Genomic Markers Research Articles

Genome-wide association studies of grain yield and quality traits under optimum and low-nitrogen stress in tropical maize (Zea mays L.).

Genome-wide association study (GWAS) demonstrated that multiple genomic regions influence grain quality traits under nitrogen-starved soils. Using genomic prediction, genetic gains can be improved through selection for grain quality traits. Soils in sub-Saharan Africa are nitrogen deficient due to low fertilizer use and inadequate soil fertility management practices. This has resulted in a significant yield gap for the major staple crop maize, which is undermining nutritional security and livelihood sustainability across the region. Dissecting the genetic basis of grain protein, starch and oil content under nitrogen-starved soils can increase our understanding of the governing genetic systems and improve the efficacy of future breeding schemes. An association mapping panel of 410 inbred lines and four bi-parental populations were evaluated in field trials in Kenya and South Africa under optimum and low nitrogen conditions and genotyped with 259,798 SNP markers. Genetic correlations demonstrated that these populations may be utilized to select higher performing lines under low nitrogen stress. Furthermore, genotypic, environmental and GxE variations in nitrogen-starved soils were found to be significant for oil content. Broad sense heritabilities ranged from moderate (0.18) to high (0.86). Under low nitrogen stress, GWAS identified 42 SNPs linked to grain quality traits. These significant SNPs were associated with 51 putative candidate genes. Linkage mapping identified multiple QTLs for the grain quality traits. Under low nitrogen conditions, average prediction accuracies across the studied genotypes were higher for oil content (0.78) and lower for grain yield (0.08). Our findings indicate that grain quality traits are polygenic and that using genomic selection in maize breeding can improve genetic gain. Furthermore, the identified genomic regions and SNP markers can be utilized for selection to improve maize grain quality traits.

Comparative genomic analysis reveals the origin and global distribution of melon necrotic virus isolates

The melon necrotic spot virus (MNSV) is the family member of Tombusviridae and genus member of Gammacarmovirus. It can be widely found from almost everywhere across the globe, infecting host plants like rockmelon, cucumber, muskmelon and watermelon. In this study, we attempted to search for country-specific genome markers of MNSV via the comparison of several genome characteristics across isolates from different countries such as South Korea, China, India, Japan, Netherlands, Canada, Australia, Brazil, USA and Spain. As a result, we discovered that the whole genome length and GC content can be used to preliminarily distinguish South Korea, Japan and Australia MNSV isolates. Besides, single nucleotide polymorphism (SNP) markers and protein profiles (RNA-dependent RNA polymerase (RdRp) and coat protein (CP)) can further differentiate Japan and South Korea MNSV isolates and identify two Japan isolates that are postulated to have South Korea origin. The motif and phylogenetic analysis revealed the previously described divergent group from USA and Australia regions. Interestingly, the RdRp and CP protein models for all isolates are identical and this is a strong indication of the high MNSV functional conservation. In short, the information yielded from this study will in turn aid in future virulence-SNP association studies essential for cost-effective biological control of this virus via genetic engineering in order to reduce harvest loss.

Tourniquet use following blast-associated complex lower limb injury and traumatic amputation promotes end organ dysfunction and amplified heterotopic ossification formation

