The purpose of the presented investigation is to evaluate the resulting torque on loaded 3D printed springs using different coil thickness and length.Specimens were designed and printed using the 3D printer MAX  with 3D printable, experimental, flexible material . The specimens were divided into three groups according to spring coil design. Control group (n = 18), length group (n = 19) and thickness group (n = 22). Groups were tested using a Sauter Machine for torque calculation  in conjunction with a universal testing machine  for clock-wise and anti-clockwise testing. Statistical analysis was performed using the Steel–Dwass test to compare median values of the three groups in both testing directions (p < 0.001).The highest torque value was determined in the thickness group for both clockwise and anti-clockwise testing directions, achieving 44.00 N/mm and 39.62 N/mm respectively. The length group ranged from 21.65 to 11.04 N/mm in clockwise direction and from 18.04 to 11.38 N/mm in counter-clockwise testing. The control group ranged from 22.72 to 17.18 N/mm in the clock-wise direction while in the anti-clock wise testing it ranged from 21.34 to 16.02 N/mm.The amount of torque produced from the computer aided designing/computer aided manufacturing (CAD/CAM) springs is being affected by diameter more than the length design parameter in comparison to the control group. The values of the thickness group are significantly higher than those of the length group (P < 0.001). The advances in dentistry and malocclusion corrections for development of a healthy occlusion have led to an increased demand of orthodontic treatment. Malocclusion can vary depending on certain factors including the dental angle classification, skeletal classification and genetic factors –3. OrthoThe usage of different orthodontic spring designs as the T or delta loops made of titanium-molybdenum or super elastic nickel titanium material could be appropriate for producing a clinically usable forces which could provide mechanical teeth retraction –8. The uThe 3D printing resin material for orthodontic springs is a recent development with neither data nor studies available for its efficiency.Accordingly, the aim of the present investigation was to mechanically evaluate the torque of the digitally designed and 3D printed orthodontic springs in vitro by having differently designed orthodontic spring groups prior to clinical application.In the present investigation, spring specimens were 3D printed using MAX  with DLP  technology via the experimental flexible printable material  which contains a 3D resin material as its main ingredient. The specimens were designed by the computer aided program Autodesk Netfabb . The post processing of the specimens was performed by GC-Europe according to the manufacturer’s instructions. Unheated ultrasonic bath  reusable isopropanol solution with a concentration of 96% was utilized to clean the specimens for 2 min followed by 2 more minutes of a clean isopropanol bath with the same concentration.The specimens were withdrawn from the solution and dried with compressed air in-between the two cleaning cycles. Surface polymerization was done using Labolight DUO  with double wavelength LED technology in a range of 380–510 nm with spectrum range peaks of 465–485 nm (12 Blue LED’s) and 390–400 nm (3 Violet LED’s). Two periods of 3-min durations each were applied, and the samples were turned for curing from both sides. After post curing, carbide bur and nipper were used to remove supports.After eliminating all specimens’ printable supports, the specimens were divided into three different groups according to their different design parameters, control group n = 18), length group (n = 19) and thickness group n = 22)  and the diameter group was defined by two coils with different radius (Δr = 0.15 mm).The control group consisted of the same material and design parameters used in recent study conducted by Othman et al. who found that the 3D resin used material provides an acceptable force in comparison to NiTi material . AccordiA reference key was designed using Autodesk Netfabb  and printed with the Varseowax Model material using Varseo S 3D printer , which acted as a spring’s attachment to the torque measuring device  in which the values were automatically recorded with an implemented software in the device itself. In order to assure pointed force exertion upon the springs base, a C shaped 3D printed key was loaded into the universal testing machine . The C shaped key was designed using Autodesk Netfabb  and printed with the Varseowax Model material using Varseo S 3D printer  .The compiled data that was gathered from the measurements was to be compared and analyzed for statistical analysis between the groups. The values of both clockwise and counter clock-wise data sets were calculated separately Tables , 3.TableThe statistical analysis showed significant difference between the three groups of springs (p < 0.001). Clockwise and counter-clock wise mechanical testing both proved significant differences for the diameter group (p < 0.001). The highest measured values found for the diameter group was 44.00 N/mm. The lowest scores noted was within the length group 11.04 N/mm.The clock and counter clockwise for the diameter group showed a significant value when compared to both control and length groups using the steel–Dwass test (p < 0.001).Finally, there are significant differences within each group between clock-wise and counter-clockwise directions (p < 0.05).The aim of the present investigation was to mechanically evaluate the torque resulted from the digitally designed and 3D printed orthodontic springs with different design parameters by mechanical loading in clock-wise and counter-clockwise direction. It was proven, that the thickness group had a highly significant torque value in clock wise than in counter-clockwise direction.The study protocol followed the manufacturer’s instructions for post processing, which ensured uniform results. Furthermore, torque testing was used to find out whether the designed springs can develop sufficient deflection processes to evaluate the tooth movement possibility with a 3D printed spring. Contrary to the current metallic springs, where a simple deflection process results in an acceptable force for orthodontic usage .Printed springs may need a deflection processes to develop sufficient forces to enable tooth movement possibility with a 3D printed material, which could lead to a customized usage of the springs depending on the malocclusion problem and treatment aims and objectives. However even with increased effort, torque and force still aid orthodontic treatment , 18. CliThis study is in line with Chudasama et al.  who deteA study conducted by Ubirajara et al. showed that spring parameters are greatly influencing the mechanical effect . It is dThis study is preliminary a feasibility testing of the 3D printed material in which the parameters should be evaluated in depth before any clinical consideration.The findings showed a higher torque value for the diameter group compared to the control group and a clear increase of value in clockwise direction for diameter group with mechanical testing. The length group did not show a significant difference between the two testing directions.


The persistence of newly acquired memories is supported by the activity of PKMζ, an atypical isoform of protein kinase C (PKC). Whether the activity of conventional and atypical PKC isoforms contributes to reactivated memories to persist is still unknown. Similarly, whether memory reactivation is a prerequisite for interventions to be able to change memory persistence is scarcely investigated. Based on the above, we examined the role of conventional and atypical PKC isoforms in the prelimbic cortex in reconsolidation and persistence of a reactivated contextual fear memory in male Wistar rats. It is shown that (i) inhibiting the PKC activity with chelerythrine or the PKMζ activity with ZIP impaired the persistence of a reactivated memory for at least 21 days; (ii) ZIP given immediately after memory reactivation affected neither the reconsolidation nor the persistence process. In contrast, when given 1 h later, it impaired the memory persistence; (iii) chelerythrine given immediately after memory reactivation impaired the reconsolidation; (iv) omitting memory reactivation prevented the chelerythrine- and ZIP-induced effects: (v) the ZIP action is independent of the time elapsed between its administration and the initial memory test. The results indicate that prelimbic cortex PKC and PKMζ are involved in memory reconsolidation and persistence. The role of each one of these PKC isoforms is currently under investigation. Accumulating evidence suggests that persistent activation of an atypical protein kinase C isoform (PKMζ) at the late phase of memory consolidation is responsible for maintaining long-term potentiation and long-term memory5. PKMζ has also been reported to be necessary for the persistence of newly acquired procedural, spatial, appetitive (rewarding), and aversive memories8. The overexpression of PKMζ in the prelimbic (PL) cortex has been shown to potentiate the aversive memory persistence9. On the other hand, its DNA methylation in the PL cortex, or its aggregation with neurofibrillary tangles in the hippocampus, has been associated with memory decline or impairment in animal models of aging-related diseases11.The family of the protein kinase C (PKC) includes conventional , novel , and atypical  isoforms12. Accordingly, a protein synthesis inhibitor given 9.5 h after reactivation has been reported to impair the persistence of a reactivated fear memory13. Similarly, PKC inhibition 6 or 9 h after fear memory reactivation has impaired its persistence without affecting the reconsolidation process14. There is some similarity between fear memory consolidation and reconsolidation, such as their time-window of occurrence17. The mechanisms supporting memory persistence during and after memory consolidation are relatively more investigated19. The investigation of memory maintenance mechanisms during and after the reconsolidation time-window is still incipient. It would be of scientific and therapeutic relevance to advance our knowledge about the latter question.It has been proposed that a brief memory reactivation induces a late phase (beyond the reconsolidation time-window) of protein synthesis that might underlie the persistence of