Objective: The purpose of this study was to investigate the cortical activity and digit classification performance during tactile imagery (TI) of a vibratory stimulus at the index, middle, and thumb digits within the left hand in healthy individuals. Furthermore, the cortical activities and classification performance of the compound TI were compared with similar compound motor imagery (MI) with the same digits as TI in the same subjects. Methods: Twelve healthy right-handed adults with no history of upper limb injury, musculoskeletal condition, or neurological disorder participated in the study. The study evaluated the event-related desynchronization (ERD) response and brain–computer interface (BCI) classification performance on discriminating between the digits in the left-hand during the imagery of vibrotactile stimuli to either the index, middle, or thumb finger pads for TI and while performing a motor activity with the same digits for MI. A supervised machine learning technique was applied to discriminate between the digits within the same given limb for both imagery conditions. Results: Both TI and MI exhibited similar patterns of ERD in the alpha and beta bands at the index, middle, and thumb digits within the left hand. While TI had significantly lower ERD for all three digits in both bands, the classification performance of TI-based BCI (77.74 ± 6.98%) was found to be similar to the MI-based BCI (78.36 ± 5.38%). Conclusions: The results of this study suggest that compound tactile imagery can be a viable alternative to MI for BCI classification. The study contributes to the growing body of evidence supporting the use of TI in BCI applications, and future research can build on this work to explore the potential of TI-based BCI for motor rehabilitation and the control of external devices. Motor imagery (MI) is the mental practice of a specific motor activity without the concomitant physical movement . MI actiImagining a tactile sensation has been reported to elicit similar cortical excitations to MI in the form of ERD/ERS . Unlike Although conventional simple imagery involving left and right side discrimination may exhibit higher BCI accuracy due to the clear neurophysiological differences between the two sides of the body ); this sTherefore, the purpose of this study was to investigate the effect of a compound TI on the cortical oscillatory activity during imagery of a vibratory stimulus at the index, middle, and thumb digits within the left hand in healthy individuals. TI-induced cortical activities and BCI digit classification performance was compared to a similar compound MI involving imagining finger movements with the same digits as TI on the left hand in the same subjects. To achieve this, we examined the brain activities with the help of an electroencephalogram (EEG) during both TI and MI. A supervised machine learning technique was applied to discriminate between the digits within the same given limb. It was hypothesized that the compound TI would show similar oscillatory patterns and perform similarly to its MI counterpart in the digit discrimination.Twelve healthy adults  with a mean age of 27.18 ± 5.79 years volunteered in the study. The number of subjects was chosen based on recent EEG studies with similar small sample sizes ,30,31. AThe ERD and BCI classification performance on discriminating between the digits in the left hand during tactile imagery of a vibratory stimuli to either the index, middle, or thumb finger pads was evaluated and compared to a MI task involving imagining a button push using the same digits as TI.EEG signals were recorded using the Allengers’ VIRGO-32 EEG system  in accordance with the international 10–20 system. The VIRGO-32 system consisted of a 20-electrode EEG cap placed on the subject’s scalp, with electrodes at FP1, FPz, FP2, F7, F3, Fz, F4, F8, T3, C3, Cz, C4, T4, T5, P3, Pz, P4, T6, O1, and O2. The reference electrode was placed at Fz, and the ground electrode was placed at FPz. Prior to the electrode placement, the skin was cleaned, and a conductive gel was applied to ensure that the impedance was below 5 kΩ.A sensory-motor box a was conTo provide a visual cue to let the subjects know when to perform the imagery and offer action observation, a 3D left hand was modeled and animated in Blender software  to perform finger movement tasks using the index, middle, and thumb digits. A 3D virtual environment was built using the Unity game engine  where the hand and its animation was gamified. A computer monitor was placed in front of the subject in which the 3D hand animations and cues to perform the imagery were displayed.The experiment was conducted in a quiet room with minimal distractions. Subjects were comfortably seated with their arms placed on the armrests and were instructed to avoid any movement including eye blinking to reduce motion artifacts during EEG recording. The experiment comprised two imagery conditions, namely, TI and MI, with each condition consisting of one block per digit , making a total of six blocks (2 imagery conditions × 3 digits).Each block of TI consisted of five sessions, with each session beginning with five training trials followed by 10 imagery trials, making it a total of 15 trials per session. For the training trials, a brief vibrotactile stimulation lasting 150 ms was provided at the start of the trial to either the index, middle, or thumb digit pad, depending on the block. The imagery trials began with a text cue displayed over the 3D hand, instructing the subjects to imagine the same vibrotactile stimulation on the corresponding digit pad. After the text cue disappeared, the subject imagined the vibratory stimulation for a brief duration of 150 ms. Each trial had a 4-s rest period following the vibrotactile stimulation and its imagination b. A textA single block of MI consisted of five sessions of 10 trials per session, where a text reminded the subject of the digit being tested for 3 s before the block started. At the start of the trial, the 3D animation showed the digit pressing down on a button, holding the position for 2 s, releasing the button, and returning to the initial position followed by a 4-s rest c. SubjecThe order of the imagery conditions and the order of the digits within each imagery condition was randomized for each subject. EEG signals were continuously recorded at a sampling rate of 250 Hz throughout the experiment.MATLAB  was used for the ERD and digit-discrimination analysis. The EEGLAB toolbox was utilized for ERD analysis, and an artificial neural network from the Neural Network toolbox in MATLAB was used for the discrimination analysis. The EEG signals were filtered using a bandpass filter between 0.5 and 50 Hz to remove the line noise and re-referenced to a common average reference, where the average of the signal at all the electrodes was computed and subtracted from the EEG at each electrode at each time point. The ADJUST algorithm  is an auTo investigate brain activity during the imagery tasks, nine electrodes, namely, F3, Fz, F4, C3, Cz, C4, P3, Pz, and P4, were chosen since they encompass the frontal, central, and parietal regions of the scalp, respectively. The electrodes were chosen to encompass the entire sensory and motor area and to account for any of the expected contralateral activation as well as any possible ipsilateral activation. The EEG data were analyzed using time–frequency analysis to generate event-related spectral perturbations (ERSPs). ERSPs reflect the fluctuations in frequency power over time. This was achieved by dividing the mean event-related spectrum at each time–frequency point by the mean spectral estimate obtained during the pre-stimulus baseline period at the same frequency as follows:B(f) is the mean spectral estimate for all baseline points at frequency f given by the formulaμThe epochs were uniformly divided into 200 time points, and the ERSP values for each epoch were normalized to its baseline spectra. Then, the averaged ERSP values of all the task epochs were calculated. The average alpha and beta band ERSP were calculated by averaging the amplitude values within the alpha (8–12 Hz) and beta (13–30 Hz) frequency ranges, respectively. ERD was seen as a decrease in ERSP power during the performance of the imagery task. The time–frequency analysis utilized a decibel (dB) scale to provide a more comprehensible display of power alterations over time, since it represents power variations in relation to the baseline. Consequently, the dB scale allows for an enhanced perception of frequency band fluctuations compared to just examining the raw power spectrum.To evaluate neural activity discrimination of three digits, an artificial neural network (ANN) was constructed using the nrptool module in MATLAB. The toolbox employed a two-layer feedforward network with a learning procedure based on the scaled conjugate gradient backpropagation algorithm. By integrating a scaling procedure that dynamically determines the direction of the gradient during optimization, the algorithm unites the conjugate gradient (CG) approach. This integration can result in quicker convergence and superior precision when compared to alternative optimization techniques such as gradient descent or Gauss–Newton.Feature extraction was performed on the ERD data to extract discriminating features associated with the three digits during the imagery tasks. For both TI and MI, a 1-s time period between 0 ms and 1000 ms in the epoch where the ERD occurred was extracted from the ERSP data in each trial within the alpha-band frequency (8–12 Hz) and beta-band frequency (13–30 Hz) for each of the nine electrodes . The extracted ERD data for each frequency band  were concatenated and fed into the ANN, which had an input layer with 150 neurons (N) corresponding to the 50 imagery trials per digit after removing the vibratory training trials, an output layer with three neurons (M) corresponding to the three digit classes, and a hidden layer with 22 neurons calculated using the formula N×M .To train the ANN, a “training set” of 70% of the data was randomly selected. A total of 15% of the remaining data was held back and used as “validation data” to validate the model, and the remaining 15% was held back as “testing data” to evaluate the model. The neural network training was repeated 100 times to minimize the influence of random fluctuations from the training set during each iteration. The accuracy rates from the 100 runs were averaged to obtain the final accuracy rate.t-tests. Additionally, a paired t-test was used to compare the classification accuracy between the two imagery conditions. The statistical analysis was conducted with SigmaStat 4.0 , and the statistical significance was set at α = 0.05.To compare the ERD elicited during TI vs. MI, a two-way repeated measures ANOVA was performed on the digit task-related average ERD for the alpha and beta band separately. Specifically, the alpha and beta bands of the ERD were ERSP averaged over the 1-s imagery period immediately after the rest cue for each of the two frequency bands. The ERD was calculated from the ERSP at the C4 electrode. C4 was chosen since they are placed over the contralateral sensorimotor area. The analysis included the imagery condition (TI vs. MI) and digit  as independent variables. Pairwise comparisons were performed using post hoc Bonferroni The present study involved conducting trials to investigate tactile and motor imagery using digits on the left hand. The experiment measured brain activity through EEG in order to study the activation of the sensorimotor area during these tasks. Moreover, to further understand the activation of the sensorimotor area, the average energy distribution of the combined alpha and beta frequency bands (8–30 Hz) of each channel was calculated and plotted into a topology map based on the channel positions . The resp > 0.05, as indicated by a Shapiro–Wilk normality test. Additionally, the Brown–Forsythe test was conducted to assess whether the variances of the differences between the vibration conditions were equivalent, with the results indicating equal variances for all groups (p > 0.05).A repeated-measures ANOVA was applied to study the differences between TI vs. MI. The assumptions of the ANOVA on