Mark E. Sorrells

Association mapping of kernel size and milling quality in wheat (Triticum aestivum L.) cultivars.

Association mapping is a method for detection of gene effects based on linkage disequilibrium (LD) that complements QTL analysis in the development of tools for molecular plant breeding. In this study, association mapping was performed on a selected sample of 95 cultivars of soft winter wheat. Population structure was estimated on the basis of 36 unlinked simple-sequence repeat (SSR) markers. The extent of LD was estimated on chromosomes 2D and part of 5A, relative to the LD observed among unlinked markers. Consistent LD on chromosome 2D was <1 cM, whereas in the centromeric region of 5A, LD extended for ∼5 cM. Association of 62 SSR loci on chromosomes 2D, 5A, and 5B with kernel morphology and milling quality was analyzed through a mixed-effects model, where subpopulation was considered as a random factor and the marker tested was considered as a fixed factor. Permutations were used to adjust the threshold of significance for multiple testing within chromosomes. In agreement with previous QTL analysis, significant markers for kernel size were detected on the three chromosomes tested, and alleles potentially useful for selection were identified. Our results demonstrated that association mapping could complement and enhance previous QTL information for marker-assisted selection.

Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach

Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)n blocks was estimated to be 3.6 x 104 and the number of (GT)n blocks to be 2.3 x 104 per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.

A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome

A map of the barley genome consisting of 295 loci was constructed. These loci include 152 cDNA restriction fragment length polymorphism (RFLP), 114 genomic DNA RFLP, 14 random amplified polymorphic DNA (RAPD), five isozyme, two morphological, one disease resistance and seven specific amplicon polymorphism (SAP) markers. The RFLP-identified loci include 63 that were detected using cloned known function genes as probes. The map covers 1,250 centiMorgans (cM) with a 4.2 cM average distance between markers. The genetic lengths of the chromosomes range from 124 to 223 cM and are in approximate agreement with their physical lengths. The centromeres were localized to within a few markers on all of the barley chromosomes except chromosome 5. Telomeric regions were mapped for the short (plus) arms of chromosomes 1, 2 and 3 and the long (minus) arm of chromosomes 7.

Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat.

Plant genomics projects involving model species and many agriculturally important crops are resulting in a rapidly increasing database of genomic and expressed DNA sequences. The publicly available collection of expressed sequence tags (ESTs) from several grass species can be used in the analysis of both structural and functional relationships in these genomes. We analyzed over 260 000 EST sequences from five different cereals for their potential use in developing simple sequence repeat (SSR) markers. The frequency of SSR-containing ESTs (SSR-ESTs) in this collection varied from 1.5% for maize to 4.7% for rice. In addition, we identified several ESTs that are related to the SSR-ESTs by BLAST analysis. The SSR-ESTs and the related sequences were clustered within each species in order to reduce the redundancy and to produce a longer consensus sequence. The consensus and singleton sequences from each species were pooled and clustered to identify cross-species matches. Overall a reduction in the redundancy by 85% was observed when the resulting consensus and singleton sequences (3569) were compared to the total number of SSR-EST and related sequences analyzed (24 606). This information can be useful for the development of SSR markers that can amplify across the grass genera for comparative mapping and genetics. Functional analysis may reveal their role in plant metabolism and gene evolution.

Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

DNA Markers in Plant Improvement

Publisher Summary Genetic markers are held for a detailed investigation of complex questions in quantitative biology. The advent of DNA markers, greatly expanding the number of genetic markers available, is allowing researchers to begin to tap the potential of this technology, to the benefit of both basic biology and agricultural productivity. Genetic markers make a major contribution to the biological sciences, especially agriculture, for the foreseeable future. DNA markers can significantly accelerate many breeding endeavors. They may provide new approaches to some objectives, which have proven difficult to achieve with classical techniques, such as introgression of valuable traits from exotic germplasm into domestic cultivars. This chapter addresses the applications of DNA markers to plant breeding. Genetic markers represent genetic variation, permitting one to estimate relatedness between different genotypes and to predict which matings might produce new and superior gene combinations.

Phytochemical Profiles and Antioxidant Activity of Wheat Varieties

Consumption of whole grains has been associated with reduced risk of developing major chronic diseases. These health benefits have been attributed in part to their unique phytochemicals. Little is known about the complete profiles of phytochemicals and antioxidant activities of different adlay varieties. The objectives of this study were to determine the phytochemicals profiles of the three adlay varieties, including both free and bound of total phenolics and total flavonoids, and to determine the total antioxidant activity of adlay. The free, bound, and total phenolic contents of adlay samples ranged from 31.23 to 45.19 mg of gallic acid equiv/100 g of sample, from 28.07 to 30.86 mg of gallic acid equiv/100 g of sample, and from 59.30 to 76.04 mg of gallic acid equiv/100 g of sample, respectively. On average, the bound phenolics contributed 45.3% of total phenolic content of the adlay varieties analyzed. The free, bound, and total flavonoid contents of adlay samples ranged from 6.21 to 18.24 mg of catechin equiv/100 g, from 18.68 to 35.27 mg of catechin equiv/100 g, and from 24.88 to 52.86 mg of catechin equiv/100 g, respectively. The average values of bound flavonoids contributed 71.1% of total flavonoids of the adlay varieties analyzed. The percentage contribution of flavonoid content to phenolic content of free, bound, and total ranged from 11.6 to 35.2%, from 50.5 to 66.8%, and from 24.6 to 50.5%. The free, bound, and total oxygen radical absorbance capacity (ORAC) values of adlay samples ranged from 231.9 to 316.6 mg of Trolox equiv/100 g, from 209.0 to 351.4 mg of Trolox equiv/100 g, and from 440.9 to 668.0 mg of Trolox equiv/100 g, respectively. The average ORAC values of bound phytochemicals contributed 48.1% of total antioxidant activity of the adlay varieties analyzed. The content of total polyphenol and the antioxidant capacity are obviously different among different species. Liaoning 5 adlay and Longyi 1 adlay are significantly better than Guizhou heigu adlay. The adlay extracts have obvious proliferate inhibition on human liver cancer cells, and substantially in the experimental concentration range, the adlay sample itself has no cytotoxicity. Knowing the phytochemical profiles and antioxidant activity of adlay gives insights to its potential application to promote health.

Quantitative trait locus effects and environmental interaction in a sample of North American barley germ plasm

Quantitative trait locus (QTL) and QTL x environment (E) interaction effects for agronomic and malting quality traits were measured using a 123-point linkage map and multi-environment phenotype data from an F1-derived doubled haploid population of barley (Hordeum vulgare). The QTL × E interactions were due to differences in magnitude of QTL effects. Highly significant QTL effects were found for all traits at multiple sites in the genome. Yield QTL peaks and support intervals often coincided with plant height and lodging QTL peaks and support intervals. QTL were detected in the vicinity of a previously mapped Mendelian maturity locus and known function probes forα- andβ-amylase genes. The average map density (9.6 cM) should be adequate for molecular marker-assisted selection, particularly since there were few cases of alternative favorable alleles for different traits mapping to the same or adjacent intervals.

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat.

Abstract  Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10−12) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity.