Transcriptional Profile of Human Pancreatic Acinar Ductal Metaplasia.

BACKGROUND AND AIMS: Aberrant acinar to ductal metaplasia (ADM), one of the earliest events involved in exocrine pancreatic cancer development, is typically studied using pancreata from genetically engineered mouse models. METHODS: We used primary, human pancreatic acinar cells from organ donors to evaluate the transcriptional and pathway profiles during the course of ADM. RESULTS: Following 6 days of three-dimensional culture on Matrigel, acinar cells underwent morphological and molecular changes indicative of ADM. mRNA from 14 donors' paired cells (day 0, acinar phenotype and day 6, ductal phenotype) was subjected to whole transcriptome sequencing. Acinar cell specific genes were significantly downregulated in the samples from the day 6 cultures while ductal cell-specific genes were upregulated. Several regulons of ADM were identified including transcription factors with reduced activity (PTF1A, RBPJL, and BHLHA15) and those ductal and progenitor transcription factors with increased activity (HNF1B, SOX11, and SOX4). Cells with the ductal phenotype contained higher expression of genes increased in pancreatic cancer while cells with an acinar phenotype had lower expression of cancer-associated genes. CONCLUSION: Our findings support the relevancy of human in vitro models to study pancreas cancer pathogenesis and exocrine cell plasticity.

A genomic data archive from the Network for Pancreatic Organ donors with Diabetes.

The Network for Pancreatic Organ donors with Diabetes (nPOD) is the largest biorepository of human pancreata and associated immune organs from donors with type 1 diabetes (T1D), maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes (CFRD), type 2 diabetes (T2D), gestational diabetes, islet autoantibody positivity (AAb+), and without diabetes. nPOD recovers, processes, analyzes, and distributes high-quality biospecimens, collected using optimized standard operating procedures, and associated de-identified data/metadata to researchers around the world. Herein describes the release of high-parameter genotyping data from this collection. 372 donors were genotyped using a custom precision medicine single nucleotide polymorphism (SNP) microarray. Data were technically validated using published algorithms to evaluate donor relatedness, ancestry, imputed HLA, and T1D genetic risk score. Additionally, 207 donors were assessed for rare known and novel coding region variants via whole exome sequencing (WES). These data are publicly-available to enable genotype-specific sample requests and the study of novel genotype:phenotype associations, aiding in the mission of nPOD to enhance understanding of diabetes pathogenesis to promote the development of novel therapies.

Pharmacological inhibition and reversal of pancreatic acinar ductal metaplasia.

Pancreatic acinar cells display a remarkable degree of plasticity and can dedifferentiate into ductal-like progenitor cells by a process known as acinar ductal metaplasia (ADM). ADM is believed to be one of the earliest precursor lesions toward the development of pancreatic ductal adenocarcinoma and maintaining the pancreatic acinar cell phenotype suppresses tumor formation. The effects of a novel pStat3 inhibitor (LLL12B) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were investigated using 3-D cultures from p48Cre/+ and p48Cre/+LSL-KrasG12D/+ (KC) mice. LLL12B and TSA inhibited ADM in both KC and p48Cre/+ mouse pancreatic organoids. Furthermore, treatment with LLL12B or TSA on dedifferentiated acini from p48Cre/+ and KC mice that had undergone ADM produced morphologic and gene expression changes that suggest a reversal of ADM. Validation experiments using qRT-PCR (p48Cre/+ and KC) and RNA sequencing (KC) of the LLL12B and TSA treated cultures showed that the ADM reversal was more robust for the TSA treatments. Pathway analysis showed that TSA inhibited Spink1 and PI3K/AKT signaling during ADM reversal. The ability of TSA to reverse ADM was also observed in primary human acinar cultures. We report that pStat3 and HDAC inhibition can attenuate ADM in vitro and reverse ADM in the context of wild-type Kras. Our findings suggest that pharmacological inhibition or reversal of pancreatic ADM represents a potential therapeutic strategy for blocking aberrant ductal reprogramming of acinar cells.

