SUMMARYFlax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94· raw, approximately 69· filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N 50 = 694 kb, including contigs with N 50 = 20.1 kb. The contig assembly contained 302 Mb of nonredundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43 384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K s ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on wholegenome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.

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.

Relatively little is known about the genetic control of agronomic traits in common wheat (Triticum aestivum L.) compared with traits that follow Mendelian segregation patterns. A doubled-haploid population was generated from the cross RL4452x'AC Domain' to study the inheritance of the agronomic traits: plant height, time to maturity, lodging, grain yield, test weight, and 1000-grain weight. This cross includes the genetics of 2 western Canadian wheat marketing classes. Composite interval mapping was conducted with a microsatellite linkage map, incorporating 369 loci, and phenotypic data from multiple Manitoba environments. The plant height quantitative trait loci (QTLs), QHt.crc-4B and QHt.crc-4D, mapped to the expected locations of Rht-B1 and Rht-D1. These QTLs were responsible for most of the variation in plant height and were associated with other agronomic traits. An additional 25 agronomic QTLs were detected in the RL4452x'AC Domain' population beyond those associated with QHt.crc-4B and QHt.crc-4D. 'AC Domain' contributed 4 alleles for early maturity, including a major time to maturity QTL on 7D. RL4452 contributed 2 major alleles for increased grain yield at QYld.crc-2B and QYld.crc-4A, which are potential targets for marker-assisted selection. A key test weight QTL was detected on 3B and prominent 1000-grain weight QTLs were identified on 3D and 4A.

A major fusarium head blight (FHB) resistance gene Fhb1 (syn. Qfhs.ndsu-3BS) was fine mapped on the distal segment of chromosome 3BS of spring wheat (Triticum aestivum L.) as a Mendelian factor. FHB resistant parents, Sumai 3 and Nyubai, were used as sources of this gene. Two mapping populations were developed to facilitate segregation of Qfhs.ndsu-3BS in either a fixed resistant (Sumai 3*5/Thatcher) (S/T) or fixed susceptible (HC374/3*98B69-L47) (HC/98) genetic background (HC374 = Wuhan1/Nyubai) for Type II resistance. Type II resistance (disease spread within the spike) was phenotyped in the greenhouse using single floret injections with a mixture of macro-conidia of three virulent strains of Fusarium graminearum. Due to the limited heterogeneity in the genetic background of the crosses and based on the spread of infection, fixed recombinants in the interval between molecular markers XGWM533 and XGWM493 on 3BS could be assigned to discrete "resistant" and "susceptible" classes. The phenotypic distribution was bimodal with progeny clearly resembling either the resistant or susceptible parent. Marker order for the two maps was identical with the exception of marker STS-3BS 142, which was not polymorphic in the HC/98 population. The major gene Fhb1 was successfully fine mapped on chromosome 3BS in the same location in the two populations within a 1.27-cM interval (S/T) and a 6.05-cM interval (HC/98). Fine mapping of Fhb1 in wheat provides tightly linked markers that can reduce linkage drag associated with marker-assisted selection of Fhb1 and assist in the isolation, sequencing and functional identification of the underlying resistance gene.

The decreasing cost along with rapid progress in next-generation sequencing and related bioinformatics computing resources has facilitated large-scale discovery of SNPs in various model and nonmodel plant species. Large numbers and genome-wide availability of SNPs make them the marker of choice in partially or completely sequenced genomes. Although excellent reviews have been published on next-generation sequencing, its associated bioinformatics challenges, and the applications of SNPs in genetic studies, a comprehensive review connecting these three intertwined research areas is needed. This paper touches upon various aspects of SNP discovery, highlighting key points in availability and selection of appropriate sequencing platforms, bioinformatics pipelines, SNP filtering criteria, and applications of SNPs in genetic analyses. The use of next-generation sequencing methodologies in many non-model crops leading to discovery and implementation of SNPs in various genetic studies is discussed. Development and improvement of bioinformatics software that are open source and freely available have accelerated the SNP discovery while reducing the associated cost. Key considerations for SNP filtering and associated pipelines are discussed in specific topics. A list of commonly used software and their sources is compiled for easy access and reference.

