Visual experiences can be triggered externally, by signals coming from the outside world during perception; or internally, by signals from memory during mental imagery. Imagery and perception activate similar neural codes in sensory areas, suggesting that they might sometimes be confused. In the current study, we investigated whether imagery influences perception by instructing participants to imagine gratings while externally detecting these same gratings at threshold. In a series of three experiments, we showed that imagery led to a more liberal criterion for reporting stimulus presence, and that this effect was both independent of expectation and stimulus-specific. Furthermore, participants with more vivid imagery were generally more likely to report the presence of external stimuli, independent of condition. The results can be explained as either a low-level sensory or a high-level decision-making effect. We discuss that the most likely explanation is that during imagery, internally generated sensory signals are sometimes confused for perception and suggest how the underlying mechanisms can be further characterized in future research. Our findings show that imagery and perception interact and emphasize that internally and externally generated signals are combined in complex ways to determine conscious perception. •Mental imagery leads to more presence responses in a perceptual detection task.•This effect is independent of expectation and is stimulus-specific.•Imagery vividness is correlated with perceptual detection criterion.•These findings can be explained by imagery sometimes being mistaken for perception. Different shapes, colours and textures are processed by our visual system to create the technicolour perception we experience every day. At the same time, while we are caught up in thinking about past or future events, our brain internally generates a rapid stream of mental images . VariousAn interaction between mental imagery and perception has been investigated within different contexts. One line of research has shown that imagining one of two interpretations of an ambiguous stimulus prior to its presentation increases the probability of subsequently perceiving that same stimulus . HoweverAnother line of research has investigated the influence of imagery on the detection of unambiguous but near-threshold external stimuli. Some studies found that simultaneous imagery increased the likelihood that an external stimulus was detected  whereas In the current study, we combined near-threshold psychophysics with large, robust sample sizes afforded by web-based data collection to investigate how imagery affects external perceptual detection. In a series of experiments, we investigated the influence of imagery on signal detection theoretic measures of detection and whether this effect was influenced by expectation, was stimulus-specific and whether individual differences in imagery vividness played a role. Participants detected gratings at threshold while simultaneously imagining either the same gratings, orthogonal gratings, or nothing at all. If imagery merely causes non-specific cognitive effects, such as distraction or a general bias to respond ‘present’, we would expect it to affect detection rates independent of whether the imagined and detected stimuli were congruent or not. In contrast, if imagery was sometimes confused for perception, or vice versa, we would expect imagery to bias presence reports in a stimulus-specific way. Furthermore, the direction of this confusion might depend on the likelihood of stimulus presentation: participants might be more likely to confuse imagery for perception when they expect stimulus presence and more likely to confuse perception for imagery when they expect stimulus absence. Finally, if this effect is indeed specific to imagery rather than a general top-down effect we would expect a correlation with imagery vividness.22.12.1.1SD = 8.3) were recruited using Prolific (www.prolific.co) and completed the study online. Data were collected on an institutional server managed by the JATOS tool  and consisted of sinusoidal gratings tilted at an orientation of 45te noise . The viste noise . The thrDuring stimulus-present trials, twenty stimulus images ranging from zero visibility to 70% detection threshold were presented over the course of 2 s, giving the impression that the stimulus was gradually ramping up. This ramping up was done to mimic the gradual nature of mental image generation . During 2.1.3d’) and criterion (c) were calculated separately for the imagery and no-imagery trials as follows:We used signal detection theory to analyse the data . Detectiz indicates the inverse of the cumulative normal distribution, H is the hit rate , and FA is the false alarm rate . Detection sensitivity d′ is a measure of detection performance, with greater values indicating better performance. Criterion c is a measure of participant's bias towards responding ‘yes’ (present) or ‘no’ (absent), irrespective of whether a stimulus is present or not. Greater values of c indicate a more conservative criterion, indicating a greater tendency towards reporting absence. Hit rates of 1 or false alarm rates of 0 lead to biased estimations of d′ and c. To correct for this, in those cases of extreme values we added a count of 0.5 to the relevant cell  compared to the no-imagery condition  = 7.693, p = 1.33e−11), indicating a higher tendency to report stimulus presence during imagery. Theoretically, criterion and detection sensitivity are independent, so a change in criterion does not necessarily lead to a change in d′. However, detection sensitivity d′ was also significantly lower in the imagery condition  compared to the no-imagery condition  = 3.933, p = 0.0002) indicating that performance was also worse during imagery. This effect on d′ can be explained by the fact that the imagery-induced increase in false alarm rate  was greater than the increase in hit rate  = 3.169, p = 0.002). Together, these results indicate that imagining a stimulus leads to an increase in the tendency to report the stimulus as present, especially when the stimulus was actually absent.The results of experiment 1 are shown in p-values >0.07). However, we did observe strong significant correlations between several SDT measures and mean imagery vividness ratings during the imagery practice trials. There were 12 SDT measures . We corrected for multiple comparisons using a Bonferroni correction such that only correlations with a p-value lower than 0.05/12 = 0.0042 were considered significant.We also investigated individual differences in SDT measures. We calculated Spearman correlations to be more sensitive to potential non-linear effects in our data and to be less sensitive to outliers. There were no significant correlations between any of the questionnaire scores and any of the SDT measures , imagery condition  or the difference between them . There was a significant negative correlation between imagery vividness and criterion c during both the no-imagery  and the imagery condition  but not the difference between them . There was a significant positive correlation between imagery vividness and FA during both the no-imagery  and the imagery condition  but not the difference between them . Finally, there was also a significant positive correlation between imagery vividness and H during the imagery condition  but not the no-imagery condition  or the difference between them . Together, these results indicate that higher imagery vividness was associated with a more liberal criterion and increase in false alarm rate during both imagery and no-imagery conditions, despite there being no relationship between vividness and the magnitude of the effect of imagery on detection.There was no significant correlation between vividness and In summary, this first experiment showed that imagining a stimulus increased the tendency to report seeing that stimulus, and that imagery vividness also positively correlated with the tendency to report external presence. One explanation for these results is in terms of source confusion: during imagery blocks participants sometimes confused their imagery for external stimulation, leading to an increase in presence reports. However, we did not observe the reverse: external stimulation sometimes being confused for imagery, leading to an imagery-induced increase in misses. One reason for this might be that participants were more likely to attribute imagery to perception than vice versa because there was an expectation of external stimulus presence . In contrast, if external stimulus presence is less likely, perception might be more likely to be attributed to imagery . In the next study we investigate whether the imagery effect is influenced by such expectations.33.13.1.1SD = 10.54).150 participants were recruited and data were collected in the same way as in experiment 1 (see Section 2.1.1). Data from 12 participants were not obtained due to technical issues. Experimental blocks were excluded based on the same criteria as in experiment 1. Due to the addition of an experimental factor (expectation) and the resulting lower number of blocks per condition, we now excluded participants if they had less than 1 usable block  in each condition. This led to the exclusion of 40 participants. Together, 98 participants were included in the final analysis  x imagery (no-imagery/imagery).3.2c with a lower criterion in the imagery  compared to the no-imagery condition  = 29.578, p < 0.0001, eta2 = 0.243), replicating the findings of experiment 1. There was also a main effect of expectation on c with a lower criterion in the expect-presence  compared to the expect-absence condition  = 53.497, p < 0.0001, eta2 = 0.368). However, there was no significant interaction between imagery and expectation  = 0.891, p = 0.348), indicating that the effect of imagery was not influenced by expectation. Furthermore, in contrast to experiment 1, in experiment 2 there was no significant main effect of imagery on d′  = 3.200, p = 0.077). There was also no significant main effect of expectation on d′  = 0.130, p = 0.719) or an interaction between these two factors  = 0.611, p = 0.437).The results of experiment 2 are shown in p-value cut-off of 0.05/4 = 0.0125. Similar to experiment 1, there was no significant correlation between imagery vividness and d′  but there was a significant negative correlation between vividness and c . Furthermore, there was a significant positive correlation between vividness and FA , but not between vividness and H . Together, these findings suggest that the more vivid participants' imagery was, the more likely they were to respond present during the detection task, especially when there was no actual stimulus present.We again investigated individual differences in SDT measures by correlating them with the mean imagery vividness ratings obtained during the imagery practice trials. Because we did not observe any significant correlations between vividness and the difference between imagery and no-imagery in experiment 1 and because the effects were similar in both imagery conditions, here we investigated SDT measures calculated over all trials together. We performed 4 tests, giving a Bonferroni corrected N = 51), ‘Maybe’  and ‘No’ . A repeated-measures ANOVA with expectation and imagery as within-subject variables and awareness of influence as a between-subject variable revealed a significant interaction between imagery and awareness on c  = 3.560, p = 0.033, eta2 = 0.073): the effect of imagery on c was largest for ‘yes' responders , smaller for ‘sometimes' responders  and smallest for ‘no’ responders . This shows that as a group, participants had metacognitive insight into the effect of imagery on their visual detection responses: those participants who showed the strongest effects on the decision criterion also tended to notice it and respond ‘yes'.To investigate whether participants' awareness of imagery influencing their responses was related to the magnitude of the imagery effect, we asked participants at the end of the experiment “Do you feel like imagining the gratings influenced your responses on the task?”