An island of receptor-like genes at the Rrs13 locus on barley chromosome 6HS co-locate with three novel sources of scald resistance.

Three Hordeum spontaneum-derived resistances (referred to as 145L2, 41T1 and 40Y5) have demonstrated long-term effectiveness against barley scald, caused by Rhynchosporium commune, in western Canada. Genetic mapping of these resistances in three populations, and the use of five barley genome assemblies, revealed they co-located to a narrowly defined 0.58-1.2 Mbp region of chromosome 6HS containing the Rrs13 scald resistance gene. Differential disease reactions among the three resistances and a Rrs13 carrier (AB6) to a panel of 24 scald isolates indicated that the four resistances were unique from one another. A marker created to target the 6HS scald locus was screened across a panel of barley germplasm that included H. vulgare, H. spontaneum and H. bulbosum lines. The marker showed specificity to H. vulgare lines known to carry the 6HS scald resistances and to two H. spontaneum lines that trace their origins to Jordan. Within the 0.58-1.2 Mbp region were 2-7 tandemly repeated leucine-rich repeat receptor-like proteins (LRR-RLP) and one lectin receptor-like kinase (Lec-RLK) genes with abundant sequence variation between them. The well-defined role that RLP and RLK genes play in plant defense responses make them logical candidate resistance genes, with one possible hypothesis being that each unique scald resistance may be encoded by a different RLP that interacts with a common RLK. It is suggested the three scald resistances be temporarily named Rrs13145L2, Rrs1341T1 and Rrs1340Y5 to recognize their co-location to the Rrs13 locus until it is determined whether these resistances represent unique genes or alleles of the same gene.

Determining the Impact of Genotype × Environment on Oat Protein Isolate Composition Using HPLC and LC-MS Techniques.

The effect of genotype and environment on oat protein composition was analyzed through size exclusion-high-performance liquid chromatography (SE-HPLC) and liquid chromatography-mass spectrometry (LC-MS) to characterize oat protein isolate (OPI) extracted from three genotypes grown at three locations in the Canadian Prairies. SE-HPLC identified four fractions in OPI, including polymeric globulins, avenins, glutelins, and albumins, and smaller proteins. The protein composition was dependent on the environment, rather than the genotype. The proteins identified through LC-MS were grouped into eight categories, including globulins, prolamins/avenins, glutelins, enzymes/albumins, enzyme inhibitors, heat shock proteins, grain softness proteins, and allergenic proteins. Three main globulin protein types were also identified, including the P14812|SSG2-12S seed storage globulin, the Q6UJY8_TRITU-globulin, and the M7ZQM3_TRIUA-Globulin-1 S. Principal component analysis indicated that samples from Manitoba showed a positive association with the M7ZQM3_TRIUA-Globulin-1 S allele and Q6UJY8_TRITU-globulin, while samples from Alberta and Saskatchewan had a negative association with them. The results show that the influence of G × E on oat protein fractions and their relative composition is crucial to understanding genotypes' behavior in response to different environments.

Genome-wide association analysis provides insights into the genetic basis of photosynthetic responses to low-temperature stress in spring barley.

Low-temperature stress (LTS) is among the major abiotic stresses affecting the geographical distribution and productivity of the most important crops. Understanding the genetic basis of photosynthetic variation under cold stress is necessary for developing more climate-resilient barley cultivars. To that end, we investigated the ability of chlorophyll fluorescence parameters (FVFM, and FVF0) to respond to changes in the maximum quantum yield of Photosystem II photochemistry as an indicator of photosynthetic energy. A panel of 96 barley spring cultivars from different breeding zones of Canada was evaluated for chlorophyll fluorescence-related traits under cold acclimation and freeze shock stresses at different times. Genome-wide association studies (GWAS) were performed using a mixed linear model (MLM). We identified three major and putative genomic regions harboring 52 significant quantitative trait nucleotides (QTNs) on chromosomes 1H, 3H, and 6H for low-temperature tolerance. Functional annotation indicated several QTNs were either within the known or close to genes that play important roles in the photosynthetic metabolites such as abscisic acid (ABA) signaling, hydrolase activity, protein kinase, and transduction of environmental signal transduction at the posttranslational modification levels. These outcomes revealed that barley plants modified their gene expression profile in response to decreasing temperatures resulting in physiological and biochemical modifications. Cold tolerance could influence a long-term adaption of barley in many parts of the world. Since the degree and frequency of LTS vary considerably among production sites. Hence, these results could shed light on potential approaches for improving barley productivity under low-temperature stress.

