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NRC publication: Yes

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.

Summary Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.

NRC publication: Yes

It is only recently, with the advent of long-read sequencing technologies, that we are beginning to uncover previously uncharted regions of complex and inherently recursive plant genomes. To comprehensively study and exploit the genome of the neglected oilseed Brassica nigra, we generated two high-quality nanopore de novo genome assemblies. The N50 contig lengths for the two assemblies were 17.1 Mb (12 contigs), one of the best among 324 sequenced plant genomes, and 0.29 Mb (424 contigs), respectively, reflecting recent improvements in the technology. Comparison with a de novo short-read assembly corroborated genome integrity and quantified sequence-related error rates (0.2%). The contiguity and coverage allowed unprecedented access to low-complexity regions of the genome. Pericentromeric regions and coincidence of hypomethylation enabled localization of active centromeres and identified centromere-associated ALE family retro-elements that appear to have proliferated through relatively recent nested transposition events (<1 Ma). Genomic distances calculated based on synteny relationships were used to define a post-triplication Brassica-specific ancestral genome, and to calculate the extensive rearrangements that define the evolutionary distance separating B. nigra from its diploid relatives.

Background Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F 1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC 1 and 3R:1S in the F 2 , which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6 . Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis. Results Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8–15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes ( Bra010552 , Bra010588 , Bra010589 , Bra010590 and Bra010663 ) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra , where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1–6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6 . Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819 . Conclusion Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study.

Background: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement. Description: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184–191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR. Conclusion: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/.

Perennial shrub willow are increasingly being promoted in short-rotation coppice systems as biomass feedstocks, for phytoremediation applications, and for the diverse ecosystem services that can accrue. This renewed interest has led to widespread willow cultivation, particularly of non-native varieties. However, Canadian willow species have not been widely adopted and their inherent diversity has not yet been thoroughly investigated. In this study, 324 genotypes of Salix famelica and Salix eriocephala collected from 33 sites of origin were analyzed using 26,016 single nucleotide polymorphisms to reveal patterns of population structure and genetic diversity. Analyses by Bayesian methods and principal component analysis detected five main clusters that appeared to be largely shaped by geoclimatic variables including mean annual precipitation and the number of frost-free days. The overall observed ( H O ) and expected ( H E ) heterozygosity were 0.126 and 0.179, respectively. An analysis of molecular variance revealed that the highest genetic variation occurred within genotypes (69%), while 8% of the variation existed among clusters and 23% between genotypes within clusters. These findings provide new insights into the extent of genetic variation that exists within native shrub willow species which could be leveraged in pan-Canadian willow breeding programs.

Camelina sativa (L.) Crantz an oilseed crop of the Brassicaceae family is gaining attention due to its potential as a source of high value oil for food, feed or fuel. The hexaploid domesticated C. sativa has limited genetic diversity, encouraging the exploration of related species for novel allelic variation for traits of interest. The current study utilized genotyping by sequencing to characterize 193 Camelina accessions belonging to seven different species collected primarily from the Ukrainian-Russian region and Eastern Europe. Population analyses among Camelina accessions with a 2n = 40 karyotype identified three subpopulations, two composed of domesticated C. sativa and one of C. microcarpa species. Winter type Camelina lines were identified as admixtures of C. sativa and C. microcarpa. Eighteen genotypes of related C. microcarpa unexpectedly shared only two subgenomes with C. sativa, suggesting a novel or cryptic sub-species of C. microcarpa with 19 haploid chromosomes. One C. microcarpa accession (2n = 26) was found to comprise the first two subgenomes of C. sativa suggesting a tetraploid structure. The defined chromosome series among C. microcarpa germplasm, including the newly designated C. neglecta diploid née C. microcarpa, suggested an evolutionary trajectory for the formation of the C. sativa hexaploid genome and re-defined the underlying subgenome structure of the reference genome.

Seed quality traits of oilseed rape, Brassica napus (B. napus), exhibit quantitative inheritance determined by its genetic makeup and the environment via the mediation of a complex genetic architecture of hundreds to thousands of genes. Thus, instead of single gene analysis, network-based systems genomics and genetics approaches that combine genotype, phenotype, and molecular phenotypes offer a promising alternative to uncover this complex genetic architecture. In the current study, systems genetics approaches were used to explore the genetic regulation of lignin traits in B. napus seeds. Four QTL (qLignin_A09_1, qLignin_A09_2, qLignin_A09_3, and qLignin_C08) distributed on two chromosomes were identified for lignin content. The qLignin_A09_2 and qLignin_C08 loci were homologous QTL from the A and C subgenomes, respectively. Genome-wide gene regulatory network analysis identified eighty-three subnetworks (or modules); and three modules with 910 genes in total, were associated with lignin content, which was confirmed by network QTL analysis. eQTL (expression quantitative trait loci) analysis revealed four cis-eQTL genes including lignin and flavonoid pathway genes, cinnamoyl-CoA-reductase (CCR1), and TRANSPARENT TESTA genes TT4, TT6, TT8, as causal genes. The findings validated the power of systems genetics to identify causal regulatory networks and genes underlying complex traits. Moreover, this information may enable the research community to explore new breeding strategies, such as network selection or gene engineering, to rewire networks to develop climate resilience crops with better seed quality.