Deciphering genetic factors contributing to enhanced resistance against Cercospora leaf blight in soybean (Glycine max L.) using GWAS analysis.

Cercospora leaf blight (CLB), caused by Cercospora cf. flagellaris, C. kikuchii, and C. cf. sigesbeckiae, is a significant soybean [Glycine max (L.) Merr.] disease in regions with hot and humid conditions causing yield loss in the United States and Canada. There is limited information regarding resistant soybean cultivars, and there have been marginal efforts to identify the genomic regions underlying resistance to CLB. A Genome-Wide Association Study was conducted using a diverse panel of 460 soybean accessions from maturity groups III to VII to identify the genomic regions associated to the CLB disease. These accessions were evaluated for CLB in different regions of the southeastern United States over 3 years. In total, the study identified 99 Single Nucleotide Polymorphism (SNPs) associated with the disease severity and 85 SNPs associated with disease incidence. Across multiple environments, 47 disease severity SNPs and 23 incidence SNPs were common. Candidate genes within 10 kb of these SNPs were involved in biotic and abiotic stress pathways. This information will contribute to the development of resistant soybean germplasm. Further research is warranted to study the effect of pyramiding desirable genomic regions and investigate the role of identified genes in soybean CLB resistance.

A chromosome-scale assembly for 'd'Anjou' pear.

Cultivated pear consists of several Pyrus species with Pyrus communis (European pear) representing a large fraction of worldwide production. As a relatively recently domesticated crop and perennial tree, pear can benefit from genome-assisted breeding. Additionally, comparative genomics within Rosaceae promises greater understanding of evolution within this economically important family. Here, we generate a fully phased chromosome-scale genome assembly of P. communis 'd'Anjou.' Using PacBio HiFi and Dovetail Omni-C reads, the genome is resolved into the expected 17 chromosomes, with each haplotype totaling nearly 540 Megabases and a contig N50 of nearly 14 Mb. Both haplotypes are highly syntenic to each other and to the Malus domestica 'Honeycrisp' apple genome. Nearly 45,000 genes were annotated in each haplotype, over 90% of which have direct RNA-seq expression evidence. We detect signatures of the known whole-genome duplication shared between apple and pear, and we estimate 57% of d'Anjou genes are retained in duplicate derived from this event. This genome highlights the value of generating phased diploid assemblies for recovering the full allelic complement in highly heterozygous crop species.

Deciphering the genetic architecture of resistance to Corynespora cassiicola in soybean (Glycine max L.) by integrating genome-wide association mapping and RNA-Seq analysis.

Target spot caused by Corynespora cassiicola is a problematic disease in tropical and subtropical soybean (Glycine max) growing regions. Although resistant soybean genotypes have been identified, the genetic mechanisms underlying target spot resistance has not yet been studied. To address this knowledge gap, this is the first genome-wide association study (GWAS) conducted using the SoySNP50K array on a panel of 246 soybean accessions, aiming to unravel the genetic architecture of resistance. The results revealed significant associations of 14 and 33 loci with resistance to LIM01 and SSTA C. cassiicola isolates, respectively, with six loci demonstrating consistent associations across both isolates. To identify potential candidate genes within GWAS-identified loci, dynamic transcriptome profiling was conducted through RNA-Seq analysis. The analysis involved comparing gene expression patterns between resistant and susceptible genotypes, utilizing leaf tissue collected at different time points after inoculation. Integrating results of GWAS and RNA-Seq analyses identified 238 differentially expressed genes within a 200 kb region encompassing significant quantitative trait loci (QTLs) for disease severity ratings. These genes were involved in defense response to pathogen, innate immune response, chitinase activity, histone H3-K9 methylation, salicylic acid mediated signaling pathway, kinase activity, and biosynthesis of flavonoid, jasmonic acid, phenylpropanoid, and wax. In addition, when combining results from this study with previous GWAS research, 11 colocalized regions associated with disease resistance were identified for biotic and abiotic stress. This finding provides valuable insight into the genetic resources that can be harnessed for future breeding programs aiming to enhance soybean resistance against target spot and other diseases simultaneously.

Comparative Transcriptome Profiling Unfolds a Complex Defense and Secondary Metabolite Networks Imparting Corynespora cassiicola Resistance in Soybean (Glycine max (L.) Merrill).

