Bartonella species are globally important emerging pathogens that were not known to infect animals or humans in North America prior to the human immunodeficiency virus (HIV) epidemic. Ongoing improvements in diagnostic testing modalities have allowed for the discovery of Bartonella species (spp.) DNA in blood; cerebrospinal fluid; and the skin of patients with cutaneous lesions, fatigue, myalgia, and neurological symptoms. We describe Bartonella spp. test results for participants reporting neuropsychiatric symptoms, the majority of whom reported the concurrent development of cutaneous lesions. Study participants completed a medical history, a risk factor questionnaire, and provided cutaneous lesion photographs. Bartonella spp. serology and Bartonella alpha proteobacteria enrichment blood culture/PCR were assessed. Within a 14-month period, 33 participants enrolled; 29/33 had serological and/or PCR evidence supporting Bartonella spp. infection, of whom 24 reported concurrent cutaneous lesions since neuropsychiatric symptom onset. We conclude that cutaneous lesions were common among people reporting neuropsychiatric symptoms and Bartonella spp. infection or exposure. Additional studies, using sensitive microbiological and imaging techniques, are needed to determine if, or to what extent, Bartonella spp. might contribute to cutaneous lesions and neuropsychiatric symptoms in patients.  Bartonella henselae [Fever, lymphadenopathy, and a history of a cat scratch/bite are the prototypical manifestations of Cat Scratch Disease (CSD), an acute onset illness caused by henselae ,2. A cuthenselae . Althoughenselae . Thus, aBartonella spp. has been reported in patients with cardiovascular, neurological, and rheumatological diseases [Bartonella spp. infection might result in cutaneous manifestations in patients with neuropsychiatric symptoms has not been determined. To our knowledge, linear cutaneous lesions were first clinically associated with B. henselae infection in 2002 in 10 pediatric patients with a history of abdominal pain, skin rash, mesenteric adenitis, gastritis, and duodenitis following a cat scratch or tick bite [B. henselae infection in a boy whose symptoms included linear cutaneous lesions—at the time, also referred to as “striae”—as well as headaches, memory loss, disorientation, and peripheral neuropathic pain; B. henselae deoxyribonucleic acid (DNA) was amplified and sequenced from a biopsy of one cutaneous lesion, as well as several blood and serum specimens [B. henselae blood stream infection in another boy diagnosed with Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) who had skin lesions described as “Bartonella-associated cutaneous lesions (BACL)”. In this patient, the cutaneous lesions and neuropsychiatric symptoms resolved with antimicrobial therapy [With the advent of increasingly sensitive diagnostic modalities, chronic blood stream infection with diseases ,2,4. If,ick bite . These cick bite . In 2013pecimens . In 2019 therapy . In 2020Bartonella spp. has been reported in families residing in Australia, Canada, Denmark, the Netherlands, and the United States [In retrospect, it is unfortunate that the term “striae” was initially applied to lesions reported in association with clinically suspected or microbiologically confirmed cases of Bartonellosis. In a 2018 retrospective case series of 12 adolescent boys with striae distensae, the authors concluded that horizontal striae distensae of the lower back in adolescent boys is associated with a rapid growth spurt, tall stature, and family (genetic) history of striae distensae . As onlyd States .Bartonella exposure/infection occurs in patients reporting neuropsychiatric symptoms and a spectrum of cutaneous lesions.The purpose of this investigation was to describe the largest case series to date involving microbiological testing for Bartonelloses in individuals with self-reported neuropsychiatric symptoms, the majority of whom developed concurrent cutaneous lesions. Dermatologists, neurologists, and psychiatrists should be aware that Bartonella Species in the blood of healthy and sick people”. A cross-sectional study was performed to determine the seroprevalence to six Bartonella species/genotypes. Bacteremia was concurrently assessed by means of a Bartonella alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture platform. Twenty-nine of 33 participants with self-reported neuropsychiatric symptoms and serological and/or PCR evidence supporting Bartonella spp. infection were included in the study. There were 12 males and 17 females, ranging in age from 12 to 58 years. The mean age for males and females was 23.3 years and 28.1 years, respectively. Fourteen participants were students (two veterinary students). Nine students were adolescents ; the remaining five students were 21 to 29 years old. Eight participants were veterinary workers (veterinarians or technicians). One participant each was a landscaper, teacher, and psychologist. The remaining four participants, all in their 20s, were medically disabled. Twenty-eight participants were from the United States, residing in sixteen states. One participant only lived in Australia. Only 10/24 participants reported consulting a dermatologist, whereas 18/29 and 21/29 reported consultation with a neurologist or psychiatrist, respectively. Participants reported median illness durations of 57.7 months (range four months to 13 years).Between August 2018 and October 2019, participants experiencing neurological or neuropsychiatric symptoms were enrolled after contacting the investigators requesting inclusion in a research study entitled “Detection of The frequencies of self-reported neuropsychiatric symptoms are summarized in Twenty-four of 29 participants (83%) reported cutaneous lesions during their illness. Ten participants had only diffuse skin eruptions, 2 only linear lesions, and 12 had both diffuse skin eruptions and linear lesions. Linear lesions were characteristically serpiginous pink-to-red patches and/or depressed or slightly elevated plaques . Seven pBartonella spp. indirect fluorescent assay (IFA) seroreactive and/or polymerase chain reaction (PCR)-positive ; and 7 were positive from both blood and BAPGM enrichment culture . The paroehlerae  was blooBartonella-positive ddPCR droplets varied between one and three per sample. All qPCR and ddPCR -negative controls remained negative throughout the study. Overall, 22 participants were PCR-positive, four by quantitative polymerase chain reaction (qPCR) and 21 by droplet digital polymerase chain reaction (ddPCR). Samples that were qPCR/DNA sequence-positive exhibited a Ct value  ranging from 35.8 to 41.2, indicating a very low bacteria gene load in blood or enrichment blood cultures for the four qPCR+ samples . Samples positive by ddPCR also showed a very low bacterial gene load. The number of B. henselae and one with Bartonella koehlerae (B. henselae Houston I strain (GenBank CP020742) or 110/110 bp (100%) with the B. henselae San Antonio 2 (SA2) strain (GenBank AF369529). A longer sequence could be amplified from one of the three B. henselae qPCR-positives, which were 549/549-bp (100%) homologous with B. henselae SA2 (GenBank AF369529). The B. koehlerae amplicon shared 100% sequence identity (110/110 bp) with B. koehlerae (GenBank accession AF312490). Based upon DNA sequencing, three participants were infected with oehlerae . Three on = 27, cats n = 22, pet birds n = 8, horses n = 7, pet reptiles n = 9, pet rabbits n = 11, pet rodents n = 16, cattle n = 5, goats n = 3, poultry n = 6, and swine n = 4. Pets slept in beds with 19/29 participants, and 23/29 participants reported allowing pets to lick their faces and hands. None of the animals to whom exposure was reported were tested for Bartonella spp. Reported known vector exposures included bed bugs n = 3, biting flies n = 17, fleas n = 23, lice n = 15, mosquitos n = 29, scabies mites n = 10, spiders n = 20, and ticks n = 26. None of the vectors to whom exposure was reported were tested for Bartonella spp.As reported in their medical surveys, the 29 participants included in the study described the following animal exposures: dogs Bartonella spp. exposure or infection, the majority of whom (83%) had cutaneous lesions accompanying their illness onset. Combining an enrichment blood culture with ddPCR substantially improved the detection of Bartonella spp. DNA in this study. Diagnostically, ddPCR is a powerful molecular technique that uses a water–oil emulsion technology driven by microfluidics and surfactant chemistry to massively partition samples into 15,000 to 20,000 1-ηL-sized droplets prior to performing DNA amplification [This case series describes 29 people with self-reported neuropsychiatric symptoms and evidence of fication . DNA tarB. henselae or other Bartonella spp. can induce cutaneous lesions or neuropsychiatric symptoms remains unclear. However, there is increasing case-based data to support an association between Bartonella spp. and cutaneous lesions, including urticarial papules and plaques along with erythematous, serpiginous patches, and plaques, reported as BACL in this case series. Maculopapular rash [Bartonella spp. infections and cutaneous lesions.The frequency and extent to which lar rash ,14, septlar rash , and vaslar rash ,17 have B. henselae case reports [B. henselae, despite treatment for environmental allergies/rash with prednisone for one year prior to study entry. However, in the context of medical complexity, she was examined by 11 medical specialists, including an allergist, dermatologist, and neurologist.In the context of chronicity, histopathology confirmed mild, nonspecific lymphoplasmacytic infiltrates in lesional cutaneous biopsies, consistent with a chronic antigenic immune response in two  reports ,8. As deBartonella spp. [Bartonella spp. [B. henselae was documented in an elderly man with ulcerated nodular skin lesions and in his dog with histopathologically confirmed panniculitis [B. henselae DNA were amplified and sequenced from the man’s enrichment blood culture and from the dog’s panniculitis biopsy. Ulcerative skin lesions and B. henselae infection were also documented by PCR amplification and DNA sequencing in a beagle with vasculitis and superficial erosions of the ear tip [Previously, we proposed that dogs may serve as sentinels for human exposure to lla spp. ,19. Addilla spp. . In the iculitis . Identic ear tip . These a ear tip . Bartonella spp. transmission is via arthropod vectors, including, but not limited to, fleas, lice, sandflies, and ticks [Borrelia burgdorferi infection [Rickettsia rickettsii and ehrlichiosis [Ehrlichia chaffeensis and Ehrlichia ewingii. In contrast, acrodermatitis chronica atrophica (ACA), most commonly caused by Borrelia afzelii, occurs months to years after infection [B. henselae and B. koehlerae are predominantly flea-borne pathogens, flea (and other arthropod) exposures should be a component of each patient’s dermatological and neuropsychiatric histories. In addition, when dermatologists consult with families experiencing flea infestations, Bartonella spp. testing should be considered if symptoms subsequently develop during the ensuing months or years.Based upon questionnaire responses, animal and vector exposures were reported frequently by study participants. The primary mode of nd ticks ,2,4. Othnd ticks . To facind ticks . Despitenfection , the macnfection  caused bichiosis  caused bnfection . Since BBartonella spp. has been recently reported in blood donors and other healthy individuals [Bartonella spp. DNA in blood or enrichment blood cultures from the majority (22/29) of the study participants, it is not possible to confirm causation for the neuropsychiatric symptoms or the cutaneous lesions. Controlled treatment trials are likely necessary to determine if or to what extent infections with Bartonella spp. contribute to the clinical presentations described in this case series. The resolution of skin lesions and neuropsychiatric symptoms with antimicrobial therapy, as described in the boy diagnosed with PANS [Bartonella spp. seroreactive. The reason for this discrepancy remains unclear. The detection of antibody reactivity only indicates exposure to one or more Bartonella spp.; however, seronegative infection is not unusual. Thus, serology, enrichment blood culture, and PCR should be used concurrently prior to and during the treatment of suspected Bartonelloses patients.Blood stream infection with ividuals ,29. Thusith PANS , provideith PANS ,29,30, aBartonella PCR, or immunohistochemical imaging of Bartonella organisms.The limitations of this study included participant self-selection and self-reporting, as these factors may have positively biased the findings. Participant age, duration and severity of illness, and prior treatment regimens likely influenced the microbiological serology and enrichment blood culture/PCR results. Due to the individual’s concerns over neuropsychiatric symptoms, most participants did not consult a dermatologist; nonprofessional photographs were provided by participants or parents, and skin biopsies were not obtained for histopathology, Bartonella spp. might contribute to concurrent cutaneous lesions in patients with neuropsychiatric symptoms. Mechanistically, could long-standing microvascular injury induced by this endotheliotropic bacteria result in chronic vascular inflammation, local mast cell activation, collagen injury, and the development of a spectrum of BACL [Bartonella spp. are predominantly vector-borne pathogens that can localize to the skin, dermatologists should assess vector and animal exposures in patients with unusual or unexplained cutaneous lesions and consider testing patients with BACL and neuropsychiatric symptoms for Bartonella spp. infection.Based upon the limited case reports and this case series, it is imperative to design studies that will determine if, or to what extent,  of BACL ,32? As BBartonella Species in the blood of healthy and sick people.” A cross-sectional study was performed to determine the seroprevalence to six Bartonella species/genotypes. Bacteremia was concurrently assessed by means of a Bartonella alpha-Proteobacteria growth medium (BAPGM)  enrichment blood culture platform. Participants were considered to have Bartonella spp. exposure or infection if they were Bartonella spp. seroreactive at an IFA titer of ≥1:64, qPCR or ddPCR positive, or positive by more than one testing modality.Between August 2018 and October 2019, participants experiencing neurological or neuropsychiatric symptoms were enrolled after contacting investigators requesting inclusion in a research study entitled “Detection of A standardized questionnaire, including demographic information, symptoms experienced, domestic and wild animal bites, scratches or exposures, and travel history, was completed. Exposure to, or bites by, different arthropods  was recorded. This study was carried out in accordance with all relevant guidelines applying to human study participation. Most participants were enrolled after publication of the case report involving the boy diagnosed with PANS . In the Bartonella spp. serological or molecular test results were included in the study. Participants/parents provided cutaneous lesion photographs for dermatologist review and written permission for publication. The dermatological description of cutaneous lesions was determined by the dermatologist author. Striae distensae risk factors were determined retrospectively via an email questionnaire provided to all participants reporting cutaneous lesions.Only participants with positive Bartonella spp. enrichment blood culture/PCR sensitivity [Blood was collected aseptically into ethylenediaminetetraacetic acid (EDTA) anti-coagulated and serum separator tubes for shipment to the Intracellular Pathogens Research Laboratory (IPRL), Comparative Medicine Institute, College of Veterinary Medicine, North Carolina State University (NCSU) for Bartonella testing. With one exception (two collection dates), each participant provided three blood and serum specimens collected on alternate days to increase the sitivity . ApproxiBartonella species or subspecies. Bartonella vinsonii subsp. berkhoffii, B. henselae, B. koehlerae, and B. quintana antibodies were determined in the IPRL at NCSU  using cell culture-grown bacteria as antigens and following standard IFA techniques [B. vinsonii subsp. berkhoffii genotype I , B. vinsonii subsp. berkhoffii genotype II , and B. vinsonii subsp. berkhoffii genotype III (NCSU 06CO-01 Klara); feline isolates of B. henselae SA2 strain , B. koehlerae , and B. quintana (NCU11-MO-01 Monkey origin) were passed from agar plate-grown cultures into Bartonella-permissive cell lines, i.e., the DH82 (a canine monocytoid) cell line for strains B. henselae SA2, B. quintana, B. vinsonii subsp. berkhoffii I, and B. koehlerae and Vero cells  for B. vinsonii subsp. berkhoffii II and III to obtain antigens for IFA testing. For each antigen, heavily infected cell cultures were spotted onto 30-well Teflon-coated slides , air-dried, acetone-fixed, and stored frozen. Fluorescein-conjugated goat anti-human immunoglobulin G (IgG)  was used to detect bacteria within cells using a fluorescent microscope . Serum samples diluted in a phosphate-buffered saline (PBS) solution containing normal goat serum, Tween-20, and powdered nonfat dry milk to block nonspecific antigen-binding sites were tested with two-fold dilutions out to 1:8192. To avoid confusion with possible nonspecific binding found at low dilutions, a cutoff of 1:64 was selected as a seroreactive antibody titer.As described in previous studies from our research group ,30,34, eBartonella alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture/PCR was performed as previously described. Briefly, qPCR and ddPCR amplifications were performed by targeting the 16S-23S intergenic transcribed spacer (ITS) region [) region ,34. For ) region . PositivSummary statistics were calculated for participant age and illness duration; distributions for each of these measures were examined visually for normality.In accordance with the 1964 Helsinki Declaration, written informed consent to be a participant and to be part of a publication if printed was obtained from the study participant or parent (in the case of a minor). This study was carried out in accordance with all relevant guidelines applying to human study participation. No nonhuman animals were tested. The protocol was approved by the North Carolina State University (NCSU) Sponsored Programs and Regulatory Compliance, Institutional Review Board (IRB) . Specimen handling was performed following regional and national guidelines and regulations.


Microsaccades are involuntary, small, jerk-like eye-movements with high-velocity that areobserved during fixation. Abnormal microsaccade rates and characteristics have been observed in a number of psychiatric and developmental disorders. In this study, we examinemicrosaccade differences in 43 non-clinical participants with high and low levels of ADHDlike traits, assessed with the Adult ADHD Self-Report Scale . A simpl Our ability to see depends partly on being able toalign our eyes with a visual target. Eye-movement behaviour is highly optimized to satisfy these needs; most ofthe time our eyes scan visual scenes in sequences of saccades and fixations. Saccades are rapid, ballistic eyemovements that bring the fovea to specific portions of thevisual field. Fixations, on the other hand, maintain thevisual gaze to a specific location keeping a target relatively stable with respect to the retina. Even though thedefinition of a fixation might suggest that the eyes remainstable, in reality the eyes are moving continuously. Morespecifically, when fixating on a stationary object our eyesperform tiny, seemingly erratic fixational eye movements. These eye movements are not performed voluntarily and we are generally not aware of their existence.The most prominent contribution to fixational eyemovements is generated by small (a few arc min to 1.0º),jerk-like eye-movements with high-velocity that are embedded into slower drifting movements . On averAnimal studies have found that the superior colliculus(SC), a multimodal laminar structure located in the midbrain that belongs to a distributed network of areas mediating saccadic eye movements, and shifts of attention,plays a key role in the microsaccade generation . Hafed  investigThe SC has recently garnered interest as one potentialsite of dysfunction in Attention Deficit HyperactivityDisorder ,the mosVariations in microsaccade rates across participantsare a common finding in studies examining eye movements . A studyA number of previous studies have reported gaze instability and increased eye movements in children andadults with ADHD . Only onHowever, the disadvantage of working with a clinicalADHD population is that the majority of individuals withADHD have been treated chronically with psychostimulant medication , which 43 participants  were recruited from thevolunteers’ list of the University of Sheffield. The ages ofthe participants varied from 18 to 30 . All subjects had normal or corrected-to-normalvision and were naïve as to the purpose of the experiment. Four participants were left-handed. All the participants were healthy and none were previously diagnosedwith ADHD or any other major mental illness.They were all awarded for their time with creditsneeded for the completion of their undergraduate degree.The subjects all gave their informed consent to take partin the experiment and the procedures were conducted inaccordance with the Code of Ethics of the World MedicalAssociation (Declaration of Helsinki).The Adult ADHD Self-Report Scale  was admiThe participant was seated in front of an the Eyelink1000 video-based eye tracker , in a padded chair located about 100 cm awayfrom the screen. The eyetracker was mounted on a headand-chin rest. Horizontal and vertical eye positions forboth eyes were sampled at a rate of 500 Hz  and stored foroffline analysis. A keyboard was positioned within easyreach in front of the subject. Subjects were asked tomaintain a stable posture and head position during thecourse of the experiment.Before the main task begun, calibration and validationwere performed. Calibration was manual and based on anumber of 9 (grid) points. Participants were required toproduce saccades towards 9 fixation points sequentiallyappearing at random on the screen. After calibration wasperformed, validation was performed by re-presenting thetargets and determining the magnitude of the calibrationerror. In case the validation was unsuccessful, calibrationwas repeated. The process took approximately 2 min foreach participant. Drift correction was performed beforeevery trial. During the drift correction, the participant waspresented with a blank screen and the same black markerpresented in the calibration and they were instructed tofocus on the black marker fixation point. Calibration wasperformed twice for each participant; at the beginning ofeach block.Once the calibration and validation were successfullyperformed, the main task was administered. It consistedof a simple sustained fixation task Overall, 2 blocks of 10 trials were presented to eachparticipant. There was a break between each trial; theexperiment was self-paced and the participant was askedto press the space-bar to begin each trial. Each trial waspreceded by the instructions screen. The stimuli werepresented with OpenSesame  usingthMicrosaccades were detected using the algorithm ofEngbert and Kliegl  adapted All the trials from all the sessions were collapsed andincluded in the analysis. First, the position data was transformed to velocities using a moving average of 3 datasamples (6 ms) for each eye. Second, the velocity threshold was computed and then multiplied by the relativevelocity threshold (6.0). If the average velocity exceededthe velocity threshold in at least three consequent samples, the movement was defined as monocular microsaccade. The microsaccades extracted from the algorithmshowed a strong correlation between peak velocity andamplitude ). These results areconsistent with the standard main sequence effect in theliterature . MicrosaData from three participants were excluded from theanalysis as they failed to complete all 20 trials. Anotherparticipant was excluded from the analysis due to highlevels of noise in the data  in the sample). As a result, data from 38 participants were included in the final analysis. The meanproportion of excluded data from the analysis due to theissues described above was 16.8% (SD=15.7). A minimum of 16 trials were included in the analysis for eachparticipant. No relationship was found between numberof excluded trials and ADHD traits (r(38)=.11, p=.47)Scores on the ASRS checklist varied from 23 to 58and the mean score was 35.74 (SD= 9.6). The mean scoreon the inattention subscale was 20.29 (SD= 4.8) and thehyperactivity subscale 15.45 (SD= 6.39). The two subscales were correlated, r(38)=.459, p<.01. The overallADHD score was correlated with both the inattention(r(38)=.807, p<.01) and the hyperactivity subscale(r(38)=.896, p<.01). Since the two subscales were strongly correlated, only overall ASRS scores were used in theanalysis.Microsaccades have often been shown to follow themain sequence; an approximately linear relation betweenthe amplitude of the eye movement and the peak velocity.The peak microsaccade velocities were plotted againsttheir amplitudes in our data. A significant linear relationship was observed A positive correlation was found between overallASRS scores and binocular microsaccade rate No correlation was found between ASRS scores andmean microsaccade peak velocity, r(38)=-.04, p=.821.There was also no correlation between ADHD traits andmicrosaccade amplitudes, r(38)=.213, p=.21.A way to investigate tracking noise in the detection ofmicrosaccades involves examining the relationship between saccade amplitude and peak velocity, known as themain sequence . NocorrThis study examined microsaccade rates in nonclinical participants with varying levels of ADHD-liketraits during a prolonged sustained fixation paradigm. Wefound that a higher level of ADHD-like traits as measured in the ASRS was associated with increased rates ofmicrosaccades. Participants who reported more inattention and hyperactivity traits made more microsaccades.This is the second study showing a connection betweenADHD symptoms and microsaccade rate , and extA deficit in top-down regulation could also explainthe relationship between ADHD traits and microsaccadefrequency. A number of ADHD symptoms such as distractibility and inattention have been attributed to deficitsin executive functions and the frontal lobes . In particular, regions such as the frontal eye fieldshave been shown to regulate microsaccade generation. As a reDeficits in response inhibition when fixating on a static target could also lead to an increased rate of microsaccades. Response inhibition has been identified as one ofthe core deficits of ADHD . Studies havedemonstrated that children with ADHD have difficultyinhibiting automatic responses and carrying out goaloriented behaviour .Our paradigm employed trials with fixed timing. Ithas been shown that fixed timing is a critical property andthat people with ADHD have a specific impairment in thetransient allocation of attention for anticipated and regular events . TherefoIndividual measures of eye-movement behaviour havebeen shown to be consistent across tasks . For exaADHD diagnosis is currently based on interviews andparent/guardian/teacher reports. All these measures arecharacterized by high subjectivity. The subjective natureof ADHD diagnosis hinders ADHD research and its effective treatment. Since there is no objective test forADHD, other conditions with similar symptoms often getmisdiagnosed as ADHD . SeveralHere we found a statistically significant correlationbetween the frequency of microsaccades executed duringa sustained fixation task and ADHD traits. Participantswith a higher level of ADHD-like traits, as assessed onthe ASRS, made more microsaccades while fixating on atarget. We acknowledge that the results presented hereare preliminary and their diagnostic power at the individual level remains to be further developed and tested. Asimple sustained fixation task requires minimum cognitive effort from the participants. In addition to this, eyetracking systems are becoming increasingly popular andtheir cost is decreasing. It is estimated that in the nearfuture eye tracking will be integrated in gadgets we useon a regular basis. A biomarker based on eye movementscould be a very effective way of testing for ADHD traits.Future research should focus on microsaccades.The authors declare that there is no conflict of interestregarding the publication of this paper.


