. aethiopica genomes, i.e. excluding the other Old World Leishmania species and the interspecific hybrids LEM3469 and L86. This resulted in a total of 94,581 high-quality INDELs and 284,776 high-quality SNPs called across 20 L. aethiopica genomes. Despite the high genome-wide density of SNPs (median 89 SNPs and average 91 SNPs per 10 kb window), we found a low number of heterozygous sites (median 6 SNPs and average 7 SNPs per 10 kb window). The allele frequency spectrum was dominated by low-frequency variants, with 58% of SNPs  occurring at <0.1% of the population. A total of 116,140 SNPs  are found within the coding region of the genome, with an approximately equal number of synonymous  and non-synonymous  mutations. A total of 267 mutations had a high impact on protein coding genes, such as the gain of a stop codon (176 SNPs) or the loss of a start codon (41 SNPs). About two third of the high impact mutations (183 SNPs) occurred at low frequency (<= 0.1%) in our panel of 20 genomes. Of the remaining 84 mutations, 66 mutations occurred within hypothetical proteins and only 18 mutations occurred within coding regions with predicted function  and the number of homozygous  and heterozygous  SNP counts  or two (L100 and L100cl1) sites. Here, we randomly excluded 1 genome per group (678-82 and L100cl1) for inference of population structure with ADMIXTURE. This is because the program ADMIXTURE assumes populations are in Hardy-Weinberg Equilibrium and Linkage Equilibrium, an assumption that may be violated when including near-identical genomes that are likely the result of clonal propagation. ADMIXTURE analyses suggested the possible presence of two (K=2) to three (K=3) populations within our panel of L. aethiopica genomes, although the 5-fold cross validation was approximately similar for K=1  were assigned to one of the three ancestry components with >99% probability  . In addition, LD levels were low with r2 descending to 0.2 at 21.9kb. These results suggest frequent genetic exchange in L. aethiopica.The phylogenetic network also revealed a reticulated pattern and long terminal branches, indicative of recombination , which should be centered around 0.5 in diploid organisms. The genome-wide distribution of ARDF was unimodal and centered around 0.5 for all L. aethiopica genomes that were largely diploid . The RD estimates revealed that chromosome 31 was at least tetrasomic in all genomes Figure 4omosomes .L. aethiopica genomes. The majority of these windows were only 2kb long  and the mean (1.1 HCN) and median (0.79 HCN) differences in HCN across our panel were minor, suggesting that our sample of L. aethiopica displays little local copy number variation. Apart from this observation, most notable was a 46 kb telomeric genomic region on chromosome 29 in strain 1561-80 showing a 10-fold increase in HCN compared to the other L. aethiopica genomes. This region covers a total of 18 protein coding genes, including a ribonuclease inhibitor-like protein (LAEL147_000545200), an actin-like protein (LAEL147_000544400) and a putative inosine-adenosine-guanosine-nucleoside hydrolase (LAEL147_000545100)  as estimated in non-overlapping 2kb windows. We identified a total of 379 genomic regions with decreased or amplified HCN across our panel of L. aethiopica - L. donovani hybrid LEM3469 and its derived clones, we detected a 6kb genomic region in chromosome 29 showing a 2-fold increase in HCN and covering a Leucine Rich Repeat gene (LAEL147_000538100)  or L. donovani (in case of the LEM3469 hybrid strain) as the other parental species. Strain LEM3469 has already been described as a potential L. aethiopica/L. donovani hybrid based on microsatellite data and single-gene sequences  hybrid strain was near diploid, being disomic at 27/36 chromosomes, suggesting balanced segregation of the chromosomes during hybridization. Trisomic chromosomes in L86 contained either one or two copies of the L. tropica parent, which may suggest that these trisomies arose after the hybridization event through multiplication of one of the parental chromosomes, possibly due to culturing in vitro (L. donovani/L aethiopica (LEM3469) hybrid strain was near triploid, being trisomic at 21/36 chromosomes. Triploid hybrids have been routinely recovered from experimental matings in Leishmania eruviana  hybrids)L. aethiopica species is genetically diverse, despite its restricted geographic distribution  but twice the number observed in the demographically bottlenecked L. peruviana (41 SNPs per 10kb)  observed in a set of 151 globally sampled genomes of the L. donovani species complex  . By comper 10kb) . Also, t complex . In cont samples  should aL. aethiopica species consists of both asexually evolving strains  and groups of parasites that show signatures of recombination (as indicated by linkage decay). In addition, parasite populations were genetically structured, suggesting that L. aethiopica consists of divergent populations, although lack of geographic/ecological data precluded us from studying its evolutionary history in greater detail. A remarkable observation was the extensive LOH in some L. aethiopica strains. Such LOH likely arose from gene conversion/mitotic recombination and indicates that L. aethiopica strains may evolve asexually over long time periods. This is exemplified by two near-identical genomes (678-82 and LEM2358cl3) that were sampled nine years from each other. Interestingly, we observed four additional LOH regions in LEM2358cl3 (sampled in 1991) impacting an additional 50 kb of the nuclear genome compared to 678-82 (sampled in 1982), suggesting that nine years of clonal evolution has resulted in LOH across 50kb of the genome.Population genomic analyses revealed that the Daphnia pulex  was isolated in 1972 and has - to the best of our knowledge - only been cultured in vitro for about 30 passages before sequencing. Hence, the extensive loss of heterozygosity cannot be explained by maintenance in vitro, and is probably due to long-term mitotic recombination in the wild, as described for the asexually evolving T. b. gambiense  under accession number PRJNA924694.Genomic sequence reads of all sequenced genomes are available on the European Nucleotide Archive (J-CD and MD conceived the original idea. IM performed the lab experiments. FB and SH supervised the work of AH. AH, SH, and FB performed the computational analyses and wrote the manuscript. All authors discussed the results. All authors contributed to the article and approved the submitted version.