Target spot is caused by Corynespora cassiicola, which heavily affects soybean production areas that are hot and humid. Resistant soybean genotypes have been identified; however, the molecular mechanisms governing resistance to infection are unknown. Comparative transcriptomic profiling using two known resistant genotypes and two susceptible genotypes was performed under infected and control conditions to understand the regulatory network operating between soybean and C. cassiicola. RNA-Seq analysis identified a total of 2571 differentially expressed genes (DEGs) which were shared by all four genotypes. These DEGs are related to secondary metabolites, immune response, defense response, phenylpropanoid, and flavonoid/isoflavonoid pathways in all four genotypes after C. cassiicola infection. In the two resistant genotypes, additional upregulated DEGs were identified affiliated with the defense network: flavonoids, jasmonic acid, salicylic acid, and brassinosteroids. Further analysis led to the identification of differentially expressed transcription factors, immune receptors, and defense genes with a leucine-rich repeat domain, dirigent proteins, and cysteine (C)-rich receptor-like kinases. These results will provide insight into molecular mechanisms of soybean resistance to C. cassiicola infection and valuable resources to potentially pyramid quantitative resistance loci for improving soybean germplasm.

Establishment of an Efficient Agrobacterium-Mediated Genetic Transformation System to Enhance the Tolerance of the Paraquat Stress in Engineering Goosegrass (Eleusine Indica L.).

Eleusine indica (goosegrass) is a problematic weed worldwide known for its multi-herbicide tolerance/resistance biotype. However, a genetic transformation method in goosegrass has not been successfully established, making a bottleneck for functional genomics studies in this species. Here, we report a successful Agrobacterium-mediated transformation method for goosegrass. Firstly, we optimized conditions for breaking seed dormancy and increasing seed germination rate. A higher callus induction rate from germinated seeds was obtained in N6 than in MS or B5 medium. Then the optimal transformation efficiency of the gus reporter gene was obtained by infection with Agrobacterium tumefaciens culture of OD600 = 0.5 for 30 min, followed by 3 days of co-cultivation with 300 µmol/L acetosyringone. Concentrations of 20 mg L-1 kanamycin and 100 mg L-1 timentin were used to select the transformed calli. The optimal rate of regeneration of the calli was generated by using 0.50 mg L-1 6-BA and 0.50 mg L-1 KT in the culture medium. Then, using this transformation method, we overexpressed the paraquat-resistant EiKCS gene into a paraquat-susceptible goosegrass biotype MZ04 and confirmed the stable inheritance of paraquat-resistance in the transgenic goosegrass lines. This approach may provide a potential mechanism for the evolution of paraquat-resistant goosegrass and a promising gene for the manipulation of paraquat-resistance plants. This study is novel and valuable in future research using similar methods for herbicide resistance.

QTL mapping and identification of candidate genes linked to red rot resistance in sugarcane.

Sugarcane (Saccharum species hybrid) is one of the most important commercial crops cultivated worldwide for products like white sugar, bagasse, ethanol, etc. Red rot is a major sugarcane disease caused by a hemi-biotrophic fungus, Colletotrichum falcatum Went., which can potentially cause a reduction in yield up to 100%. Breeding for red rot-resistant sugarcane varieties has become cumbersome due to its complex genome and frequent generation of new pathotypes of red rot fungus. In the present study, a genetic linkage map was developed using a selfed population of a popular sugarcane variety CoS 96268. A QTL linked to red rot resistance (qREDROT) was identified, which explained 26% of the total phenotypic variation for the trait. A genotype-phenotype network analysis performed to account for epistatic interactions, identified the key markers involved in red rot resistance. The differential expression of the genes located in the genomic region between the two flanking markers of the qREDROT as well as in the vicinity of the markers identified through the genotype-phenotype network analysis in a set of contrasting genotypes for red rot infection further confirmed the mapping results. Further, the expression analysis revealed that the plant defense-related gene coding 26S protease regulatory subunit is strongly associated with the red rot resistance. The findings can help in the screening of disease resistant genotypes for developing red rot-resistant varieties of sugarcane. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03481-7.

Identification of small effect quantitative trait loci of plant architectural, flowering, and early maturity traits in reciprocal interspecific introgression population in cotton.