Optimization of Ustilago nuda Inoculum Concentration for Screening Un8-Mediated Loose Smut Resistance in Barley Reveals a Resistance Reaction that Disrupts Seed Germination and Suggests a Role for Abscisic Acid in Disease Development.

Barley loose smut has been effectively controlled for decades through resistance conferred by the Un8 gene. However, evaluation of loose smut reaction using floret inoculation at the standard inoculum concentration is associated with the production of small, discolored seeds in Un8 carriers and susceptible genotypes. Interestingly, Un8 carriers also displayed significantly poorer germination than susceptible genotypes and produce short-lived seedlings following inoculation. To understand these observations, a Un8 carrier (TR11698) and susceptible non-Un8 carrier (CDC Austenson) were assessed for seed traits, Ustilago nuda biomass in the seed, infection rate, and phytohormone profile across a range of lower inoculum concentrations. At lower inoculum concentrations, seed appearance and weight improved in both genotypes, and infection rate increased in CDC Austenson. Pathogen load in the seed was similar in both genotypes and was positively correlated with the CDC Austenson infection rate. No infection was ever observed in TR11698. Significantly, germination rate improved in CDC Austenson, whereas the very low germination rate and short-lived seedlings remained associated with TR11698. It appears that poor seed appearance in both genotypes and low germination rate in the susceptible genotype can be improved by lowering the inoculum concentration. However, the very low germination rates and seedling death associated with the Un8 carrier TR11698 are indicative of Un8-mediated resistance to loose smut. Finally, profiling of 38 phytohormones revealed that larger seeds observed at some inoculum concentrations compared with mock inoculation had higher abscisic acid concentrations. This could represent a pathogen survival strategy by ensuring better growth of the host.

Identification of Putative SNP Markers Associated with Resistance to Egyptian Loose Smut Race(s) in Spring Barley.

Loose smut (LS) disease is a serious problem that affects barley yield. Breeding of resistant cultivars and identifying new genes controlling LS has received very little attention. Therefore, it is important to understand the genetic basis of LS control in order to genetically improve LS resistance. To address this challenge, a set of 57 highly diverse barley genotypes were inoculated with Egyptian loose smut race(s) and the infected seeds/plants were evaluated in two growing seasons. Loose smut resistance (%) was scored on each genotype. High genetic variation was found among all tested genotypes indicating considerable differences in LS resistance that can be used for breeding. The broad-sense heritability (H2) of LS (0.95) was found. Moreover, genotyping-by-sequencing (GBS) was performed on all genotypes and generated in 16,966 SNP markers which were used for genetic association analysis using single-marker analysis. The analysis identified 27 significant SNPs distributed across all seven chromosomes that were associated with LS resistance. One SNP (S6_17854595) was located within the HORVU6Hr1G010050 gene model that encodes a protein kinase domain-containing protein (similar to the Un8 LS resistance gene, which contains two kinase domains). A TaqMan marker (0751D06 F6/R6) for the Un8 gene was tested in the diverse collection. The results indicated that none of the Egyptian genotypes had the Un8 gene. The result of this study provided new information on the genetic control of LS resistance. Moreover, good resistance genotypes were identified and can be used for breeding cultivars with improved resistance to Egyptian LS.

Genetic diversity and population structure assessment of Western Canadian barley cooperative trials.

Studying the population structure and genetic diversity of historical datasets is a proposed use for association analysis. This is particularly important when the dataset contains traits that are time-consuming or costly to measure. A set of 96 elite barley genotypes, developed from eight breeding programs of the Western Canadian Cooperative Trials were used in the current study. Genetic diversity, allelic variation, and linkage disequilibrium (LD) were investigated using 5063 high-quality SNP markers via the Illumina 9K Barley Infinium iSelect SNP assay. The distribution of SNPs markers across the barley genome ranged from 449 markers on chromosome 1H to 1111 markers on chromosome 5H. The average polymorphism information content (PIC) per locus was 0.275 and ranged from 0.094 to 0.375. Bayesian clustering in STRUCTURE and principal coordinate analysis revealed that the populations are differentiated primarily due to the different breeding program origins and ear-row type into five subpopulations. Analysis of molecular variance based on PhiPT values suggested that high values of genetic diversity were observed within populations and accounted for 90% of the total variance. Subpopulation 5 exhibited the most diversity with the highest values of the diversity indices, which represent the breeding program gene pool of AFC, AAFRD, AU, and BARI. With increasing genetic distance, the LD values, expressed as r2, declined to below the critical r2 = 0.18 after 3.91 cM, and the same pattern was observed on each chromosome. Our results identified an important pattern of genetic diversity among the Canadian barley panel that was proposed to be representative of target breeding programs and may have important implications for association mapping in the future. This highlight, that efforts to identify novel variability underlying this diversity may present practical breeding opportunities to develop new barley genotypes.