Microsatellite (SSR) markers with known precise intrachromosomal locations are widely used for mappinggenes in rye and for the investigation of wheat-rye translocation lines and triticale highly demanded formapping economically important genes and QTL-analysis. One of the sources of novel SSR markers in rye aremicrosatellites transferable from the wheat genome. Broadening the list of available SSRs in rye mapped to chromosomesis still needed, since some rye chromosome maps still have just a few microsatellite loci mapped. Thegoal of the current study was to integrate wheat EST-SSRs into the existing rye genetic maps and to constructa consensus rye microsatellite map. Four rye mapping populations  weretested with CFE (EST-SSRs) primers. A total of 23 Xcfe loci were mapped on rye chromosomes: Xcfe023, -136 and-266 on chromosome 1R, Xcfe006, -067, -175 and -187 on 2R, Xcfe029 and -282 on 3R, Xcfe004, -100, -152, -224 and-260 on 4R, Xcfe037, -208 and -270 on 5R, Xcfe124, -159 and -277 on 6R, Xcfe010, -143 and -228 on 7R. With theexception of Xcfe159 and Xcfe224, all the Xcfe loci mapped were found in orthologous positions considering multipleevolutionary translocations in the rye genome relative to those of common wheat. The consensus map wasconstructed using mapping data from the four bi-parental populations. It contains a total of 123 microsatellites,12 SNPs, 118 RFLPs and 2 isozyme loci. Several linkage maps of rye carrying RFLP, AFLP, SSR, DArTand SNP markers are available to date .SSRs (microsatellites) are among the most widely usedDNA-markers in rye genetics. For example, SSR markerswere used for mapping the sy1, sy9, sy18 and sy19 asynapticgenes , thegene mo1 for supernumerary spikelets , several anthocyanin biosynthesis genes , Ddw1  and Ddw3  dwarfing genes, the powderymildew resistance locus , the Elm-R1gene related with embryo lethality in wheat-rye hybrids as wellas the hybrid dwarfness gene Hdw-R1 , aluminum tolerance loci in rye andtriticale  and several QTL for agronomic traits includinggrain yield .SSRs can be suitable for marker-assisted breeding , detection of the genetic variability in rye andtriticale  as wellas for marker-assisted identification of rye genetic materialin wheat cultivars and lines .In spite of several sets of microsatellite markers available inrye, broadening a list of SSRs mapped to rye chromosomes isstill needed, since some rye chromosome maps still have just afew microsatellite loci mapped . Thegoal of the current study was to integrate wheat EST-SSRs,expressed sequence tag SSR ) into the existing rye microsatellite map and constructthe consensus microsatellite map of rye genome.2 mapping populations ) wereused in PCR assays with CFE primers available at GrainGenesdatabase (http://wheat.pw.usda.gov). DNA was available fromprevious studies .PCR and analysis of the amplified fragments length was performedas described in L.Y. Zhang et al. (2005). Chromosomearm location of homologous sequences carrying the CFEs(http://wheat.pw.usda.gov) was performed using BLASTanalysis of the corresponding wheatESTs given at http://wheat.pw.usda.gov against wheat chromosomesurvey sequences available at https://urgi.versailles.inra.fr/blast/blast.php. Linkage maps were constructed with MAPMAKER2.0  using Kosambi function, based on genotyping data obtained inthe current study and previously . The consensus mapwas constructed using JoinMap 2.0 program .Four rye FDespite the possibility of a high‐throughput marker analysisusing SNPs , microsatellites remain convenientand low‐cost markers for mapping genes and markerassisted selection in rye and triticale. For these purposes microsatellitemarkers with known precise intrachromosomal locationare needed. The sources for mapping novel SSR loci inrye were rye EST-SSRs , or wheat genomic microsatellites . In the current study, we used wheat EST-SSRs forgenotyping rye mapping populations.The parents of the four rye mapping populations  were tested with 301 CFE primerpairs. Thirty-two pairs revealed polymorphism between theparents of one or more mapping populations: 10 betweenP87 and P105, 13 between N6 and N2, 11 between N7 andN2 and 15 between N7 and N6. The portion of polymorphicCFE markers (10.6 %) is comparable with that described forgenomic wheat SSRs GWM transferred to the same set ofmapping populations parents (9.2 %) .1.Twenty-three of the 32 markers were segregating in themapping populations, while nine pairs produced monomorphicPCR-products, that can be explained by rye heterogeneity.Twenty-three Xcfe loci were genetically mapped on ryechromosomes 1Supplementary Materials are available in the online version of the paper:http:/www.bionet.nsc.ru/vogis/download/pict-2020-24/appx5.pdfA consensus map was constructed using mapping data forthe four populations. The consensus map contains 11 microsatellite markers on chromosome1R, 23 on 2R, 10 on 3R, 15 on 4R, 29 on 5R, 17 on 6R, 18 on7R . In addition to these 123 SSR markers theconsensus map contains 12 SNPs (Xgbs…), 118 RFLP markers(other X… names), and two isozyme loci. The former ryeconsensus map constructed in 2009 contained 10 microsatellitemarkers only .Most of the microsatellites mapped in the current studyconsist of 3 bp repeats (15 loci), 5 of the mapped SSRs weredinucleotide, 2 sequences carried tetra- and 1 hexanucleotiderepeat (see the Table).Twenty-one of the 23 Xcfe loci mapped in orthologouspositions (see the Table) considering multiple evolutionarytranslocations in the rye genome relative to those of commonwheat, as described in detail by K.M. Devos et al. (1993).Two loci Xcfe159-6R and Xcfe224-4R have no orthologywith wheat Xcfe159  and Xcfe224 (5B). The portionof the Xcfe loci showing orthology between wheat and rye (91 %) is higher than that found previously for genomicSSR loci Xgwm (73 %) . This mayreflect conservatism of the coding portion of plant genome, inparticular that of the regions complementary to the primers,flanking microsatellites.Usually the markers mapped to 7RS are found in a comprehensiveregion of the chromosome 7R corresponding toancient translocation, while just a few markers are availablefor the small proximal region not involved in this translocation. The Xcfe010-7R locus mapped in the current study islocated in this region  and can be used for taggingthe part of chromosome 7RS, which is orthologous to theshort arm of chromosome 7 of Triticeae .The Xcfe loci mapped can be recommended for various applicationsin rye genetics and breeding. Some of them locatein the regions carrying known rye genes and therefore have apotential for marker-assisted selection. For example, comparisonof the consensus map  with data availablefrom previous gene mapping studies suggests Xcfe270-5R tobe close to the dwarfing gene Ddw1 ), while the Xcfe006-2R locus is mapped in the region highly comparablewith location of the asynaptic genes sy9 and sy18 on chromosome2R .Overall, the consensus map of rye contains 123 microsatellites.The list of mapped SSRs can be broaden in the futurebased on 856 SSRs recently found in rye genome shotgunsurvey sequences .The consensus map constructed in the current study containsa total of 123 microsatellites (including 23 SSRs transferredin our study from wheat to rye map), 12 SNPs, 118 RFLPs and 2 isozyme loci. Co-linearity between rye and wheat chromosomeregions carrying these microsatellite loci was shownusing 21 from 23 SSRs. These markers can be useful for bothcomparative mapping between wheat, rye and triticale as wellas for marker-assisted breeding.The authors declare no conflict of interest.Adonina I.G., Orlovskaya O.A., Tereshchenko O.Y., Koren L.V., KhotylevaL.V., Shumny V.K., Salina E.A. Development of commerciallyvaluable traits in hexaploid triticale lines with Aegilops introgressionsas dependent on the genome composition. Russ. J. Genet.2011;47:453-461. DOI 10.1134/S1022795411040028.Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basiclocal alignment search tool. J. Mol. Biol. 1990;215:403-410. DOI10.1016/S0022-2836(05)80360-2.Bauer E., Schmutzer T., Barilar I., Mascher M., Gundlach H., MartisM.M., Twardziok S.O., Hackauf B., Gordillo A., Wilde P.,Schmidt M., Korzun V., Mayer K.F.X., Schmid K., Schön C.-C.,Scholz U. Towards a whole-genome sequence for rye . Plant J. 2017;89:853-869. DOI 10.1111/tpj.13436.Bednarek P.T., Masojc P., Lewandowska R., Myskow B. Saturating ryegenetic map with amplified fragment length polymorphism (AFLP)and random amplified polymorphic DNA (RAPD) markers. J. Appl.Genet. 2003;44:21-33.Benito C., Silva-Navas J., Fontecha G., Hernández-Riquer M.V.,Eguren M., Salvador N., Gallego F.J. From the rye Alt3 and Alt4aluminum tolerance loci to orthologous genes in other cereals. PlantSoil. 