Daryl J. Somers

A Physical Map of the 1-Gigabase Bread Wheat Chromosome 3B

As the staple food for 35% of the worlds population, wheat is one of the most important crop species. To date, sequence-based tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17–billion–base-pair hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)–based integrated physical map of the largest chromosome, 3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps in large, complex, polyploid genomes with a chromosome-based approach.

Molecular detection of QTLs for agronomic and quality traits in a doubled haploid population derived from two Canadian wheats (Triticum aestivum L.)

Development of high-yielding wheat varieties with good end-use quality has always been a major concern for wheat breeders. To genetically dissect quantitative trait loci (QTLs) for yield-related traits such as grain yield, plant height, maturity, lodging, test weight and thousand-grain weight, and for quality traits such as grain and flour protein content, gluten strength as evaluated by mixograph and SDS sedimentation volume, an F1-derived doubled haploid (DH) population of 185 individuals was developed from a cross between a Canadian wheat variety “AC Karma” and a breeding line 87E03-S2B1. A genetic map was constructed based on 167 marker loci, consisting of 160 microsatellite loci, three HMW glutenin subunit loci: Glu-A1, Glu-B1 and Glu-D1, and four STS-PCR markers. Data for investigated traits were collected from three to four environments in Manitoba, Canada. QTL analyses were performed using composite interval mapping. A total of 50 QTLs were detected, 24 for agronomic traits and 26 for quality-related traits. Many QTLs for correlated traits were mapped in the same genomic regions forming QTL clusters. The largest QTL clusters, consisting of up to nine QTLs, were found on chromosomes 1D and 4D. HMW glutenin subunits at Glu-1 loci had the largest effect on breadmaking quality; however, other genomic regions also contributed genetically to breadmaking quality. QTLs detected in the present study are compared with other QTL analyses in wheat.

Integrated Metabolo-Proteomic Approach to Decipher the Mechanisms by Which Wheat QTL (Fhb1) Contributes to Resistance against Fusarium graminearum

Background Resistance in plants to pathogen attack can be qualitative or quantitative. For the latter, hundreds of quantitative trait loci (QTLs) have been identified, but the mechanisms of resistance are largely unknown. Integrated non-target metabolomics and proteomics, using high resolution hybrid mass spectrometry, were applied to identify the mechanisms of resistance governed by the fusarium head blight resistance locus, Fhb1, in the near isogenic lines derived from wheat genotype Nyubai. Findings The metabolomic and proteomic profiles were compared between the near isogenic lines (NIL) with resistant and susceptible alleles of Fhb1 upon F. graminearum or mock-inoculation. The resistance-related metabolites and proteins identified were mapped to metabolic pathways. Metabolites of the shunt phenylpropanoid pathway such as hydroxycinnamic acid amides, phenolic glucosides and flavonoids were induced only in the resistant NIL, or induced at higher abundances in resistant than in susceptible NIL, following pathogen inoculation. The identities of these metabolites were confirmed, with fragmentation patterns, using the high resolution LC-LTQ-Orbitrap. Concurrently, the enzymes of phenylpropanoid biosynthesis such as cinnamyl alcohol dehydrogenase, caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, flavonoid O-methyltransferase, agmatine coumaroyltransferase and peroxidase were also up-regulated. Increased cell wall thickening due to deposition of hydroxycinnamic acid amides and flavonoids was confirmed by histo-chemical localization of the metabolites using confocal microscopy. Conclusion The present study demonstrates that the resistance in Fhb1 derived from the wheat genotype Nyubai is mainly associated with cell wall thickening due to deposition of hydroxycinnamic acid amides, phenolic glucosides and flavonoids, but not with the conversion of deoxynivalenol to less toxic deoxynivalenol 3-O-glucoside.

Molecular mapping of quantitative trait loci for yield and yield components in spring wheat (Triticum aestivum L.)

