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The genomic prediction of phenotypes and breeding values in animals and plants has developed rapidly into its own research field. Results of genomic prediction studies are often difficult to compare because data simulation varies, real or simulated data are not fully described, and not all relevant results are reported. In addition, some new methods have been compared only in limited genetic architectures, leading to potentially misleading conclusions. In this article we review simulation procedures, discuss validation and reporting of results, and apply benchmark procedures for a variety of genomic prediction methods in simulated and real example data. Plant and animal breeding programs are being transformed by the use of genomic data, which are becoming widely available and cost-effective to predict genetic merit. A large number of genomic prediction studies have been published using both simulated and real data. The relative novelty of this area of research has made the development of scientific conventions difficult with regard to description of the real data, simulation of genomes, validation and reporting of results, and forward in time methods. In this review article we discuss the generation of simulated genotype and phenotype data, using approaches such as the coalescent and forward in time simulation. We outline ways to validate simulated data and genomic prediction results, including cross-validation. The accuracy and bias of genomic prediction are highlighted as performance indicators that should be reported. We suggest that a measure of relatedness between the reference and validation individuals be reported, as its impact on the accuracy of genomic prediction is substantial. A large number of methods were compared in example simulated and real (pine and wheat) data sets, all of which are publicly available. In our limited simulations, most methods performed similarly in traits with a large number of quantitative trait loci (QTL), whereas in traits with fewer QTL variable selection did have some advantages. In the real data sets examined here all methods had very similar accuracies. We conclude that no single method can serve as a benchmark for genomic prediction. We recommend comparing accuracy and bias of new methods to results from genomic best linear prediction and a variable selection approach (e.g., BayesB), because, together, these methods are appropriate for a range of genetic architectures. An accompanying article in this issue provides a comprehensive review of genomic prediction methods and discusses a selection of topics related to application of genomic prediction in plants and animals.

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50-60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.

The Lophophora genus of the Cactaceae family includes Lophophora diffusa and Lophophora williamsii , which has traditionally been used as a natural analgesic; however, its use is now under strict regulation worldwide as it contains mescaline, a unique psychotropic agent. Recently, non-medical and illegal distribution and abuse of L. williamsii have increased worldwide; thus, effective species identification methods are urgently needed. Here, we identified a new variable number tandem repeat (VNTR) marker in the trnL intron region to identify and characterize species in forensic analyses. The VNTR marker has a unique structure of tandem repeats, each with 13 nucleotides; one repeat unit was found in L. williamsii and two in L. diffusa . Phylogenetic and length polymorphism analyses confirmed that this novel VNTR marker could distinguish between Lophophora species. Furthermore, our newly developed TaqMan genotyping assay utilizes two probes; the color and position of dots on the discrimination plot differ according to the tandem repeat count within the VNTR marker. The limits of detection of the assay were 0.000063 ng (LW-VNTR probe-1) and 0.000066 ng (LW-VNTR probe-2), indicating high sensitivity. Moreover, when crime scene samples of 16 presumed L. williamsii species were analyzed, the results coincided with those of gas chromatography–mass spectrometry, confirming the applicability of our marker for Lophophora species identification. Thus, the tandem repeats within the trnL intron region can be exploited as a VNTR marker to identify L. williamsii and L. diffusa . Our dual TaqMan genotyping assay based on a novel marker demonstrates potential for forensic applications.

The opium poppy acts as an important natural pain reliever but is also responsible for increased rates of severe drug abuse and addiction owing to its characteristic psychoactive effect. Non-medical illicit use of the poppy plant is markedly increasing worldwide, thereby highlighting the need for a robust species identification strategy. In this study, we identified SNPs within the region of two universal DNA barcodes, matK (maturase K) and the trnL-trnF (tRNA-Leu [3′exon]-tRNA-Phe [exon] intergenic spacer, that are forensically applicable for distinguishing opium poppy species based on a genetic analysis of 164 samples of family Papaveraceae obtained from locations spanning Jeolla-do and Jeju Island, Republic of Korea. A comparative analysis of the DNA barcode sequences for two narcotic types of the Papaver species (Papaver somniferum, Papaver somniferum subs. setigerum) to eight non-narcotic species revealed three unique nucleotide substitution events. Newly identified SNPs were located at position 255 of matK and at positions 305 and 306 of trnL-trnF; the narcotic species contained C, A, and T, whereas non-narcotic species contained T, G, and C at these positions. Phylogenetic analysis demonstrated that newly identified SNPs, which we named PsMAT255 and PsLF305/306, could be used to clearly differentiate between the narcotic and non-narcotic types of Papaver species based on the patterns of nucleotide variation. These results indicate that the nucleotide differences between the narcotic and non-narcotic species may influence genetic markers. We, therefore, developed a novel SNP-based allelic genotyping assay using the RT-PCR system that can reliably differentiate the narcotic type of the Papaver species. In summary, our findings suggest that the newly identified species-specific SNPs of both matK and trnL-trnF can be used as identification markers of narcotic Papaver species. Furthermore, a newly developed TaqMan allelic discrimination assay may be used as a practically applicable diagnostic method to survey several illicit narcotic specimens carrying the type-specific SNP. •SNP markers were analyzed to differentiate narcotic and non-narcotic Papaver species.•The novel SNP genotyping assay discriminated between narcotic types of Papaver species.•Identified SNP markers for 133 suspected to be illegal poppy plants were validated.

Background: In livestock species like the chicken, high throughput single nucleotide polymorphism (SNP) genotyping assays are increasingly being used for whole genome association studies and as a tool in breeding (referred to as genomic selection). To be of value in a wide variety of breeds and populations, the success rate of the SNP genotyping assay, the distribution of the SNP across the genome and the minor allele frequencies (MAF) of the SNPs used are extremely important. Results: We describe the design of a moderate density (60k) Illumina SNP BeadChip in chicken consisting of SNPs known to be segregating at high to medium minor allele frequencies (MAF) in the two major types of commercial chicken (broilers and layers). This was achieved by the identification of 352,303 SNPs with moderate to high MAF in 2 broilers and 2 layer lines using Illumina sequencing on reduced representation libraries. To further increase the utility of the chip, we also identified SNPs on sequences currently not covered by the chicken genome assembly (Gallus_gallus-2.1). This was achieved by 454 sequencing of the chicken genome at a depth of 12x and the identification of SNPs on 454-derived contigs not covered by the current chicken genome assembly. In total we added 790 SNPs that mapped to 454-derived contigs as well as 421 SNPs with a position on Chr_random of the current assembly. The SNP chip contains 57,636 SNPs of which 54,293 could be genotyped and were shown to be segregating in chicken populations. Our SNP identification procedure appeared to be highly reliable and the overall validation rate of the SNPs on the chip was 94%. We were able to map 328 SNPs derived from the 454 sequence contigs on the chicken genome. The majority of these SNPs map to chromosomes that are already represented in genome build Gallus_gallus-2.1.0. Twenty-eight SNPs were used to construct two new linkage groups most likely representing two micro-chromosomes not covered by the current genome assembly. Conclusions: The high success rate of the SNPs on the Illumina chicken 60K Beadchip emphasizes the power of Next generation sequence (NGS) technology for the SNP identification and selection step. The identification of SNPs from sequence contigs derived from NGS sequencing resulted in improved coverage of the chicken genome and the construction of two new linkage groups most likely representing two chicken micro-chromosomes.

