Nicholas A. Tinker

Biplot analysis of multi-environment trial data: Principles and applications

Biplot analysis has evolved into an important statistical tool in plant breeding and agricultural research. Here we review the basic principles of biplot analysis and recent developments in its application in analyzing multi-environment trail (MET) data, with the aim of providing a working guide for breeders, agronomists, and other agricultural scientists on biplot analysis and interpretation. The review is divided into four sections. The first section is a complete but succinct description of the principles of biplot analysis. The second section is a detailed treatment of biplot analysis of genotype by environment data. It addresses environment and genotype evaluation from all perspectives. The third section deals with biplot analysis of various two-way tables that can be generated from a three-way MET dataset, which is an integral and essential part to a fuller understanding and exploration of MET data. The final section discusses questions that are frequently asked about biplot analysis. Methods descri...

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

BackgroundGenetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.ResultsBased on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.ConclusionsThe high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

Using Genotyping-By-Sequencing (GBS) for Genomic Discovery in Cultivated Oat

Advances in next-generation sequencing offer high-throughput and cost-effective genotyping alternatives, including genotyping-by-sequencing (GBS). Results have shown that this methodology is efficient for genotyping a variety of species, including those with complex genomes. To assess the utility of GBS in cultivated hexaploid oat (Avena sativa L.), seven bi-parental mapping populations and diverse inbred lines from breeding programs around the world were studied. We examined technical factors that influence GBS SNP calls, established a workflow that combines two bioinformatics pipelines for GBS SNP calling, and provided a nomenclature for oat GBS loci. The high-throughput GBS system enabled us to place 45,117 loci on an oat consensus map, thus establishing a positional reference for further genomic studies. Using the diversity lines, we estimated that a minimum density of one marker per 2 to 2.8 cM would be required for genome-wide association studies (GWAS), and GBS markers met this density requirement in most chromosome regions. We also demonstrated the utility of GBS in additional diagnostic applications related to oat breeding. We conclude that GBS is a powerful and useful approach, which will have many additional applications in oat breeding and genomic studies.

Proteomic analyses of Fusarium graminearum grown under mycotoxin-inducing conditions

Non‐gel‐based quantitative proteomics technology was used to profile protein expression differences when Fusarium graminearum was induced to produce trichothecenes in vitro. As F. graminearum synthesizes and secretes trichothecenes early in the cereal host invasion process, we hypothesized that proteins contributing to infection would also be induced under conditions favouring mycotoxin synthesis. Protein samples were extracted from three biological replicates of a time course study and subjected to iTRAQ (isobaric tags for relative and absolute quantification) analysis. Statistical analysis of a filtered dataset of 435 proteins revealed 130 F. graminearum proteins that exhibited significant changes in expression, of which 72 were upregulated relative to their level at the initial phase of the time course. There was good agreement between upregulated proteins identified by 2‐D PAGE/MS/MS and iTRAQ. RT‐PCR and northern hybridization confirmed that genes encoding proteins which were upregulated based on iTRAQ were also transcriptionally active under mycotoxin producing conditions. Numerous candidate pathogenicity proteins were identified using this technique. These will provide leads in the search for mechanisms and markers of host invasion and novel antifungal targets.

Genetic diversity among oat varieties of worldwide origin and associations of AFLP markers with quantitative traits

One hundred and fourteen oat (Avena sativa L.) varieties of worldwide origin were evaluated for genetic diversity based on 77 molecular polymorphisms produced by eight selective AFLP primer combinations. Genetic similarity, calculated using the DICE coefficient, was used for cluster analysis and principal component analysis was applied. In addition population structure was explored to identify discrete subpopulations based on allele frequency. Although clustering and population structure showed relationships with region and country of origin, there was no obvious relationship to hull presence or hull colour. Oat varieties originating from European breeding programs showed less diversity than varieties originating from North and South America. Associations between AFLP markers and agronomic traits (grain yield, groat yield, panicle emergence, plant height, and lodging) as well as kernel quality traits (kernel weight, test weight, screening percent and groat percent) were also investigated. Marker-trait associations were tested using a naïve simple regression model and five additional models that account for population structure. Significant associations were found for 23 AFLP markers, with many of these affecting multiple traits. This study demonstrates that diversity can be significantly enhanced using a global collection, and provides evidence for marker-trait associations that can be validated in segregating populations and exploited through marker-assisted selection.

