Single Nucleotide Polymorphism Genotyping Arrays Research Articles

Molecular Mapping of a Recessive Gene for Stripe Rust Resistance at the YrCf75 Locus Using Bulked Segregant Analysis Combined with Single Nucleotide Polymorphism Genotyping Arrays and Bulked Segregant RNA-Sequencing.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases in wheat worldwide. Planting resistant varieties is the most economical, effective, and environment-friendly measure to control wheat stripe rust. Changfeng 75, a Chinese winter wheat variety, shows high stripe rust resistance in both seedling and adult-plant stages. The seedling tests of F1, F2, and F2:3 populations derived from Mingxian 169/Changfeng 75 inoculated with Chinese predominant Pst race CYR34 showed that the stripe rust resistance of Changfeng 75 was controlled by a single recessive gene. The locus was temporarily designated as YrCf75. Bulked segregant analysis (BSA) combined with the wheat 660K single-nucleotide polymorphism (SNP) array and bulked segregant RNA-sequencing indicated that the proportion of polymorphic SNPs on wheat chromosome 2A was the highest, which suggested that YrCf75 was likely located on chromosome 2A. Two hundred and twenty-five Kompetitive allele-specific PCR (KASP) and 75 simple sequence repeat (SSR) markers on chromosome 2A were used to map YrCf75 using the BSA approach. Linkage analysis indicated that 31 KASP markers and one SSR marker were linked to YrCf75, and the genetic distances of the two closest flanking KASP markers, AX-1110060462 and AX-111004763, were 1.2 and 2.7 cM, respectively. YrCf75 was located on wheat chromosome 2AL. The molecular detection, resistance specificity, and chromosome location showed that YrCf75 is likely a new gene that is different from the known stripe rust resistance genes (Yr1 and Yr32) on wheat chromosome 2AL.

Development of a high-throughput SNP array for sea cucumber (Apostichopus japonicus) and its application in genomic selection with MCP regularized deep neural networks

High-throughput single nucleotide polymorphism (SNP) genotyping assays are powerful tools for genetic studies and genomic breeding applications for many species. Though large numbers of SNPs have been identified in sea cucumber (Apostichopus japonicus), but, as yet, no high-throughput genotyping platform is available for this species. In this study, we designed and developed a high-throughput 24 K SNP genotyping array named HaishenSNP24K for A. japonicus, based on the multi-objective-local optimization (MOLO) algorithm and HD-Marker genotyping method. The SNP array exhibited a relatively high genotyping call rate (> 96%), genotyping accuracy (>95%) and exhibited highly polymorphic in sea cucumber populations. In addition, we also assessed its application in genomic selection (GS). Deep neural networks (DNN) that can capture the complicated interactions of genes have been proposed as a promising tool in GS for SNP-based genomic prediction of complex traits in animal breeding. To overcome the problem of over-fitting when using the HaishenSNP24K array as high-dimensional DNN input, we developed minmax concave penalty (MCP) regularization for sparse deep neural networks (DNN-MCP) that finds an optimal sparse structure of a DNN by minimizing the square error subject to the non-convex penalty MCP on the parameters (weights and biases). Compared to two linear models, namely RR-GBLUP and Bayes B, and the nonlinear model DNN, DNN-MCP has greatly improved the genomic prediction ability for three quantitative traits (e.g., wet weight, dry weight and survival time) in the sea cucumber population. To the best of our knowledge, this is the first work to develop a high-throughput SNP array for A. japonicus and a new model DNN-MCP for genomic prediction of complex traits in GS. The present results provide evidence that supports the HaishenSNP24K array with DNN-MCP will be valuable for genetic studies and molecular breeding in A. japonicus.

188-LB: Deep Learning–Based Population Screening of Type 1 Diabetes and Celiac Disease Genetic Risk from Blood Spots at Birth