. Participants were categorized into three categories based on their responses: ‘Yes’ .Based on the size of the effects in the previous two experiments, we performed a power calculation to determine the number of participants for this experiment. Assuming a medium effect of 0.5, 34 participants would be required to reach a power of 80% . Taking 4.1.2c).The main experimental design was similar to experiment 1 . In this4.2c was significantly reduced during congruent imagery  compared to no-imagery  = −2.966, p = 0.0054), but not during incongruent imagery  compared to no-imagery (t(35) = 0.474, p = 0.639). Accordingly, the imagery-effect on c was larger for congruent  than incongruent imagery  = −3.018, p = 0.0047). In contrast to experiment 1 but in line with experiment 2, there was no significant difference in d′ during congruent imagery  compared to no-imagery  = −0.311, p = 0.758). However, there was a significant decrease in d′ during incongruent imagery  compared to no-imagery (t(35) = −5.458, p = 0.000004), indicating that incongruent imagery hampered performance. Accordingly, the imagery-effect on d′ was larger for incongruent  compared to congruent imagery  = −3.994, p = 0.0003). This double dissociation between the effect of congruent and incongruent imagery on c and d′ is caused by the different effects on false alarm and hit rates. While there was no significant difference in the imagery-induced increase in false alarms between congruent  and incongruent imagery  = −0.034, p = 0.973), hit rate was increased during congruent imagery  but decreased during incongruent imagery  = 4.517, p = 0.00007), leading to a decrease in sensitivity for incongruent imagery and a decrease in criterion for congruent imagery.The results for experiment 3 are shown in c and d′ can be explained by source confusion only occurring during congruent imagery, when the internal and external stimuli are similar. Under this explanation, during congruent imagery participants confuse their mental image to reflect perception, leading to a heightened tendency to report stimuli are present. This is unlikely to occur during incongruent imagery, when participants know that any external stimuli would be orthogonally oriented to the one they are imagining. In contrast, the decrease in d′ during incongruent imagery may have been caused by imagery disrupting the processing of congruent input, leading to both an increase in false alarms together with a decrease in hits. This explanation would mean that an increase in FA observed in the two imagery conditions , suggesting that there is a relationship between the mechanisms underlying these effects and potentially challenging either the source confusion and distraction account. However, it could also be that participants sometimes thought they perceived the imagined stimuli during incongruent imagery, such that the FA effect here actually also represented source confusion. Indeed, in experiment 2 a few participants reported that, despite the instructions, they believed the other orientation was also sometimes presented. Alternatively, both source confusion and distraction could be driven by the strength of mental imagery as a third common factor, with stronger imagery leading to both more source confusion and more distraction.Together, these results indicate that the imagery-induced decrease in criterion is specific to the content of the mental image. In contrast, a decrease in d’ might be partly due to the dual-task nature of the imagery compared to no-imagery conditions. Therefore, the double dissociation between nditions  is causec , indicating that more vivid imagery was correlated with a higher tendency to report presence. However, in contrast to the previous findings, we also observed a significant negative correlation between vividness and d′ , suggesting that more vivid imagery was also associated with worse detection performance (possibly due to the effect of imagery on the size of the incongruent distraction effect). Furthermore, there was a strong significant positive correlation between vividness and FA , but not between vividness and H .We again investigated the relationship between imagery vividness and external signal detection. In line with experiment 1 and 2, there was a significant negative correlation between imagery vividness and 5All three experiments showed that imagining the to-be-detected stimulus during external detection leads to a higher likelihood of reporting stimulus presence. In contrast, detection sensitivity, measured by d’, showed a significant decrease due to imagery only in Experiment 1. Furthermore, higher imagery vividness was associated with a more liberal detection criterion but was not related to d’ in two out of three experiments. To further investigate the differences between (congruent) imagery and no-imagery on criterion and d’, we pooled together the data from these conditions from all three experiments for follow-up analyses.t(229) = −9.72, p = 6.81e-19), with a medium effect size (Cohen's d = 0.532). Furthermore, when pooling all data together, there was also a significant decrease in detection sensitivity (d’) due to imagery t(229) = −3.67, p = 0.0003), with a small effect size (Cohen's d = 0.230). If the decrease in criterion and the decrease in d’ were driven by the same underlying mechanisms, we might expect them to correlate between participants. However, there was no significant correlation between the decrease in criterion and the decrease in d’ , suggesting that these effects may have different causes. Furthermore, in this pooled analysis, imagery vividness significantly correlated with the shift in criterion  but not with the shift in d’ .As expected, the pooled analysis also revealed a significant decrease in criterion due to imagery , and that such internal generation is occasionally mistakenly inferred to be due to an external stimulus. During stimulus-absent trials, this internally generated sensory activation then sometimes crosses the detection threshold, leading to an increase in false alarms. During stimulus-present trials, signals that would have been too weak to cross the threshold in the absence of imagery, are now boosted by the addition of internally-generated activation, leading to an increase in hits. Neuroimaging studies showing that imagery activates sensory representations in a perception-like way are in favour of an explanation in terms of source confusion ; howeverFinally, imagery causing a decrease in decision threshold would not be in line with source confusion. In the second experiment we also found that if participants were aware that imagery might influence detection, the imagery effect was bigger. This finding could mean that the belief that imagery influences detection made participants respond accordingly, suggesting our results might (partly) reflect demand characteristics e.g. . Demand There are substantial differences between individuals in the vividness of mental imagery . In the Interestingly, in all three experiments we only observed one direction of source confusion: imagined stimuli being mistaken for real, leading to a decrease in detection criterion. Early studies of mental imagery showed that when participants were asked to imagine certain objects and describe what they experienced, external presentation of these same objects at threshold was often missed . This obAnother important issue is whether the imagery effects observed here could be (partly) due to imagery increasing visual attention to the external stimulus. Indeed, spatial attention has also been shown to sometimes decrease criterion , leadingTaken together, we have shown that imagining a stimulus while detecting that same stimulus at threshold leads to an increased tendency to say that external stimuli are present. This effect may be explained by internally generated sensory signals during imagery being erroneously attributed to an external source. This is in line with the idea that the neural correlates of imagined and perceived stimuli are highly similar such that perceptual reality monitoring is a non-trivial process that is prone to error in specific circumstances . Our stuThe authors declare that there are no competing interests.


Apparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio  or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH).To detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension.In this observational case–control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (β-2 microglobulin) gene was selected as the reference housekeeping gene.p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression.Among family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels  that progressively decreased in 221 AG heterozygous with hypertension , 221 AG heterozygous normotensives , and wild-type 221 AA subjects . Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (HSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension. Apparent mineralocorticoid excess (AME) is a rare autosomal recessive disorder caused by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme deficiency that consequently leads to an activation of the mineralocorticoid receptor (MR) not mediated by aldosterone . An impaper se although it was hypothesized that a mild reduction of 11β-HSD2 activity can have a role also in low renin essential hypertension (EH). Milder forms of AME, recently named as “non-classic” AME, are at higher prevalence than classic AME, have a different phenotype and genotype, and are mainly related only to a partial enzymatic deficiency  hypertensive patients and (2) members of a family affected by AME as previously described .Blood sample for biochemical routine parameters, 24-h urine, and second morning urine samples were collected for each subject. Second morning urine was obtained the same day of the collection of the 24-h urine vessel. A sterile container was given to patients who provided the second morning void directly at the outpatient clinic.Measurement of biochemical, hormonal, and routine laboratory tests were performed at the laboratory of the Clinical Chemistry Institute of the Verona University Hospital. Plasma renin and aldosterone levels were measured by commercially available methods , as previously described .The study was approved by the Ethics Committee of our Institution  and patients gave their informed written consent after full explanation of the study.Patients were enrolled among those referring to the Hypertension Unit of the Verona University Hospital for resistant hypertension or for possible secondary causes of hypertension. After the exclusion of secondary forms of hypertension  only patients for whom a diagnosis of EH was made were included in the study. Similarly, patients currently treated with glucocorticoids, or with a clinical history of previous glucocorticoid treatment, were excluded. None was taking licorice-containing sweets, or 11β-HSD2 inhibitors, such as cancer treatment dithiocarbamates (DTCs) or fungicides such as itraconazole, hydroxyitraconazole (OHI), and posaconazole , 20.According to the study protocol, all patients had not taken any hypotensive drugs other than verapamil and/or alpha-blockers over the previous 4 weeks. Plasma samples for aldosterone and renin were obtained after at least 2 h in the upright position and a subsequent period of 10 min in the seated position. Blood samples for hormonal and routine parameters were collected after overnight fasting between 8:00 and 9:00 a.m. Biochemical parameters were determined and 24-h urine cortisol/cortisone metabolite assay was performed on 24-h collected urine.Fourteen subjects with a definite diagnosis of EH  were selected based on the availability of biological samples for the evaluation of HSD211B exosomal mRNA.Fourteen members of a family previously studied for a story of AME syndrome  were enrThe two homozygous probands were not taking antihypertensive drugs at the time of enrolment whereas the other family members with a previous diagnosis of hypertension were already on treatment. The two probands were followed over time, with clinical and biochemical evaluation.Urinary free cortisol (F) and cortisone (E) quantification was performed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method, as extensively described elsewhere . BrieflyTotal tetrahydro-cortisol (THF), 5α-tetrahydrocortisol (5α-THF), and tetrahydro-cortisone (THE) were quantified by gas chromatography–mass spectrometry (GC-MS) method, as previously described , 