the ERD outcomes were first verified. No significant outliers were detected in the data, and the results were normally distributed with no violations of normality observed in any group with a p = 0.014), but not by digit  (p = 0.647), or their interactions (p = 0.771). Post hoc Bonferroni tests demonstrated that ERD (p = 0.023), middle (p = 0.041), or thumb (p = 0.011) digits.In the alpha band, a repeated measures ANOVA showed that ERD significantly differed by the main effect of imagery condition (TI vs. MI) (that ERD  was signp = 0.001), but not by digit (p = 0.613). The interactions between the imagery conditions and digits were not significant (p = 0.412). As shown by the post hoc Bonferroni tests, the ERD was significantly higher in MI compared to TI (p = 0.001), middle (p = 0.027), and thumb (p = 0.019) digits.In the beta band, the repeated measures ANOVA showed that ERD significantly differed between the imagery conditions (ed to TI  for the t-test was conducted. The results showed that there was no significant difference (p = 0.821) between the digit classification accuracy in the tactile imagery (mean ± SD = 77.74 ± 7.89%) and motor imagery (mean ± SD = 78.36 ± 6.51%). The percentage accuracy and the mean absolute percentage error are shown in In addition to the ERD analysis, a digit discrimination analysis was performed using a neural network for both tactile and motor imagery conditions. The results of the classification accuracy percentage for both the tactile and motor imagery for each subject is presented in The current study investigated the effect of tactile imagery on cortical oscillatory activity during tactile imagery of a vibratory stimulus at the index, middle, and thumb digits within the left hand in healthy individuals. The cortical activities and BCI performance from the compound tactile imagery involving digits from the same hand were compared to compound motor imagery involving imagining finger movements with the same digits as TI in the same subjects. Both TI and MI induced a clear oscillatory power decrease in the alpha and beta bands in the sensorimotor areas, as seen from the EEG data. The presence of an identifiable pattern of ERD, seen as an oscillatory power decrease in the alpha and beta bands, is an indicator of the performance of imagery . FurtherTactile imagery of a short vibratory burst in the left-hand digits induced an EEG power decrease in the form of ERD in both the alpha (8–12 Hz) and beta (13–30 Hz) bands in the contralateral right hemisphere, focused on the C4 electrode over the sensorimotor cortex. The ERD induced during TI were in accordance with other studies that have shown similar ERD with vibratory stimulus applied to the hand ,36. The Although the ERD induced by TI was significantly lower than MI for all three digits, the TI-induced ERD still showed a high discriminative spatial pattern between the three digits from the same hand. Previous studies using BCI based on steady-state somatosensory evoked potentials (SSSEP) to repeated tactile stimuli reported that over 80% of the subjects showed an accuracy below 70% , which mIn the current study’s experimental setup, both action observation (AO) and MI were used. It has been demonstrated that AO + MI causes a greater desynchronization . Clear iThe current study proposes a methodology that was developed based on previous work on MI and TI, which introduced subthreshold vibration as a method to improve BCI performance , and useThe major limitation of this study was the presence of event-related potential (ERP) components from the action observation that may have interacted with the ERD elicited by the kinesthetic motor imagery. The experimental design in the current study made it impossible to separate the AO related ERP components from the MI related ERD. Future studies could run practice sessions with an AO component to train the subjects on the pacing and timing of the performed kinesthetic MI, and for the actual experiment, the AO was turned off and the subjects were made to perform MI without a visual cue. While the oscillatory dynamics induced by TI were comparable to those induced by MI, they might have different cortical activation sources. Future studies should employ functional near-infrared spectroscopy (fNIRS) combined with EEG to offer better spatial resolution and help increase the understanding of TI and MI. The study mainly consisted of young adult subjects, and future studies should aim to broaden the age range to evaluate the age effect. Additionally, the current subjects did not undergo extensive BCI training, which has been demonstrated to enhance BCI performance. A longer duration study involving multiple sessions of TI would be necessary to evaluate the effect of TI-based BCI training on improving BCI-illiteracy. Finally, due to the limited sample size and observed variations in individual subject performances, caution should be exercised when interpreting the results of this study, as these factors may have impacted the statistical power.In summary, the current study evaluated the effect of a novel compound tactile imagery of a vibratory stimulus on the digits from a single given limb on the cortical response and BCI discrimination performance. The proposed compound tactile imagery showed a reduced ERD response but a similar BCI performance to a similar compound MI. The current approach also provides a novel paradigm to increase the number of BCI commands.


Early identification of genetic risk factors for complex diseases can enable timely interventions and prevent serious outcomes, including mortality. While the genetics underlying many Mendelian diseases have been elucidated, it is harder to predict risk for complex diseases arising from the combined effects of many genetic variants with smaller individual effects on disease aetiology. Polygenic risk scores (PRS), which combine multiple contributing variants to predict disease risk, have the potential to influence the implementation for precision medicine. However, the majority of existing PRS were developed from European data with limited transferability to African populations. Notably, African populations have diverse genetic backgrounds, and a genomic architecture with smaller haplotype blocks compared to European genomes. Subsequently, growing evidence shows that using large-scale African ancestry cohorts as discovery for PRS development may generate more generalizable findings. Here, we (1) discuss the factors contributing to the poor transferability of PRS in African populations, (2) showcase the novel Africa genomic datasets for PRS development, (3) explore the potential clinical utility of PRS in African populations, and (4) provide insight into the future of PRS in Africa.The online version contains supplementary material available at 10.1186/s13073-023-01245-9. Clinical risk can be evaluated from the analysis of blood biomarkers, symptoms, and prevailing family history. However, recent work has suggested that risk prediction for common chronic diseases can be improved using genetic data [Disease prevalence varies largely across the world, and some diseases are often specific to certain geographic locations. Lifestyle, diet, and environmental determinants, as well as genetic factors, explain pathological conditions in diverse settings and are likely to impact on the severity in different individuals and populations (Genome-wide association studies (GWAS) have significantly contributed to identifying a huge number of loci associated to a variety of complex diseases and traits. However, most genetic association discoveries have been made in European ancestry individuals –5. The sPCSK9 missense mutations and their impact on plasma low-density lipoprotein cholesterol levels across diverse ancestries. This breakthrough discovery exemplifies how African ancestry individuals have contributed to advancing medical knowledge, thereby benefiting the entire human race. The rich genetic variation in African populations provides so many opportunities that extends well beyond the scope of PRS. In this review article, the aim is to (1) review factors contributing to poor transferability of PRS in African populations, (2) showcase the novel genomic datasets that could enhance PRS transferability in continental Africa, and (3) explore the potential clinical utility of PRS in African populations.The poor transferability of PRS derived from European ancestry dataset to diverse African populations is a cause of concern. This is likely to be due to unique differences in genetic architecture and environmental exposures of the different populations . The lacThere are many factors contributing to poor transferability of PRS in African populations. This includes genetic factors such as minor allele frequencies, difference in linkage disequilibrium patterns, and their interactions with environmental considerations like diet, exercise, age, gender, and variability in phenotype measurement.PRS are calculated by aggregating the effect of many common variants that are associated with the diseases of interest. Given that Africa is the continent where all humans originated, it has the highest genetic diversity in the whole world. However, the current lack of diversity in genomic studies have implications on the predictive power of the methods that are trained and developed on euro-centric datasets. PRS constructed with such method may differ primarily on how the weight of the effect size is generated and how the number of single nucleotide polymorphism (SNPs) to be included in the PRS calculation is determined. For example, the interrogation of high-risk variants may involve inclusion of a causal variant from a population, whereas PRS estimates may incorporate variants that are not perfectly correlated with the causal genetic factor . The impA linkage disequilibrium (LD) reference panel and data on allele frequencies are prerequisites for application of PRS methods in a heterogeneous background. These factors are important for PRS development. For example, allele frequency differences may cause predicted risks of a disease to vary across populations. Given that LD blocks are shorter in African populations, the SNPs which are in high LD can be removed as they inflate the score. Several studies have shown lower levels of LD in African populations compared to other populations  which maThe phenomenon of pleiotropy is an indication of the complexity of how mutations in one locus may influence several pathways or functions. One gene may be responsible for different unrelated traits. SNP markers that are selected for PRS calculations may well impact on another phenotype(s) than the one for which the risk is being calculated. Graff et al. found patterns of pleiotropy when investigating PRS in Europeans for 16 different types of cancers . PositivMany PRS methods have attempted to solve the problem of different LD pattern using LD clumping or/and penalised regression. Ge and colleagues  and Bake4 show that environmental factors such as diet, exercise, age, gender, and living in rural or urban community can influence PRS portability [In addition to genetic contributions to lower PRS accuracy in African population, environmental exposures can also play a major role in contributing to poor transferability of PRS across populations . As mosttability . The restability . They altability  shows a tability .15 verifies that when population-specific SNPs are included in the calculations, the risk calculation is improved. Considerable efforts to understand GxE interaction effects is key for transferability of PRS as the effects of genetic variants on phenotype can be different between populations as demonstrated by Chikowore and colleagues [In the study by Reisberg and colleagues , SNPs frlleagues .In addition, research strategies and medical procedures are not always consistent across all countries in Africa. This is critical for diseases such as psychiatric disorders, where phenotype reporting requires intricate and complicated procedures. As an alternative, minimal phenotyping, which has recourse to hospital records, self-reporting symptoms, or prescription of medications, is used for identification of cases. This approach consists of sampling based on heterogeneous self-reported symptoms and not on the recommended criteria for diagnosis. GWAS based on minimal phenotyping produce a large number of associated loci which are however of lower heritability and have non-specific effects. Cai et al. show that when minimal phenotyping is used, for major depressive disorder (MDD), the genetic architecture is different from when the strictly defined MDD is used .Collectively, genetic factors such as differences in effect sizes, allele frequencies, LD patterns, phenomenon of pleiotropy, and phenotyping in addition to environmental exposures are limiting the generalizability of genetic predictions of diseases and traits to African populations. Pereira, et al. discussed in detail these factors that influence PRSs and limit transferability including highlighting the complex scenarios of the importance of using genomic data from multiple populations to develop appropriate population-specific applications .To do genomic research, biobanks are essential. As a result, national biobanks have been established by several governments globally to support scientific research and advance precision medicine. One notable example is UKBioBank , a well-APOL1, PCSK9, and G6PD for kidney diseases, lipid traits, and diabetes respectively [Growing evidence shows that using large-scale African ancestry cohorts as discovery for PRS development may generate more generalizable findings. Data from GWAS are fundamental as they are used for developing PRS. To date, GWAS has increasingly identified a large number of genetic variants which are associated with a range of complex traits , 24, 25.ectively . In TablOne key factor to determine the accuracy and predictive power of PRS is the power of the discovery GWAS data to avoid reaching misleading conclusions . To imprIn order to improve the representation of African genomic data in the global context for discovery and genetic risk prediction in the last decade, some initiatives have been initiated in Africa including the Africa America Diabetes Mellitus (AADM) , the UgaHundreds of PRS studies have been carried out including those on its clinical utility mainly in European populations –65. A reThe potential utility of PRS in African populations is limited by many factors. First, the current PRS methods limit the general utility of PRS as they have mostly been developed and optimised in European populations. Unless sufficient research is also undertaken to optimise the application of PRS in African populations, there is a risk of inequitable distribution of health benefits from future clinical utility of PRS. PRS calculations cannot, for now, capture the full spectrum of disease risk because of allele types, their frequencies, and their effect sizes. For precise estimates to be possible, a complete representation of all contributing loci is desirable . CurrentLack of infrastructure and difficulties with accurate phenotyping are major barriers for conducting genomic research in resource limit settings like Africa. However, to ensure collection of more accurate phenotype, a standardised data collection instrument known as the H3Africa Standard Case Report Form (CRF) was developed by H3Africa , which eFor a sustainable solution to some lack of infrastructure in the continent for genomic research, H3Africa  and otheEven with the most accurate PRS, addition of conventional risk factors to PRS would be central to potential clinical utility in Africa , 73. SucUltimately, as shown by a few African PRS studies, PRS may have clinical utility in Africa when combined with traditional risk factors for some diseases, such as cardiometabolic traits, but first, right healthcare system and genomic infrastructure must be in place, and large-scale African genomic studies are required to demonstrate the utility of polygenic risk estimation. This might require the development of multiple models for every disease given the broad genetic diversity within Africa.https://www.nature.com/articles/d44148-023-00052-z). The initiative, which carries a significant price tag of US$200 million, is built from the 3MAG programme and seeks to obtain financial support from many sources globally. These sources include African governments, industrial partners, the US government, and other funding agencies. It is imperative that Africa actively participates in the genomic medicine revolution, ensuring that it does not lag behind in harnessing the transformative potential it offers.PRS currently have limited transferability, caused mainly lack diversity in genomic studies. To improve the prediction accuracy of PRS in African ancestry individuals, it is most important to include ethnically diverse individuals from continental Africa in genomic studies. Wonkam  recommenSuch large-scale African genomic studies like 3MAG can reveal novel genes including causal genetic variants not found in previous Eurocentric studies. In addition, it would offer the opportunity to develop regional PRS within Africa to cater for genetic differences within Africans which is even larger than between Africans and Eurasians. Invariably, this would largely solve many barriers poised by difference in allele frequencies, effect sizes, and LD patterns when developing PRS. Leveraging the greater genetic diversity in Africa, within representative genomic data from Africa, PRS derived from African population may be more predictive to all global populations .Currently, PRS statistical models are trained with Eurocentric datasets. While representation of African genomics is being improved which might take decades, statistical model could be trained to estimate the projected effect sizes and allele frequency of those unknown African GWAS loci for genetic risk prediction. With current advances in machine learning and artificial intelligence, expanding the PRS models is a more practical solution to addressing the effect of genetics and its interaction with environmental exposure . This meConsidering the current poor state of many healthcare settings in Africa, even with best models and perfect PRS transferability, the prospect of clinical utility of PRS is slim in resource-limited medical settings. It is most likely that PRS would first be accessible across Africa via direct-to-consumer (DTC) company and specialist private hospitals for only those who could afford it, but there are concerns about ethical legal and social issues (ELSI) and how PRS will be regulated. Regulatory bodies should consider limiting power in the hands of PRS service providers to use their discretion to test and report any conditions or traits; otherwise, the easy access to PRS may also lead to inappropriate use and abuse. For example, the use of PRS for embryo selection, intelligence, and other psychiatric and socio-behavioural traits is strongly recommended to be restricted.Collectively, in the future, with increased representation of Africans in genomics, sophisticated predictive PRS models which account for both genetic and non-genetic factors, it may well be possible for PRS to be utilised in the medical practice for some diseases with multiple polygenic scores generated for different diseases or traits in combination with conventional risk factors. This would need to be guided with robust ethical framework, but more translational research is needed.Additional file 1: Table S1. Different methods for the calculation of PRS and their relevant parameters.


Microsporidia (phylum Microspora) is a large, diverse group of obligate intracellular, spore-forming parasites related to fungi that can infect animals, including humans . The compacted 8.8 Mbp genome consists 2,280 protein-coding genes. Various transporters are annotated, supporting the resource transfer from the host. N. ceranae maintained a complete RNAi pathway, which is absent in most microsporidia . The haplotype analysis suggests that P. alburnus have intergenomic variation and recombination. The phylogenetic analysis suggests a minor variation among geographically distinct populations. High genetic diversity is also found in N. ceranae. Wei et al. revealed allelic oscillation through transgenerational analysis. The dynamic of the allele frequency supports the red queen hypothesis during N. ceranae and honey bee co-evolution, where the essential genes regulating ATP binding and apoptosis are constantly under selection.High genetic diversity is found in the parasite Orlansky and Ben-Ami find that the parasite regulates host competition. Daphnia magna competitively excludes Daphnia similis when they coexist in a habitat. Once the parasite Hamiltosporidium tvaerminnensis engages, the competitive advantage of D. magna dramatically decreases. The results suggest that H. tvaerminnensis shapes the host community, allowing the coexistence of the hosts, which provides broader insight into the ecological importance of the parasite.It is common that the hosts share habitats. This topic covers comparative genomics, genetic diversity, and host regulation of microsporidian parasites. We hope this topic provides a state-of-the-art reference to the emerging field of microsporidian genetics and ecology.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Prunus genus encompasses a group of economically important and closely related crops, sharing an essentially common genome and, thereby, a high level of conserved and transferable microsatellite (SSR) loci. In Southern Italy, many of the local and/or neglected varieties are abandoned and at risk of extinction due to the high degree of urbanization and agricultural intensification, despite their value as genetic resources for crop improvement. This research aimed to genetically and morphologically characterize the traditional apricot (P. armenica) and peach (P. persica) germplasms collected in old family orchards. Most of the official descriptor categories were scored, thus revealing a rather high level of phenotypic variation in both collections. Genetic data allowed the discovery of diversity masked by morphological traits. Genotyping in 15 and 18 SSRs, eight of which were transferable across both species, showed an average polymorphic informativeness (PIC) of 0.44 and 0.59 for apricot and peach, respectively, and a total of 70 and 144 alleles. A reliable identification of each genotype was achieved, and the presence of possible mislabeling and/or erroneous denominations was solved. These results are encouraging for the valorization of the still poorly explored Italian Prunus germplasm, with significant economic consequences for bioresource conservation and management.The  Prunus persica (L.) Batsch) and common apricot (Prunus armenica L.) belong to the Rosaceae family of the Prunus genus [The peach  [Several studies have analyzed the morphological and genetic diversity of apricot and peach collections, although few have directly compared these two closely related species. Recent works have underlined the great potential of some apricot accessions to be directly cultivated or employed in breeding programs thanks to their high level of phenotypic  and geneunis L.) ,37,38, hPrunus species [Prunus genus [Prunus species and result in polymorphism. Thus, SSRs have been widely applied for genotyping cultivars, especially in traditional mixed orchards, often characterized by the simultaneous cultivation of several related species.Microsatellite markers (SSRs) are an invaluable tool for germplasm characterization thanks to some advantageous properties, such as their codominance, multi-allelism, high polymorphism rate, robustness and analytical simplicity. Moreover, the extensive synteny existing among the  species  makes pous genus ,41, i.e.In this study, 15 and 18 SSRs were used to assess the genetic diversity of apricot and peach populations, respectively; among those, 8 SSRs have been proven to be transferable across the two species, providing successful amplification profiles. Most loci were highly polymorphic and informative, and the allele lengths were consistent with the literature. Considering the whole collection of apricots and peaches analyzed with eight common microsatellites, a total of 89 alleles were detected, ranging from 8 for the CPPCT033 marker to 14 for the BPPCT025 marker . MoreoveWhen comparing these results within the single species, some