Volume 44, Issue 2, Pages 192-203.e5 (January 2018) Variation in Splicing Efficiency Underlies Morphological Evolution in Capsella  Ushio Fujikura, Runchun Jing, Atsushi Hanada, Yumiko Takebayashi, Hitoshi Sakakibara, Shinjiro Yamaguchi, Christian Kappel, Michael Lenhard  Developmental Cell  Volume 44, Issue 2, Pages 192-203.e5 (January 2018) DOI: 10.1016/j.devcel.2017.11.022 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2018 44, 192-203. e5DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Phenotypic Effect of the Chromosome 6 Petal-Size QTL (A–D) Petal size (A), petal-cell size (B), shoot dry weight (C), and mature-plant height (D) of NIL and qIL plants segregating for the chromosome 6 petal-size QTL. Values from plants homozygous for the C. grandiflora QTL allele (gg) are shown by gray bars here and throughout; those from plants homozygous for the C. rubella QTL allele (rr) are shown by white bars. The C. grandiflora allele causes a pleiotropic reduction in organ growth. Values are mean ± SD from 40 petals (4 petals/plant) (A and B) and ten plants (C and D). ∗p < 0.05, significantly different based on Student's t test. (E–H) Average leaf size (E) and representative leaf outline (F), stamen length (G), and sepal size (H) of qIL plants with the indicated genotypes. Values are mean ± SD from more than ten organs. ∗p < 0.05, significantly different based on Student's t test. (I–K) Representative petals and petal cells from the indicated genotypes. Scale bars, are 1 cm (F), 1 mm (I), and 50 μm (J; applies also to K). See also Figure S1. Developmental Cell 2018 44, 192-203.e5DOI: (10.1016/j.devcel.2017.11.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Identification of CYP724A1 as the Causal Gene (A–C) Representative petals (A), petal size (B), and leaf size (C) of transgenic A. thaliana plants expressing the C. grandiflora or C. rubella alleles of CYP724A1 (pCgCYP::CgCYP, pCrCYP::CrCYP) or the corresponding promoter swap constructs pCrCYP::CgCYP and pCgCYP::CrCYP. Schematic drawings of constructs are shown within the bars, with the promoter indicated by the arrow and the transcribed sequence by the box. Sequences derived from the C. grandiflora allele are shown by gray fill and those from the C. rubella allele by white fill. WT denotes non-transgenic wild-type (n > 10); bars with schematic constructs inside represent average value of eight independent transgenic lines per construct, with more than 12 plants per line. The C. grandiflora transcribed sequence increases organ growth irrespective of the promoter used. Values are mean ± SD. ∗p < 0.05, significantly different based on Student's t test. (D–F) Representative petals (D) and petal size (E and F) from qILgg and qILrr plants transformed with the C. rubella allele of CYP724A1 (D and E) or with the chimeric pCgCYP724A1::CrCYP724A1 construct (F). Constructs are indicated as in (B) and (C). The C. rubella transcribed sequence reduces petal growth in a qIL_gg background but not in qIL_rr background. Values are mean ± SD from 40 petals (4 petals/plant). ∗p < 0.05, significantly different based on Student's t test. (G–I) Representative petals (G), petal size (H), and petal-cell size (I) from qILrr plants with downregulated CYP724A1 expression in petals using the petal-specific APETALA3 (AP3) promoter. Downregulation of CYP724A1 expression increases petal size in a qIL_rr background. See Figure S3D for quantification of CYP724A1 expression. Values are mean ± SD from 40 petals (4 petals/plant) and from more than 400 cells from ten petals in (H) and (I). ∗p < 0.05, significantly different from non-transgenic qILrr based on Student's t test. Scale bars, 1 mm (A, D, and G). See also Figures S1–S3. Developmental Cell 2018 44, 192-203.e5DOI: (10.1016/j.devcel.2017.11.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Differential Splicing Efficiency of Alternative CYP724A1 Alleles (A) CYP724A1 mature mRNA levels quantified by qRT-PCR in inflorescences of the indicated genotypes relative to ACTIN2. Values are mean ± SD from three biological replicates. (B) Overall CYP724A1 transcript levels, spliced levels, and unspliced levels as determined by qRT-PCR in inflorescences of the indicated genotypes relative to ACTIN2. See Figure S4A for details of primer placement for the different forms. Values are mean ± SD from three biological replicates. (C) Differential splicing efficiency of CYP724A1 transcript from the C. rubella (left) and the C. grandiflora (right) alleles in the indicated NILs as determined by RT-PCR in inflorescences. Three biological replicates are shown. (D) Schematic representation of the constructs used for protoplast transformation and RT-PCR products for CYP724A1 using primers spanning intron 1 (left, “primer1”) and intron 6 (right, “primer2”). Exons are indicated by boxes, introns by the black line; the 35S promoter and nos terminator sequences are indicated. Sequences derived from the C. grandiflora allele are shown by gray fill in exons and those from the C. rubella allele by white fill in