Skip to main content
. 2016 Sep 6;5:e17956. doi: 10.7554/eLife.17956

Figure 2. Co-segregation of CYP734A50 with the style phenotype.

(A–C) PCR-genotyping of individuals with the indicated phenotypes of P. veris (A), P. vulgaris (B) and P. forbesii (C). ‘Long homo (UK)’ are naturally occurring long homostyles from England; ‘long homo mutant’ are long homostyles from the commercially grown population. ‘Long homo’ P. forbesii are long-homostylous putative recombinants found in our experimental population. Multiplex PCR amplified CYP734A50 exons (arrowheads) and ITS as an internal control. Enlarged arrowhead marks absence of exon 4 in the mutant. Asterisk in (B) marks a segregating L-morph individual. (D) Expression of CYP734A50 in dissected floral organs of naturally occurring long homostyles, S- and L-morphs of P. vulgaris, normalized to α-TUBULIN. Values are mean ± SD from three biological replicates. (E,F) PCR-genotyping of individuals with the indicated phenotypes from species in Primula sect. Primula (E) and Primula sect. Aleuritia (F). Long homostylous species are indicated in bold. Multiplex PCR was performed using primers to amplify exon 3 of CYP734A50 (arrowhead) and ITS as an internal control. Full gel images are shown in Figure 2—figure supplement 2. Specificity of PCR amplification for CYP734A50 is shown in Figure 2—figure supplement 3. Each set of plants was genotyped at least twice independently.

DOI: http://dx.doi.org/10.7554/eLife.17956.008

Figure 2—source data 1. Read counts from Illumina whole-genome sequencing of S- and L-morph plants of P. vulgaris and P. forbesii.
Reads were mapped to the exons of CYP734A50 and CYP734A51 including the 200 surrounding intronic nucleotides (± 100 bp). Absolute read counts are given. Due to the very similar target lengths for mapping against both genes, absolute read counts can be directly compared. All P. vulgaris samples are from the SRA project deposited under http://www.ncbi.nlm.nih.gov/bioproject/PRJEB9683.
DOI: http://dx.doi.org/10.7554/eLife.17956.009
elife-17956-fig2-data1.docx (11.9KB, docx)
DOI: 10.7554/eLife.17956.009

Figure 2.

Figure 2—figure supplement 1. Co-segregation of CYP734A50 with the style phenotype.

Figure 2—figure supplement 1.

PCR-genotyping of individuals with the indicated phenotypes of P. veris (left), P. vulgaris (middle) and P. forbesii (right). Multiplex PCR amplified CYP734A50 and ITS as an internal control. Arrowhead marks 300 bp band in DNA-size standard.
DOI: http://dx.doi.org/10.7554/eLife.17956.010

Figure 2—figure supplement 2. Loss of CYP734A50 from natural long homostylous species.

Figure 2—figure supplement 2.

PCR-genotyping of individuals with the indicated phenotypes from species in Primula sect. Primula (top) and Primula sect. Aleuritia (bottom). Long homostylous species are indicated in bold. Multiplex PCR was performed using primers to amplify exon 3 of CYP734A50 and ITS as an internal control.
DOI: http://dx.doi.org/10.7554/eLife.17956.011

Figure 2—figure supplement 3. Specificity of PCR-genotyping in different Primula species.

Figure 2—figure supplement 3.

Multiple-sequence alignments at the DNA (top) or protein (bottom) levels of the CYP734A50-specific fragments (see Figure 2E,F) amplified from the indicated species. PCR-products from two S-morph individuals were sequenced from each species. The corresponding region of CYP734A51 is included for comparison to show specificity of amplification of the CYP734A50 orthologues.
DOI: http://dx.doi.org/10.7554/eLife.17956.012

Figure 2—figure supplement 4. Naturally occurring long homostyles of P. vulgaris.

Figure 2—figure supplement 4.

Phenotypes of naturally occurring long homostyles, L- and S-morph plants of P. vulgaris collected near Wanstrow (UK). Flowers were pressed before photographing. Blue arrowheads indicate the position of the stigma, yellow arrowheads indicate the position of anthers. Scale bar is 5 mm.
DOI: http://dx.doi.org/10.7554/eLife.17956.013

Figure 2—figure supplement 5. Characterization of long-homostylous mutants of P. vulgaris and P. x pruhoniciana.

Figure 2—figure supplement 5.

(A) Phenotype of long homostyle mutant of P. x pruhoniciana. Blue arrowheads indicate the position of the stigma, yellow arrowheads indicate the position of anthers. Scale bar is 5 mm. (B) PCR-genotyping of S- and L-morph P. x pruhoniciana individuals, three long homostyle mutant P. x pruhoniciana plants and one long homostyle mutant P. vulgaris individual. Multiplex PCR was performed using primers to amplify the indicated CYP734A50 exons and ITS as an internal control. Arrowheads mark CYP734A50 specific bands; asterisks indicate exon-4 deletion in the mutants. Three different primer pairs were used to test for the presence of exon 4. Each genotyping PCR was performed twice independently. (C) Expression of full-length CYP734A50 transcript and of TUBULIN control in S- and L-morph plants, as well as the long homostyle exon-4 deletion mutant from the commercially grown P. vulgaris population. Fragment lengths of DNA-size standard are indicated. RT: reverse transcription.
DOI: http://dx.doi.org/10.7554/eLife.17956.014

Figure 2—figure supplement 6. Virus-induced gene silencing in P. forbesii.

Figure 2—figure supplement 6.

(A) Phenotypes of P. forbesii seedlings injected with empty TRV-vectors as negative control (left) and with PDS-containing TRV vectors (right) showing bleaching due to silencing of phytoene desaturase (PDS) expression. No phenotypic alteration is seen in the negative control seedlings compared to untreated plants. Scale bar is 1 cm. (B) Flower phenotypes from the indicated treatments. Mock indicates injection of the inflorescence stems with water. (C,D) Anther height (C) and style length (D) of plants from the indicated treatments. Numbers of flowers for stamen and style-length measurements are indicated. Values are mean ± SD. Means of indicated samples were compared using a two-sided t-test assuming unequal variance, followed by Bonferroni correction. ***p<0.001. (E) Expression of CYP734A50 in dissected styles of plants from the indicated treatments and phenotypes as determined by qRT-PCR, normalized to α-TUBULIN. Each sample (#1, #2, etc) is derived from pooling styles of one individual plant. Values are mean ± SD from three technical replicates per sample.
DOI: http://dx.doi.org/10.7554/eLife.17956.015