BackgroundTraumatic heterotopic ossification (tHO) is characterized by ectopic bone formation in extra-skeletal sites leading to impaired wound healing, entrapment of neurovascular structures, pain, and reduced range of motion. HO has become a signature pathology affecting wounded military personnel who have sustained blast-associated traumatic amputations during the recent conflicts in Iraq and Afghanistan and can compound recovery by causing difficulty with prosthesis limb wearing. Tourniquet use to control catastrophic limb hemorrhage prior to surgery has become almost ubiquitous during this time, with the recognition the prolonged use may risk an ischemia reperfusion injury and associated complications. While many factors influence the formation of tHO, the extended use of tourniquets to limit catastrophic hemorrhage during prolonged field care has not been explored.MethodsUtilizing an established pre-clinical model of blast-associated complex lower limb injury and traumatic amputation, we evaluated the effects of tourniquet use on tHO formation. Adult male rats were subjected to blast overpressure exposure, femur fracture, and soft tissue crush injury. Pneumatic tourniquet (250–300 mmHg) applied proximal to the injured limb for 150-min was compared to a control group without tourniquet, before a trans-femoral amputation was performed. Outcome measures were volume to tHO formation at 12 weeks and changes in proteomic and genomic markers of early tHO formation between groups.ResultsAt 12 weeks, volumetric analysis with microCT imaging revealed a 70% increase in total bone formation (p = 0.007) near the site of injury compared to rats with no tourniquet time in the setting of blast-injuries. Rats subjected to tourniquet usage had increased expression of danger-associated molecular patterns (DAMPs) and end organ damage as early as 6 h and as late as 7 days post injury. The expressions of pro-inflammatory cytokines and chemokines and osteochondrogenic genes using quantitative RT-PCR similarly revealed increased expression as early as 6 h post injury, and these genes along with hypoxia associated genes remained elevated for 7 days compared to no tourniquet use.ConclusionThese findings suggest that tourniquet induced ischemia leads to significant increases in key transcription factors associated with early endochondral bone formation, systemic inflammatory and hypoxia, resulting in increased HO formation.

Drug Response Prediction Based on 1D Convolutional Neural Network and Attention Mechanism.

There are multiple methods based on gene expression, copy number variation, and methylation biomarkers for screening drug response have been developed. On the other hand, many machine learning algorithms have been applied in recent years to predict drug response, such as neural networks and random forests for the discovery of genomic markers of drug sensitivity for individual drugs in cancer cell lines. In this paper, we propose a drug response prediction algorithm based on 1D convolutional neural networks with attention mechanism and combined with pathway networks, which combines the individual histological data affecting drug response and considers the topological nature of the pathways to find the subpathways highly correlated with drug response and use this as a feature to predict drug response by training using convolutional neural networks. Thus, the output values will represent the probability of occurrence of each of these two categories. In this experiment, using five-fold cross-validation, the identification accuracy reached an average of 84.6%, which is 4.5% higher than the direct random forest approach for drug prediction with an AUC value. This proves that the use of the one-dimensional1D convolutional neural network with attention mechanism to predict the response of low-grade glioma patients and drugs has better prediction results.

Abstract PR002: Antigen presentation and processing pathway is associated with early relapse after neoadjuvant immune checkpoint blockade (ICB) in dedifferentiated liposarcomas (DDLPS)