2010;327:107-120. DOI 10.1007/s11104-009-0035-9.Bolibok H., Rakoczy-Trojanowska M., Hromada A., Pietrzykowski R.Efficiency of different PCR-based marker systems in assessing geneticdiversity among winter rye  inbred lines.Euphytica. 2005;146:109-116. DOI 10.1007/s10681-005-0548-0.Bolibok-Brągoszewska H., Heller-Uszyńska K., Wenzl P., Uszyński G.,Kilian A., Rakoczy-Trojanowska M. DArT markers for the ryegenome-genetic diversity and mapping. BMC Genomics. 2009;10:578. DOI 10.1186/1471-2164-10-578.Devos K.M., Atkinson M.D., Chinoy C.N., Francis H.A., Harcourt R.L.,Koebner R.M.D., Liu C.J., Masojc P., Xie D.X., Gale M.D. Chromosomalrearrangements in the rye genome relative to that of wheat.Theor. Appl. Genet. 1993;85:673-680. DOI 10.1007/BF00225004.Dobrovolskaya O., Martinek P., Voylokov A.V., Korzun V., Röder M.S.,Börner A. Microsatellite mapping of genes that determine supernumeraryspikelets in wheat (T. aestivum) and rye . Theor.Appl. Genet. 2009;119:867-874. DOI 10.1007/s00122-009-1095-1.Dolmatovich T.V., Malyshev S.V., Sosnikhina S.P., Tsvetkova N.V.,Kartel N.A., Voylokov A.V. Mapping of meiotic genes in rye : Localization of sy18 mutation with impaired homologoussynapsis using microsatellite markers. Russ. J. Genet. 2013a;49:411-416. DOI 10.1134/S1022795413040030.Dolmatovich T.V., Malyshev S.V., Sosnikhina S.P., Tsvetkova N.V.,Kartel N.A., Voylokov A.V. Mapping of meiotic genes in rye : Localization of sy19 mutation with impaired homologoussynapsis using microsatellite markers. Russ. J. Genet. 2013b;49:511-516. DOI 10.1134/S1022795413030058.Fontecha G., Silva-Navas J., Benito C., Mestres M.A., Espino F.J.,Hernández-Riquer M.V., Gallego F.J. Candidate gene identificationof an aluminum-activated organic acid transporter gene at theAlt4 locus for aluminium tolerance in rye . Theor.Appl. Genet. 2007;114:249-260. DOI 10.1007/s00122-006-0427-7.Gustafson J.P., Ma X.F., Korzun V., Snape J.W. A consensus map ofrye integrating mapping data from five mapping populations. Theor.Appl. Genet. 2009;118:793-800. DOI 10.1007/s00122-008-0939-4.Hackauf B., Haffke S., Fromme F.J., Roux S.R., Kusterer B., MusmannD., Kilian A., Miedaner T. QTL mapping and comparativegenome analysis of agronomic traits including grain yield in winterrye. Theor. Appl. Genet. 2017;130:1801-1817. DOI 10.1007/s00122-017-2926-0.Hackauf B., Wehling P. Development of microsatellite markers in rye:map construction. Plant Breed. Seed Sci. 2003;48:143-151.Khlestkina E.K., Dobrovolskaya O.B., Leonova I.N., Salina E.A. Diversificationof the duplicated F3h genes in Triticeae. J. Mol. Evol.2013;76:261-266. DOI 10.1007/s00239-013-9554-3.Khlestkina E.K., Myint Than M.H., Pestsova E.G., Röder M.S., MalyshevS.V., Korzun V., Börner A. Mapping of 99 new microsatellitederivedloci in Rye  including 39 expressed sequencingtags. Theor. Appl. Genet. 2004;109:725-732. DOI 10.1007/s00122-004-1659-z.Khlestkina E.K., Salina E.A., Matthies I., Leonova I.N., Börner A.,Röder M.S. Comparative molecular marker-based genetic mappingof flavanone 3-hydroxylase genes in wheat, rye and barley. Euphytica.2011;179:333-341. DOI 10.1007/s10681-010-0337-2.Khlestkina E.K., Tereshchenko O.Yu., Salina E.A. Anthocyanin biosynthesisgenes location and expression in wheat-rye hybrids. Mol.Genet. Genom. 2009;282:475-485. DOI 10.1007/s00438-009-0479-x.Korzun V., Malyshev S., Voylokov A.V., Börner A. A genetic map ofrye  combining RFLP, isozyme, protein, microsatelliteand gene loci. Theor. Appl. Genet. 2001;102:709-717. DOI10.1007/s001220051701.Kosambi D.D. The estimation of map distances from recombinationvalues. Ann. Eugen. 1944;12(1):172-175. DOI 10.1111/j.1469-1809.1943.tb02321.x.Lander E.S., Green P., Abrahamson J., Barlow A., Daly M.J., LincolnS.E., Newburg I. MAPMAKER: an interactive computer packagefor constructing primary genetic linkage maps of experimentaland natural populations. Genomics. 1987;1:174-181.Lapitan N.L.V., Peng J., Sharma V. A high-density map and PCR markersfor Russian wheat aphid resistance gene on chromosome 1RS/1BL.Crop Sci. 2007;47:811-818. DOI 10.2135/cropsci2006.08.0529.Li J., Zhou R., Endo T.R., Stein N. High-throughput development ofSSR marker candidates and their chromosomal assignment in rye. Plant Breed. 2018;137:561-572. DOI 10.1111/pbr.12619.Malyshev S.V., Dolmatovich T.V., Voylokov A.V., Sosnikhina S.P.,TsvetkovaN.V., Lovtsus A.V., Kartel N.A. Molecular genetic mappingof the sy1 and sy9 asynaptic genes in rye using microsatellite and isozyme markers. Russ. J. Genet. 2009;45:1444-1449. DOI 10.1134/S1022795409120060.Milczarski P., Bolibok-Brągoszewska H., Myśków B., Stojałowski S.,Heller-Uszyńska K., Góralska M., Brągoszewski P., Uszyński G.,Kilian A., Rakoczy-Trojanowska M. A high density consensus mapof rye  based on DArT markers. PLoS One. 2011;6:e28495. DOI 10.1371/journal.pone.0028495.Milczarski P., Hanek M., Tyrka M., Stojałowski S. The application ofGBS markers for extending the dense genetic map of rye  and the localization of the Rfc1 gene restoring male fertilityin plants with the C source of sterility-inducing cytoplasm. J. Appl.Genet. 2016;57:439-451. DOI 10.1007/s13353-016-0347-4.Niedziela A., Bednarek P.T., Labudda M., Mańkowski D.R., Anioł A.Genetic mapping of a 7R Al tolerance QTL in triticale . J. Appl. Genet. 2014;55:1-14. DOI 10.1007/s13353-013-0170-0.Philipp U., Wehling P., Wricke G. A linkage map of rye. Theor. Appl.Genet. 1994;88:243-248. DOI 10.1007/BF00225904.Schlegel R. Hybrid breeding boosted molecular genetics in rye. Russ.J. Genet.: Appl. Res. 2016;6(5):569-583. DOI 10.1134/S2079059716050105.Schlegel R., Korzun V. Notes on the origin of 4BL-5RL rye translocationsin common wheat (Triticum aestivum L.). Cereal Res. Commun.2008;36:373-385. DOI 10.1556/CRC.36.2008.3.2.Schneider A., Molnár-Láng M. Detection of the 1RS chromosome armin Martonvásár wheat genotypes containing 1BL.1RS or 1AL.1RStranslocations using SSR and STS markers. Acta Agron. Hung.2009;57:409-416. DOI 10.1556/AAgr.57.2009.4.3.Senft P., Wricke G. An extended genetic map of rye . Plant Breed. 1996;115:508-510. DOI 10.1111/j.1439-0523.tb00966.x.Silkova O.G., Dobrovolskaya O.B., Dubovets N.I., Silkova O.G., AdoninaI.G., Kravtsova L.A., Röder M.S., Salina E.A., Shchapova A.I.,Shumny V.K. Production of wheat-rye substitution lines and identificationof chromosome composition of karyotypes using C-banding,GISH, and SSR markers. Russ. J. Genet. 2006;42:645-653. DOI10.1134/S1022795406060093.Silkova O.G., Leonova I.N., Krasilova N.M., Dubovets N.I. Preferentialelimination of chromosome 5R of rye in the progeny of 5R5Ddimonosomics. Russ J. Genet. 2011;47:942-950. DOI 10.1134/S1022795411080151.Stam P. Construction of integrated linkage maps by means of a newcomputer package: Join Map. Plant J. 1993;5:739-744. DOI10.1046/j.1365-313x.1993.03050739.Tenhola-Roininen T., Tanhuanpää P. Tagging the dwarfing gene Ddw1in a rye population derived from doubled haploid parents. Euphytica.2010;172:303-312. DOI 10.1007/s10681-009-9982-8.Tikhenko N., Tsvetkova N., Priyatkina S., Voylokov A., Börner A. Genemutations in rye causing embryo lethality in hybrids with wheat: allelismand chromosomal localization. Biol. Plant. 2011;55(3): 448-452. DOI 10.1007/s10535-011-0109-4.Tsvetkova N.V., Tikhenko N.D., Hackauf B., Voylokov A.V. Two ryegenes responsible for abnormal development of wheat–rye hybridsare linked in the vicinity of an evolutionary translocation on chromosome6R. Plants. 2018;7:55. DOI 10.3390/plants7030055.Varshney R.K., Beier U., Khlestkina E.K., Kota R., Korzun V., GranerA., Börner A. Single nucleotide polymorphisms in rye : discovery, frequency, and applications for genome mappingand diversity studies. Theor. Appl. Genet. 2007;114:1105-1116.DOI 10.1007/s00122-007-0504-6.Vyhnánek T., Nevrtalová E., Slezáková K. Detection of the geneticvariability of triticale using wheat and Rye SSR markers. CerealRes. Commun. 2009;37:23-29. DOI 10.1556/CRC.37.2009.1.3.Wang D., Zhuang L., Sun L., Feng Y., Pei Z., Qi Z. Allocation of a powderymildew resistance locus to the chromosome arm 6RL of Secalecereale L. cv. ‘Jingzhouheimai’. Euphytica. 2010;176:157-166. DOI10.1007/s10681-010-0199-7.Xu H., Yin D., Li L., Wang Q., Li X., Yang X., Liu W., An D. Developmentand application of EST-based markers specific for chromosomearms of rye . Cytogenet. Genome Res. 2012;136:220-228. DOI 10.1159/000336478.Yang S., Zhu H., Yu J., Zhong Y.Y., Zhao L.-B., Jiang Y.-F., Hao M.,Zhang L., Ning S., Chen X.J., Liu D., Yuan Z. Using a wheat-ryeamphihaploid population to map a rye gene responsible for dwarfness.Euphytica. 2018.214:166. DOI 10.1007/s10681-018-2247-7.Zhang L.Y., Bernard M., Leroy P., Feuillet C., Sourdille P. Hightransferability of bread wheat EST-derived SSRs to other cereals.Theor. Appl. Genet. 2005;111:677-687. DOI 10.1007/s00122-005-2041-5.


