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.

Development of barley (Hordeum vulgare L.) lines with altered starch granule size distribution.

Microscope analysis of starches prepared from 139 barley genotypes identified a Japanese genotype, Kinai Kyoshinkai-2 (KK-2), with altered starch granule size distribution. Compared to normal barley starch, KK-2 produced consistently higher volumes of starch granules with 5-15 µm diameter and reduced volumes of starch granules with >15 µm diameter when grown in different environments. A cross between KK-2 and normal starch cultivar CDC Kendall was made and led to the production of 154 F5 lines with alterations to the normal 7:3:1 distribution for A-:B-:C-type starch granule volumes. Three F5 lines showed unimodal starch granule size distribution due to apparent lack of very small (<5.0 µm diameter) C-type starch granules, but the phenotype was accompanied by reduced grain weight and total starch concentration. Five F5 lines produced a significantly larger population of large (>15 µm diameter) A-type starch granules as compared to normal starch and showed on average a 10:4:1 distribution for A-:B-:C-type starch granule volumes. The unusual starch phenotypes displayed by the F5 lines confirm starch granule size distribution in barley can be genetically altered.

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.

Polymorphism in the barley granule bound starch synthase 1 (gbss1) gene associated with grain starch variant amylose concentration.

Granule bound starch synthase 1 (GBSS1) accumulation within starch granules and structure of Gbss1 alleles were determined for nine barley ( Hordeum vulgare L.) genotypes producing amylose-free (undetectable), near-waxy (1.6-4.5%), normal (25.8%), and increased (38.0-40.8%) amylose grain starches. Compared to normal starch granules, GBSS1 accumulation was severely reduced in three near-waxy, slightly reduced in two waxy, and slightly elevated in three increased amylose starches. Gbss1 nucleotide sequence analysis for the nine genotypes distinguished them into three Gbss1 groups with several single-nucleotide polymorphisms. A new unique Q312H substitution within GBSS1 was discovered in near-waxy genotype SB94912 with reduced amylose (1.6%) concentration relative to the other two near-waxy lines, CDC Rattan and CDC Candle (4.5%). The two waxy genotype GBSS1 showed a previously described D287V change for CDC Alamo and a new G513W change for CDC Fibar. Both amino acid alterations are conserved residues within starch synthase domains involved in glucan interaction. The increased amylose genotypes showed several unique nucleotide changes within the second and fourth Gbss1 introns, but only SB94893 GBSS1 showed a unique amino acid substitution, A250T in exon 6. The Gbss1 nucleotide differences were used to design genetic markers to monitor Gbss1 alleles in genotypes with various amylose grain starches.

Influence of different carbohydrate composition in barley varieties on Salmonella Typhimurium var. Copenhagen colonisation in a "Trojan" challenge model in pigs.

Based on previously performed in vitro studies, which showed that hulless barley varieties could reduce large intestinal Salmonella Typhimurium var. Copenhagen proliferation in pigs, two in vivo experiments were conducted to prove these observations. In Experiment (Exp.) 1, 126 weaning piglets were randomly allocated into pens of seven animals each and fed one of six experimental diets. Three diets contained (75% as-fed) one of three hulless barley varieties with beta-glucan (BG) contents ranging from 5 to 11% and amylose from 5 to 40%, and two diets contained a low BG and amylose hulless barley supplemented with isolated barley BG or raw potato starch. A hulled barley diet served as a control. Two piglets per pen ("Trojan" pigs) were orally infected with Salmonella Typhimurium var. Copenhagen (ST). The remaining five pigs per pen were designated "Contact" pigs. The ST shedding was determined over one week after infection. On day 6, the two Trojans and two random Contacts from each pen were euthanised and intestinal contents and mesenteric lymph nodes cultured for ST. Intestinal volatile fatty acids and microbial composition were determined. In Exp. 2, 126 piglets were assigned to one of three diets based on hulled or hulless barleys. The timeline, infection, sampling and analyses were similar as in Exp. 1 except samples were taken from four Contact pigs. Hulless barley varieties with high BG and amylose tended to decrease ST persistence in Exp. 1. Clostridia from cluster I in the colon were reduced with high amylose hulless barley or diets supplemented with potato starch (p < 0.05), whereas other microbial groups were not. Propionate increased (p < 0.05) and acetate decreased (p < 0.05) with hulless barley inclusion. Exp. 2 revealed a reduced ST shedding and reduced number of clostridia for high BG hulless barley as compared to common hulled barley and a low BG variety (p < 0.05). In conclusion, high BG hulless barley do not prevent ST colonisation but might help to reduce transmission in pigs, likely by supporting an intestinal environment limiting growth of this zoopathogen.

Barley grain constituents, starch composition, and structure affect starch in vitro enzymatic hydrolysis.