differences among the SSR features were found . For exaPrunus genotypes (15.14) [In particular, the genetic profiles of 65 apricot genotypes by 15 SSRs allowed a total of 70 alleles to be detected, 20 of which were rare and found in 30 genotypes, while 8 were unique and found in 5 genotypes. The highest numbers of rare and unique alleles were recorded for marker CPDCT025 and for markers UDP98409 and CPPCT006, respectively. The Na mean value in the Apulian apricot collection was five alleles per locus , which i (15.14) . Accordi (15.14) ,46.P. armenica (2.5) [Generally, the discrimination power of most microsatellites ranged from 0.06 to 0.73, with a mean value of 0.44 , which ica (2.5) .The analysis of the peach collection that was constituted of 64 genotypes  allowed the detection of a total of 144 alleles across 18 microsatellite loci, with an average of 8 Na and 3 Ne per locus . This grThe genetic results of this investigation are generally comparable to those obtained in similar studies about peach germplasm characterization ,29,30,47In order to investigate the putative cases of synonymy in both collections, an LRM (pairwise relatedness) analysis was performed by fixing 0.50 as the maximum value for identical genetic profiles. For apricots, several cases of complete genetic identity were observed . With reThe relationships among the apricot genotypes were elucidated by a cluster analysis of the genetic and morphological distance matrices.The phylogenetic tree divided the Apulian apricot collection into two main clusters A: clustePhylogenetic clustering confirmed most of the LRM results since the cultivars with high genetic similarity were located in the same branch of the tree, such as in the case of the genotypes belonging to the groups named “Ananassa” and “Picocca”. In addition, erroneous cases of homonymy have been solved since cultivars with the same name but clearly with different genetic profiles were distinguished. For example, the cultivars “Barese_1” and “Barese_2”, or “Mandorla_Dolce_1” and “Mandorla_Dolce_2” or “Natalicchio_1” and “Natalicchio_2” resulted genetically distinguishable from each other. Similarly, the group of “Sant’Antonio” cultivars showed high molecular heterogeneity A.The principal coordinate analysis (PCoA) was not as informative as expected because no well-defined clusters were clearly identified. However, the genetic distance for the reference cultivars and most cultivars with LRM values of 0.5 was respected. A slight discrepancy with the LRM results and the phylogenetic clustering was observed in some cases, such as those for the couples “Natalicchio_2” and “San_Michele”, “Petrelli” and “San_Nicola”, “Picocchina” and “Spasimato”, and “Cafona” and “Due_maschere” that did not perfectly overlap in the PCoA plot .A subset of the apricot collection (50 samples) was subjected to a phenotypic characterization by means of 33 morphological and phenological traits . The pheThe considered phenotypic traits resulted polymorphic with at least two different categories each . In geneThe phylogenetic tree divided the Apulian peach collection into two main clusters: G1 and G2, which, in turn, included at least two sub-clusters each, called G1A and G1B, and G2A and G2B A. Most gThe clustering of Apulian local peaches supported the LRM results because cultivars with high genetic similarity were found to belong to the same tree branch A. The clThe PCoA plot was consistent with the genetic clustering tree, confirming the allelic similarity of the cultivars with high LRM values and the resolution of wrong homonymous cases. The first two components explained 16.63% of the total genetic variation . According to that, the cultivars were divided into three main groups: A, B and C . In detaThe phenotypic variation in a subset of peach genotypes (49) was assessed by 36 morphological and phenological traits . Again, Phenotypic traits displaying substantial differences in their range of variation were related to phenology and fruits, as reported by other studies ,60 Figu, while aSimilar to apricots, the characteristics of peaches can attract potential consumers, too, whose acceptance and satisfaction boost the market value of the fruits themself. Additionally, the fruit’s size and shape can also affect post-harvest handling. Thereby, considering consumer trends, global climate warming and environmental concerns, stakeholders generally prioritize some important traits, such as the fruit’s size, shape and color, texture and sweetness, fruit developmental stage and harvest time.This study was conducted on 57 apricot and 59 peach genotypes collected from different orchards spread in the Apulia region . All genotypes belonged to the ex situ collection of the CRSFA Institute , located in Locorotondo (Bari). They were considered ancient, rare or neglected varieties to be preserved from the risk of extinction. In addition, some commercial apricot (8) and peach (5) varieties provided by CRSFA were included in the analysis and used as references.Genomic DNA was extracted from powdered young leaves, according to . DNA conThe amplification reactions were performed, as in . All forhttp://biology-assets.anu.edu.au/GenAlEx, accessed on 6 June 2022) [The following statistical parameters were calculated for each SSR marker by the use of GenALEX v. 6.51b2 software (ne 2022) : Ne, I [ne 2022) , Ho, He ne 2022) . Moreovene 2022) . The PIChttp://darwin.ci-rad.fr, accessed on 7 June 2022). The robustness of branches was tested using 1000 bootstraps [In order to evaluate the genetic relationship between the varieties, both in the individual collections and in the group as a whole, the simple matching dissimilarity index was applied. A weighted neighbor-joining tree  was compotstraps . Moreoveotstraps  using thSeveral phenotypic traits were analyzed for each genotype over two consecutive years. Specifically, 30 and 32 morphological traits and 3 and 4 phenological traits were coded for a subset of apricot and peach genotypes, respectively , accordiIn this study, an investigation of the genetic and phenotypic diversity of apricot and peach germplasms in the Apulian region  has been illustrated. The relevance of the combined dual approach is highlighted for the accurate and proper identification of genotypes, especially those neglected or never studied before. A rather extensive variation has been found among the cultivars of the examined species with both approaches. All genotypes were clearly identified and distinguished, and a cluster analysis enabled the establishment of similar groups of genotypes. The possibility of separating morphologically related accessions by applying genetic tools has also been demonstrated, which is an important issue, particularly for local germplasms subject to possible genetic erosion and worthy of being preserved. Thus, these results have underlined how the local genotypes with interesting values, in terms of fruit quality traits and/or productivity, can have the potential to be used for direct cultivation as well as in breeding programs, especially in the current scenario of severe climate change.


This review article highlights the significance of donkeys as valuable livestock, especially in arid and semi-arid regions, and underscores the importance of implementing breed conservation programs to safeguard genetic diversity among donkey populations. Recent advancements in sequencing and analyzing complete mitochondrial DNA (mtDNA) molecules have revolutionized the evaluation of genetic diversity, with a focus on the mitochondrial D-loop region, which serves as a highly effective genetic marker. Furthermore, extensive research has explored the association between genetic markers at the DNA and RNA levels and production and reproductive traits in donkeys. This article presents a comprehensive review of the evolution of mitochondrial DNA as a tool for assessing biodiversity and genetic markers at the DNA and RNA levels that are linked to productive and reproductive attributes in donkeys.Equus asinus) play a pivotal role as essential livestock in arid and semi-arid regions, serving various purposes such as transportation, agriculture, and milk production. Despite their significance, donkey breeding has often been overlooked in comparison to other livestock species, resulting in limited genetic improvement programs. Preserving donkey genetic resources within each country necessitates the establishment of breed conservation programs, focusing on managing genetic diversity among populations. In recent years, significant strides have been made in sequencing and analyzing complete mitochondrial DNA (mtDNA) molecules in donkeys. Notably, numerous studies have honed in on the mitochondrial D-loop region, renowned for its remarkable variability and higher substitution rate within the mtDNA genome, rendering it an effective genetic marker for assessing genetic diversity in donkeys. Furthermore, genetic markers at the RNA/DNA level have emerged as indispensable tools for enhancing production and reproduction traits in donkeys. Traditional animal breeding approaches based solely on phenotypic traits, such as milk yields, weight, and height, are influenced by both genetic and environmental factors. To overcome these challenges, genetic markers, such as polymorphisms, InDel, or entire gene sequences associated with desirable traits in animals, have achieved widespread usage in animal breeding practices. These markers have proven increasingly valuable for facilitating the selection of productive and reproductive traits in donkeys. This comprehensive review examines the cutting-edge research on mitochondrial DNA as a tool for assessing donkey biodiversity. Additionally, it highlights the role of genetic markers at the DNA/RNA level, enabling the informed selection of optimal production and reproductive traits in donkeys, thereby driving advancements in donkey genetic conservation and breeding programs.Donkeys ( Equus asinus) are a significant domesticated animal that is used for transportation, agriculture, and tourism in many parts of the world, including China [Equus species [Donkeys . The studies were sourced from reputable outlets such as SpringerLink, Scopus, Web of Science, PubMed, Google Scholar, and ScienceDirect. The search was conducted using several major keywords, including donkeys, genetic resources, biodiversity, mitochondrial DNA, GWAS, RNA-sequencing, genetic marker, polymorphism, and production traits. Only data published from 2000 to the present in well-reputed, peer-reviewed journals in English were considered for discussion. Conference abstracts, books and book chapters were excluded from the review.Equus africanus africanus); and clade II, believed to have originated from an extinct African wild ass population, possibly similar to the Somali wild ass  [A summary of studies employing mitochondrial DNA as a tool for assessing biodiversity in donkeys is provided in liensis) ,4.The Huaibei Grey donkey mitochondrial genome is 16,617 bp long and contains 37 genes, including 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes . PhylogeEquus asinus) was meticulously investigated, alongside its evolutionary connections to other equids. Analysis of the Dezhou donkey’s mitochondrial genome, spanning a length of 16,655 bp, unveiled the presence of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. Notably, phylogenetic examinations highlighted a significant proximity between the Dezhou donkey, Somali wild ass, and Nubian wild ass, while domestic horse and other donkey breeds displayed comparatively more distant relationships [Equus hemionus). Additionally, the analysis of the non-d2 control region revealed significant nucleotide diversity among individuals, indicating potential applicability in population genetics and conservation efforts [In a comprehensive study conducted by Guo et al. , the mitionships ,53. Anot efforts  investigIn addition to China, several other countries, including India, Turkey, Spain, Brazil, Denmark, Italy, France, and Pakistan, have conducted studies on the genetic diversity of donkeys. In line with this trend, Earnist et al.  conducteA study conducted by Xia et al.  reportedIn a study conducted by Rosenbom et al. , the genTranscriptomic analysis reveals