Secale cereale L., 2n = 2x = 14, RR), a relative of common wheat, is a large gene resource pool for wheat improvement. Accurate and convenient identification of the rye chromatin in wheat background will facilitate the transfer and utilization of elite genes derived from rye in wheat breeding.Rye  to develop large-scale rye-specific markers. Based on SLAF-seq and bioinformatics analyses, a total of 404 universal PCR-based and a whole set of Kompetitive allele-specific PCR (KASP) markers specific for the 14 individual rye chromosome arms were developed and validated. Additionally, two KASP markers specific for 1RS and 2RL were successfully applied in the detection of 1RS translocations in a natural population and 2RL chromosome arms in wheat-rye derived progenies that conferred adult resistance to powdery mildew.The 404 PCR-based markers and 14 KASP markers specific for the 14 individual rye chromosome arms developed in this study can enrich the marker densities for gene mapping and accelerate the utilization of rye-derived genes in wheat improvement. Especially, the KASP markers achieved high-throughput and accurate detection of rye chromatin in wheat background, thus can be efficiently used in marker-assisted selection (MAS). Besides, the strategy of rye-specific PCR-based markers converting into KASP markers was high-efficient and low-cost, which will facilitate the tracing of alien genes, and can also be referred for other wheat relatives. Triticum aestivum L.) is a major grain crop worldwide. With the expanding global population to nine billion by 2050, wheat production is facing a challenge of about 70% growth to meet the demands in the future [Common wheat , a naturally cross-pollinated relative of common wheat, can be used as a huge gene donor for wheat improvement. For example, the wheat-rye T1RS·1BL translocation derived from rye cultivar Petkus carries the powdery mildew resistance gene Pm8, stripe rust resistance gene Yr9, leaf rust gene Lr26 and stem rust resistance gene Sr31 [Pm7 and Lr25 on 2RL from rye cultivar Rosen [Lr45 on 2RL from rye cultivar Petkus [Sr59 on 2RL from triticale VTB28041 [Sr27 on 3RS from rye cultivar Imperial [Pm56 on 6RS from rye cultivar Qinling [Pm20 on 6RL from rye cultivar Prolific [Rye  and fluorescence in situ hybridization (FISH), especially multicolor FISH (mc-FISH), are widely-used detection methods owing to intuitive and accurate specialty , 22. MeaWith rapid advancement of next-generation sequencing technology (NGS) and low-cost genome sequencing, an increasing number of single nucleotide polymorphism (SNP) markers were developed attributing to their high stability, high resolution and low cost, and therefore are suitable for large-scale genotyping –33. On tIn this study, an efficient NGS method specific-locus amplified fragment sequencing (SLAF-seq) and bioinformatics analyses were combined to develop a large number of universal PCR-based markers distributed on all the chromosome arms of rye. Then, these markers were analyzed to generate a set of KASP markers specific for each arm of the rye chromosome. Furthermore, these KASP markers were validated and applied in MAS on scale. The strategy for development of KASP markers adopted in this study can be a good reference for other wheat relatives.Based on the results of high-throughput sequencing method SLAF-seq for the five rye cultivars, including Imperial, German White, Jingzhouheimai, Baili and Guyuan, a total of 653,144 SLAFs were acquired. The average Q30 ratio was 86.79%, indicating that the data has high quality. By sequence alignment between the five rye cultivars and Chinese Spring , 3871 seThen, a complete set of wheat-rye disomic and ditelosomic addition lines of ‘Holdfast-KingII’ and a set of wheat-rye disomic addition lines of ‘Chinese Spring-Imperial’ were used for the location and verification of these 667 markers. All of the wheat-rye addition lines were clearly identified to contain the two corresponding rye chromosome arms or chromosomes by GISH and non-denaturing FISH (ND-FISH) analyses, such as the 3RS ditelosomic addition line of ‘Holdfast-KingII’ identified by the probes of Oligo-pSc119.2–1 and Oligo-pTa535–2  substitution line which was previously confirmed to exhibit adult resistance to powdery mildew on 2RL chromosome. To further transfer the resistance into susceptible commercial cultivar, WR91 has been crossed with Shixin633 and generated FRye is an important gene donor for disease resistance, superior agronomic traits and environment adaptation in wheat breeding. A great number of desirable wheat-rye progenies from various rye accession have been developed by distant hybridization and chromosome engineering, such as wheat-rye 6R disomic addition line WR49 carrying powdery mildew resistance derived from rye cultivar German White , wheat-rPreviously, developing markers was low-efficient due to the lack of genome reference information of the wheat relatives. SLAF-seq is an efficient next-generation sequencing method that can rapidly obtain a large amount of high-quality enzymatic fragments . Therefog segments in wheat-Aegilops geniculate introgression lines by combining chromosome flow-sorting and next-generation sequencing technology, while the flow-sorting technology is expensive and not yet widespread. Rahmatov et al. [Agropyron cristatum chromosome based on the RNA-seq data. Recently, Grewal et al. [Recent advances in NGS promoted the construction of reference sequence assemblies and development of high-density DNA genotyping platforms for crops. Winfield et al.  developev et al.  developev et al.  developel et al.  developeS. cereale L. Lo7 scaffolds database [S. cereale L. Lo7 scaffolds [Compared with the reported studies, the strategy of developing alien specific KASP markers in this study was low-cost and high-efficient. In this study, 14 specific markers distributed on the 14 arms were selected and finally a whole set of rye chromosomal arm-specific KASP markers were developed and valuated. Firstly, SLAF-seq was used to develop PCR-based markers. Subsequently, the original SLAF sequences of the specific markers or the expanded sequences obtained by the database  were comcaffolds  have proDasypyrum villosum translocation line T6VS·6AL harboring Pm21 [The whole set of 14 rye chromosome arm-specific KASP markers could clearly distinguish three genotypes for wheat, rye, and wheat-rye introgression. Meanwhile, the targeted single unique SNPs with wheat were sequenced to be conserved among five rye cultivars as well it was turned out in six other rye cultivars, indicating the universality of these KASP markers in various rye cultivars. For the detection of 1RS translocations in a natural population with 161 wheat cultivars/lines, SWK5282 was more time-saving and convenient than PCR-based marker SW5282. The result also demonstrated that this KASP marker can be applied to screen wheat-rye derived populations on large scale, unconstrained by different wheat and rye backgrounds. In addition, the introduction of 1RS arms into common wheat leads to defects in bread-making quality while improving yield , the deting Pm21  and wheaBased on SLAF-seq and bioinformatics analyses, a total of 404 universal rye chromosome arm-specific PCR-based markers and a set of KASP markers were developed and validated. These markers can enrich the rye-specific markers and accelerate the large-scale screening of various rye germplasm resources and high-throughput detection of rye chromatin in wheat background. Two KASP markers specific for 1RS and 2RL were successfully applied in the detection of 1RS translocations in a natural population and 2RL chromosome arms that conferred resistance to powdery mildew. In addition, the present study provided an efficient strategy for wheat relatives to develop specific KASP markers, which would improve the efficiency of genetic studies and MAS for alien chromosome engineering breeding.Five rye cultivars including Imperial, German White, Jingzhouheimai, Baili and Guyuan were sequenced by SLAF-seq to develop rye chromosome arm-specific markers. Rye cultivar Baili, wheat cultivar Holdfast, and a complete set of wheat-rye disomic addition lines of ‘Holdfast-KingII’ were kindly provided by Dr. Zongxiang Tang . Rye cultivar Imperial and a complete set of wheat-rye disomic addition lines of ‘Chinese Spring-Imperial’ were kindly provided by Dr. John Raupp . Rye cultivar Jingzhouheimai was kindly provided by Dr. Lifang Zhuang . A complete set of ditelosomic addition lines of ‘Holdfast-KingII’ were kindly provided by Dr. Adrian Turner . Rye cultivars KingII, CIse 1, CIse 12, CIse14, CIse17, CIse53 and CIse54 were kindly provided by Dr. Yiwen Li .The following materials maintained in our laboratory including wheat cultivars Chinese Spring, Shixin633, Shixin733, Shixin828, Gao8901, Jishi02–1, Shimai15, Kenong199, Heng5471 and Jimai22, two octoploid triticale lines  09R1–38 and 09R1–100, and a wheat-rye T1RS·1BL translocation line Lovrin10, were used for the location and verification of rye chromosome arm-specific markers. The wheat-rye lines used for the verification also included wheat-rye 4R disomic addition line WR35, T4BL·4RL and T7AS·4RS translocation line WR41, 6R disomic addition line WR49, 2RL ditelosomic addition line WR56 and 2R (2D) disomic substitution line WR91, all these lines were developed from ‘Xiaoyan 6 × German White’, and previously identified by using GISH and mc-FISH analyses .2 individuals derived from the cross of WR91 and Shixin633 susceptible to powdery mildew were used to validate the practical utilization of the rye chromosome arm-specific KASP markers. The wheat cultivar Mingxian169 without Pm genes that maintained in our laboratory was used as susceptible control in assessment of reaction to powdery mildew at the adult stage. All the seedlings were used for extracting total genomic DNA by the CTAB method [One hundred and sixty-one wheat cultivars/lines maintained in our laboratory and the FB method . GenomicGenomic DNA samples of rye cultivars Imperial, German White, Jingzhouheimai, Baili and Guyuan were, respectively, sequenced by SLAF-seq  as previously described with some modifications . FirstlyAll SLAF pair-end reads generated from SLAF-seq raw reads were filtered according to the quality, and then high-quality clean reads with over 90% identity were clustered into one SLAF locus. In order to obtain the rye-specific fragments, all the SLAFs derived from the five rye cultivars were compared with the wheat genome using BWA software , leavingGISH analyses were performed to confirm the existence of the rye chromosomes or chromosome arms in the complete set of wheat-rye disomic and ditelosomic addition lines of ‘Holdfast-KingII’ and a set of disomic addition lines of ‘Chinese Spring-Imperial’. The root tip cells were prepared for mitotic chromosome observation and hybridization as previously described . Total ghttp://www.primer3plus.com/primer3web/) for the amplification of five rye cultivars Imperial, German White, Jingzhouheimai, Baili, Guyuan and wheat cultivar Holdfast. All the primers were synthesized by Shanghai Sangon Bio-technology Co., Ltd. . Three steps of PCR analysis were performed to develop rye-specific markers: firstly, those markers whose specific band absented in Holdfast but presented in all the six rye cultivars were considered as universal rye-specific markers. Secondly, a set of wheat-rye disomic addition lines of ‘Holdfast-KingII’ were used to assign the markers to individual rye chromosomes. Finally, a set of wheat-rye ditelosomic addition lines of ‘Holdfast-KingII’ were used to locate the rye chromosome-specific markers onto rye chromosome arms. The frequency and number of markers assigned to the individual chromosome arms were showed in a circos plot drawn by the Circos-0.69-9 software [According to the obtained conserved rye-specific sequences, PCR primers were designed using Primer 3 software . The PCR procedure was as follow: 94 °C for 5 min, followed by 36 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 40 s, and a final extension at 72 °C for 10 min. The PCR products were separated in 8% non-denaturing polyacrylamide gels with 29:1 ratio of acrylamide and bis-acrylamide, and silver-stained prior to visualizing the banding patterns [The PCR amplification system  contains 1 μl of