Plant architecture, flowering time and maturity traits are important determinants of yield and fiber quality of cotton. Genetic dissection of loci determining these yield and quality components is complicated by numerous loci with alleles conferring small differences. Therefore, mapping populations segregating for smaller numbers and sizes of introgressed segments is expected to facilitate dissection of these complex quantitative traits. At an advanced stage in the development of reciprocal advanced backcross populations from crosses between elite Gossypium hirsutum cultivar 'Acala Maxxa' (GH) and G. barbadense 'Pima S6' (GB), we undertook mapping of plant architectural traits, flowering time and maturity. A total of 284 BC4F1 and BC4F2 progeny rows, 120 in GH and 164 in GB background, were evaluated for phenotype, with only 4 and 3 (of 7) traits showing significant differences among progenies. Genotyping by sequencing yielded 3,186 and 3,026 SNPs, respectively, that revealed a total of 27 QTLs in GH background and 22 in GB, for plant height, days to flowering, residual flowering at maturity and maturity. More than of 90% QTLs identified in both backgrounds had small effects (%PV < 10), supporting the merit of this population structure to reduce background noise and small effect QTLs. Germplasm developed in this study may serve as potential pre-breeding material to develop improved cotton cultivars.

Improved Upland Cotton Germplasm for Multiple Fiber Traits Mediated by Transferring and Pyramiding Novel Alleles From Ethyl Methanesulfonate-Generated Mutant Lines Into Elite Genotypes.

Ethyl methanesulfonate (EMS) mutagenesis offers important advantages for improving crops, such as cotton, with limited diversity in elite gene pools. EMS-induced point mutations are less frequently associated with deleterious traits than alleles from wild or exotic germplasm. From 157 mutant lines that have significantly improved fiber properties, we focused on nine mutant lines here. A total of eight populations were developed by crossing mutant lines in different combinations into GA230 (GA2004230) background. Multiple lines in each population were significantly improved for the fiber trait that distinguished the donor parent(s), demonstrating that an elite breeding line (GA230) could be improved for fiber qualities using the mutant lines. Genotypes improved for multiple fiber traits of interest suggesting that allele pyramiding is possible. Compared to midparent values, individual progeny in the population conferred fiber quality improvements of as much as 31.7% (in population O) for micronaire (MIC), 16.1% (in population P) for length, 22.4% (in population K) for strength, 4.1% (in population Q) for uniformity, 45.8% (in population N) for elongation, and 13.9% (in population O) for lint percentage (lint%). While further testing for stability of the phenotype and estimation of yield potential is necessary, mutation breeding shows promise as an approach to reduce the problem of the genetic bottleneck of upland cotton. The populations developed here may also contribute to identifying candidate genes and causal mutations for fiber quality improvement.

Insights into the Genomic Architecture of Seed and Pod Quality Traits in the U.S. Peanut Mini-Core Diversity Panel.

Traits such as seed weight, shelling percent, percent sound mature kernels, and seed dormancy determines the quality of peanut seed. Few QTL (quantitative trait loci) studies using biparental mapping populations have identified QTL for seed dormancy and seed grade traits. Here, we report a genome-wide association study (GWAS) to detect marker-trait associations for seed germination, dormancy, and seed grading traits in peanut. A total of 120 accessions from the U.S. peanut mini-core collection were evaluated for seed quality traits and genotyped using Axiom SNP (single nucleotide polymorphism) array for peanut. We observed significant variation in seed quality traits in different accessions and different botanical varieties. Through GWAS, we were able to identify multiple regions associated with sound mature kernels, seed weight, shelling percent, seed germination, and dormancy. Some of the genomic regions that were SNP associated with these traits aligned with previously known QTLs. For instance, QTL for seed dormancy has been reported on chromosome A05, and we also found SNP on the same chromosome associated with seed dormancy, explaining around 20% of phenotypic variation. In addition, we found novel genomic regions associated with seed grading, seed germination, and dormancy traits. SNP markers associated with seed quality and dormancy identified here can accelerate the selection process. Further, exploring the function of candidate genes identified in the vicinity of the associated marker will assist in understanding the complex genetic network that governs seed quality.

Detection of subgenome bias using an anchored syntenic approach in Eleusine coracana (finger millet).

BACKGROUND: Finger millet (Eleusine coracana 2n = 4x = 36) is a hardy, nutraceutical, climate change tolerant, orphan crop that is consumed throughout eastern Africa and India. Its genome has been sequenced multiple times, but A and B subgenomes could not be separated because no published genome for E. indica existed. The classification of A and B subgenomes is important for understanding the evolution of this crop and provide a means to improve current and future breeding programs. RESULTS: We produced subgenome calls for 704 syntenic blocks and inferred A or B subgenomic identity for 59,377 genes 81% of the annotated genes. Phylogenetic analysis of a super matrix containing 455 genes shows high support for A and B divergence within the Eleusine genus. Synonymous substitution rates between A and B genes support A and B calls. The repetitive content on highly supported B contigs is higher than that on similar A contigs. Analysis of syntenic singletons showed evidence of biased fractionation showed a pattern of A genome dominance, with 61% A, 37% B and 1% unassigned, and was further supported by the pattern of loss observed among cyto-nuclear interacting genes. CONCLUSION: The evidence of individual gene calls within each syntenic block, provides a powerful tool for inference for subgenome classification. Our results show the utility of a draft genome in resolving A and B subgenomes calls, primarily it allows for the proper polarization of A and B syntenic blocks. There have been multiple calls for the use of phylogenetic inference in subgenome classification, our use of synteny is a practical application in a system that has only one parental genome available.