Abstract Background: We evaluated the activity of neoadjuvant ICB in localized resectable DDLPS (n=17) and undifferentiated pleomorphic sarcomas (UPS; n=10). DDLPS and UPS patients were randomized to neoadjuvant nivolumab or ipilimumab+nivolumab, with UPS patients receiving concurrent radiotherapy. We assessed genomic markers of early relapse (progression before surgery or relapse within 52 weeks following surgery) using longitudinally acquired tumor samples. Methods: RNA sequencing (RNAseq) and whole genome sequencing (WGS) were performed on longitudinally acquired samples (baseline biopsies and surgical specimens). Differential gene expression between any two groups of patients (i.e., non-early relapse [non-relapsers] vs early relapse [relapsers]) were selected (fold change>1.5 and p value<0.05). Gene set enrichment analyses (GSEA) of KEGG pathways were performed and a network-based approach used to identify genes/pathways associated with MHC-I. Results: At a median follow-up of 23 months, 12 patients (9 DDLPS, 3 UPS) relapsed, including 7 early relapses (relapsers: 5 DDLPS, 2 UPS). The median relapse-free survival was 22 months in DDLPS patients (6 months in relapsers; not reached [NR] in non-relapsers) and NR in UPS patients. At baseline, the most differentially upregulated pathways in non-relapsers compared to relapsers were “graft versus host disease” (GSEA Normalized Enrichment Score[NES]=2.25; False Discovery Rate[FDR] q= 0.009), “natural killer cell mediated cytotoxicity” (NES=2.17; FDR q=0.009), “antigen processing and presentation” (NES=2.16; FDR q=0.009), “allograft rejection” (NES=1.99; FDR q=0.019) and “B-cell receptor signaling pathway” (NES=1.87; FDR q=0.018). In DDLPS patients, the antigen presentation and processing pathway was the most upregulated pathway in non-relapsers compared to relapsers (NES=2.01; FDR q=0.025) while it was not significantly upregulated in UPS (NES=1.15; FDR q=0.62). When looking at pathways longitudinally, the antigen presentation and processing pathway was significantly upregulated at surgery compared to baseline in DDLPS. As antigen presentation and processing was significantly upregulated in DDLPS patients and associated with relapse, we looked for expressed neoantigens that may be processed and presented. Using WGS, we detected 5712 rearrangements at baseline in DDLPS, of which 230 were found in more than one tumor specimen. We also sought to identify genes associated with MHC-I. We selected genes upregulated during ICB comparing baseline to surgical specimens in DDLPS relapsers and looked at the top 10% of genes associated with MHC-I in order to identify potential therapeutic targets for combination. We identified 41 genes upregulated during ICB and associated with MHC-I in relapsers, for which up to 275 inhibitory compounds were found in drug databases. Conclusion: Antigen presentation and processing is a major driver of response to immunotherapy. Future efforts should focus on identifying which antigens are presented to find synergizing compounds in order to increase the clinical benefit of ICB. Citation Format: Elise F. Nassif, Chia-Chin Wu, Kadir Akdemir, Russell G. Witt, Raymond Traweek, Brandon Cope, Prapassorn Thirasastr, Taylor Tate, Grace Mathew, Shadarra Crosby, Randy Chu, Mohammad Mohammad, Kenna Shaw, Ingram Davis, Khalida Wani, Alexander J. Lazar, Wei-Lien Wang, Sheila Duncan, Ashleigh B. Guadagnolo, Andrew J. Bishop, Valerae Lewis, Justin E. Bird, Keila E. Torres, Kelly K. Hunt, Barry W. Feig, Christopher P. Scally, Ravin Ratan, Shreyaskumar Patel, Robert S. Benjamin, Robert Satcher, Kevin McBride, Wolf H. Fridman, Ignacio Wistuba, Andrew Futreal, Jennifer A. Wargo, Neeta Somaiah, Christina L. Roland, Emily Z. Keung. Antigen presentation and processing pathway is associated with early relapse after neoadjuvant immune checkpoint blockade (ICB) in dedifferentiated liposarcomas (DDLPS) [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR002.

GNOSIS: an R Shiny app supporting cancer genomics survival analysis with cBioPortal.

Exploratory analysis of cancer consortia data curated by the cBioPortal repository typically requires advanced programming skills and expertise to identify novel genomic prognostic markers that have the potential for both diagnostic and therapeutic exploitation. We developed GNOSIS (GeNomics explOrer using StatistIcal and Survival analysis in R), an R Shiny App incorporating a range of R packages enabling users to efficiently explore and visualise such clinical and genomic data. GNOSIS provides an intuitive graphical user interface and multiple tab panels supporting a range of functionalities, including data upload and initial exploration, data recoding and subsetting, data visualisations, statistical analysis, mutation analysis and, in particular, survival analysis to identify prognostic markers. GNOSIS also facilitates reproducible research by providing downloadable input logs and R scripts from each session, and so offers an excellent means of supporting clinician-researchers in developing their statistical computing skills.

Microevolution and subspecific taxonomy of Trypanosoma cruzi

Trypanosoma cruzi, the agent of Chagas disease, is a highly polymorphic species, subdivided into 6 main evolutionary lineages or near-clades (formerly discrete typing units or DTUs). An additional near-clade (TC-bat) has recently been evidenced. This pattern is considered to be the result of predominant clonal evolution (PCE). PCE is compatible with occasional mating/hybridization, which do not break the prevalent pattern of clonal evolution, the main trait of it being the presence of Multigene Bifurcating Trees (MGBTs) at all evolutionary levels ("clonal frame"). The development of highly resolutive genetic (microsatellites*) and genomic (sequencing and multi-single nucleotide polymorphism {SNP}* typing) markers shows that PCE also operates at a microevolutionary* level within each of the near-clades ("Russian doll pattern"), in spite of occasional meiosis and hybridization events. Within each near-clade, one can evidence widespread clonal multilocus genotypes*, linkage disequilibrium*, Multigene Bifurcating Trees and lesser near-clades. The within near-clade population structure is like a miniature picture of that of the whole species, suggesting gradual rather than saltatory evolution. Additional data are required to evaluate the stability of these lesser near-clades in the long run and to evaluate the need for an adequate nomenclature for this microevolutionary level.

Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selection in parasitic nematodes and on how best to detect them. This study addresses these knowledge gaps by using field populations of Haemonchus contortus with well-defined benzimidazole treatment histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 genomic markers to genotype 20 individual adult worms from each of four H. contortus populations: two from closed sheep flocks with an approximate 20 year history of frequent benzimidazole treatment, and two populations with a history of little or no treatment. Sampling occurred in the same geographical region to limit genetic differentiation and maximise the detection sensitivity. A clear signature of selection was detected on chromosome I, centred on the isotype-1 β-tubulin gene. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I spanning 3.75 Mbp and 259 annotated genes, and one on chromosome II spanning a region of 3.3 Mbp and 206 annotated genes, including the isotype-2 β-tubulin locus. We also assessed how sensitivity was impacted by sequencing depth, worm number, and pooled versus individual worm sequence data. This study provides the first known direct genome-wide evidence for any parasitic nematode, that the isotype-1 β-tubulin gene is quantitatively the single most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole resistance loci of secondary importance. This study provides an experimental framework to maximise the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes.

1490MO CDK4/6 inhibition in locally advanced/metastatic chordoma (NCT PMO-1601)

Chordoma is a rare bone tumor with an unmet therapeutic need. In chordoma cell lines and patient biopsies, the p16 (cyclin-dependent kinase inhibitor 2a, encoded by CDKN2A) tumor suppressor is consistently inactivated, resulting in aberrant cyclin D-CDK4/6-RB pathway activity, which can be efficiently inhibited by the CDK4/6 inhibitor palbociclib (PMID 26183925). Patients (pts) ≥18 years (yrs) with locally advanced or metastatic chordoma, at least one measurable tumor lesion per RECIST 1.1, ECOG PS 0–2, adequate organ function, loss of p16 (by immunohistochemistry [IHC]) or CDKN2A (by genomic analysis) and presence of CDK4/6 and RB1 (by IHC or RNA-seq) and not amenable to curative surgery or radiotherapy, are eligible. Palbociclib 125 mg q.d. is given in a 21-days-on/7-days-off schedule. Based on a Simon optimal 2-stage design, disease control rate (DCR) is the primary endpoint. Response is defined as complete response (CR), partial response (PR), or stable disease (SD) per RECIST 1.1 after 6 cycles. For sample size calculation, a poor or good response were assumed to be 10% and 25%, respectively (α, 5 %; β, 20%), resulting in a 1st stage of 18 pts and, if ≥3 pts respond, study continuation to a total of 43 pts. Between 12/2017 and 04/2022, 26 pts (female, n=6; median age, 59 yrs [range, 31–84]) were enrolled. Primary chordoma sites were sacrococcygeal (n=14), skull base (n=4), and other parts of the spine (n=8). Palbociclib was well tolerated. Adverse events were mostly mild (CTC-AE °1–2) cytopenias; one CTC-AE °5 was recorded (neutropenic sepsis). The interim analysis revealed 6/18 pts (33%) with SD and none with PR or CR. Recruitment was continued in 11/2021 as initially planed. Median progression-free survival was 5.75 months (95% confidence interval [CI], 3.55–not reached), and the median number of applied cycles was 6 (range, 2–42). Overall survival after 2 yrs was 74% (95% CI, 56–97%). Correlative biomarker studies, including WGS/WES and RNA-seq, are ongoing. This is the 1st proof-of-concept trial in chordomas using a genomic marker to predict palbociclib activity. Therapy with palbociclib resulted in a DCR of 33% with manageable toxicity. Although no PR or CR were yet achieved, single-agent palbociclib showed anti-tumor activity.