template DNA (100 ng/μl), 4 μl 2 × Taq Master Mix , 4 μl ddHpatterns .S. cereale L. Lo7 scaffolds [http://www.primer3plus.com/primer3web/). The wheat sequences were referred to the IWGSC Ref Seq v1.0 [The PCR-based markers specific for rye chromosome arms were analyzed to convert to KASP markers. Firstly, the sequence alignment of the original SLAF sequence with the data of caffolds  was condSeq v1.0 .Subsequently, these KASP markers were validated their specificity, stability, and universality by testing 12 rye cultivars including KingII, Imperial, German White, Jingzhouheimai, Baili, Guyuan, CIse 1, CIse 12, CIse14, CIse17, CIse53 and CIse54, a complete set of wheat-rye disomic and ditelosomic addition lines of ‘Holdfast-KingII’, a set of disomic addition lines of ‘Chinese Spring-Imperial’, and eleven wheat cultivars including Holdfast, Chinese Spring, Shixin633, Shixin733, Shixin828, Gao8901, Jishi02–1, Shimai15, Kenong199, Heng5471 and Jimai22.2O and 2.4 μl of template DNA (50 ng/μl). The reaction was performed in a Bio-Rad CFX real-time PCR system , PCR procedure was as follow: 94 °C for 15 min, 94 °C for 20 s, followed by 10 touchdown cycles of 64 to 58 °C (decreasing 0.6 °C per cycle), 38 cycles of 94 °C for 20s, 58 °C for 60s, and fluorescence was detected using Bio-Rad CFX Manage 3.1 software.The KASP amplification system  contains 4.5 μl high rox KASP master mix, 0.13 μl Assay mix, 0.97 μl ddH2 seeds. WR91, Shixin633 and their F2 seeds were planted in early October at the Luancheng Agro-Ecological Experimental Station, Chinese Academy of Sciences, Shijiazhuang, China. Twenty plants were grown in each 1.5-m long row, spaced 25 cm apart. Wheat cultivar Mingxian169 was planted around the F2 population as susceptible control and inoculum spreader. In late March of the next year, the spreader rows were artificially inoculated by the mixture of Blumeria graminis f. sp. tritici isolates prevalent in northern China. In May, when the susceptible control Mingxian169 exhibited serious disease symptoms, the evaluation of the powdery mildew reactions at heading and grain filling stages was recorded using a 0–9 scale, where 0–4 was considered as resistant and 5–9 as susceptible [A wheat-rye 2R (2D) substitution line WR91, derived from ‘Xiaoyan 6 × German White’, was previously confirmed to confer the adult resistance to powdery mildew that was located on the chromosome arm 2RL. To further transfer the resistance into susceptible wheat cultivar, WR91 has been crossed with Shixin633 and generated F2 individuals of ‘WR91 × Shixin 633’. Meanwhile, the corresponding PCR-based marker SW252224 was also used to test the F2 population to confirm the result of SWK252224. Chinese Spring and Imperial were used as the KASP marker genotyping controls.The 1RS-specific KASP marker SWK5282 was used to screen a natural population with 161 wheat cultivars/lines to determine the distribution of 1RS translocation lines therein. For the verification of the accuracy and stability of the KASP marker, the corresponding PCR-based 1RS-specific marker SW5282 was also used to identify these 161 wheat cultivars/lines. In addition, the KASP marker SWK252224 specific for 2RL was applied in detecting chromosome 2RL in FAdditional file 1: Table S1. Primer sequences of the 404 molecular markers specific for 14 rye chromosome arms.Additional file 2: Table S2. Detection of 1RS translocations in 161 wheat cultivars/ lines.


We also assessed subtype-specific self-esteem rating in ADHD subtypes and explored its association with cognitive correlates.Attention-deficit hyperactivity disorder (ADHD) is a major neurodevelopmental disorder with heterogeneous symptoms, subtypes, and cognitive deficits. Cognitive deficits are central to ADHD pathophysiology and one potential source of heterogeneity in ADHD. Subtype-specific cognitive correlates are not, however, well-studied. We explored cognitive correlates of ADHD One hundred thirty-nine children with ADHD  were categorized into the predominantly “hyperactive (ADHD-H)”, “inattentive (ADHD-I)” and “combined (ADHD-C)” subtype based on their symptoms and scores on the Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS-PL) and Conners Parent-Rating Scale (CPRS-RS). They were then individually administrated the WISC-IV and completed a self-esteem inventory. Group differences in the WISC-IV indices and their predictability in discriminating ADHD subtypes were analyzed.quantitative differentiation of cognitive abilities among ADHD subtypes with “working memory” as the most compromised cognitive domain. ADHD-I had the poorest cognitive profile while ADHD-H scored highest in all cognitive domains. Importantly, cognitive abilities were negatively correlated with inattention and positively correlated with hyperactive symptoms. Moreover, self-esteem ratings were positively correlated with the cognitive domains and were rated differently based on the subtypes. ADHD-H, with the highest cognitive strength, reported the highest level of self-esteem among all subtypes.We found a ADHD subtype-specific symptoms, cognitive deficits, and self-esteem problems should be considered for precise diagnosis and effective and personalized treatment in ADHD in light of further supporting evidence and assessments. Cognitive interventions might be more compatible with and effective in inattentive and combined subtypes of ADHD. Working memory improving-based interventions can benefit all ADHD subtypes. A supportive educational system in school and providing adjunct supportive interventions should be considered for children with ADHD as well. A precise description of the pathophysiology underlying ADHD is difficult due to its neuropsychological heterogeneity [Attention-deficit hyperactivity disorder (ADHD) is a major neurodevelopmental disorder with ogeneity  and subsogeneity . Cognitiogeneity  and amonogeneity  and noveogeneity , 6. Thesogeneity . One aspogeneity , 9 whichogeneity , howeverogeneity .Furthermore, it is still elusive whether a specific subtype is critical to consider when examining treatment effects. For example, a recent study suggests that the effectiveness of neurofeedback treatment on the executive functioning of children with ADHD is subtype-specific . ADHD-I subtypes are not adequately explored in the previous studies in ADHD while several neuropsychological distinctions in ADHD subtypes  have been identified [Previous studies showed that the Wechsler Intelligence Scale for Children (WISC) provides a relatively comprehensive profile of cognitive strengths and weaknesses and is commonly used for cognitive evaluation in the clinical pediatric population. Application of WISC in ADHD also confirms that it reliably differentiates between ADHD patients and healthy controls , 16. Morentified .Similar to subtype-specific cognitive deficits, self-esteem and social functioning in ADHD subtypes are not well-studied and the available studies are even more limited. Lower ratings of self-esteem in ADHD patients compared to the healthy control, regardless of ADHD subtypes, have been reported in previous studies –28. Thesn = 139). We also performed a discriminative analysis to evaluate if the cognitive profile of each ADHD subtype can predict group membership. We further assessed the level of self-esteem in each ADHD subtypes to determine any associations between subtype-specific cognitive correlates and self-esteem ratings in ADHD.The purpose of the present study was, therefore, to explore cognitive correlates of ADHD subtypes based on the WISC-IV scores in a relatively large sample size , referred to the Fatemi Hospital at Ardabil University of Medical Sciences, were included in this study. The data was collected in the child psychiatry division of the hospital which is dedicated to the diagnosis and treatment of child and adolescence psychiatric disorders, including neurodevelopmental disorders . Participants’ recruitment took place from mid-2017 until the end of 2019. The inclusion criteria were: (1) ADHD diagnosis according to the DMS-5 criteria by a licensed psychiatrist and a child psychologist, (2) moderate to severe score in the parent or teacher version of the Conners’ Parent Rating Scale (CPRS) in addition to an independent diagnosis made by psychiatrist/psychologist, (3) being 6–15 years old, (4) and no current and history of psychiatric, neurodevelopmental disorders and chronic physical illness. All participants were on medication treatment when recruited for the experiment however, there were prevented from the medication 24 h before testing to ensure that WISC-IV performance was not affected by medication as suggested by previous studies , 29, 30.The K-SADS-PL (3) is a semi-structured interview for assessing psychiatric diagnoses in children and adolescents from 6 to 18 years old. It assesses the present, history of psychiatric disorders, and the severity of symptoms based on the DSM-IV. The K-SADS-PL-P is especially sensitive at diagnosing patients with ADHD, major depressive disorder, bipolar disorder, general anxiety disorder, post-traumatic stress disorder, and substance use disorder. The test-retest reliability and kappa coefficients are reported excellent for the present and lifetime diagnosis of major psychiatric disorders. A native-language version of the scale used in this study with good-to-excellent concurrent validity in diagnosing current major disorders . The KapThe revised version of Conners’ Rating Scale  has threThe WISC-IV is an individually administered test battery that assesses intelligence in school-aged children (from 6 to 16 years of age) , 36. TheThe 58-items CSE with high reliability of 0.88 and validity  was used139 children in this study were consecutive referrals to our child psychiatry hospital who were diagnosed with ADHD. After we received institutional review board approval, children with ADHD and their parents were interviewed by a psychiatrist based on the CPRS-RS and K-SADS-PL and were categorized into ADHD-I, ADHD-H, and ADHD-C accordingly. Twenty-four hours before the testing day, the patients that were on medication stopped taking the medication. They were then administrated the WISC-IV and completed the self-esteem inventory. All tests were conducted in the same testing room. The order of tests was randomized.p < .05 was used for all statistical comparisons.We used IBM Statistical Package for the Social Sciences (SPSS) for Windows, version 24  for data analysis. A one-way analysis of variance (ANOVA) and Bonferroni-corrected t-tests were applied to examine group differences for the major WISC-IV indices, including the VCI, PRI, WMI, PSI, and the full-scale intelligence quotient (FSIQ). It is of note that the potential covariate effect of age was controlled as FSIQ and indices scores are estimated by age-scaled scores and thus age was not entered a covariate in the analysis. A separate similar ANOVA was conducted on the rating of self-esteem domains. Our data met the ANOVA linear assumptions and Leven’s test was used to examine the homogeneity of variances. Additionally, we performed a discriminant analysis to explore whether WISC-IV indices scores, as predictor variables, can predict grouping of ADHD patients into ADHD-I, ADHD-H, and ADHD-C subtypes. Correlational analyses between the outcome measures were calculated using the Pearson correlation (two-tailed). A significance level of t = 4.21, p < 0.001) and all indices of VCI , PRI , WMI , and PSI . Moreover, ADHD-I scores in the VCI  and PSI  indices were significantly lower than ADHD-C. No significant difference between ADHD-H vs ADHD-C was found in any of the indices. In sum, WMI was the weakest (regardless of ADHD subtype), VCI was the strongest index, and FSIQ score was 3 points lower than 90  that accounted for 90.7% of the discriminative ability of the WISC-IV subtests in predicting ADHD group membership . The canonical correlation between predictor variables and grouping was R = 0.727. Correct grouping of the function for ADHD-I, ADHD-H, and ADHD-C was 57.1, 84.5, and 45.5% respectively. Moreover, the discriminant function could correctly classify 68.3% of the individuals or identified the group to which the individuals belong  and ADHD-C . Similarly, ADHD-H patients had a significantly higher global self-esteem compared with ADHD-I  and ADHD-C . No significant differences