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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 eightythree 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. [ABSTRACT FROM AUTHOR]

SUMMARY: Polyploidy is a persistent phenomenon in angiosperm genome evolution that is hypothesized to have contributed to the diversity of extant flowering plants. Brassica napus, one of the world's most important angiosperm oilseed species, originated from the interspecific hybridization of Brassica rapa (An) and Brassica oleracea (Cn). While the trends of genome dominance in transcriptomics are beginning to emerge, less is known about the epigenetic and small RNA landscapes in polyploids during reproductive development. The seed is the pivotal developmental transition into the new sporophytic generation, and experiences substantial epigenetic modifications over time. Here, we investigated the prevalence of bias in the contexts of DNA methylation and small interfering (si)RNA profiles in both subgenomes (An and Cn), as well as the ancestral fractionated genomes across B. napus seed development. We report ubiquitous Cn subgenome bias of siRNA expression and cytosine methylation, with DNA methylation being particularly abundant on gene promoters in the Cn subgenome. Further, we provide evidence that siRNA transcriptional patterns were conserved within the ancestral triplicated subgenomes of B. napus, but not across the An and Cn subgenomes. We discuss how methylation patterns in the B. napus seed relate to genes, promoter regions, siRNA loci and transposable elements through the lens of genome fractionation and polyploidization. Taken together we provide evidence for epigenetic regulation selectively silencing the Cn subgenome during seed development, and explore the impact of genome fractionation on the epigenetic components of the B. napus seed. Significance Statement: Brassica napus (canola) is one of many globally important polyploid crops. As an amphidiploid species that underwent an ancestral whole‐genome triplication event, B. napus has both distinct subgenomes from interspecific hybridization between B. rapa and B. oleracea in addition to fractionated ancestral genomes. Here, we analyzed both DNA methylation and siRNA profiles of B. napus and its two subgenomes, and report on the dynamic epigenetic landscape underpinning seed development. [ABSTRACT FROM AUTHOR]

A genome‐wide association study (GWAS) was carried out by using a Brassica napus population of 175 lines, developed from six B. napus × B. oleracea interspecific crosses, and 5,743 single nucleotide polymorphism (SNP) markers to identify quantitative trait loci (QTL) for agronomic and seed quality traits and to understand the effect of B. oleracea alleles on these traits. Heritability of these traits varied from 52.9 to 84.1%. About 79% of the SNPs were positioned to the nine C genome chromosomes, while only 21% to the 10 A genome chromosomes; this was largely due to little genetic variation in the A genome of this population, as expected. However, the SNPs were distributed throughout the entire length of the chromosomes suggesting their usefulness in GWAS. The C genome SNPs detected nine genomic regions affecting these traits. This included the genomic regions of C2 and C5 affecting days to flowering, C1 affecting the duration of grain‐filling period, C1, C5, and C8 affecting oil content, and C1, C2, and C6 affecting glucosinolate content; among these, some of the loci has not been reported previously. The QTL alleles of B. oleracea which can be beneficial in oilseed B. napus, such as the C5 QTL allele for the earliness of flowering, were also identified. Several putative candidate genes were identified in the QTL regions. Thus, the results provided evidence of the utility of the B. oleracea gene pool for use in unveiling the unidentified QTL in B. napus as well as its use in breeding. Core Ideas: C genome alleles from six vegetable Brassica oleracea accessions were introgressed into oilseed B. napus.Quantitative trait loci for agronomic and seed quality traits were mapped and the effect of Brassica oleracea alleles in B. napus were detected.Beneficial quantitative trait loci allele of Brassica oleracea were identified for use in B. napus breeding.C genome quantitative trait loci that has not been reported previously were disclosed. [ABSTRACT FROM AUTHOR]

SUMMARY: We profiled the global gene expression landscape across the reproductive lifecycle of Brassica napus. Comparative analysis of this nascent amphidiploid revealed the contribution of each subgenome to plant reproduction. Whole‐genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive‐lethal phenotype. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome‐specific transcription factor networks identified distinct transcription factor families enriched in each of the An and Cn subgenomes early in seed development. Analysis of laser‐microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat, whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm and seed coat of the developing seed. Transcriptional networks spanning both the An and Cn genomes of the whole B. napus seed can identify valuable targets for seed development research and that ‐omics level approaches to studying gene regulation in B. napus can benefit from both broad and high‐resolution analyses. Significance Statement: Global gene expression profiling and laser microdissection of the B. napus seed identifies subgenome bias and spatial distribution of biological processes and transcription factor networks. [ABSTRACT FROM AUTHOR]