The Genetic Architecture of Milling Quality in Spring Oat Lines of the Collaborative Oat Research Enterprise.

Most oat grains destined for human consumption must possess the ability to pass through an industrial de-hulling process with minimal breakage and waste. Uniform grain size and a high groat to hull ratio are desirable traits related to milling performance. The purpose of this study was to characterize the genetic architecture of traits related to milling quality by identifying quantitative trait loci (QTL) contributing to variation among a diverse collection of elite and foundational spring oat lines important to North American oat breeding programs. A total of 501 lines from the Collaborative Oat Research Enterprise (CORE) panel were evaluated for genome-wide association with 6 key milling traits. Traits were evaluated in 13 location years. Associations for 36,315 markers were evaluated for trait means across and within location years, as well as trait variance across location years, which was used to assess trait stability. Fifty-seven QTL influencing one or more of the milling quality related traits were identified, with fourteen QTL mapped influencing mean and variance across location years. The most prominent QTL was Qkernel.CORE.4D on chromosome 4D at approximately 212 cM, which influenced the mean levels of all traits. QTL were identified that influenced trait variance but not mean, trait mean only and both.

Genome Assembly of the Canadian two-row Malting Barley cultivar AAC Synergy.

Barley (Hordeum vulgare L.) is one of the most important global crops. The six-row barley cultivar Morex reference genome has been used by the barley research community worldwide. However, this reference genome can have limitations when used for genomic and genetic diversity analysis studies, gene discovery, and marker development when working in two-row germplasm that is more common to Canadian barley. Here we assembled, for the first time, the genome sequence of a Canadian two-row malting barley, cultivar AAC Synergy. We applied deep Illumina paired-end reads, long mate-pair reads, PacBio sequences, 10X chromium linked read libraries, and chromosome conformation capture sequencing (Hi-C) to generate a contiguous assembly. The genome assembled from super-scaffolds had a size of 4.85 Gb, N50 of 2.32 Mb, and an estimated 93.9% of complete genes from a plant database (BUSCO, benchmarking universal single-copy orthologous genes). After removal of small scaffolds (< 300 Kb), the assembly was arranged into pseudomolecules of 4.14 Gb in size with seven chromosomes plus unanchored scaffolds. The completeness and annotation of the assembly were assessed by comparing it with the updated version of six-row Morex and recently released two-row Golden Promise genome assemblies.

Localization of the Stem Rust Resistance Gene Pg2 to Linkage Group Mrg20 in Cultivated Oat (Avena sativa).

Stem rust is an important disease of cultivated oat (Avena sativa) caused by Puccinia graminis f. sp. avenae. In North America, host resistance is the primary strategy to control this disease and is conferred by a relatively small number of resistance genes. Pg2 is a widely deployed stem rust resistance gene that originates from cultivated oat. Oat breeders wish to develop cultivars with multiple Pg genes to slow the breakdown of single gene resistance, and often require DNA markers suited for marker-assisted selection. Our objectives were to (i) construct high density linkage maps for a major oat stem rust resistance gene using three biparental mapping populations, (ii) develop Kompetitive allele-specific PCR (KASP) assays for Pg2-linked single-nucleotide polymorphisms (SNPs), and (iii) test the prediction accuracy of those markers with a diverse panel of spring oat lines and cultivars. Genotyping-by-sequencing SNP markers linked to Pg2 were identified in an AC Morgan/CDC Morrison recombinant inbred line (RIL) population. Pg2-linked SNPs were then analyzed in an AC Morgan/RL815 F2 population and an AC Morgan/CDC Dancer RIL population. Linkage analysis identified a common location for Pg2 in all three populations on linkage group Mrg20 of the oat consensus genetic map. The most predictive markers were identified and converted to KASP assays for use in oat breeding programs. When used in combination, the KASP assays for the SNP loci avgbs2_126549.1.46 and avgbs_cluster_23819.1.27 were highly predictive of Pg2 status in panel of 54 oat breeding lines and cultivars.