exons. (E and F) RT-PCR performed on RNA from transiently transformed protoplasts to detect splicing defect in intron 1 (E) or intron 6 (F). Protoplasts were transformed with the constructs shown to the left of the gel images in (D). Cr-Cg indicates the construct with the first exons from C. rubella and the later ones from C. grandiflora, and Cg-Cr indicates the complementary combination. PCR products of spliced and unspliced transcripts are indicated on the right. The origin of exon 1-intron 1-exon 2 determines the splicing efficiency not just of intron 1 (E) but also of intron 6 (F). (G and H) Splicing assay of CrCYP724A1 constructs carrying the C. grandiflora allele at the indicated SNP (left) or of CgCYP724A1 constructs carrying the C. rubella allele at the indicated SNP (right). Results from protoplasts expressing truncated CYP724A1 fused to CFP (G) or full-length CYP724A1 (H) are shown. (I) Splicing assay in stably transformed A. thaliana suspension cell cultures. Constructs are as in (G). Right-hand panels show cultures expressing constructs with both SNP2 and SNP4 exchanged as indicated, as well as ACT control to demonstrate the absence of DNA contamination. (J) RT-PCR products for CYP724A1 from eight C. grandiflora accessions from different geographical regions assayed using primers spanning intron 1. Weak lower band (arrowhead) represents the spliced product. (K) Petal sizes of transgenic Arabidopsis plants expressing the indicated Capsella CYP724A1 genomic coding sequences under the control of the 35S promoter. Constructs are shown schematically within bars, with the arrow indicating 35S promoter and the box the transcribed sequence; gray is C. grandiflora-derived sequence, white is C. rubella-derived sequence. “Control” is non-transgenic Columbia-0 wild-type. Values are mean ± SD of 80 petals of 10 plants from 4 to 8 independent transgenic lines per construct and the same for controls. Asterisk indicates significant difference from the control as determined by ANOVA followed by Tukey’s HSD test (∗p < 0.05). See also Figures S4 and S5. Developmental Cell 2018 44, 192-203.e5DOI: (10.1016/j.devcel.2017.11.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Effect of Exogenous Brassinosteroid Treatment on Petal Growth (A and B) Quantification of castasterone (CS) and brassinolide (BL) levels in flowers of the alternative NIL (A) and qIL genotypes (B). Values are mean ± SD from three biological replicates. ∗p < 0.05, significantly different based on Student’s t test. (C) Petal size of qILgg and qILrr plants treated with epi-brassinolide (EBL) (100 nM, twice a day). Values are mean ± SD from 40 petals (4 petals/plant). ∗p < 0.05, significantly different based on Student's t test. (D) Petal-cell sizes in qILgg plants after EBL treatment. Values are mean ± SD from more than ten petals. (E) Representative petals (top) and petal cells (bottom) from plants with the indicated genotypes treated with H2O or EBL. Scale bars, 1 mm (top) and 50 μm (bottom). (F and G) Petal sizes of A. thaliana wild-type (F) and Capsella qIL_gg plants (G) treated with the indicated concentrations of epi-brassinolide (EBL) once a day after the start of flowering. Values are mean ± SD from more than 48 petals (4 petals/plant). ∗p < 0.05, significantly different from mock control based on ANOVA followed by Tukey's HSD test. See also Figure S3. Developmental Cell 2018 44, 192-203.e5DOI: (10.1016/j.devcel.2017.11.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Population-Genetic Analysis of CYP724A1 Locus in Capsella (A) Putative recent C. rubella CYP724A1 introgression in the C. grandiflora population was estimated by counting the presence of C. rubella reference like k-mers of different lengths (in bp) in genome resequencing raw data of a C. grandiflora population (CgPop) and in a species-wide sample of C. rubella (CrDK) around the CYP724A1 locus (transcribed sequence ±1,000 bp). Each line represents an individual. The C. grandiflora population contains 13 samples (shown in red; CgPop_ig) with a signal of recent introgression of the CYP724A1 locus from C. rubella, i.e., a high proportion of long C. rubella reference k-mers. (B) Density distribution of samples with the given presence of C. rubella reference 63-mers. Colors are as in (A). (C) Haplotype clustering and genotype at polymorphisms in the 5′ region of CYP724A1 of all the resequenced individuals. Analysis was restricted to all SNPs polymorphic between the two alleles segregating in our RIL population. The red squares indicate C. grandiflora individuals with a signal of more recent introgression from C. rubella (CgPop_ig) as identified in (A) and (B). This dataset includes sequences from all C. grandiflora resequenced individuals (CgPop), from the parental NILgg (Cg926) and NILrr (Cr1504) alleles, and from all publicly available C. rubella genomes (CrDK). Arrowhead indicates individual Cg152P. See also Figure S5. Developmental Cell 2018 44, 192-203.e5DOI: (10.1016/j.devcel.2017.11.022) Copyright © 2017 Elsevier Inc. Terms and Conditions