Introduction: Screening for type 1 diabetes (T1D) genetic risk in early life can prevent life threatening complications such as diabetic ketoacidosis and allow cost-effective recruitment into intervention and immunotherapy trials. Celiac disease (CD) frequently presents as a comorbidity in those with T1D and shares a strong genetic basis with T1D. Single nucleotide polymorphism (SNP) based genetic risk scores (GRS) for both T1D and CD have shown to be very predictive of future disease but are difficult to generate and typically require SNP array genotyping. We aimed to develop a combined GRS screening panel that could be genotyped from a single dried blood spot at birth. Methods: We developed assays for proxy SNPs of common HLA-DQ haplotypes reported in previous GRS and additional loci from the most recent available genome-wide association studies (GWAS) . We used backwards stepwise regression to identify a subset of variants able to be genotyped with DNA eluted from 800 6mm dried blood spots. We developed and validated neural network models to quantify genetic risk of both T1D and CD using T1DGC and UK Celiac case-control SNP array data, validated in UK Biobank. Assays were developed with LGC Genomics and validated on 675 Seattle area samples. Results: The complete panel consisted of 71 validated SNP assays, including 11 backup variants for key loci. We generated neural nework models which demonstrate equivalent or greater predictive power (AUC: T1D=0.914, CD=0.893) to previously published GRS yet require much less expertise to apply. We have developed an algorithm for preparation of raw genotyping data and subsequent generation of GRS requiring little expertise to apply. Conclusion: A 71 SNP blood-spot screening panel is highly effective at screening genetic risk associated with T1D and CD at birth. Using a neural network model the panel enables widely available, easy to generate and inexpensive population screening of genetic risk for T1D and CD. Disclosure S. A. Sharp: None. J. M. Locke: None. Y. Xu: None. D. P. Fraser: None. L. A. Ferrat: None. M. N. Weedon: None. M. Inouye: None. R. A. Oram: Consultant; Janssen Research & Development, LLC, Research Support; Randox R & D. W. Hagopian: Research Support; Janssen Research & Development, LLC. Funding Diabetes UK (16/0005529) JDRF (3-SRA-2019-827-S-B)

SoySNP618K array: A high-resolution single nucleotide polymorphism platform as a valuable genomic resource for soybean genetics and breeding.

Innovations in genomics have enabled the development of low-cost, high-resolution, single nucleotide polymorphism (SNP) genotyping arrays that accelerate breeding progress and support basic research in crop science. Here, we developed and validated the SoySNP618K array (618,888 SNPs) for the important crop soybean. The SNPs were selected from whole-genome resequencing data containing 2,214 diverse soybean accessions; 29.34% of the SNPs mapped to genic regions representing 86.85% of the 56,044 annotated high-confidence genes. Identity-by-state analyses of 318 soybeans revealed 17 redundant accessions, highlighting the potential of the SoySNP618K array in supporting gene bank management. The patterns of population stratification and genomic regions enriched through domestication were highly consistent with previous findings based on resequencing data, suggesting that the ascertainment bias in the SoySNP618K array was largely compensated for. Genome-wide association mapping in combination with reported quantitative trait loci enabled fine-mapping of genes known to influence flowering time, E2 and GmPRR3b, and of a new candidate gene, GmVIP5. Moreover, genomic prediction of flowering and maturity time in 502 recombinant inbred lines was highly accurate (>0.65). Thus, the SoySNP618K array is a valuable genomic tool that can be used to address many questions in applied breeding, germplasm management, and basic crop research.

Transfer of the Dominant Virus Resistance Gene AV-1 pro From Asparagus prostratus to Chromosome 2 of Garden Asparagus A. officinalis L.

An introgression breeding programme was carried out to transfer the virus resistance gene AV-1pro from the wild species Asparagus prostratus to the garden asparagus Asparagus officinalis. Serious crossing barriers caused by genetic distance and different ploidy levels of the crossing parents have been overcome using embryo rescue for the F1, BC1, and BC2 generations. The male and female fertility was widely restored in BC2 and was shown to be comparable to the cultivated asparagus. Five AV-1 resistant diploid (2n = 2x = 20) BC2 plants were selected and reciprocally backcrossed with asparagus cultivars. Segregation analyses of fourteen seedborne BC3 progenies suggested a monogenic dominant inheritance of the AV-1 resistance. Genotyping by sequencing analysis gave a strong hint for location of the resistance gene on asparagus Chromosome 2. Using an Axiom single nucleotide polymorphism (SNP) genotyping array for the analysis of three BC3 families with 10 AV-1 resistant and 10 AV-1 susceptible plants each, as well as 25 asparagus cultivars, the AV-1pro locus on Chromosome 2 was further narrowed down. The SNP with the highest LOD score was converted to a kompetitive allele specific PCR (KASP) marker, shown to be useful for the further backcross programme and serving as the starting point for the development of a diagnostic marker.