reactivated aversive memories21. PKMζ expression in this medial prefrontal cortex sub-region also seems to support the maintenance of newly acquired aversive memories22. Based on these facts, we hypothesized that not only PKC but also the PKMζ isoform could support the persistence of a reactivated aversive memory. Therefore, the main objective of the present work was to investigate the role of PL cortex PKC and PKMζ in the persistence of contextual fear memory in rats. Initially, we focused on specific time points after the end of the memory reconsolidation time-window (from 6 to 18 h after memory reactivation). Next, by omitting memory reactivation, we assessed the requirement of this process to induce memory persistence mechanisms. Finally, we addressed whether PKC and/or PKMζ is also important for memory reconsolidation and/or persistence within the reconsolidation time-window (0 and/or 1 h after memory reactivation).The PL cortex has been implicated in aversive memory reconsolidation = 33.7; P < 0.0001], the treatment , and the interaction between these factors , for freezing time. As shown in Fig. post-hoc test showed a significant difference between control and chelerythrine groups  during Test A2 , but not the reactivation session  or Test A1 .We tested this pharmacological intervention 6, 9, 12, and 18 h after memory reactivation Fig. . At the  = 25.1; P < 0.0001], the treatment , and their interaction . As shown in Fig. 2 , but not the reactivation session  or Test A1 .At the second time point selected (9 h), a mixed ANOVA showed significant effects of the sessions , the treatment , and their interaction . As shown in Fig. 2 , but not the reactivation session  or Test A1 .At the third time point selected (12 h), a mixed ANOVA showed significant effect of the sessions , and an interaction between sessions and treatment , but not the treatment . As shown in Fig. 2 and the reactivation session in animals treated with vehicle , but not chelerythrine . There were no significant differences between groups during any session performed.At the fourth time point selected (18 h), a mixed ANOVA showed significant effects of the sessions , but not the treatment  or their interaction . As shown in Fig. 1 and A2 than in the no reactivation session (P < 0.0001), confirming that this conditioned behavior is observed predominantly when the animals are re-exposed to the conditioning context. Further, no treatment effect was observed, indicating that the memory reactivation is essential for chelerythrine-induced impairments in its persistence.Changes in memory persistence induced by chelerythrine were similar when this drug was given 6, 9 or 12 h after the reactivation session. Based on this, the first time point was selected to conduct the next experiment in which the session of memory reactivation was omitted Fig.  to inves9. Based on this and preceding chelerythrine findings, we investigated whether PKMζ activity in the PL cortex 6 h after reactivating a contextual fear memory is involved in its persistence  = 31.4; P < 0.0001], the treatment , and their interaction . As shown in Fig. 2 , and Test A3 , but not the reactivation session  or Test A1 . Altogether, the results associate the PL cortex PKMζ activity 6 h after reactivating an aversive memory with its persistence.The overexpression of PKMζ in the PL cortex has been shown to potentiate the aversive memory persistencence Fig. . A mixed = 202; P = 0.00001], but not the treatment  or their interaction . As shown in Fig. 1, A2 and A3 than in the no reactivation session (P < 0.0001), confirming that fear expression is more selective to the conditioning context. Moreover, no treatment effect was observed,, indicating that memory reactivation is necessary for the ZIP-induced changes in the memory persistence.To investigate whether memory reactivation is a prerequisite for ZIP to affect the process of persistence, in the next experiment animals were exposed to a neutral and unpaired Context B (the no reactivation session) and 6 h later treated with vehicle (n = 6) or ZIP (n = 9) Fig. . A mixedζ may impair memory expression rather than permanently interfering with its persistence23. To investigate whether drug-induced effects on persistence of a reactivated memory depends on the time elapsed between treatment and Test A1 . A mixed ANOVA showed significant effects of the sessions , the treatment , and their interaction . As shown in Fig. 1 , but not the reactivation session . These results corroborate that PL cortex PKMζ activity 6 h after reactivating a contextual fear memory is involved in its persistence, and indicate the ZIP action is independent of the time elapsed between its administration and Test A1.It has been argued that inhibiting PKM A1 Fig. , in the  = 22.5; P < 0.0001], the treatment , and their interaction . As shown in Fig. 1 , Test A2 , and Test A3 , but not the reactivation session . These results indicate that PL cortex PKC activity immediately after reactivating a contextual fear memory is involved in its reconsolidation, and the drug-induced reconsolidation impairment was still present 21 days later.We tested this pharmacological intervention immediately after the memory reactivation session Fig. . A mixed = 379.7; P = 0.0001], but not the treatment , or their interaction . As shown in Fig. 1 and A2 than in the no reactivation session (P < 0.0001), confirming that fear expression is more selective to the conditioning context. Moreover, there was no treatment effect, indicating that memory reactivation is also necessary for drug-induced effects in memory reconsolidation.To investigate whether memory reactivation is a prerequisite for chelerythrine to affect the reconsolidation process, in the next experiment the reactivation session was omitted because the animals were exposed to context B and then treated with vehicle or chelerythrine  = 4.33; P < 0.303], the treatment , and their interaction . As shown in Fig. We tested this pharmacological intervention immediately after the memory reactivation session and 1 h later. At the first time point selected Fig. , a mixed = 42.2; P < 0.0001], the treatment , and their interaction . As shown in Fig. 2 , and Test A3 , but not the reactivation session  or Test A1 . Altogether, the results associate the PL cortex PKMζ activity as early as 1 h after reactivating an aversive memory with its persistence.At the second time point selected Fig. , a mixedThe present study sought to investigate the role of PL cortex PKC and PKMζ in the reconsolidation and persistence of a reactivated contextual fear memory in rats. It was shown that: 1) activity of both PKC and PKMζ are necessary for the persistence of a reactivated memory; 2) effects of PKC or PKMζ inhibition require prior memory reactivation; 3) effects of PKMζ inhibition are independent of the initial test 1 day after ZIP infusion; 4) activity of PKC, but not PKMζ, is also involved in reconsolidation of a reactivated memory; 5) activity of PKMζ during the reconsolidation time-window is important for memory persistence. These findings indicate that PL cortex PKC and PKMζ have a differential involvement in the processes examined.1). This result corroborates prior evidence showing that various experimental interventions delivered at this time point are no longer able to interfere with aversive memory reconsolidation24. However, after seven days (Test A2) there was a difference between groups, suggesting that PKC activity in the PL cortex is required for the reactivated aversive memory to persist. This pattern of results (reduced freezing time 7 days but not 24 h after drug treatment) is in line with those reported after infusing a protein synthesis inhibitor into the basolateral amygdala13, an ERK pathway inhibitor into the dorsal hippocampus25, and a systemic administration of a non-selective PKC inhibitor14.The chelerythrine-induced PKC inhibition 6 h after memory reactivation had no effects on freezing time when the animals were tested one day later , then one would expect no changes in memory persistence in animals infused intra-PL cortex with chelerythrine 6 h after their exposure to a non-conditioned context. Indeed, no effects of PKC inhibition were observed when memory reactivation was omitted, a result agreeing with those showed that the induction of persistence-associated mechanisms is triggered by memory reactivation29. Recently, the focus has been on the potential involvement of certain atypical PKC isoforms, particularly PKMζ, in the persistence of newly acquired and reactivated aversive memories33. Considering that chelerythrine has an affinity for both atypical and conventional PKC isoforms35, an additional experiment was performed in which the selective PKMζ inhibitor ZIP was infused intra-PL cortex 6 h after memory reactivation. There was a drug-induced reduction in freezing time when compared with controls during both Tests A2 and A3, which indicates a PKMζ requirement for the persistence of a reactivated memory. This result agrees with those showing that infusing ZIP into other discrete brain regions affects the persistence of various types of newly acquired memories38. Importantly, since chelerythrine and ZIP effects on memory persistence were similar, chelerythrine action was probably mediated, at least in part, by the inhibition of PKMζ activity. Whatever the case, as shown with chelerythrine, ZIP effects required memory reactivation. This result is of particular relevance since PKMζ inhibition in the absence of memory reactivation has been reported to impair its persistence39. The specificity of ZIP has also been challenged, as it was able to impair the LTP maintenance in PKMζ knockout mice40, and inhibit the activity of an atypical PKC isoform termed PKCι/λ5, which currently has only been associated with early phases of memory consolidation and early LTP5. Thus, one could argue that ZIP effects only are partially associated with PKMζ inhibition. In fact, as a compensatory mechanism, knockout mice for PKMζ increased the expression of PKCι/λ, which in turn mediated the process memory persistence39. Besides, in our study the animals are not transgenic, making it less probable that ZIP-induced effects depend on mechanisms other than those mediated by PKMζ.The role of conventional PKC isoforms has long been investigated in aversive learning paradigms23. Here, the intra-PL infusion of ZIP 6 h after memory reactivation reduced the freezing expression when the animals were