The vaginal microbiome is associated with endometrial cancer grade and histology.

The human microbiome has been strongly correlated with disease pathology and outcomes, yet remains relatively underexplored in patients with malignant endometrial disease. In this study, vaginal microbiome samples were prospectively collected at the time of hysterectomy from 61 racially and ethnically diverse patients from three disease conditions: 1) benign gynecologic disease (controls, n=11), 2) low-grade endometrial carcinoma (n=30), and 3) high-grade endometrial carcinoma (n=20). Extracted DNA underwent shotgun metagenomics sequencing, and microbial α and ß diversities were calculated. Hierarchical clustering was used to describe community state types (CST), which were then compared by microbial diversity and grade. Differential abundance was calculated, and machine learning utilized to assess the predictive value of bacterial abundance to distinguish grade and histology. Both α- and ß-diversity were associated with patient tumor grade. Four vaginal CST were identified that associated with grade of disease. Different histologies also demonstrated variation in CST within tumor grades. Using supervised clustering algorithms, critical microbiome markers at the species level were used to build models that predicted benign vs carcinoma, high-grade carcinoma versus benign, and high-grade versus low-grade carcinoma with high accuracy. These results confirm that the vaginal microbiome segregates not just benign disease from endometrial cancer, but is predictive of histology and grade. Further characterization of these findings in large, prospective studies is needed to elucidate their potential clinical applications.

Digital Pathology in Cameroon.

PURPOSE: Cancer is becoming increasingly prevalent among the group of treatable diseases in African countries. There is a shortage of clinicians and pathologists available for cancer diagnosis and treatment. These limited resources must be efficiently used to maximize the number of patients treated. One of the critical factors in treatment efficiency is the correct and timely diagnosis of specimens by pathologists. However, there is currently a significant shortage of cancer care clinicians in Africa and an even more considerable shortage of pathologists. This article presents an example in which telepathology was used to mitigate the lack of pathologists in Cameroon. METHODS: The telepathology workaround was implemented in a district hospital based in Cameroon's Adamawa region, where a European surgeon provides cancer treatment. A small histology laboratory there is run by one histotechnologist who processes surgical biopsies into histology slides. As there are no pathologists on site, these slides are digitally scanned using a mobile phone and a whole slide imaging (WSI) scanner. The slides are then shared electronically with a volunteering pathologist in Europe who provides a diagnostic report. RESULTS: From 2018 to July 2019, specimens for 101 patients were photographed through an iPhone connected to a microscope eyepiece producing several individual images per specimen. From July 2019 to December 2020, slides from 282 patients were scanned using WSI and digitally transmitted. CONCLUSION: WSI on hematoxylin and eosin histology slides for remote diagnosis can increase cancer treatment efficacy and reduce overtreatment of tumors clinically suspicious for malignancy in under-resourced countries with a lack of pathologists.

The Hologic Aptima SARS-CoV-2 assay enables high ratio pooling saving reagents and improving turnaround time.

BACKGROUND: The Hologic Aptima™ TMA SARS-CoV-2 assay was employed to test pooled nasopharyngeal (NP) samples to evaluate the performance of pooled sample testing and characterize variables influencing results. METHODS: Results on 1033 previously tested NP samples were retrieved to characterize the relative light units (RLU) of SARS-CoV-2-positive samples in the tested population. The pooling strategy of combining 10 SARS-CoV-2 samples into one pool (10/1) was used in this study. The results were compared with neat sample testing using the same Aptima™ TMA SARS-CoV-2 assay and also the CDC RT-PCR and the Cepheid SARS-CoV-2 assays. RESULTS: The Aptima assay compares favorably with both CDC RT-PCR and the Cepheid SARS-CoV-2 assays. Once samples are pooled 10 to 1 as in our experiments, the resulting signal strength of the assay suffers. A divide opens between pools assembled from strong-positive versus only weak-positive samples. Pools of the former can be reliably detected with positive percent agreement (PPA) of 95.2%, while pools of the latter are frequently misclassified as negative with PPA of 40%. When the weak-positive samples with kRLU value lower than 1012 constitute 3.4% of the total sample profile, the assay PPA approaches 93.4% suggesting that 10/1 pooled sample testing by the Aptima assay is an effective screening tool for SARS-CoV-2. CONCLUSION: Performing pooled testing, one should monitor the weak positives with kRLU lower than 1012 or quantification cycle (Cq) value higher than 35 on an ongoing basis and adjust pooling approaches to avoid reporting false negatives.