Genetic gain and G×E interaction in bread wheat cultivars representing 105 years of breeding in Pakistan

AbstractIt is important to understand the genetic gain achieved through selection of key yield traits for planning future breeding strategies in developing high yielding wheat (Triticum aestivum L.) cultivars. The aim of this study was to characterize the genetic changes and genotype × environment (G×E) interaction by additive main effect and multiplicative interactions (AMMI) for morphological, physiological, and yield component traits under five environments using 24 wheat cultivars released from 1911 to 2016 in Pakistan. There was a significant increase in grain yield (9.03 kg ha−1 yr−1, 0.37%), and plant height was reduced linearly (−0.26 cm yr−1, −0.33%). The traits waxiness, leaf rolling, harvest index, spike length, and grains per spike significantly increased but the gain was only 0.16–0.2% per year. Analysis of variance revealed that genotype, environment, and G×E interaction were highly significant (P < .01) for all traits except relative chlorophyll content, biomass, days to maturity, and number of spikes. Gene‐specific markers identified the durable resistance gene Lr67/Yr46/Sr55/Pm46 in obsolete cultivars as early as 1911, whereas the photoperiod‐insensitive allele Ppd‐D1a and reduced height alleles Rht‐B1b and Rht‐D1b were present only in the post‐1965 cultivars. Diversity analysis based on a 50K single nucleotide polymorphism genotyping array clearly differentiated temporal patterns in 24 cultivars, which was correlated with the agronomic performance of the cultivars. This dataset provided detailed insight into the performance of historical wheat cultivars and could help in devising wheat breeding strategies to focus on the traits contributing to grain yield and have slower rate of genetic progress.

Novel Design of Imputation-Enabled SNP Arrays for Breeding and Research Applications Supporting Multi-Species Hybridization.

Array-based single nucleotide polymorphism (SNP) genotyping platforms have low genotype error and missing data rates compared to genotyping-by-sequencing technologies. However, design decisions used to create array-based SNP genotyping assays for both research and breeding applications are critical to their success. We describe a novel approach applicable to any animal or plant species for the design of cost-effective imputation-enabled SNP genotyping arrays with broad utility and demonstrate its application through the development of the Illumina Infinium Wheat Barley 40K SNP array Version 1.0. We show that the approach delivers high quality and high resolution data for wheat and barley, including when samples are jointly hybridised. The new array aims to maximally capture haplotypic diversity in globally diverse wheat and barley germplasm while minimizing ascertainment bias. Comprising mostly biallelic markers that were designed to be species-specific and single-copy, the array permits highly accurate imputation in diverse germplasm to improve the statistical power of genome-wide association studies (GWAS) and genomic selection. The SNP content captures tetraploid wheat (A- and B-genome) and Aegilops tauschii Coss. (D-genome) diversity and delineates synthetic and tetraploid wheat from other wheat, as well as tetraploid species and subgroups. The content includes SNP tagging key trait loci in wheat and barley, as well as direct connections to other genotyping platforms and legacy datasets. The utility of the array is enhanced through the web-based tool, Pretzel (https://plantinformatics.io/) which enables the content of the array to be visualized and interrogated interactively in the context of numerous genetic and genomic resources to be connected more seamlessly to research and breeding. The array is available for use by the international wheat and barley community.

Application of a novel 50K SNP genotyping array to assess the genetic diversity and linkage disequilibrium in a farmed Pacific white shrimp (Litopenaeus vannamei) population

The inclusion of genomic information became a reality in shrimp breeding and it is expected to accelerate the genetic gain over time. The decay of linkage disequilibrium (LD) between single nucleotide polymorphisms (SNPs) is an important measure to evaluate the feasibility of implementing genomic selection. The aim of this study was to evaluate the use of a novel 50 K SNP array tool to characterize the genomic diversity, LD and effective population size (Ne) in a farmed shrimp population. A total of 96 animals (40 sires and 56 dams) were genotyped using the novel Illumina AquaArray HD (50 K) vannamei®. Quality control (QC) of genomic data was performed and three different minor allele frequency (MAF) exclusion thresholds were applied: < 0.10 (QC1), < 0.05 (QC2) and < 0.01 (QC3). After QC, 34,425, 39,091 and 42,789 SNPs were retained for QC1, QC2 and QC3, respectively, validating the high informativeness of this SNP array to this particular shrimp breeding population. The population showed a considerable high overall heterozygosity in comparison to other aquaculture species meaning that genetic diversity is stable despite selection. The principal component analysis revealed three genetically distant groups with the first two principal components explaining 27.7 % of total variation. LD decayed rapidly in the first 30Kb of distance between markers from 0.20 to 0.07 and then decreased to 0.02 in the long-range distance. These results suggest a relatively recent incorporation of animals from different populations in the broodstock. Ne size reduced from 7,871 to 301 animals in 827 generations for QC1, 8,253 to 305 animals for QC2 and 8,957 to 315 animals for QC3, both in 899 generations. Contemporary Ne was close to 86 for all QCs. The level of LD estimated suggests that genomic selection and genome-wide association studies are feasible in shrimp by using this SNP array.

