Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.

Genetic mapping of the wheat leaf rust resistance gene Lr2a and its importance in Canadian wheat cultivars.

KEY MESSAGE: Leaf rust resistance gene Lr2a was located to chromosome arm 2DS in three mapping populations, which will facilitate map-based cloning and marker-assisted selection of Lr2a in wheat breeding programs. Incorporating effective leaf rust resistance (Lr) genes into high-yielding wheat cultivars has been an efficient method of disease control. One of the most widely used genes in Canada is the multi-allelic resistance gene Lr2, with alleles Lr2a, Lr2b, Lr2c, and Lr2d. The Lr2a allele confers complete resistance to a large portion of the Puccinia triticina (Pt) population in Canada. In this study, Lr2a was genetically mapped in two doubled haploid populations developed from the crosses Superb/BW278 and Superb/86ISMN 2137, and an F2 population developed from the cross Chinese Spring/RL6016. Seedlings were tested with the Lr2a avirulent Pt races 74-2 MGBJ (Superb/BW278) and 12-3 MBDS (Superb/86ISMN 2137 and Chinese Spring/RL6016) in greenhouse assays and were genotyped with 90K wheat Infinium SNP and kompetitive allele-specific PCR (KASP) markers. Lr2a was mapped to a collinear position on chromosome arm 2DS in all three populations, within a 1.00 cM genetic interval between KASP markers kwm1620 and kwm1623. This corresponded to a 305 kb genomic region of chromosome 2D in Chinese Spring RefSeq v2.1. The KASP marker kwh740 was predictive of Lr2a in all mapping populations. A panel of 260 wheats were tested with three Pt isolates, which revealed that Lr2a is common in Canadian wheats. The KASP markers kwh740 and kwm1584 were highly associated with resistance at the Lr2 locus, while kwm1622 was slightly less correlated. Genetic mapping of the leaf rust resistance gene Lr2a and DNA markers developed here will facilitate its use in wheat breeding programs.

Genetic analysis of oviposition deterrence to orange wheat blossom midge in spring wheat.

KEY MESSAGE: A major QTL for oviposition deterrence to orange wheat blossom midge was detected on chromosome 1A in the Canadian breeding line BW278 that was inherited from the Chinese variety Sumai-3. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin, Diptera: Cecidomyiidae) is an important insect pest of wheat (Triticum aestivum L.) that reduces both grain yield and quality. Oviposition deterrence results in a reduction of eggs deposited on spikes relative to that observed on a wheat line preferred by OWBM. Quantification of oviposition deterrence is labor-intensive, so wheat breeders require efficient DNA markers for the selection of this trait. The objective of this study was to identify quantitative trait loci (QTL) for oviposition deterrence in a doubled haploid (DH) population developed from the spring wheat cross Superb/BW278. The DH population and check varieties were evaluated for OWBM kernel damage from five field nurseries over three growing seasons. QTL analysis identified major effect loci on chromosomes 1A (QSm.mrc-1A) and 5A (QSm.mrc-5A). Reduced kernel damage was contributed by BW278 at QSm.mrc-1A and Superb at QSm.mrc-5A. QSm.mrc-1A mapped to the approximate location of the oviposition deterrence QTL previously found in the American variety Reeder. However, haplotype analysis revealed that BW278 inherited this oviposition deterrence allele from the Chinese spring wheat variety Sumai-3. QSm.mrc-5A mapped to the location of awn inhibitor gene B1, suggesting that awns hinder OWBM oviposition. Single-nucleotide polymorphisms (SNPs) were identified for predicting the presence or absence of QSm.mrc-1A based upon haplotype. Functional annotation of candidate genes in 1A QTL intervals revealed eleven potential candidate genes, including a gene involved in terpenoid biosynthesis. SNPs for QSm.mrc-1A and fully awned spikes provide a basis for the selection of oviposition deterrence to OWBM.

Genetic analysis of loose smut (Ustilago tritici) resistance in Sonop spring wheat.