Wrong Tissue in Block.

OBJECTIVES: Maintaining specimen identity during surgical pathology tissue processing is critical. Epic Beaker Laboratory Information System requires sequential scanning of specimen label and grossed blocks (block confirmation) to ensure specimen identity. We report our institution's experience with wrong tissue in block (WTIB) grossing errors before and after adopting block confirmation. METHODS: During the first 18 months of Beaker implementation, block confirmation was not required. We then mandated block confirmation for a 3-month period. To ensure compliance, we then built a "hard stop" feature that prevents scanning any unconfirmed blocks onto a packing list. We reviewed WTIB incidents pre- and postimplementation of these solutions. RESULTS: Before using block confirmation, we had WTIB incidents involving 17 (0.043%) of 38,848 cases. When we mandated block confirmation use, we had WTIB involving 2 (0.043%) of 4,646 cases. After implementing the hard stop feature, we had WTIB incidents involving 2 (0.005%) of 42,411 cases. Overall, there was an 88.4% (0.043% vs 0.005%; P < .001) reduction in WTIB incidents using block confirmation with a hard stop. CONCLUSIONS: Beaker is a customizable platform that can be tailored to a laboratory's workflow. By using barcoding, implementing custom-built features, and improving workflow protocols, we significantly reduced WTIB errors.

vcf2fhir: a utility to convert VCF files into HL7 FHIR format for genomics-EHR integration.

BACKGROUND: VCF formatted files are the lingua franca of next-generation sequencing, whereas HL7 FHIR is emerging as a standard language for electronic health record interoperability. A growing number of FHIR-based clinical genomics applications are emerging. Here, we describe an open source utility for converting variants from VCF format into HL7 FHIR format. RESULTS: vcf2fhir converts VCF variants into a FHIR Genomics Diagnostic Report. Conversion translates each VCF row into a corresponding FHIR-formatted variant in the generated report. In scope are simple variants (SNVs, MNVs, Indels), along with zygosity and phase relationships, for autosomes, sex chromosomes, and mitochondrial DNA. Input parameters include VCF file and genome build ('GRCh37' or 'GRCh38'); and optionally a conversion region that indicates the region(s) to convert, a studied region that lists genomic regions studied by the lab, and a non-callable region that lists studied regions deemed uncallable by the lab. Conversion can be limited to a subset of VCF by supplying genomic coordinates of the conversion region(s). If studied and non-callable regions are also supplied, the output FHIR report will include 'region-studied' observations that detail which portions of the conversion region were studied, and of those studied regions, which portions were deemed uncallable. We illustrate the vcf2fhir utility via two case studies. The first, 'SMART Cancer Navigator', is a web application that offers clinical decision support by linking patient EHR information to cancerous gene variants. The second, 'Precision Genomics Integration Platform', intersects a patient's FHIR-formatted clinical and genomic data with knowledge bases in order to provide on-demand delivery of contextually relevant genomic findings and recommendations to the EHR. CONCLUSIONS: Experience to date shows that the vcf2fhir utility can be effectively woven into clinically useful genomic-EHR integration pipelines. Additional testing will be a critical step towards the clinical validation of this utility, enabling it to be integrated in a variety of real world data flow scenarios. For now, we propose the use of this utility primarily to accelerate FHIR Genomics understanding and to facilitate experimentation with further integration of genomics data into the EHR.