An F1 derived doubled haploid (DH) population of 402 lines from the adapted spring wheat cross Superb (high yielding)/BW278 (low yielding) was developed to identify quantitative trait loci (QTL) associated with yield and yield components. A subset of the population (186 lines) was evaluated in replicated field trials in 2001 and 2002 at six locations in Manitoba and Saskatchewan, Canada. Agronomic parameters, grain yield and yield components including 1,000 grain weight, harvest index, average seed weight spike−1, seed number spike−1 and spikes number m−2 were measured. A genetic map was constructed with 268 microsatellite marker loci and included two morphological genes, reduced plant height, Rht-B1b, and the presence/absence of awns, B1. Composite interval mapping was conducted to estimate the location and effect of QTL associated with the evaluated traits. A total of 53 QTL were identified on 12 chromosomes for the 9 evaluated traits with the coefficient of determination ranging from 0.03 to 0.21 of the total variation. The increase in yield and yield components ranged from 4.5 to 17.1% over the population mean. The five grain yield QTL were detected on chromosomes 1A, 2D, 3B, and 5A and showed a combined increase of 34.4%, over the population mean. The alleles from Superb were associated with increased yield for four of the five QTL. This study identified potential chromosome segments for use in marker-assisted selection to improve yield and yield components in spring wheat.

Quantitative trait loci for lodging resistance, plant height and partial resistance to mycosphaerella blight in field pea ( Pisum sativum L.)

With the development of genetic maps and the identification of the most-likely positions of quantitative trait loci (QTLs) on these maps, molecular markers for lodging resistance can be identified. Consequently, marker-assisted selection (MAS) has the potential to improve the efficiency of selection for lodging resistance in a breeding program. This study was conducted to identify genetic loci associated with lodging resistance, plant height and reaction to mycosphaerella blight in pea. A population consisting of 88 recombinant inbred lines (RILs) was developed from a cross between Carneval and MP1401. The RILs were evaluated in 11 environments across the provinces of Manitoba, Saskatchewan and Alberta, Canada in 1998, 1999 and 2000. One hundred and ninety two amplified fragment length polymorphism (AFLP) markers, 13 random amplified polymorphic DNA (RAPD) markers and one sequence tagged site (STS) marker were assigned to ten linkage groups (LGs) that covered 1,274 centi Morgans (cM) of the pea genome. Six of these LGs were aligned with the previous pea map. Two QTLs were identified for lodging resistance that collectively explained 58% of the total phenotypic variation in the mean environment. Three QTLs were identified each for plant height and resistance to mycosphaerella blight, which accounted for 65% and 36% of the total phenotypic variation, respectively, in the mean environment. These QTLs were relatively consistent across environments. The AFLP marker that was associated with the major locus for lodging resistance was converted into the sequence-characterized amplified-region (SCAR) marker. The presence or absence of the SCAR marker corresponded well with the lodging reaction of 50 commercial pea varieties.

Genome-wide linkage disequilibrium analysis in bread wheat and durum wheat.

Bread wheat and durum wheat were examined for linkage disequilibrium (LD) using microsatellite markers distributed across the genome. The allele database consisted of 189 bread wheat accessions genotyped at 370 loci and 93 durum wheat accessions genotyped at 245 loci. A significance level of p < 0.001 was set for all comparisons. The bread and durum wheat collections showed that 47.9% and 14.0% of all locus pairs were in LD, respectively. LD was more prevalent between loci on the same chromosome compared with loci on independent chromosomes and was highest between adjacent loci. Only a small fraction (bread wheat, 0.9%; durum wheat, 3.2%) of the locus pairs in LD showed R2 values > 0.2. The LD between adjacent locus pairs extended (R2 > 0.2) approximately 2-3 cM, on average, but some regions of the bread and durum wheat genomes showed high levels of LD (R2 = 0.7 and 1.0, respectively) extending 41.2 and 25.5 cM, respectively. The wheat collections were clustered by similarity into subpopulations using unlinked microsatellite data and the software Structure. Analysis within subpopulations showed 14- to 16-fold fewer locus pairs in LD, higher R2 values for those pairs in LD, and LD extending further along the chromosome. The data suggest that LD mapping of wheat can be performed with simple sequence repeats to a resolution of <5 cM.

Allelic reduction and genetic shift in the Canadian hard red spring wheat germplasm released from 1845 to 2004

Analysis of genetic diversity changes in existing gene pools of cultivated crops is important for understanding the impact of plant breeding on crop genetic diversity and developing effective indicators for genetic diversity of cultivated plants. The objective of this study was to assess genetic diversity changes in 75 Canadian hard red wheat (Triticum aestivum L.) cultivars released from 1845 to 2004 using 31 simple sequence repeats (SSRs) markers. A total of 267 SSR alleles were detected, and their allelic frequencies ranged from 0.01 to 0.97, with an average of 0.14. Significant allelic reduction was observed at only four SSR loci for the cultivars released from 1970 onwards. However, 51 alleles (about 19%) present in pre-1910 cultivars were undetected in cultivars released after 1990 and were spread over 27 SSR loci. The proportion of SSR variation accounted for by six breeding periods was 12.5%, by four ancestral families, 16.5%, and by eight breeding programs, 8.4%. The average genetic diversity measured by three different band-sharing methods did not change significantly among cultivars released from different breeding periods, breeding programs, and ancestral families. However, genetic shift was obvious in the cultivars released over the six breeding periods, reflecting well the various breeding efforts over years. These results clearly show the allelic reduction and genetic shift in the Canadian hard red spring wheat germplasm released over time. Consequently, more effort needs to be made to broaden the wheat breeding base and conserve wheat germplasm.

