D. E. Mather

Characterization of polyploid wheat genomic diversity using a high-density 90 000 single nucleotide polymorphism array

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker–trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence–absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.

Random amplified polymorphic DNA and pedigree relationships in spring barley.

SummaryWe investigated random amplified polymorphic DNA (RAPD) in 27 inbred barley lines with varying amounts of common ancestry and in 20 doubled-haploid (DH) lines from a biparental cross. Of 33 arbitrary 10 base primers that were tested, 19 distinguished a total of 31 polymorphisms. All polymorphisms were scored as dominant genetic markers except for 1, where Southern analysis indicated the presence of two codominant amplification products. The inheritance of 19 RAPD polymorphisms and one morphological trait was studied in the DH lines. There was no evidence for segregation distortion, but a group of four tightly linked loci was detected. The frequencies of RAPD polymorphism in pairs of inbred lines were used to compute values of genetic distance (d), which were compared to kinship coefficients (r) between the same pairs of lines. A linear relationship between r and d was evident, but low values of r gave poor predictions of d. Cluster analysis showed that groups of inbred lines based on r were similar to those based on d with some notable exceptions. RAPD markers can be used to gain information about genetic similarities or differences that are not evident from pedigree information.

The Genetics and Transcriptional Profiles of the Cellulose Synthase-Like HvCslF Gene Family in Barley

Cellulose synthase-like CslF genes have been implicated in the biosynthesis of (1,3;1,4)-β-d-glucans, which are major cell wall constituents in grasses and cereals. Seven CslF genes from barley (Hordeum vulgare) can be divided into two classes on the basis of intron-exon arrangements. Four of the HvCslF genes have been mapped to a single locus on barley chromosome 2H, in a region corresponding to a major quantitative trait locus for grain (1,3;1,4)-β-d-glucan content. The other HvCslF genes map to chromosomes 1H, 5H, and 7H, and in two cases the genes are close to other quantitative trait loci for grain (1,3;1,4)-β-d-glucan content. Spatial and temporal patterns of transcription of the seven genes have been defined through quantitative polymerase chain reaction. In developing barley coleoptiles HvCslF6 mRNA is most abundant. Transcript levels are maximal in 4- to 5-d coleoptiles, at a time when (1,3;1,4)-β-d-glucan content of coleoptile cell walls also reaches maximal levels. In the starchy endosperm of developing grain, HvCslF6 and HvCslF9 transcripts predominate. Two peaks of transcription are apparent. One occurs just after endosperm cellularization, 4 to 8 d after pollination, while the second occurs much later in grain development, more than 20 d after pollination. Marked varietal differences in transcription of the HvCslF genes are observed during endosperm development. Given the commercial importance of cereal (1,3;1,4)-β-d-glucans in human nutrition, in stock feed, and in malting and brewing, the observation that only two genes, HvCslF6 and HvCslF9, are transcribed at high levels in developing grain is of potential relevance for the future manipulation of grain (1,3;1,4)-β-d-glucan levels.

Effect of population size on the estimation of QTL: a test using resistance to barley stripe rust

The limited population sizes used in many quantitative trait locus (QTL) detection experiments can lead to underestimation of QTL number, overestimation of QTL effects, and failure to quantify QTL interactions. We used the barley/barley stripe rust pathosystem to evaluate the effect of population size on the estimation of QTL parameters. We generated a large (n=409) population of doubled haploid lines derived from the cross of two inbred lines, BCD47 and Baronesse. This population was evaluated for barley stripe rust severity in the Toluca Valley, Mexico, and in Washington State, USA, under field conditions. BCD47 was the principal donor of resistance QTL alleles, but the susceptible parent also contributed some resistance alleles. The major QTL, located on the long arm of chromosome 4H, close to the Mlo gene, accounted for up to 34% of the phenotypic variance. Subpopulations of different sizes were generated using three methods—resampling, selective genotyping, and selective phenotyping—to evaluate the effect of population size on the estimation of QTL parameters. In all cases, the number of QTL detected increased with population size. QTL with large effects were detected even in small populations, but QTL with small effects were detected only by increasing population size. Selective genotyping and/or selective phenotyping approaches could be effective strategies for reducing the costs associated with conducting QTL analysis in large populations. The method of choice will depend on the relative costs of genotyping versus phenotyping.

Chapter 10 - Genetic diversity for quantitatively inherited agronomic and malting quality traits

This chapter reviews diversity in agronomic traits, diversity in malting quality traits, and the current status of Quantitative Trait Loci (QTL) analysis in barley and the application of QTL tools to the analysis of genetic diversity in barley and crop improvement. Agronomic and quality traits were undoubtedly key issues for the domesticators of barley. Crop productivity would clearly have been an attribute of key interest, and the selection of shattering-resistant mutants probably led to a quantum leap in yield. Because barley has been used both as a food and as a principal ingredient of fermented beverages from the earliest times, there may well have been conscious selection for end-use properties. The selection of hull-less mutants in areas of the world where barley was a principal foodstuff underscores the importance of end-use properties in domestication. The malting and brewing properties of wild barley accessions and landraces have not been welldescribed and are, in fact, extremely difficult to measure. Plant breeding efforts are directed primarily at traits exhibiting quantitative variation. Breeders and geneticists were now able to collaborate in developing and testing hypotheses regarding the number, location, effect, and interactions of genes influencing quantitative traits.