Mapping of the Oat Crown Rust Resistance Gene Pc39 Relative to Single Nucleotide Polymorphism Markers.

Crown rust, caused by Puccinia coronata f. sp. avenae Eriks. (Pca), is among the most important oat diseases resulting in significant yield losses in many growing regions. A gene-for-gene interaction is well established in this pathosystem and has been exploited by oat breeders to control crown rust. Pc39 is a seedling crown rust resistance gene that has been widely deployed in North American oat breeding. DNA markers are desired to accurately predict the specific Pc genes present in breeding germplasm. The objectives of the study were as follows: (i) to map Pc39 in two recombinant inbred line (RIL) populations (AC Assiniboia/MN841801 and AC Medallion/MN841801) and (ii) to identify single nucleotide polymorphism (SNP) markers for postulation of Pc39 in oat germplasm. Pc39 was mapped to a linkage group consisting of 16 SNP markers, which placed the gene on linkage group Mrg11 (chromosome 1C) of the oat consensus map. Pc39 cosegregated with SNP marker GMI_ES01_c12570_390 in the AC Assiniboia/MN841801 RIL population and was flanked by the SNP markers avgbs_126086.1.41 and GMI_ES15_c276_702, with genetic distances of 1.7 and 0.3 cM, respectively. In the AC Medallion/MN841801 RIL population, similar results were obtained but the genetic distances of the flanking markers were 0.4 and 0.4 cM, respectively. Kompetitive Allele-Specific PCR assays were successfully designed for Pc39-linked SNP loci. Two SNP loci defined a haplotype that accurately predicted Pc39 status in a diverse panel of oat germplasm and will be useful for marker-assisted selection in oat breeding.

Chromosomal location of the crown rust resistance gene Pc98 in cultivated oat (Avena sativa L.).

KEY MESSAGE: SNP loci linked to the crown rust resistance gene Pc98 were identified by linkage analysis and KASP assays were developed for marker-assisted selection in breeding programs. Crown rust is among the most damaging diseases of oat and is caused by Puccinia coronata var. avenae f. sp. avenae (Urban and Marková) (Pca). Host resistance is the preferred method to prevent crown rust epidemics. Pc98 is a race-specific, seedling crown rust resistance gene obtained from the wild oat Avena sterilis accession CAV 1979 that is effective at all growth stages of oat. Virulence to Pc98 has been very low in the Pca populations that have been tested. The objectives of this study were to develop SNP markers linked to Pc98 for use in marker-assisted selection and to locate Pc98 on the oat consensus map. The Pc98 gene was mapped using F2:3 populations developed from the crosses Pc98/Bingo and Pc98/Kasztan, where Pc98 is a single-gene line carrying Pc98. Both populations were evaluated in seedling inoculation experiments. Pc98 was mapped relative to Kompetitive Allele-Specific PCR SNP markers in both populations, placing Pc98 on the Mrg20 linkage group of the consensus map. Pc98 was bracketed by two SNP markers GMI_ES22_c3052_382_kom399 and GMI_ES14_lrc18344_662_kom398 in the Pc98/Bingo mapping population with genetic distances of 0.9 cM and 0.3 cM, respectively. Pc98 co-segregated with four SNP markers in the Pc98/Kasztan population, and the closest flanking markers were GMI_DS_LB_6017_kom367 and avgbs2_153634.1.59_kom410 with genetic distances of 0.7 cM and 0.3 cM, respectively. Two SNP loci defined a haplotype that accurately predicted Pc98 status in a diverse group of oat germplasm, which will be valuable for marker-assisted selection of Pc98 in breeding of new oat cultivars.

Mapping of the stem rust resistance gene Pg13 in cultivated oat.

KEY MESSAGE: The widely deployed, oat stem rust resistance gene Pg13 was mapped by linkage analysis and association mapping, and KASP markers were developed for marker-assisted selection in breeding programs. Pg13 is one of the most extensively deployed stem rust resistance genes in North American oat cultivars. Identification of markers tightly linked to this gene will be useful for routine marker-assisted selection, identification of gene pyramids, and retention of the gene in backcrosses and three-way crosses. To this end, high-density linkage maps were constructed in four bi-parental mapping populations using SNP markers identified from 6K oat Infinium iSelect and genotyping-by-sequencing platforms. Additionally, genome-wide associations were identified using two sets of association panels consisting of diverse elite oat lines in one set and landrace accessions in the other. The results showed that Pg13 was located at approximately 67.7 cM on linkage group Mrg18 of the consensus genetic map. The gene co-segregated with the 7C-17A translocation breakpoint and with crown rust resistance gene Pc91. Co-segregating markers with the best prediction accuracy were identified at 67.7-68.5 cM on Mrg18. KASP assays were developed for linked SNP loci for use in oat breeding.

