Rapeseed (Brassica napus) is the second most important oilseed crop in the world but the genetic diversity underlying its massive phenotypic variations remains largely unexplored. Here, we report the sequencing, de novo assembly and annotation of eight B. napus accessions. Using pan-genome comparative analysis, millions of small variations and 77.2–149.6 megabase presence and absence variations (PAVs) were identified. More than 9.4% of the genes contained large-effect mutations or structural variations. PAV-based genome-wide association study (PAV-GWAS) directly identified causal structural variations for silique length, seed weight and flowering time in a nested association mapping population with ZS11 (reference line) as the donor, which were not detected by single-nucleotide polymorphisms-based GWAS (SNP-GWAS), demonstrating that PAV-GWAS was complementary to SNP-GWAS in identifying associations to traits. Further analysis showed that PAVs in three FLOWERING LOCUS C genes were closely related to flowering time and ecotype differentiation. This study provides resources to support a better understanding of the genome architecture and acceleration of the genetic improvement of B. napus. The assembly of eight high-quality rapeseed genomes allows identification of presence and absence variations (PAVs) and small variations. PAV-based genome-wide association analysis uncovered causal variations for agronomic traits and ecotype differentiation.

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Eight high-quality genomes reveal pan-genome architecture and ecotype differentiation of Brassica napus.

Flowering time regulation in crops—what did we learn from Arabidopsis?

Crop Breeding Chips and Genotyping Platforms: Progress, Challenges, and Perspectives

A key step during crop domestication is the loss of seed shattering. Here, we show that seed shattering in sorghum is controlled by a single gene, Shattering1 (Sh1), which encodes a YABBY transcription factor. Domesticated sorghums harbor three different mutations at the Sh1 locus. Variants at regulatory sites in the promoter and intronic regions lead to a low level of expression, a 2.2-kb deletion causes a truncated transcript that lacks exons 2 and 3, and a GT-to-GG splice-site variant in the intron 4 results in removal of the exon 4. The distributions of these non-shattering haplotypes among sorghum landraces suggest three independent origins. The function of the rice ortholog (OsSh1) was subsequently validated with a shattering-resistant mutant, and two maize orthologs (ZmSh1-1 and ZmSh1-5.1+ZmSh1-5.2) were verified with a large mapping population. Our results indicate that Sh1 genes for seed shattering were under parallel selection during sorghum, rice and maize domestication.

Parallel Domestication of the Shattering1 Genes in Cereals

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species.

An Atlas of over 90,000 Conserved Noncoding Sequences Provides Insight into Crucifer Regulatory Regions

Brassica species, including crops such as cabbage, turnip and oilseed, display enormous phenotypic variation. Brassica genomes have all undergone a whole-genome triplication (WGT) event with unknown effects on phenotype diversification. We resequenced 199 Brassica rapa and 119 Brassica oleracea accessions representing various morphotypes and identified signals of selection at the mesohexaploid subgenome level. For cabbage morphotypes with their typical leaf-heading trait, we identified four subgenome loci that show signs of parallel selection among subgenomes within B. rapa, as well as four such loci within B. oleracea. Fifteen subgenome loci are under selection and are shared by these two species. We also detected strong subgenome parallel selection linked to the domestication of the tuberous morphotypes, turnip (B. rapa) and kohlrabi (B. oleracea). Overall, we demonstrated that the mesohexaploidization of the two Brassica genomes contributed to their diversification into heading and tuber-forming morphotypes through convergent subgenome parallel selection of paralogous genes.

Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea

Background: Flowering time is an important trait in Brassica rapa crops. FLOWERING LOCUS C (FLC) is a MADS-box transcription factor that acts as a potent repressor of flowering. Expression of FLC is silenced when plants are exposed to low temperature, which activates flowering. There are four copies of FLC in B. rapa. Analyses of different segregating populations have suggested that BraA.FLC.a (BrFLC1) and BraA.FLC.b (BrFLC2) play major roles in controlling flowering time in B. rapa. Results: We analyzed the BrFLC2 sequence in nine B. rapa accessions, and identified a 57-bp insertion/deletion (InDel) across exon 4 and intron 4 resulting in a non-functional allele. In total, three types of transcripts were identified for this mutated BrFLC2 allele. The InDel was used to develop a PCR-based marker, which was used to screen a collection of 159 B. rapa accessions. The deletion genotype was present only in oil-type B. rapa, including ssp. oleifera and ssp. tricolaris, and not in other subspecies. The deletion genotype was significantly correlated with variation in flowering time. In contrast, the reported splicing site variation in BrFLC1, which also leads to a non-functional locus, was detected but not correlated with variation in flowering time in oil-type B. rapa, although it was correlated with variation in flowering time in vegetable-type B. rapa. Conclusions: Our results suggest that the naturally occurring deletion mutation across exon 4 and intron 4 in BrFLC2 gene contributes greatly to variation in flowering time in oil-type B. rapa. The observed different relationship between BrFLC1 or BrFLC2 and flowering time variation indicates that the control of flowering time has evolved separately between oil-type and vegetable-type B. rapa groups.

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A naturally occurring InDel variation in BraA.FLC.b (BrFLC2) associated with flowering time variation in Brassica rapa.

A splicing site mutation in BrFLC5, a non-syntenic paralogue of FLOWERING LOCUS C, was demonstrated to be related to flowering time variation in Brassica rapa. Flowering time regulation in Brassica rapa is more complex than in Arabidopsis, as there are multiple paralogues of flowering time genes in B. rapa. Brassica rapa contains four FLOWERING LOCUS C (FLC) genes, three of which are syntenic orthologues of AtFLC, while BrFLC5 is not. BrFLC1, BrFLC2, and BrFLC3 have been reported to be involved in flowering time regulation. However, BrFLC5 has thus far been deemed a pseudogene. We detected two alternative splicing patterns of BrFLC5 resulting from a nucleotide mutation (G/A) at the first nucleotide of intron 3 (named as Pi3+1(G/A)). Genotyping of BrFLC5Pi3 + 1(G/A) for 301 B. rapa accessions showed that this single nucleotide polymorphism was significantly related to flowering time variation (p < 0.001). In the collection, the frequency of the functional G allele (35.2%) was much lower than that of the nonfunctional A allele (59.1%); however, the frequency of the G allele was very high among the turnips (83.6%). An F2 population segregating at this locus was developed to analyze the genetic effect of BrFLC5. The result showed that the G allele individuals began to bolt two days later than the A allele individuals, indicating that BrFLC5 is a weak regulator of flowering time. BrFLC5 was expressed at the lowest level among the three analyzed BrFLCs. The late allele (G allele) was dominant to the early allele (A allele) at the BrFLC5 locus, which was in contrast to that of BrFLC1 and BrFLC2. This characteristic suggests that BrFLC5 would be more efficient for breeding premature bolting resistance in B. rapa.

Keyun Wei

Papers

Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea

A naturally occurring InDel variation in BraA.FLC.b (BrFLC2) associated with flowering time variation in Brassica rapa.

BrFLC5: a weak regulator of flowering time in Brassica rapa