Building a precision oncology workforce by multidisciplinary and case-based learning.

BACKGROUND: Participants in two recent National Academy of Medicine workshops identified a need for more multi-disciplinary professionals on teams to assist oncology clinicians in precision oncology. METHODS: We developed a graduate school course to prepare biomedical students and pharmacy students to work within a multidisciplinary team of oncology clinicians, pathologists, radiologists, clinical pharmacists, and genetic counselors. Students learned precision oncology skills via case-based learning, hands-on data analyses, and presentations to peers. After the course, a focus group session was conducted to gain an in-depth student perspective on their interprofessional training experience, achievement of the course learning outcomes, ways to improve the course design in future offerings, and how the course could improve future career outcomes. A convenience sampling strategy was used for recruitment into the focus group session. A thematic content analysis was then conducted using the constant comparative method. RESULTS: Major themes arising from student feedback were (1) appreciation of a customized patient case-based teaching approach, (2) more emphasis on using data analysis tools, (3) valuing interdisciplinary inclusion, and (4) request for more student discussion with advanced preparation materials. CONCLUSIONS: Feedback was generally positive and supports the continuation and expansion of the precision oncology course to include more hands-on instruction on the use of clinical bioinformatic tools.

Integrative analyses of TEDDY Omics data reveal lipid metabolism abnormalities, increased intracellular ROS and heightened inflammation prior to autoimmunity for type 1 diabetes.

BACKGROUND: The Environmental Determinants of Diabetes in the Young (TEDDY) is a prospective birth cohort designed to study type 1 diabetes (T1D) by following children with high genetic risk. An integrative multi-omics approach was used to evaluate islet autoimmunity etiology, identify disease biomarkers, and understand progression over time. RESULTS: We identify a multi-omics signature that was predictive of islet autoimmunity (IA) as early as 1 year before seroconversion. At this time, abnormalities in lipid metabolism, decreased capacity for nutrient absorption, and intracellular ROS accumulation are detected in children progressing towards IA. Additionally, extracellular matrix remodeling, inflammation, cytotoxicity, angiogenesis, and increased activity of antigen-presenting cells are observed, which may contribute to beta cell destruction. Our results indicate that altered molecular homeostasis is present in IA-developing children months before the actual detection of islet autoantibodies, which opens an interesting window of opportunity for therapeutic intervention. CONCLUSIONS: The approach employed herein for assessment of the TEDDY cohort showcases the utilization of multi-omics data for the modeling of complex, multifactorial diseases, like T1D.

Disparities in Pancreatic Ductal Adenocarcinoma-The Significance of Hispanic Ethnicity, Subgroup Analysis, and Treatment Facility on Clinical Outcomes.

BACKGROUND: Disparities exist among patients with pancreatic ductal adenocarcinoma (PDAC). Non-White race is regarded as a negative predictor of expected treatment and overall survival. Data suggest that Academic Research Programs (ARP) provide better outcomes for minorities, but ethnic/minority outcomes are underreported. We hypothesize that outcomes among racially/ethnically diverse PDAC patients may be influenced by treatment facility. METHODS: The National Cancer Database was used to identify 170,327 patients diagnosed with PDAC between 2004 and 2015. Cox proportional-hazard regression was used to compare survival between race/ethnic groups across facilities. RESULTS: In unadjusted models, compared to non-Hispanic Whites (NHW), non-Hispanic Blacks (NHB) had the worst overall survival (HR = 1.05, 95%CI: 1.03-1.06, P < .001) and Hispanics had the best overall survival (HR = 0.92, 95%CI: 0.90-0.94, P < .001). After controlling for socioeconomic and clinical covariates, NHB (HR = 0.95, 95%CI: 0.93-0.96, P < .001) had better overall survival compared to NHW, and Hispanics continued to have the best comparative outcomes (HR = 0.84, 95%CI: 0.82-0.86, P < .001). Among Hispanics, Dominicans and South/Central Americans lived the longest, at 10.25 and 9.82 months, respectively. The improved survival in Hispanics was most pronounced at ARP (HR = 0.80, 95%CI: 0.77-0.84, P < .001) and Integrated Network Cancer Programs (HR = 0.78, 95%CI: 0.73-0.84, P < .001). NHB had improved survival over NHW at Comprehensive Community Care Programs (HR = 0.96, 95%CI: 0.93-0.98, P = .002) and ARP (HR = 0.96, 95%CI: 0.94-0.98, P = .001), which was influenced by income, education, and surgical resection. CONCLUSION: Survival was improved at ARP for all populations. Hispanics had the best comparative overall survival. NHB had improved overall survival at higher volume centers, but this was dependent upon income, education, and surgical resection.

