1
Experimental Procedures:
Accession numbers. Short read sequence data from this article can be found in the NCBI
Sequence Read Archive under accession number SRR882054.
Plant material and cultivation. C. hirsuta of the reference Ox accession (specimen
voucher Hay 1 (OXF) (Hay and Tsiantis 2006) and A. thaliana Col-0 were used unless
otherwise stated. The DR5::VENUS line was previously described (Barkoulas et al.
2008). Plants were grown in long day conditions in a greenhouse (18h light at 22°C, 6h
dark at 20°C) or in a controlled environment room (16h light at 22°C, 8h dark at 20°C)
unless otherwise stated. Dry seeds were either sown on well-watered soil (2:1
peat:vermiculite) in 7x7 cm pots, or sterilized by ethanol washes and sown on plates
containing Murashige and Skoog (MS) solid agar medium, covered, and placed at 4°C to
stratify for 2-10 days before transfer to culture conditions.
Plant transformation efficiency. The percentage of A. thaliana Col-0 and C. hirsuta Ox
seedlings that were hygromycin resistant was determined following transformation by
floral dip with Agrobacterium tumefaciens (strain GV3101) containing the Gateway
vector pMDC32 with a uidA gene insert.
Microscopy. Scanning electron microscopy was performed as previously described (Hay
and Tsiantis 2006) and an Olympus BX50 was used for light microscopy. Confocal laser
scanning microscopy (CLSM) was performed with a Zeiss 510 Meta microscope.
BAC libraries. A bacterial artificial chromosome (BAC) library was constructed by
Southern Illinois University, Carbondale, IL 62901-6899, USA, with large genomic DNA
inserts of C. hirsuta Ox in the vector pIndigoBAC5 (Hind III). A second BAC library
was constructed by the Arizona Genomics Institute, University of Arizona, Tucson, AZ
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2
85721, USA, in the vector pAGIBAC1, which is a modification of pIndigoBAC5 by the
addition of a SwaI site, according to the following protocol (Grotewold 2003). A full
description of library construction will be given elsewhere.
Recombinant inbred line (RIL) population. To initiate a C. hirsuta RIL population, the
Wa accession from Clark County, WA, USA (gift from K. Marhold) was selfed twice and
then crossed to the Ox accession, which had been selfed eight times, with Ox as the
maternal parent. A single F1 progeny was selected and allowed to self-fertilize to
generate an F2 population. A total of 195 F2 progeny were propagated through selfing
and single-seed descent to generate the F8 RIL population. DNA was extracted from
single individuals of all 195 lines when the majority had reached F8 (8 lines were F7
generation). Results of genotyping the F8 showed that 8 lines had unexpectedly high
levels of heterozygosity and were permanently discarded to prevent contamination of the
population due to inadvertent cross-pollination. To maintain the allele frequency present
in this F8 population of 187 RILs, seed from 6 progeny plants was bulked for future use
(for 66 lines, less than 6 plants were bulked).
Molecular marker design and genotyping. To identify nucleotide polymorphisms for
use as molecular markers, primers were designed based on Ox BAC-end sequences to
sequence the respective regions in Wa (BigDye® Terminator v3.1). In addition, EST
sequence libraries of both accessions were screened for single nucleotide polymorphisms
(SNPs). Sequenom assays were designed from selected SNPs and used to genotype each
individual from the Ox x Wa RIL population (Welcome Trust Center for Human
Genetics, High Throughput Genomics, Oxford, UK). The RILs were genotyped for 46
microsatellite markers by amplifying the locus with a fluorescently labelled primer pair
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3
and subsequent analysis by ABI3100 capillary electrophoresis and for an additional set of
SNPs by pyrosequencing (TraitGenetics GmbH, Gatersleben, Germany). Four PCR based
dCAPS markers were added to the genetic map using these primer pairs and restriction
enzymes: marker STM_322_1: primers 5'-TTGTTCCTTTTGGCTAGTG-3' and 5'CAAAGATCATGGCTCATCC-3', enzyme
Hpy188I;
marker
AP1:
primers 5'-
TCCCTAAAACCGCTCTTAGC-3' and 5'-AGAGAGATAAAGAAGAGTTCAGGC-3',
enzyme AluI; marker TCP4: primers 5'-TGAGCTTCCTCCTTGGAATC-3' and 5'ACCGAACTGAAGCTGGTGTTGCAG-3', enzyme AluI; marker PSL: primers 5'ATCTTCACGTTGGAGAAGCAGGG-3' and 5'-TTCGATCTTGCAGAACAACTGTA3', enzyme RsaI.