retested either 1 or 10 days later, indicating that the time elapsed between drug administration and initial testing is not a pivotal factor influencing the ZIP action. Moreover, it was reported that ZIP infused into the insular cortex impaired the taste aversion memory persistence 1 month after its infusion36. We investigated the PL cortex ZIP effects on memory persistence by using a protocol of contextual fear conditioning with a familiarization session, in which the contribution of the medial prefrontal cortex to long-term memory is greater than in protocols without pre-exposure to the context to-be-conditioned41, as used in those works36. Thus, differences in protocols and the brain areas where ZIP was infused may account for the mixed findings observed.A study reported that ZIP-induced effects depend on the initial test that occurred 1 day after its infusion into the basolateral amygdala when the fear-potentiated startle was used1, suggesting that PKC activity also influences the contextual fear memory reconsolidation. This result agrees with those showing the importance of the PKC activity during memory reconsolidation in other brain regions42, and the PL cortex contribution to reconsolidate aversive memories44. It is worth mentioning that the chelerythrine group also expressed lower freezing levels than respective controls when tested 7 and 21 days later (Tests A2 and A3), a result in line with studies showing that interventions targeting the reconsolidation are not associated with extinction-related features, such as reinstatement and spontaneous recovery of original fear memory46. Moreover, it was shown that chelerythrine effects on reconsolidation require prior memory reactivation.It is currently unknown whether reconsolidation and persistence mechanisms overlap in the PL cortex. To start to address this question, chelerythrine was given immediately after memory reactivation. There was a drug-induced reduction in freezing time relative to controls during Test A1, A2 and A3 when it was given immediately after memory reactivation, suggesting that inhibiting the PKMζ at this time point in the PL cortex affects neither the reconsolidation nor the persistence of a reactivated memory. It has been proposed that memory retrieval and reactivation mechanisms may be partially inhibited by ZIP. For instance, the AMPA receptor GluR2A subunit trafficking into synapses is induced by memory retrieval and is necessary for memory reconsolidation49. Since ZIP’s action is correlated with an inhibition of GluR2A trafficking, this drug could have impaired the memory reactivation and, therefore, the reconsolidation process was not sufficiently induced, which in turn prevented the action of ZIP to occur. It has been shown that PKMζ inhibition into CA1 impaired the reconsolidation of spatial memory50. Besides, a reconsolidation-induced enhancement of PKMζ activity in the amygdala was related to the maintenance of olfactory fear memory in juvenile rats51. It is plausible that differences in paradigms used  may account for the mixed findings reported. Indeed, it has been suggested that PKMζ maintains fear memory in the basolateral amygdala51 and the PL cortex9, but not in the dorsal hippocampus52, although this issue is still under debate. Further, the animals’ age may also influence the outcome since juvenile rats present less memory retention than adult ones53. Here, to further address the PKMζ role in memory reconsolidation, another group of rats received ZIP infusion into the PL cortex 1 h after memory reactivation. As depicted in Fig. 2 and A3, suggesting that PL cortex PKMζ activity mediates aspects specifically related to memory persistence as early as 1 h after memory reactivation. This result is consistent with those from the study by Krawczyk et al. (2016)25. where the inhibition of ERK1/2 in the dorsal hippocampus 3 h after memory reactivation kept the fear memory intact one day later but impaired it when the animals were retested 7 days later. Remarkably, it was recently shown that memory reactivation induces mechanisms related to both memory reconsolidation and persistence25.The potential ZIP effects on memory reconsolidation were also investigated. There were no drug-induced changes in freezing time relative to controls during Tests ATogether, the chelerythrine-induced effects on memory reconsolidation and the lack of ZIP effects on this memory phase suggest a differential contribution of conventional PKC in memory reconsolidation and atypical PKC, such as PKMζ, for memory persistence following reactivation. Future studies could address which PKC isoforms are involved in each memory process in the PL cortex. Altogether, present findings indicate that PL cortex PKC and PKMζ are involved in the reconsolidation and persistence of a reactivated contextual fear memory. Moreover, present findings demonstrated that after the end of the reconsolidation time-window there is an extended opportunity to mitigate the fear memory.ad libitum, and maintained on a 12-hour light/dark cycle (lights on at 7:00 am and off at 7:00 pm) and controlled temperature of 22 ± 2 °C. All experiments were performed after the approval of the experimental protocol by the Ethical Committee for the care and use of laboratory animals of the Biological Sciences Sector of Federal University of Parana (authorization number 1011) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals 54.Adult male Wistar rats weighing 290–320 g  were kept in plastic cages in groups of five per cage with access to food and water 55.Chelerythrine , a selective PKC inhibitor, was dissolved in saline containing 5% of polyoxyethylene sorbitan monooleate (Tween 80), which alone served as vehicle solution. The PKMζ inhibitor ZIP  and the scrambled-ZIP , were dissolved in phosphate buffer saline (PBS). The dose selection of each drug was based on previously published studies56 and fixed to the skull with two screws and dental acrylic. To avoid possible occlusion, a stylet was introduced inside each guide cannula. Immediately after the surgery, the animals received 0.4 ml of ibuprofen orally . After ten days, the experiments were initiated. At different moments after memory reactivation, the animals received a bilateral infusion with dental needles introduced through the guide cannulas until their tips were 1.5 mm below the cannula end. During 1 min, 0.2 µl/side of either vehicle or drug was injected using two 5.0-µl syringes connected to an infusion pump . A polyethylene catheter was interposed between the upper end of the dental needles and the syringes. The displacement of an air bubble inside the polyethylene was used to monitor drug flow. The needles were removed 45 s after the end of injections.Rats were anesthetized with ketamine  and xylazine , associated with local anesthesia , and positioned in a stereotaxic frame. After anesthesia, the animals were placed in the stereotaxic frame. Two stainless-steel guide cannulas  were implanted bilaterally aiming at the PL cortex following the coordinates  of the rat brain Atlas of Paxinos and Watson (2009)After the end of experiments, animals were intraperitoneally anesthetized using 1.0 mL/kg of a solution containing xylazine  and chloral hydrate  intraperitoneally (i.p.). Evans Blue (0.2 µl/ hemisphere) was injected through the guide cannulas for the subsequent evaluation of the locations where vehicle, chelerythrine, ZIP or scrambled-ZIP was infused. Soon after, the brain was removed and immersed in a 10% formalin solution. Brain slices (50 µm thick) were obtained in a vibratome (Leica), mounted on glass microscope slides, and the site of injection was determined. Animals were included in the analysis when both sides of the PL cortex were tagged by Evans Blue Fig. .Figure 6Contextual fear conditioning was performed in a chamber , with sidewalls made of aluminum, the front wall and top cover made of transparent acrylic. The floor was made of stainless steel bars (3 mm in diameter and spaced 0.9 mm) connected to a shock-generating font . A neutral chamber  with transparent plexiglass walls and a black cover to provide contextual cues as different as possible from those of Context A was used. Context B was used to assess fear generalization or as a context unable to induce memory reactivation.57, and were performed between 1:00 and 5:00 PM to minimize possible circadian influences on learning and memory processing. All animals were acclimated to the experimenter and the experimental room for 30 min before each session. The experimental rooms were kept under controlled temperature (22 ± 2 °C) and brightness (~ 80 lux). The contextual fear conditioning consisted of the following sessions: on day 1 the animals were placed in Context A for 3 min, where they were allowed to explore it freely and then returned to their home cages. After 24 h, the animals were submitted to the conditioning session in Context A. After the initial 30 s, the animals received three footshocks , the unconditioned stimulus (US). After the last shock, the animal remained for an additional 30 s in the conditioning chamber and then returned to the home cage. After 24 h, during the reactivation session, the animals were re-exposed to Context A for 3 min without the US presentation. Immediately, 1, 6, 9, 12 or 18 h after the reactivation session the treatment was bilaterally infused into the PL cortex. After 24 h, the animals were re-exposed to Context A for 3 min (Test A1); they were again re-exposed to Context A (3 min) 7 and/or 21 days later .The experiments were conducted similarly to previous studies1 and A2, the animals were exposed to the unpaired and neutral Context B (Test B) for 3 min. Since no fear generalization was observed, this data was omitted. Moreover, Context B exposure was also used to omit memory retrieval. The chambers were cleaned with a 10% ethanol/water solution after each session.In all experiments, to assess the possible expression of generalized fear, 24 h after Tests ATo investigate whether PKC inhibition by chelerythrine interferes with the persistence of a reactivated memory (experiment 1), contextually fear-conditioned animals were randomly allocated to receive a bilateral infusion of vehicle or chelerythrine (3.0 nmol) intra-PL cortex 6, 9, 12 or 18 h after the reactivation session , contextually fear-conditioned animals were randomly allocated to receive a bilateral infusion of vehicle or chelerythrine (3.0 nmol) intra-PL cortex 6 h after Context B exposure