22. InsEither F/E or THF+allo-THF/THE was measured on 24-h collected urine.g and 4°C for 1 h. The obtained pellet was used for analysis after careful removal of the supernatant. Vesicle size was checked by electron microscop.Second morning urine samples from AME family members and patients affected by EH were collected, processed as previously described with minor modifications  and storTotal exosomal RNA was extracted by a commercial kit . According to the manufacturer’s instructions, RNA was extracted, purified, and eluted in a final volume of 15 µl and stored at −80°C.Synthesis of cDNAs was carried out using a commercial kit  and a preamplification step was included  to improve mRNA detection and measurement. Preamplification primer mix included the genes HSD11B2 and β-2 microglobulin (B2M).t-test. Reproducibility was tested with three independent samples and intra-assay coefficients of variation (CV) calculated for HSD11B2 and B2M mRNA were <5%. Statistical significance was achieved with p-value < 0.05. More details on RNA quantification are available in the Supplementary Material.Droplet Digital PCR was performed at the Genomics and Transcriptomics Platform of “Centro Piattaforme tecnologiche” of the University of Verona by the use of Bio-Rad QX200 Droplet digital PCR system (Bio-Rad). Reactions and processing of the samples were obtained following the manufacturer’s instructions. In brief, the mix completed with cDNA samples were loaded with a multichannel pipette into a droplet generator cartridge and 70 µl of oils was added to the lower wells and the cartridge or reaction plates containing the samples was placed into a QX200 Automatic Droplet Generator (Bio-Rad) to produce the individual droplets. Each reaction was partitioned into ~20,000 nanoliter-sized droplets. Procedures were standardized and performed with high care. After the PCR was completed, the sealed plate containing the droplets was loaded in a QX200 droplet reader for the detection of completed PCR reaction individual droplets. Data were analyzed with Quanta Soft Software version 1.7.4.0917 (Bio-Rad) with the thresholds for detection set manually based on results from negative control wells containing water instead of RNA. During all the preparation, and in particular for the steps of droplet generation and droplet handling, high accuracy was required to preserve droplet integrity. The detection of HSD11B2 RNA was assumed valid only if the housekeeping gene B2M was present. Optimization was evaluated by the separation between positives and negatives. qPCR results were calculated according to the standard curves. Mean values and standard deviations of ddPCR and qPCR were assessed using Student’s Data analysis for comparison of clinical/biochemical data was performed using the SPSS 24.0 for Windows . Quantitative data are expressed as median values with minimum–maximum range, while categorical variables were expressed as percentage. Continuous variables showing a skewed distribution were analyzed on log-transformed values. Correlations between continuous variables were assessed by Pearson’s test. Taking into account the very limited sample size of the different study subgroups , all the comparisons are shown only as descriptive and not quantitative statistics.r = 0.9134, p < 0.0001).Subjects enrolled in the study were characterized in terms of hormonal and biochemical parameters. In particular, the members of the AME family presented the features shown in n = 2, 7.66, range 6.41–8.91) than those in the other groups, 221 AG_H , 221 AG_N , and 221 AA   and the lowest values were found in 221 AA subjects  , 221 AG_N   . In AME, the mRNA exosomal copies of HSD11B2 significantly inversely correlated with aldosterone and renin , respectively  and an inverse but non-significant correlation with renin  in the AME family  while there was no significant correlation in EH patients   is widely distributed but it is mostly abundant in liver and adipose tissue, whereas the 11β-HSD2 iso-enzyme (inactivating cortisol to cortisone) is primarily expressed in MR target tissues, i.e., kidney, colon, and salivary gland. To study the functional role of 11β-HSD2 in the regulation of water balance, the most critical issue is that the kidney cannot be easily available unless a renal biopsy, i.e., an invasive procedure is made. The 11β-HSD2 enzyme activity is, therefore, usually estimated by a surrogate marker, i.e., the urinary cortisol-to-cortisone metabolite ratios. As a proof of this assumption in a cohort of patients undergoing renal biopsy because of suspected underlying renal disease, it was demonstrated that renal 11β-HSD2 mRNA expression relates to both urinary cortisol metabolite ratios (THF+aTHF/THE and F/E). Moreover, a decreased HSD11B2 expression was observed among the patients with worse renal function . In diffWe demonstrated that HSD11B2 mRNA is detectable in urinary exosomes both in patients with AME syndrome and in patients with EH. The relationship with clinical–biochemical parameters are different in the two conditions. In the AME family, the expression of HSD11B2 mRNA varied according to the 662 C>G genotype: the homozygous probands showed the highest levels, the heterozygous subjects showed intermediate levels, and the wild-type subjects showed the lowest levels. Among heterozygous carriers, hypertensive subjects had higher HSD11B2 exosomal mRNA than normotensives. These data suggest the following: (i) higher expression of the gene when the enzymatic activity is mostly reduced, at least as estimated by urinary steroid ratios (urinary THF/THE and THF+5αTHF/THE ratios); (ii) 11β-HSD2 mRNA is inversely related to the renin and aldosterone concentrations, mirroring a pronounced inhibition of the renin–aldosterone axis. The first point may be explained by an increased rate of transcription of the mutated gene as a feedback mechanism to compensate for the reduced enzymatic activity. However, this effect could be specific for the 662 C>G variant, while other mutations at different points of the gene sequence could have inhibitory consequences on the transcription process with reduced or absent mRNA.The mechanisms previously investigated of the modulation of 11β-HSD2 gene expression were at pre- and post-transcriptional levels: the presence of polymorphisms, variations in microsatellite regions , 34, andDNA methylation is the main epigenetic feature in mammalian cells, leading to a gene transcriptional regulation by transcriptional repression when the gene promoter site is hypermethylated . In a coA further indirect demonstration of the role of a gene expression modulation of HSD11B2 related to the entity of MR activation derives from the data of the two probands at follow-up. At baseline, when the two patients were free from specific therapy, with a florid disease characterized by low-renin, low-aldosterone hypokalemic hypertension, we observed a high exosomal HSD11B2 mRNA expression. After a long period of antihypertensive therapy including MR antagonist (eplerenone), along with a good blood pressure control and normalization of renin and kalemia, we observed a clear reduction in exosomal HSD11B2 mRNA. If HSD11B2 gene expression at baseline was increased in order to compensate for the excessive MR activation, a therapy-mediated MR inhibition also reduced HSD11B2 mRNA. Interestingly, the higher THF+aTHF/THE ratio as compared to baseline observed after eplerenone treatment in the two boys is consistent with the finding of a lower exosomal HSD11B2 gene expression.The enzymatic activity of 11β-HSD2 is estimated by either urinary THF+aTHF/THE or serum or urinary F/E ratio. It was hypothesized that F/E may be a better marker of the activity of 11β-HSD2 isoenzyme, while THF+5αTHF/THE may represent a proxy of global HSD11B2 activity  , 41–46. A further strength of the study is the accuracy of the method to quantify the urinary HSD11B2 mRNA expression since we utilized an advanced and sensitive novel technology, namely, the ddPCR  that is The present study has some limitations that need to be acknowledged. The study sample is very limited and, therefore, mostly descriptive, which implies that they should be interpreted with caution. We also recognize the substantial overlap in exosomal HSD11B2 RNA copy number between different subgroups, including 221 GG homozygotes and hypertensive 221 AG heterozygotes, which limits the diagnostic value of such assay. Moreover, the comparison between AME family members and EH subjects is limited by the lack of age and sex matching. Digital PCR has proven to be the right tool for investigating HSD11B2 mRNA, as other approaches were not successful in allowing the detection of such a minimal amount of material such as the mRNA present in UVs, a limitation to get enough mRNA to analyze all samples, even after the necessary step of preamplification. Further studies are indeed required to confirm these findings in a larger data set.Despite the study limitations, our results appear biologically plausible and could be seen as a potential proof of concept about the role of 11βHSD2 activity in mineralocorticoid hypertension.HSD11B2 mRNA is detectable and quantifiable in urinary exosomes by ddPCR technology. In AME family members (662 C>G genotype), we observed an increased HSD11B2 expression in homozygous as compared to heterozygous and wild types and a positive correlation between exosomal mRNA and the 11βHSD2 enzyme activity as estimated by the urinary steroid ratios, i.e., opposite to the trend observed in EH patients. The overexpression of HSD11B2 mRNA is a possible mechanism of compensation of the enzyme deficiency. The study of exosomal expression is a useful tool to investigate 11β-HSD2 functional activity in kidney through the analysis of exosomal mRNA cargo in mineralocorticoid hypertension.The present study, moreover, adds to the current knowledge and understanding that it is possible to perform a kidney tissue gene expression analysis simply by evaluating a urine sample. It is also possible to speculate that also other genes of interest related to diseases with a renal involvement could be investigated and provide novel insights for novel perspectives in clinical approaches.francesca.pizzolo@univr.it.The datasets generated for this study will not be made publicly available due to privacy policy. Requests to access the datasets should be directed to FP, The studies involving human participants were reviewed and approved by Ethics Committee of our Institution . The patients/participants provided their written informed consent to participate in this study.Conceived and designed research: SF, FP, and OO. Performed experiments: LB, FM DD, ED, SU, and MV. Analyzed data: LB, DD, AC, FM, and SU. Interpreted results of experiments: OO, FP, SF, NM, and ED. Prepared figures: DD and NM. Drafted the manuscript: FP, AC, and DD. All authors contributed to the article and approved the submitted version.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This research was funded by the University of Verona independent funding project “Ricerca di Base” to FP.