In hexaploid wheat, leaf rust resistance gene Lr1 is located at the distal end of the long arm of chromosome 5D. To clone this gene, an F(1)-derived doubled haploid population and a recombinant inbred line population from a cross between the susceptible cultivar AC Karma and the resistant line 87E03-S2B1 were phenotyped for resistance to Puccinia triticina race 1-1 BBB that carries the avirulence gene Avr1. A high-resolution genetic map of the Lr1 locus was constructed using microsatellite, resistance gene analog (RGA), BAC end (BE), and low pass (LP) markers. A physical map of the locus was constructed by screening a hexaploid wheat BAC library from cultivar Glenlea that is known to have Lr1. The locus comprised three RGAs from a gene family related to RFLP marker Xpsr567. Markers specific to each paralog were developed. Lr1 segregated with RGA567-5 while recombinants were observed for the other two RGAs. Transformation of the susceptible cultivar Fielder with RGA567-5 demonstrated that it corresponds to the Lr1 resistance gene. In addition, the candidate gene was also confirmed by virus-induced gene silencing. Twenty T (1) lines from resistant transgenic line T (0)-938 segregated for resistance, partial resistance and susceptibility to Avr1 corresponding to a 1:2:1 ratio for a single hemizygous insertion. Transgene presence and expression correlated with the phenotype. The resistance phenotype expressed by Lr1 seemed therefore to be dependant on the zygosity status. T (3)-938 sister lines with and without the transgene were further tested with 16 virulent and avirulent rust isolates. Rust reactions were all as expected for Lr1 thereby providing additional evidence toward the Lr1 identity of RGA567-5. Sequence analysis of Lr1 indicated that it is not related to the previously isolated Lr10 and Lr21 genes and unlike these genes, it is part of a large gene family.

A set of 146,611 expressed sequence tags (ESTs) were generated from 10 flax cDNA libraries. After assembly, a total of 11,166 contigs and 11,896 singletons were mined for the presence of putative simple sequence repeats (SSRs) and yielded 806 (3.5%) non-redundant sequences which contained 851 putative SSRs. This is equivalent to one EST-SSR per 16.5 kb of sequence. Trinucleotide motifs were the most abundant (76.9%), followed by dinucleotides (13.9%). Tetra-, penta- and hexanucleotide motifs represented <10% of the SSRs identified. A total of 83 SSR motifs were identified. Motif (TTC/GAA)n was the most abundant (10.2%) followed by (CTT/AAG)n (8.7%), (TCT/AGA)n (8.6%), (CT/AG)n (6.7%) and (TC/GA)n (5.3%). A total of 662 primer pairs were designed, of which 610 primer pairs yielded amplicons in a set of 23 flax accessions. Polymorphism between the accessions was found for 248 primer pairs which detected a total of 275 EST-SSR loci. Two to seven alleles were detected per marker. The polymorphism information content value for these markers ranged from 0.08 to 0.82 and averaged 0.35. The 635 alleles detected by the 275 polymorphic EST-SSRs were used to study the genetic relationship of 23 flax accessions. Four major clusters and two singletons were observed. Sub-clusters within the main clusters correlated with the pedigree relationships amongst accessions. The EST-SSRs developed herein represent the first large-scale development of SSR markers in flax. They have potential to be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping and fingerprinting cultivars for example.

Wheat grain quality is a complex group of traits of tremendous importance to wheat producers, end-users and breeders. Quantitative trait locus (QTL) analysis studied the genetics of milling, mixograph, farinograph, baking, starch and noodle colour traits in the spring wheat population RL4452/'AC Domain'. Forty-seven traits were measured on the population and 99 QTLs were detected over 18 chromosomes for 41 quality traits. Forty-four of these QTLs mapped to three major QTL clusters on chromosomes 1B, 4D, and 7D. Fourteen QTLs mapped near Glu-B1, 20 QTLs mapped near a major plant height QTL on chromosome 4D, and 10 QTLs mapped near a major time to maturity QTL on chromosome 7D. Large QTLs were detected for grain and flour protein content, farinograph absorption, mixograph parameters, and dietary fibre on chromosome 2BS. QTLs for yellow alkaline noodle colour parameter L* mapped to chromosomes 5B and 5D, while the largest QTL for the b* parameter mapped to 7AL.