Fusarium graminearum is an important pathogen causing Fusarium head blight (FHB) on wheat and barley and Fusarium ear rot (FER) on maize, and harvested grains often are contaminated with trichothecenes such as deoxynivalenol (DON) and nivalenol (NIV) that are a major health and food safety concern due to their toxicity to humans and farm animals. In this study, species identity and trichothecene toxin potential of 294 members of the Fusarium graminearum species complex (FGSC) collected from wheat, barley and maize in France in 2011 was determined using a microsphere-based multilocus genotyping assay. F. graminearum was predominant on all three hosts, but three isolates of F. cortaderiae and two isolates representing F. graminearum × F. boothii hybrids were also identified from maize. The 15-ADON trichothecene chemotype predominated on all three hosts, representing 94.7 %, 87.8 % and 85.4 % of the strains on barley ( N  = 19), wheat ( N  = 90), and maize ( N  = 185), respectively. However, the NIV chemotype was found in 12.2 % of the wheat isolates and in 14.6 % of the maize isolates. Only a single FGSC isolate from this study, originating from barley, was found to have the 3-ADON chemotype. Regional differences could be observed in the distribution of the 15-ADON and NIV chemotypes, with the NIV producing-isolates being present at higher frequency (21.2 %) in the South of France compared to the rest of the country (4.4 %). Such information is critical because of the increased concern associated with NIV contamination of cereals. In addition, these results are needed to develop management strategies for FHB and FER in France and to improve understanding of the distribution and significance of FGSC diversity in Europe and worldwide.

Spanish bunch groundnut varieties occupy most of the cultivated area in Asia and Africa, and these varieties lack required 2-3 weeks of fresh seed dormancy (FSD) hampering kernel quality. Genomic breeding can help to improve commercial groundnut cultivars for FSD in a shorter time with greater precision. In this regard, a recombinant inbred line (RIL) population from the cross ICGV 02266 (non-dormant) × ICGV 97045 (dormant) was developed and genotyped with a 5 K mid-density genotyping assay. A linkage map was constructed with 325 SNP loci spanning a total map length of 2335.3 cM and five major QTLs were identified on chromosomes Ah01, Ah11, Ah06, Ah16 and Ah17. Based on differential gene expression using transcriptomic information from dormant (Tifrunner) and non-dormant (ICGV 91114) genotypes, histone deacetylases, histone-lysine N-methyltransferase, cytochrome P450, protein kinases, and ethylene-responsive transcription factor were identified as key regulators involved in the hormonal regulation of dormancy. Six Kompetitive Allele Specific PCR (KASP) markers were successfully validated in the diverse panel including selected RILs of the same population and germplasm lines. These validated KASP markers could facilitate faster breeding of new varieties with desired dormancy using marker-assisted early generation selection.

Background Herbicide tolerance is an important trait that allows effective weed management in wheat crops in dryland farming. Genetic knowledge of metribuzin tolerance in wheat is needed to develop new cultivars for the industry. Here, we investigated gene effects for metribuzin tolerance in nine crosses of wheat by partitioning the means and variances of six basic generations from each cross into their genetic components to assess the gene action governing the inheritance of this trait. Metribuzin tolerance was measured by a visual senescence score 21 days after treatment. The wheat 90 K iSelect SNP genotyping assay was used to identify the distribution of alleles at SNP sites in tolerant and susceptible groups. Results The scaling and joint-scaling tests indicated that the inheritance of metribuzin tolerance in wheat was adequately described by the additive-dominance model, with additive gene action the most significant factor for tolerance. The potence ratio for all the crosses ranged between − 1 and + 1 for senescence under metribuzin-treated conditions indicating a semi-dominant gene action in the inheritance of metribuzin tolerance in wheat. The number of segregating genes governing metribuzin tolerance was estimated between 3 and 15. The consistent high heritability range (0.82 to 0.92) in F 5–7 generations of Chuan Mai 25 (tolerant) × Ritchie (susceptible) cross indicated a significant contribution of additive genetic effects to metribuzin tolerance in wheat. Several genes related to photosynthesis (e.g. photosynthesis system II assembly factor YCF48), metabolic detoxification of xenobiotics and cell growth and development (cytochrome P450, glutathione S-transferase, glycosyltransferase, ATP-binding cassette transporters and glutathione peroxidase) were identified on different chromosomes (2A, 2D, 3B, 4A, 4B, 7A, 7B, 7D) governing metribuzin tolerance. Conclusions The simple additive–dominance gene effects for metribuzin tolerance will help breeders to select tolerant lines in early generations and the identified genes may guide the development of functional markers for metribuzin tolerance.

For rapid detection and tracking of SARS-CoV-2, a simple screening method alternative to laborious and expensive sequencing is highly desirable. Here, we evaluated performance characteristics of TaqMan SARS-CoV-2 mutation panel genotyping molecular assay for detection of most common reported SARS-CoV-2 variants using specific RT-PCR assays targeting single nucleotide polymorphisms (SNP). A total of 150 SARS-CoV-2 positive samples from March to July were included for this study. In addition, five controls comprised of synthetic RNA B.1.1.7_601443, B.1.351_678597, P.1_792683, B.1.617.1_1662307 and MN908947.3-Wuhan-hu-1 from Twist bioscience and B.1.1.7 (England/204820464/2020) and B.1.351 (South Africa/KRISP-K005325/2020) from Zeptometrix, NY, USA were used for validation. Total RNA from specimens was extracted using Omega Bio-Tek Mag-Bind Viral RNA Xpress Extraction Kit and tested for known SARS-CoV2 variants using ThermoFisher TaqMan SARS-CoV-2 mutation panel molecular assay on the QuantStudio 12K Flex. Nine representative samples have been compared with sequencing. Data were analyzed by genotype calling using QuantStudio™ design and analysis software v 2.5 with the genotyping analysis module. All validation controls were tested in triplicate and repeated in singlet on three different days and all reported variants were matched as expected. Out of 150 SARS-CoV-2 positive specimens, 69 (46%) were B.1.617.2, 49 (32.7%) were B.1.1.7, P.1 and P.2 were 4 (2.7%) each and B.1.351 and B.1.427/B.1429 were 2 (1.3%) each. Three (2%) were B.1.526, and 17 (11.3%) have a mutation in D614G. Genotyping results from the present study showing B.1.617.2, B.1.1.7, and B.1.526 variants and their mutation genes were concordant with sequencing results. Our study indicates that TaqMan SARS-CoV-2 mutation panel molecular genotyping assays detect and differentiate all published common variants B.1.617.2 (Delta), B.1.1.7 (Alpha), B.1.526 (Iota), B.1.351 (Beta), P.1 (Gamma), P.2 (Zeta), B.1.617.1 (Kappa) and B.1.427/B.1.429 (Epsilon) that can be used for surveillance and epidemic control and prevention.

Key MessageThe tendency of somatic embryogenesis to regenerate plants only from the L1 layer, associated with the spread of chimerism in grapevine, must be carefully considered in the framework of biotechnological improvement programmes.Grapevine is an important fruit crop with a high economic value linked to traditional genotypes that have been multiplied for centuries by vegetative propagation. In this way, somatic variations that can spontaneously occur within the shoot apical meristem are fixed in the whole plant and represent a source of intra-varietal variability. Previously identified inconsistencies in the allelic calls of single nucleotide variants (SNVs) suggested that the Vitis vinifera ‘Nebbiolo’ CVT185 clone is a potential periclinal chimera. We adopted the somatic embryogenesis technique to separate the two genotypes putatively associated with the L1 and L2 layers of CVT185 into different somaclones. Despite the recalcitrance of ‘Nebbiolo’ to the embryogenic process, 58 somaclones were regenerated and SNV genotyping assays attested that the genotype of all them differed from that of the mother plant and was only attributable to L1. The results confirmed that L2 has low or no competence for differentiating somatic embryos. After one year in the greenhouse, the somaclones showed no phenotypic alterations in comparison with the mother plant; however further analyses are needed to identify potential endogenous sources of variation. The tendency of somatic embryogenesis to regenerate plants only from L1 must be carefully considered in the framework of biotechnological improvement programmes in this species.