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Sorghum bicolor L. (Moench)] and with unique ecogeographic features, possesses a large number of sorghum landraces that have not been well studied. Increased knowledge of this diverse germplasm through large-scale genomic characterization may contribute for understanding of evolutionary biology, and adequate use of these valuable resources from the center of origin. In this study, we characterized genetic diversity, population structure and selection signature in 304 sorghum accessions collected from diverse sorghum growing regions of Ethiopia using genotyping-by-sequencing. We identified a total of 108,107 high-quality single-nucleotide polymorphism (SNPs) markers that were evenly distributed across the sorghum genome. The average gene diversity among accessions was high (He = 0.29). We detected a relatively low frequency of rare alleles (26%), highlighting the potential of this germplasm for subsequent allele mining studies through genome-wide association studies. Although we found no evidence of genetic differentiation among administrative regions , population structure and cluster analyses showed clear differentiation among six Ethiopian sorghum populations  adapting to different environments. Analysis of SNP differentiation between the identified genetic groups revealed a total of 40 genomic regions carrying signatures of selection. These regions harbored candidate genes potentially involved in a variety of biological processes, including abiotic stress tolerance, pathogen defense and reproduction. Overall, a high level of untapped diversity for sorghum improvement remains available in Ethiopia, with patterns of diversity consistent with divergent selection on a range of adaptive characteristics.Ethiopia, the probable center of origin and diversity for sorghum [ Sorghum bicolor L. (Moench)], native to the dry regions of northeast Africa  that are adapted to temperate regions represent partial of the genetic diversity in breeding programs, the diversity underlying traits of economic and adaptive importance remains trapped within the tropical germplasm  is increasingly being used for profiling genome-wide nucleotide variation in many species  markers in a collection of 304 sorghum accessions. The objectives of this study were to (1) assess the extent and patterns of genetic diversity among sorghum accessions collected from major sorghum growing regions of Ethiopia, (2) determine the population structure of the accessions, and explore their potential for future genome-wide association studies, and (3) identify genomic regions and genes potentially subjected to selection.A total of 304 sorghum accessions used in this study were collected from farmers’ fields of major sorghum growing administrative regions of Ethiopia (see Supplementary File S1). Accessions from regions with sample size <10 were included in adjacent regions to reduce bias due to small sample size. In doing so, six, two, and two accessions from Gambella, Afar, and Somali regions were, respectively, placed under the Southern Nations, Amhara, and Oromia regions. This reduced the seven regions from which the accessions were originally collected to four major sorghum producing regions . These regions include a broad swath of the range of sorghum cultivation that account for 94% of the total sorghum production in the country . During ApeKI enzyme system. In brief, each DNA sample was digested with ApeKI , then ligated to a unique barcoded adapter. For each library, 96 samples were pooled, and fragments with 200–500 base pair (bp) in length were extracted from a 2% agarose gel after electrophoresis and purified using a Qiagen Gel Extraction Kit . The purified DNA was PCR amplified using GoTaq Colorless Master Mix , and the PCR product was extracted as above to eliminate primer–dimers. All libraries were sequenced on a NextSeq platform  with 150 bp single‐end reads.Prior to DNA extraction, the accessions were grown under field conditions and subjected to one cycle of controlled self-fertilization for purification. Leaf samples from a single representative plant per accession were collected from 15-day-old plants grown in small pots in a greenhouse. DNA was then extracted from lyophilized leaf tissues following a modified cetyltrimethyl ammonium bromide protocol , ADMIXTURE was run with a fivefold cross-validation (CV) procedure for K ranging from 1 to 20, and the K value with the lowest CV error was selected (q) estimated with ADMIXTURE program using the optimum number of subpopulations (K = 6) suggested by CV procedure , observed heterozygosity (Ho), gene diversity  and polymorphism information content (PIC) were estimated using the allele frequencies of each SNP. The above genetic diversity estimates were also computed for pooled accessions within each administrative region and ADMIXTURE inferred subpopulation. However, a comparison of diversity estimates in big populations compared that in small populations could be largely biased by the different sample sizes. To account for differences in population size, we used a subsampling scheme by taking into account the required level of precision (α = 0.05), the variances and average differences in allele frequencies between populations  for each population. In addition, allelic richness (Rs) and number of private alleles for each population were computed with the rarefaction method, that adjusts for differences in sample sizes across populations  among populations was estimated based on the method of Nm) as (1−FST)/4FST as described by To estimate the components of variance among and within populations, analysis of molecular variance (AMOVA) was performed as described in STF outliers were detected based on SNPs with MAF > 5% using BayeScan 2.1  of 0.05 as implemented in the “plot_bayescan” function in R. Genes found within 100 kb of the genomic regions detected in the above test were also searched using the most recently annotated version of the sorghum genome v3.1 (www.phytozome.net). The distance 100 kb was based on the average genome-wide linkage disequilibrium decay of 100 kb (data not shown).To detect signatures of selection among ADMIXTURE subpopulations, Scan 2.1 . To reduK = 6. Supplementary File S4 contains detailed description of FST outlier SNPs and candidate genes located in the vicinity of these SNPs based on BAYESCAN results for outlier prediction. Supplementary Figure S1 contains genomic distribution of 108,107 high-quality SNPs across the 10 sorghum chromosomes, and their corresponding density. Supplementary Table S1 contains AMOVA and FST test results for accessions by geographic/administrative region and ADMIXTURE subgroup analyses.Supplementary File S1 contains detailed descriptions of Ethiopian sorghum accessions, their regions of origin and geographic information. Supplementary File S2 contains SNP ID numbers, locations and SNP genotypes for all accessions and SNPs. Supplementary File S3 contains co-ancestry coefficient matrix of 304 Ethiopian sorghum accessions based on ADMIXTURE analysis at https://doi.org/10.25387/g3.12813224.Supplementary material is available at figshare DOI: Materials and Methods’ for criteria) produced a total of 115,501 high-quality SNPs (see Supplementary File S2). Overall, 32.81% of the SNP calls were missing and imputed using Beagle V4.0  in plant genomes  revealed that the highest number of these SNPs were physically mapped on chromosome 2 . The highest and lowest marker densities were observed on chromosome 7 (7.41 kb) and chromosome 5 (5.31 kb), respectively, with an average marker density of 6.13 kb per chromosome. The identified SNPs were also categorized according to nucleotide substitutions as either transitions (A↔G or C↔T) or transversions . Our analysis of transitions (Ts) and transversions (Tv) SNPs showed a Ts/Tv ratio of 1.7:1  well-differentiated genetic groups and some admixtures. Therefore based on ADMIXTURE analysis, we assigned 234 (77%) accessions to one of the six subgroups with an ancestry membership coefficient probability of >0.60 (q > 0.60), whereas the remaining 23% showed evidence of mixed population ancestry (see Supplementary File S3).ADMIXTURE analysis using a fivefold CV for il K = 6 . For exato SG-VI . Althougto SG-VI , also suAccording to i.e., 58,673 SNPs), whereas only 18–41% of such SNPs were common among all ADMIXTURE subgroups .Based on the allele frequency distribution of this collection, 26% of the SNPs were rare  ranged from 0.09 to 0.50, and its value average all loci was 0.29 (Ho = 0.12) of the entire panel was similar with that observed in previous studies of sorghum landraces , and the lowest in the Oromia region . Allelic richness based on rarefaction was relatively higher in the Southern Nations (RS = 1.81) and lower in the Oromia region  was found in SG-V  and harbored many private alleles (NPA = 1430) compared with other subgroups  among the regions, leading to a low genetic differentiation between the regions. The pairwise fixation index (FST) among ADMIXTURE subgroups ranged from 0.11 to 0.48, indicating a relatively high level of genetic differentiation (Nm = 0.64) among the populations.AMOVA showed that 98% of the total variation was found within regions, whereas 28.06% and 71.94% of the total variation was found among and within ADMIXTURE subgroups, respectively (see Supplementary Table S1). The number of migrants per generation as indirect estimate of gene flow was also very high  and two corresponded to balancing selection (α < 0).The identification of functional genomic regions that might be targets of selection provides information useful for the discovery of candidate genes of breeding importance in sorghum . This apFST outlier SNPs (see Supplementary File S4). These candidate genes represented different categories of biological processes, including regulation of biotic and abiotic stress tolerance , signal transduction (similar to low temperature-responsive RNA-binding protein), plant cell wall synthesis (Glycosyl transferase 1) and ion transport.We also identified a total of 47 candidate genes in the vicinity of the genomic regions containing these Ethiopia, the probable center of origin and diversity for sorghum and with unique ecogeographic features, possesses a large number of landraces in the gene bank as well as under subsistence agriculture. The germplasm from this region represents one of the most important sources of useful genes for sorghum improvement efforts around the world . In addiIn this study, a high-throughput GBS technology was used to explore genetic diversity, population structure, and selection signature in sorghum accessions collected across the center of origin and domestication, Ethiopia. Indeed, the lower frequency of rare alleles  observedOur results (see Supplementary Table S1) support previous reports of low level of regional differentiation for cultivated sorghum in Ethiopia  could beWhile regional differentiation is lacking, analysis of genotypic data revealed clear differentiation among six Ethiopian populations  and 3. Pi.e., cool/humid, cool/subhumid, cool/semiarid, warm/subhumid, warm/semiarid, and warm/arid) of Ethiopian sorghum may indicate a strong contribution of agro-ecological variation to genetic groupings observed in this study. Further studies that combine population analyses with environmental and phenotypic trait variables should provide a more complete understanding of sorghum genetic structure in Ethiopia and facilitate breeding of locally adapted sorghum varieties.Overall, the observation that the six distinct genetic groups identified equaled the number of different agro-ecological zones . Of the 40 outlier SNPs identified, 31 were located at <100 kb from annotated genes (see Supplementary File S4), which provides the first support for their putative relevance.We expect higher genetic population differentiation for adaptive SNP than neutral SNP if adaptation to local environments is the principal source of genetic differentiation . To idenFST outlier SNP  with a signature of diversifying selection, was located at ∼24.42 kb from Sobic.005G033801, a candidate gene which encodes flavonol 3-O-glucosyltransferase protein known to be involved in anthocyanin biosynthesis pathway  for human consumption (Sobic.005G041200 and Sobic.007G135301) located at 64.13 and 17.33 kb from S5_3724645 and S7_56063576 on chromosome 5 and 7, respectively. The Sobic.005G041200 gene encodes F-box protein known to be associated with sorghum response to various stresses including drought (Sobic.007G135301 gene encodes MADS box protein which has been associated to smut resistance in sorghum (Sobic.003G269600), for which evidence of association with sorghum phenotypic diversity for plant height had been reported (For instance, the top  pathway . Anthocy pathway , and prosumption . The divreported , where dThis study reported a high level of genetic diversity and differentiation among Ethiopian sorghum accessions, which provides a great opportunity for developing new cultivars with desirable characteristics. Our results illustrate how populations adapting to different environments become structured genetically on small spatial scales. We found genomic regions of potential interest, with further large-scale phenotypic and geographic characterization can provide multiple lines of evidence for the putative importance of these particular loci in the genetic control of traits of economic and adaptive importance in sorghum. Overall, this study contributes to the genomic resources available for sorghum improvement efforts around the world.