The Ligon lintless -2 Short Fiber Mutation Is Located within a Terminal Deletion of Chromosome 18 in Cotton.

Extreme elongation distinguishes about one-fourth of cotton (Gossypium sp.) seed epidermal cells as "lint" fibers, useful for the textile industry, from "fuzz" fibers (<5 mm). Ligon lintless-2 (Li 2 ), a dominant mutation that results in no lint fiber but normal fuzz fiber, offers insight into pathways and mechanisms that differentiate spinnable cotton from its progenitors. A genetic map developed using 1,545 F2 plants showed that marker CISP15 was 0.4 cM from Li 2 , and "dominant" simple sequence repeat (SSR) markers (i.e. with null alleles in the Li 2 genotype) SSR7 and SSR18 showed complete linkage with Li 2 Nonrandom distribution of markers with null alleles suggests that the Li 2 phenotype results from a 176- to 221-kb deletion of the terminal region of chromosome 18 that may have been masked in prior pooled-sample mapping strategies. The deletion includes 10 genes with putative roles in fiber development. Two Glycosyltransferase Family 1 genes showed striking expression differences during elongation of wild-type versus Li 2 fiber, and virus-induced silencing of these genes in the wild type induced Li 2 -like phenotypes. Further, at least 7 of the 10 putative fiber development genes in the deletion region showed higher expression in the wild type than in Li 2 mutants during fiber development stages, suggesting coordinated regulation of processes in cell wall development and cell elongation, consistent with the hypothesis that some fiber-related quantitative trait loci comprise closely spaced groups of functionally diverse but coordinately regulated genes.

Role of Endobronchial Ultrasound-guided Transbronchial Needle Aspiration in the Diagnosis and Management of Mediastinal Cyst.

BACKGROUND: Foregut cysts account for >50% of cystic lesions in the mediastinum, of which bronchogenic cysts are most common. Surgical resection is the most definitive approach for its diagnosis and treatment. A recent systematic review, however, suggests that endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has a role in the management of bronchogenic cyst. We report our experience with EBUS-TBNA in the diagnosis and management of bronchogenic cysts. METHODS: Medical records of patients with evidence of mediastinal cysts who underwent EBUS-TBNA between 2008 and 2016 were reviewed.The primary aims of this study were to assess EBUS-TBNA diagnostic yield of peri-bronchial cysts and their specific type/origin and to determine its short-term and long-term drainage efficacy. RESULTS: A total of 26 patients met the inclusion criteria. The cytopathology diagnosis was compatible with bronchogenic cyst in 4 cases, pleural-pericardial cyst in 3 cases, and 19 were indeterminate cysts. Successful long-term treatment occurred in 5.5% of the subjects. One patient developed inflammatory pericarditis after EBUS-TBNA. CONCLUSION: Diagnostic and therapeutic yield of EBUS-TBNA for mediastinal cysts is limited and surgical resection remains the treatment of choice.

A novel mutation A212T in chloroplast Protoporphyrinogen oxidase (PPO1) confers resistance to PPO inhibitor Oxadiazon in Eleusine indica.

BACKGROUND: Protoporphyrinogen oxidase (PPO) with two isoforms, chloroplast-targeted (PPO1) and mitochondrial-targeted (PPO2), catalyzes a step in the biosynthesis of chlorophyll and heme. PPO1 and PPO2 are herbicide target sites of PPO-inhibiting herbicides. Target-site mutations conferring resistance to PPO inhibitors have all thus far been in PPO2. Oxadiazon is a unique PPO inhibitor utilized for preemergence Eleusine indica control. In this research, we evaluated the response of two previously confirmed oxadiazon-resistant and susceptible E. indica biotypes to other PPO inhibitors and identified the resistance mechanism in two oxadiazon-resistant E. indica biotypes. RESULTS: Two E. indica biotypes were resistant to oxadiazon, but not to other structurally unrelated PPO inhibitors, such as lactofen, flumioxazin and sulfentrazone. A novel mutation A212T was identified in the chloroplast-targeted PPO1, conferring resistance to oxadiazon in a heterologous expression system. Computational structural modeling provided a mechanistic explanation for reduced herbicide binding to the variant protein: the presence of a methyl group of threonine 212 changes the PPO1 active site and produces repulsive electrostatic interactions that repel oxadiazon from the binding pocket. CONCLUSION: The novel A212T mutation in PPO1 conferring resistance specifically to PPO inhibitor oxadiazon was characterized. This is the first evidence of the direct role of PPO1 in the PPO mode of action, and the first evidence of evolved resistance in PPO1. © 2019 Society of Chemical Industry.