Genetic map. The genetic map was produced with Joinmap® 4.0 (Van Ooijen 2006),
using allelic information from all F8 RILs.
Transmission distortion. 163 F8 seeds from RIL46 were sown and 146 seeds
germinated. The number of seeds that failed to germinate did not fit the expectation of
25% for zygotic lethality (Χ2; p<0.001). These plants were genotyped with markers 705_
and 475_3 located at 64.2 and 66.6 cM in a distorted region on chromosome 4 (SSLP
marker
705_:
primers
5'-GGTTTGTTGATATTGATGGG-3'
TGCAGTATAATTGCCTCCTT-3';
dCAPS
marker
475_3:
and
5'-
primers
5'-
CACAGAATCGGTACACAAAGGA-3' and 5'-CGTGTGAACTTAGACTGCGATG-3',
enzyme apoI).
Chromosome preparation and probes for comparative chromosome painting (CPP)
and BAC fluorescence in situ hybridization (FISH). C. hirsuta whole inflorescences
were fixed in freshly prepared ethanol : acetic acid (3:1) overnight and stored in 70%
3
4
ethanol at –20°C until used. Microscopic preparations with meiotic (pachytene) and
mitotic chromosomes were obtained from young anthers as previously described (Lysak
and Mandakova 2013, Mandakova and Lysak 2008). Suitable slides were post-fixed in
4% formaldehyde in deionized water for 10 min, air-dried and stored at 4°C until used. A.
thaliana BAC clones were obtained from the Arabidopsis Biological Resource Center
(ABRC, Columbus, OH) and used as probes for CCP in C. hirsuta. Chromosome-specific
BAC contigs were arranged to represent 24 ancestral genomic blocks (A to X) of the
putative Ancestral Crucifer Karyotype (Schranz et al. 2006). On average, every third
BAC clone was used for each contig from the full list of BAC clones previously
described (Mandakova and Lysak 2008). For BAC FISH in C. hirsuta, 40 C. hirsuta
BAC clones corresponding to seven genomic blocks (O, P, Q, R, V, W, and X) of CH6
and CH8 were used (Table 1 below). DNA of individual BAC clones was isolated using a
standard alkaline extraction omitting the phenol:chloroform purification step. BAC DNA
was labeled by biotin-, digoxigenin-, and Cy3- dUTP via nick translation as previously
described (Lysak and Mandakova 2013, Mandakova and Lysak 2008).
CCP and BAC FISH. Selected slides were treated by RNase (AppliChem; 100µg/mL in
water) at 37°C for 1 h, and washed in 2  SSC for 2–5 min. To remove cytoplasm, the
slides were treated with pepsin (Sigma-Aldrich; 0.1 mg/mL) in 0.01M HCl at 37°C for 10
min, followed by a wash in 2  SSC for 2–5 min. Subsequently, the slides were postfixed in 4% formaldehyde in 2  SSC for 10 min, washed in 2  SSC (2–5 min), and
dehydrated in an ethanol series (70, 80, and 96%). Labeled BAC DNAs were pooled and
ethanol precipitated to reduce the probe volume; 500 ng of labeled DNA per BAC clone
were used. For one slide, the probe was dissolved in 20 µL of hybridization mix (50%
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5
formamide, 10% dextran sulfate in 2  SSC) at 37°C overnight. The probe and
chromosomes were denatured together on a hot plate at 80°C for 2 min and incubated in a
moist chamber at 37°C for 48 h. Post-hybridization washing was performed in 20%
formamide in 2  SSC for 3  5 min at 42°C. Detection of labeled DNA was done as
previously described (Lysak and Mandakova 2013). Chromosome preparations were
counterstained with DAPI (2 µg/mL) in Vectashield (Vector Laboratories), observed and
photographed using an Olympus BX-61 epifluorescence microscope. Monochromatic
images were acquired separately for all fluorochromes using appropriate excitation and
emission filters (AHF Analysentechnik) using an AxioCam CCD camera (Zeiss), and
pseudo-coloured and merged using Adobe Photoshop CS2 software (Adobe Systems).
Pachytene chromosomes in Figure 3 were straightened using the “straighten-curvedobjects” plugin in the Image J software (Kocsis et al., 1991).