Brucella infection and health seeking behaviour in response to brucellosis-like symptoms, amongst cattle farm workers and veterinary officials in South Africa, are unknown. Farm workers and veterinary officials (N = 230) were screened for brucellosis using commercial Rose Bengal Test (RBT®), IgM Enzyme-linked Immunoassay (ELISA)®, IgG ELISA® and the BrucellaCapt® test. Knowledge of brucellosis and risk factors for exposure to Brucella were also investigated. Seroprevalence varied according to test used: 10.1% (RBT®), 20.9% (IgG ELISA®) and 6.5% (BrucellaCapt®). Only 22.2% (6/27) of veterinary officials opt to visit a clinic, doctor, or hospital in response to self-experienced brucellosis-like symptoms, compared to 74.9% (152/203) of farm workers (p < 0.001). Of the BrucellaCapt® seropositive participants, 53% (7/15) did not visit a clinic in response to brucellosis-like symptoms. Weak evidence of an association between the handling of afterbirth or placenta and infection of a short evolution  was found , and strong evidence of an association between this outcome and the slaughter of cattle . There was strong evidence of a positive association between inactive/resolved infection and veterinary officials vs. farm workers exposed to seropositive herds , with a simultaneous negative association with the handling of afterbirth or placenta . Findings suggest a proportion of undetected clinical cases of brucellosis amongst workers on cattle farms in Gauteng.Brucellosis in humans is under-detected and underreported in sub-Saharan Africa. Risk factors associated with  Brucellosis is a neglected zoonotic disease of global health and economic importance ,2,3. SymBrucella bacteria through contaminated milk . The antibody index was calculated as [(sample O.D./cut off serum mean O.D.) × 10]. Samples were classified as negative, equivocal, or positive if the antibody index was <9, 9–11, and >11, respectively. The IgM ELISA® was conducted, and results interpreted in a similar manner as the IgG ELISA®, except for the initial preparation of the wells, which required 25 μL of human IgG sorbent to be added to each well to remove excess IgG antibodies or rheumatoid factor.For the IgG ELISA® test was carried out as follows: all reagents were brought to room temperature before use. A total of 50 μL of serum diluent was added into Well A, after which 50 μL of serum diluent was added into all wells (A–H). A total of 5 μL of each serum, negative and positive control were added to Well A. Doubling dilutions with 50 μL of each well was made from A to H. A total of 50 μL of the provided bacterial suspension (well homogenized by prior vigorous shaking) was added into all wells. Wells were sealed with adherent tape and incubated for 24 h at 37 °C in a chamber. Titre results were read after this and interpreted as follows: Row A—1:40, Row B—1:80, Row C—1:160, Row D—1:320, Row E—1:640, Row F—1:1280, Row G—1:2560, Row H—1:5120. Due to the paucity of information on the prevalence of endemic brucellosis in people in South Africa [The BrucellaCapt® (n = 2) were excluded from the analysis. All samples were tested with the RBT®, IgG ELISA® and BrucellaCapt® tests. Samples that were seropositive on the ELISA IgG® were tested further using the IgM ELISA®. Samples seronegative on the IgG ELISA®, but seropositive using the RBT®, were also subjected to an IgM ELISA® test. This selective testing of samples using the IgM ELISA® was due a limited budget. The purpose was to detect the presence or absence of Brucella IgM antibodies in these selected samples to better understand the evolutionary stage of infection in the farm workers and veterinary officials. Stages of infection were considered along a continuum from a short evolution of infection  to a long evolution of infection . As such, each seropositive person fell into one of five mutually exclusive groups depending on the outcome of a combination of tests: (i) RBT®, IgM ELISA® positive and IgG ELISA® negative, (ii) RBT® negative and IgM, IgG ELISA® positive, (iii) RBT®, IgM ELISA® positive and IgG ELISA® positive, (iv) RBT®, IgG ELISA® positive and IgM ELISA® negative, and (v) RBT®, IgM ELISA® negative and IgG ELISA® positive. Seropositive reactors on the BrucellaCapt® test were allocated to the group defined by the outcomes of the RBT®, IgM ELISA® and IgG ELISA®.Subjects with insufficient blood for the RBT® that were classified as equivocal (n = 3) were removed from the analysis. Titres were determined using the BrucellaCapt® test. A titre of greater or equal to 1:320, was considered positive.Subjects with test results for the IgG ELISA® and IgM ELISA® were used in series to increase the specificity of RBT® to detect Brucella IgM, as an indication of infection of a short evolution. To detect Brucella IgG, an indication of a possibly longer evolution, we used the IgG ELISA® test. The BrucellaCapt® test was used, with the recommended cut-off titre of 1:320, for the detection of possible clinical brucellosis with either a short or long evolution. All tests were performed according to the manufacturer’s guidelines.The RBT® Access® (2013), for Microsoft 365, Redmond, WA, USA. Laboratory results were captured into the appropriate record, by matching the unique identifiers of the samples.Completed questionnaires were captured into the electronic form function of Microsoft® Excel® for Microsoft 365, Redmond, WA, USA. Univariable analyses were conducted in STATA 14® StataCorp College Station Texas 77845 United States, for outcomes (1) RBT®, IgM ELISA® positive and IgG ELISA® positive, (2) RBT®. IgG ELISA® positive and IgM ELISA® negative, and (3) RBT®, IgM ELISA® negative and IgG ELISA® positive and (4) BrucellaCapt® seropositivity amongst farm workers and veterinary officials (N = 230).Descriptive statistics were done in Microsoftp < 0.20 were selected for inclusion into mixed effects multivariable logistic regression models. Farm was included as a random effect in all three models. Veterinary officials were clustered into three groups, according to the State Vet Area they serviced. Each cluster was allocated a unique number and added to the farm variable. Three separate mixed effects logistic regression models were fit to identify risk factors for increasing evolution of infection: (1) RBT®, IgM ELISA® positive and IgG ELISA® positive (short evolution), (2) RBT®, IgG ELISA® positive and IgM ELISA® negative (long evolution), and most likely inactive or resolved infection but indicative of exposure to Brucella spp (3) RBT®, IgM ELISA® negative and IgG ELISA® (exposure/inactive or resolved infection). Variables with p > 0.05 in the models, were systematically removed by backward elimination [Univariable associations between each variable and the outcomes were assessed using Fisher’s exact test. Variables with mination ,58.Brucella on cattle farms participating in the bovine brucellosis control programme in Gauteng varies depending on the serological screening test used. However, when tests results were combined to illuminate the evolution of infection in this group, significant risk factors and symptoms were found to be associated with infection of short and long evolution. This, in addition to the finding of poor health seeking behaviour in response to brucellosis-like symptoms among farm workers and veterinary officials with these antibody profiles, strongly suggest the presence of undetected cases of human brucellosis on cattle farms.Evidence of cattle handler exposure to ® test as described in Diaz et al. (2011) to facilitate an early detection and response to brucellosis in these occupationally exposed persons and their families [® should be used on plain serum and, if positive, RBT® on serum dilutions up to 1/32. Dilutions should be contrasted with clinical symptoms (if any). It is also recommended that medical practitioners in SA be made aware of the clinical symptoms of both short and long evolution brucellosis and the risk of brucellosis amongst persons occupationally exposed to cattle herds in Gauteng province. With greater awareness, medical practitioners can monitor the endemic seroprevalence of brucellosis to adapt cut-off points for commercially available serological tests, such as the BrucellaCapt®. Awareness programmes to increase knowledge of human and cattle symptoms of brucellosis are recommended to be part of the routine veterinary regulatory service to these farms. Occupational health and safety measures to protect the health of veterinary officials should be implemented and monitored.Since this study was undertaken as a pilot study, our first recommendation is to establish a representative sampling frame of occupationally exposed persons to attain a sample large and representative enough to determine the true endemic seroprevalence of brucellosis in this group and to obtain a better measure of odds ratios and confidence intervals for risk factors . To achifamilies . In brie


Carassius auratus fish, which is widespread in East Asian fresh waters and exhibits one of the most extensive distribution among asexual vertebrates despite its dependence on host sperm. Our analyses of genetic composition using dozens of genetic markers and genome-wide transcriptome sequencing uncover admixed genetic composition of Japanese asexual triploid Carassius consisting of both the diverged Japanese and Eurasian alleles, suggesting the involvement of Eurasian lineages in its origin. However, coexisting sexual diploid relatives and asexual triploids in Japan show regional genetic similarity in both mitochondrial and nuclear markers. These results are attributed to a unique unidirectional gene flow from diploids to sympatric triploids, with the involvement of occasional sexual reproduction. Additionally, the asexual triploid shows a weaker population structure than the sexual diploid, and multiple triploid lineages coexist in most Japanese rivers. The generated diversity via repeated interploidy gene flow as well as an increased establishment of immigrants is assumed to offset the cost of asexual reproduction and might contribute to the successful broad distribution of this asexual vertebrate.Asexual vertebrates are rare and at risk of extinction due to their restricted adaptability through the loss of genetic recombination. We explore the mechanisms behind the generation and maintenance of genetic diversity in triploid asexual (gynogenetic)  A large body of literature aimed at explaining the maintenance of sex has generally treated sexual and asexual reproduction as mutually exclusive strategies and has predicted that asexuality is an evolutionary dead end5. However, recent findings regarding long-surviving asexual lineages and their genetic diversity have changed the view of the evolutionary potential of asexual taxa6. The genetic diversity in these long-surviving asexual species is facilitated by rare sexual reproduction8; transmission of genetic elements from related sexual taxa 12; or in some cases, mitotic recombination13. Exploring the mechanisms underlying the generation and maintenance of genetic diversity in asexual organisms