Haplotype diversity at fusarium head blight resistance QTLs in wheat.

Fusarium head blight (FHB) reduces grain yield and quality in common and durum wheat. Host FHB resistance is an effective control measure that is achieved by stacking multiple resistance genes into a wheat line. Therefore, breeders would benefit from knowing which resistance sources carry different resistance genes. A diverse collection of FHB-resistant and -susceptible wheat lines was characterized with microsatellite markers linked to FHB resistance quantitative trait loci (QTLs) on chromosomes 2DL, 3BS (distal to the centromere), 3BSc (proximal to the centromere), 4B, 5AS and 6BS identified in wheat lines Maringa, Sumai 3 and Wuhan 1. Putative Sumai 3 QTLs were commonly observed in advanced breeding lines, whereas putative Maringa and Wuhan 1 QTLs were relatively rare. Marker data suggested the 3BS, 3BSc and 5AS QTLs in the Brazilian cv. Maringa were derived from Asian germplasm and not from Frontana or other Brazilian lines. Haplotype diversity was reduced near the 5AS QTL, which might impact the deployment of this QTL. Finally, Brazilian germplasm was not closely related to other resistance sources and might be useful for pyramiding with Asian wheat-derived FHB resistance.

Marker Assisted Selection of Fusarium Head Blight Resistance Genes in Two Doubled­ Haploid Populations of Wheat

Fusarium head blight, caused primarily by Fusarium graminearum, is the most important wheat disease in Canada causing both grain yield and quality losses. Selection for resistance to Fusarium head blight in breeding programs has been difficult because of the complex inheritance of resistance and the environmental effect on disease development and expression. The present study was conducted to examine microsatellite markers associated with resistance to Fusarium head blight and evaluate the effectiveness of these microsatellite markers in selecting for resistance to Fusarium head blight in two doubled-haploid populations segregating for Sumai 3-derived resistance genes. Both doubled-haploid populations were evaluated for resistance to Fusarium head blight by inoculation with F. graminearum in the greenhouse. Eight microsatellite markers from chromosomes 3BS, 6B and 5AL were applied to both doubled-haploid populations. The most significant microsatellite markers were found on the short arm of chromosome 3B, explaining 12% and 36% of phenotypic variation for resistance in the DH181/AC Foremost and AC Foremost/93FHB 21 doubled-haploid populations, respectively. Another important microsatellite marker, gwm644 on 6B, explained 21 % of the phenotypic variation for resistance to Fusarium head blight in the DH181/AC Foremost doubled-haploid population. There was a general lack of marker polymorphism on 5AL for the parents used in this study. Microsatellite markers on chromosome 3BS in addition to microsatellite markers on 6B have the potential for accelerating the development of wheat cultivars with improved Fusarium head blight resistance through the use of marker-assisted selection.

Molecular markers linked to white rust resistance in mustard Brassica juncea

Abstract White rust, caused by Albugo candida (Pers.) Kuntze, is an economically important disease of Brassica juncea (L.) Czern. and Coss mustard, particularly in India. The most efficient and cost-effective way of protecting mustard plants from white rust disease is through genetic resistance. The objective of this study was to identify RAPD markers for white rust resistance in an F1-derived doubled-haploid (DH) population originating from a cross between white rust-susceptible and white rust-resistant breeding lines of B. juncea from the canola-quality B. juncea breeding project of the Agriculture and Agri-Food Canada-Saskatoon Research Centre. The DH population was used to screen for RAPD markers associated with white rust resistance/susceptibility using bulked segregant analysis. Two markers, WR2 and WR3, linked to white rust resistance, flanked the resistance locus Ac21 and were highly effective in identifying the presence or absence of the resistance gene in the DH population. These two markers were shown to be specific to the Russian source of white rust resistance utilized in this project. It is concluded that the availability of these RAPD markers will enhance the breeding for white rust resistance in B. juncea.