Liquid chromatography-tandem mass spectrometry reveals an active response to DNA damage in human spermatozoa

To investigate how endogenously elevated DNA fragmentation alters the human sperm proteome, and whether this fragmentation contributes to genomic deletions. Research study. Commercial fertility clinic. Men with low (0%-4%, n = 7) or high (≥16%, n = 6) sperm DNA fragmentation, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. None. Global sperm proteome, single-nucleotide polymorphism genotyping array. A total of 78 significantly differentially abundant proteins (30 decreased, 48 increased) were observed in control vs. high DNA damage samples. DNA damage resulted in robust proteomic responses, including markers of oxidative stress and apoptosis, DNA damage repair proteins, and transcription/translation and protein turnover machinery. Several key sperm functional proteins were significantly decreased in ejaculates with high DNA damage. We were unable to substantiate a link between increased DNA fragmentation and genomic deletions in human spermatozoa. Developing human spermatozoa initiate an active transcriptional response to endogenous DNA damage, which manifests as alterations in the sperm proteome.

Concordance rate in cattle and sheep between genotypes differing in Illumina GenCall quality score.

Proper quality control of data prior to downstream analyses is fundamental to ensure integrity of results; quality control of genomic data is no exception. While many metrics of quality control of genomic data exist, the objective of the present study was to quantify the genotype and allele concordance rate between called single nucleotide polymorphism (SNP) genotypes differing in GenCall (GC) score; the GC score is a confidence measure assigned to each Illumina genotype call. This objective was achieved using Illumina beadchip genotype data from 771 cattle (12428767 genotypes in total post-editing) and 80 sheep (1557360 SNPs genotypes in total post-editing) each genotyped in duplicate. The called genotype with the lowest associated GC score was compared to the genotype called for the same SNP in the same duplicated animal sample but with a GC score of >0.90 (assumed to represent the true genotype). The mean genotype concordance rate for a GC score of <0.300, 0.300-0.549, and ≥0.550 in the cattle (sheep in parenthesis) was 0.9467 (0.9864), 0.9707 (0.9953), and 0.9994 (0.99997) respectively; the respective allele concordance rate was 0.9730 (0.9930), 0.9849 (0.9976), and 0.9997 (0.99998). Hence, concordance eroded as the GC score of the called genotype reduced, albeit the impact was not dramatic and was not very noticeable until a GC score of <0.55. Moreover, the impact was greater and more consistent in the cattle population than in the sheep population. Furthermore, an impact of GC score on genotype concordance rate existed even for the same SNP GenTrain value; the GenTrain value is a statistical score that depicts the shape of the genotype clusters and the relative distance between the called genotype clusters.

Development of a highly efficient 50K single nucleotide polymorphism genotyping array for the large and complex genome of Norway spruce (Picea abies L. Karst) by whole genome resequencing and its transferability to other spruce species.

Norway spruce (Picea abies L. Karst) is one of the most important forest tree species with significant economic and ecological impact in Europe. For decades, genomic and genetic studies on Norway spruce have been challenging due to the large and repetitive genome (19.6 Gb with more than 70% being repetitive). To accelerate genomic studies, including population genetics, genome‐wide association studies (GWAS) and genomic selection (GS), in Norway spruce and related species, we here report on the design and performance of a 50K single nucleotide polymorphism (SNP) genotyping array for Norway spruce. The array is developed based on whole genome resequencing (WGS), making it the first WGS‐based SNP array in any conifer species so far. After identifying SNPs using genome resequencing data from 29 trees collected in northern Europe, we adopted a two‐step approach to design the array. First, we built a 450K screening array and used this to genotype a population of 480 trees sampled from both natural and breeding populations across the Norway spruce distribution range. These samples were then used to select high‐confidence probes that were put on the final 50K array. The SNPs selected are distributed over 45,552 scaffolds from the P. abies version 1.0 genome assembly and target 19,954 unique gene models with an even coverage of the 12 linkage groups in Norway spruce. We show that the array has a 99.5% probe specificity, >98% Mendelian allelic inheritance concordance, an average sample call rate of 96.30% and an SNP call rate of 98.90% in family trios and haploid tissues. We also observed that 23,797 probes (50%) could be identified with high confidence in three other spruce species (white spruce [Picea glauca], black spruce [P. mariana] and Sitka spruce [P. sitchensis]). The high‐quality genotyping array will be a valuable resource for genetic and genomic studies in Norway spruce as well as in other conifer species of the same genus.

Towards Identifying the Target of Autoimmunity in Aplastic Anemia

Acquired aplastic anemia (AA) is an autoimmune bone marrow aplasia caused by T cell-mediated destruction of hematopoietic stem and progenitor cells. The antigenic target in AA remains unknown. Recently, we and others identified frequent somatic loss of function of several human leukocyte antigen (HLA) class I alleles in hematopoietic cells that survive the AA immune attack, suggesting these alleles ("risk alleles") present autoantigen in the affected patients. We hypothesize that risk alleles share structural features and peptide-binding characteristics that may inform our understanding of the immune mechanism of AA. To identify additional AA HLA class I risk alleles, we have partnered with two multi-institutional consortia, the North American Pediatric Aplastic Anemia Consortium (NAPAAC) and the Center for International Blood and Marrow Transplant Research (CIBMTR), to evaluate 507 AA patients for somatic HLA loss. Using a combination of targeted massively parallel sequencing of HLA class I genes and single nucleotide polymorphism array genotyping, we identified HLA loss in 19% of the NAPAAC cohort (30 of 156 unselected AA patients) and 13% of the CIBMTR cohort (46 of 351 patients selected to maximize discovery of less common risk alleles). Copy number-neutral loss of heterozygosity of chromosome arm 6p (6p CN-LOH) was the most common cause of HLA loss, occurring in 50 patients (10% of total cohort, 12% NAAPAC and 9% CIBMTR). Somatic mutations were present in 45 patients (9% of total cohort, 12% of NAPAAC and 7% CIBMTR). Patients with HLA loss had a median of 1 mutant clone per patient (range 1-7). Of the somatic mutations, 80% were predicted to disrupt expression of the affected allele (through loss of start, nonsense, or frameshift mutations). The other 20% were missense mutations affecting residues in the peptide binding groove (6 mutations) and the α3 domain (9 mutations) of the HLA class I protein. A total of 19 distinct AA HLA risk alleles were identified, of which 13 were newly identified in this study. Several risk alleles (HLA-B*14:02, HLA-B*40:02, HLA-A*02:01) were enriched in AA patients compared to ethnicity matched controls. Among the 507 patients in the study, at least one risk allele was present in 436/493 (88%) of AA patients and in 34/39 (87%) of the 6p CN-LOH events. Mutations clustered in several groups of alleles (supertypes) known to bind overlapping peptide repertoires, with alleles in B27 and B44 supertypes being most commonly affected. There were no mutations in A01, A01A03, A01A24, A24, B58, B62, or in any of the HLA-C alleles analyzed. Using several metrics to estimate the strength of the autoimmune selection on a given allele, including the frequency of somatic loss and similarity to other HLA risk alleles, we developed an AA HLA risk allele pathogenicity index ranging from very high (B*14:02 and B*40:02) and high pathogenicity (A*33:03, B*08:01, B*13:02, B*14:01, B*27:03, B*27:05, B*38:02, B*41:02 and B*49:01) to those unlikely to be pathogenic. Interestingly, even within high risk supertypes such as B44, there was a wide variation in predicted pathogenicity, ranging from high (for B*40:02 and B*41:02) to low pathogenicity (for B*44:02 and B*44:03), suggesting differences in autopeptide binding. Our study provides a comprehensive analysis of AA HLA risk alleles in a large diverse cohort of AA patients. Our results suggest that HLA risk alleles have shared autoantigen binding specificities that define their pathogenicity in AA, which can be used to identify candidate AA autoantigens. HLA risk allele pathogenicity may have future clinical utility as an adjunctive diagnostic test, as well as for prognostic assessment and haploidentical donor selection. Disclosures Lee: AstraZeneca: Research Funding; Kadmon: Research Funding; Takeda: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Syndax: Research Funding; Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding. Monos:Omixon: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

Identification of Genetic Susceptibility Factors Associated with Canine Gastric Dilatation-Volvulus.

Canine gastric dilatation-volvulus (GDV) is a common life-threatening condition occurring primarily in large and giant breeds with a 3.9% to 36.7% lifetime risk. The genetic correlates of GDV have not previously been systematically explored. We undertook an inter-breed genome-wide association analysis (GWAS) of 253 dogs from ten breeds including 106 healthy dogs and 147 dogs with at least one GDV episode. SNP array genotyping followed by imputation was conducted on 241 samples to identify GDV-associated single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). A subset of 33 dogs (15 healthy dogs and 18 GDV patients from the three most represented breeds) was characterized by whole genome sequencing (WGS). After genome-wide Bonferroni correction, we identified a significant putatively protective intergenic SNP (rs851737064) across all breeds. The signal was most significant in Collies, German Shorthaired Pointers, and Great Danes. Subsequent focused analysis across these three breeds identified 12 significant additional putatively protective or deleterious SNPs. Notable significant SNPs included those occurring in genes involved in gastric tone and motility including VHL, NALCN, and PRKCZ. These data provide important new clues to canine GDV risk factors and facilitate generation of hypotheses regarding the genetic and molecular underpinnings this syndrome.

Identification of quantitative trait loci governing early germination and seedling vigor traits related to weed competitive ability in rice

Weed competitive ability (WCA) is vital for the improvement of grain yield under direct-seeded and aerobic rice ecosystems where weeds are a major limiting factor. Early seed germination (ESG) and early seedling vigor (ESV) are the crucial traits for WCA. This study attempted to map the quantitative trait loci (QTLs) and hotspot regions governing ESG and ESV traits. A total of 167 BC1F5 selective introgression lines developed from an early backcross population involving Weed Tolerant Rice 1 (WTR-1) as the recipient parent and Y-134 as the donor parent were phenotyped for ESG and ESV traits. Analysis of variance revealed significant differences in ESG-related traits except for root length and in ESV-related traits except for plant height at 7 days after sowing. A total of 677-high quality single nucleotide polymorphism (SNP) markers were used to analyze the marker-trait association from a 6 K SNP genotyping array. Forty-three QTLs were identified on all chromosomes, except on chromosomes 4 and 8. Thirty QTLs were contributed by a desirable allele from Y-134, whereas 13 QTLs were from WTR-1. Twenty-eight of the identified genetic loci associated with ESG and ESV traits were novel. Two QTL hotspot regions were mapped on chromosomes 11 and 12. The genomic regions of QTL hotspots were fine-tuned and a total of 13 putative candidate genes were discovered on chromosomes 11 and 12 collectively. The mapped QTLs will be useful in advancing the marker aided-selection schemes and breeding programs for the development of rice cultivars with WCA traits.

SCREENING SLC2A1 GENE FOR SEQUENCE AND COPY NUMBER VARIATIONS ASSOCİATED WITH GLUT-1 DEFICIENCY SYNDROME

Objective: Glucose transporter-1 deficiency syndrome (GLUT1- DS) is defined as a metabolic encephalopathy that is associated with heterozygous and usually de novo pathogenic variations in the SLC2A1 (solute carrier family2 member1) gene. Materials and Methods: In this study, all coding exons and neighboring intronic regions of SLC2A1 were Sanger sequenced in 12 patients with clinically suspected GLUT1-DS. For de novo variations revealed after sequencing and segregation analysis, we also performed genome wide Single Nucleotide Polymorphism (SNP) genotyping to confirm parental relatedness with the proband. In patients without any sequence variations, real-time quantitative real-time polymerase chain reaction (qPCR) was applied to determine the presence of any copy number variations (CNV). Results: Sanger sequencing followed by bioinformatics analysis, segregation in the family and SNP array genotyping revealed two novel and de novo pathogenic variations associated with the GLUT1-DS phenotype in 2 patients. qPCR results were compatible with one copy loss of SLC2A1 gene in another patient. All variations identified herein are likely to have caused null alleles and resulted in GLUT1-DS through haplo insufficiency. Disscussion: In this study we used a series of molecular genetic approaches in order to identify all possible variations in SLC2A1 that may be associated with GLUT1-DS. This collective effort facilitated diagnosis in 3 patients.