were found in the subdomains of self-esteem between the groups  and all of the self-esteem domains except for the family self-esteem Table . FurtherIn the present study, we explored subtype-specific cognitive correlates in 139 children with ADHD based on the WISC-IV. We also assessed self-esteem ratings in ADHD subtypes. Our results show that ADHD-I has the most impaired cognitive profile among all ADHD subtypes and is mostly discriminated with ADHD-H, with the least impaired cognitive functions. Our results further suggest a quantitative differentiation of cognitive profiles among ADHD subtypes with working memory as the most compromised cognitive domain with the lowest value in ADHD-I. Moreover, we found a converging pattern of ADHD subtype-specific differences in self-esteem rating with a significantly higher-rated self-esteem in ADHD-H compared to ADHD-I and ADHD-C.With regard to subtype-specific cognitive correlates, we found a quantitative differentiation of cognitive profiles among ADHD subtypes regardless of WISC-IV domains in line with previous works , 44, 45.subtypes. The quantitative pattern observed in cognitive profiles of ADHD subtypes might also suggest that WM is the underlying basic executive function [An important aspect of our findings was that WM is the poorest cognitive domain in all ADHD subtypes especially in the ADHD-I. Previous studies using the Wechsler Intelligence Scale in both children and adults with ADHD, regardless of subtype, showed that working memory and processing speed are usually among the most impaired domains in ADHD patients compared to healthy controls , 29, 47.function  that afffunction . More imfunction  and implfunction , 6 also In addition to the cognitive correlates, we found a subtype-specific pattern in self-esteem ratings with ADHD-H reporting a higher level of global and total self-esteem compared to other subtypes. Previous studies documented social impairment in ADHD and emphasized on the need for further investigation of subtype-specific social deficits in ADHD . In lineOne important point to be noted here is the distribution of ADHD subtypes in our sample. Participants with the ADHD-H subtype constitute the majority of our sample (51.1%) while in other studies, the ratio of ADHD-I and ADHD-C has been relatively reported higher , 56. OneTaken together, the results of the present study show that there is subtype-specific cognitive profile, measured by WISC-IV, in ADHD confirming a cognitive heterogeneity in ADHD in line with recent evidence . ADHD suIt is necessary to note that although the focus of this study is on subtype-specific differences in cognitive and self-esteem profiles of children with ADHD, such subtype specification is not confirmed in all studies. Some studies have shown an instability or shift in subtypes over years in children with ADHD , 62. FinThe following limitations should be considered. First, we did not have a control group consisting of typically developing children because the purpose of this study was to determine subtype-specific cognitive differences in ADHD. Nevertheless, comparison with typically developing children can also reveal insightful differences of children with ADHD compared to their healthy peers. Second, because we did not intend to focus on gender differences, the number of girls in the study was small and need to be explored in larger samples. Third, the WISC-IV indices might not examine specific aspects of cognition in ADHD and cognitive profile of ADHD subtypes needs to be explored with more specific cognitive measures of executive functions, such as cold vs hot executive functions . FinallyWe found a subtype-specific quantitative difference in cognitive correlates and self-esteem ratings of children with ADHD which can be considered for precise diagnosis and individualized interventions. Further assessments and neuroimaging findings in support of a subtype-specific distinction are needed. Our findings also support the notion that ADHD is characterized by neurocognitive heterogeneity. Cognitive interventions might be more compatible with and effective in inattentive and combined subtypes of ADHD but working memory improving-based interventions can benefit all subtypes. Association of cognitive performance and self-esteem ratings indicates the importance of educational support system in school for children with ADHD and/or providing adjunct supportive interventions in addition to cognitive ones.


Alopecia areata (AA) is an autoimmune disease that causes sudden hair loss. Although few studies have reported the association between AA and attention-deficit/hyperactivity disorder (ADHD), the impact of methylphenidate (MPH) on AA has not been examined. This study examined whether AA risk is higher in children with ADHD than in those without ADHD as well as the impact of MPH use on AA risk in children with ADHD. From the Taiwan Maternal and Child Health Database, we enrolled all 1,750,456 newborns from 2004 to 2017 in Taiwan. Of them, 90,016 children received a diagnosis of ADHD whereas the remaining 1,660,440 did not. To compare AA risk in ADHD and the impact of MPH treatment on it, multiple Cox regression with adjustments for covariates  was performed. The results indicated that 88 (0.098%) children with ADHD and 1191 (0.072%) children without ADHD had AA. Nevertheless, after adjustment for the covariates, AA risk was higher in children with ADHD than in those without ADHD . Our data indicated a considerable reduction in AA risk (aHR: 0.64) among children with ADHD who received MPH than among those who did not receive MPH; however, this difference was nonsignificant, indicated by a wide 95% CI (0.32–1.25). In conclusion, ADHD and AA may share some underlying mechanisms. Alopecia areata (AA) is an autoimmune condition that attacks the hair follicles, causing nonscarring hair loss . In healAttention-deficit/hyperactivity disorder (ADHD) is more likely to be observed in individuals with AA; however, the findings of the association between AA and ADHD are limited and inconsistent ,8. For eMethylphenidate (MPH), a stimulant that blocks dopamine and norepinephrine transporters and increases extracellular dopamine and norepinephrine, is used to treat ADHD . ApproxiA case series study recruiting three ADHD cases found that stimulant treatment in individuals with ADHD caused alopecia: all cases demonstrated sudden hair loss (patchy and diffuse hair loss) during the dose-increasing period of MPH (20–30 mg per day), and the hair loss stopped and hair resumed growing after the children were switched from MPH to atomoxetine or lisdexamfetamine . Animal To address the aforementioned research gaps, this nationwide population-based cohort study examined whether AA risk was higher in children with ADHD than in those without ADHD and whether MPH use in children with ADHD affected this risk. We hypothesized that AA risk was higher in children with ADHD than in those without ADHD and that AA risk was lower in children with ADHD who received MPH than in those who did not receive MPH.From the Taiwan Maternal and Child Health Database, we extracted the data of all liveborn children from 1 January 2004 to 31 December 2017 including complete information on gestational age at birth and parents’ identities. We recruited children aged >4 years because AA and ADHD are more common and recognizable from that age. The aforementioned database includes data of 99.78% of all births nationwide since 2004 in Taiwan . This stThe exposure in this study was ADHD diagnosis in children based on the ambulatory care expenditures by visits and the inpatient expenditures by admissions. Participants were considered to have ADHD if they received at least one inpatient diagnosis or more than two outpatient diagnoses of ADHD  during the study period.MPH treatment in children with ADHD was defined according to dispensation records of the Details of Ambulatory Care Orders, which covers all medication dispensations and accompanying prescriptions for outpatients written in Taiwan since July 1996. MPH use was identified on the basis of the Anatomical Therapeutic Chemical Classification code for MPH (N06BA04) from 1 January 2004 to 31 December 2017. Participants were considered to be taking MPH if they were prescribed it for >3 consecutive months .The study outcome was AA diagnosis in children based on the ambulatory care expenditures by visits and the inpatient expenditures by admissions. Participants were considered to have AA if they received at least one inpatient diagnosis or more than two outpatient diagnoses of AA  during the study period.Here, the covariates were children’s age, sex, and common mental disorders in children, namely autistic spectrum disorder , anxiety disorders , tic disorder , and major depressive disorder .SAS (version 9.4)  was used for data management and analysis. Descriptive statistics are presented as numbers with proportions and means with standard deviations for categorical and continuous variables, respectively. To compare AA risk between children with and without ADHD, multiple Cox regression was conducted with or without adjustment for age, sex, and comorbid disorders . Age was used as the time scale used in the Cox regression. Furthermore, to clarify the impact of MPH on AA in children with ADHD, multiple Cox regression was conducted with or without adjustments for age, sex, and comorbid disorders in children with ADHD receiving or not receiving MPH. The risks without and with adjustments were presented as crude hazard ratio (cHR) and adjusted hazard ratio (aHR), with their 95% confidence intervals (CIs). If the CI included the null value of 1, the difference was considered nonsignificant. The effect size of HR has been reported based on Azuero’s suggestion, wherein small, medium, and large HRs comparing two groups would be approximately 1.3, 1.9, and 2.8 for categorical risk factors (RR > 1) and 0.77, 0.53, and 0.36 for the categorical protective factors (RR < 1), respectively .In total, 90,016 records of children with ADHD and 1,660,440 records of children without ADHD were retrieved from the Taiwan Maternal and Child Health Database. The findings of this national population-based cohort study revealed that children with ADHD had a higher AA risk than those without ADHD. Some shared inflammatory mechanisms and genetic pathogenesis may explain this association. Individuals with AA had significantly higher levels of inflammatory markers and oxidative stress indicators including serum C-reactive protein (2.52-to 2.73-fold greater), 8-hydroxy deoxyguanosine (1.28-fold greater), and high mobility group box 1 protein (14.87-fold greater) than those without AA ,24. SimiMajor histocompatibility complex genes, particularly the polymorphisms of HLA-DR4 and HLA-DRB1, have been reported to be the potential genes of both AA ,27 and AIn addition to AA, the comorbid association of ADHD with other autoimmune diseases including psoriasis, ankylosing spondylitis, autoimmune thyroid diseases, and inflammatory bowel disease has been reported ,32. FurtAlthough we found that AA risk in children with ADHD receiving MPH  was lower than that in all children with ADHD , the effect of MPH treatment on reducing AA risk was non-significant. AA incidence in children  was extremely low in the present study; although this study used a national dataset with approximately two million children, only 88 children with ADHD had AA. A study involving children with ADHD indicated that MPH normalizes the immunological characteristics of glial function . MoreoveThis study had some limitations. First, our sample comprised children who were 4–14 years old; thus, the associations between ADHD, MPH, and AA may not be generalizable to other age groups. Second, AA incidence in children  was extremely low in the present study. Third, because of the low number, we excluded children with ADHD taking atomoxetine.This national population-based cohort study revealed that children with ADHD had a higher AA risk than those without ADHD. The association between ADHD and AA indicates a possible hypothesis of a shared etiology of immune function between them. The difference in the AA risk between children with ADHD receiving and not receiving MPH was non-significant. The role of MPH treatment on AA in children with ADHD warrants further research.