Transcriptome Analysis Reveals Unique Relationships Among Eleusine Species and Heritage of Eleusine coracana.

Relationships in the genus Eleusine were obtained through transcriptome analysis. Eleusine coracana (E. coracana ssp. coracana), also known as finger millet, is an allotetraploid minor crop primarily grown in East Africa and India. Domesticated E. coracana evolved from wild E. africana (E. coracana ssp. africana) with the maternal genome donor largely supported to be E. indica; however, the paternal genome donor remains elusive. We developed transcriptomes for six Eleusine species from fully developed seedlings using Illumina technology and three de novo assemblers (Trinity, Velvet, and SOAPdenovo2) with the redundancy-reducing EvidentialGene pipeline. Mapping E. coracana reads to the chloroplast genes of all Eleusine species detected fewer variants between E. coracana and E. indica compared to all other species. Phylogenetic analysis further supports E. indica as the maternal parent of E. coracana and E. africana, in addition to a close relationship between E. indica and E. tristachya, and between E. floccifolia and E. multiflora, and E. intermedia as a separate group. A close relationship between E. floccifolia and E. multiflora was unexpected considering they are reported to have distinct nuclear genomes, BB and CC, respectively. Further, it was expected that E. intermedia and E. floccifolia would have a closer relationship considering they have similar nuclear genomes, AB and BB, respectively. A rethinking of the labeling of ancestral genomes of E. floccifolia, E. multiflora, and E. intermedia is maybe needed based on this data.

Development of a goosegrass (Eleusine indica) draft genome and application to weed science research.

BACKGROUND: Genomes are vital to the study of genomics, population genetics, and evolution of species. To date, only one genome (Echinochloa crus-galli) for C4 annual weedy grass species has been sequenced. Research was conducted to develop a draft genome of goosegrass (Eleusine indica; 2n = 2x = 18), one of the most common and troublesome weeds in the world. RESULTS: A draft assembly of an approximately 492 Mb whole-genome sequence of goosegrass was obtained by de novo assembly of paired-end and mate-paired reads generated by Illumina sequencing of total genomic DNA. The genome was assembled into 24,072 scaffolds with N50 = 233,459 bp. More than 99% of transcriptome sequences were mapped to the goosegrass draft genome, and 95% of the commonly conserved plant genes were present. The assembled genome contains 25,467 unique protein-coding genes. Genes associated with herbicide resistance were obtained and variant calling allowed the detection of 754,409 single nucleotide polymorphisms. In addition, we also report 115,417 simple sequence repeats which can be deployed in population genetics and phylogenetic analysis. CONCLUSION: This is the first report of genome sequence of goosegrass. Our assembly was able to identify all major herbicide-resistance related genes and develop a useful tool for other genomic and evolutionary analysis. © 2019 Society of Chemical Industry.

Genetic Analysis of Gossypium Fiber Quality Traits in Reciprocal Advanced Backcross Populations.

In mapping populations segregating for many loci, the large amount of variation among genotypes often masks small-effect quantitative trait loci (QTL). This problem can be reduced by development of populations with fewer chromosome segments segregating. Here, we report early QTL detection in reciprocal advanced backcross populations from crosses between elite Gossypium hirsutum L. 'Acala Maxxa' (GH) and G. barbadense L. 'Pima S6' (GB). A total of 297 BCF and BCF progeny rows-127 segregating for GB chromosome segments in GH background and 170 segregating for GH chromosome segments in GB background-were evaluated in three environments. Totals of 3186 and 3026 polymorphic single-nucleotide polymorphisms (SNPs) in GH and GB backgrounds, respectively, were identified and used for trait mapping. Small-effect QTL (<10% variance explained) made up 87 and 100% of QTL in GH and GB backgrounds, respectively. In both species, favorable alleles were found with effects being masked or neutralized by unfavorable alleles, with greater scope for improvement of GH than GB by introgressive breeding. A total of three stable QTL-two in GH background for fiber elongation (ELO) and micronaire (MIC) and one in GB background for upper-half mean length (UHM)-were identified in two out of three environments. Curiously, only four QTL-three for UHM and one for ELO-showed the expected opposite effects in reciprocal backgrounds, perhaps reflecting the combined consequences of epistasis, small phenotypic effects, and low coverage of some genomic regions. Along with new information for marker-assisted breeding, this study adds to knowledge that can be used to unravel complex genetic networks governing fiber quality traits.

