Diverse regulatory factors associate with flowering time and yield responses in winter-type Brassica napus

Background: Flowering time, plant height and seed yield are strongly influenced by climatic and day-length adaptation in crop plants. To investigate these traits under highly diverse field conditions in the important oilseed crop Brassica napus, we performed a genome-wide association study using data from diverse agroecological environments spanning three continents. Methods: A total of 158 European winter-type B.napus inbred lines were genotyped with 21,623 unique, single-locus single-nucleotide polymorphism (SNP) markers using the Brassica 60 K-SNP Illumina® Infinium consortium array. Phenotypic associations were calculated in the panel over the years 2010–2012 for flowering time, plant height and seed yield in 5 highly diverse locations in Germany, China and Chile, adding up to 11 diverse environments in total. Results: We identified 101 genome regions associating with the onset of flowering, 69 with plant height, 36 with seed yield and 68 cross-trait regions with potential adaptive value. Within these regions, B.napus orthologs for a number of candidate adaptation genes were detected, including central circadian clock components like CIRCADIAN CLOCK- ASSOCIATED 1 (Bna.CCA1) and the important flowering-time regulators FLOWERING LOCUS T (Bna.FT) and FRUITFUL (Bna.FUL). Discussion: Gene ontology (GO) enrichment analysis of candidate regions suggested that selection of genes involved in post-transcriptional and epigenetic regulation of flowering time may play a potential role in adaptation of B. napus to highly divergent environments. The classical flowering time regulators Bna.FLC and Bna.CO were not found among the candidate regions, although both show functional variation. Allelic effects were additive for plant height and yield, but not for flowering time. The scarcity of positive minor alleles for yield in this breeding pool points to a lack of diversity for adaptation that could restrict yield gain in the face of environmental change. Conclusions: Our study provides a valuable framework to further improve the adaptability and yield stability of this recent allopolyploid crop under changing environments. The results suggest that flowering time regulation within an adapted B. napus breeding pool is driven by a high number of small modulating processes rather than major transcription factors like Bna.CO. In contrast, yield regulation appears highly parallel, therefore yield could be increased by pyramiding positively associated haplotypes

Capturing sequence variation among flowering-time regulatory gene homologs in the allopolyploid crop species Brassica napus

Flowering, the transition from the vegetative to the generative phase, is a decisive time point in the lifecycle of a plant. Flowering is controlled by a complex network of transcription factors, photoreceptors, enzymes and miRNAs. In recent years, several studies gave rise to the hypothesis that this network is also strongly involved in the regulation of other important lifecycle processes ranging from germination and seed development through to fundamental developmental and yield-related traits. In the allopolyploid crop species Brassica napus, (genome AACC), homoeologous copies of flowering time regulatory genes are implicated in major phenological variation within the species, however the extent and control of intraspecific and intergenomic variation among flowering-time regulators is still unclear. To investigate differences among B. napus morphotypes in relation to flowering-time gene variation, we performed targeted deep sequencing of 29 regulatory flowering-time genes in four genetically and phenologically diverse B. napus accessions. The genotype panel included a winter-type oilseed rape, a winter fodder rape, a spring-type oilseed rape (all B. napus ssp. napus) and a swede (B. napus ssp. napobrassica), which show extreme differences in winter-hardiness, vernalization requirement and flowering behaviour. A broad range of genetic variation was detected in the targeted genes for the different morphotypes, including non-synonymous SNPs, copy number variation and presence-absence variation. The results suggest that this broad variation in vernalisation, clock and signaling genes could be a key driver of morphological differentiation for flowering-related traits in this recent allopolyploid crop species

Different copies of SENSITIVITY TO RED LIGHT REDUCED 1 show strong subfunctionalization in Brassica napus