for 3 min Fig. .1 (experiment 3 C), contextually fear-conditioned animals were randomly allocated to receive a bilateral infusion of Scr-ZIP (10 nmol) or ZIP (10 nmol) into the PL cortex 6 h after Context A re-exposure or 6 h after Context B exposure for 3 min. The groups were tested after 1 and 7 , whether impairments in memory persistence induced by ZIP require memory reactivation (experiment 3B), or whether impairments in memory persistence induced by ZIP depend on the initial Test Ad 7 Fig.  or 10 dad 7 Fig. .To investigate whether chelerythrine-induced PKC inhibition also interferes with the reconsolidation of a reactivated memory (experiment 4 A), or whether possible impairments in memory reconsolidation induced by chelerythrine require memory reactivation (experiment 4B), contextually fear-conditioned animals were randomly allocated to receive a bilateral infusion of vehicle or chelerythrine (3.0 nmol) intra-PL immediately after memory reactivation Fig.  or immedTo investigate whether ZIP-induced PKMζ inhibition also interferes with the reconsolidation of a reactivated memory (experiment 5), contextually fear-conditioned animals were randomly allocated to receive a bilateral infusion of Scr-ZIP (10 nmol) or ZIP (10 nmol) intra-PL 0 Fig.  or 1 h  after me58, was used as an index of fear memory. Animal behavior was recorded, and freezing time was quantified in seconds by a trained observer blind to the experimental groups and expressed as the percentage of total session time.Freezing behavior, defined as the total absence of body and head movements except for those associated with breathing1, A2, and A3 were submitted to a mixed analysis of variance (ANOVA). The factors evaluated were the treatment and sessions (exposures to Context A and/or Context B). The interaction between treatment and sessions was also assessed. The statistical significance level was set at P < 0.05. The Tukey’s test was used for post-hoc comparisons when F values achieved statistical significance. GraphPad Prism 8.3  was used for statistical analysis and graphing.The results are expressed as mean ± S.E.M. The percentages of freezing time in the no reactivation (Context B), the reactivation session, and Tests Aa priori sample size determined by power analysis was of eight animals per group . The group sizes were equal by design, but due to experimental losses (e.g. when treatment was infused outside the target brain region), in a few cases, they were slightly unequal.The g to reflect the mean differences between two groups (n ≤ 20 per group) that could be dissimilar in size. A g ≥ 0.8 was considered a large effect size59.The effect size was calculated using the formula for Hedges’


Specifications table•These data provide information on the transcriptional effects on whole aorta after treatment with alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist AZ6983 in the atherosclerosis-prone ApoE-/- mouse.•Researchers interested in atherosclerosis, as well as, α7nAChR signaling will find these data a valuable resource.•The information provided here may be used for future studies on how α7nAChR stimulation influence the vascular transcriptome.•These data can generate hypothesis for new studies investigating the α7nAChR-related transcriptomic profiles, as well as signaling pathways, in other tissues•The present data on α7nAChR signaling is predominantly available from cell culture experiments, using cell lines, this data set provides additional information on the signaling pathways in tissue from long-term treatment in vivo.1To investigate the effects of alpha 7 nicotinic acetylcholine receptor (α7nAChR) stimulation on atherosclerosis in apolipoprotein E deficient (ApoE-/-) mice, mice were treated with α7nAChR agonist AZ6983 for 8 weeks. Thoracic aortas were used for RNA sequencing analysis. 22.1ad libitum. All procedures involving mice were approved by the Regional Animal Ethics Committee at the University of Gothenburg, in accordance with the European Communities Council Directives of 22 September 2010 (2010/63/EU).Male apoE-/- mice  were kept at the Laboratory for Experimental Biomedicine, Gothenburg, Sweden. At 10 weeks of age, mice were anesthetized using isoflurane and subcutaneously implanted with osmotic minipumps  delivering vehicle , or α7nAChR agonist AZ6983 (50 µmol kg-1 per day) for 8 weeks. Due to the duration of the minipumps, they were replaced after 4 weeks. From 10 weeks of age and throughout the experiment, mice were fed a high fat, cholesterol enriched diet . All animals were housed at 21–24 °C in a room with 12 h light/ 12 h dark cycle. Water and food were available 2.2Ⓡ Fibrous Tissue Mini Kit  according to the manufacturer's protocol. Concentration and quality was analyzed using a NanoDrop  and electrophoresis .RNA of thoracic aorta was extracted by using the RNAeasyAortic RNA from mice treated with AZ6983 (n=6) or controls (n=6) was isolated as described above and Stranded Total RNA Sample preparations were performed using the Illumina TrueSeq Stranded Total RNA Sample Preparation Kit with Ribo Sero Gold according to the TruSeq Stranded Total RNA Sample Preparation Guide (15031048 Rev. E). Sequencing of the enriched libraries was performed on Illumina Nextseq500 (2x75bp). The quality of the data was analyzed with FastQC and reads with an average quality score of >30 were included in the sequencing. Differentially expressed genes (DEGs) were identified using the DESeq2-method with Benjamini Hochberg adjusted p-values  Ⓡ Pathway Analysis  was used to study potential functions of AZ6983 treatment in the aorta Ⓡ Knowledge Base. Networks are ranked according to their degree of relevance to the genes in the data set. Functional analysis identified the top ranked biological functions and diseases that were enriched in the dataset by calculating the number of molecules that cohere to a functional category and was estimated by Fisher's exact test (q<0.05). Activation Z-score predicts if a specific function is activated (≥ 2) or inhibited (≤ −2) and is supported by one or more references from IngenuityⓇ Knowledge Base.QIAGEN's Ingenuity-/- mice treated with AZ6983 compared with controls. Major functions of the networks are indicated in A and B, followed by the network score. Networks are ranked according to their degree of relevance to the eligible network molecules in the data set and the score is calculated with an algorithm based on p-scores derived from q-values. The up- (red) or down– (green) regulation of genes are indicated by the intensity of node color, and the functional class of the gene product is indicated by different symbols. Relationship between genes are supported by one or more references and illustrated with a connecting line.Top two networks identified with Ingenuity Pathway Analysis (IPA) software for genes that were differently expressed in aorta of ApoE


Epithelial bending is a fundamental process that shapes organs during development. Previously known mechanisms involve cells locally changing shape from columnar to wedge-shaped. Here we report a different mechanism that occurs without cell wedging. In mammalian salivary glands and teeth, we show that initial invagination occurs through coordinated vertical cell movement: cells towards the periphery of the placode move vertically upwards while their more central neighbours move downwards. Movement is achieved by active cell-on-cell migration: outer cells migrate with apical, centripetally polarised leading edge protrusions but remain attached to the basal lamina, depressing more central neighbours to “telescope” the epithelium downwards into underlying mesenchyme. Inhibiting protrusion formation by Arp2/3 protein blocks invagination. FGF and Hedgehog morphogen signals are required, with FGF providing a directional cue. These findings show that epithelial bending can be achieved by a morphogenetic mechanism of coordinated cell rearrangement quite distinct from previously recognised invagination processes. Mechanisms that regulate epithelial bending mainly link to cell shape changes, for example, the formation of wedge shaped cells. Here, the authors identify a different cell behaviour in the salivary glands and teeth where initial invagination arises by a coordinated vertical cell movement. We previously showed that in mammalian tooth germs, mammary glands and hair follicles, the epithelium invaginates when it stratifies and cells in the suprabasal layers  migrate centripetally and pull radially inwards on a ring of underlying basal layer cells, both bending the epithelium and narrowing the “neck” of the bud , as well as the other glandular ectodermal organs, initially invaginate with a hollow space and no canopy above the invagination  and are all affected simultaneously by congenital ectodermal dysplasiasbud Fig. . In thision Fig. . Close iion Fig. . The absIn this work we show that SG and early molar placodes invaginate by a mechanism we call “vertical telescoping” in which essentially columnar cells migrate upwards relative to their immedately more central neighbours, effectively pushing the latter down, but involving centrally directed apical protrusions and centripetal polarisation similar to that seen in canopy contraction in other ectodermal organs.4). We found that neither F-actin nor diphosphomyosin was detectably enriched in invaginating salivary germs compared to nearby flat oral epithelium   Fig.. This imode Fig. .14. Vertical telescoping definitively predicts that cells will lean or tilt outwards from the centre of the placode relative to the normal to the lamina . To test for vertical telescoping in the tooth, we examined molar primordia at their early initiation stages, when stratification has barely begun. Transverse sections revealed vertical cells on the inclined slope of the lamina . Thus, the protrusions are consistent with the model depicted in Fig. If vertical telescoping is epithelially driven, then it implies active cell movement consisting of vertical cell migration of cells relative to their neighbours—in effect a “mesenchymoid” behaviour. By analogy with migration of cells on substrates, one might expect that the cells move with a leading-edge protrusionsal Fig. , b, althsal Fig. 7. Live iTo determine the function of the apical protrusions and signals that induce and orient them, we applied small molecule cytoskeletal inhibitors to cultured SG placode explants. We titrated the concentrations of the actin polymerisation inhibitor cytochalasin