Formation and diversification of a paradigm biosynthetic gene cluster in plants

Numerous examples of biosynthetic gene clusters (BGCs), including for compounds of agricultural and medicinal importance, have now been discovered in plant genomes. However, little is known about how these complex traits are assembled and diversified. Here, we examine a large number of variants within and between species for a paradigm BGC (the thalianol cluster), which has evolved recently in a common ancestor of the Arabidopsis genus. Comparisons at the species level reveal differences in BGC organization and involvement of auxiliary genes, resulting in production of species-specific triterpenes. Within species, the thalianol cluster is primarily fixed, showing a low frequency of deleterious haplotypes. We further identify chromosomal inversion as a molecular mechanism that may shuffle more distant genes into the cluster, so enabling cluster compaction. Antagonistic natural selection pressures are likely involved in shaping the occurrence and maintenance of this BGC. Our work sheds light on the birth, life and death of complex genetic and metabolic traits in plants.

Data supporting the findings of this work are available within the paper and its Supplementary Information files. A reporting summary for this Article is available as a Supplementary Information file. The datasets and plant materials generated and analyzed during the current study are available from the corresponding author upon request. The source data underlying Figure 2b as well as Supplementary Figure 3b are provided as a Source Data file. Source data are provided with this paper.
There are nine OSC-centric genomic neighborhoods (GNs) in Arabidopsis thaliana, four of which contain BGCs and the rest do not. We used two-tailed Student's t test to compare the non-random associated biallelic sites between clustered and non-clustered GNs.
No data were excluded.
For LC-MS analysis, shown are representatives out of three biological replicates. All replicates showed similar results.

Selection of individual plants for LC-MS analysis was random.
Yes, all relevant data were treated blindly before analysis.
Briefly describe the study type including whether data are quantitative, qualitative, or mixed-methods (e.g. qualitative cross-sectional, quantitative experimental, mixed-methods case study).
State the research sample (e.g. Harvard university undergraduates, villagers in rural India) and provide relevant demographic information (e.g. age, sex) and indicate whether the sample is representative. Provide a rationale for the study sample chosen. For studies involving existing datasets, please describe the dataset and source.
Describe the sampling procedure (e.g. random, snowball, stratified, convenience). Describe the statistical methods that were used to predetermine sample size OR if no sample-size calculation was performed, describe how sample sizes were chosen and provide a rationale for why these sample sizes are sufficient. For qualitative data, please indicate whether data saturation was considered, and what criteria were used to decide that no further sampling was needed.
Provide details about the data collection procedure, including the instruments or devices used to record the data (e.g. pen and paper, computer, eye tracker, video or audio equipment) whether anyone was present besides the participant(s) and the researcher, and whether the researcher was blind to experimental condition and/or the study hypothesis during data collection.

nature research | reporting summary
April 2020 Ecological, evolutionary & environmental sciences study design All studies must disclose on these points even when the disclosure is negative. Note the sampling procedure. Describe the statistical methods that were used to predetermine sample size OR if no sample-size calculation was performed, describe how sample sizes were chosen and provide a rationale for why these sample sizes are sufficient.
Describe the data collection procedure, including who recorded the data and how.
Indicate the start and stop dates of data collection, noting the frequency and periodicity of sampling and providing a rationale for these choices. If there is a gap between collection periods, state the dates for each sample cohort. Specify the spatial scale from which the data are taken If no data were excluded from the analyses, state so OR if data were excluded, describe the exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.
Describe the measures taken to verify the reproducibility of experimental findings. For each experiment, note whether any attempts to repeat the experiment failed OR state that all attempts to repeat the experiment were successful.
Describe how samples/organisms/participants were allocated into groups. If allocation was not random, describe how covariates were controlled. If this is not relevant to your study, explain why.
Describe the extent of blinding used during data acquisition and analysis. If blinding was not possible, describe why OR explain why blinding was not relevant to your study.
Describe the study conditions for field work, providing relevant parameters (e.g. temperature, rainfall).
State the location of the sampling or experiment, providing relevant parameters (e.g. latitude and longitude, elevation, water depth).
Describe the efforts you have made to access habitats and to collect and import/export your samples in a responsible manner and in compliance with local, national and international laws, noting any permits that were obtained (give the name of the issuing authority, the date of issue, and any identifying information).
Describe any disturbance caused by the study and how it was minimized. Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Wild animals

Field-collected samples
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Not applicable
Describe the validation of each primary antibody for the species and application, noting any validation statements on the manufacturer's website, relevant citations, antibody profiles in online databases, or data provided in the manuscript.

Not applicable
Describe the authentication procedures for each cell line used OR declare that none of the cell lines used were authenticated.
Confirm that all cell lines tested negative for mycoplasma contamination OR describe the results of the testing for mycoplasma contamination OR declare that the cell lines were not tested for mycoplasma contamination.
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.

Not applicable
Indicate where the specimens have been deposited to permit free access by other researchers.
If new dates are provided, describe how they were obtained (e.g. collection, storage, sample pretreatment and measurement), where they were obtained (i.e. lab name), the calibration program and the protocol for quality assurance OR state that no new dates are provided.
Identify the organization(s) that approved or provided guidance on the study protocol, OR state that no ethical approval or guidance was required and explain why not.

Not applicable
Provide details on animals observed in or captured in the field; report species, sex and age where possible. Describe how animals were caught and transported and what happened to captive animals after the study (if killed, explain why and describe method; if released, say where and when) OR state that the study did not involve wild animals.
For laboratory work with field-collected samples, describe all relevant parameters such as housing, maintenance, temperature, photoperiod and end-of-experiment protocol OR state that the study did not involve samples collected from the field.
Identify the organization(s) that approved or provided guidance on the study protocol, OR state that no ethical approval or guidance was required and explain why not.

Not applicable
Describe how participants were recruited. Outline any potential self-selection bias or other biases that may be present and how these are likely to impact results.
Identify the organization(s) that approved the study protocol. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots. Describe the antibodies used for the ChIP-seq experiments; as applicable, provide supplier name, catalog number, clone name, and lot number.
Specify the command line program and parameters used for read mapping and peak calling, including the ChIP, control and index files used.
Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.
Describe the software used to collect and analyze the ChIP-seq data. For custom code that has been deposited into a community repository, provide accession details.

Not applicable
Identify the instrument used for data collection, specifying make and model number.
Describe the software used to collect and analyze the flow cytometry data. For custom code that has been deposited into a community repository, provide accession details.
Describe the abundance of the relevant cell populations within post-sort fractions, providing details on the purity of the samples and how it was determined.
Describe the gating strategy used for all relevant experiments, specifying the preliminary FSC/SSC gates of the starting cell population, indicating where boundaries between "positive" and "negative" staining cell populations are defined.

Not applicable
Specify the number of blocks, trials or experimental units per session and/or subject, and specify the length of each trial or block (if trials are blocked) and interval between trials.
State number and/or type of variables recorded (e.g. correct button press, response time) and what statistics were used to establish that the subjects were performing the task as expected (e.g. mean, range, and/or standard deviation across subjects).

Specify in Tesla
Specify the pulse sequence type (gradient echo, spin echo, etc.), imaging type (EPI, spiral, etc.), field of view, matrix size, slice thickness, orientation and TE/TR/flip angle.
State whether a whole brain scan was used OR define the area of acquisition, describing how the region was determined.