Physiology. For auxin sensitivity experiments, seedlings were grown on vertical MS
plates supplemented with indole-3-acetic acid (IAA, Sigma) at the concentrations
indicated in Figure 5. Alternatively, seedlings were transferred to IAA-containing media
at 4 DAG and (1) stained with propidium iodide (25 μg/ml) after 40 minutes and
analysed for DR5::VENUS expression by CLSM, or (2) root meristem size measured
after 9-and 24-hr. Root meristem size is expressed as the number of cortex cells in a file
extending from the quiescent center to the first elongated cortex cell, as previously
described (Dello Ioio et al. 2008). To assay hypocotyl elongation response to simulated
shade, A. thaliana and C. hirsuta seedlings were germinated and grown on horizontal MS
plates for 3 days under white light (W) and then either kept in W or transferred to far-red
supplemented lighting for 4 more days. At day 7, hypocotyls were measured from at least
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6
30 seedlings for each treatment and species. To assay hypocotyl elongation response to
gibberellic acid (GA3, Sigma), seedlings were germinated and grown in W for 10 days on
horizontal MS plates supplemented with GA3 at the concentrations indicated in Figure 5.
At least 10 seedlings were measured for each treatment and species.
Morphology and Histology. Petal shape was determined from measurements of petals at
floral stages 13-15 viewed under light microscopy. Seed area was measured by fitting an
ellipse to the contour of photographed seeds using the Image-J macro "DrawEllipse"
(National Institutes of Health) and weight was determined from batches of counted seeds.
Roots of C. hirsuta and A. thaliana seedlings 5 days after germination (DAG) were (1)
fixed, embedded in paraffin, and 8 μm sections through the differentiation zone stained
with toluidine blue-0 as previously described (Grigg et al. 2005), (2) stained with a
modified pseudo-Schiff propidium iodide method as previously described (Rast and
Simon 2012) and root meristems viewed with a 40x lens by CLSM. A scoring diagram
for shoot branching is described in Figure S7.
QTL analysis. QTL analysis of stamen number variation was performed in the Ox x Wa
RIL population. Three progeny plants of the genotyped lines and 15 replicates of each
founder accession were grown in the greenhouse. Flowers were removed from the plant
when they opened and the number of stamens counted with the assistance of a dissecting
microscope. At least 24 flowers were sampled per RIL with the exception of 3 RILs for
which fewer flowers were scored. Average stamen number was calculated per plant and
used to determine the broad sense heritability (H2) of the trait. QTL analysis was
performed on the mean average stamen number per RIL, including the 8 F7 lines, using
Genstat 15th edition (Payne 2010). Genetic predictors were calculated with no more than
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7
2cM distance between them. A genome-wide significance threshold for the QTL scan
was determined to be 3.39 –log10(P) for α=0.05, using the method for Meff described in
(Li and Ji 2005). The QTL mapping methodology involved fitting a mixed effects model
in which the QTL were fixed effects and the genotypes were random effects. A simple
interval mapping genome scan was followed by several rounds of composite interval
mapping during which co-factors were added or removed until no further improvement
could be made. A final QTL model was fitted that included all detected QTL to determine
the allelic effects and the total phenotypic variance explained. The variance explained per
QTL was estimated by sequentially dropping each QTL from the model. The difference
in variance explained by the reduced model versus the full model was attributed to the
respective QTL. Epistatic interactions between the detected QTL were analysed by
backwards selection from a model with all additive QTL and all possible pairwise
combinations between them. At each round of selection the least significant interaction
was removed until only significant interactions (α=0.05) remained. A final epistatic QTL
model was fitted that contained all QTL as additive terms plus the significant interactions
between them to estimate the variance explained.
High-throughput sequence analysis. RNA was isolated from C. hirsuta shoots of Ox
and Wa accessions following floral induction. The Ox and Wa samples were used for
sequencing on the Illumina HiSeq 2000 platform (WTCHG Oxford Genomics Centre).
RNAseq reads from Ox and Wa accessions were mapped to A. thaliana reference genome
(TAIR10) using STAR (Dobin et al. 2013). Raw counts for each gene were measured
with HTSeq version 0.5.4p3 (http://www-huber.embl.de/users/anders/HTSeq/) using the
function "-stranded=no -mode=intersection-strict -t CDS". Differential expression was
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8
called
using
these
counts
with
DESeq
version
1.10.1
(http://www-
huber.embl.de/users/anders/DESeq) with a FDR of 5% (Figure S8). Fold changes in
expression were reported after correcting for differences in library sizes. The RNAseq
alignment files for two biological duplicates of the same accession, generated using
STAR in the preceding analysis, were merged together to generate accession based
alignment files in bam format. A preliminary scan was performed separately on each
accession's bam file to identify SNPs against TAIR10 using samtools (Li et al. 2009).
The SNP sites identified in both accessions were pooled together as candidate sites. At
each candidate site, the read count supporting each allele was compared between the Ox
and Wa accessions using Fisher’s exact test and calls that were not significantly different
were rejected. The program "mcmerge dscmp" from the software package IMR/DENOM
was used for this analysis with a cutoff of Phred score at 20 (Gan et al. 2011).