is essential for understanding the ecological and evolutionary relevance of the alternative reproductive modalities, leading to the identification of the proximate forces shaping the evolution of sex.The ubiquity of sexual reproduction in the eukaryotes is one of the great puzzles in evolutionary biology because the costs of sexual reproduction  are immediate, whereas its potential benefits  are delayed17. One type of asexual reproduction found in vertebrates is gynogenesis, in which offspring are formed parthenogenetically, yet egg development cannot be completed without sperm from a related bisexual species that degenerates without fusing with the egg nucleus3. Sperm-dependent asexual species can only persist in sympatry with a closely related sexual host that offers a continuous supply of males. Thus, gynogenesis suffers from certain disadvantages of sexuality  along with those of asexuality, which should make it difficult for these lineages to show long-term persistence18.Asexual vertebrates are of particular interest to biologists due to both a fascination with their rarity and anticipation that such exceptions deepen our understanding of sexual reproductionCarassius (Teleostei: Cyprinidae) is a good model for studying the evolution of asexual vertebrates. This genus is taxonomically confusing, but at least three species are recognized: the wild goldfish Carassius auratus-complex , the Japanese white crucian carp (C. cuvieri), and the crucian carp (C. carassius). Among them, much attention has been focused on the C. auratus-complex exhibiting sexual and gynogenetic reproduction associated with ploidy polymorphism 21. This gynogenetic triploid is one of the most widespread asexual vertebrates, which is distributed in and around Eurasia, including Taiwan and the Japanese islands, and uses sympatric sexual diploid Carassius fish as sperm donors22. Substantial morphological and genetic variability has been reported in the gynogenetic triploid Carassius25. Intriguingly, phylogenetic studies on mitochondrial DNA reported that sexual diploids and gynogenetic triploids shared various mitochondrial haplotypes over diverged clades in wide ranges of East Asian freshwaters27. These results indicate underlying mechanisms that facilitate genetic diversity or multiple autotriploidization-like origins from sexual diploid populations despite the extreme rarity of asexual vertebrates3.The gynogenetic triploid found in the genus Carassius fish has spread widely and avoided extinction by examining the origin of this fish in the Japanese archipelago and the mechanisms facilitating its genetic diversity. Integrating the dataset of nuclear loci obtained by target resequencing and transcriptomes, mitochondrial sequences, microsatellites and morphological measurements generated from geographically comprehensive specimens , and the other is autotriploidization, i.e., the occurrence of triploid individuals from a single diploid population29. We examined the genetic composition of the Japanese gynogenetic triploid to test for presumable admixtures between Japanese and Eurasian lineages , including mitochondrial DNA, from related sympatric sexual species, as suggested for several invertebrates8 and plants30. Under the autopolyploidization scenario, sympatric pairs of diploid and triploid are expected to have high genetic similarity and they should be almost indistinguishable. Conversely, in the case of hybrid origin with gene flow, although the sympatric pairs of diploid and triploid are also expected to have some geographical genetic cohesion, they would be distinguished by traces of ancestral parent alleles in triploids  between diploid and triploid Carassius. We further evaluated the frequency of coexistence of genetically distinct triploids and clarified the population structure of Carassius fishes to evaluate the contributions of selection and migration in the maintenance of the diversity as possible key factors enabling triploid Carassius to thrive.The objective of this research was to elucidate how the gynogenetic triploid Carassius, we conducted target resequencing on available nuclear markers used in Cyprinidae phylogeny31 and previous studies on Carassius33. The PC1 axis of principal component analysis (PCA) based on 1,100 informative single nucleotide polymorphisms (SNPs) from 30 nuclear markers of 15 sexual diploid and 19 gynogenetic triploid Carassius  spanning 14 markers between the assumed parental lineages  between the putative parental lineages, Japanese diploids and Eurasian diploids/triploids. We used RNA-seq data conducted on six diploids and four triploids  values of mtDNA between sampling localities with discriminating ploidy, and IBD pattern was detected between and within diploid and triploid fish around Japan  was inferred from the 933 23 , as expected from their clonal reproduction, supporting robustness of our ploidy identificationK = 10), whereas a subtle regional structure with K = 6 was inferred for triploids . A more plausible cause is natural hybridization following secondary contact between the Japanese and Eurasian groups. Paleogeographic studies have suggested that a land bridge between mainland Asia and the Japanese islands emerged repeatedly during glacial periods of the Pleistocene36. We found the current coexistence of the Japanese and the unique Eurasian mtDNA lineage in the land bridge area  colonized Eurasian triploids hybridized with Japanese diploids via interploidy gene flow, and/or (ii) triploidization by hybridization between Japanese and Eurasian diploids. Such events likely happened when a land bridge was formed during the glacial period. The gynogenetic lineages have persisted since it invaded the Japanese islands, where they dispersed throughout the archipelago and diversified.Carassius fishes in Lake Biwa23, and mature triploid and tetraploid males have been reported in natural habitats38). These tetraploids could result from the fertilization of unreduced eggs produced by a triploid female (3n) with haploid sperm (n) from a diploid male39. If this rare tetraploid male produces diploid sperm (2n), fertilization by such sperm of haploid eggs (n) from diploid females would lead to gynogenetic triploid individuals  the occurrence of males in the triploid lineage and (ii) reorigination of gynogenetic triploids from a cross between a male from the triploid lineage and a sexual diploid female. One plausible scenario is the involvement of tetraploid males. There is a low but significant proportion of mature tetraploids (0.44% of 8), plants (dandelion7 and Boehmeria30), and vertebrates . In triploid Carassius, the combination of interploidy gene flow and gynogenesis is assumed to make a large contribution to its genetic characteristics. The genome-wide F1-like hybrid genomic compositions of western and central Japanese Carassius triploids revealed by our RNA-seq are likely explained as a maintenance of the hybrid genome constitution via clonal inheritance or biased inheritance/selection in the process of interploidy gene flow. Moreover, other mechanisms such as recombination in the process of interploidy gene flow , and the following negative frequency-dependent selection45 can be attributed to the maintenance of clonal lineage diversity in asexual organisms. These types of mechanisms, e.g., mechanisms associated with food habits46 and resistance to infection47, may facilitate diversity and the coexistence of multiple gynogenetic triploid lineages of Carassius. The genetic diversity in triploids could be reinforced by their enhanced connectivity of populations resulting from the increased establishment of immigrants after dispersal. For gynogenetic triploids, rare immigrants from geographically isolated populations maintain genetic independence from individuals of a recipient population, and the immigrants could be advantageous due to the anticipated negative frequency-dependent selection. By contrast, for sexual populations, alleles from such immigrants, even selectively favorable alleles, are often eliminated through genetic drift and recombination. This explanation is supported by the nuclear and mitochondrial less structured populations of triploids than those of diploids in Carassius despite their presumed similarity in ecological niches and dispersal ability  inherited from once-established gynogenetic triploid likely contribute to the observed frequent occurrence of new triploid clones. This inheritability of gynogenetic phenotype in natural populations of vertebrates is, to our knowledge, the first such evidence. The genetic diversity of Japanese triploids, generated by this interploidy gene flow, is considered to be maintained by the increased establishment of immigrants and probable negative frequency-dependent selection. Regarding the other gynogenetic animals, there is accumulating evidence of elevated heterozygosity counteracting the genetic degeneration process of Muller’s ratchet53, such as occasional leakages of a portion of the paternal genetic element12, balancing selection47, and selection on usage of hybrid genome 54. Similarly, gynogenetic Carassius could receive some of the benefits of sexual reproduction, counteracting the accumulation of deleterious mutations, as well as those of asexual reproduction. Importantly, geographically cohesive genetic similarity between ploidies and the expression of Japanese alleles in triploids suggest that gynogenetic Carassius utilize acquired alleles from their sexual hosts. The gene pool of the sexual host could become a bypassed venue for gynogenetic triploids to obtain alleles related to local adaptation  and sexual selection . Hence, occasional sexual reproduction and the effective use of acquired alleles, as well as increased migration, are considered key to avoiding extinction due to the genetic degradation and successful proliferation of this asexual vertebrate.The results of the present study suggest the presence of unique interploidy gene flow from sexual diploid to triploid 55, ARRIVE guidelines56, and approved by the Animal Experimentation Committee of Kyoto University. A total of 933 Carassius auratus-complex were collected from 77 river systems in Japan and 4 river systems in China from 2007 to 2016 . The ploidy of C. auratus-complex specimens was estimated using the amplified number of alleles from 12 microsatellite loci33. This method had previously achieved > 97% matching with the result of flow cytometry in three geographically distant Japanese populations23. Genotypes were scored against the size standard ROX 400HD or LIZ 500 on an ABI 3130xl sequencer (Applied Biosystems) using GeneMapper software 3.0 (Applied Biosystems). The primers used for PCR amplification and the microsatellite genotypes are given in Supplementary Tables All procedures were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments31 and previous studies on Carassius33. Target resequencing of 30 well-amplified loci was conducted for 15 diploid and 19 triploid geographically representative C. auratus-complex specimens, in addition to C. cuvieri and Cyprinus carpio  