D and the Arp2/3 actin-branching inhibitor CK666 to the lowest concentrations that had a phenotypic effect. At these levels of cytochalasin D, the actin cytoskeleton is affected  and fibroblast growth factor (FGF) pathways drive early tooth morphogenesis. We therefore tested the effect of a Hedgehog signalling inhibitor (cyclopamine) and an inhibitor of FGF receptor (SU5402) in SG explants. At minimum concentrations known to inhibit expression of pathways targets, cyclopamine reduced invagination and SU5402 essentially abolished it  daughters, allowing them to migrate even more centripetally. They remain mechanically linked to their basal sisters, thus creating a contractile canopy coupled to the basal layer. The simple sum of the migratory and oriented cell division behaviours achieves the difference between these two types of ectodermal organ. The mechanical forces of vertical telescoping, as distinct from the kinematics, and the adhesive and cytoskeletal structures that are involved, remain to be established: mechanical modelling and experimental measurement of forces in this system is a current focus of further investigation.Comparing the SG with the tooth Fig.  illustraUnderstanding mechanisms of physical morphogenesis is a necessary basis for improving the fidelity of organoids to their natural counterparts and for precision regeneration in vivo. Vertical telescoping is one such mechanism, a new type of fold in the art of epithelial origami and a mechanistic module that can be combined with others to generate phenotypic diversity.Gt(ROSA)26Sortm4Luo/J (Jackson Laboratories strain 007576)] crossed with a tamoxifen-inducible Cre male B6.Cg-Tg(CAG-cre/Esr1)5Amc/J (Jackson Laboratories strain 004682)3. To induce mosaic expression of membrane green flourescent protein (GFP), 2 mg/animal Tamoxifen (Sigma) in corn oil was injected I.P. Twenty-four hour prior to the experiment into pregnant mT/mG females, together with 1 mg progesterone (Sigma) to reduce foetal resorption. Automated cell orientation analysis, immunofluorescent staining, and inhibitor treatment were performed with CD-1 mice. Embryos were staged by nominal age .All animal experiments were conducted under license from the United Kingdom Home Office and approval of the institutional Ethical Review Board. Live imaging of mosaically labelled cells, cell shape analyses in fixed materials, were performed using mT/mG mice [17 for confocal imaging.Specimens were fixed in 4% paraformaldehyde for 3 h at room temperature (RT). Fixed whole-mount tissues were washed 3 times with phosphate-buffered saline-triton (0.1%) and blocked with 20% goat serum (Sigma G6767) for 1 h at RT. Antibody and chemical staining were performed in blocking solution at the following dilutions: anti-β-catenin 1:500 (Sigma C2206), anti-laminin 1:500 (Sigma L9393), anti-phosphorylated myosin 2 light chain , Alexa 488/568 anti-mouse/rabbit/rat 1:500 (Life Technologies), fluorescent phalloidin (Life Technologies) 1:200, DAPI (Life Technologies) 1:10,000. Validations of antibodies can be found on manufacturers’ websites. Incubations for both primary and secondary antibodies were at 4 °C overnight. For specimens >80 μm thick, post-fixation  was performed after immunostaining, and stained tissues were cleared using Scale A2 and Scale B4 solutions3D image stacks were captured on a Leica SP5 confocal microscope with an HCX PL APO CS 40× oil (N.A. 1.25) or an HCX PL APO 63×/1.3 GLYC CORR CS (21 °C) objective. For 3D cell shape and orientation analyses, placodes were imaged en face at optimal (isotropic voxel) z resolution.z the GFP cell boundary started and ended. These two positions were marked and slice numbers of a quarter, half, three quarters and 9/10 cell height from the base were calculated. Areas of the cell inside the boundary at each of these heights were drawn manually using the freehand tool in Fiji to return the area enclosed. “Base” area for Fig. z resolution under a 63× confocal objective. The centre of the nucleus and the height of each cell were measured by viewing the cell in a transverse digital section containing the largest section area of the cell. The line tool in Fiji was used to manually measure the height of the nucleus and the cell. ANOVA test was performed to determine whether there was any significant difference between groups.For cell shape analysis, GFP-positive embryos were dissected and submandibular glands were imaged en face at optimal z resolution under the 63× objective. Isolated GFP-positive cells contacting the basal lamina were identified by digitally reslicing the 3D stack frontally and viewing in frontal section. To measure cell shapes, along the apicobasal axis, the apical and basal limits of a cell were determined by scrolling up and down the original image stack to find where in z resolution on a Leica SP5 40× or 63× objective. Fiji macro and MATLAB were used to segment epithelial cells in 3D and compute cell orientations, respectively (scripts available upon request). Essential steps in the pipeline are as follows: an entire image was divided in x–y as a grid (each square 20 × 20 pixels), keeping the z depth. The 3D coordinates of the local lamina were registered as the peak of the laminin staining intensity in z and the x–y coordinates of the square. The mesenchyme was blacked out from the image below the lamina. Cells were segmented in 3D using our self-developed iterative thresholding Fiji macro and the 3D suite Fiji plugin14 based on the cell boundary staining, and the segmented objects were filtered for DAPI staining to confirm them as real cells. Ellipsoids were fitted to each segmented cell volume using the Fiji ellipsoid-fitting macro14 and information of cell centroids and axes of the ellipsoids were extracted. These cell data together with the lamina grid positions were imported into MATLAB. The plane of the local lamina of a cell was determined as that defined by the xyz coordinates of the lamina at the centre points of the three 20 × 20 pixel regions closest to the centroid of the cell. Orientation of a cell was computed in MATLAB as the angle between the major axis (as a vector) of the cell and the normal vector to the local lamina plane. Displacements of these two vectors were plotted as a vector map to show the “leaning” of columnar cells on a bending lamina. A map of the depth of the basal lamina was plotted as a heat map in R18. For scatter plots in Supplementary Fig. Whole-mount mandibles were stained for β-catenin (for cell boundary), α-laminin  and DAPI (for nucleus), and a 3D stack of the placode was imaged en face at optimal 15. Concentrations of drugs used in culture were: 20 μM cyclopamine , 5 μg/ml SU5402  0.05 μg/ml cytochalasin D  and 250 μM CK666 . Ethanol or DMSO were used as vehicle, respectively. Culture medium was phenol red-free Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) + 15% FCS + 0.1 g/L Ascorbic Acid + Pen/Strep. Sample sizes of each experiment are detailed in each figure legend.Explant culture of salivary placodes was performed as described in ref. z resolution. The culture was supplied with humidified 5% CO2 in air in a 37 °C incubation chamber throughout the imaging.GFP-positive mT/mG embryos were selected for live imaging. Submandibular gland explants were dissected at E11.25 in DMEM/F12, immobilised apical-side-down in a glass bottom dish by overlaying with a piece of transparent membrane from a porous cell culture insert  anchored to the glass at its edges with small blobs of Vaseline, and imaged on an inverted Leica SP5 with a 63× objective with optimal To assess the effect of drugs on cell protrusions in live tissue, mandibular explants were dissected and cultured with drugs in concentrations listed above, in a glass bottom dish and shallow medium so that the surface of the tissue was in contact with air . Explants were cultured at least 3 h before setting up for imaging.Where sample numbers are not stated explicitly in figure legends, e.g. for micrographs, data are representative of at least three different embryos from at least three different litters.Further information on research design is available in the Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary InformationDescription of Additional Supplementary FilesReporting Summary


The monitoring site was deployed at “El Soldado” estuary (27°57′14.4″ N and 110°58′19.2″ W) located in the southern coast of the Mexican State of Sonora. UAV flight path was programmed to flight in autonomous mode with an altitude of 30 m, a velocity of 5 m/s and a frontal and side overlap of 85 and 75% respectively. This dataset was created to support mapping surveys for surface classification and site description. This dataset is aimed to support researchers, stakeholders and general public interested in coastal areas, natural resources management and ecosystem conservation. Finally, this dataset could be also used for those interested in digital photogrammetry and 3D reconstruction as benchmark example to develop high resolution orthomosaics.It is well known that remote sensing is a series of procedures which detects physical characteristics of the earth surface by remotely-measuring its reflected and emitted radiation using cameras or sensors. Lately, the increasing use of unmanned aerial vehicles (UAVs) as remote sensing platforms and the development of small-size sensors have resulted in the expansion of continuous monitoring of earth surface at smaller spatial scales. For this reason, the integration of UAV- and consumer-grade cameras can be useful to acquire surface characteristics at plot or footprint scale. This dataset contains 314 aerial images covering an area of aproximately 18,800 m Specifications table•Database is aimed to those interested in ecosystem conservation, natural resources management and studies in coastal ecosystems by applying digital photogrammetry and 3D reconstruction.•Dataset can be used as a benchmark example to improve digital photogrammetry methods that helps to develop accurate high resolution orthomosaics . However, several ground control points (GCP) are suggested to reduce geometric distortion.•Dataset can be used as a mapping resource to identify objects of interest and to estimate the spatial extension of surface features in coastal environments.•The dataset can be used as a baseline for a long-term monitoring schemes at El Soldado estuary. In addition, this dataset can be used for comparison in coastal landforms evolution with similar intertidal coastal environments in Northwestern Mexico or semiarid coasts in the world”.This article describes a coastal survey routine by means of aerial images acquisition using an unmanned aerial vehicle and a commercial-grade RGB camera over an intertidal coastal strip within “El Soldado” estuary, in Guaymas, Sonora, Mexico  [1]. 314This dataset contains 314 aerial photographs taken in March 02, 2019 at 9 a.m. local time (UTC - 16:00) over an experimental site instrumented with an eddy covariance tower and meteorological sensors at "El Soldado" estuary located at the South-eastern margin of the Gulf of California (27°57′14.4″ N and 110°58′19.2″ W). The images were taken with a 16 MP MAPIR Survey2 RGB camera pointed downwards at 90° with a field of view (FOV) of 82°, a shooting interval of two seconds, focal aperture of f/2.8 (default for MAPIR Survey2 Cameras), shutter speed set to 1/125 and ISO 50. Image quality was estimated based on image sharpness using the Agisoft Metashape's image quality tool. All images with a sharpness score lower than 0.5 were discarded


