Dataset Information

Refining bulk segregant analyses: ontology-mediated discovery of flowering time genes in Brassica oleracea.

ABSTRACT:

Background

Bulk segregant analysis (BSA) can help identify quantitative trait loci (QTLs), but this may result in substantial bycatch of functionally irrelevant genes.

Results

Here we develop a Gene Ontology-mediated approach to zoom in on specific genes located inside QTLs identified by BSA as implicated in a continuous trait. We apply this to a novel experimental system: flowering time in the giant woody Jersey kale, which we phenotyped in four bulks of flowering onset. Our inferred QTLs yielded tens of thousands of candidate genes. We reduced this by two orders of magnitude by focusing on genes annotated with terms contained within relevant subgraphs of the Gene Ontology. A pathway enrichment test then led to the circadian rhythm pathway. The genes that enriched this pathway are attested from previous research as regulating flowering time. Within that pathway, the genes CCA1, FT, and TSF were identified as having functionally significant variation compared to Arabidopsis. We validated and confirmed our ontology-mediated results through genome sequencing and homology-based SNP analysis. However, our ontology-mediated approach produced additional genes of putative importance, showing that the approach aids in exploration and discovery.

Conclusions

Our method is potentially applicable to the study of other complex traits and we therefore make our workflows available as open-source code and a reusable Docker container.

SUBMITTER: Vos RA

PROVIDER: S-EPMC9252076 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPlants adopt different reproductive strategies as an adaptation to growth in a range of climates. In Arabidopsis thaliana FRIGIDA (FRI) confers a vernalization requirement and thus winter annual habit by increasing the expression of the MADS box transcriptional repressor FLOWERING LOCUS C (FLC). Variation at FRI plays a major role in A. thaliana life history strategy, as independent loss-of-function alleles that result in a rapid-cycling habit in different accessions, appear to have evolved many times. The aim of this study was to identify and characterize orthologues of FRI in Brassica oleracea.ResultsWe describe the characterization of FRI from Brassica oleracea and identify the two B. oleracea FRI orthologues (BolC.FRI.a and BolC.FRI.b). These show extensive amino acid conservation in the central and C-terminal regions to FRI from other Brassicaceae, including A. thaliana, but have a diverged N-terminus. The genes map to two of the three regions of B. oleracea chromosomes syntenic to part of A. thaliana chromosome 5 suggesting that one of the FRI copies has been lost since the ancient triplication event that formed the B. oleracea genome. This genomic position is not syntenic with FRI in A. thaliana and comparative analysis revealed a recombination event within the A. thaliana FRI promoter. This relocated A. thaliana FRI to chromosome 4, very close to the nucleolar organizer region, leaving a fragment of FRI in the syntenic location on A. thaliana chromosome 5. Our data show this rearrangement occurred after the divergence from A. lyrata. We explored the allelic variation at BolC.FRI.a within cultivated B. oleracea germplasm and identified two major alleles, which appear equally functional both to each other and A. thaliana FRI, when expressed as fusions in A. thaliana.ConclusionsWe identify the two Brassica oleracea FRI genes, one of which we show through A. thaliana complementation experiments is functional, and show their genomic location is not syntenic with A. thaliana FRI due to an ancient recombination event. This has complicated previous association analyses of FRI with variation in life history strategy in the Brassica genus.