Clinical and radiological outcome of arthrocentesis followed by autologous blood injection for treatment of chronic recurrent temporomandibular joint dislocation.

BACKGROUND: This study was conducted to evaluate the functional outcome and MRI findings of arthrocentsis followed by autologous blood injection (ABI) into the joint space for management of chronic recurrent TMJ dislocation. MATERIAL AND METHODS: Total ten patients with bilateral chronic recurrent condylar dislocation were included in the study. Arthrocentesis of both TMJ was performed on each patient, followed by the injection of 2 ml of autologous blood into the superior joint compartment and 1 ml onto the outer surface of the joint capsule. Preoperative and postoperative assessment included; thorough history, clinical examination of TMJ, maximal mouth opening, frequency of dislocation, TMJ radiographs (open and closed mouth position), MRI, recurrence and presence of facial nerve paralysis. RESULTS: At the end of 3 months follow-up 8 patients (80%) had successful outcome with no further episodes of dislocation, whereas two patients reported with recurrence. Post-operative MRI showed significant improvement after ABI, compared to pre-operative MRI. There were no degenerative changes to the bony and soft tissue components of TMJ. CONCLUSIONS: ABI is a simple, safe, minimally invasive and cost-effective technique for treatment of chronic recurrent TMJ dislocation. MRI evaluation showed an improvement in the anatomical and spatial relationship of the osseous and soft tissue components of the TMJ. Key words:TMJ lavage, luxation, fibrosis, magnetic resonance imaging.

Dental, dermatological and radiographic findings in a case of Gorlin-Goltz Syndrome: report and review.

Gorlin-Goltz syndrome (GGS) is a rare autosomal dominant disorder. The disease shows multiple organ involvement with variable clinical presentation. Thus a multidisciplinary approach is required for its prompt clinical diagnosis and management of this condition. This paper highlights a case of GGS presenting in a young male patient with cranial, facial, dermatological, dental and skeletal involvement. The diagnosis of the syndrome was based on its clinical presentation, radiological features and histopathological findings. A review of the diagnostic criteria is also presented.

Interobserver agreement in the cytologic grading of atypia in neoplastic pancreatic mucinous cysts with the 2-tiered approach.

BACKGROUND: The accurate cytologic grading of epithelial atypia in fine-needle aspirates of pancreatic mucinous cysts has important implications for clinical management. The Papanicolaou Society of Cytopathology has recommended a 2-tiered system of low-grade (LG) and high-grade (HG) for grading this atypia. Using this approach, this study examined the interobserver agreement within a group of cytopathologists at the Cleveland Clinic. METHODS: Twenty cases of fine-needle aspiration of pancreatic neoplastic mucinous cysts with documented histologic follow-up and representative lesional cells were selected. Blinded to the histologic outcome, 4 cytopathologists were independently asked to assign the highest grade of atypia with the 2-tiered system of LG and HG atypia for these cases. The interobserver agreement was calculated with the κ statistic. RESULTS: The overall raw agreement in the grading of atypia was 60%. The overall chance-adjusted agreement was fair (κ = 0.28). On the basis of the histologic outcomes, the cases were stratified into group A (HG dysplasia or worse) and group B (LG or intermediate-grade [IG] dysplasia on follow-up). Group A (n = 12) showed good chance-adjusted agreement (κ = 0.65). For group B, the chance-adjusted agreement among the observers was poor (κ = 0.03). CONCLUSIONS: This study shows that the cytologic recognition of HG dysplasia or worse as HG atypia in pancreatic mucinous cysts has a good degree of interobserver reproducibility among cytopathologists. In contrast, a problematic area with a lack of agreement appears to be the cytologic recognition of LG and IG dysplasia as LG atypia. Additional studies with the development of reproducible criteria and educational tools may help with this challenging distinction. Cancer Cytopathol 2016;124:909-916. © 2016 American Cancer Society.