Schiessl S, Williams N, Specht P, Staiger D, Johansson M. Different copies of SENSITIVITY TO RED LIGHT REDUCED 1 show strong subfunctionalization in Brassica napus. BMC Plant Biology. 2019;2019(19): 372.#### Background Correct timing of flowering is critical for plants to produce enough viable offspring. In Arabidopsis thaliana (Arabidopsis), flowering time is regulated by an intricate network of molecular signaling pathways. Arabidopsis srr1–1 mutants lacking SENSITIVITY TO RED LIGHT REDUCED 1 (SRR1) expression flower early, particularly under short day (SD) conditions (1). SRR1 ensures that plants do not flower prematurely in such non-inductive conditions by controlling repression of the key florigen FT. Here, we have examined the role of SRR1 in the closely related crop species Brassica napus. #### Results Arabidopsis SRR1 has five homologs in Brassica napus. They can be divided into two groups, where the A02 and C02 copies show high similarity to AtSRR1 on the protein level. The other group, including the A03, A10 and C09 copies all carry a larger deletion in the amino acid sequence. Three of the homologs are expressed at detectable levels: A02, C02 and C09. Notably, the gene copies show a differential expression pattern between spring and winter type accessions of B. napus. When the three expressed gene copies were introduced into the srr1–1 background, only A02 and C02 were able to complement the srr1–1 early flowering phenotype, while C09 could not. Transcriptional analysis of known SRR1 targets in Bna.SRR1-transformed lines showed that CYCLING DOF FACTOR 1 (CDF1) expression is key for flowering time control via SRR1. #### Conclusions We observed subfunctionalization of the B. napus SRR1 gene copies, with differential expression between early and late flowering accessions of some Bna.SRR1 copies. This suggests involvement of Bna.SRR1 in regulation of seasonal flowering in B. napus. The C09 gene copy was unable to complement srr1–1 plants, but is highly expressed in B. napus, suggesting specialization of a particular function. Furthermore, the C09 protein carries a deletion which may pinpoint a key region of the SRR1 protein potentially important for its molecular function. This is important evidence of functional domain annotation in the highly conserved but unique SRR1 amino acid sequence

“Doubled-haploid” allohexaploid Brassica lines lose fertility and viability and accumulate genetic variation due to genomic instability

Microspore culture stimulates immature pollen grains to develop into plants via tissue culture and is used routinely in many crop species to produce "doubled haploids": homozygous, true-breeding lines. However, microspore culture is also often used on material that does not have stable meiosis, such as interspecific hybrids. In this case, the resulting progeny may lose their "doubled haploid" homozygous status as a result of chromosome missegregation and homoeologous exchanges. However, little is known about the frequency of these effects. We assessed fertility, meiosis and genetic variability in self-pollinated progeny sets (the MDL2 population) resulting from first-generation plants (the MDL1 population) derived from microspores of a near-allohexaploid interspecific hybrid from the cross (Brassica napus × B. carinata) × B. juncea. Allelic inheritance and copy number variation were predicted using single nucleotide polymorphism marker data from the Illumina Infinium 60K Brassica array. Seed fertility and viability decreased substantially from the MDL1 to the MDL2 generation. In the MDL2 population, 87% of individuals differed genetically from their MDL1 parent. These genetic differences resulted from novel homoeologous exchanges between chromosomes, chromosome loss and gain, and segregation and instability of pre-existing karyotype abnormalities. Novel karyotype change was extremely common, with 2.2 new variants observed per MDL2 individual. Significant differences between progeny sets in the number of novel genetic variants were also observed. Meiotic instability clearly has the potential to dramatically change karyotypes (often without detectable effects on the presence or absence of alleles) in putatively homozygous, microspore-derived lines, resulting in loss of fertility and viability

Inherited allelic variants and novel karyotype changes influence fertility and genome stability in Brassica allohexaploids

Synthetic allohexaploid Brassica hybrids (2n\ua0=\ua0AABBCC) do not exist naturally, but can be synthesized by crosses between diploid and/or allotetraploid Brassica species. Using these hybrids, we aimed to identify how novel allohexaploids restore fertility and normal meiosis after formation. Chromosome inheritance, genome structure, fertility and meiotic behaviour were assessed in three segregating allohexaploid populations derived from the cross (B.\ua0napus\ua0×\ua0B.\ua0carinata)\ua0×\ua0B.\ua0juncea using a combination of molecular marker genotyping, phenotyping and cytogenetics. Plants with unbalanced A-C translocations in one direction (where a C-genome chromosome fragment replaces an A-genome fragment) but not the other (where an A-genome fragment replaces a C-genome fragment) showed significantly reduced fertility across all populations. Genomic regions associated with fertility contained several meiosis genes with putatively causal mutations inherited from the parents (copies of SCC2 in the A genome, PAIR1/PRD3, PRD1 and ATK1/KATA/KIN14a in the B genome, and MSH2 and SMC1/TITAN8 in the C genome). Reduced seed fertility associated with the loss of chromosome fragments from only one subgenome following homoeologous exchanges could comprise a mechanism for biased genome fractionation in allopolyploids. Pre-existing meiosis gene variants present in allotetraploid parents may help to stabilize meiosis in novel allohexaploids