A molecular linkage map with associated QTLs from a hulless × covered spring oat population

In spring-type oat (Avena sativa L.), quantitative trait loci (QTLs) detected in adapted populations may have the greatest potential for improving germplasm via marker-assisted selection. An F6 recombinant inbred (RI) population was developed from a cross between two Canadian spring oat varieties: ‘Terra’, a hulless line, and ‘Marion’, an elite covered-seeded line. A molecular linkage map was generated using 430 AFLP, RFLP, RAPD, SCAR, and phenotypic markers scored on 101 RI lines. This map was refined by selecting a robust set of 124 framework markers that mapped to 35 linkage groups and contained 35 unlinked loci. One hundred one lines grown in up to 13 field environments in Canada and the United States between 1992 and 1997 were evaluated for 16 agronomic, kernel, and chemical composition traits. QTLs were localized using three detection methods with an experiment-wide error rate of approximately 0.05 for each trait. In total, 34 main-effect QTLs affecting the following traits were identified: heading date, plant height, lodging, visual score, grain yield, kernel weight, milling yield, test weight, thin and plump kernels, groat β-glucan concentration, oil concentration, and protein. Several of these correspond to QTLs in homologous or homoeologous regions reported in other oat QTL studies. Twenty-four QTL-by-environment interactions and three epistatic interactions were also detected. The locus controlling the covered/hulless character (N1) affected most of the traits measured in this study. Additive QTL models with N1 as a covariate were superior to models based on separate covered and hulless sub-populations. This approach is recommended for other populations segregating for major genes. Marker-trait associations identified in this study have considerable potential for use in marker-assisted selection strategies to improve traits within spring oat breeding programs.

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

A SNP Genotyping Array for Hexaploid Oat

Recognizing a need in cultivated hexaploid oat (Avena sativa L.) for a reliable set of reference single nucleotide polymorphisms (SNPs), we have developed a 6000 (6K) BeadChip design containing 257 Infinium I and 5486 Infinium II designs corresponding to 5743 SNPs. Of those, 4975 SNPs yielded successful assays after array manufacturing. These SNPs were discovered based on a variety of bioinformatics pipelines in complementary DNA (cDNA) and genomic DNA originating from 20 or more diverse oat cultivars. The array was validated in 1100 samples from six recombinant inbred line (RIL) mapping populations and sets of diverse oat cultivars and breeding lines, and provided approximately 3500 discernible Mendelian polymorphisms. Here, we present an annotation of these SNPs, including methods of discovery, gene identification and orthology, population‐genetic characteristics, and tentative positions on an oat consensus map. We also evaluate a new cluster‐based method of calling SNPs. The SNP design sequences are made publicly available, and the full SNP genotyping platform is available for commercial purchase from an independent third party.

Identification of molecular markers for aluminium tolerance in diploid oat through comparative mapping and QTL analysis

The degree of aluminium tolerance varies widely across cereal species, with oats (Avena spp.) being among the most tolerant. The objective of this study was to identify molecular markers linked to aluminium tolerance in the diploid oat A. strigosa. Restriction fragment length polymorphism markers were tested in regions where comparative mapping indicated the potential for orthologous quantitative trait loci (QTL) for aluminium tolerance in other grass species. Amplified fragment length polymorphism (AFLP) and sequence-characterized amplified region (SCAR) markers were used to provide additional coverage of the genome. Four QTL were identified. The largest QTL explained 39% of the variation and is possibly orthologous to the major gene found in the Triticeae as well as Alm1 in maize and a minor gene in rice. A second QTL may be orthologous to the Alm2 gene in maize. Two other QTL were associated with anonymous markers. Together, these QTL accounted for 55% of the variation. A SCAR marker linked to the major QTL identified in this study could be used to introgress the aluminium tolerance trait from A. strigosa into cultivated oat germplasm.

A consensus map in cultivated hexaploid oat reveals conserved grass synteny with substantial subgenome rearrangement

We constructed a hexaploid oat consensus map from 12 populations representing 19 parents. The map represents the most common physical chromosome arrangements in oat. Deviations from the consensus map may indicate physical rearrangements. Large chromosomal translocations vary among different varieties. There is regional synteny with rice but considerable subgenome rearrangement.