Nonlinear interferometers with correlated photons hold promise to advance optical characterization and metrology techniques by improving their performance and affordability. These interferometers offer subshot noise phase sensitivity and enable measurements in detection-challenging regions using inexpensive and efficient components. The sensitivity of nonlinear interferometers, defined by the ability to measure small shifts of interference fringes, can be significantly enhanced by using multiple nonlinear elements, or crystal superlattices. However, to date, experiments with more than two nonlinear elements have not been realized, thus hindering the potential of nonlinear interferometers. Here, we build a nonlinear interferometer with up to five nonlinear elements, referred to as superlattices, in a highly stable and versatile configuration. We study the modification of the interference pattern for different configurations of the superlattices and perform a proof-of-concept gas sensing experiment with enhanced sensitivity. Our approach offers a viable path towards broader adoption of nonlinear interferometers with correlated photons for imaging, interferometry, and spectroscopy. Ultra-sensitive interferometric measurements can be made by using correlated photons generated in multiple nonlinear crystals, researchers in Singapore have shown. Correlated photons, linked through a form of quantum entanglement, are useful for studying materials in detection challenging spectral ranges. For example, when infrared photons are affected by striking a sample, the same changes will happen to their correlated photons of visible light, which are easier to detect. Anna Paterova and Leonid Krivitsky at A*STAR in Singapore developed an interferometer that generates correlated photons using up to five nonlinear elements which form crystal superlattices – periodic layers of different materials. Compared to previous systems that used only two nonlinear elements, the new system allowed the researchers to observe smaller changes in interference patterns, and therefore showed greater sensitivity when detecting carbon dioxide gas in tests. For example, strong temporal correlations between photons were used for a single-photon calibration of the efficiency of retinal cells3 and enhancing the nonlinear response of biological samples4. Furthermore, two-photon interference effects have formed the basis for dispersion-free optical coherence tomography7, microscopy with enhanced phase contrast9, and noise-robust spectroscopy of nanostructures10 to name a few.Optical characterization and metrology techniques benefit from using correlated photons, particularly in studies of light-sensitive and fragile biological and chemical samples15. A nonlinear interferometer is composed of two nonlinear elements, which produce pairs of correlated photons  under coherent excitation. The signal (in the visible range) and idler (in the IR range) photons are mixed in the interferometric setup, and as long as one cannot distinguish which nonlinear element produced the photons, interference fringes are observed. The interference pattern of signal photons depends on the phases and amplitudes of the signal, idler, and pump photons. When idler photons interact with a sample, its properties in the IR range can be inferred from the interference pattern of signal photons in the visible range. Thus, this technique addresses practical challenges of generation and detection of IR light since the sample response is obtained using accessible components for visible light.Recently, the nonlinear interference of correlated photons has attracted particular interest in the context of infrared (IR) metrology and sensing18, gas cells19, fiberized networks21, and nonlinear waveguides23. Additionally, nonlinear interferometers have been used for imaging24, spectroscopy27, optical coherence tomography29, super-resolution interferometry19, and polarimetry30. All these techniques are intrinsically interferometric. Hence, their sensitivity is defined by the ability to detect small changes in the interference pattern, such as a shift of the fringes or change in the fringe visibility.Nonlinear interferometers have been realized using numerous physical platforms, including bulk nonlinear crystals31, who considered a setup with N identical nonlinear elements separated by linear gaps, referred to here as a crystal superlattice. He showed that with an increase in the number of crystals, bright interference fringes in the frequency domain narrow, yet the spacing between fringes remains unchanged. This idea was theoretically expanded in more recent works33; however, to the best of our knowledge, there are no reports on the experimental realization of nonlinear interferometers with more than two nonlinear elements. The major challenges in practical realization are associated with (1) the necessity of superimposing signal and idler modes from multiple nonlinear elements while preserving the quantum indistinguishability, and (2) the necessity to align and stabilize increasingly complex setups.One possible way to enhance the sensitivity of nonlinear interferometers was outlined by D. KlyshkoHere, for the first time, we realize a nonlinear interferometer with a crystal superlattice consisting of up to five nonlinear elements. In our setup, nonlinear elements are arranged sequentially and are pumped by a single coherent laser. By careful design and alignment, we achieve a robust mode overlap of signal and idler photons with remarkable stability. We observe the interference pattern in the frequency-angular spectrum with full flexibility of crystal arrangements and theoretically describe this effect. We also perform a proof-of-concept gas sensing experiment with enhanced sensitivity.35. Analysis of the interference in both the frequency and spatial domains was limited to only two nonlinear crystals37. Here, we analyse the frequency-angular spectrum obtained in a nonlinear interferometer with N crystals. Let us consider N identical nonlinear crystals of length l separated by N−1 equal linear gaps l'; see Fig. s) and idler (i) photons via spontaneous parametric down-conversion (SPDC). The down-converted photons from each crystal are redirected to the next crystal. The state of the two-photon field produced by a single crystal is given by34:n-th crystal n = , n-th crystal with wavevectors 31.Earlier works theoretically analysed the multicrystal interference in the frequency domain for a single spatial mode38:n-th nonlinear crystal; Ep is the field of the pump; and j = s, i, p):k and Δk' are the wavevector mismatches inside the nonlinear crystal and in the linear gap, respectively. For N identical crystals, the two-photon field amplitude is given by the sum of contributions from individual crystals as follows:φ = (Δkl + Δk'l'). Then, from Eq.  and idler photons at 4.14 μm (bandwidth 92 nm). The normalized frequency-angular spectra of signal photons for two and five nonlinear crystals are shown in Fig. s = 610.4 nm for the interferometer with two and five crystals, respectively. The cross sections are taken by averaging the intensity across the bandwidth of Δλs = 0.4 nm. To achieve the same flux of the photons for the two- and five-crystal configurations, the acquisition time is set to 360 and 144 s, respectively.First, we set the phase-matching angle of the crystal to θc = ±0.02°. We found that a slight misalignment in setting θc becomes crucial for the interferometer with an increasing number of crystals. A detailed analysis of the sensitivity of the interferometer to various experimental parameters is presented in Sections 3 and 4 of the Supplementary Materials.The solid curves in Fig. N-crystal interferometer δθsN and two-crystal interferometer δθs2. The key observation is consistent with the theory: the interference fringes become narrower with increasing number N of nonlinear crystals. The green dots in Fig. θc = ±0.02°, which is consistent with our experimental data, shown by black squares. Note that a stronger dependence on the uncertainty of the experimental parameters in the interferometer with a crystal superlattice is a manifestation of the common property of multielement interferometers.We experimented with sets of two, three, four, and five crystals in the superlattice. In each case, we fit the experimental data by Eq.  and deteθc = 50.23° ± 0.02° and obtain signal photons at λs = 609.3 nm and idler photons in the vicinity of the absorption resonance of CO2 at λi = 4.19 μm. The frequency-angular spectra of signal photons from an interferometer with two and five crystals are shown in Fig. 2 gas is injected in the gaps (concentration (35 ± 3.5) × 103 ppm). Because of the absorption of idler photons by the gas, the interference pattern of signal photons experiences a phase shift and reduction in visibility.Next, we set the phase-matching angle to s = 609.3 nm (λi = 4.19 μm) when the wavelength of idler photons is detuned from the absorption resonance of CO2 by ~72 nm. In this case, the gas causes a phase shift of the interference fringes without a significant change in the fringe visibility. Figure Figure 2 detection in the current five-crystal configuration is 1.8 × 103 ppm.From the fitting of the experimental data, we find that the relative shift of the interference fringes for the two-crystal interferometer is 40.The sensitivity of this method can be further improved by using more nonlinear crystals and increasing the interaction length. In practice, such a device can be realized using a high-finesse cavity with just a single crystal. We estimate that for a cavity with a finesse of 150 and a base of 80 mm, the theoretical value of the sensitivity reaches a level of a few tens of ppm, which is comparable with that of compact commercial optical sensorsOur experimental setup allows full flexibility in investigating nonlinear interferometers with variable crystal configurations. To demonstrate this, we remove the third crystal from the interferometer and observe the interference from the first, second, fourth, and fifth crystals . We use Eq.  to calcu35.The theoretical interference pattern given by Eq.  is shownWe realized a nonlinear interferometer with a crystal superlattice with up to five nonlinear elements. We experimentally demonstrated that with an increase in the number of nonlinear elements, the interference fringes become narrower, which directly translates to improved sensitivity in metrological and sensing applications. The observed effect originates from the constructive interference of wavefunctions of down-converted photons, which are coherently generated in different crystals. The effect is clearly analogous with classic multi-source interference. We also found that an interferometer with a crystal superlattice becomes increasingly dependent on the accuracy in setting the experimental parameters, in particular, the phase-matching angles of the crystals. This reflects the common property of multiple-beam interferometers, which are more demanding for the settings of individual elements.The presented configuration allows flexibility in the realization of unconventional crystal configurations, for example, by setting different gaps between crystals and using crystals of different sizes, which opens an interesting possibility for quantum state engineering.41. We believe that the presented concept will provide a viable path towards high-performance devices for sensing, metrology, and quantum state engineering. When finalizing this work, we became aware of relevant work on the realization of a three-stage nonlinear interferometer42.We anticipate that our work will trigger more than one creative design in the realization of complex nonlinear interferometers with correlated photons, such as by using mirrors, integrated photonics, or fibre platforms. It is also of interest to investigate this scheme for the high-gain regime of parametric down-conversion3, l = 1 mm, cut angle of 48.5°, Eksma Optics). Each crystal is coated with broadband AR coating, which introduces less than 1% loss for pump and signal photons and less than 5% loss for idler photons. The crystals are separated by the distance l’ = 8.2 mm. They are mounted on a kinematic prism mount (KM100PM Thorlabs) and clamped by a small adjustable clamping arm . The mount provides 0.45 deg adjustment per revolution. The estimated experimental accuracy in setting the phase-matching angle of the crystal is Δθc = ±0.02°.Our experimental setup is shown in Fig. f = 300 mm). The interference pattern of signal photons in frequency-angular coordinates is recorded by a CCD camera for visible light (Andor iXon 897) at the output of the spectrometer. The camera has 512 × 512 pixels and a pixel size of 16 μm, the gain of the camera is set to 290, and the temperature of the camera sensor is kept at −80 °C. The optical noise is measured independently and taken into account at the stage of data processing.Photon pairs are generated in each nonlinear crystal in the type-I quasi-collinear frequency nondegenerate regime. A notch filter NF and a polarizer V are used to filter out the pump. Signal photons are focused on the slit of the imaging spectrograph (Acton) using the lens F (l + l’ʹ)tan(θs) ≪ d, which links the scattering angle θs, pump diameter d, and parameters of the superlattice l, lʹ(ref. 26). To satisfy this condition, we set d ~ 3 mm using the beam expander (LS) and detect angles up to θs = ±0.85°.To ensure the indistinguishability of photon pairs produced in every crystal of the superlattice, all the SPDC photons should be generated and propagate within the interaction volume defined by the pump beam. This requirement is expressed in the condition . The wavelength of the idler photons is set to match the absorption peak at ~4.27 μm. The gas homogeneously fills the volume between the crystals. Its concentration in the enclosure is controlled by a commercial CO2 sensor . The experiments are conducted at a room temperature of 22 °C.In the gas sensing experiments, the interferometer is placed in an airtight enclosure (marked by a dashed rectangle) with an input socket for carbon dioxide gas (COSupplementary Information for Nonlinear interference in crystal superlattices