The Ox sample was also used for one sequencing run on a Roche Genome
Sequencer FLX (Liverpool Centre for Genomic Research) and gave 515,571 clean
sequence reads with mean read length of 246.2-bp. De novo assembly yielded 22,496
contigs longer than 200-bp and 7665 of these contigs were longer than 500-bp (N50 =
585). To improve the assembly we used the 22,496 contigs as a BLAST query against the
annotated A. thaliana coding sequences from TAIR9. This gave 12,072 alignments with
A. thaliana unigenes and a distribution of much longer C. hirsuta cDNA contigs (Figure
S9). GO annotation of C. hirsuta cDNAs was close to 100% for most contigs, but
considerably lower for shorter sequences (Figure S10). Nucleotide divergence between C.
hirsuta and A. thaliana cDNAs differed dramatically between the 1st, 2nd and 3rd codon
positions, as expected for protein coding genes (Figure S11).
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9
Phylogeography. 163 accessions from the putative native range of C. hirsuta and 15
accessions from the introduced range were sampled from herbarium specimens, field
collected silica-dried material, fresh material from plants grown from seed and 4 DNA
extractions (see Table S2 for details). A. thaliana herbarium specimens included two
Ethiopian specimens from Wageningen University (de Wilde, J.J.F.E. 6945 and
Wieringa, J.J. 4966) and two Moroccan specimens from the University of Reading (Jury,
S.L. 14175 and Monserrat, J.M. 2379/94). Total genomic DNA was extracted using the
DNeasy Plant Mini Kit (Qiagen, Valencia, CA). PCR amplification of the chloroplast
ndhF-rpl32 intergenic spacer was performed using the previously described primers ndhF
and rpL32-R (Shaw et al. 2007) and internal primers designed for this study: ndhFrpl32_FI
5’-AGAATAKAACAAAGATTTACAC-3’
and
ndhF-rpl32_RI
5’-
TCACTTAGTGTACTGRAAGACAA-3’. The primers were used in the following
combinations: ndhF-rpl32_FI and rpL32-R; ndhF and ndhF-rpl32_RI to amplify two
fragments with 167-bp overlap in C. hirsuta (Ox). A portion of the 5’ flanking region of
the nuclear Atmyb2 gene was amplified using the primers ATM1 and ATM4 from (Beck
et al. 2008). The IVS1 intron of the nuclear MADS-box gene PISTILLATA was amplified
using the primers PI-ITF and PI-ITR from (Lee et al. 2002). PCR was performed in a
total volume of 15 μL, containing 1x PCR buffer, 2.3 mM MgCl2, 0.2 μM of each primer,
0.3 mM of each dNTP, 0.67 mg/ml bovine serum albumin, 0.4 μL Mango Taq (Bioline),
and 1.5 μL template DNA. PCR conditions were as follows: for chloroplast primers;
80°C for 5 min followed by 30 cycles each consisting of 95°C for 1 min, 47-49°C for 1
min, a ramp of 0.3°C/s to 65°C, 65°C for 4 min, followed by a final extension step at
65°C for 5 min; for Atmyb2 primers: 94 °C for 3 min then 35 cycles of 94 °C for 1 min,
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10
50 °C for 1 min and 72 °C for 2 min, then a final extension for 6 min at 72 °C; and for
PISTILLATA primers: 94°C for 2 min followed by 35 cycles each consisting of 94°C for
30 s, 55-58°C for 30 s, 68°C for 1 min; and a final extension step at 68°C for 5 min. Both
strands were sequenced using BigDye v 3 (Applied Biosystems) and products were
analysed on a 3730x1 DNA Analyzer (Applied Biosystems). Sequences were assembled
using Sequencher v 4.5 (Gene Codes Corporation), A. thaliana sequences were trimmed
to
sequences
downloaded
from
GenBank
(Atmyb2
EF552847-EF553318
and
PISTILLATA EF594536-EF594739), and aligned by eye in MEGA 5.05 (Tamura et al.
2011). Length variable mononucleotide repeats were removed from the alignment as they
are potentially homoplastic. Indels longer than 1 bp were treated as single-step events and
recoded as single characters. The relationship among haplotypes was reconstructed using
statistical parsimony with a 95% connection limit (Templeton et al. 1992) and gaps as a
5th state in TCS v 1.21 (Clement et al. 2000). One reticulation was deleted from the
haplotype network in Figure 7 between haplotype 17 and an intermediate haplotype
between haplotypes 5 and 11, following the criteria in (Crandall and Templeton 1993).
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