Phenotyping is considered a significant bottleneck impeding fast and efficient crop improvement. Similar to many crops, Brassica napus , an internationally important oilseed crop, suffers from low genetic diversity, and will require exploitation of diverse genetic resources to develop locally adapted, high yielding and stress resistant cultivars. A pilot study was completed to assess the feasibility of using indoor high-throughput phenotyping (HTP), semi-automated image processing, and machine learning to capture the phenotypic diversity of agronomically important traits in a diverse B. napus breeding population, SKBnNAM, introduced here for the first time. The experiment comprised 50 spring-type B. napus lines, grown and phenotyped in six replicates under two treatment conditions (control and drought) over 38 days in a LemnaTec Scanalyzer 3D facility. Growth traits including plant height, width, projected leaf area, and estimated biovolume were extracted and derived through processing of RGB and NIR images. Anthesis was automatically and accurately scored (97% accuracy) and the number of flowers per plant and day was approximated alongside relevant canopy traits (width, angle). Further, supervised machine learning was used to predict the total number of raceme branches from flower attributes with 91% accuracy (linear regression and Huber regression algorithms) and to identify mild drought stress, a complex trait which typically has to be empirically scored (0.85 area under the receiver operating characteristic curve, random forest classifier algorithm). The study demonstrates the potential of HTP, image processing and computer vision for effective characterization of agronomic trait diversity in B. napus , although limitations of the platform did create significant variation that limited the utility of the data. However, the results underscore the value of machine learning for phenotyping studies, particularly for complex traits such as drought stress resistance. [ABSTRACT FROM AUTHOR]

Phenotyping crop performance is critical for line selection and variety development in plant breeding. Canola (Brassica napus L.) flowers, the bright yellow flowers, indeterminately increase over a protracted period. Flower production of canola plays an important role in yield determination. Yellowness of canola petals may be a critical reflectance signal and a good predictor of pod number and, therefore, seed yield. However, quantifying flowering based on traditional visual scales is subjective, time-consuming, and labor-consuming. Recent developments in phenotyping technologies using Unmanned Aerial Vehicles (UAVs) make it possible to effectively capture crop information and to predict crop yield via imagery. Our objectives were to investigate the application of vegetation indices in estimating canola flower numbers and to develop a descriptive model of canola seed yield. Fifty-six diverse Brassica genotypes, including 53 B. napus lines, two Brassica carinata lines, and a Brassica juncea variety, were grown near Saskatoon, SK, Canada from 2016 to 2018 and near Melfort and Scott, SK, Canada in 2017. Aerial imagery with geometric and radiometric corrections was collected through the flowering stage using a UAV mounted with a multispectral camera. We found that the normalized difference yellowness index (NDYI) was a useful vegetation index for representing canola yellowness, which is related to canola flowering intensity during the full flowering stage. However, the flowering pixel number estimated by the thresholding method improved the ability of NDYI to detect yellow flowers with coefficient of determination (R 2) ranging from 0.54 to 0.95. Moreover, compared with using a single image date, the NDYI-based flowering pixel numbers integrated over time covers more growth information and can be a good predictor of pod number and thus, canola yield with R 2 up to 0.42. These results indicate that NDYI-based flowering pixel numbers can perform well in estimating flowering intensity. Integrated flowering intensity extracted from imagery over time can be a potential phenotype associated with canola seed yield. [ABSTRACT FROM AUTHOR]

Summary: Ensuring faithful homologous recombination in allopolyploids is essential to maintain optimal fertility of the species. Variation in the ability to control aberrant pairing between homoeologous chromosomes in Brassica napus has been identified. The current study exploited the extremes of such variation to identify genetic factors that differentiate newly resynthesised B. napus, which is inherently unstable, and established B. napus, which has adapted to largely control homoeologous recombination.A segregating B. napus mapping population was analysed utilising both cytogenetic observations and high‐throughput genotyping to quantify the levels of homoeologous recombination.Three quantitative trait loci (QTL) were identified that contributed to the control of homoeologous recombination in the important oilseed crop B. napus. One major QTL on BnaA9 contributed between 32 and 58% of the observed variation. This study is the first to assess homoeologous recombination and map associated QTLs resulting from deviations in normal pairing in allotetraploid B. napus.The identified QTL regions suggest candidate meiotic genes that could be manipulated in order to control this important trait and further allow the development of molecular markers to utilise this trait to exploit homoeologous recombination in a crop. See also the Commentary on this article by Sourdille & Jenczewski, 229: 3041–3043. [ABSTRACT FROM AUTHOR]