Macadamia has a recent history of domestication as a commercial nut crop. We aimed to establish the first sequence-based haploid-correlated reference genetic linkage maps for this primarily outcrossing perennial tree crop, with marker density suitable for genome anchoring. Four first generation populations were used to maximise the segregation patterns available within full-sib, biparental and self-pollinated progeny. This allowed us to combine segregation data from overlapping subsets of >4,000 informative sequence-tagged markers to increase the effective coverage of the karyotype represented by the recombinant crossover events detected. All maps had 14 linkage groups, corresponding to the Macadamia haploid chromosome number, and enabled the anchoring and orientation of sequence scaffolds to construct a pseudo-chromosomal genome assembly for macadamia. Comparison of individual maps indicated a high level of congruence, with minor discrepancies satisfactorily resolved within the integrated maps. The combined set of maps significantly improved marker density and the proportion (70%) of the genome sequence assembly anchored. Overall, increasing our understanding of the genetic landscape and genome for this nut crop represents a substantial advance in macadamia genetics and genomics. The set of maps, large number of sequence-based markers and the reconstructed genome provide a toolkit to underpin future breeding that should help to extend the macadamia industry as well as provide resources for the long term conservation of natural populations in eastern Australia of this unique genus.The Proteaceae genus  Macadamia was the first genus in the family to be domesticated although others are also cultivated for edible kernel (Gevuina) or for ornamental use . The long-lived Australian subtropical rainforest trees M. integrifolia (Maiden & Betche) and M. tetraphylla (L.A.S. Johnson) and their hybrids underpin the macadamia ‘nut’ industry. Although commercially important, there is limited understanding of genome structure and the genetic basis of adaptive traits2. Macadamia is diploid, with a reported haploid chromosome number of 144. Although mass flowers are produced on racemes, less than 5% of these set fruit5, and partial self-incompatibility has been observed7. World macadamia production has expanded faster than any other tree nut crop over the past ten years and currently accounts for ~3% of the global nut trade8. There is growing need to develop new cultivars to meet yield and quality standards, as well as increased cultivation for a wider range of cultivation conditions9. The ability of breeders to improve yield, pest and disease resistance, and tolerance to a variety of environmental conditions is greatly enhanced by aligning trait loci and underlying genetic variation with the genome10.The Gondwanan plant family Proteaceae comprises 83 genera and over 1,600 species14. Given that linkage maps represent the distribution of chiasmatic crossovers (COs) resulting from parental meiotic recombination, they provide valuable insights into the contribution that each set of parental alleles may make to subsequent progeny. They may also be used for map-based cloning, to estimate the rate of introgression of specific alleles, and to determine the likelihood of linkage drag between loci15.Genetic linkage maps are a powerful resource that can improve breeding efficiency by identifying the relative chromosomal location of genes underlying key phenotypic traits, and assisting in the development of selective markers, which may be of particular value in perennial crops with long generation times. More specifically, dense linkage maps remain valuable for anchoring and orienting whole-genome sequence scaffolds, mapping of quantitiative trait loci (QTL), and informing comparative genomic and evolutionary studies16. Inconsistencies in marker order between maps are inevitable as each map is based on the segregation of alleles resulting from independent parental meioses that arise from the particular cross used to generate the map.It is widely accepted that the marker order and the length of genetic linkage maps can vary within species, and the notion that a single linkage map represents an accurate distribution of variation in recombination for a species is flawed17. The introduction of next generation DNA sequencing (NGS) and genotyping by sequencing (GBS) marker generation has led to the production of many revised linkage maps with increased marker density and resolution that has improved the downstream applications of linkage maps.Many early linkage maps for a range of species were constructed using a limited number of molecular markers, and were often not of sufficient density or resolution to achieve fine mapping of QTLs, MAS or genome anchoring1 progeny of HAES ‘246’ x HVP ‘A16’18. However, due to low marker density and a lack of sequence-based markers they were unable to be used for QTL detection or genome anchoring. This highlights the need for robust, dense linkage maps developed using sequence-based markers to provide an accurate reflection of parental meioses, comprehensive coverage of each chromosome, and selective markers of value for future breeding programs.The first macadamia genetic linkage maps were constructed using 328 arbitrary amplified RAF and RAPD markers and a single tagged STMS marker from 56 FM. integrifolia cultivar HAES ‘741’ has been generated, consisting of 193,493 scaffolds with a combined length of 518 megabases (Mb)2. Following subsequent improvements, the most recent ‘741’ assembly comprises 4,098 scaffolds with a combined length of 744 Mb19. However, there is no information on the relative location or orientation of these scaffolds with respect to the individual chromosomes karyotype.A draft genome assembly of 21. We compared the ability of multiple parental maps and integrated cultivar-specific maps to increase marker density and accuracy of marker order when compared with individual linkage maps. We also explored parental contributions to observed variation in recombination rate and the relative reliability and congruence of individual and integrated maps. Overall, this approach allowed us to develop a series of genetic linkage maps which captured the range of recombination from four populations and provided a suitable density of sequence based markers to anchor genome assembly sequences. To our knowledge, these platform genomic resources are the first sequence-based haploid-correlated genetic linkage and physical maps for macadamia, and will contribute significantly to future breeding programs and macadamia research.There is evidence that SNP-based genetic linkage maps from multiple populations can, in combination, maximise genome anchoring and orientationM. integrifolia parental cultivar (‘741’). This cultivar had previously been used as the reference genotype for genome sequencing and assembly2 and originated from the Hawaiian Agricultural Experiment Station (HAES). Populations comprised unselected progeny from self-pollinated ‘741’, as well as biparental crosses of ‘741’ and the M. integrifolia - M. tetraphylla hybrid cultivars ‘A268’ and ‘A4’ from Hidden Valley Plantations (HVP), Australia  from approximately 20 mg of dry leaf material following methods previously reportedMature, sclerophyllous leaf material was processed in individual tubes as the required concentration and quality of DNA was difficult to obtain using the DNeasy 96 microtiter plate method. In some cases, individual extracts or pooled multiple extractions were concentrated using an Eppendorf Concentrator 5301  in order to obtain minimum concentration, 50 ng/µl, required for downstream analysis. For a small number of samples multiple lysates were loaded onto columns to increase concentration. DNA was quantified using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific Inc) and a Qubit dsDNA BR assay .28. DArTseq is is a rapid, cost-effective platform for genetic marker discovery and genotyping that targets non-repetitive regions and has been used extensively for genetic linkage mapping of plant crops29. Methods followed those previously optimized and described for macadamia using PstI + HhaI genome complexity reduction to generate co-dominant SNP and dominant presence-absence variant (PAV) sequence markers of 64 base pair (bp) in length30.Marker genotyping was conducted by Diversity Array Technologies Ltd. using DArTseq methodologyet al.29 were applied, and markers with reproducibility >90%, call rate >90% and average polymorphic information content (PIC) > 0.25 were retained for genetic linkage mapping.Exclusion of markers from the data set was based on the following parameters. Markers were excluded if there was: inconsistency between biological or technical replicates of parental genotypes, or greater than 10% missing values in progeny, or ambiguous or impossible segregation . Quality control parameters provided by DArT, as outlined by Barilli, 32 was used to construct all linkage maps. A ‘741’ self map was constructed using heterozygous SNP markers coded for a cross pollination (CP) population. Phase information generated in JoinMap for each marker was then used to recode markers for an F2 population. An F2 map was generated using the multipoint likelihood mapping algorithm, the Kosambi mapping function with default settings, recombination frequency calculated with a maximum of 