all expressed genes in a specific tissue or cell type, aiding in the identification of differentially expressed genes associated with animal production traits ,62. UsinRecently, several studies have employed transcriptomic analysis to identify genetic markers associated with production traits in donkeys ,67,68,69A study conducted by Dong et al. explores the genetic diversity and coat color of the Asiatic wild ass and its hybrids through whole-genome sequencing. Furthermore, the study found that hybridization has led to the introduction of novel genetic variants in the wild ass population, resulting in increased genetic diversity. Additionally, this study reveals new insights into the genetic basis of coat color in the wild ass population. Similarly, another study conducted a genome-wide analysis of the Liangzhou donkey to identify selection signatures associated with body size and drought adaptation . In addiThe candidate gene approach is a strategy used to screen genetic markers associated with a particular trait of interest by focusing on specific genes that are known or hypothesized to be involved in the expression of that trait ,74. ThisThe assessment of meat production phenotypic traits encompasses parameters such as body size, carcass weight, skin thickness, body length, rump height, vertebrae length and count, chest circumference, and cannon circumference. Among these, body size holds paramount importance within the livestock industry as a pivotal economic trait. Remarkably, the length and count of vertebrae emerge as influential determinants of both meat quantity and quality. Notably, extended body lengths often correlate with an increased rib count, while the genetic underpinning of vertebral number variation underscores its high heritability. This emphasis on the vertebral count is particularly pronounced within the thoracic and lumbar regions, as evidenced by concentrated multi-vertebral numbers therein. Noteworthy research ,79 underAs an important member of the nuclear receptor family, nuclear receptor subfamily 6, group A, member 1 (NR6A1) plays an important role in regulating growth, metabolism, and the differentiation of embryonic stem cells. Recently, a study noted a positive association of NR6A1 with body lengths and body heights in Guanzhong and Dezhou donkeys. Liu, Z. et al.  investigA comprehensive study was conducted to examine the lactation performance of Xinjiang donkeys, resulting in the development of a Kompetitive Allele-Specific PCR (KASP) marker specifically tailored to milk traits within this particular donkey population . The resIn order to promote the growth of the entire donkey species, it is crucial to examine the reproductive characteristics of male animals and engage in effective breeding practices. Comprehensive research into their reproductive traits and diligent breeding efforts are vital for the advancement of the industry as a whole. Recently, Yu et al.  investigThe following section offers some general insights into potential reasons why genetic markers associated with milk and reproductive traits have been ignored in donkey research.Lack of interest: Donkeys are primarily used as working animals in many parts of the world, and their milk and reproductive traits may not be considered as important as their ability to perform work. Therefore, researchers may not prioritize the study of genetic markers associated with milk and reproductive traits in donkeys.Limited research funding: Funding for research on donkeys may be limited compared to other livestock species, such as cattle and sheep, which are more commonly used for milk production. As a result, researchers may not have the resources to study genetic markers associated with milk and reproductive traits in donkeys.Lack of available data: Studying genetic markers associated with milk and reproductive traits requires large amounts of data, including phenotypic information and genomic data. The availability of such data for donkeys may be limited, making it difficult for researchers to study genetic markers associated with these traits.Technical challenges: Studying genetic markers associated with complex traits such as milk and reproductive traits can be challenging, and the methods used for other livestock species may not be directly applicable to donkeys. Developing new methods and techniques specific to donkeys may require additional resources and expertise.Limited commercial interest: The commercial value of donkeys is primarily based on their work performance rather than their milk or reproductive traits. Therefore, there may be limited commercial interest in determining genetic markers associated with these traits in donkeys.https://david.ncifcrf.gov/tools.jsp; accessed on PMC1933169, 1 July 2007) [Bioinformatics analysis was performed to discover the significantly regulated biological signaling pathways of the above-mentioned genes. For this purpose, we collected all the genes (mentioned in ly 2007) . ThroughIn the future, research efforts regarding donkey production traits can focus on several strategies. First, comprehensive genetic resources should be developed, such as reference genomes and gene expression datasets, to facilitate the identification of genetic markers associated with production traits. Second, integrating multi-omics data, including genomic, transcriptomic, and proteomic data, can facilitate a deeper understanding of the biological mechanisms underlying these traits and enable the identification of markers linked to broader biological processes. Third, validating and applying genetic markers in diverse donkey populations is crucial to ensure their effectiveness in improving production traits. This requires the collection of accurate phenotypic data and tailored breeding programs. Additionally, implementing genomic selection can accelerate genetic gains, but it requires accurate prediction models, validated markers, and high-quality phenotypic data. Finally, future studies should also consider the role of structural variations (SVs), including copy number variations (CNVs), inversions, and translocations , as theyIn conclusion, mtDNA analysis in donkeys has yielded valuable insights into their genetic diversity, evolution, and phylogenetic relationships. Donkey populations demonstrate limited genetic diversity, likely due to domestication and selective breeding. The identification of distinct lineages in domestic donkeys, found in Africa and Eurasia, has significant implications for conservation and breeding programs. However, selecting suitable genetic markers for donkeys based on RNA/nuclear DNA remains challenging due to the limited genomic information available. Based on the published data, it appears that there is still a significant amount of work to be done in order to identify the genetic markers associated with various production traits, including growth, meat production, milk production, and reproductive performance. Further research is needed to fully exploit the potential of genetic markers in donkey breeding. Future studies should focus on identifying additional markers linked to economically important production traits and developing practical tools for implementing marker-assisted selection in donkey breeding programs.


Knowledge about the genetic diversity of the available common bean germplasm can help breeders properly direct the choice of genetic material in the breeding process. The aim of the present work was to estimate the usefulness of 10 RAPD and 10 SCoT markers in genetic diversity detection among 33 common bean genotypes. Both molecular marker systems were able to generate high levels of polymorphism in the genetic material, which was supported by the relatively high polymorphic information content (PIC) values observed for the used markers. The Diversity Detection Index (DDI) and Marker Index (MI) were used to compare the effectiveness of RAPD and SCoT markers. For both techniques, high values of MI and DDI were calculated, representing their effectivity. The SCoT markers showed higher values of the parameters used  than the RAPD markers , indicating their higher efficiency in the detection of molecular variability. Three constructed dendrograms and PCoA plots were created using RAPD and SCoT, and both methods combined confirmed sufficient separation of the bean genotypes from each other. At the same time, a higher efficiency of SCoT markers compared to RAPD markers in the detection of the genetic diversity of beans was also proven. The results may be of future interest in the choice of genetically distant material for breeding purposes. Phaseolus vulgaris L., 2n = 22) is a significant vegetable and grain legume of worldwide importance [The common bean  legume with 11 pairs of relatively small and morphologically similar chromosomes (around 2 µm) . The gen legume wEach marker type has its advantages and disadvantages. The combination of several systems can be instrumental in getting a better understanding of the genetic diversity in the analysed species. The study of the genetic diversity of the common bean in the germplasm collections may enrich the existing knowledge of the genetic material stored in gene banks and direct its best use in breeding programmes. Therefore, the main aim of this study was to analyse the genetic variability in the collection of common bean genotypes by two different molecular marker systems (RAPD and SCoT) and to estimate their effectiveness in the genetic diversity studies of the common bean.For the genetic variability analysis, altogether 20 RAPD and SCoT markers were used to explore the level of DNA polymorphism in the collection of 33 common bean genotypes (Table 4). Both sets of DNA primers (10 RAPD and 10 SCoT listed in Table 5) produced distinct and polymorphic patterns in the genotypes.Ten RAPD primers (Table 5) amplified a total of 96 PCR products. Out of these, 70 fragments (72.91%) were polymorphic, and 26 fragments (27.09%) were monomorphic . The numThe hierarchical cluster analysis based on the RAPD polymorphism  grouped The second main cluster (two) separated 24 genotypes into two subclusters . Subcluster 2a consisted of the genotype Goliat (8) originating from Poland. Subcluster 2b subdivided 23 genotypes into two subgroups of four and nineteen common bean genotypes, respectively. The genetic similarity according to the Jaccard coefficient varied from 0.494 to 0.875. Based on the RAPD polymorphism, the genotypes Canada (six) originating from Canada and Marika (nine) originating from the Czech Republic were the most genetically similar, with a value of Jaccard’s similarity coefficient of 0.875. On the other hand, the genotype Marika (nine) and genotype Atlanta (three) originating from The Netherlands were the most genetically distant. Therefore, these two genotypes are the most suitable candidates for marker-assisted breeding.The ScoT primers amplified a slightly higher amount of scorable PCR products than the RAPD primers. Among the 117 amplified fragments, which were produced by 10 selected ScoT primers (Table 5), a total of 94 fragments (80.34%) were polymorphic, and 23 (19.66%) fragments were monomorphic . The numBased on the SCoT data, the hierarchical cluster analysis separated common bean genotypes into two main clusters (one and two). Cluster one included genotype Enso (21) originating from Sweden, and cluster two grouped the remaining 32 genotypes , which wThe combined RAPD and SCoT molecular data were also used for the construction of a joint dendrogram. The hierarchical cluster analysis  grouped The data from the combined RAPD and SCoT binary matrix were also used to construct the three-dimensional plot based on the PCoA analysis . The resTo compare the effectivity of the RAPD and SCoT markers used in the genetic diversity analysis of the common bean genotypes, different indicators were calculated . RAPD-PCThe study of genetic diversity is an integral part of the crop improvement process. The discovery of genetic relationships among plant genotypes based on DNA polymorphism may contribute to important knowledge leading not only to the simplification of the selection of germplasm for the breeding process but also to efficient conservation strategies ,38 of geThe individual marker techniques