Flowering Time Gene Variation in Brassica Species Shows Evolutionary Principles

Flowering time genes have a strong influence on successful reproduction and life cycle adaptation. However, their regulation is highly complex and only well understood in diploid model systems. For crops with a polyploid background from the genus Brassica, data on flowering time gene variation are scarce, although indispensable for modern breeding techniques like marker-assisted breeding. We have deep-sequenced all paralogs of 35 Arabidopsis thaliana flowering regulators using Sequence Capture followed by Illumina sequencing in two selected accessions of the vegetable species Brassica rapa and Brassica oleracea, respectively. Using these data, we were able to call SNPs, InDels and copy number variations (CNVs) for genes from the total flowering time network including central flowering regulators, but also genes from the vernalisation pathway, the photoperiod pathway, temperature regulation, the circadian clock and the downstream effectors. Comparing the results to a complementary data set from the allotetraploid species Brassica napus, we detected rearrangements in B. napus which probably occurred early after the allopolyploidisation event. Those data are both a valuable resource for flowering time research in those vegetable species, as well as a contribution to speciation genetics

The role of genomic structural variation in the genetic improvement of polyploid crops

Many of our major crop species are polyploids, containing more than one genome or set of chromosomes. Polyploid crops present unique challenges, including difficulties in genome assembly, in discriminating between multiple gene and sequence copies, and in genetic mapping, hindering use of genomic data for genetics and breeding. Polyploid genomes may also be more prone to containing structural variation, such as loss of gene copies or sequences (presence–absence variation) and the presence of genes or sequences in multiple copies (copy-number variation). Although the two main types of genomic structural variation commonly identified are presence–absence variation and copy-number variation, we propose that homeologous exchanges constitute a third major form of genomic structural variation in polyploids. Homeologous exchanges involve the replacement of one genomic segment by a similar copy from another genome or ancestrally duplicated region, and are known to be extremely common in polyploids. Detecting all kinds of genomic structural variation is challenging, but recent advances such as optical mapping and long-read sequencing offer potential strategies to help identify structural variants even in complex polyploid genomes. All three major types of genomic structural variation (presence–absence, copy-number, and homeologous exchange) are now known to influence phenotypes in crop plants, with examples of flowering time, frost tolerance, and adaptive and agronomic traits. In this review, we summarize the challenges of genome analysis in polyploid crops, describe the various types of genomic structural variation and the genomics technologies and data that can be used to detect them, and collate information produced to date related to the impact of genomic structural variation on crop phenotypes. We highlight the importance of genomic structural variation for the future genetic improvement of polyploid crops. Keywords: Presence–absence variation, Copy-number variation, Homeologous exchanges, Genome structure, Pan-genom

Additional file 3: Table S2. of Diverse regulatory factors associate with flowering time and yield responses in winter-type Brassica napus

Candidate genes for associating regions for BOF-ABS, BOF-REL, HEI-ABS, YIE-ABS and COMP. Each gene has a unique identifier according to [20] and a short sequence description. The table names the highest associating marker in the defined region in its LD block with r 2  > 0.5 as well as the SNP marker most closely linked to the respective gene with its r 2 value to the highest associating SNP marker, and the closest markers distance from the gene. It also gives the value of the highest association among the respective region. The next columns show the strength of association in the respective environment; * stands for –log10(p) > 2, ** for -log10(p) > 3 and *** for –log10(p) > 4, and the last columns show the absolute effect of the highest associating marker on the respective trait. (XLSX 553 kb

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets

Rationale: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome