This work explores the clinical significance of Delphian lymph nodes (DLN) in thyroid papillary carcinoma (PTC). At the same time, a nomogram is constructed based on clinical, pathological, and ultrasonic (US) features to evaluate the possibility of DLN metastasis (DLNM) in PTC patients. This is the first study to predict DLNM using US characteristics.A total of 485 patients, surgically diagnosed with PTC between February 2017 and June 2021, all of whom underwent thyroidectomy, were included in the study. Using the clinical, pathological, and US information of patients, the related factors of DLNM were retrospectively analyzed. The risk factors associated with DLNM were identified through univariate and multivariate analyses. According to clinical + pathology, clinical + US, and clinical + US + pathology, the predictive nomogram for DLNM was established and validated.p = 0.039). However, there was no significant difference between clinical + pathology + US diagnostic model and clinical + US diagnostic model .Of the 485 patients with DLN, 98 (20.2%) exhibited DLNM. The DLNM positive group had higher positive rates of central lymph node metastasis (CLNM), lateral lymph node metastasis (LLNM), and T3b–T4b thyroid tumors than the negative rates. The number of CLNM and LLNM lymph nodes in the DLNM+ group was higher as compared to that in the DLNM- group. Multivariate analysis demonstrated that the common independent risk factors of the three prediction models were male, bilaterality, and located in the isthmus. Age ≥45 years, located in the lower pole, and nodural goiter were protective factors. In addition, the independent risk factors were classified as follows: (I) P-extrathyroidal extension (ETE) and CLNM based on clinical + pathological characteristics; (II) US-ETE and US-CLNM based on clinical + US characteristics; and (III) US-ETE and CLNM based on clinical +US + pathological features. Better diagnostic efficacy was reported with clinical + pathology + US diagnostic model than that of clinical + pathology diagnostic model (AUC 0.872 vs. 0.821, This study found that DLNM may be a sign that PTC is more invasive and has extensive lymph node metastasis. By exploring the clinical, pathology, and US characteristics of PTC progression to DLNM, three prediction nomograms, established according to different combinations of features, can be used in different situations to evaluate the transfer risk of DLN. Papillary thyroid carcinoma (PTC) is the most common histological type of thyroid malignancy, prone to early lymph node metastasis. Although it has an ideal prognosis, lymph node metastasis increases the risk of long-term recurrence , 2. The DLN, also known as prelaryngeal lymph node or precricoid lymph node, is a group of lymph nodes located in front of the cricoid membrane between cricoid cartilage and thyroid cartilage. It mainly receives lymphatic drainage from the throat and thyroid. Studies have revealed a positive correlation of DLN metastasis (DLNM) with a poor prognosis due to extensive regional lymph node metastasis, recurrence, and death of head and neck tumors  –12. AlthSome researchers have reported clinical risk factors for DLNM in PTC, though the results vary across studies. None of the studies has included ultrasonic (US) features in preoperative analysis. US examination is the most used and successful imaging approach for thyroid nodules. US allows for vivid evaluation of the morphological characteristics of thyroid nodules and cervical lymph nodes. Therefore, the present study aims to explore the significance of DLN in PTC invasion and metastasis. Then, establish multiple nomographic prediction models based on clinical, pathological, and US characteristics to predict the risk of DLNM. The findings aims to enrich the clinicians with a preoperative understanding of the condition and guide clinical decision-making to reduce the recurrence and metastasis probability of PTC.This is a retrospective study. The treatment records of 1,279 PTC patients who underwent the first thyroidectomy in the First Affiliated Hospital of Nanchang University between February 2017 and June 2021 were reviewed. All patients underwent preoperative thyroid ultrasonography. DLN was detected in 485 (37.9%) patients during operation. The clinical, US, and pathological information of the 485 patients were collected, and the patients were divided randomly into the training cohort  and the validation cohort . The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013).Three radiologists  with 5–10 years of experience in ultrasonic diagnosis of thyroid, performed US image acquisition using Philips iu22 and Philips EPIQ7  US diagnostic instrument. The linear probe was 5–12 MHz, with the condition set to “thyroid” mode.Three radiologists explored the thyroid region of the 1,279 patients. The complete thyroid US images were loaded into the US system, and clear nodule images were obtained. The position of the nodule  was recorded, and measurements of the vertical and horizontal diameters of the nodule were taken. The maximum diameter of the nodule and the presence of suspected cervical lymph node metastasis were recorded. After review of 1,279 cases, the thyroid US image features of 485 patients with DLN were independently recorded by two radiologists (PX and AZ) with more than 8 years of experience in US diagnosis of thyroid. The two reviewers compared and discussed their results in case of dispute to reach a consensus. The US image features recorded in this study include the following: nodule size (maximum diameter of nodule), nodule location , nodule composition (solid or non-solid), nodule echo , nodule shape , nodule margin , aspect ratio , microcalcification, multifocality, bilaterality, US-extrathyroidal extension (US-ETE), US-CLNM, nodular thyroid, and Hashimoto thyroiditis (HT). Lobulated/microneedle nodules were classified as “irregularly shaped”; A/T was classified as >1 and ≤1; the presence of calcifications ≤1 mm within the nodule was classified as “microcalcifications”; the presence of multiple PTCs in the entire thyroid gland was considered “multifocality”; here, the nodule with the largest diameter was recorded first; the presence of PTCs in both thyroid lobes was classified as “bilaterality”. The diagnosis of “multifocality”, “bilaterality”, and “nodular goiter” was according to ultrasonography and pathology. According to guidelines by the Joint Commission on Ultrasound for Cancer , US-ETE Total thyroidectomy with bilaterality central node dissection (CND) was performed for PTCs present in both thyroid lobes. Besides, unilateral lobectomy with isthmus resection or total thyroidectomy with unilateral or bilaterality CND was performed for unilateral lobar PTC. Total thyroidectomy was considered for patients with unilateral PTC but specifically to those who met one or more of the following conditions: tumor size >4 cm, multiple tumors in a single lobe, Pathological-ETE (P-ETE) (referring to the invasion of the primary tumor into adjacent tissues beyond the gland), or distant metastases. Lymph node located anterior to the thyroid membrane above the thyroid isthmus and between the cricothyroid muscle was labeled as DLN. Any evidence of LLNM on preoperative examination necessitated lateral lymph node dissection (LLND).http://www.r-project.org). Continuous variables were expressed as mean ± standard deviation, whereas categorical variables were expressed as number [percent (%)]. Categorical variables were compared by the χ2 test or Fisher’s exact test. ICC (intraclass correlation coefficient) was used to evaluate inter-observer agreement. Factors independently associated with DLNM and CLNM were established via logistic regression. Three nomograms were established according to the binary logistic regression results to evaluate risk of DLNM preoperatively. The diagnostic efficacy was evaluated using the receiver operating characteristic (ROC) curves. The ROC curves were compared by the DeLong test. The calibration curves were used to determine the prediction compliance. The clinical application value of diagnostic models was evaluated by the decision curve analysis (DCA). A p < 0.05 represented a statistically significant difference.All statistical data were analyzed in SPSS version 26.0  or R  were analyzed in this study. Patients with DLNM were 98 (20.2%). Patients with CLNM were 233 (48.0%). A total of 87 patients were found with LLNM, of which 25 (5.2%) did not have CLNM. Detailed data are shown in p < 0.001). Larger PTC size was reported in the DLNM+ group (p = 0.008). Patients in the DLNM+ group were more likely to exhibit multifocality, bilaterality, P-ETE, and T3b–T4b grading . A higher incidence of nodular goiter was revealed in the DLNM- group (p < 0.001). Regarding US features, the DLNM+ group was more likely to develop PTCs characterized by irregular shape, microcalcifications, and US-ETE . Regarding LNM, the DLNM+ group exhibited a higher incidence of CLNM, US-CLNM, and LLNM than in the DLNM- group . For CLNM, US-CLNM demonstrated 34.3% sensitivity, 95.2% specificity, a positive predictive value of 87.0%, a negative predictive value of 61.1%, and 66.0% accuracy. No significant differences were observed in Hashimoto, Margin, and A/T between the two groups . Additionally, distant metastases were present in 6 patients (4 patients with DLNM and 2 patients without DLNM).The clinical, US, and pathological characteristics of the DLNM+ group vs. the DLNM- group are outlined in p < 0.001). The number of CLNMs was higher in the DLNM+ group than in the DLNM- group . Notably, 137 patients underwent LLND. The incidence of LLNM positivity was higher than LLNM negativity  in the DLNM+ group. More LLNM was reported in the DLNM+ group than in the DLNM- group . Among 485 patients, a higher probability of DLNM was reported in T3b–T4b thyroid tumors than in non-T3b–T4b thyroid tumors . DLNM+ patients had a higher incidence of CLNM, LLNM, and T3b–T4b than DLNM- patients   Table 1.p < 0.001).Consistency analysis was conducted for the original data of the two doctors. The ICC value of each US feature was as follows: 0.878 (echogenicity), 0.972 (aspect ratio), 0.893 , 0.954 (margin), 0.957 (shape), 0.922 (location), and 0.956 (US-ETE) with good repeatability , age , location , multiple (p < 0.001), bilaterality (p < 0.001), PTC size , shape , microcalcification (p = 0.002), US-ETE (p < 0.001), P-ETE (p < 0.001), no nodular goiter (p = 0.002), US-CLNM (p < 0.001), and CLNM (p < 0.001) exhibited a significant association with DLNM , clinical + US features (II), and clinical + US + pathological features (III), respectively.p < 0.001), bilaterality , P-ETE , CLNM , and Isthmus in PTC location  were potential independent risk factors for DLNM in PTC patients. Age ≥45 years , lower in PTC location , and nodular goiter  were potential independent protective factors for DLNM in patients with PTC , bilaterality , shape Irregular , US-ETE , US-CLNM , and Isthmus in PTC location  were potential independent risk factors for DLNM in PTC patients. Age ≥45 years , lower in PTC location , and nodular goiter  were potential independent protective factors for DLNM in patients with PTC , bilaterality , shape Irregular , US-ETE , CLNM , and Isthmus in PTC location  were potential independent risk factors for DLNM in PTC patients. Age ≥45 years , lower in PTC location , and nodular goiter  were potential independent protective factors for DLNM in patients with PTC , clinical + US features (II), and clinical + US + pathological features (III), respectively, and draw interactive nomograms. The study subjects were divided randomly into a training group  and a validation group . The training data were used to construct the diagnostic models. The model performance was evaluated using AUC from the validation group. Each factor corresponded to a specific score, and then the value of all the scores were added to obtain the DLNM rate.Seven risk factors were included in the nomogram Figure 3Eight risk factors were included in the nomogram Figure 3Eight risk factors were included in the nomogram Figure 3Of note, “PTC located in the lower” and “age ≥45 years “contributed least to the model in all three models.p = 0.636, clinical + US, p = 0.480, clinical + US + pathology, p = 0.993) (p = 0.039). No significant difference was reported between the clinical + US diagnostic model and the clinical + US + pathology diagnostic model (p = 0.724).The calibration plots show excellent consistency of the three prediction models between the predicted metastasis probability and the actual metastasis probability  Figure 4After verifying the accuracy of the models, DCA curves were applied to the validation cohort. As illustrated in Mounting evidence indicates that PTC is prone to early lymph node metastases and 30%–80% of patients are approximated to develop cervical lymph node metastases. Preoperative imaging has a probability of missing cervical lymph node metastases. In a previous study, cN0 patients were reported to have a 53.71% probability of having CLNM . DelphiaConsistent with previous reports, the present study revealed the following: (i) the incidence of CLNM in DLNM+ patients was approximately twice that of DLNM- patients; (ii) the incidence of LLNM in DLNM+ patients was approximately 1.5 times that of DLNM- patients; (iii) the incidence of DLNM+ in CLNM+ patients was three times that of CLNM- patients; and (iv) the incidence of DLNM+ in LLNM+ patients was twice that of LLNM- patients. Moreover, more CLNM and LLNM metastatic lymph nodes were reported in the DLNM+ group than in the DLNM- group. Regarding tumor aggressiveness, the incidence of T3b–T4b was approximately three times higher in DLNM+ patients than in DLNM- patients, and the incidence of T3b–T4b was 2.5 times higher than that of T1–T3a in DLNM+ patients. Four of the six patients with distant metastases had DLNM, whereas two did not have DLNM. In view of these findings, DLNM may predict whether PTC is more aggressive and is characterized by extensive lymph node metastasis. Huang et al. suggested that DLN could be used as a first step approach to determine the appropriate extent of lymph node dissection before thyroidectomy with routine intraoperative frozen section consultation . The finStudies found that the incidence of DLNM was 19.4%–24.5% at the time of DLN detection, which is in agreement with our finding (20.2%) , 9, 17. Compelling evidence demonstrates that the majority of anterior laryngeal lymph node drainage originates from the superior thyroid lobe, the isthmus, and the conus of the thyroid. As such, anterior laryngeal lymph node metastasis is hypothesized to have a close association with the lesion location, especially in the isthmus and upper third of the thyroid gland , 18, 20.p < 0.001). In the clinical + pathology group, P-ETE was an independent risk factor. However, in the clinical + pathology + US and clinical + US groups, where both US-ETE and P-ETE were present, only US-ETE was an independent risk factor for DLNM. Furthermore, P-ETE was about 80 points in nomogram I, while US-ETE was about 100 points in both nomograms II and III. With US, the relationship between the location of the tumor and the envelope is evident. On the other hand, it becomes challenging to determine whether the tumor invades the envelope in the imaging picture, especially when the thyroid envelope is in contact with the lesion without significant breakthrough. Nevertheless, US provides information about tumor location and the thyroid envelope, and perhaps the proximity and contact with the envelope also contribute to DLN metastasis. The 2015 American Thyroid Association (ATA) guidelines recommend US for preoperative examination of cervical lymph nodes in PTC patients  was similar to that of the model by Li et al.  , the Postgraduate Innovation Special Fund Project of Jiangxi Province (YC2021-B043), and the Interdisciplinary Innovation Fund of Natural Science, Nanchang University (9167-28220007-YB2110).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. 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Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder. Advances in diffusion magnetic resonance imaging (MRI) acquisition sequences and analytic techniques have led to growing body of evidence that abnormal white matter microstructure is a core pathophysiological feature of ADHD. This systematic review provides a qualitative assessment of research investigating microstructural organisation of white matter amongst children and adolescents with ADHD. This review included 46 studies in total, encompassing multiple diffusion MRI imaging techniques and analytic approaches, including whole-brain, region of interest and connectomic analyses. Whole-brain and region of interest analyses described atypical organisation of white matter microstructure in several white matter tracts: most notably in frontostriatal tracts, corpus callosum, superior longitudinal fasciculus, cingulum bundle, thalamic radiations, internal capsule and corona radiata. Connectomic analyses, including graph theory approaches, demonstrated global underconnectivity in connections between functionally specialised networks. Although some studies reported significant correlations between atypical white matter microstructure and ADHD symptoms or other behavioural measures there was no clear pattern of results. Interestingly however, many of the findings of disrupted white matter microstructure were in neural networks associated with key neuropsychological functions that are atypical in ADHD. Limitations to the extant research are outlined in this review and future studies in this area should carefully consider factors such as sample size, sex balance, head motion and medication status. It is oDiffusion magnetic resonance imaging is a technique that enables the assessment of the underlying architectural organisation of white matter tracts through the measurement of restricted diffusion of water molecules in tissue . In earlThere are many different methods of diffusion analysis, which can be broadly categorised as follows: whole brain, region of interest, and connectomic. Whole-brain analyses evaluate local voxel-wise differences across the whole brain. A common whole-brain analysis technique is tract-based spatial statistics, an automated analysis for evaluating diffusion metrics in major white matter tracts on a voxel-wise level across groups of subjects . Region Connectomic analyses is a technique which models the human brain as a complex network (connectome) and evaluates the topological property of this network enabling the investigation of white matter organisation at the macroscopic level . Typicalmeta-analyses of diffusion MRI research in children with ADHD reported wide-spread abnormalities in white matter microstructure. These abnormalities were in brain regions including the corpus callosum, cingulum, inferior and superior longitudinal fasciculus, inferior fronto-occipital fasciculus, uncinate fasciculus, internal capsule, cerebellum, basal ganglia and areas of the frontal, temporal, parietal and occipital lobe A systematic literature search of the EMBASE, Medline, PsychINFO, Web of Science, and the Cochrane Library databases was conducted on the 18th of June 2021. Reference lists of retrieved studies were also searched manually to screen for additional papers. The search strategy was prospectively registered to PROSPERO, where full details and breakdown of the search strategy are available After de-duplication, the title and abstract of 1538 papers were screened, and relevant studies were selected and reviewed. Inclusion criteria were: human research that investigated between-group white matter differences using diffusion-weighted MRI, in children aged 3–18, who had a formal diagnosis of ADHD according to DSM-4, DSM-4-TR, DSM-5 or ICD-10. Studies were included only if they included a typically developing comparison group aged 3–18, were published in English and in a peer-reviewed journal. After screening, 1323 records were excluded, and two authors (MC and JM) independently reviewed 215 studies that met inclusion criteria to confirm eligibility. 46 studies met inclusion criteria see .Fig. 1FlThe following information was extracted: study population characteristics , diffusion MRI modelling technique, diffusion MRI analysis technique, main findings (Table 1). Data extraction was completed independently by the two authors MC and JM, and disagreements regarding extracted data or study inclusion were resolved by a mediator (EOH/RW) see . A quali33.13.1.1There were nine studies that examined frontostriatal tracts. Seven studies reported reduced white matter microstructure in all four frontostriatal tracts   in children and adolescents with ADHD, characterised by reduced generalised fractional anisotropy . Two stu3.1.2Twelve studies investigated the corpus callosum in ADHD. Eight studies reported decreased organisation of white matter microstructure in regions of the corpus callosum in young people with ADHD which was characterised by reduced fractional anisotropy  and high3.1.3Of the sixteen studies that examined the superior longitudinal fasciculus, twelve reported reduced white matter microstructural organisation in the superior longitudinal fasciculus in children and adolescents with ADHD. These studies included reports of decreased generalised fractional anisotropy , decreas3.1.4Fourteen studies investigated white matter microstructure of the cingulum. Six studies described reduced white matter organisation in the cingulum in children with ADHD characterised by reduced generalised fractional anisotropy , fractio3.1.5Ten studies examined thalamic white matter; four of these reported a reduction in microstructural organisation of the thalamic radiation in participants with ADHD characterised by lower generalised fractional anisotropy , lower f3.1.6Of the eight studies examining the internal capsule, seven found disrupted organisation of white matter in children with ADHD. Five reported decreased fractional anisotropy in the internal capsule  and post3.1.7Eight studies investigated the corona radiata in children and adolescents with ADHD. Five of these reported disrupted organisation of corona radiata white matter. Reduced fractional anisotropy was reported in all regions of the corona radiata  , and inc3.1.8Other white matter tracts have not been as extensively studied, and for many tracts there has been mixed findings relating to white matter microstructure with some studies finding between-group differences but others failing to find a difference. Reduced organisation of white matter microstructure has been reported in the arcuate fasciculus , inferio3.2Five studies were identified that used graph theory analysis to investigate both global and regional white matter microstructure in children with ADHD. In graph theory analysis of whole brain networks, children with ADHD displayed the same small-world network organisation seen in a neurotypical population , but dec3.3Several of the diffusion MRI studies included in this systematic review investigated the relationship between white matter organisation and ADHD symptom severity.3.3.1Greater severity of ADHD symptoms has been correlated with increased fractional anisotropy in the cingulum bundle  and left3.3.2Higher inattention scores were significantly associated with reduced generalised fractional anisotropy in the left striatum-orbitofrontal cortex , right s3.3.3Higher scores on measures of hyperactivity/impulsivity have been associated with increased fractional anisotropy in the right inferior longitudinal fasciculus , corpus 3.3.4Reduced organisation of white matter microstructure in young people with ADHD has also been significantly associated with deficits in a variety of neuropsychological functions including executive function , vigilan44.1The results of this systematic review highlight widespread abnormalities of white matter microstructure in both discrete white matter tracts and neural networks in children and adolescents with ADHD. Whole-brain and region of interest approaches reported atypical organisation of white matter microstructure in several white matter tracts, with the most prominent findings in the frontostriatal tracts, corpus callosum, superior longitudinal fasciculus, cingulum bundle, thalamic radiations, internal capsule and corona radiata. Connectomic approaches suggested global underconnectivity in connections between functionally specialised networks as well as regional reductions in network efficiency in frontal, parietal, striatal, occipital, and cerebellar regions. In some white matter tracts however, increased connectivity was reported and it appears that ADHD is not simply characterised by underconnectivity within neural networks, highlighting the complexity of this neurodevelopmental disorder.From a behavioural perspective, many studies have reported significant correlations between disrupted white matter organisation and a variety of behavioural measures. However, few studies have investigated the association between the same behavioural measure and diffusion metric in the same white matter tract, and replication is therefore required. In many studies it was not clear whether correlation analyses were exploratory or whether there was correction for multiple comparisons. In addition, a number of studies did not report the direction of the association rendering the information less clinically meaningful. Consequently, there is not yet a clear consensus on the overall impact white matter pathology has on core features of ADHD or other behavioural measures.Research in ADHD has consistently reported deficits across a wide range of neurocognitive domains. Several neuropsychological theories postulate that the core deficits of ADHD are underpinned primarily by executive dysfunction , atypica4.1.1The executive dysfunction theory of ADHD holds that deficits in executive function underpin the core symptoms of ADHD . There i4.1.2Altered sensitivity to reward is considered a core element in the pathophysiology of ADHD . Behavio4.1.3The default mode network (DMN) theory of ADHD suggests that many problems associated with ADHD arise from periodic lapses in attention due to spontaneous intrusions of DMN activation . The DMN4.1.4The delay aversion theory holds that a desire to avoid delay underpins the core deficits in ADHD , delay aIn summary, this systematic review is the first review paper to synthesise evidence of atypical white matter microstructure in children and adolescents with ADHD in relation to the neuropsychological theories of ADHD . Disrupted organisation of white matter may be a neurobiological feature that could potentially provide a unifying pathophysiological account for the diverse neuropsychological theories of ADHD.4.2Key limitations include variance in sample demographics, sample size, head motion and medication status.4.2.1In relation to study populations, it is important to consider sex and age range and a recent study using normative modelling to investigate white matter in ADHD and autism spectrum disorder suggested that some of the inconsistencies in findings might be explained by confounders of age and sex . There w4.2.2Small sample sizes, typical for brain-wide association studies , may be a key element in the widespread replication failure of brain-wide association studies . It is h4.2.3Many diffusion MRI studies in children with ADHD have not controlled for head motion which may lead to false positive findings . Head mo4.2.4Another factor that may contribute to the differences in findings between studies is the impact of ADHD medication on brain structure. It has been suggested that unmedicated children with ADHD display reduced white matter volume compared to both neurotypical controls and medicated children with ADHD . A recen5This paper was a systematic review of diffusion MRI research in children and adolescents with ADHD. Our results showed that white matter microstructural organisation was disrupted in many major fibre tracts in young people with ADHD, however there is heterogeneity in the literature that may stem from a variety of methodological limitations. There is not yet a clear consensus about the impact of white matter pathology on core features of ADHD or other behavioural measures, but this review has shown that numerous studies have reported aberrant white matter in the neural networks associated with four key neuropsychological theories of ADHD. Atypical white matter microstructure appears to be a core neurobiological feature of ADHD which could provide a unifying pathophysiological explanation for major neuropsychological theories of ADHD.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Several recent studies confirmed that Attention Deficit Hyperactivity Disorder (ADHD) has a negative influence on peer relationship and quality of life in children. The aim of the current study is to investigate the association between prosocial behaviour, peer relationships and quality of life in treatment naïve ADHD samples. The samples included 79 children with ADHD  and 54 healthy control children . Measurements included: The “Mini International Neuropsychiatric Interview Kid; Strengths and Difficulties Questionnaire” and the “Inventar zur Erfassung der Lebensqualität bei Kindern und Jugendlichen”. The ADHD group showed significantly lower levels of prosocial behaviour and more problems with peer relationships than the control group. Prosocial behaviour has a weak positive correlation with the rating of the child’s quality of life by the parents, both in the ADHD group and in the control group. The rating of quality of life and peer relationship problems by the parents also showed a significant negative moderate association in both groups. The rating of quality of life by the child showed a significant negative weak relationship with peer relationships in the ADHD group, but no significant relationship was found in the control group. Children with ADHD and comorbid externalizing disorders showed more problems in peer relationships than ADHD without comorbid externalizing disorders. Based on these results, we conclude that therapy for ADHD focused on improvement of prosocial behaviour and peer relationships as well as comorbid externalizing disorders could have a favourable effect on the quality of life of these children. Prosocial behaviour does not have a generally accepted, unified definition, but researchers tend to agree to use it as an umbrella term for several behaviours, including helper, supportive, sharing, cooperative and politeness behaviour, without the expectation of a possible reward ,2. The fAttention-Deficit/Hyperactivity Disorder (ADHD) is one of most common neurodevelopmental disorders, affecting 4–6% of the primary school population ,9. Its oADHD is associated with cognitive, social and emotional impairments ,21 and nMeasuring quality of life could be important for the investigation of function impairment and therapeutic effectiveness among childhood psychiatric disorders, including ADHD ,30,31,32Peer relationships are important for the social development of children . ChildreBased on these findings, we examined if a better understanding of peer relationship and prosocial behavior can improve the quality of life of children with ADHD. Additionally, the assessment of prosocial skills and prosocial behavior in treatment naïve children with ADHD can serve as a baseline measurement for monitoring the efficacy of therapies. Although several previous studies have examined the functional impairment in children with ADHD, including such aspects as social functions, peer functioning and prosocial behaviour, the effectiveness of treatment in clinical trials is not filtered out, which is important when we want to evaluate functional impairment ,27,28. TThe aim of the current study was to investigate the levels of prosocial behaviour and peer relationship problems among samples which were carefully selected: 1. a treatment-naïve ADHD group of children who were diagnosed both by a child psychiatrist and a structured diagnostic interview, and 2. a control group of children with no previously recognized psychiatric disorders or any psychiatric disorders currently diagnosed by a structured diagnostic interview. Moreover, we wanted to investigate the relationship between prosocial behaviour, peer relationships and quality of life (both the parents’ and the child’s ratings) in both the control sample and the treatment naïve ADHD sample. Finally, our goal was to explore the differences, in terms of prosocial behaviour and peer relationships, between those in the treatment naïve ADHD sample who had comorbid externalising disorders  and those who did not have comorbid externalising disorders.Hypothesis 1.Treatment naïve children with ADHD show a lower level of prosocial behaviour and a higher level of peer relationship problems than healthy children.Hypothesis 2.A higher level of prosocial behaviour is associated with a higher level of quality of life in treatment naïve children with ADHD and healthy children based both on parental proxy- and children’s self-reports.Hypothesis 3.A higher level of peer relationship problems is associated with a lower level of quality of life in the treatment naïve children with ADHD and healthy children based both on parental proxy and children’s self-report.Hypothesis 4.A lower level of prosocial behaviour is associated with a higher level of peer relationship problems in treatment naïve children with ADHD and healthy children.Hypothesis 5.Treatment naïve children diagnosed with ADHD and comorbid externalizing disorders show a lower level of prosocial behaviour and a higher level of peer relationship problems than treatment naïve children with ADHD and without comorbid externalizing disorders.We admitted into our study a treatment-naïve ADHD group and a healthy control group of children aged 6 to 18 years. The ADHD group of children was recruited from the Vadaskert Child and Adolescent Psychiatric Hospital and Outpatient Clinic, Budapest, Hungary. We used the following inclusion criteria for the treatment-naive ADHD group: children with a diagnosis of ADHD according to a structured diagnostic interview (see below), no previous psychological and/or psychiatric treatment including both psychotherapy and pharmacotherapy in the medical history. We enrolled these children into our study after their psychiatrists diagnosed them with ADHD in the hospital/out-patient clinic, but before their treatment started.In the clinical group, the child was not included into the study if the child’s psychiatrist indicated that intellectual disability had been suspected or confirmed previously or during the current hospital examination. From the clinical group, two different groups were created: one with comorbid externalizing problems, and one without comorbid externalizing problems. Among the ADHD group with externalizing problems, besides the ADHD diagnoses, CD and/or ODD had to be present. Among the group without externalizing problems, neither of these two diagnoses were present.To create the control group, twelve schools were randomly selected from a list of public primary schools of Budapest. Furthermore, two schools from countryside were included, which were selected by researchers. Only primary schools educating children with average intelligence were included. Special schools that educate children with mental retardation have been excluded. Children with any ongoing or previous psychological or psychiatric treatment were also excluded. The absence of any psychiatric disorders was confirmed by a structured psychiatric interview (see below).χ2(1) = 0.879, p = 0.349).The treatment naïve ADHD group consisted of 79 children: 64 (81%) boys and 15 (19%) girls. The mean age of children with ADHD was 10.24 years . Among the ADHD group, 49 (62.8%) children were diagnosed with an externalizing comorbid disorder (CD and/or ODD), while 29 (37.2%) children were not diagnosed with any comorbid externalizing disorders. Gender distribution in the ADHD group without externalizing disorders: 25 (86.2%) boys, 4 (13.8%) girls; while, in the ADHD group with externalizing disorders: 38 (77.6%) boys, 11 (22.4%) girls. The gender and the group  revealed no significant relationship (p = 0.344). The gender and the group (ADHD-control) revealed a significant relationship (χ2(1) = 8.758, p = 0.003). The percentage of boys in the clinical group was 81% (64 boys); in the control group, it was 57.4% (31 boys). There was no gender and age difference in any of the variables examined (see The control group consisted of 54 children: 31 (57.4%) boys and 23 (42.6%) girls. The mean age of children in the control group was 9.66 years . There was a non-significant difference in age between the ADHD and the control group , project identification code: 26182/2011-EKU. The parents of each child and adolescent were provided written informed consent after being informed of the nature of the study. Children/adolescents participated in a diagnostic interview recorded by a psychologist and then completed questionnaires related to the study. No compensation was provided to the participants.To measure psychopathology and diagnoses both in the clinical and healthy control groups, the modified version of the Hungarian Mini International Neuropsychiatric Interview for Children and Adolescents (MINI Kid) 2.0 ,42,43,44We used the Hungarian version of the Strengths and Difficulties Questionnaire (SDQ) ,46, whicThe Quality of Life Questionnaire , or “InvAfter data recording, a 10% inspection followed by data cleaning was performed to create a valid database. Salient cases, i.e., cases where the response exceeded the minimum or maximum score of the questionnaire, have been excluded. The distribution of relative frequencies and the descriptive analysis with means and standard deviations were calculated to describe the sample characteristics and the used measurements; Cronbach’s alpha was used to measure the internal consistency of the measurements. The Shapiro–Wilk test was applied to test the normality of peer relationships problems, prosocial behaviour and quality of life (ILK). As none of the variables show normal distribution, non-parametrical tests were applied. In order to examine the differences in prosocial behaviour and peer relationship problems between the clinical group and the control group, and between the ADHD group with externalizing problems and the ADHD group without externalizing problems Mann–Whitney U tests were performed. Spearman rank correlation coefficients were calculated for evaluating monotonous associations between prosocial behaviour, peer relation problems and self- and parent-rated quality of life. Post hoc power analysis was calculated using G*Power software for detecting significant effects of the results analysed . StatistThe treatment naïve ADHD group showed a significantly lower value in prosocial behaviour than the control group (power × 0.77). The treatment naïve ADHD group’s value in peer relationship problems was significantly higher than that of the control group (power × 1.00) see .Prosocial behaviour had a weak positive relationship with the parent’s evaluation of the child’s quality of life both in the ADHD and in the control group. The parent’s rating of quality of life and peer relationship problems also showed a significant, negative moderate association in the ADHD and in the control group. Between prosocial behaviour and peer relationship problems, a negative weak association was detected in the ADHD group, and a negative moderate association in the control group. Furthermore, in the ADHD group, the child’s evaluation of quality of life showed a significant negative weak relationship with peer relationships, and there was no significant relationship with prosocial behaviour. In the control group, the child’s view of life quality did not show a significant relationship with the other items.The two treatment naïve ADHD groups—i.e., (1) children diagnosed with externalizing comorbid disorders (CD and/or ODD); and (2) children not diagnosed with externalizing comorbid disorders—showed no significant difference in prosocial behaviour (power × 0.37). The two groups showed a significant difference in peer relationship problems. The ADHD group with externalizing comorbid disorders showed higher values in peer relationship problems than the ADHD group without externalizing disorders (power × 0.81) see .To our knowledge, the current study was the first to investigate the association between prosocial behaviour and peer relationship problems and quality of life among treatment naïve children diagnosed with ADHD. Furthermore, the present study compared a carefully selected homogeneous treatment-naïve ADHD group with a control group of children with no previously recognized psychiatric disorders or any psychiatric disorders currently diagnosed by a structured diagnostic interview.Based on our results, we can establish that treatment-naïve children with ADHD show lower levels of prosocial behaviour than healthy children; moreover, they have more problems with their peers. These results are consistent with the findings of Paap et al. , which sThe present study explored the association between prosocial behaviour, peer relationship problems and parents’ and child’s evaluation of life quality in a treatment naïve ADHD group and a control group. A notable finding of our research was that a weak positive relationship exists between prosocial behaviour and the parental evaluation of quality of life both in the treatment naïve ADHD group and the control group, while the self-reported quality of life did not reveal an association with prosocial behaviour in either group. This finding suggests that higher prosocial values have a positive correlation with the child’s quality of life, as evaluated by their parents, in both groups, while, in contrast, they do not have this positive correlation with quality of life evaluated by the child itself. Previous studies have also highlighted that there may be a difference in perception of quality of life between parents and their children ,31. WhenSince most children with ADHD can be diagnosed with a comorbid psychiatric disorder ,14,15, tThe findings of the current study must be interpreted in light of certain limitations. First, our study was cross-sectional, which does not allow for any causational conclusions. Based on our results, we can, however, state that we found significant differences between healthy children and treatment naïve children with ADHD in the studied variables, which highlights the importance of learning more about prosociality and peer relationships through further research. Second, there was a difference in gender distribution between the clinical and control groups. This difference, however, reflects the general difference in gender ratio between children with ADHD, especially in clinical practice , and heaThe results of the present study indicate that peer relationship problems, prosocial behaviour, and their relationship to quality of life of children with ADHD are important areas for future research, preferably in a longitudinal design. Understanding which factors play a role in prosociality and peer relationships in children diagnosed with ADHD, could provide valuable insights in the development of ADHD symptoms, as well as their quality of life.In summary, our research points out that treatment-naïve children diagnosed with ADHD have a lower level of prosocial behaviour and have more peer relationship problems than children not diagnosed with ADHD. Focusing on prosocial behaviour in ADHD therapy could have a favourable effect both on peer relationship problems and on quality of life, since prosocial behaviour has a positive relationship with quality of life, while peer relationship problems have a negative relationship with quality of life. Moreover, based on our study, therapy focusing on comorbid externalizing diagnoses could contribute to reducing peer relationship problems, and enhance quality of life. Social support from parents, teachers, as well as peers is important for all patients with psychiatric disorders, including ADHD. Psychoeducation can be an important factor for parents and teachers to improve acceptance and support towards children with ADHD. However, peers cannot be expected to be more accepting and tolerant to children with ADHD as a result of psycho-education, because they are themselves children as well. Therefore, children diagnosed with ADHD may need extra support to improve their relationship with their peers. Reducing social exclusion and improving peer relationships in addition to effective medication and non-medication therapies can help to protect children diagnosed with ADHD from future loneliness or deviations.


The lack of differences was confirmed by a Cox regression demonstrating that the ADHD diagnosis according to DIVA, gender, education, civil status, presence of psychiatric comorbidity, and psychiatric and ADHD familiarity; severity of symptomatological scales as evaluated by WHODAS, BPRS, BARRAT, DERS, HSRS, and ASRS did not influence treatment drop-out . Our A-ADHD-SUD patients have the same treatment retention rate as A-ADHD patients without SUD, so it seems that substance use comorbidity does not influence this clinical parameter.Attention-Deficit/Hyperactivity Disorder (ADHD) is the most widespread neurodevelopmental disorder, and it still persists into adulthood in 2–6% of the population. Psychiatric comorbidities are very common in adult ADHD (A-ADHD) patients; in particular, Substance Use Disorder (SUD) is found in 40% of these patients. Co-occurrence of ADHD and SUD is described as detrimental to clinical outcome by many authors, while only a few studies describe good clinical results in A-ADHD-SUD patients when they were treated for ADHD, both for the efficacy and the compliance of patients. In this study we tested to determine whether SUD can influence the treatment outcome of A-ADHD patients by correlating lifetime, past and current substance use in A-ADHD patients with their outcome (retention rate) during a 5-year follow-up of patients treated with stimulant and non-stimulant medications, using Kaplan–Meier survival analysis with overall and pairwise comparison. The association between demographic, symptomatological and clinical aspects with retention in treatment, adjusting for potential confounding factors, was summarized using Cox regression. After 5 years of observation, the cumulative treatment retention was 49.0%, 64.3% and 41.8% for A-ADHD patients without lifetime SUD (NSUD/A-ADHD), A-ADHD with past SUD (PSUD/A-ADHD) and A-ADHD with current SUD (CSUD/A-ADHD), respectively. Overall comparisons were not significant  Test = 1.48; df = 2;  Attention-Deficit/Hyperactivity Disorder (ADHD) is the most common neurodevelopmental disease, and about 2–6% of the adult population fulfill the criteria for the disorder . HyperacOn the other hand, ADHD in individuals with SUD complicates the diagnosis and treatment . IdeallyAs the whole question is still controversial, the aim of the present study is to better understand if the co-occurrence of SUD in a population of A-ADHD patients can influence treatment retention and/or rate of dropout from medication treatment in an outpatient psychiatric setting that has extensive experience in the treatment of Dual Disorder patients.Most of our SUD/A-ADHD patients were recruited at the Second Psychiatric Unit of Santa Chiara University Hospital, Pisa, which includes a specialized Dual Disorder Unit where some patients have only SUDs.The setting of the SUD treatment featured the following characteristics: outpatient treatment; easy access to therapy; delivering various different types of intervention for addictive disorders and related problems ; participation of patients in the determination of medication dose and knowledge of the dose dispensed; urine specimens collected on a weekly basis, and analyzed for morphine and cocaine (availability of 1–3 results per month).Research information on patients included in the present study came from the A-ADHD Clinic (A-ADHD outpatient clinic) study, a cohort study including information on patients admitted to outpatient treatment for A-ADHD at the Second Psychiatric Unit of Santa Chiara University Hospital, University of Pisa, and at the Department of Neurology and Psychiatry of the Sapienza University of Rome, Italy. For the purposes of the present study, data collected at the time of enrolment into the study (baseline) and at the last available evaluation, including the status of retention in treatment, were extracted from the A-ADHD-Clinic dataset . Once exAll the subjects examined signed an informed consensus document that allowed them to participate in the A-ADHD-Clinic study. Both the consent form and the experimental procedures were approved by the ethics committee of the University of Pisa , in accordance with internationally accepted criteria for ethical research. Aims, data gathered, and the future perspectives of this study are summarized in The sample consisted of 118 A-ADHD outpatients according to the DSM-5 criteria; 84 (71.2%) patients were males and 34 (28.8%) females. At the time of recruitment, the average age of the sample was 27.19 ± 10.2 years . The duration of the education received was 13.55 ± 3.2 years; 106 (89.8%) patients were single.Sixty-seven (56.8%) patients have been treated with methylphenidate (MPH) , while 51 (43.2) have taken non-stimulant medications (Atomoxetine (ATM) or Bupropion). Atomoxetine was used at mean dose of 40 mg/die . Bupropion was used in 5 (4.2%) subjects at dosages of 150 mg/die. At the baseline, treatment was decided based on the clinical status of the patients. Tendentially to the more impulsive ones and with SUD MPH was preferred having a greater effect on the ventral striatum (more effective on craving). Ninety-five (80.5%) patients showed a psychiatric comorbidity, 86 (72.9%) first degree psychiatric familiarity, 19 (16.1%) first degree SUD familiarity, and 45 (38.1%) first degree ADHD familiarity.Forty-nine (41.5%) had never had a comorbid SUD (NSUD/A-ADHD); 14 (11.9%) had only had that condition in the past (PSUD/A-ADHD), and 55 (46.6%) had it at treatment entry (CSUD/A-ADHD). So current/past comorbid SUD was reported for 69 (58.5%) A-ADHD patients.The diagnosis of ADHD was made according to DSM-5 criteria and confirmed using the Italian version of the semi-structured, clinician-administered Diagnostic Interview for ADHD in adults (DIVA 2.0) . WheneveThe clinical evaluation was based on the following scales:Adult ADHD Self-Report Scale (ASRS) : this 18Barratt Impulsiveness Scale (BIS-11) : this isBrief Psychiatric Rating Scale (BPRS) ,28: thisDifficulties in Emotion Regulation Scale (DERS) : this isHypomania Check List-32 (HCL-32) .: this sWHO Disability Assessment Schedule (WHODAS 2.0) : this 36Patients were divided into two groups for the first analysis, based on SUD co-occurrence and no SUD co-occurrence. Comparison of retention in treatment was performed using the Kaplan–Meier survival analysis and the Wilcoxon Rank-Sum  Test for comparison between the survival curves. For the purpose of this analysis, the term ‘censored observations’ (positive outcome) refers to patients who were still in treatment at the observation point, or were leaving treatment for reasons unrelated to the treatment itself . We considered it to be a negative outcome  when a patient dropped out of the treatment not presenting to the scheduled follow-up. For positive outcome the following characteristics have been added: “if symptoms are present, they are transient and expectable reactions to psychosocial stressors : no more than slight impairment in social, occupational, or school functioning . In the “negative” outcome these criteria were not met. We compared survival rates for study participants according to the presence of a comorbid SUD. The association between SUD comorbidity and retention in treatment was summarized using Cox regression. In our model we included sociodemographic, clinical, and symptomatological variables that may act as confounding factors.During the first year of follow-up 39 out of 118 patients dropped out of the treatment, so that cumulative survival was 0.67. After a further 20 subjects had dropped out during the second year, cumulative retention was 0.46. Three patients left the program during the third year of follow-up, leaving cumulative survival at 0.41. Notably, none of the patients who reached the third year of follow-up dropped out at any time in the fourth or fifth year.Patients still in treatment at the end of the 5-year follow-up were 24 out of 49 (49.0%), 9 out of 14 (64.3%), and 23 out of 55 (41.8%) for NSUD/A-ADHD, PSUD/A-ADHD, and CSUD/A-ADHD, respectively . No statKaplan–Meier survival analysis again showed the lack of statistically significant differences in dropout rates during the 5 years of observation. Trends over time for patients with ADHD with and without SUD are shown in 2 22.30; df = 20 p = 0.324).Following the addition of demographic, symptomatological and clinical aspects as potential confounding factors in a Cox model of proportional hazards, once again no association was found between the presence of a SUD and the retention in treatment of our patients  , and compulsive behavior in facing dilemmas on the use of substances ,37,38,39What is more, various studies highlight the fact that the chronic use of psychoactive substances reduces the therapeutic efficacy of drugs commonly used for ADHD treatment and the length of the treatment itself ,42. One Generally speaking, the efficacy of ADHD treatment is negatively influenced by the additional burden of psychiatric comorbidity ,46,47. HFirst, this study has several limitations. First of all, the sample size was not big enough to generalize our findings. All the patients evaluated were never diagnosed or treated for ADHD before adulthood and presented with a severe clinical picture. To confirm our data a wider sample should be enrolled. The second limitation is the self-assessment of several features, such as impulsivity, emotional dysregulation, and overall functioning, which may lead to reporting biases.The strength is that our sample included patients treated in a dual disorder unit, very different from locations in which psychiatric units and addiction units work independently, resulting in poor communication between the two. The collaboration should be encouraged and the treatment of both aspects of patient psychopathology should be addressed also in non-dual disorder settings.In our Dual Disorder Unit, A-ADHD-SUD patients have the same treatment retention rate as A-ADHD patients without SUD, so substance use comorbidity seems to exert no influence on this clinical parameter, at least when patients are treated in a dual disorder therapeutic setting.


Secale cereal L.) genotypes of different origins and winter-hardiness levels. A total of 10,244 single nucleotide polymorphism (SNP) markers were identified by genotyping by sequencing and 259 marker-trait-associations  were revealed by association mapping. The ten most significant SNPs (p < 1.49e−04) associated with WFS corresponded to nine strong candidate genes: Inducer of CBF Expression 1 (ICE1), Cold-regulated 413-Plasma Membrane Protein 1 (COR413-PM1), Ice Recrystallization Inhibition Protein 1 (IRIP1), Jasmonate-resistant 1 (JAR1), BIPP2C1-like protein phosphatase, Chloroplast Unusual Positioning Protein-1 (CHUP1), FRIGIDA-like 4 (FRL4-like) protein, Chalcone Synthase 2 (CHS2), and Phenylalanine Ammonia-lyase 8 (PAL8). Seven of the candidate genes were also significant for one or several of the sub-traits supporting the hypothesis that WFS, LTT, FLN, and PGH are genetically interlinked. The winter-hardy rye genotypes generally carried additional allele variants for the strong candidate genes, which suggested allele diversity was a major contributor to cold acclimation efficiency and consistent high WFS under varying field conditions.Winter field survival (WFS) in autumn-seeded winter cereals is a complex trait associated with low temperature tolerance (LTT), prostrate growth habit (PGH), and final leaf number (FLN). WFS and the three sub-traits were analyzed by a genome-wide association study of 96 rye ( Secale cereale L.) is a member of the Triticeae tribe within the Pooideae subfamily, which belongs to the Poaceae grass family. Similar to the Triticeae crops barley (Hordeum vulgare L.) and bread wheat (Triticum aestivum L.), rye is mainly cultivated for its grain, but can also be grown for its biomass used as forage or production of bioenergy. The main rye producing areas are located in North America, China, Russia, and Europe, where the grain is used for production of bread, alcoholic beverages, and feed1.Rye (Triticeae genomes are likely derived from a common progenitor genome which explains the high sequence synteny between the the seven chromosomes of the diploid rye R genome (2n = 2x = 14), diploid H genome of barley and the three genomes  of hexaploid wheat2. Since the wheat and rye lineages diverged, several translocations have occurred within the rye genome as revealed by transcript mapping and genome sequence assemblies4. The haplotype diversity in rye is high5 as it is an open-pollinated species, whereas current wheat and barley cultivars have lower genetic diversity due to self-pollination and extensive breeding during the last century. Although some genetic erosion has occurred in improved rye germplasm, the landraces and wild rye lines display a relatively high allelic variation that is available for mining6.The Triticeae species, as many autumn-seeded rye varieties developed for northern latitudes have the highest winter-hardiness among cereal crops7. Most of the temperate grasses are inherently chilling tolerant, but acquire frost tolerance following weeks of acclimation to low but non-freezing temperatures8. For autumn-seeded cereals, the cold acclimation process occurs prior to winter and is characterized by two major physiological processes: (a) accumulation of increased freezing tolerance and (b) fulfilment of vernalization requirement causing shift from vegetative to reproductive growth at the shoot apical meristem (SAM). Upon vernalization saturation, further development of floral organs is paused until new growth from the crown tissue is stimulated by inductive photoperiod and temperature in the spring9.Rye can serve as a good model for winter hardiness studies in 11. An early start and long acclimation period result in higher winter hardiness for winter cereals, and thus, the timing of floral transition impacts the total amount of accumulated freezing tolerance12. Cold acclimated cereals further increase their freezing tolerance when exposed to slightly below 0 °C temperatures, which stimulate a second hardening process13. Transcriptional analyses show different sets of genes are induced during the above-zero and sub-zero cold hardening processes14.Cold acclimation initiates when plants integrate and respond to environmental signals received from gradual reductions in photoperiod, daily temperature, light intensity, and red/far-red ratio in incoming light8. The morphological changes observed in cereals generally include a switch to prostrate growth habit (PGH), compact growth, strengthening of cell walls, changes in membrane structures, and increased number of leaf initials formed at SAM18. The biochemical changes involve production of various cryoprotectants, antioxidants, and antifreeze proteins to aid protection against future frost damage20. An enhanced photosynthetic performance is observed for the most winter-hardy cereals and results in higher biomass production and reduced susceptibility to photoinhibition as compared to tender genotypes21. Through the modification of photosynthesis, winter-hardy cereals can effectively increase their carbon pools in crown tissues to be retrieved during deacclimation incidents throughout winter and regrowth in the spring. Cold acclimation also causes epigenetic changes at the DNA and histone levels with effects on gene activities23. Overall, cold responses are coordinated by an extensive cross-talk between cold-induced Ca2+, reactive oxygen species, phytohormone, and light signaling pathways26, of which retrograde/anterograde stress signaling between plastids and nucleus are suggested to play a central role27.Winter-hardy plants undergo a multitude of developmental adjustments during cold acclimation to build up frost resistanceVernalization 1 (VRN1) and Frost resistance-2 (FR-2) loci on homoeologous group 5 chromosomes are the major determinants for freezing tolerance in Triticeae species28. Allelic differences at VRN1, Fr-A2 and their interactions relate to cold acclimation efficiency and duration29. In addition, VRN1 and FR-2 interactions with genes controlling plant development is required for optimal cold acclimation31. Large clusters of AP2/ERF transcription factor genes denoted C-repeat Binding Factors (CBFs) are present at FR loci, where variations in CBF expression levels, copy numbers, and haplotypes affect freezing tolerance and WFS levels in hexaploid wheat33, barley34 and rye36. Cold-induced CBFs bind to C-repeat/dehydration-responsive elements present in promoters of many cold-regulated (COR) genes and thereby induce their expression during cold stress37. Besides the CBF-COR regulon, additional pathways respond to cold stress38 as demonstrated by triple CBF mutants in Arabidopsis showing reduced frost tolerance but retained cold-response for several COR genes39.The 40. In addition, the developmental traits FLN and PGH were highly correlated with both WFS and LTT . Estimations of heritability for WFS, FLN and PGH revealed moderate to high values (h2 = 0.45 to 0.81), which allowed us in this study to perform a genome-wide association study (GWAS) with the aim to find genes influencing WFS and associated traits. GWAS is useful for identification of candidate genes for traits showing variation in a population of unrelated genotypes41. The genetic mapping technology has greatly benefitted from recently developed next generation sequencing and genotyping by sequencing (GBS) technologies, which allow identification of large numbers of single nucleotide polymorphism (SNP) markers42. Rye is well suited for association mapping as it’s out-crossing nature generates a low linkage disequilibrium (LD)5. In addition, a near full-length annotated genome (6.74 Gb of estimated 7.9 Gb) sequence is available for genome mapping4. The association study of rye genotype panel revealed several candidate genes were shared between WFS and one or more of the three associated traits; thus, suggesting the traits were genetically interconnected. The characteristics of nine strong candidate genes affecting WFS are discussed.In a previous study, we analyzed WFS in a rye population of 96 genotypes of different origins and winter hardiness levels and showed WFS was almost entirely determined by the cold acclimation processSecale cereale L.) accessions (96 genotypes) used for association mapping are listed in Table 40. The genotypes were maintained through generations by crossing five plants per pollination bag. All experimental research and field studies on plants (cultivated or wild) including collection of plant material complied with the institutional and funding agencies, national and international guidelines and legislation. The permission to collect seeds and plant material was granted under the material transfer agreements with international gene banks and Universities.Rye 43 for WFS for each genotype using the statistical analysis Multi Environment Trial Analysis with R (META-R) software version 6.04 . Based on the BLUE scores, WFS for the population was divided into five groups: very high , high , moderate , low  and very low  WFS (Table Five years (2014–2019) of field trials were conducted at the University of Saskatchewan Experimental Farm, Saskatoon, Saskatchewan, Canada  as previously reportedFS Table .40. The freezing temperature at which 50% of the plants regrew two weeks upon exposure to freezing was noted as the LT50 temperature. Negative LT50 values represented the LTT score.Freeze tests of cold acclimated plants were performed by a step wise decrease in temperature using an EPZ-4H test chamber  as described40. PGH was rated upon completion of cold-acclimation in growth chamber. The visual PGH rating was based on three types of growth habits: (1) erect, (2) intermediate, and (3) prostrate. To determine FLN, plant leaves were labeled numerically as they developed from the primary stem. The flag leaf number was recorded as FLN. BLUE scores for the two developmental traits were calculated to determine the overall FLN and PGH scores for each genotype.The two developmental traits PGH and FLN were analyzed in four separate trials with five plants per genotype in each trial as previously described® dsDNA Assay Kit . Uniform presence of high molecular weight DNA in the samples was confirmed by agarose (0.8%) gel electrophoresis. Normalized DNA preparations at 10 ng µL−1  were subjected to library preparations and GBS analysis, a service provided by Plateforme d’analyses Génomiques of the Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, Canada. Reduced-representation libraries were prepared from PstI and MspI double-digested DNA as described44 using an Ion CHEF System, Hi-Q reagents, and P1 V3 chips  for the library and chip preparations45. Sequencing analysis was done on an Ion Proton Sequencer .Leaf tissue (~ 130 mg) was collected from five two-week-old plants of each genotype and ground to a fine powder in liquid nitrogen using a mortar and pestle. DNA was extracted from the homogenized leaf material using a standard hexadecetyltrimethylammonium bromide-based method. The concentration of isolated DNA was determined using a Quant-iT™ PicoGreen46 with a four-base window setting and a minimum average quality score of 16. Barcodes, adaptors, and reads less than 36 bp were eliminated from the sequences before alignment to scaffold sequences of rye inbred line Lo7–version 23 using Bowtie 2.0 version 2.2.6 software47. Duplicates were removed using the rmdp function48 and SNP variants including insertions/deletions (InDels) were detected by Sequence Alignment/Map tools version 0.1.1948. The markers were filtered using the Variant Call Format tool version 0.1.1449, a minor allele frequency > 5%, and a minimum reading depth of 6.0. A total of 10,244 SNP markers were identified and numbered according to matching scaffold of rye inbred line Lo7–version 23. The selected markers contained no missing data. BLASTn analyses against the 2021 rye Lo7 v1 genome assembly4 were done using default settings and e-value threshold set to ≤ 1e−70. SNP marker distances and squared correlations of allele frequencies (r2) for 9547 mapped markers for the population were determined by the TASSEL version 5.2.20 software50. The generated data was displayed as linkage disequilibrium (LD) decay plots using the RStudio package version 3.5.1 software51. LD value at r2 = 0.20 was estimated from each plot’s trend line.Raw FASTQ files obtained from GBS analyses of the 96 rye genotypes were processed by Trimmomatic version 0.3652. The analysis was made using genetic information for all 10,244 SNP markers. Based on an admixed model and allele frequencies correlated, the number of subpopulations (K) ranging from 1 to 9 were tested with burn-in time set to 50,000 and Markov Chain Monte Carlo replication number to 100,000. A total of 20 runs were performed for each data set to compute the log likelihood for each simulated K value. The STRUCTURE HARVESTER version 0.6.94 software53 was used to establish the optimal number of sub-populations according to the ΔK method54.The genetic relationship among the 96 rye genotypes was analyzed using a model-based clustering method implemented by the STRUCTURE version 2.3.4 software55. The evolutionary relationships among the rye genotypes were calculated by the centered JBS method provided by the TASSEL version 5.2.20 software 56, and the result was displayed as a neighbor-joining dendrogram tree.To visualize the relative genetic distances between the rye genotypes, the SNP data was subjected to a principal component analysis (PCA) conducted using RStudio package version 3.5.1 software40. The population structure obtained from STRUCTURE version 2.3.4, kinship data, genotype, and phenotype data for the rye population were used to determine p and R2 (explained variation) for MTAs by the mixed linear model available in TASSEL version 5.2.20 software50. The minor allele frequency was set to > 5%. Significant SNP variants were initially tested based on a false discovery rate (FDR)-adjusted p value of 0.05 following a step-wise procedure57, and lowest adjusted p values (threshold p < 1.49e−04) was identified after a Bonferroni correction58. To illustrate the relationships between the allele variations and trait values for the 96 genotypes, phenotype and genotype data for the population was subjected to a principal component analysis (PCA) using RStudio package version 3.5.1 software55. The results of the analyses were displayed as bi-plots.Phenotypic data for WFS, LTT, FLN, and PGH of the rye population was determined by replicated trials in a previous studySecale cereale Lo7 version 1 pseudomolecules hosted by Graingenes (https://wheat.pw.usda.gov/blast/) and the IPK Galaxy Blast Suite  databases to identify matches to the latest assembly of rye Lo7 genome and its annotated genes4. BLAST searches were also made against the National Center for Biotechnology Information database (https://blast.ncbi.nlm.nih.gov). Gene orthologues in hexaploid wheat were identified by BLAST searches against the EnsemblPlants (http://plants.ensembl.org) wheat RefSeqv2.0 assembly constructed by the International Wheat Genome Sequencing Consortium59. The DNASTAR software package version 15.3.0  was used to analyze gene sequences. A physical map of rye was drawn using the MapChart software version 2.360. Transmembrane, signal peptide and phosphorylation site predictions in proteins were done using TMHMM, SignalP 5.0, and NetPhos 3.1 programs available at https://services.healthtech.dtu.dk/.The marker sequences carrying SNP of interest underwent BLASTN searches (default settings) against the The manuscript was reviewed, edited by all authors and approved for submission.3 produced 252,158 SNP markers, of which 10,244 SNP markers with no missing data remained upon data filtering. BLASTn analyses of the marker sequences to the 2021 rye genome assembly4 assigned a total of 9,547 markers (93.2%) to chromosome positions. The markers were relatively evenly distributed on the genome, with an average of 1364 markers per chromosome  associated with one or several of the WFS, LTT, FLN, and PGH trait values  (Table p < 0.001), of which 81 matched genes  Table . A totales Table . Of the , FLN, and PGH  located on chromosome 7R  from Triticum dicoccoides (XP_037426067). BIPP2C1 proteases are members of the Mg2+/Mn2+-dependent Ser/Thr phosphatase family subclade K in Arabidopsis, rice, and wheat64. No function has been assigned for wheat BIPP2C164, whereas clade K member OsBIPP2C1/PP45 from rice is cold-induced63, protects against abiotic and biotic stresses65, and has a negative role in abscisic acid signaling66. The T/G SNP variation for rye BIPP2C1 caused a V185 to G185 change within a glycine-rich region located outside of the catalytic domain for the phosphatase (data not shown). All 38 rye genotypes with low or very low WFS encoded only the G185 variant for the phosphatase , and erect growth habit  or only V185 variant for BIPP2C1 had overall the highest WFS, LTT and FLN values  was associated with all four traits, WFS, LTT6258 was se Table , and thiues Fig. .Figure 2SECCE7Rv1G0458910.1 located on chromosome 7R  domain, similar to those of phytosulfokine receptor tyrosine kinases, and an ice-binding domain at the C-terminal end68. During exposure to freezing, IRIPs present in the apoplastic space bind to small ice crystals and thereby inhibit further ice recrystallization and frost damage of the plasma membrane69. The proteins may also prevent bacteria from serving as ice-nucleators in the extra-cellular space70. The Xuos530120 SNP encoded a G36/R36 variation within one of two preserved cysteine loops preceding the LRR-like region, which may be proteolytically separated from the ice-binding domain in the apoplastic space as suggested by studies in Brachypodium distachyon69. Thus, the amino acid substitution for the rye IRIP was not expected to influence protein binding to ice crystals, but could affect the function of the N-terminal region  arranged in tandem with SECCE7Rv1G0458910.The high-significance marker Xuos530120 associated with WFS, LTT, and PGH  and SECCE5Rv1G0354880.1 (89.2%), respectively. The two COR genes are arranged in tandem ~ 11 Mb distal of the vernalization locus (ScVRN1) on chromosome 5R  gene (SECCE3Rv1G0209310.1) positioned on chromosome 3R  is present at corresponding site for ICE1 isoforms XI and X2 produced by Aegilops tauschii subsp. tauschii (XP_020168495 and XP_020168503), AenICE2 of Aegilops neglecta, and AeuICE2 of Aegilops umbellulata, respectively77. Among 38 rye genotypes with low or very low WFS, 33 encoded only the H283 ICE1 variant (Table 283 or both variants (L283/H283) for ICE1  on chromosome 1R  to an Aegilops tauschii subsp. tauschii, Jasmonate-resistant 1 (JAR1) mRNA isoform X3. JAR1 is a cytosolic auxin-inducible enzyme catalyzing reversible conversion of Jasmonic Acid (JA) to JA-Ile, which is the active growth regulator in JA signaling78. Both JA and methyl jasmonate (MeJA) can be converted to JA-Ile and participate in the activation of systemic defense; thus, JA is involved in several stress responses79. The more significant SNP for JAR1 (Xuos75199) caused an N11/T11 variation, of which the T11 variant was common among genotypes with low or very low WFS  variants , Oryza sativa (XP_025879725), and Triticum urartu (EMS54431.1). Interestingly, SECCE5Rv1G0358500.1 located 1,343 bp upstream and oriented in the same direction as SECCE5Rv1G0358510.1 codes for a XRN4-like protein without the FRIGIDA-like domain. As evidence is lacking for a rye transcript coding for both XRN4 and FRIGIDA-like domains, we assume the gene targeted by Xuos372616 only encoded the FRIGIDA-like protein. Amino acid alignment to FRIGIDA-like proteins characterized in Arabidopsis80 revealed the rye FRIGIDA-like protein had highest similarity  to subfamily IV represented by AtFRL4a and AtFRL4b. AtFRL4b is co-regulated with genes encoding chromatin remodeling factors and proteins involved in floral transition81 and maize FRL4 also participates in a chromatin remodeling network involving MADS box factors controlling floral development82. However, no information regarding FRL4 action among Triticeae species is available. For the rye FRL4-like protein, the Xuos372616 SNP did not alter the encoded amino acid (A235). Homozygous A/A alleles at SNP site dominated among genotypes with low or very low WFS (36 out of 38), whereas both (C/A) alleles were frequent among genotypes with very high and high WFS (21 out of 39). . Fig. . FRL4-liSECCE1Rv1G0013960.1 gene positioned on chromosome 1R  gene, for which SNP variation (A/G) was synonymous (R310/R310). CHUP1, which is localized in the chloroplast envelope, regulates polymerization and/or maintenance of chloroplast actin (cp-actin), which mediates blue light-regulated translocation of chloroplasts to specific sites on the plasma membrane depending on the intensity of incoming light83. WEB1 has a role in blue-light-induced reorganization of cp-actin filaments during the avoidance response and is associated with the speed chloroplast move84. The identification of highly significant SNPs targeting two different genes involved in chloroplast relocation suggest positioning of chloroplasts could be an important factor for WFS in rye.Two of the markers identified in the study coincided with genes controlling chloroplast movement. Marker Xuos2264 associated with WFS, FLN, and PGH was of higher significance (Table FS Table . The secFS Table  and matcSECCE6Rv1G0399250.1) located on chromosome 6R  by two markers (Xuos76228a and Xuos76228b) on chromosome 2R , whereas the reference F291 allele was primarily encoded by the most winter-hardy genotypes  gene , CBFIVa-2.2 (SECCE5Rv1G0340460.1), and CBFIIIa-6.2 (SECCE5Rv1G0340660.1), respectively , whereas SNP variation for CBFIVa-2.2 caused an H113/P113 change within encoded AP2 DNA-binding region and immediately prior to signature motif CMIII-1 , and CBFIIIa-6 (H224) was found for genotypes with low or very low WFS (data not shown). Besides CBFs at FR-A2, Xuos328096 marker associated with FLN matched SECCE4Rv1G0278790.1 encoding CBFII-5 from chromosome 4R  and PC2 (9.7 to 11.3%) in the four trait bi-plots  significant SNP markers mapped to annotated genes (Table The LD decay rate is the major factor limiting mapping resolution in GWAS and it is largely determined by the mating system (self or out-crossing), recombination rate, distribution of recombination hot spots on the genome, and population structurekb Table  or less5es Table .6, it has still been possible to select/develop very cold hardy rye varieties with winter hardiness far exceeding that of wheat and barley cultivars, which have undergone loss of diversity for genes conferring high WFS. Thus, the key genetic factors required to improve winter hardiness in tender winter cereals are present in certain rye genotypes.The PCA and STRUCTURE analyses grouped the rye population of 96 genotypes into three main subpopulations Figs. . Many ofCHUP1 and WEB1 encoding proteins involved in chloroplast movement90 were associated with WFS levels within the rye population  encoded from 7R affects PGH and possibly SAM development during cold acclimation.Unexpectedly, our study suggested IRIP sequence variation affected PGH Table , which iTriticeae members, rye Fr-R2 carries a cluster of CBF genes104, for which haplotype variation is suggested to underlie differences in freezing tolerance among rye accessions36. However, the significant associations between WFS and three different CBF genes  at FR-R2 were not among the strongest MTAs  present in promoters of CBF genes and several other cold-regulated genes74. Alternatively, the H283 ICE1 variation, which is also located at the start of the ZIP domain, could affect dimer formation76. Through heterodimers formed with various bHLH factors, both ICE1 and ICE2 have a role in the establishment and differentiation of stomata in dicot plants109 and stomata lineage formation in monocot plants110. Through similar heterodimer formations, additional developmental roles for rye ICE1 may exist at SAM as ICE1 allele variation in this study was associated with both FLN and PGH besides WFS and LTT  genome assembly of Chinese rye line Weining with a low heterozygosity rate of 0.26%118. Similar to the expansion of CBF and IRIP gene families that occurred when Pooideae lineages adapted to extreme temperatures during the Eocene–Oligocene transition119, the extra alleles for some of the cold-regulated candidate genes identified in this study may have evolved during periods of cold stress in the history of rye. Gene expansion is not unique to cereals, but has occurred throughout the evolutionary history of plants and is predominantly observed for stress-responsive genes120. Whether the additional gene copy number for cold-responsive genes identified by our study provides that extra winter-hardiness observed for some of the Canadian rye cultivars when compared to other rye genotypes needs to be explored further.Rye genotypes with high and very high WFS showed an overall higher frequency of allele variants for candidate genes than genotypes with low or very low cold hardiness Fig. . This teh2 = 0.45 to 0.81), can be used to dissect a complex trait like WFS40. With the abundance of SNP markers provided by GBS combined with a draft genome sequence for rye, the association mapping conducted on the rye population identified in total 90 annotated candidate genes (p < 0.01). The accuracy of the mapping is strengthened by finding seven of the nine strongest candidate genes have roles in plant adaptation during cold acclimation and contribute to enhanced winter hardiness in cereals and other plants. For two strong candidate genes, BIPP2C and FRL4-like, there are no obvious associations to WFS in Triticeae species. As similar genes in other monocot species are implicated in floral transition, studies of the rye BIPP2C and FRL4-like is likely to advance our knowledge of the cold tolerance process in winter cereals. Thus, future studies are planned with a diverse panel of rye genotypes with varying LTT and/or WFS to validate all the candidate genes identified in this study, many of which seem promising but were not discussed here. The long-term objective is to verify gene variants that enhance winter hardiness in rye and use this information to increase WFS in temperate winter cereals of interest for the Northern hemisphere. Identification of genetic factors contributing to WFS in winter cereals is of particular importance considering the immediate challenges of global warming increasing the demand for crops with higher resilience to variable weather patterns.The phenotypic relationships reported earlier for the rye population studied here showed that sub-traits like LTT, FLN, and PGH with high heritability estimates (Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.