High-Resolution Mapping of the Novel Early Leaf Senescence Gene Els2 in Common Wheat.

Early leaf senescence negatively impacts the grain yield in wheat (Triticum aestivum L.). Induced mutants provide an important resource for mapping and cloning of genes for early leaf senescence. In our previous study, Els2, a single incomplete dominance gene, that caused early leaf senescence phenotype in the wheat mutant LF2099, had been mapped on the long arm of chromosome 2B. The objective of this study was to develop molecular markers tightly linked to the Els2 gene and construct a high-resolution map surrounding the Els2 gene. Three tightly linked single-nucleotide polymorphism (SNP) markers were obtained from the Illumina Wheat 90K iSelect SNP genotyping array and converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers. To saturate the Els2 region, the Axiom® Wheat 660K SNP array was used to screen bulked extreme phenotype DNA pools, and 9 KASP markers were developed. For fine mapping of the Els2 gene, these KASP markers and previously identified polymorphic markers were analyzed in a large F2 population of the LF2099 × Chinese Spring cross. The Els2 gene was located in a 0.24-cM genetic region flanked by the KASP markers AX-111643885 and AX-111128667, which corresponded to a physical interval of 1.61 Mb in the Chinese Spring chromosome 2BL containing 27 predicted genes with high confidence. The study laid a foundation for a map-based clone of the Els2 gene controlling the mutation phenotype and revealing the molecular regulatory mechanism of wheat leaf senescence.

Rapid mapping of a stripe rust resistance gene YrZl31 using bulked segregant analysis combined with high-throughput single-nucleotide polymorphism genotyping arrays

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat. Identification and utilization of new resistance genes is one of the most effective way for achieving durable disease control. Zhongliang 31, a cultivar of common wheat (Triticum aestivum L.), is highly resistant to Chinese predominant Pst races and has been grown widely in the Longnan region in Gansu Province, China. Inheritance analysis of F1, F2, and F2:3 populations indicated that the stripe rust resistance of Zhongliang 31 was controlled by a single dominant gene, temporarily designated as YrZl31. Bulked segregant analysis (BSA) combined with wheat 660K single-nucleotide polymorphism (SNP) array showed the highest proportion of polymorphic SNPs located on chromosome 2B. One hundred forty-one kompetitive allele-specific PCR (KASP) and 165 simple sequence repeat (SSR) markers on chromosome 2B were used to map YrZl31. Linkage analysis indicated that 31 markers were linked to YrZl31, and the genetic distances of the two closest flanking KASP markers AX-111650565 and AX-110196738 were 2.8 and 4.1 cM, respectively. YrZl31 was located on wheat chromosome 2BL. Pedigree, resistance specificity, chromosome location, molecular and allelism tests suggested that YrZl31 may be a new gene and can be pyramided with other effective stripe rust resistance genes using the closely linked KASP markers developed in this study in wheat breeding programs.

Identification of Ancestry Informative Marker (AIM) Panels to Assess Hybridisation between Feral and Domestic Sheep.

Simple SummaryOnce present in the entirety of Europe, mouflon (wild sheep) became extinct due to intense hunting, but remnant populations survived and became feral on the Mediterranean islands of Corsica and Sardinia. Although now protected by regional laws, Sardinian mouflon is threatened by crossbreeding with domestic sheep causing genetic hybridisation. The spread of domestic genes can be detrimental for wild populations as it dilutes the genetic features that characterise them. This work aimed to identify diagnostic tools that could be applied to monitor the level of hybridisation between mouflon and domestic sheep. Tens of thousands of genetic markers known as single nucleotide polymorphisms (SNPs) were screened and we identified the smallest number of SNPs necessary to discriminate between pure mouflon and sheep. We produced four SNP panels of different sizes which were able to assess the hybridisation level of a mouflon and we verified that the SNP panels efficacy is independent of the domestic sheep breed involved in the hybrid. The implementation of these results into actual diagnostic tools will help the conservation of this unique and irreplaceable mouflon population, and the methodology applied can easily be transferred to other case studies of interest. Hybridisation of wild populations with their domestic counterparts can lead to the loss of wildtype genetic integrity, outbreeding depression, and loss of adaptive features. The Mediterranean island of Sardinia hosts one of the last extant autochthonous European mouflon (Ovis aries musimon) populations. Although conservation policies, including reintroduction plans, have been enforced to preserve Sardinian mouflon, crossbreeding with domestic sheep has been documented. We identified panels of single nucleotide polymorphisms (SNPs) that could act as ancestry informative markers able to assess admixture in feral x domestic sheep hybrids. The medium-density SNP array genotyping data of Sardinian mouflon and domestic sheep (O. aries aries) showing pure ancestry were used as references. We applied a two-step selection algorithm to this data consisting of preselection via Principal Component Analysis followed by a supervised machine learning classification method based on random forest to develop SNP panels of various sizes. We generated ancestry informative marker (AIM) panels and tested their ability to assess admixture in mouflon x domestic sheep hybrids both in simulated and real populations of known ancestry proportions. All the AIM panels recorded high correlations with the ancestry proportion computed using the full medium-density SNP array. The AIM panels proposed here may be used by conservation practitioners as diagnostic tools to exclude hybrids from reintroduction plans and improve conservation strategies for mouflon populations.