was conducted using the R package pcaMethods63.Reads from target resequencing were mapped against the target marker sequences of 64 and interspecific heterozygosity were estimated from 103 commonly observed SNPs  using est.h (fixed = FALSE) and calc.intersp.het functions, respectively, implemented in the R package Introgress65. The est.h function uses a maximum likelihood method to estimate the proportion of genome with alleles derived from one of the assumed ancestry populations, whereas the calc.intersp.het function estimate the proportion of genome with alleles inherited from both ancestry populations. For the analysis of hybrid genetic composition, 12 Japanese diploids and eight Eurasian diploids/triploids were used as potential admixed ancestries based on the result of PCA. FST values for each SNP between the putative ancestry lineages were calculated using vcftools.To test the hybrid genetic composition of Japanese triploids, we calculated a hybrid index by mapping against interspecific heterozygosity. The hybrid indexC. auratus-complex specimens belonging to three major Japanese and one Eurasian mtDNA lineages34 using the RNeasy Lipid Tissue Mini Kit (Qiagen). These specimens were different from those used for the target resequencing, but they were collected from the same river systems except for the Ryukyu islands  between their putative parent lineages . We used RNA-seq to obtain these genome-wide dSNPs. Liver total RNA was extracted from the pairs of diploid and triploid mature female C. auratus-complex specimens were mapped to the goldfish genome68 using STAR v2.7.9a69. We follow the GATK Best Practices workflow for RNA-seq70 with slight modification. First, to facilitate mapping reads, a splice junction table was created by mapping all RNA-seq reads to the goldfish genome with the options “alignSJDBoverhangMin 1 and outFilterMismatchNmax 999.” Then, with the aid of the created splice junction database, we mapped the reads from each Carassius specimen with the parameters “alignSJoverhangMin 8, alignSJDBoverhangMin 1, and outFilterMismatchNmax 999.” After filtering out multi-mapping reads using samtools v1.1071, SNPs were called using HaplotypeCaller implemented in GATK with the option “–dont-use-soft-clipped-bases,” and then jointly genotyped. Next, SNPs were selected and filtered out with “FS > 30, QD < 2.0, -window 35, -cluster 3, DP > 20, GQ > 20,” resulting 66,451 bi-allelic SNPs. Then, dSNPs located on the goldfish chromosomes-level assembly were developed by calculating FST and exploring fixed alleles (FST = 1) between the putative parents (three lineages of Japanese diploids vs. four Eurasian diploids/triploids specimens) using vcftools. Genotypes of triploids from the resulting 2772 dSNPs were summarized for each chromosome and visualized using the R package ggplot272.After removal of adapter and low-quality bases using fastp with the option “-3 -q 15 -l 50,” the RNA-seq reads from the diploid and triploid b (cytb) (885 bp) for all specimens. Representative samples of diploid and triploid C. auratus-complex specimens from the subgroups were further analyzed for the partial mitochondrial control region (CR) (297–316 bp) and the complete cytb region (1141 bp). The primers used for PCR amplification and direct sequencing are given in Supplementary Table b, LC337605–LC337605; CR, LC337606–LC337660).We sequenced partial gene regions of mitochondrial cytochrome b regions, with 218 operational taxonomic units (211 unique sequences of C. auratus-complex and 7 outgroup sequences), using BEAST version 1.8.473. We imposed the minimum and maximum age constraints on the coalescence of the Carassius as 3.1–19.4 million years (Myr) ago in 95% interval  based on the fossil records of the Carassius (3.6–4.0 Myr ago74) and the estimated divergence time between Carassius and Cyprinus (11–20 Myr ago75). We adopted the strict clock model, giving the mean cytb molecular clock rate of 0.76%/Myr/lineage  covering the range of cytb clock rates in cyprinid fishes76. Further, we imposed the topological constraint of monophyly for C. auratus-complex26. The optimal substitution models for CR and cytb were HKY + I + G and GTR + G + I, respectively, based on the Bayesian information criterion (BIC) in jModelTest v2.1.777, and the Birth and Death model78 was adopted as the tree prior. The BEAST MCMC run was performed for 50 million generations, sampling every 2,000th generation with 25% discarded as burn-in. The consensus tree was calculated using TreeAnnotator v1.8.4 in the BEAST package.Genealogical reconstruction and estimation of the divergence time were conducted with mtDNA sequences of the CR and cytC. auratus-complex, regional genetic similarity between the ploidies were compared. The relationships among mitochondrial haplotypes of partial cytb sequences were assessed by constructing a statistical parsimony network with a 95% connection limit using TCS version 1.2179. Fixation index (ФST) values between sampling localities with discriminating ploidy . To further evaluate the frequency of coexistence of genetically distinct triploid lineages and clarify the population structure of C. auratus-complex, STRUCTURE analysis (v2.3.4)86 was carried out. Diploids and triploids populations were analyzed separately due to their difference in inheritance. For the triploid dataset, an ambiguity in allele copy number derived from a partial heterozygote was treated as two distinct alleles and a missing allele. Ten simulation runs were conducted for each of K = 3 to 20 using a length of burn-in of 106 and 106 replicates of MCMC. The optimum K was estimated by using the delta K method87 implemented in Structure Harvester88.Nuclear genetic similarity among diploid and triploid C. auratus-complex individuals collected from 11 localities that represented the population structure in Japan were used for the analysis. Eleven landmarks and the standard body length (SL) were recorded on each picture (c.f., Supplementary Fig. 89. Aligned Procrustes coordinates were used for generating a covariance matrix and were subjected to PCA using MorphoJ90.As complementary evidence of gene flow, morphological characteristics were also compared among ploidies across regions using landmark-based geometric morphometrics. A total of 219 Supplementary Information 1.Supplementary Information 2.


FST statistical measurements, and further analyzed the results to find genomic regions under selection for hot adaptation and immune response-related traits.Understanding how evolutionary forces relating to climate have shaped the patterns of genetic variation within and between species is a fundamental pursuit in biology. Iranian indigenous chickens have evolved genetic adaptations to their local environmental conditions, such as hot and arid regions. In the present study, we provide a population genome landscape of genetic variations in 72 chickens representing nine Iranian indigenous ecotypes  and two commercial lines (White Leghorn and Arian). We further performed comparative population genomics to evaluate the genetic basis underlying variation in the adaptation to hot climate and immune response in indigenous chicken ecotypes. To detect genomic signatures of adaptation, we applied nucleotide diversity (θπ) and HSP70, HSPA9, HSPH1, HSP90AB1 and PLCB4 that have been previously unknown to be involved in environmental-adaptive traits. In addition, we found some other candidate loci on different chromosomes probably related with hot adaptation and immune response-related traits.By generating whole-genome data, we assessed the relationship between the genetic diversity of indigenous chicken ecotypes and their genetic distances to two different commercial lines. The results of genetic structure analysis revealed clustering of indigenous chickens in agreement with their geographic origin. Among all studied chicken groups, the highest level of linkage disequilibrium (LD) (~ 0.70) was observed in White Leghorn group at marker pairs distance of 1 Kb. The results from admixture analysis demonstrated evidence of shared ancestry between Arian individuals and indigenous chickens, especially those from the north of the country. Our search for potential genomic regions under selection in indigenous chicken ecotypes revealed several immune response and heat shock protein-related genes, such as The work provides crucial insights into the structural variation in the genome of Iranian indigenous chicken ecotypes, which up to now has not been genetically investigated. Several genes were identified as candidates for drought, heat tolerance, immune response and other phenotypic traits. These candidate genes may be helpful targets for understanding of the molecular basis of adaptation to hot environmental climate and as such they should be used in chicken breeding programs to select more efficient breeds for desert climate.The online version contains supplementary material available at 10.1186/s12864-022-08434-7. Gallus gallus domesticus), as the most extensively distributed of the poultries, have long been bred after its initial domestication in Southeast Asia, probably from the red junglefowl , around 9500 ± 3300 years ago [Domestic animals are excellent models for genetic studies of phenotypic evolution, due to strong phenotypic selection over many generations . They haears ago . Today, ears ago . SimilarDuring recent decades, observed global climate data clearly display a warming trend in most part of the globe, resulting in a wide range of climatic impacts , 6. IranIn the last decade, some genomic studies in Iranian indigenous chickens based on limited microsatellite loci reported genetic variability between different ecotypes –14. OnlyIn this study, for the first time, whole genome sequencing (WGS) data from most Iranian indigenous ecotypes (nine different populations) and two commercial lines  were used to characterize the genetic diversity, population structure and signatures of selection analysis. Results from comparative genomic analysis uncovered novel candidate genomic regions related to hot climate adaptation and immune response traits that could be under natural selection in indigenous chicken population. Our findings further provide new insights to better understanding the genetic relationship between indigenous chicken populations and commercial lines and will be helpful in the selection of superior native chickens.n = 7), Isfahan (n = 5), Lari (n = 6), Marand (n = 4), Mashhad (n = 7), Naked neck (n = 5), Sari (n = 5), Shiraz (n = 7) and Yazd (n = 5). The samples were collected from different provinces as indicated in Fig. n = 11) and Arian (n = 10), were used in order to perform a comparison between different groups. For all studied individuals, venous blood samples (2 mL) were collected under the wing of the chickens for genomic DNA extraction method. No bird individuals died in this study, and all chickens stayed healthy after collecting blood samples. Total genomic DNA was isolated from whole blood samples using salting out protocol. The quality and quantity of the extracted DNA were tested