Mapping Oat Crown Rust Resistance Gene Pc45 Confirms Association with PcKM.

Molecular mapping of crown rust resistance genes is important to effectively utilize these genes and improve breeding efficiency through marker-assisted selection. Pc45 is a major race-specific crown rust resistance gene initially identified in the wild hexaploid oat Avena sterilis in the early 1970s. This gene was transferred to cultivated oat (Avena sativa) and has been used as a differential for identification of crown rust races since 1974. Previous research identified an association between virulence to Pc45 and PcKM, a crown rust resistance gene in the varieties 'Kame' and 'Morton'. This study was undertaken to reveal the relationship between Pc45 and PcKMPc45 was studied in the crosses 'AC Morgan'/Pc45 and 'Kasztan'/Pc45, where Pc45 is the differential line carrying Pc45 F2 progenies and F2:3 families of both populations were inoculated with the crown rust isolate CR258 (race NTGG) and single gene segregation ratios were observed. SNP markers for PcKM were tested on these populations and linkage maps were generated. In addition, 17 newly developed SNP markers identified from genotyping-by-sequencing (GBS) data were mapped in these two populations, plus another three populations segregating for Pc45 or PcKMPc45 and PcKM mapped to the same location of Mrg08 (chromosome 12D) of the oat chromosome-anchored consensus map. These results strongly suggest that Pc45 and PcKM are the same resistance gene, but allelism (i.e., functionally different alleles of the same gene) or tight linkage (i.e., two tightly linked genes) cannot be ruled out based on the present data.

Effect of fibrolytic enzymes on lactational performance, feeding behavior, and digestibility in high-producing dairy cows fed a barley silage-based diet.

The objectives of this study were to evaluate the effects of pretreating dairy cow rations with a fibrolytic enzyme derived from Trichoderma reesei (FETR; mixture of xylanase and cellulase; AB Vista, Wiltshire, UK) on lactation performance, digestibility, and feeding behavior in response to feeding a barley silage-based diet. Before starting the dairy trial, in vitro incubations were conducted to determine whether the addition of FETR would have an effect on these animal performance characteristics when applied to a barley silage-based diet for dairy cows. The dairy trial was performed using 8 Holstein dairy cows. The cows were blocked by parity and assigned randomly to 1 of 4 treatments: 0, 0.5, 0.75, and 1 mL of FETR/kg of dry matter (DM) diet in a replicated Latin square design. The pretreatment was applied to the complete diet during the mixing process. The experimental period continued for 22 d, with each experimental period consisting of a 16-d adaptation period and a 6-d sampling period. The daily feed intake of each individual cow was monitored using Insentec feed bins (RIC system, Insentec, Marknesse, the Netherlands). Feeding behavior characteristics were measured during the entire sampling period using the feed bin attendance data. Milk samples were collected in the last 3 d of each experimental period. The addition of FETR linearly increased the in vitro DM digestibility and tended to improve the in vitro digestibility of barley silage. There was a cubic effect of the enzyme levels on the total-tract DM and neutral detergent fiber digestibility. Maximal digestibility was reached at 0.75 mL of FETR/kg of TMR. The milk fat yield, fat-corrected milk, and energy-corrected milk quadratically responded to the incremental levels of FETR. The milk protein percentage linearly improved in response to FETR. Increasing FETR levels resulted in a quadratic effect on feed efficiency. There was no effect of FETR level on feeding behavior. In conclusion, pretreating dairy cow barley silage-based diet with 0.75 mL of FETR/kg of TMR increased the milk production efficiency of dairy cows fed diet containing 34% barley silage (DM basis). The positive effect of adding FETR could benefit the dairy industry in western Canada, where barley silage-based diets are common.

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-ß-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.

Genome-Wide Association Mapping of Crown Rust Resistance in Oat Elite Germplasm.