Genetic Differentiation of the Two Types of Polish Cold-blooded Horses Included in the National Conservation Program.

Simple SummaryIn this study we investigated the degree of genetic differentiation among two types of Polish cold-blooded horses, namely Sokólski and Sztumski. Both these horse types are a subject of conservation breeding as their populations have been dramatically reduced during recent decades. These cold-blooded horses are considered as separate breeds, with separate stud books, but yet still their genetic differentiation has not been precisely determined, especially on the genome–wide level. In this study, we characterized genetic differentiation of these two horse populations as well as their genetic variation, admixture patterns, and level of relatedness within populations with the use of single nucleotide polymorphism (SNP) genotyping arrays. We also attempted to detect genome regions with the largest genetic differences between those horses by the detection of so-called diversifying selection signals. The results of this study provide initial evidence supporting decisions made during conservation breeding program design and answer some of the questions raised by the breeders of Sokólski and Sztumski horses.The current role of the horse as a companion animal resulted in a decrease of interest in breeding and usage of draft horses. This meant that the population of cold-blooded horses in Poland has been dramatically reduced during the last decades. To avoid impoverishment of the gene pool of the local horse population, a conservation program was established which involves draft horses and other local horse breeds. The draft horses bred in Poland can be subdivided in a few horse types of which the most widespread and consolidated are Sztumski and Sokólski horses. These two subpopulations are phenotypically diversified, however, the overall level of their genetic differentiation seems to be relatively low and not precisely determined, especially with the use of molecular markers. In reference to this, in this study we used Illumina genotyping arrays to describe in detail the genetic differentiation of these two cold-blooded horse populations. We describe the genetic distance between them, as well as within-population variation, admixture patterns, and level of relatedness within populations. We also made an attempt to detect genome regions divergently selected between those horses by the detection of diversifying selection signals. The results of this study provide initial evidence supporting breeding decisions that were made during conservation breeding program design and answer questions raised by the breeders of Sokólski and Sztumski horses concerning the level of their genetic variation and differentiation.

Genetic Diversity and Population Structure in a Vitis spp. Core Collection Investigated by SNP Markers

Single nucleotide polymorphism (SNP) genotyping arrays are powerful tools to measure the level of genetic polymorphism within a population. The coming of next-generation sequencing technologies led to identifying thousands and millions of SNP loci useful in assessing the genetic diversity. The Vitis genotyping array, containing 18k SNP loci, has been developed and used to detect genetic diversity of Vitis vinifera germplasm. So far, this array was not validated on non-vinifera genotypes used as grapevine rootstocks. In this work, a core collection of 70 grapevine rootstocks, composed of individuals belonging to Vitis species not commonly used in the breeding programs, was genotyped using the 18k SNP genotyping array. SNP results were compared to the established SSR (Simple Sequence Repeat) markers in terms of heterozygosity and genetic structure of the core collection. Genotyping array has proved to be a valuable tool for genotyping of grapevine rootstocks, with more than 90% of SNPs successfully amplified. Structure analysis detected a high degree of admixed genotypes, supported by the complex genetic background of non-vinifera germplasm. Moreover, SNPs clearly differentiated non-vinifera and vinifera germplasm. These results represent a first step in studying the genetic diversity of non-conventional breeding material that will be used to select rootstocks with high tolerance to limiting environmental conditions.