by agarose gel (1%) electrophoresis and NanoDrop spectrophotometer analysis, and high-quality DNA samples were utilized for the subsequent whole genome resequencing.In this study, we sampled a total of 51 indigenous chickens from nine different ecotypes, including Creeper (n = 51) and two commercial lines (n = 21) were sequenced on an Illumina Hiseq 2000 platform with a read length of 125 bp and ~ 10.2 × coverage , and adapters and poor quality base pairs were removed by Trimmomatic software (version 0.36) [https://www.ncbi.nlm.nih.gov/assembly/GCF_000002315.5/) using Burrows Wheeler Aligner (BWA mem Version 0.7.10) [http://broadinstitute.github.io/picard). To improve the alignment accuracy, base quality score recalibration (BQSR) and local realignment around indels were performed using Genome Analysis Toolkit (GATK) tools [The individual genomes from nine indigenous chicken ecotypes n = 5 and two ines n =  were seq 0.7.10) . The SAMK) tools . Final vK) tools . After fines n = 1 were sehttps://itol.embl.de/) was used to visualize the topological structure.The maximum-likelihood (ML) method was applied to reconstruct phylogenetic tree. We first converted filtered VCF file into consensus FASTA files using vcf2fq in vcfutils.pl from Samtools , and theK) size ranging from 2 to 6 and 10,000 iterations for each run [Ho), expected heterozygosity (He) and proportion of polymorphic SNPs (Pn) using PLINK with the default settings. Additionally, we used the '–het' flag in PLINK to estimate the inbreeding coefficient (F) for each individual and each chicken group [Since linkage disequilibrium (LD) could modify the genetic structure analysis , the SNPeach run . Additioeach run . The deceach run . Runs ofeach run . For eacen group .FST [FST values were set to 0.210 and 0.195 , to determine outliers between indigenous chicken ecotypes and two commercial lines, White Leghorn and Arian groups, respectively. We then estimated nucleotide diversity θπ by using VCFtools  [FST and log2  values of SNPs in each window were calculated.For the selective sweep analysis, two different methods were used to detect regions under selection. We calculated the genome-wide weighted FST , as it iFST . The thr 25,000) . For theFST and log2 θπ ratio), all candidate regions were annotated using the Variant Effect Predictor (VEP) available at (http://asia.ensembl.org/info/docs/tools/vep/index.html). Functional enrichment analysis, based on biological process Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways, was then performed by using the ‘g:Profiler’ (https://biit.cs.ut.ee/gprofiler/) enrichment analysis tool, to uncover their biological functions. Finally, the P-value (P < 0.01) of the gene enrichment was corrected by Benjamini–Hochberg False Discovery Rate (FDR-BH).To explore the possible pathways related to identified regions by the above-mentioned methods  inferred the ancestry-component for all samples studied Fig. D. The K  to K6 in2 values were the highest for all studied groups at marker pairs distance of 1 Kb  with a gradual decline with increasing physical distance between SNPs (up to 60 Kb) and then stable trend (> 60 Kb). The highest and lowest r2 values were observed in White Leghorn (0.61) and Marand (0.52) ecotypes at marker pairs distance of 100 Kb, respectively. We further found that from marker pairs distance of 1 Kb to ≤ 60 Kb, the decay of LD was more rapid in White Leghorn group than other birds, reaching an average r2 values from 0.70 to 0.61   followed by Arian chickens (n = 4974)  than both commercial groups . In all studied chicken groups, Pn ranged from 0.634 (White Leghorn) to 0.912 (Marand), with a mean of 0.768  of genetic differentiation between both commercial lines and indigenous chickens  and log2 θπ ratio values (1% cutoff), respectively  and log2 ratio of θπ tests (1% cutoff), respectively  between native ecotypes and White Leghorn genomes  that are involved in heat shock tolerance and/or immune activity related traits  commercial lines.Information about population structure and genetic diversity among native chicken ecotypes is of fundamental importance for genetic improvement, understanding of environmental adaptation as well as for conservation and sustainable management programs . Our stuHo values (ranged from 0.181 to 0.211) for native chicken ecotypes in the present study were relatively similar to those reported in native chicken ecotypes in Thailand and Jordan [Ho and He values in all chicken ecotypes. However, we found lower Ho (ranged from 0.124 to 0.145) than He values (ranged from 0.168 to 0.192) in both two commercial lines, showing a departure from Hardy–Weinberg equilibrium and therefore possibility of selection, non-random mating and/or inbreeding pressure within populations [Ho = 0.124), which is in agreement with previous studies [Iranian indigenous chickens are typically kept by smallholder farmers under extensive management systems and without controlled breeding programs . In lined Jordan –61, but d Jordan –64. We oulations . We furt studies  and coul studies .Knowledge about the patterns of LD decay can provide important insights into population history, demographic processes and breeding systems , 69. The2 values obtained in this study for native ecotypes were in the ranges of 0.52 to 0.57 at marker pairs distance of 100 Kb, which agreed with those reported for Chinese and Canadian native chicken populations [The rulations , 72. We ulations . In addiulations  and wholulations . Our resulations , displayFST and 1% log2 θπ ratio values) that may be involved in heat tolerance and immune response traits  5 (53.058–53.060 MB). The expression/activity of this gene in response to different thermal challenges has been extensively investigated in chicken and mammals [HSP70 variants with the survival ability of cells affected by heat tolerance. It was reported a silent mutation in coding region of HSP70 could possibly be used as a heat tolerance marker in indigenous and commercial chickens [HSP70 mRNA in the heart and liver of young White Leghorn was positively correlated with body temperature [The native ecotypes are more likely to be a good model for exploring the genetic mechanisms underlying adaptation to local cultural conditions and disease. To investigate this, all indigenous chickens were combined and compared to two different commercial lines to uncover loci subjected to natural selection over a long number of generations. Previous reports showed that the heat tolerance negatively influences the health and production of livestock species, and today the genetic selection for heat-tolerant is a challenge in poultry industry –79. Majoen Table A. HSPs a mammals , 86, 87.chickens . The genperature . Most reperature .IL3, IL13, IL5) and interferon (IRF1) genes that are responsible in immune response. Interleukins are a group of cytokines that expressed by leukocytes [We further found some candidate genes on different chromosomes to be potentially involved in chicken immune system. For example, we identified a group of interleukin  and reduced nucleotide diversity , we found some candidate genes associated with heat tolerance  and oxidative stress response (eg PLCB4) (Table Hsp105 or Hsp110) is a high molecular weight protein belonging to the HSP family that functions in cellular stress response and apoptosis pathways [HSPH1 gene was also observed in chicken macrophage-like cell line under acute heat tolerance [HSPH1 gene is differentially expressed in liver, brain and leg muscle of broiler chickens that exposed to heat tolerance [HSPH1 selected region (Chr1: 176.34–176.36 Mb) showed that the indigenous Iranian chicken haplotypes are mostly identical at all of the SNPs across this gene  Table B. HSPH1 pathways . This prpathways . High leolerance . Previouolerance . FurtherFST) and gene diversity  between nine Iranian indigenous chicken ecotypes and two commercial lines. Multiple lines of evidence suggest that there is a high level of genetic distance between native chicken ecotypes and White Leghorn group. However, we observed a relatively low genetic variability between Arian group and native chicken ecotypes, especially those from northern parts of the country. We further found relatively high levels of LD decay and ROH in both two commercial lines, which could be a consequence of artificial selection for desired traits such as egg or meat production performances. In addition, we detected several candidate genes associated with adaptation to arid environment, immune response activation and parasite resistance in Iranian indigenous chicken ecotypes. Our finding will help improve our understanding of the mechanisms and identify the targets of selection in indigenous chickens that live in desert regions and also facilitate future genome wide association studies and investigations into genomic targets of selection [This study represents the first attempt to investigate the genetic diversity and population structure of most Iranian indigenous chicken ecotypes by using whole-genome sequencing data. We estimated and compared several genetic diversity parameters such as LD, ROH, fixation index  used in this study. Table S2. Chromosome-length and number of detected variants before and after filtering. Table S3. Mean number of ROHs longer than different classes (Kb).Table S4. Summary  diversity results. Proportion of polymorphic SNPs (PN ), Observed heterozygosity (Ho), Expected heterozygosity (He), and average inbreeding coefficient (F) for each studied group. Table S5: Positively selected genes (top %5) identified between indigenous group and White-Leghorn identified by FST method. Table S6. Positively selected genes identified by top 1% highest log2 (θπ·Native-ecotypes/θπ·White-Leghorn). Table S7. Positively selected genes (top %5) identified by FST method between indigenous group and Arian. Table S8: Positively selected genes identified by top 1% highest log2 (θπ·Native-ecotypes/θπ·Arian). Table S9. Overrepresented GO categories among genes showing high Fst values between indigenous group and  White-Leghorn. Table S10. Overrepresented GO categories among genes showing high log2 (θπ·Native-ecotypes/θπ·White-Leghorn). Table S11. Overrepresented GO categories among genes showing high Fst values (indigenous group versus Arian). Table S12. Overrepresented GO categories among genes showing high log2 (θπ·Native-ecotypes/θπ·Arian).Additional file 2: Figure S1.Venndiagrams summarize unique and common variants among groups. Fig. S2. Theheat map of ChromoPainter’s coancestry matrix. Each row corresponds to therecipient genomes and columns represent the donor individuals. Fig. S3. Cross validationerror (CV) plot from ADMIXTURE. Fig. S4. Box plots of nucleotide diversity, calculated in 50kb sliding window with 20 kb increments across the genome. Fig. S5. Genomic regions with selection signals in nativechickens by π method. (A) Indigenousgroup versus Arian chickens. (B) Indigenous group versus White Leghornchickens. Fig. S6. Haplotype network based on pairwise differenceswithin the selective sweep region (Chr1: 176.34-176.36) inHSPH1.