200 iterations, and a recombination tolerance of 1.0 e−0.8. Markers that showed significantly distorted segregation (P < 0.01) were removed if adjacent markers segregating for the same parent did not show distortion.The program JoinMap 5.0Maternal and paternal maps were constructed using either filtered segregating SNP and PAV markers for the biparental crosses (‘741’ x ‘A4’), (‘741’ x ‘A268’) and (‘A268’ x ‘741’). These maps were generated for each parent individually in JoinMap using the parameters already described.Map integration for the four ‘741’ maps and two ‘A268’ maps was performed using the regression mapping algorithm with default settings in JoinMap.In order to determine marker order consistency across the maps, Spearman’s Rank correlation coefficients were calculated. Each of the parental ‘741’ maps was compared to the ‘741’ self map, while both the ‘A268’ maps were compared to the ‘A4’ map to test for map congruence.33 was used to illustrate regions of segregation distortion (SD) on the ‘741’ self-map.Marker distortion was estimated by Chi–square goodness-of-fit test with a threshold p-value of 0.01 in JoinMap. The number of SNP markers showing segregation distortion was counted for each of the parental maps and the ‘741’ self map. MapChart v2.3The number and distribution of apparent crossovers (COs) was assessed by visual interrogation of the colour coded  JoinMap genotyping data output. Apparent COs, based on the number of observed exchanges for each genotype, were recorded for all progeny in each linkage group across the six parental maps. Individuals with no apparent COs per linkage group and proportion of distal COs (within the end 10% of linkage group length) were also recorded.34. Multiple linkage maps can be included in a single run and scaffolds are ordered and orientated to generate sequences that are concordant with the input linkage maps.ALLMAPS is a method that configures sequence scaffolds to maximise the collinearity of markers located on physical and genetic linkage mapsM. integrifolia ‘741’ v2 genome assembly  using ALLMAPS software34. The software required two datasets as input: (1) the genetic position in centimorgans (cM) of markers on all nine maps, and (2) the physical location of markers used in the genetic map on the v2 genome assembly, obtained via marker sequence alignment to the scaffolds. These datasets were tabulated and saved as a comma-separated value text files for use by ALLMAPS. The ALLMAPS output generated for each map provided ordered and anchored scaffolds for each linkage group and identified disagreements in marker order between linkage maps.To create the chromosome-scale physical genome assembly of macadamia, genetic linkage map information was used to order and orient the 4,098 scaffolds from the merge function from the linkage information of the ‘741’ self map and the six parental maps. This integrated the scaffold order based on marker order from the input maps, and increased the number of markers available to order and anchor genome scaffolds. Four different weighting schemes were tested. The optimal weighting regime was selected based on map confidence, congruence and number of anchored and orientated scaffolds. Chromosomal scale sequence was constructed in ALLMAPS with adjacent scaffolds joined with gaps of 100 Ns. Maps were merged with the following weights applied: weight 3 for the ‘741’ self-map, weight 2 for parental ‘741’ maps and weight 1 for the ‘A4’ and ‘A268’ hybrid cultivar parental maps. Linkage groups were subsequently ordered into pseudo-chromosomes based on congruence between genetic and physical maps, relative size in megabases and consistency of marker distribution across maps.A consensus physical map was produced in ALLMAPS, using the −6. The genetic position of markers on both the integrated ‘741’ map and the integrated ‘A268’ map were then plotted against the physical location in base pairs (bp) of the markers along the same pseudo-chromosome.In order to determine the relationship between genetic and physical location of the markers from the genetic linkage maps, marker sequences were aligned this time against the pseudo-chromosomes of the consensus map generated by ALLMAPS, with marker position determined at alignment thresholds of ≥80% query coverage, >90% sequence identity, bit score ≥40 and e-value <1e7. The proportion of progeny not assigned to the expected cross ranged from 5.6% to 43.3%  and were excluded from further analysis. In addition, between 3–25% of markers had missing data and a further 7.5–24% were removed based on PIC, reproducibility or call rate. After quality filtering to remove low quality markers 901 (‘741’ self), 3,805 (‘741’x’A4’), 3,533 (‘741’x ‘A268’), and 2,650 (‘A268’ x ‘741’) informative markers were identified for the respective populations Table .The ‘741’ self-map was constructed from the largest population (n = 116) using heterozygous SNP markers. The biparental populations allowed independent investigation of male and female meioses. Similar proportions (38–40%) of pre-filtered markers were polymorphic in each of the populations, with similar SNP heterozygosity within the parents of 0.18 (‘741’ and ‘A268’) and 0.16 (‘A4’). After quality filtering, between 2–4% of available markers in the biparental populations segregated with a co-dominant F2 ratio of 1:2:1, 16–18% segregated with a backcross ratio of 1:1 and 80% segregated with a dominant 3:1 ratio. Overall, 20% of the filtered markers available for mapping were SNP and 80% were PAV Table . ApproxiMacadamia3. The ‘741’ self-map was constructed exclusively with codominant SNP markers, whilst the parental maps incorporated both SNP and PAV markers.Access to segregation data from different types of populations provided an opportunity to develop a range of maps, capturing recombination behaviour in different contexts. In total, nine genetic linkage maps were constructed, including one self, three maternal, three paternal, and integrated maps for each of the cultivars ‘741 and ‘A268’  was similar to each of the ‘741’ maternal maps, it included a total of 1,445 markers, of which 1,277 (88%) represented unique map positions. This resulted in a higher marker density (1.5 markers per cM) than the parental ‘741’ maps  and the ’741’ self map (0.66). Integrating both parental ‘A268’ maps reduced the map length  by 28%, and increased the marker density to 1.93 markers per cM in comparison to 0.33 and 0.42 markers/cM for the parental ‘A268’ maps (Table Congruence of marker locus order between maps is an indication of map reliability and accuracy of order. Congruence was determined using Spearman’s Rank correlation (ρ) Table  and was For the hybrid maps congruence was highest  between the ‘A4’ paternal map (Map 5) and the ‘A268’ paternal map (Map 6). However, for LG12 a correlation could not be established due to a lack of common markers. Average congruence was higher  between Map 7 and Map 6 than between Map 5 and Map 7 . Where congruence was low it reflected the smaller number of tightly linked markers on one of the maps Fig. .The ‘741’ self map (Map 1) was particularly valuable for detecting variation in SD, as it was constructed exclusively using co-dominant SNP markers. Regions of SD were identified using the Chi-squared test to detect departures from expected Mendelian segregation Fig. . In totaBased on evidence from multiple maps, it appeared that there was considerable variation in the ability of SNP markers to detect SD in different maps. The proportion of such markers varied from 1–1.5% in the ‘741’ parental maps, and increased in the ‘A268’ male (3.3%), ‘A4’ male (4.3%) and ‘A268’ female (8.8%) maps.Apparent crossover (CO) frequency was calculated by counting the predicted genetic COs for each linkage group in each parental map Fig. . Across In each population, counts of one or zero COs were recorded amongst the 14 linkage groups for between 50% and 84% of progeny Table . CrossovThe highest apparent CO frequency detected was in the ‘A268’ x ‘741’ population. Crossover counts ≥2 per linkage group were recorded for 31% of progeny contributing information to the ‘741’ male map (Map 4) and 50% for the ‘A268’ female map (Map 7). Crossover counts >4 were recorded for over 20% of progeny for five linkage groups in Map 7 Fig. . In addiBased on BLASTn analysis, a total of 4,266 marker sequences mapped to 1,667 different scaffolds, with most mapping to a single unique location within a unique genome scaffold . A further 12% (512 markers) mapped to two, 3% (128 markers) to three scaffolds and 2% mapped to more than three scaffolds. Two anomalous markers, mapped to 140 and 92 scaffolds respectively Fig. , suggestM. integrifolia ‘741’ v2 genome scaffolds. The consensus map included 4,184 markers  attributed to individual chromosomes by anchoring the marker sequences to the The ‘741’ self map alone anchored 33.3% of the genome assembly. Although constructed entirely with heterozygous SNP markers from the single cultivar, this map anchored 550 scaffolds, of which 246 were N50 scaffolds. Of these, 176 scaffolds included ≥2 markers Table .Successively combining multiple male and female maps from the biparental populations led to a progressive increase in the proportion of the genome assembly that was anchored. The ‘741’ paternal map anchored 29% of the genome, including 213 N50 scaffolds and 121 scaffolds anchored with ≥2 markers. Maternal maps from the ‘741’ x ‘A4’ and ‘741’ x ‘A268’ crosses anchored 39.9% and 32.8% of the assembly with 687 and 543 scaffolds respectively.M. integrifolia ‘741’ maps, parental maps derived from the Macadamia interspecific hybrid cultivars ‘A4’ and ‘A268’ included relatively more markers and anchored and oriented a greater proportion of the genome assembly  for each of 14 linkage groups and  for ten linkage groups Table .Table 2SThe weakest average congruence was observed for LG12 (ρ = 0.78), and appeared due largely to incongruence of the hybrid ‘A268’ maps  and with Map 3 (ρ = 0.78). The lowest congruence with the consensus map was LG02 of paternal ‘741’ map (ρ = 0.20). The maternal ‘A268’ map had the weakest mean correlation overall with the consensus map (ρ = 0.82) primarily due to low correlations at LG03, LG07 and LG12 Table .ALLMAPS was also used to anchor the ‘741’ v2 assembly by using both of the integrated cultivar maps from ‘741’ and ‘A268’. The integrated ‘741’ map anchored 42.8%  and the integrated ‘A268’ map anchored 51.7%  Table .M. integrifolia cultivar ‘741’  variation and informative markers available, making it difficult to produce high density genetic linkage maps36. While larger population sizes are expected to increase the number of detectable recombination events, the use of multiple populations with differing parentage can increase the numbers of markers detected and map density13.In order to generate accurate information to meet the requirements for marker-assisted trait breeding and genome anchoring, contemporary linkage maps require high marker density to increase map resolution and statistical support for marker orderArabidopsis37 and rice39, as well as outcrossing crops including apple41, strawberry42 and peach13. This includes Multi-parent Advanced Generation Intercross (MAGIC) populations which have been developed for a number of inbreeding crops such as tomato43, wheat44 and sorghum45 in order to increase genetic diversity and sample additional recombination events and subsequently improve map resolution.The value of including multiple populations into mapping projects to capture allelic diversity and increase representation of recombination has been recognised and demonstrated both for inbreeding species such as The relatively small population sizes used in this study (n = 35 to 116) limited the number of COs detected for each population. However, the inclusion of three biparental populations with different sets of informative markers increased the overall allelic diversity in the dataset thereby increasing marker density and coverage of the genome.48. Combining information from all six parental maps and the ‘741’ self map generated sufficiently high level of congruence  to allow the generation of integrated maps. The high frequency (>80%) of common markers between parental maps allowed map integration increasing marker density by 63% and 78% in the ‘741’ and ‘A268’ integrated maps compared to individual maps. This is consistent with previous studies that have shown that integrated maps generated from multiple populations can provide higher marker density and therefore greater genome coverage compared to individual maps49.Strong congruence between component maps has led to successful map integration in other studies51, which has been detected along chromosomes in many plant species53. Variation in the distribution of CO along chromosomes has been found to arise due to a range of factors, including the distribution of transposable elements, DNA methylation and histone marks, heterozygosity, domestication, environment, age, sex and pathogen infection58.Discrepancies between linkage maps, including length and crossover frequency, may reflect variation in recombination rates61. Cultivar ‘741’ is relatively self-fertile and paternity testing of the progeny used in this study identified self-fertilised progeny (3.3–5.6%) from controlled crosses where ‘741’ was the female parent but not where ‘A268’ was the female parent  and 0.16 (‘A4’) were observed in the parental genotypes. Although this was comparable to previous reports also based on codominant DArT data for macadamia breeding populations30, it is significantly lower than SNP-based estimates for other fruit crops including olive (0.44), apple (0.36) and peach (0.28)77. For macadamia, the relatively low level of heterozygosity detected in this study may reflect the bottleneck in recent domestication78. Based on data from randomly amplified RAMiFi markers, heterozygosity was reported as being lower in the Hawaiian compared to hybrid macadamia cultivars27. Interestingly, our results based on 5,793 (‘741’), 4,727 (‘A4’) and 4,867 (‘A268’) SNP markers, excluding those with missing data, indicated little difference in heterozygosity between the Hawaiian ‘741’ and Australian ‘A268’ and ‘A4’ hybrid cultivars.The distribution of gametic COs is also known to be affected by variation in heterozygosity, with localised recombination frequency being dependent on factors including chromatin structure and repair mechanismsM. integrifolia ‘741’ genotype were relatively consistent. For instance, the proportion of distorted markers and CO frequencies were consistent in the three parental ‘741’ maps. Likewise, the ‘741’ maps are consistently shorter than the parental maps from the hybrid species ‘A268’ and ‘A4’ irrespective of whether the map was derived from maternal or paternal segregation.The mapping populations used in this study included three different parental cultivars , with populations established from different crosses of these cultivars. The maps generated provide a range of different map lengths and CO frequencies, depending on the cross. In general, the maps generated from the 79. Likewise, differences in recombination rate attributed to different Eucalytptus parents has been found to be statistically significant80.In contrast, greater variation in map length, CO frequency and marker distortion was detected in parental maps from the hybrid cultivars. Map length was relatively longer for maternal  and paternal  ‘A268’ maps. Similarly, the male ‘A4’ map was longer than the corresponding ‘741’ map derived from the same cross. This may suggest that differences in the genetic background may be contributing to the variations between the maps. In grape, variations in recombination rate were detected based on crosses using a range of parents, interspecific crosses and a F1 population generated from two half-sib parents81.Both of the parental maps from the ‘A268’ x ‘741’ cross had a relatively high frequency of at least two apparent COs per linkage group compared with the other crosses. We also observed that maps generated from crosses involving hybrid genotypes (’A268’ and’A4’) had a higher level of marker clustering, with fewer unique marker positions , selection and even chromosome loss or rearrangements91. Markers displaying SD can be useful in QTL mapping92 as they are often linked to candidate genes for specific traits. For instance, it was found that regions of SD in sweet cherry, where 8% of markers showed distortion, are linked with hotspots containing fruit and bloom time traits, as well as a self-incompatibility modifier locus93. In cotton, several important agronomic traits have been recorded on chromosome 18 where 34% of markers showed distortion90.Markers showing distortion have been recorded in a number of crops94. Peace, et al.18 identified SD in four regions compared to seven across six linkage groups in the current study. They also reported a higher proportion of distorted markers in the hybrid cultivar ‘A16’ compared to M. integrifolia cultivar ‘246’. Likewise, in our study a higher proportion of distorted markers were detected when ‘A268’ was the female parent. The relatively higher levels of distorted markers in the ‘741’ self and ‘A268’ maternal maps may indicate selection against deleterious loci. For regions within some pseudo-chromosomes it appears that there may be evidence for gamete-specific skews in SD.Within the ’741’ self map we found that many of the markers displaying SD clustered into seven distinct regions Fig. . The pro19.We have generated the first sequence-based linkage maps for macadamia. Compared with the individual maps, the combination of genetic maps generated from different mapping populations improved the proportion of the genome sequence assembly anchored Table . The hig34 and has been used for anchoring genome assemblies using linkage maps for a range of crops including capsicum, cucumber, foxtail millet and cocao97. The genetic linkage maps developed in this study anchored 1,465 scaffolds covering 69.7% of the macadamia genome assembly. This facilitated the construction of 14 chromosome-scale sequence assemblies with a total length of 519.3 Mbp and anchoring 85% of N50 scaffolds. Scaffold orientation requires at least two markers per scaffold. Of the 1,465 scaffolds anchored in the consensus physical map 58% contained ≥2 markers, including all scaffolds over 1 Mbp in length.The ALLMAPS scaffold ordering method maximises collinearity to generate assemblies that are consistent with multiple mapsArabidopsis98, banana99 and cotton100.As expected, congruence of marker order was generally high between each of the self and parental maps and the consensus physical map. However, a relatively lower congruence 0.89, 0.82) between the consensus and maps generated from the ‘A268’ x ‘741’ population may indicate local structural variations in the ‘A268’ genome  where a section of the ‘A268’ map forms a close to perpendicular line from the genome diagonal indicating a possible terminal inversion on Chr06  and the hybrids: ‘A268’, M integrifolia (50%) x tetraphylla (50%), and ‘A4’, with parents ‘Own Choice’, M. integrifolia (100%) x ‘Renown’, M. integrifolia (50%) x M. tetraphylla (50%).SSR-based estimates of genetic distance within the macadamia species-complex indicate historical separation between the The strategy of amalgamating information from multiple populations and linkage maps was successful in increasing marker density and detecting variation in CO frequency between parental genotypes. Compared with a single-species biparental population, the use of four populations involving a common parent and two additional parents of diverse origin both increased the number of recombination events and informative markers. Generating integrated cultivar-specific maps also enabled us to resolve inconsistencies in marker order and reduce gaps in individual maps. Combining genetic maps to facilitate generation of a consensus physical map was successful and improved the proportion of the assembly anchored in comparison to individual maps.In conclusion, we demonstrate the value of establishing a series of genetic linkage maps and reconstructed genome as a platform for future genetic and genomic studies in the perennial outcrossing nut tree macadamia. The genetic linkage maps generated in this study were successfully utilised for anchoring and orientation of genome scaffolds and construction of the first pseudochromosome-scale assembly for macadamia. This represents a significant increase in our understanding of the genetic landscape and genome for this nut crop. The set of maps, large number of sequence-based markers and the reconstructed genome provide a toolkit to underpin future breeding that should help to extend the macadamia industry as well as provide resources for the long term conservation of natural populations in eastern Australia of this unique Proteaceous genus.Supplementary Information.