The relationship between starch physical properties and enzymatic hydrolysis was determined using ten different hulless barley genotypes with variable carbohydrate composition. The ten barley genotypes included one normal starch (CDC McGwire), three increased amylose starches (SH99250, SH99073, and SB94893), and six waxy starches (CDC Alamo, CDC Fibar, CDC Candle, Waxy Betzes, CDC Rattan, and SB94912). Total starch concentration positively influenced thousand grain weight (TGW) (r(2) = 0.70, p < 0.05). Increase in grain protein concentration was not only related to total starch concentration (r(2) = -0.80, p < 0.01) but also affected enzymatic hydrolysis of pure starch (r(2) = -0.67, p < 0.01). However, an increase in amylopectin unit chain length between DP 12-18 (F-II) was detrimental to starch concentration (r(2) = 0.46, p < 0.01). Amylose concentration influenced granule size distribution with increased amylose genotypes showing highly reduced volume percentage of very small C-granules (<5 µm diameter) and significantly increased (r(2) = 0.83, p < 0.01) medium sized B granules (5-15 µm diameter). Amylose affected smaller (F-I) and larger (F-III) amylopectin chains in opposite ways. Increased amylose concentration positively influenced the F-III (DP 19-36) fraction of longer DP amylopectin chains (DP 19-36) which was associated with resistant starch (RS) in meal and pure starch samples. The rate of starch hydrolysis was high in pure starch samples as compared to meal samples. Enzymatic hydrolysis rate both in meal and pure starch samples followed the order waxy > normal > increased amylose. Rapidly digestible starch (RDS) increased with a decrease in amylose concentration. Atomic force microscopy (AFM) analysis revealed a higher polydispersity index of amylose in CDC McGwire and increased amylose genotypes which could contribute to their reduced enzymatic hydrolysis, compared to waxy starch genotypes. Increased ß-glucan and dietary fiber concentration also reduced the enzymatic hydrolysis of meal samples. An average linkage cluster analysis dendrogram revealed that variation in amylose concentration significantly (p < 0.01) influenced resistant starch concentration in meal and pure starch samples. RS is also associated with B-type granules (5-15 µm) and the amylopectin F-III (19-36 DP) fraction. In conclusion, the results suggest that barley genotype SH99250 with less decrease in grain weight in comparison to that of other increased amylose genotypes (SH99073 and SH94893) could be a promising genotype to develop cultivars with increased amylose grain starch without compromising grain weight and yield.

Physicochemical characteristics, hydroxycinnamic acids (ferulic acid, P-coumaric acid) and their ratio, and in situ biodegradability: comparison of genotypic differences among six barley varieties.

Barley contains hydroxycinnamic acids, mainly ferulic acid (FA; 3-methoxy-4-hydroxycinnamic acid) and p-coumaric acid (PCA; 4-hydroxycinnamic acid). Ferulic acid is produced via the phenylpropanoid biosynthetic pathway and covalently cross-linked to polysaccharides by ester bonds and to components of lignin mainly by ether bonds. Various studies have consistently indicated that FA is among the factors most inhibitory to the biodegradability of cell wall polysaccharides. p-Coumaric acid is also covalently linked to polysaccharides (minor) and lignin (major), but does not form the inhibitory cross-linkages as FA does and is considered to represent cell wall lignification. The objectives in this study were to (1) determine genotypic differences in physicochemical characteristics in terms of (a) two major low molecular weight hydroxycinnamic acid profiles (FA, PCA, PCA-to-FA ratio, which are associated with digestion and lignification), (b) particle size distributions (mean, median), (c) hull content, and (d) digestion-resistant fiber fractions and (2) determine genotypic differences in in situ solubilization kinetics of FA and PCA. The barley varieties grown during three consecutive years (2003, 2004, and 2005) included AC Metcalfe, CDC Dolly, McLeod, CDC Helgason, CDC Trey, and CDC Cowboy. These barleys were grown at the Kernen Crop Research Farm (KCRF, University of Saskatchewan) and managed using standard agronomic production practices. Results showed that there were significant differences in hull content (P < 0.05) among the barley varieties, with Mcleod having the highest (11% DM) and CDC Dolly and CDC Helgason the lowest hull content (9% DM). Ferulic acid ranged from 555 to 663 microg/g of DM (P < 0.05). p-Coumaric acid ranged (P < 0.05) from 283 to 345 microg/g of DM. PCA-to-FA ratios ranged (P < 0.05) from 0.49 to 0.56. Mean particle size ranged (P < 0.05) from 3.06 to 3.66 mm, and median particle size ranged (P < 0.05) from 2.71 to 3.04 mm. In situ DM degradability ranged from 44 to 49%. In situ solubilized FA fractions ranged (P < 0.05) from 60 to 72% and of PCA ranged (P < 0.05) from 71 to 81%. In conclusion, CDC Dolly was best and McLeod barley was poorest as feed barley in terms of hull and FA contents. There were significant genotypic differences in FA, PCA and their ratio, hull content, particle size distribution, and in situ solubilization of FA and PCA among the barley varieties.

New DArT markers for oat provide enhanced map coverage and global germplasm characterization.

BACKGROUND: Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). RESULTS: Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' x 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. CONCLUSION: These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.

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.

The altered pattern of amylose accumulation in the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5'-non-coding region.

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5'-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxy ancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.