BACKGROUND: The genetics of resistance to loose smut of wheat (Triticum aestivum L.) caused by the fungus Ustilago tritici (Pers.) Rostr. is not well understood. This study examines loose smut resistance in Sonop (TD-14), a South African spring wheat variety. A doubled haploid (DH) population of 163 lines derived from the cross Diamont/TD-14 was studied. The parents and progenies were inoculated with U. tritici races T2, T9, and T39 individually in growth facilities at Morden and Swift Current, Canada. Loose smut incidence (LSI) and partial loose smut resistance (PLSR) were assessed. RESULTS: A whole genome linkage map was developed consisting of 11,519 SNP loci found on 31 linkage groups spanning 2845 cM. A new major resistance gene Ut11 was located to the distal end of chromosome arm 7BS. Ut11 conferred resistance to U. tritici race T2, but not races T9 and T39. Quantitative trait locus (QTL) mapping identified four QTL controlling LSI in the Diamont/TD-14 DH population on chromosomes 3B, 4B, 5B, and 7B (at Ut11) with TD-14 contributing the resistance alleles at three of these loci. The major QTL QUt.mrc-5B was effective against all three races and explained up to 81% of the phenotypic variation. The only QTL identified for PLSR coincided with the LSI QTL QUt.mrc-5B indicating that this locus affected both loose smut incidence and partial smutting of spikes. CONCLUSIONS: A race-specific resistance gene Ut11 and a broadly effective resistance QTL QUt.mrc-5B were the main loci controlling loose smut resistance in the differential line TD-14 (cultivar Sonop). This study provides insight into the genetics of loose smut resistance in spring wheat Sonop and the single nucleotide polymorphism (SNP) markers linked to the resistance gene Ut11 and QTL QUt.mrc-5B will be useful for selecting loose smut resistance in breeding programs.

Genetic analyses of native Fusarium head blight resistance in two spring wheat populations identifies QTL near the B1, Ppd-D1, Rht-1, Vrn-1, Fhb1, Fhb2, and Fhb5 loci.

KEY MESSAGE: QTL analyses of two bi-parental mapping populations with AC Barrie as a parent revealed numerous FHB-resistance QTL unique to each population and uncovered novel variation near Fhb1. Fusarium head blight (FHB) is a destructive disease of wheat worldwide, leading to severe yield and quality losses. The genetic basis of native FHB resistance was examined in two populations: a recombinant inbred line population from the cross Cutler/AC Barrie and a doubled haploid (DH) population from the cross AC Barrie/Reeder. Numerous QTL were detected among the two mapping populations with many being cross-specific. Photoperiod insensitivity at Ppd-D1 and dwarfing at Rht-B1 and Rht-D1 was associated with increased FHB susceptibility. Anthesis date QTL at or near the Vrn-A1 and Vrn-B1 loci co-located with major FHB-resistance QTL in the AC Barrie/Reeder population. The loci were epistatic for both traits, such that DH lines with both late alleles were considerably later to anthesis and had reduced FHB symptoms (i.e., responsible for the epistatic interaction). Interestingly, AC Barrie contributed FHB resistance near the Fhb1 locus in the Cutler population and susceptibility in the Reeder population. Analyses of the Fhb1 candidate genes PFT and TaHRC confirmed that AC Barrie, Cutler, and Reeder do not carry the Sumai-3 Fhb1 gene. Resistance QTL were also detected at the expected locations of Fhb2 and Fhb5. The native FHB-resistance QTL detected near Fhb1, Fhb2, and Fhb5 do not appear to be as effective as Fhb1, Fhb2, and Fhb5 from Sumai-3. The presence of awns segregated at the B1 awn inhibitor locus in both populations, but was only associated with FHB resistance in the Cutler/AC Barrie population suggesting linkage caused the association rather than pleiotropy.

Structural organization of fatty acid desaturase loci in linseed lines with contrasting linolenic acid contents.

Flax (Linum usitatissimum L.), the richest crop source of omega-3 fatty acids (FAs), is a diploid plant with an estimated genome size of ~370 Mb and is well suited for studying genomic organization of agronomically important traits. In this study, 12 bacterial artificial chromosome clones harbouring the six FA desaturase loci sad1, sad2, fad2a, fad2b, fad3a and fad3b from the conventional variety CDC Bethune and the high linolenic acid line M5791 were sequenced, analysed and compared to determine the structural organization of these loci and to gain insights into the genetic mechanisms underlying FA composition in flax. With one gene every 3.2-4.6 kb, the desaturase loci have a higher gene density than the genome's average of one gene per 7.8-8.2 kb. The gene order and orientation across the two genotypes were generally conserved with the exception of the sad1 locus that was predicted to have additional genes in CDC Bethune. High sequence conservation in both genic and intergenic regions of the sad and fad2b loci contrasted with the significant level of variation of the fad2a and fad3 loci, with SNPs being the most frequently observed mutation type. The fad2a locus had 297 SNPs and 36 indels over ~95 kb contrasting with the fad2b locus that had a mere seven SNPs and four indels in ~110 kb. Annotation of the gene-rich loci revealed other genes of known role in lipid or carbohydrate metabolic/catabolic pathways. The organization of the fad2b locus was particularly complex with seven copies of the fad2b gene in both genotypes. The presence of Gypsy, Copia, MITE, Mutator, hAT and other novel repeat elements at the desaturase loci was similar to that of the whole genome. This structural genomic analysis provided some insights into the genomic organization and composition of the main desaturase loci of linseed and of their complex evolution through both tandem and whole genome duplications.

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

Functional characterization of flax fatty acid desaturase FAD2 and FAD3 isoforms expressed in yeast reveals a broad diversity in activity.

With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed.