Common evolutionary trajectory of short life-cycle in Brassicaceae ruderal weeds

Abstract

Weed species are detrimental to crop yield. An understanding of how weeds originate and adapt to field environments is needed for successful crop management and reduction of herbicide use. Although early flowering is one of the weed trait syndromes that enable ruderal weeds to overcome frequent disturbances, the underlying genetic basis is poorly understood. Here, we establish Cardamine occulta as a model to study weed ruderality. By genome assembly and QTL mapping, we identify impairment of the vernalization response regulator gene FLC and a subsequent dominant mutation in the blue-light receptor gene CRY2 as genetic drivers for the establishment of short life cycle in ruderal weeds. Population genomics study further suggests that the mutations in these two genes enable individuals to overcome human disturbances through early deposition of seeds into the soil seed bank and quickly dominate local populations, thereby facilitating their spread in East China. Notably, functionally equivalent dominant mutations in CRY2 are shared by another weed species, Rorippa palustris, suggesting a common evolutionary trajectory of early flowering in ruderal weeds in Brassicaceae.

Fig. 1. The population structure of C. occulta in East Asia.

a Representative C. occulta plants growing in forests, paddy fields, lawns, and flower beds. The white arrows point to wild C. occulta plants. b Distribution of the C. occulta accessions used in this study. The area of each pi represents the sample size in each site, and the colors indicate C. occulta subgroups. The filled circles and the open circles represent the high-disturbance and low-disturbance habitats, respectively. c A neighbor-joining tree of 82 C. occulta accessions constructed using whole-genome SNP data. Branch colors denote subgroups. Three representative accessions, Yunnan, HANGYY8055, and Pudong, are marked with 1, 2, and 3, respectively. The colors of the dots to the right of the tree indicate vernalization requirement (Ver), haplotypes of FLC and CRY2, and degree of habitat disturbance of each accession. Short black lines, data not available. The ADMIXTURE results at k = 3, which had the lowest cross-validation error, are shown on the right. Subgroups (pop1, pop2, and pop3) are indicated by the colored bars. d PCA of 82 C. occulta accessions. The proportion of the variance explained is 51.73% for PC1 and 13.87% for PC2. e Population history inferred by SMC++. Per-generation mutation rate was assumed to be 7.1 × 10⁻⁹. The dashed vertical line indicates the time of divergence (nearly 1000 generations ago). f Nucleotide diversity (θπ per 50 kb) of pop2 (n = 15 accessions) and pop3 (n = 35 accessions). Nucleotide diversity (π) values were calculated using the whole-genome SNP data with a window size of 50 kb and a step size of 20 kb. The black lines in the box represent the median values and the lower and upper hinges correspond to the first and third quartiles. The upper and lower whiskers extend from the hinge to the values > or <1.5 × interquartile range from the hinge. g The frequency spectrum of C. occulta populations. The site frequency spectrum was estimated for 30, 15, and 30 individuals from pop1, pop2, and pop3, respectively, using the whole-genome SNP data.

Fig. 2. Adaptation of pop3 to high-disturbance environments.

a The proportion of plants from three populations in low-disturbance areas (i.e., forests and mountains) and high-disturbance areas (i.e., paddy fields, flower beds, and roadsides). See also Supplementary Data 1. b F_ST values of pop2 and pop3 accessions across the whole genome. The gray dashed line represents the top 5% threshold. c GO term analyses of the genes within the top 5% F_ST regions. The top eight enriched GO biological processes are shown. The biological processes shaded in green are associated with the transition from vegetative-to reproductive growth. See also Supplementary Data 4 and Supplementary Figs. 2a, b. d Flowering time of C. occulta accessions. The SD/LD ratio was calculated by dividing the median number of total leaves when plants started to bolt in SD by the median number of total leaves when plants started to bolt in LD. Plants were grown in a growth chamber. The accessions are grouped by subgroups, and each dot represents an accession. See also Supplementary Fig. 2c. The source data underlying Fig. 2d are provided as a Source Data file.

Fig. 3. Identification of FLC as the gene responsible for the loss of the vernalization requirement in pop3.

a, b Flowering phenotypes (a) and flowering time (b) of the Pudong and HANGYY8055 accessions and Pudong × HANGYY8055 F₁ plants grown in LD in a growth chamber. +/−V, with or without vernalization treatment. Scale bar in (a) 5 cm. c Flowering time of the Pudong and HANGYY8055 accessions and plants in the Pudong × HANGYY8055 F₂ segregating population in LD. d Genome-wide Δ(SNP index) plot from BSA of the F₂ segregating population derived from a cross between Pudong and HANGYY8055. The black lines indicate tricube-smoothed Δ(SNP index), and gray lines represent the corresponding two-sided 99% confidence intervals. e The Δ(SNP index) plot of Chr 6D from BSA of the F₂ segregating population derived from a cross between Pudong and HANGYY8055. The black line indicates tricube-smoothed Δ(SNP index), and gray lines mark the corresponding two-sided 99% confidence intervals. The blue dotted line indicates the location of C. occulta FLC (CoFLC) on Chr 6D. f The gene structure of CoFLC and the location of the mutation (blue line). Black boxes, gray boxes, and black lines represent exons, untranslated regions (UTRs), and introns, respectively. g Analysis of the transcript levels of CoFLC genes on Chr 6D in the Pudong and HANGYY8055 accessions by RNA-seq. Normalized counts and adjusted P values were both analyzed by DESeq2. The P values attained by the two-sided Wald test were corrected for multiple testing using the Benjamini and Hochberg methods. Data are mean ± s.e. of three biological replicates (open circle). ns, not significant. h Flowering time of Arabidopsis FRI^SF2 FLC, FRI^SF2 flc, and T₁ transgenic lines in LD. We generated an allele of Arabidopsis FLC (FLC^L160*) that mimics the mutation in FLC allele of pop3. Plants were grown in a growth chamber. Letters indicate significant differences as determined by ordinary one-way ANOVA. Error bars in b denote s.d. The number of examined plants (n) is given. The centers of the error bars represent the mean values. The source data underlying Fig. 3b, c, and h are provided as a Source Data file.

Fig. 4. Identification of CRY2 as the gene responsible for early-flowering in short days.

a, b Flowering phenotypes (a) and flowering time (b) of the Yunnan and Pudong accessions and Yunnan × Pudong F₁ plants grown under LD or SD conditions in a growth chamber. Error bars denote s.d. The number of examined plants (n) is given. The centers of the error bars represent the mean values. c Genome-wide Δ(SNP index) plot from BSA of the F₂ segregating population derived from a cross between Pudong and Yunnan. The black lines indicate tricube-smoothed Δ(SNP index), and the gray lines indicate corresponding two-sided 99% confidence intervals. d Δ(SNP index) plot of Chr 1 A from BSA of the F₂ segregating population derived from a cross between Pudong and Yunnan. The black line indicates tricube-smoothed Δ(SNP index), and gray lines mark the corresponding two-sided 99% confidence intervals. The orange dotted line indicates the location of C. occulta CRY2 (CoCRY2). e Gene structure of CoCRY2. Black boxes, gray boxes, and black lines represent exons, untranslated regions, and introns, respectively. The mutation site (W374M) is labeled with an orange line. f Flowering time of the Yunnan and Pudong accessions and transgenic lines in LD (upper panels) or SD (lower panels) in a growth chamber. One representative plant for each genotype is shown. The flowering time is given as mean ±s.d. The number of examined plants (n) is given in Supplementary Fig. 3g. Scale bar in (a, f), 5 cm. The source data underlying Fig. 4b, f are provided as Source Data file.

Fig. 5. CRY2^W374M contributes to the adaptation of pop3 to high-disturbance environments.

a Dominant mutations in CRY2 contribute to early-flowering in SD. The SD/LD ratio was calculated by dividing the median number of total leaves when plants started to bolt in SD by the median number of total leaves when plants started to bolt in LD. The 79 C. occulta accessions are grouped by CRY2 haplotypes. Each dot represents an accession. b The proportion of the C. occulta accessions with dominant mutations in CRY2 in high-disturbance and low-disturbance areas. The number of accessions for each CoCRY2 haplotype is given at the top. c Genome-wide F_ST values in pop2 and pop3 across the 1.5 Mb regions spanning CoCRY2 on chromosome 1 A. The gray dashed line represents the top 5% threshold. The orange dashed line marks the position of CoCRY2. The source data underlying Fig. 5a are provided as a Source Data file.

Fig. 6. Dominant mutations in CRY2 might serve as a common genetic basis for ruderality in Brassicaceae.

a Flowering time of R. palustris accessions. Plants were grown under LD, SD, or vernalization conditions in a growth chamber. The total number of leaves when plants started to bolt was counted. Asterisks indicate plants that did not flower within 1 year. The number of examined plants (n) is given. Error bars denote s.d. The centers of the error bars represent the mean values. b Flowering time of wild-type (Col-0), cry2, and T₁ transgenic Arabidopsis lines. Plants were grown in a growth chamber under SD conditions. The different versions of CRY2 (CRY2^W374M, CRY2^W367M, CRY2^V360M, CRY2^S401F, and CRY2^D393G) were expressed from the Arabidopsis CRY2 promoter. The CRY2^W374M, CRY2^V367M, and CRY2^V360M haplotypes were identified in C. occulta accessions (Fig. 1c), whereas the CRY2^S401F and CRY2^D393G haplotypes were found in R. palustris accessions (a). Letters indicate significant differences as determined by ordinary one-way ANOVA. c Y2H assays showing the interactions between different versions of CRY2 and CIB1. Transformed yeast cells were grown on SD/-Leu/-Trp/-His plates supplemented with 5–25 mM 3-amino-1,2,4-triazole (3-AT) under dark or light conditions. AD, GAL4 activation domain; BD, GAL4 DNA binding domain. d A close-up view of the CRY2 structure (PBD ID 6M79). The FAD molecule and mutated residues are displayed as sticks and colored in yellow and violet, respectively. Helices α15–α17 with mutated residues are colored in light blue. e Proposed model for the evolution of weed ruderality in Brassicaceae. Three genotypes (FLC CRY2, flc CRY2, and flc CRY2^W374M) are shown. The source data underlying Fig. 6a, b are provided as a Source Data file.