EP05.01-014 Circulating Tumor DNA to Identify Genomic Biomarkers of Radiation Sensitivity in Locally Advanced Non-Small Cell Lung Cancer

Despite our increasing understanding of the genomic heterogeneity of non-small cell lung cancer (NSCLC), patients with locally advanced inoperable disease are treated with the same dose of radiation therapy (RT). Tissue-based analyses have identified likely biomarkers of RT response. We evaluated the feasibility of prospective molecular profiling with liquid biopsy ctDNA testing to identify genomic markers of RT sensitivity and resistance.

Regulation of Molecular Diagnostics.

Molecular diagnostic tests enable rapid analysis of genomic and proteomic markers. These tests are subject to diverging premarket access and postmarket surveillance requirements and mechanisms in the United States and the European Union. Each of these jurisdictions has its own challenges in keeping the regulations up to date with technological developments. A specific area of attention is that of laboratory-developed tests in the United States and health institution in-house-produced tests in the European Union, for which the United States and the European Union have markedly different regulatory approaches. Both jurisdictions have specific but differing requirements for the use of test samples and test-related data under their rules regarding the protection of (personal) health data, which can cause complexity when moving samples or sample-related data from one jurisdiction to the other.

Distinct Predictive Immunogenomic Profiles of Response to Immune Checkpoint Inhibitors and IL2: A Real-world Evidence Study of Patients with Advanced Renal Cancer.

Next-generation IL2 agents, designed for improved tolerability over traditional HD-IL2 (aldesleukin), are in clinical development. Retrospective molecular tumor profiling of patients treated with HD-IL2 or anti-PD-1 therapy provides insights into genomic characteristics of therapy response. This study revealed common and distinct immune-related predictive response markers for IL2 and anti-PD-1 therapy which may play a role in therapy guidance, and rational combination strategies for these agents.

Discovering epistasis interactions in Alzheimer's disease using deep learning model

Alzheimer's disease (AD) is the most common form of dementia. Single Nucleotide Polymorphisms (SNPs) are single nucleotide alterations that can be used as genomic markers disclosing susceptibility to complex diseases like AD. Epistasis has long been significant for recognizing the function of genetic pathways and the evolutionary dynamics of difficult genetic systems. Discovering epistasis interactions holds a vital key to personalized medicine (PM). PM needs a better understanding of the relationship between human genetic data and complex diseases. In this proposed work, a deep neural network (DNN) is applied using SHapley Additive exPlanations (SHAP) to get top 20, 100, 300, and 500 ranking SNPs responsible for AD risk through epistasis interactions. Multi-locus interaction analysis is performed on these identified SNPs using Multifactor Dimensionality Reduction (MDR). This constructive induction algorithm is integrated with DNN for discovering epistasis interactions in a computationally effective method. The proposed framework is applied to Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset. The best accuracies are achieved using the top 500 SNPs, and the classification accuracies varied between 0.860 and 0.874 in the five-way interaction model. However, the classification accuracies of 2-way, 3-way, 4-way models varied between 0.663 and 0.670, 0.718 and 0.727, and 0.793 and 0.803, respectively. The results revealed that the reported accuracy scores of the proposed framework outperform the referenced literature work. The proposed framework presents high-ranked risk genes and promising epistasis interactions that may help in explaining the risk of AD.

Sleep disruption induces activation of inflammation and heightens risk for infectious disease: Role of impairments in thermoregulation and elevated ambient temperature

ABSTRACT Thermoregulation and sleep are tightly coordinated, with evidence that impairments in thermoregulation as well as increases in ambient temperature increase the risk of sleep disturbance. As a period of rest and low demand for metabolic resources, sleep functions to support host responses to prior immunological challenges. In addition by priming the innate immune response, sleep prepares the body for injury or infection which might occur the following day. However when sleep is disrupted, this phasic organization between nocturnal sleep and the immune system becomes misaligned, cellular and genomic markers of inflammation are activated, and increases of proinflammatory cytokines shift from the nighttime to the day. Moreover, when sleep disturbance is perpetuated due to thermal factors such as elevated ambient temperature, the beneficial crosstalk between sleep and immune system becomes further imbalanced. Elevations in proinflammatory cytokines have reciprocal effects and induce sleep fragmentation with decreases in sleep efficiency, decreases in deep sleep, and increases in rapid eye movement sleep, further fomenting inflammation and inflammatory disease risk. Under these conditions, sleep disturbance has additional potent effects to decrease adaptive immune response, impair vaccine responses, and increase vulnerability to infectious disease. Behavioral interventions effectively treat insomnia and reverse systemic and cellular inflammation. Further, insomnia treatment redirects the misaligned inflammatory- and adaptive immune transcriptional profiles with the potential to mitigate risk of inflammation-related cardiovascular, neurodegenerative, and mental health diseases, as well as susceptibility to infectious disease.

Development of SLAF-Sequence and Multiplex SNaPshot Panels for Population Genetic Diversity Analysis and Construction of DNA Fingerprints for Sugarcane.

A genetic diversity analysis and identification of plant germplasms and varieties are important and necessary for plant breeding. Deoxyribonucleotide (DNA) fingerprints based on genomic molecular markers play an important role in accurate germplasm identification. In this study, Specific-Locus Amplified Fragment Sequencing (SLAF-seq) was conducted for a sugarcane population with 103 cultivated and wild accessions. In total, 105,325 genomic single nucleotide polymorphisms (SNPs) were called successfully to analyze population components and genetic diversity. The genetic diversity of the population was complex and clustered into two major subpopulations. A principal component analysis (PCA) showed that these accessions could not be completely classified based on geographical origin. After filtration, screening, and comparison, 192 uniformly-distributed SNP loci were selected for the 32 chromosomes of sugarcane. An SNP complex genotyping detection system was established using the SNaPshot typing method and used for the precise genotyping and identification of 180 sugarcane germplasm samples. According to the stability and polymorphism of the SNPs, 32 high-quality SNP markers were obtained and successfully used to construct the first SNP fingerprinting and quick response codes (QR codes) for sugarcane. The results provide new insights for genotyping, classifying, and identifying germplasm and resources for sugarcane breeding

Predictive molecular diagnostics in breast cancer : What are the requirements for pathology today and in the future?

Mit zunehmenden zielgerichteten Optionen zur Behandlung solider Tumoren wachsen für die Pathologie die Anforderungen an die prädiktive molekulare Diagnostik. Beim Mammakarzinom war das Erfordernis der Bestimmung genomischer prädiktiver Marker für zielgerichtete Therapien bisher überschaubar (Nachweis einer PIK3CA-Mutation beim endokrin vorbehandelten Luminaltumor und nur beim sekretorischen Mammakarzinom angezeigte Suche nach NTRK-Fusionen). Spätestens bei Nichtansprechen der Erst- bzw. Zweitlinienstandardtherapien ist eine Next-Generation-Sequencing-Panel-Diagnostik sinnvoll, um Resistenzmechanismen z. B. gegen die endokrine Therapie oder „cyclin-dependent kinase 4/cyclin-dependent kinase 6“ (CDK4/CDK6) abzuklären und Ansatzpunkte für in Entwicklung befindliche Therapien zu identifizieren. Die Interpretation sollte qualitätsgesichert gemäß internationalem Standard erfolgen und der interdisziplinären Tumorkonferenz zeitnah in einem transparenten und standardisierten Report zur Verfügung stehen.

Genomic analysis of the rare British Lop pig and identification of distinctive genomic markers.