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.

Endometrial Thickness as Measured by Transvaginal Ultrasound and the Corresponding Histopathologic Diagnosis in Women With Postmenopausal Bleeding.

Endometrial thickness as measured by transvaginal ultrasound (TVUS) is being increasingly used as a first-line method to evaluate patients with vaginal bleeding. Our study aims to examine correlation between the histopathologic diagnosis and the results of TVUS and find a threshold that could reliably exclude carcinoma. We included women, age 55 years and above, who presented with postmenopausal bleeding and had a TVUS within 30 days of their endometrial biopsy. Total of 304 patients met our criteria and were divided into 4 groups. Patients in group A (n=198) had benign/atrophic endometrium, group B (n=44) had polyps, group C (n=30) had hyperplasia, and group D (n=32) had carcinoma. The endometrial thickness obtained by TVUS was compared with the histopathologic finding of the endometrial biopsy. The mean endometrial thickness was 7.5, 12.1, 14.8, and 16.9 mm for groups A to D, respectively. Statistical analysis showed that very low endometrial thickness (3 to 4 mm) would be ideal to use as a threshold to maximize sensitivity. Three of 32 patients in group D had an endometrial thickness ≤4 mm. At a threshold of 4 mm, the sensitivity is 90.6% and increases to 96.9% when decreasing the threshold to 3 mm. However, other parameters such as test accuracy, specificity, and positive predictive values are very low at these thresholds. Sensitivity can be maximized to 96.9% using a threshold of 3 mm. However, this would call into question the cost-effectiveness of this method. Postmenopausal bleeding remains the most reliable indicator of endometrial pathology.

Potential use of low-copy nuclear genes in DNA barcoding: a comparison with plastid genes in two Hawaiian plant radiations.

BACKGROUND: DNA barcoding of land plants has relied traditionally on a small number of markers from the plastid genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes because of the absence of universal primers for PCR amplification. RESULTS: From pooled-species 454 transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic haplotypes compared to plastid genes and no interspecies gaps. CONCLUSIONS: The apparently greater coalescence times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from either genome were scarce on the youngest island; a minimum age of ca. two million years may be needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a superior alternative to slowly evolving plastid genes.

Primers for low-copy nuclear genes in the Hawaiian endemic Clermontia (Campanulaceae) and cross-amplification in Lobelioideae.

PREMISE OF THE STUDY: Primers were developed to amplify 12 intron-less, low-copy nuclear genes in the Hawaiian genus Clermontia (Campanulaceae), a suspected tetraploid. • METHODS AND RESULTS: Data from a pooled 454 titanium run of the partial transcriptomes of seven Clermontia species were used to identify the loci of interest. Most loci were amplified and sequenced directly with success in a representative selection of lobeliads even though several of these loci turned out to be duplicated. Levels of variation were comparable to those observed in commonly used plastid and ribosomal markers. • CONCLUSIONS: We found evidence of a genome duplication that likely predates the diversification of the Hawaiian lobeliads. Some genes nevertheless appear to be single-copy and should be useful for phylogenetic studies of Clermontia or the entire Lobelioideae subfamily.