target various regions of the genome. The use of more than one marker system may provide a more informative approach while estimating the genetic diversity among the germplasm, as it might target different genomic regions. Therefore, the present study focuses on the comparison of the effectiveness of two molecular marker techniques (RAPD and SCoT) to detect genetic polymorphism in a set of 33 common bean genotypes. This information may serve as a significant means to access molecular variability and characterise the genetic relationships among the genotypes.Each of the 20 markers used was able to produce different and polymorphic DNA profiles. The total number of amplified fragments per primer, together with PIC, MI, and EMR (effective multiplex ratio) , is regaBoth sets of markers used in the study were able to generate considerable levels of polymorphism, indicating the suitability of these markers for the distinction of the genetic material. The RAPD markers revealed a lower average percentage of polymorphism (71.71%) than the SCoT markers (80.71%). High levels of polymorphism in the common bean germplasm, which are in accordance with our results, were earlier observed by several authors who used the RAPD and SCoT analyses ,47,48,49Fabaceae species was detected, such as soybean [The values of polymorphic information content represent the level of marker effectivity in the determination of polymorphism in the set of genotypes. The value of PIC depends not only on the number of produced fragments but also on their relative frequency ,51. On a soybean  and fiel soybean . Osman a soybean  detectedThe suitability of RAPD and SCoT markers was compared by calculating two parameters, which are often used for the evaluation of the general effectiveness of marker systems in genetic diversity studies: the Marker Index (MI) and Diversity Detecting Index (DDI) . Higher The comparison of some significant parameters , which are used for the evaluation of the marker system’s efficiency, revealed a slightly higher informative character of the SCoT markers compared to the RAPD markers. In general, based on the observed MI and DDI values as well as high polymorphism levels, we may consider the SCoT and RAPD markers to be effective tools suitable for the assessment of genetic variability in a collection of common beans. However, due to the detection of relatively low values of PIC with two of the used markers (SCoT62 and OPD-19), we would not recommend them for further analyses of common bean germplasm.The study of molecular variability in crops based on DNA markers provides some essential information that is very useful for future crop improvement . The maiIn our study, a hierarchical cluster analysis based on the RAPD, SCoT, and combined data from the RAPD and SCoT markers was able to successfully distinguish among the genotypes and was proven to be a suitable approach for the evaluation of genetic variability in the common bean germplasm. A UPGMA dendrogram, which is based on the joint RAPD and SCoT data, grouped the analysed material into two main clusters. In accordance with the hierarchical cluster analysis based on the SCoT polymorphism, cluster one (one) included only one genotype, Enso from Sweden (twenty-one), and cluster two grouped thirty-two genotypes, which were divided into subclusters according to their genetic similarity.The placement of individual genotypes in the different subclusters of the dendrogram and their mutual genetic distance may be a valuable source of knowledge for future breeding strategies. The right choice of genotypes for breeding purposes may lead to increased genetic diversity and the production of genetic material with superior traits. In current research, the UPGMA dendrogram and the PCoA plots confirmed a clear separation of the genotypes of common beans from each other using the RAPD, SCoT, and joint results of both methods.An effective use of more than one marker technique in the analysis of genetic variability was previously noted in several studies of plants ,53,57,58Indeed, our results showed that we can effectively discriminate the genetic differences and phylogenetic relationships among common bean varieties based on SCoT and RAPD markers. Furthermore, these results help establish the theoretical foundation for selection, genetic preservation, and breeding of the common bean varieties.The genotypes of the common bean used in the present study were taken from the collections of genetic resources of the Gene Bank of the Slovak Republic at the Research Institute of Plant Production in Piešťany, Slovak Republic, and the Gene Bank of the Czech Republic at the Research Institute of Plant Production in Prague-Ruzyně, Czech Republic. The seed samples were provided based on cooperation with the gene banks of the Slovak Republic and Czech Republic, with the aim of analysing the genetic relationships in the set of common bean genotypes. Information on the genetic background of the samples is not currently available. A total of 33 common bean genotypes  from difThe RAPD molecular marker analysis was performed using 10 RAPD markers . The ampAltogether, 10 SCoT markers  designed®  camera system.The PCR products of RAPD and SCoT amplifications were separated with horizontal electrophoresis in 1.5% agarose gels in a 1× TBE buffer (Tris—borate—EDTA) containing 0.5 μg/mL ethidium bromide at a constant voltage of 100 V for approximately 1 h, according to . The DNA® Purple 2-Log  1 kb DNA ladder. The binary values were used to compute pair-wise similarity based on the Jaccard coefficient for the preparation of the similarity matrices. The hierarchical cluster analysis of genotypes was performed using the unweighted pair-group method with an arithmetic averages algorithm (UPGMA), which yielded the constructed dendrograms using the software iTOL [https://itol.embl.de/upload.cgi) accessed on 10 May 2023, which is available online. The PCoA  plot was constructed using the free statistical programme R Project, version 4.0.5. The PIC (polymorphic information content) was calculated according to [Amplified RAPD and SCoT fragments were scored as present (1) or absent (0) in the agarose gel, and each of them was treated as independent. The binary data were used to assess the level of polymorphism. The size of the DNA fragments was estimated by comparison with the Quick-Loadare iTOL   across the studied genotypes in comparison to the RAPD markers, which detected an average polymorphism of 71.71%. Some of the characteristics of the markers, such as MI (RAPD = 5.323 and SCoT = 7.474) and DDI (RAPD = 1.612 and SCoT = 2.265), were proof of the efficiency of the selected markers for the study of genetic variability. Two molecular markers (OPD-19 and SCoT62) achieved lower PIC values. We may expect their lower effectivity for the detection of polymorphism in the analysed collection. The cluster analysis based on the RAPD, SCoT, and combined data was able to sufficiently differentiate among the individual bean genotypes. The dendrograms constructed using the UPGMA algorithm appropriately illustrate the genetic relationships among genotypes, which may be used as an important source of information and be of value in breeding programmes with the aim of expanding the genetic variability of germplasm.


Plasmodium knowlesi cases. This alarming trend highlighted the need for our understanding of this parasite and its associated vectors, especially considering the role of genetic diversity in the adaptation and evolution among vectors in endemic areas, which is currently a significant knowledge gap in their fundamental biology. Thus, this study aimed to investigate the genetic diversity of Anopheles balabacensis, Anopheles cracens, Anopheles introlatus, and Anopheles latens—the vectors for P. knowlesi malaria in Malaysia. Based on cytochrome c oxidase 1 (CO1) and internal transcribed spacer 2 (ITS2) markers, the genealogic networks of An. latens showed a separation of the haplotypes between Peninsular Malaysia and Malaysia Borneo, forming two distinct clusters. Additionally, the genetic distances between these clusters were high (2.3–5.2% for CO1) and (2.3–4.7% for ITS2), indicating the likely presence of two distinct species or cryptic species within An. latens. In contrast, no distinct clusters were observed in An. cracens, An. balabacensis, or An. introlatus, implying a lack of pronounced genetic differentiation among their populations. It is worth noting that there were varying levels of polymorphism observed across the different subpopulations, highlighting some levels of genetic variation within these mosquito species. Nevertheless, further analyses revealed that all four species have undergone demographic expansion, suggesting population growth and potential range expansion for these vectors in this region.Although Malaysia is considered free of human malaria, there has been a growing number of  P. knowlesi, a simian malaria parasite, is the predominant species currently occurring in the country [P. knowlesi, with the exception of Timor-Leste [P. knowlesi cases in the region. This highlights the importance of ongoing surveillance, monitoring, and control efforts to effectively address the persistence of malaria and prevent the potential reintroduction of human malaria in Malaysia and neighboring countries.Malaria remains a persistent global public health challenge, and countries in Southeast Asia have been assigned the goal of malaria elimination by 2030. This ambitious target highlights the urgency and importance of concerted efforts to combat malaria and reduce its burden in the region. Malaysia has been free of human malaria since 2018 [or-Leste  recommenP. knowlesi, other simian malarias, such as Plasmodium cynomolgi, Plasmodium inui, and Plasmodium fieldi, have been reported in Southeast Asia [Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) are the primary hosts of these simian malaria parasites. Recent studies have shown that P. cynomolgi and P. inui are the predominant species occurring in macaques [In addition to ast Asia ,12,13,14macaques . SimilarAnopheles hackeri (belonging to the Leucosphyrus Group) was the first species to be incriminated as the vector of P. knowlesi in Peninsular Malaysia [An. latens [An. cracens [An. balabacensis [An. introlatus [P. knowlesi in Malaysia. With changes in landscape and deforestation, humans, macaques, and mosquitoes are now found in the same environment, thus enabling the transmission of simian malaria to humans [Malaysia . This wa. latens ,18, An.  cracens ,20, An. bacensis , and An.trolatus , which ao humans .Anopheles, which comprises a complex of species, presents challenges in morphological identification of each species. Additionally, there is a lack of comprehensive information on the genetic studies of natural vectors of simian malaria in Malaysia. In a previous study, the genetic variation within the subpopulations of An. balabacensis in Kudat, Sabah, was examined using mitochondrial genes, revealing an expanding and growing population of An. balabacensis in Sabah. Notably, while the overall population showed low genetic diversity, the subpopulations exhibited high genetic diversity, likely due to interpopulation migration and breeding, facilitating gene flow among the subpopulations [To obtain certification as a malaria-free country, it is crucial to address the spread of simian malaria within Malaysia. However, there are currently limited data on the vectors responsible for transmitting simian malaria in the country. The Leucosphyrus Group of ulations .Hence, the information on genetic diversity of vectors in endemic areas is crucial for determining species taxonomy and the spatial limitations of natural populations . Based oAn. balabacensis, An. cracens, An. introlatus, and An. latens, using the mitochondrial and ribosomal sequences for the first time in Malaysia.Thus, this study aimed to analyze the population genetic structure and genetic diversity of the four important malaria vectors, Anopheles mosquitoes were collected from multiple states across Malaysia, including Negeri Sembilan, Johor, Kelantan, Pahang, Perak, and Sarawak. This sampling strategy ensured that the study covered the north, south, east, and west regions of the country. The