Concentration of production on a few commercial pig breeds has led to the marginalization of many native, numerically small breeds, increasing their risk of endangerment. In the UK, one such rare breed is the British Lop, a lop-eared breed, of similar origin to the Welsh breed. The objective of the present study was to address the genomic status of the British Lop and its relationship with other breeds and identify a small set of genomic markers that uniquely characterize and distinguish British Lop animals. Results have shown that the British Lop is a relatively distinct population with reduced genomic diversity and effective size consistent with its status as a rare breed. Furthermore, we demonstrated the genetic closeness of the British Lop to phenotypically similar breeds such as Landrace and Welsh as well Large White, Middle White and Pietrain. Finally, a set of 75 Single Nucleotide Polymorphisms distributed across multiple chromosomes were identified and validated as markers that can consistently distinguish British Lops from other closely related breeds. Results may inform breeding and management strategies aiming to enhance diversity as well as the development of a breed purity test.

Genetic diversity and structure of a core collection of Huangqi (Astragalus ssp.) developed using genomic simple sequence repeat markers

Genetic diversity and structure of a core collection of Huangqi (Astragalus ssp.) developed using genomic simple sequence repeat markers

Skimming for barcodes: rapid production of mitochondrial genome and nuclear ribosomal repeat reference markers through shallow shotgun sequencing.

DNA barcoding is critical to conservation and biodiversity research, yet public reference databases are incomplete. Existing barcode databases are biased toward cytochrome oxidase subunit I (COI) and frequently lack associated voucher specimens or geospatial metadata, which can hinder reliable species assignments. The emergence of metabarcoding approaches such as environmental DNA (eDNA) has necessitated multiple marker techniques combined with barcode reference databases backed by voucher specimens. Reference barcodes have traditionally been generated by Sanger sequencing, however sequencing multiple markers is costly for large numbers of specimens, requires multiple separate PCR reactions, and limits resulting sequences to targeted regions. High-throughput sequencing techniques such as genome skimming enable assembly of complete mitogenomes, which contain the most commonly used barcoding loci (e.g., COI, 12S, 16S), as well as nuclear ribosomal repeat regions (e.g., ITS1&2, 18S). We evaluated the feasibility of genome skimming to generate barcode references databases for marine fishes by assembling complete mitogenomes and nuclear ribosomal repeats. We tested genome skimming across a taxonomically diverse selection of 12 marine fish species from the collections of the National Museum of Natural History, Smithsonian Institution. We generated two sequencing libraries per species to test the impact of shearing method (enzymatic or mechanical), extraction method (kit-based or automated), and input DNA concentration. We produced complete mitogenomes for all non-chondrichthyans (11/12 species) and assembled nuclear ribosomal repeats (18S-ITS1-5.8S-ITS2-28S) for all taxa. The quality and completeness of mitogenome assemblies was not impacted by shearing method, extraction method or input DNA concentration. Our results reaffirm that genome skimming is an efficient and (at scale) cost-effective method to generate all mitochondrial and common nuclear DNA barcoding loci for multiple species simultaneously, which has great potential to scale for future projects and facilitate completing barcode reference databases for marine fishes.

Development and characterisation of SSR markers in the potato rot nematode Ditylenchus destructor

Summary The potato rot nematode, Ditylenchus destructor, causes serious disease limiting the production of many crops. This disease usually decreases sweet potato yield by 20-50%, and in heavily infested fields the crop may be completely lost. Although the nematode has economic importance in China, its transmission route and genetic diversity are unknown. In this study, a collection of 1761 contigs of the D. destructor genome was mined for simple sequence repeat (SSR) markers, which resulted in the identification of 9745 SSRs. A total of 150 pairs of SSR primers were further developed and used for validation of the amplification rate and assessment of the polymorphism. Nine SSR markers were finally identified and analysed using 96 individual specimens of D. destructor sampled from four provinces in China. These loci were found to be moderately polymorphic with 2-8 alleles per locus. The observed and expected heterozygosity across the four populations ranged from 0.000 to 0.833 and from 0.000 to 0.666, respectively. This is the first report of the development and characterisation of genomic SSR markers in D. destructor. Our study demonstrated the obvious gene differentiation among different populations of D. destructor in China. This suggests that D. destructor in China may have been introduced from multiple origins. Much more work is needed on this species to identify patterns of spread, and the microsatellite loci we develop here should be useful in many regions for modelling range expansion, studying the evolution of resistance, and increasing the effectiveness of pest management strategies.