Rapid, repeated, and clustered loss of duplicate genes in allopolyploid plant populations of independent origin.

The predictability of evolution is debatable, with recent evidence suggesting that outcomes may be constrained by gene interaction networks [1]. Whole-genome duplication (WGD; polyploidization-ubiquitous in plant evolution [2]) provides the opportunity to evaluate the predictability of genome reduction, a pervasive feature of evolution [3, 4]. Repeated patterns of genome reduction appear to have occurred via duplicated gene (homeolog) loss in divergent species following ancient WGD [5-9], with evidence for preferential retention of duplicates in certain gene classes [8-10]. The speed at which these patterns arise is unknown. We examined presence/absence of 70 homeologous loci in 59 Tragopogon miscellus plants from five natural populations of independent origin; this allotetraploid arose ~80 years ago via hybridization between diploid parents and WGD [11]. Genes were repeatedly retained or lost in clusters, and the gene ontology categories of the missing genes correspond to those lost after ancient WGD in the same family (Asteraceae; sunflower family) [6] and with gene dosage sensitivity [8]. These results provide evidence that the outcomes of WGD are predictable, even in 40 generations, perhaps due to the connectivity of gene products [8, 10, 12]. The high frequency of single-allele losses detected and low frequency of changes fixed within populations provide evidence for ongoing evolution.

Next-generation sequencing and genome evolution in allopolyploids.

PREMISE OF THE STUDY: Hybridization and polyploidization (allopolyploidy) are ubiquitous in the evolution of plants, but tracing the origins and subsequent evolution of the constituent genomes of allopolyploids has been challenging. Genome doubling greatly complicates genetic analyses, and this has long hindered investigation in that most allopolyploid species are "nonmodel" organisms. However, recent advances in sequencing and genomics technologies now provide unprecedented opportunities to analyze numerous genetic markers in multiple individuals in any organism. METHODS: Here we review the application of next-generation sequencing technologies to the study of three aspects of allopolyploid genome evolution: duplicated gene loss and expression in two recently formed Tragopogon allopolyploids, intergenomic interactions and chromosomal evolution in Tragopogon miscellus, and repetitive DNA evolution in Nicotiana allopolyploids. KEY RESULTS: For the first time, we can explore on a genomic scale the evolutionary processes that are ongoing in natural allopolyploids and not be restricted to well-studied crops and genetic models. CONCLUSIONS: These approaches can be easily and inexpensively applied to many other plant species-making any evolutionarily provocative system a new "model" system.

Characterization of duplicate gene evolution in the recent natural allopolyploid Tragopogon miscellus by next-generation sequencing and Sequenom iPLEX MassARRAY genotyping.

Tragopogon miscellus (Asteraceae) is an evolutionary model for the study of natural allopolyploidy, but until now has been under-resourced as a genetic model. Using 454 and Illumina expressed sequence tag sequencing of the parental diploid species of T. miscellus, we identified 7782 single nucleotide polymorphisms that differ between the two progenitor genomes present in this allotetraploid. Validation of a sample of 98 of these SNPs in genomic DNA using Sequenom MassARRAY iPlex genotyping confirmed 92 SNP markers at the genomic level that were diagnostic for the two parental genomes. In a transcriptome profile of 2989 SNPs in a single T. miscellus leaf, using Illumina sequencing, 69% of SNPs showed approximately equal expression of both homeologs (duplicate homologous genes derived from different parents), 22% showed apparent differential expression and 8.5% showed apparent silencing of one homeolog in T. miscellus. The majority of cases of homeolog silencing involved the T. dubius SNP homeolog (164/254; 65%) rather than the T. pratensis homeolog (90/254). Sequenom analysis of genomic DNA showed that in a sample of 27 of the homeologs showing apparent silencing, 23 (85%) were because of genomic homeolog loss. These methods could be applied to any organism, allowing efficient and cost-effective generation of genetic markers.