selection of survey locations was based on the Ministry of Health (MoH), Malaysia’s data on P. knowlesi malaria cases, allowing for a comprehensive assessment of vector populations in areas with known incidences of the disease ( disease .Mosquitoes were collected using bare-leg catch (BLC), human-baited trap, Mosquito Magnet, and CDC light trap  during tAnopheles mosquitoes from the Leucosphyrus Group obtained in this study, including some archived samples, were further molecularly characterised using the internal transcribed spacer 2 (ITS2) region and mitochondrial cytochrome c oxidase subunit 1(CO1) gene. The ITS2 was amplified by ITS2A and ITS2B primers [CO1 gene. The PCR conditions were as follows: denaturation at 95 °C for 3 min; 35 cycles of amplification at 95 °C for 1 min; annealing step at 50 °C for 1 min, with elongation step at 72 °C for 1 min; followed by final elongation step of 10 min at 72 °C and held at a temperature of 4 °C. Each reaction mixture of 25 μL contained 5 μL DNA template, 0.5 μM primers, respectively, 0.2 mM dNTP, 3 mM MgCl2, 1 × GoTaq® Flexi Buffer, and 1.0 U of GoTaq® DNA polymerase . This master mix was used for both primer sets. Amplicons were subjected to electrophoresis on 1.5% agarose gels. The amplified product was purified from the gel and outsourced for Sanger sequencing . Sequences of each species were performed using the Basic Local Alignment Search Tool (BLAST)  for similarity searches. A species was confirmed by ≥98% identity and query coverage to the deposited sequence.DNA was extracted from the mosquitoes’ legs using InstaGene Matrix  according to the manufacturers’ protocol. The extracted DNA was kept at −20 °C until required. All  primers , with th primers  were useCO1, ITS2, and combined sequences of each species were aligned using BioEdit (Version 7.2) [An. balabacensis, An. cracens, An. introlatus, and An. latens, based on their polymorphic sites, were constructed by using the median-joining method in NETWORK version 5.0.0.1 software . The number of haplotypes in the subpopulations, haplotype diversity [The study included sequences of mosquitoes collected from Peninsular Malaysia and Sarawak, while additional sequences from Sabah and Selangor were retrieved from the NCBI GenBank, providing a comprehensive representation of mosquito populations across different regions of Malaysia. ion 7.2) . Haplotyiversity , and nuciversity  were alsiversity .STF) and gene flow (Nm) values between the subpopulations of each species were tested for significance. DnaSP 5.0 software was also used in estimating gene flow using 1000 permutations [STF > 0.25 (great differentiation), 0.05 to 0.25 (moderate differentiation), and STF < 0.05 (negligible differentiation) [2 statistic of Ramos-Onsins and Rozas [Pairwise genetic differentiation (utations . The levtiation) . The levtiation) . To analtiation)  and Fu’stiation)  with 100tiation) , and thend Rozas  were useAn. introlatus had a greater number of haplotypes in the combined sequences (n = 25), followed by An. latens (n = 15) and An. cracens (n = 7), and the least was An. balabacensis (n = 5) , An. latens from Rumah Sewa Panto, Sarawak , An. cracens from Kem Sri Gading, Pahang , and An. balabacensis from Simpang Utong, Sabah  had the highest haplotype and nucleotide diversities. (n = 5) . The An.An. cracens had the least number of haplotypes in ITS2 (n = 15) and CO1 (n = 4) compared with the others. The overall subpopulation of ITS2 (Hd = 0.611 ± 0.067) showed lower haplotype diversity than CO1 (Hd = 0.702 ± 0.031). The summaries of the CO1 and ITS2 diversities are shown in ITS2 (p = 0.00165 ± 0.00035) showed higher nucleotide diversity than did CO1 (p = 0.00146 ± 0.00013) for the overall population.ITS2 (n = 21) and CO1 (n = 16) sequences of An. introlatus. The highest haplotype diversity was noticed in Sg. Sendat, Selangor (Hd = 0.833 ± 0.222), while the highest nucleotide diversity was in Kem Microwave, Johor (p = 0.00563 ± 0.00085) for the CO1 analysis. Among the subpopulations in Hulu Kalong and Sg. Sendat from Selangor, the ITS2 sequences depicted the highest haplotype diversity (Hd = 1.000 ± 0.126). Similarly, Hulu Kalong had the highest nucleotide diversity (p = 0.00513 ± 0.00124).A greater number of haplotypes were found in the ITS2 (n = 16), CO1 showed a smaller number of haplotypes, n = 14 for An. latens. Based on CO1, the Rumah Sewa Panto, Sarawak, subpopulation had the highest haplotype and nucleotide diversities . In addition, the ITS2 subpopulation of Rumah Sewa Panto, Sarawak, indicated high nucleotide diversity, p = 0.00674 ± 0.00358, but high haplotype diversity was observed in Danum Valley Field Centre, Sabah (Hd = 0.956 ± 0.033).Compared with CO1 and ITS2 sequences, respectively, for An. balabacensis. Based on CO1, the highest haplotype diversity was observed in Simpang Utong, Sarawak (Hd = 1.000 ± 0.272), and the highest nucleotide diversity was observed in Lipasu Lama, Sabah (p = 0.00200 ± 0.00094). The highest haplotype and nucleotide diversities in ITS2 were recorded in Limbuak Laut, Sabah .A total of 16 and 10 haplotypes were observed in the 2 statistic from the mismatch distribution tests and the results of the neutrality test indicated that all the vector species studied were expanding. This expansion trend was further supported by the unimodal shape of the graph observed in at least one gene dataset of the mismatch distribution analysis. These findings collectively suggest that the vector populations are undergoing growth and expansion . A higher number of haplotypes (n = 25) was observed in the combined network, with the majority originating from Johor. An. latens from Peninsular Malaysia were clustered distantly from Malaysia Borneo in all three CO1, ITS2, and combined network analyses. H4 in An. cracens held all the populations from Perlis, and the other three haplotypes included the Pahang population for CO1. Only H1 (the predominant haplotype) shared populations from Perlis and Pahang for ITS2. The haplotypes from the combined network were connected to each other, as they were from the same population (Pahang) of An. cracens. The “star-like” shape was observed across all four species. Overall, the combined network for An. balabacensis indicated fewer haplotypes (n = 5) compared with CO1 and ITS2  collected from various sites in Malaysia. Based on the combined sequences of An. introlatus, the samples from Hulu Kalong and Kongsi Balak had the highest genetic diversities among the other subpopulations, suggesting greater genetic variation and potential population differentiation in these specific locations. The population from Perak had zero diversity for all the sequences analysed. This is likely linked to the lack of variation within the population in Perak [CO1-H2, ITS2-H1, and the combined sequences-H2, creates the possibility of inter-breeding and migration among the An. introlatus subpopulations [This study represents the first attempt to investigate four simian malaria vectors  for CO1, ITS2, and the combined sequences. This hypothesis needs further attention because low genetic diversity is the typical characteristic of insects in island populations [An. latens were separated into two clusters for all three haplotype networks. The haplotype clusters contained sequences from Peninsular Malaysia separated from sequences from Malaysia Borneo. Consequently, it is unknown whether the behaviour and the capability of spreading the simian malaria parasites of these two clusters of An. latens mosquitoes are similar or different.The ulations ,42,43. TAn. latens clusters based on the CO1 and ITS2 sequences. Nonetheless, defining a species in Anopheles is challenging, even if it is well studied. Thus, multiple genes and concatenated markers were used to improve the accuracy of the assessments of the genetic population structure [CO1 is commonly used for genetic studies of malaria vectors [ITS2 is the well-known molecular marker for species identifications [An. latens in Malaysia.Additionally, this study revealed the presence of the two genetically distinct tructure ,45. It i vectors ,47,48,49ications . Thus, bAn. latens from Peninsular Malaysia and Malaysia Borneo were relatively high, ranging from 2.3% to 5.2% for CO1 and from 2.3% to 4.7% for ITS2. Notably, a cutoff point of 3% is often used as a threshold for species boundaries in insects. Given that the genetic distances observed between the An. latens populations from these different geographical regions surpassed this threshold, it strongly suggests that they may represent two distinct species. A detailed morphological examination is also warranted to confirm its species status.The genetic distances between the two clades of An. latens from the two different geographical regions. The South China Sea significantly divides East and West Malaysia, and this may be a factor leading to the intra-specific genetic discontinuities [An. latens from Peninsular Malaysia and Borneo. This is further supported by the high levels of genetic differentiation and moderate gene flow in this study.The findings demonstrated that geographic distance might have a major effect on the genetic structure of inuities ,52. TherAn. cracens in Pahang indicates its population growth, likely resulting from the accumulation of new mutations over time, with the emergence of new haplotypes within the population. Differences were observed between the CO1 and ITS2 markers, which can be attributed to the selection of different molecular markers. Each marker may have distinct mutation or evolutionary rates, varying selection pressures, and different gene constraints [An. cracens in the study were not dependent on the geographical distances. Gene flow among the vectors of malaria due to the lack of geographical distance and barriers was observed in several studies [Overall, the high haplotype diversity observed in straints ,54. Nevestraints . The gen studies ,57,58,59An. balabacensis. Nevertheless, low levels of gene flow were also observed from the overall subpopulations in the combined sequences, yet high gene flow was seen in the COI and ITS2 analyses. Although the sampling distributions varied, the ecological habit is relatively similar in Sabah and Sarawak [The samples from different study sites could belong to the same, single continuous population because of the low genetic differentiation detected among the subpopulation pairs in both genes for  Sarawak . Thus, t2 statistic from the mismatch distribution tests, along with the negative values from the neutrality tests, further support the population expansion of An. balabacensis, An. introlatus, An. latens, and An. cracens in Malaysia. Likewise, evidence of demographic expansion has also been reported in other insects, such as dragon flies, black flies, and buffalo flies, in Southeast Asia [The “star-like” network observed in all four vectors suggests population expansion ,62. Furtast Asia ,64,65.An. latens populations could be a topic of interest for future studies, as it would allow for the identification of which genotypes are more likely to be exposed to simian malaria infection in the wild. Furthermore, in this study, the collection sites covered most of the P. knowlesi malaria endemic regions in Malaysia, yet future research should conduct extensive sample collection in a wider distribution area to obtain a complete overview of the genetic structure of the vectors. Thus, in light of the elimination of malaria, it is timely for Southeast Asian nations to commit a concerted effort to study the vectors and to develop vector control strategies to prevent future outbreaks.A deeper understanding of the genetic diversity of local vectors could provide valuable information for epidemiological surveillance and malaria vector control strategies. The presence of the different lineages in the