Whole genome mapping of agronomic and metabolic traits to identify novel quantitative trait loci in bread wheat grown in a water-limited environment

Comparison of the agronomic and metabolic trait variation between drought-sensitive and drought-tolerant wheat crosses uncovers novel correlations between agronomic traits and the levels of certain metabolites as well as important regions on the wheat genome that require further investigation. Drought is a major environmental constraint responsible for grain yield losses of bread wheat (Triticum aestivum) in many parts of the world. Progress in breeding to improve complex multigene traits, such as drought stress tolerance, has been limited by high sensitivity to environmental factors, low trait heritability, and the complexity and size of the hexaploid wheat genome. In order to obtain further insight into genetic factors that affect yield under drought, we measured the abundance of 205 metabolites in flag leaf tissue sampled from plants of 179 cv Excalibur/Kukri F1-derived doubled haploid lines of wheat grown in a field experiment that experienced terminal drought stress. Additionally, data on 29 agronomic traits that had been assessed in the same field experiment were used. A linear mixed model was used to partition and account for nongenetic and genetic sources of variation, and quantitative trait locus analysis was used to estimate the genomic positions and effects of individual quantitative trait loci. Comparison of the agronomic and metabolic trait variation uncovered novel correlations between some agronomic traits and the levels of certain primary metabolites, including metabolites with either positive or negative associations with plant maturity-related or grain yield-related traits. Our analyses demonstrate that specific regions of the wheat genome that affect agronomic traits also have distinct effects on specific combinations of metabolites. This approach proved valuable for identifying novel biomarkers for the performance of wheat under drought and could facilitate the identification of candidate genes involved in drought-related responses in bread wheat.

QTL analysis of malting quality traits in two barley populations

Selection for malting quality traits is a major breeding objective for barley breeding programs. With molecular markers linked to loci affecting these traits, this selection can be undertaken at an earlier stage of the breeding program than is possible using conventional tests. Quantitative trait loci (QTLs) associated with malting quality traits were mapped in 2 populations derived from parents with elite malting quality. Progeny from an Arapiles/Franklin population grown in 4 environments and an Alexis/Sloop population grown in 5 environments were tested for grain protein percentage, α-amylase activity, diastatic power, hot water extract, wort viscosity, wort β-glucan, β-glucanase, and free α-amino acids. QTL analysis was performed using a one-stage approach, which allowed for modelling of spatial variation in the field, and in each phase of the malting quality analysis in the laboratory. QTLs for malting quality traits were detected on all chromosomes and for both populations. Few of these QTLs were significant in all of the environments, indicating that QTL × environment interactions were important. There were many coincident QTLs for traits that are expected to be related such as diastatic power and α-amylase activity, wort β-glucan and wort viscosity and for some traits that are not expected to be related such as hot water extract and malt viscosity.

Temperature Switch PCR (TSP): Robust assay design for reliable amplification and genotyping of SNPs

BackgroundMany research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs). Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP), a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays.ResultsWe demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method.ConclusionTheoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.

Genetic improvement of spring barley cultivars grown in eastern Canada from 1910 to 1988

SummarySpring barley is an important feed crop in eastern Canada, and the development of high-yielding, high grain protein cultivars is desirable. This study was conducted to assess the impact of breeding on the yield and protein aspects of cultivar development, and to identify related changes in plant characteristics which may have been altered over time. A 3-year field experiment was conducted to evaluate twenty six-rowed spring barley genotypes representing the majority of cultivars developed from 1910 to 1988 for eastern Canada. The yields of barley cultivars released from 1935 to 1988 increased at a rate of about 0.03 t ha-1 yr-1, and showed no evidence of having reached a plateau. Increases in yield were associated with higher total dry matter production and harvest index, reduced plant height and increased lodging resistance. No consistent change in main stem or tiller yield components was observed. Grain protein concentration decreased progressively with time, especially with the newer cultivars. Reduction in grain protein concentration was not associated with lower protein content on a per grain basis, but rather with an increase in the amount of non-structural carbohydrate per grain. Total plant and grain N accumulation showed positive trends with time. No trends were observed for N harvest index, apparent post-heading N uptake, N retranslocation, and retranslocation efficiency. Thus, while the newer cultivars accumulated more total and grain N, proportional N partitioning to the grain was not altered.

Quantitative trait loci for grain fructan concentration in wheat (Triticum aestivum L.)

Fructans (fructo-oligosaccharides) are prebiotics that are thought to selectively promote the growth of colonic bifidobacteria, thereby improving human gut health. Fructans are present in the grain of wheat, a staple food crop. In the research reported here, we aimed to detect and map loci affecting grain fructan concentration in wheat using a doubled-haploid population derived from a cross between a high-fructan breeding line, Berkut, and a low-fructan cultivar, Krichauff. Fructan concentration was measured in grain samples grown at two locations in Australia and one in Kazakhstan. Fructan concentration varied widely within the population, ranging from 0.6 to 2.6% of grain dry weight, and was quite repeatable, with broad-sense heritability estimated as 0.71. With a linkage map of 528 molecular markers, quantitative trait loci (QTLs) were detected on chromosomes 2B, 3B, 5A, 6D and 7A. Of these, the QTLs on chromosomes 6D and 7A had the largest effects, explaining 17 and 27% of the total phenotypic variance, respectively, both with the favourable (high-fructan concentration) alleles contributed from Berkut. These chromosome regions had similar effects in another mapping population, Sokoll/Krichauff, with the favourable alleles contributed from Sokoll. It is concluded that grain fructan concentration of wheat can be improved by breeding and that molecular markers could be used to select effectively for favourable alleles in two regions of the wheat genome.