Aaron D. Beattie

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.

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 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.

Mapping of the oat crown rust resistance gene Pc91

Crown rust is an important disease of oat caused by Puccinia coronata Corda f. sp. avenae Eriks. Crown rust is efficiently and effectively managed through the development of resistant oat varieties. Pc91 is a seedling crown rust resistance gene that is highly effective against the current P. coronata population in North America. The primary objective of this study was to develop DNA markers linked to Pc91 for purposes of marker-assisted selection in oat breeding programs. The Pc91 locus was mapped using a population of F7-derived recombinant inbred lines developed from the cross ‘CDC Sol-Fi’/‘HiFi’ made at the Crop Development Centre, University of Saskatchewan. The population was evaluated for reaction to P. coronata in field nurseries in 2008 and 2009. Pc91 mapped to a linkage group consisting of 44 Diversity Array Technology (DArT) markers. DArTs were successfully converted to sequence characterized amplified region (SCAR) markers. Five robust SCARs were developed from three non-redundant DArTs that co-segregated with Pc91. SCAR markers were developed for different assay systems, such that SCARs are available for agarose gel electrophoresis, capillary electrophoresis, and Taqman single nucleotide polymorphism detection. The SCAR markers accurately postulated the Pc91 status of 23 North American oat breeding lines.

Population Genomics Related to Adaptation in Elite Oat Germplasm

An oat association‐mapping panel contributed by active breeding programs worldwide. Characterized population structure and found subdivisions related to adaptation Characterized genome‐wide and chromosome‐specific linkage disequilibrium Performed association‐mapping and post hoc modeling of heading date Found several consistently associated QTL

A major quantitative trait locus conferring adult plant partial resistance to crown rust in oat

BackgroundCrown rust, caused by Puccinia coronata f. sp. avenae, is the most important disease of oat worldwide. Adult plant resistance (APR), based upon partial resistance, has proven to be a durable rust management strategy in other cereal rust pathosystems. The crown rust APR in the oat line MN841801 has been effective for more than 30 years. The genetic basis of this APR was studied under field conditions in three recombinant inbred line (RIL) populations: 1) AC Assiniboia/MN841801, 2) AC Medallion/MN841801, and 3) Makuru/MN841801. The populations were evaluated for crown rust resistance with the crown rust isolate CR251 (race BRBB) in multiple environments. The 6 K oat and 90 K wheat Illumina Infinium single nucleotide polymorphism (SNP) arrays were used for genotyping the AC Assiniboia/MN841801 population. KASP assays were designed for selected SNPs and genotyped on the other two populations.ResultsThis study reports a high density genetic linkage map constructed with oat and wheat SNP markers in the AC Assiniboia/MN841801 RIL population. Most wheat SNPs were monomorphic in the oat population. However the polymorphic wheat SNPs could be scored accurately and integrated well into the linkage map. A major quantitative trait locus (QTL) on oat chromosome 14D, designated QPc.crc-14D, explained up to 76% of the APR phenotypic variance. This QTL is flanked by two SNP markers, GMI_GBS_90753 and GMI_ES14_c1439_83. QPc.crc-14D was validated in the populations AC Medallion/MN841801 and Makuru/MN841801.ConclusionsWe report the first APR QTL in oat with a large and consistent effect. QPc.crc-14D was statistically significant in all environments tested in each of the three oat populations. QPc.crc-14D is a suitable candidate for use in marker-assisted breeding and also an excellent target for map-based cloning. This is also the first study to use the 90 K wheat Infinium SNP array on oat for marker development and comparative mapping. The Infinium SNP array is a useful tool for saturating oat maps with markers. Synteny with wheat suggests that QPc.crc-14D is orthologous with the stripe rust APR gene Yr16 in wheat.

Identification of Molecular Markers Linked to a Pyrenophora teres Avirulence Gene

ABSTRACT Genetic control of avirulence in the net blotch pathogen, Pyrenophora teres, was investigated. To establish an appropriate study system, a collection of 10 net form (P. teres f. teres) and spot form (P. teres f. maculata) isolates were evaluated on a set of eight barley lines to identify two isolates with differential virulence on an individual host line. Two net form isolates, WRS 1906, exhibiting avirulence on the cv. Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for reaction on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (chi(2) = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism markers closely linked to the avirulence gene (Avr(Heartland)). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model and represents an initial step toward map-based cloning of this gene.

Investigating the molecular structural features of hulless barley (Hordeum vulgare L.) in relation to metabolic characteristics using synchrotron-based fourier transform infrared microspectroscopy.