Furthermore, the corrosion resistance of the Ag-GLC coatings were evaluated. The results showed that the spontaneous escape phenomenon of silver could take place at the GLC coating, regardless of preparation condition. These three preparation factors all had an influence on the size, number and distribution of the escaped silver particles. However, in contrast with the silver target current and the addition of CH4 gas flow, only the change in deposition temperature had a significant positive effect on the corrosion resistance of the Ag-GLC coatings. The Ag-GLC coating showed the best corrosion resistance when the deposition temperature was 500 °C, which was due to the fact that increasing the deposition temperature effectively reduced the number of silver particles escaping from the Ag-GLC coating.Silver-doped graphite-like carbon (Ag-GLC) coatings were prepared on the surface of aluminum alloy and single-crystal silicon by magnetron sputtering under different deposition parameters. The effects of silver target current and deposition temperature, as well as of the addition of CH For this reason, several studies have explored the possibility of doping the coating with metallic elements, such as Cr or Ag, to increase the electrical conductivity of these amorphous carbon coatings [There is a development tendency that the traditional graphite bipolar plates are being replaced by the thinner metallic ones, particularly, in the field of proton exchange membrane fuel cells (PEMFC) ,2. Compacoatings ,11,12.In addition to the enhancement of the electrical conductivity values, the use of silver as a doping element can also improve their corrosion resistance ,13,14. YSilver surface segregation and clustering has also been observed in the development of several functional coatings, such as diamond-like carbon (DLC) ,22, TiCN4 gas flow. The main purpose of this work was to explore the influence of deposition parameters during magnetron sputtering on the silver’s spontaneous escape locations, the density of silver particles at the sample surface, the silver residue content and the corrosion performance of Ag-GLC coatings.In this work, a series of the Ag-GLC coatings were prepared by the magnetron sputtering using a different silver target current and deposition temperature as well as the addition of CHCr) 0.30 A, current of silver target (IAg) 0.10 A, and a bias voltage of −400 V). Secondly, a pure Cr layer with thickness ca. 150 nm was deposited for 3 min (ICr 2.00 A and bias voltage of −120 V). Subsequently, three groups of samples were prepared with the current of both graphite targets (Ic) at 1.50 A and the bias voltage at −60 V for 150 min. The other processing conditions were fixed as follows: (1) IAg = 0.01 A, 0.02 A, 0.03 A, and 0.05 A, deposition temperature 300 °C; (2) IAg = 0.03 A, deposition temperatures 200 °C, 400 °C and 500 °C; (3) IAg = 0.03 A, and deposition temperature 500 °C with CH4 gas flow at a rate of 20 mL/min. During the deposition process, the substrate holder was designed to separate the substrate form the target plate (ds-t) and fix it at 120 mm at a speed of 4 rev/min, while the thermocouple was also fixed at 120 mm without rotation. After the ion plating process, in order to explore the escape behaviour of silver, the specimens were exposed to the atmospheric environment for 3600 h.The silver-doped GLC coatings were prepared on the polished 6061 aluminum alloys and single-crystal silicon substrates using a MSIP 019 magnetron sputtering ion-plating system. For complete details on the preparation of targets, please refer to our previous work . FirstlyThe surface and cross-section morphologies of the silver-doped GLC coatings on single-crystal silicon substrates were characterized by field emission scanning electron microscopy , when exposed to the atmospheric environment (1 atm) for 3600 h. The content of the residual silver in the GLC coating was determined by an energy dispersive X-ray spectroscopy (EDS). Image Pro Plus 6.0 software  was used to measure the density and average particle size of silver particles on the surface of the GLC coating.2SO4 solution containing 5 ppm F− at 80 °C and bubbled O2 (cathode simulated). A platinum sheet with a surface area of 4 cm2 was used as counter electrode, while a saturated calomel electrode (SCE) was used as reference electrode. The working electrode  was sealed with epoxy resin with an exposed surface area of 1 cm2. The potentiodynamic polarization curve was recorded in the range from −0.2 V vs. open-circuit voltage to +1.0 V vs. SCE at a potential scanning rate of 1 mV/s after immersion for 0.5 h in the electrolyte solution.A CH Instrument CHI 760D electrochemical analyzer and a conventional three-electrode cell were used to conduct the electrochemical tests in 0.5 mol/L HAg at 300 °C after being exposed to air (1 atm) for 3600 h. It can be seen that the spontaneous escape phenomenon of silver could take place, regardless of IAg. The escaped silver clusters or particles did not cover the whole surface of the GLC coating. However, the value of IAg had a great impact on the distribution of the escaped silver particles on the sample surface. Ag = 0.01 A, a bimodal particle distribution was found. Most of the silver particles were smaller than 10 nm, which tended to agglomerate in clusters and were located very close to the three-point grain boundaries. Moreover, as indicated by the circle in Ag = 0.02 A  to 17.2 % (IAg = 0.05 A). This is reflected in the increase in the coating thickness that was measured in the different samples  into atmosphere (1 atm), under the huge pressure difference the gas molecules in air can easily rush into and fill up the carbon cluster interface, and the Ag atoms and clusters distributed on the outer layer of the GLC coating will escape in the case of gas influx [These phenomena in s influx . Afterwas influx . Wettergs influx  and Zhdas influx  have reps influx ,28.Ag (0.01 A and 0.02 A at 300 °C)  b, the nu 300 °C) b.When the Ag target current increases, the amount of silver particles between the carbon grains increases, resulting in a reduction in the C–C grain connectivity. In this situation, it is not only the smaller silver particles that can migrate, but also the bigger ones. These big silver particles have a lower mobility on the surface and, for this reason, shorter distances between the silver particles in the coating surface were measured and a broader size distribution was observed c.During the magnetron sputtering process, the sputtered ion energy was proportional to the deposition temperature, and the higher deposition temperature was conducive to improving the interdiffusion of atoms in the Ag-GLC coatings. Therefore, studies have proven that the more compact carbon cluster interface could be obtained at a higher substrate temperature ,26. WithcorrE) and corrosion current density (corrI) derived from the Tafel curves are listed in Ag was beneficial to improve the corrE of the Ag-GLC coatings, but had little effect on their corrI. The silver residue content inside the GLC coating increased with IAg, and thus lowered the connectivity of the GLC coating. Therefore, changing the IAg value had little effect on enhancing the corrosion resistance of the Ag-GLC coatings.The corrosion resistances of the different Ag-GLC coatings were evaluated using potentiodynamic polarization, and the results are presented in corrE and decreasing corrI. In particular, when the substrate temperature rose to 400 °C and 500 °C, the Ag-GLC coatings exhibited a better corrosion resistance than the GLC coating, which was closely related to the lower defect density of the GLC coatings prepared at a high temperature [4 gas flow during the deposition was detrimental to the corrosion resistance of the Ag-GLC coatings. This was largely due to the fact that the addition of CH4 gas flow destroyed the adherence of the GLC coating to the substrate. According to the polarization studies combined with the SEM images, the GLC coating prepared at 500 °C with IAg = 0.03 A displayed the best corrosion resistance compared with the other specimens, as shown by the lowest corrosion current density and relatively compact uniform microstructure. By analyzing the results in perature . MoreoveThe silver target current directly decided the silver content incorporated into the GLC coating and increased the coating thickness, but it played a minor role in enhancing the corrosion resistance of the GLC coatings.When the silver target current was fixed, the deposition temperature did not change the initial content of silver particles incorporated into the GLC coatings. However, increasing the deposition temperature effectively reduced the number of silver particles escaping from the GLC coatings. Meanwhile, as it rose up to 400 °C and 500 °C, the corrosion resistance of the GLC coating was clearly enhanced.4 gas flow, the escaped silver particles were obviously refined, but these Ag-GLC coatings had a poor adhesion with the substrate. Hence, the use of CH4 gas flow actually led to a worsening of the corrosion resistance of the coating.Compared to the Ag-GLC coatings prepared by argon under the same conditions, after using CHThis work has primarily investigated the effects of the deposition parameters on the spontaneous escape behavior of silver from the GLC coatings prepared by magnetron sputtering. The main conclusions that can be drawn are as follows:


Brook trout populations have been declining throughout their native range in the east coast of the United States. Many populations are now distributed in small, isolated habitat patches where low genetic diversity and high rates of inbreeding reduce contemporary viability and long‐term adaptive potential. Although human‐assisted gene flow could theoretically improve conservation outcomes through genetic rescue, there is widespread hesitancy to use this tool to support brook trout conservation. Here, we review the major uncertainties that have limited genetic rescue from being considered as a viable conservation tool for isolated brook trout populations and compare the risks of genetic rescue with other management alternatives. Drawing on theoretical and empirical studies, we discuss methods for implementing genetic rescue in brook trout that could yield long‐term evolutionary benefits while avoiding negative fitness effects associated with outbreeding depression and the spread of maladapted alleles. We also highlight the potential for future collaborative efforts to accelerate our understanding of genetic rescue as a viable tool for conservation. Ultimately, while we acknowledge that genetic rescue is not without risk, we emphasize the merits that this tool offers for protecting and propagating adaptive potential and improving species' resilience to rapid environmental change. Many brook trout populations exist in small, isolated habitats where demographic stochasticity and the lack of gene flow threaten long‐term persistence. In this article, we review existing literature to evaluate whether genetic rescue may be a viable management goal for brook trout conservation. Although brook trout were once abundant in many mountain streams and lakes, native populations have declined over the last century due to the interactive effects of habitat loss, climate change, and competition with non‐native species  isolated by anthropogenic activities may be particularly vulnerable to demographic and genetic collapse, as bottlenecked populations frequently experience higher genetic loads owing to inbreeding, loss of genetic diversity, and increased prevalence of deleterious mutations  despite the use of an adaptively divergent source population. Likewise, Wells et al.  and have lost substantial genetic diversity may be better candidates for genetic rescue. Ralls et al.  suggesteSalvelinus spp., authors have been able to disentangle the relative effects of neutral and adaptive genetic processes for shaping contemporary patterns of genetic diversity, ultimately providing insights into the colonization success of introduced brook trout populations . Likewise, regional population genetic analyses are necessary to identify source populations with enough genetic diversity to enable genetic rescue and to screen for introgression with domestic hatchery lineages ; investigation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Jacob M. Rash: Conceptualization ; supervision ; writing – original draft (supporting); writing – review and editing (supporting). David C. Kazyak: Conceptualization ; supervision (supporting); writing – original draft (supporting); writing – review and editing (supporting).None.The authors declare no conflicts of interest.