Oat crown rust, caused by f. sp. , is a major constraint to oat ( L.) production in many parts of the world. In this first comprehensive multienvironment genome-wide association map of oat crown rust, we used 2972 single-nucleotide polymorphisms (SNPs) genotyped on 631 oat lines for association mapping of quantitative trait loci (QTL). Seedling reaction to crown rust in these lines was assessed as infection type (IT) with each of 10 crown rust isolates. Adult plant reaction was assessed in the field in a total of 10 location-years as percentage severity (SV) and as infection reaction (IR) in a 0-to-1 scale. Overall, 29 SNPs on 12 linkage groups were predictive of crown rust reaction in at least one experiment at a genome-wide level of statistical significance. The QTL identified here include those in regions previously shown to be linked with seedling resistance genes , , , , , and and also with adult-plant resistance and adaptation-related QTL. In addition, QTL on linkage groups Mrg03, Mrg08, and Mrg23 were identified in regions not previously associated with crown rust resistance. Evaluation of marker genotypes in a set of crown rust differential lines supported as the identity of . The SNPs with rare alleles associated with lower disease scores may be suitable for use in marker-assisted selection of oat lines for crown rust resistance.

Identification and functional analysis of new peroxygenases in oat.

MAIN CONCLUSION: Two new peroxygenases for the biosynthesis of epoxy fatty acids in oat were identified and functionally analyzed by heterologous expression along with rationally designed site-directed mutagenesis. Oat (Avena sativa L.) contains a large family of peroxygenases, a group of heme-containing monooxygenases catalyzing hydroperoxide-dependent epoxidation of unsaturated fatty acids. Here, we report identification and functional analysis of two new peroxygenases AsPXG2 and AsPXG3 from oat. The open reading frame (ORF) of AsPXG2 contains 702 bps encoding a polypeptide of 233 amino acids, while the ORF of AsPXG3 is 627 bps coding for 208 amino acids. Both AsPXG2 and AsPXG3 comprise a single transmembrane domain, conserved histidines for heme binding and a conserved EF-hand motif for calcium binding, but they only share about 50% amino acid sequence identity with each other. When expressed in Escherichia coli and Pichia pastoris, AsPXG3 showed high epoxidation activity, while AsPXG2 exhibited no activity in E. coli and low activity in P. pastoris. AsPXG3 could effectively epoxidize both mono- and polyunsaturated fatty acids with linolenic acid being the most preferred substrate. Site-directed mutagenesis was employed to investigate the structure-function relationship of oat peroxygenase on 12 conserved residues of AsPXG3. Replacement of two conserved histidines, the ligands to the prosthetic heme group of the peroxygenase, by alanine resulted in complete loss of activity. Substitution of three conserved residues surrounding the two histidines resulted in reduction of the enzymatic activity by more than 80%. These results imply that these conserved residues might be located in or near the catalytic pocket, where the two histidine residues coordinate the heme group and the surrounding residues define the shape and size of the pocket for interaction with the heme as well as two substrates.

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-ß-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.

A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement.

Hexaploid oat ( L., 2 = 6 = 42) is a member of the Poaceae family and has a large genome (∼12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high-density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA-derived single-nucleotide polymorphism (SNP) array and genotyping-by-sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice ( L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes.

Population Genomics Related to Adaptation in Elite Oat Germplasm.

Six hundred thirty five oat ( L.) lines and 4561 single-nucleotide polymorphism (SNP) loci were used to evaluate population structure, linkage disequilibrium (LD), and genotype-phenotype association with heading date. The first five principal components (PCs) accounted for 25.3% of genetic variation. Neither the eigenvalues of the first 25 PCs nor the cross-validation errors from = 1 to 20 model-based analyses suggested a structured population. However, the PC and = 2 model-based analyses supported clustering of lines on spring oat vs. southern United States origin, accounting for 16% of genetic variation ( < 0.0001). Single-locus -statistic () in the highest 1% of the distribution suggested linkage groups that may be differentiated between the two population subgroups. Population structure and kinship-corrected LD of = 0.10 was observed at an average pairwise distance of 0.44 cM (0.71 and 2.64 cM within spring and southern oat, respectively). On most linkage groups LD decay was slower within southern lines than within the spring lines. A notable exception was found on linkage group Mrg28, where LD decay was substantially slower in the spring subpopulation. It is speculated that this may be caused by a heterogeneous translocation event on this chromosome. Association with heading date was most consistent across location-years on linkage groups Mrg02, Mrg12, Mrg13, and Mrg24.