The synchrotron-based Fourier transform infrared microspectroscopy (SR-FTIRM) technique was used to quantify molecular structural features of the four hulless barley lines with altered carbohydrate traits [amylose, 1-40% of dry matter (DM); β-glucan, 5-10% of DM] in relation to rumen degradation kinetics, intestinal nutrient digestion, and predicted protein supply. Spectral features of β-glucan (both area and heights) in hulless barley lines showed a negative correlation with protein availability in the small intestine, including truly digested protein in the small intestine (DVE) (r = -0.76, P < 0.01; r = -0.84, P < 0.01) and total metabolizable protein (MP) (r = -0.71, P < 0.05; r = -0.84, P < 0.01). Variation in absorption intensities of total carbohydrate (CHO) was observed with negative effects on protein degradation, digestion, and potential protein supply (P < 0.05). Molecular structural features of CHO in hulless barley have negative effects on the supply of true protein to ruminants. The results clearly indicated the impact of the carbohydrate-protein structure and matrix.

Whole-Grain Fiber Composition Influences Site of Nutrient Digestion, Standardized Ileal Digestibility of Amino Acids, and Whole-Body Energy Utilization in Grower Pigs

BACKGROUND Variant chemical composition and physical structure of whole grains may change the site of energy digestion from the small to the large intestine. OBJECTIVE We determined the site of nutrient digestion, standardized ileal digestibility (SID) of amino acids (AAs), and net energy (NE) value of barley cultivars that vary in nutrient composition compared with wheat. METHODS Ileal-cannulated barrows (27.7 kg initial body weight) were fed diets containing 800 g whole grains/kg alongside a basal and nitrogen-free diet for calculations in a 6 (period) × 7 (diet) Youden square. Diets included 1 of 5 whole grains-1) high-fermentable, high-β-glucan, hull-less barley (HFB); 2) high-fermentable, high-amylose, hull-less barley (HFA); 3) moderate-fermentable, hull-less barley (MFB); 4) low-fermentable, hulled barley (LFB); and 5) low-fermentable, hard red spring wheat (LFW). Intestine nutrient flow and whole-body energy utilization were tested and explained by using whole-grain and digesta confocal laser scanning. RESULTS Starch apparent ileal digestibility was 14-29% lower (P < 0.05) in HFB and HFA than in MFB, LFB, and LFW due to the unique embedding of starch within the protein-fiber matrix of HFB and the high amylose content in HFA. Starch hindgut fermentation was 50-130% higher (P < 0.05) in HFB and HFA than in MFB, LFB, and LFW. The SID of indispensable AAs was lower (P < 0.05) in HFB and HFA than in MFB, LFB, and LFW. NE value was 18% higher (P < 0.05) for HFB than for HFA and was not different from MFB, LFB, and LFW. CONCLUSIONS Whole grains high in fermentable carbohydrates shifted digestion from the small intestine to the hindgut. NE value depended on the concentration of fermentable fiber and starch and digestible protein, ranging from 2.12-1.76 Mcal/kg in barley to 1.94 Mcal/kg in wheat. High-fiber whole grains may be used as energy substrates for pigs; however, the reduced SID of AAs requires titration of indispensable AAs to maintain growth.

Whole-Grain Starch and Fiber Composition Modifies Ileal Flow of Nutrients and Nutrient Availability in the Hindgut, Shifting Fecal Microbial Profiles in Pigs

Background: Changes in whole-grain chemical composition can affect the site of nutrient digestion, which may alter substrate availability and gut microbiota composition.Objective: This study elucidated the function of whole-grain fermentable fiber composition on ileal substrate flow, hindgut substrate availability, and subsequent gut microbial profiles in pigs.Methods: Five whole grains-1) high-fermentability, high-β-glucan hull-less barley (HFB); 2) high-fermentability, high-amylose hull-less barley (HFA); 3) moderate-fermentability hull-less barley (MFB); 4) low-fermentability hulled barley (LFB); or 5) low-fermentability hard red spring wheat (LFW)-were included at 800 g/kg into diets fed to ileal-cannulated growing pigs for 9 d in a 6 (periods) × 5 (diets) Youden square. Digesta were analyzed for nutrient flow and microbial composition via 16S ribosomal RNA gene sequencing.Results: The consumption of fermentable whole grains, HFB, and HFA increased (P < 0.05) ileal starch flow by 69% and dry matter flow by 37% compared with LFB and LFW intakes. The consumption of HFB and HFA increased (P < 0.05) fecal Firmicutes phylum abundance by 26% and 21% compared with LFB intake and increased (P < 0.05) fecal Dialister genus abundance, on average, by 98% compared with LFB and LFW intakes. Fecal Sharpea and Ruminococcus genera abundances increased (P < 0.05) with HFB intake compared with LFB and LFW intakes. In contrast, the consumption of LFB increased (P < 0.05) fecal Bacteroidetes phylum abundance by 43% compared with MFB intake. Ileal starch flow and fecal Firmicutes abundance were positively correlated and determined by using principal components analysis.Conclusions: Increasing dietary fermentable fiber from whole grains can increase ileal substrate flow and hindgut substrate availability, shifting the fecal microbiota toward Firmicutes phylum members. Thus, digesta substrate flow is important to shape gut microbial profiles in pigs, which indicates that the manipulation of substrate flow should be considered as a tool to modulate gut microbiota composition.