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Summary: Genome structural variation (SV) contributes strongly to trait variation in eukaryotic species and may have an even higher functional significance than single‐nucleotide polymorphism (SNP). In recent years, there have been a number of studies associating large chromosomal scale SV ranging from hundreds of kilobases all the way up to a few megabases to key agronomic traits in plant genomes. However, there have been little or no efforts towards cataloguing small‐ (30–10 000 bp) to mid‐scale (10 000–30 000 bp) SV and their impact on evolution and adaptation‐related traits in plants. This might be attributed to complex and highly duplicated nature of plant genomes, which makes them difficult to assess using high‐throughput genome screening methods. Here, we describe how long‐read sequencing technologies can overcome this problem, revealing a surprisingly high level of widespread, small‐ to mid‐scale SV in a major allopolyploid crop species, Brassica napus. We found that up to 10% of all genes were affected by small‐ to mid‐scale SV events. Nearly half of these SV events ranged between 100 bp and 1000 bp, which makes them challenging to detect using short‐read Illumina sequencing. Examples demonstrating the contribution of such SV towards eco‐geographical adaptation and disease resistance in oilseed rape suggest that revisiting complex plant genomes using medium‐coverage long‐read sequencing might reveal unexpected levels of functional gene variation, with major implications for trait regulation and crop improvement. [ABSTRACT FROM AUTHOR]

SUMMARY: In plants, race‐specific defence against microbial pathogens is facilitated by resistance (R) genes which correspond to specific pathogen avirulence genes. This study reports the cloning of a blackleg R gene from Brassica napus (canola), Rlm9, which encodes a wall‐associated kinase‐like (WAKL) protein, a newly discovered class of race‐specific plant RLK resistance genes. Rlm9 provides race‐specific resistance against isolates of Leptosphaeria maculans carrying the corresponding avirulence gene AvrLm5‐9, representing only the second WAKL‐type R gene described to date. The Rlm9 protein is predicted to be cell membrane‐bound and while not conclusive, our work did not indicate direct interaction with AvrLm5‐9. Rlm9 forms part of a distinct evolutionary family of RLK proteins in B. napus, and while little is yet known about WAKL function, the Brassica–Leptosphaeria pathosystem may prove to be a model system by which the mechanism of fungal avirulence protein recognition by WAKL‐type R genes can be determined. Significance Statement: Rlm9 encodes a wall‐associated kinase‐like (WAKL) race‐specific resistance protein, is a member of an emerging class of race‐specific plant receptor‐like kinase resistance genes and is the first of its kind to be cloned from a dicot species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Key message: Provide evidence that the Brassica B genome chromosome B3 carries blackleg resistance gene, and also the B genome chromosomes were inherited several generations along with B. napus chromosomes. Abstract: Blackleg disease caused by fungus Leptosphaeria maculans causes significant yield losses in Brassica napus. Brassica carinata possesses excellent resistance to this disease. To introgress blackleg resistance, crosses between B. napus cv. Westar and B. carinata were done. The interspecific-hybrids were backcrossed twice to Westar and self-pollinated three times to produce BCS families. Doubled haploid lines (DH1) were produced from one blackleg resistant family. SSR markers were used to study the association between B genome chromosome(s) and blackleg resistance. The entire B3 chromosome of B. carinata was associated with blackleg resistance in DH1. A second DH population (DH2) was produced from Fs of resistant DH1 lines crossed to blackleg susceptible B. napus cv. Polo where resistance was found to be associated with SSR markers from the middle to bottom of the B3 and top of the B8 chromosomes. The results demonstrated that the B3 chromosome carried gene(s) for blackleg resistance. Genomic in situ hybridization (GISH) and GISH-like analysis of the DH2 lines revealed that susceptible lines, in addition to B. napus chromosomes, possessed one pair of B genome chromosomes (2 n = 40), while resistant lines had either one (2 n = 40) or two pairs (2 n = 42) of B chromosomes. The molecular and GISH data suggested that the B chromosome in the susceptible lines was B7, while it was difficult to confirm the identity of the B chromosomes in the resistant lines. Also, B chromosomes were found to be inherited over several generations along with B. napus chromosomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]