DNA was extracted from plants representing carrot diversity, comprising cultivated carrots of different origin and breeding history (supplementary Table 1) previously used for carrot genetic diversity study (Baranski et al. 2011). Full-length DcSto1 elements were amplified from the genomic DNA of three unrelated cultivated carrots (8503B, 493B, 70349-F2), three wild carrots (Daucus carota subsp. carota, D. carota subsp. commutatus, D. carota subsp. gummifer) and other closely related Daucus species (D. capillifolius, D. sahariensis).
DNA isolation, cloning and sequencing
Total genomic DNA was isolated from fresh, young leaves using commercial DNeasy Plant Mini Kit (Qiagen) following a manufacturer’s protocol. PCR products separated on agarose gels were cut out, purified with Wizard SV Gel and PCR Clean-Up (Promega), cloned into pGEM-T (Promega) and transformed into Escherichia coli, strain DH10B or PEAK™ Efficiency Competent Cells (GeneMate). From the transformed bacterial cells, identified by blue-white selection, plasmids were extracted using Wizard SV Miniprep kit (Promega). Sequencing reactions were set up with universal primers Sp6 and T7 or PCR-specific primers using Big Dye terminator chemistry (Applied Biosystems) or CEQ™ DTCS Quick Start Kit (Beckman Coulter) as recommended by manufacturer. Sequencing was carried out on ABI 3700 capillary sequencer (Applied Biosystems) or CEQ8000 capillary DNA sequencer (Beckman Coulter).
Amplification of DcSto1 from the Genomic DNA of Carrot
A primer complementary to the TIR sequence of a DcSto1 (DcS-TIR) was designed manually based on sequence of DcSto1 element identified in the Rs locus. To design the DcS-TIR primer, both Stowaway characteristic features, 16 bp consensus TIR sequence and the characteristic TSD, were considered. To amplify full-length DcSto1 elements the reaction was prepared in 10 μl and contained 20 ng genomic DNA, 1 mM DcS-TIR primer (5′ TAC TCC CTC CGT CCC ACC 3′), 0.25 mM dNTPs (Fermentas), 0.5 U Taq DNA polymerase (Fermentas) and 1× Taq buffer. The amplification profile was as follows: 94 °C (1 min), 30 cycles of 94 °C (30 s), 50 °C (40 s), 68 °C (1 min) and 68 °C (4 min). PCR products were separated in 1 % agarose gels, purified, cloned, and sequenced.
To identify DcSto insertion in the chxb1 gene, intron primers were designed based on the available mRNA sequence (GenBank: DQ192193). A PCR reaction was set up in 10 μl containing 10 ng genomic DNA, 1 mM CHXB1-5′F: 5′CCG AAA TGA TAG CTC GGG TA3′ primer, 1 mM CHXB1-5′R: 5′AGC CTT TGT GGA AGA AAC CA3′primer, 0.25 mM dNTPs (Fermentas), 1 U Taq DNA polymerase (Fermentas) and 1× Taq buffer. The amplification profile was as follows: 94 °C (1 min), 30 cycles of 94 °C (30 s), 56 °C (45 s), 68 °C (3 min) and a final extension step of 68 °C (8 min). Amplified products were separated in 1 % agarose gels, purified, cloned, and sequenced.
Estimation of the Copy Number of DcSto 1
Copy number of DcSto1 elements was estimated essentially as proposed by Grzebelus et al. (2006). Two rounds of amplification with DcS-TIR primer were carried out. First PCR was set up to check if at least one element is present per 384-well BAC plate and to confirm the specificity of amplification. For this purpose, pools by plate of B8503 carrot genomic BACs (Cavagnaro et al. 2009) were used. PCR reaction contained 10–30 ng BAC DNA, 1 mM DcS-TIR primer (5′ TAC TCC CTC CGT CCC ACC 3′), 0.25 mM dNTPs (Fermentas), 0.5 U Dream Taq DNA polymerase (Fermentas) and 1× Dream Taq buffer. The thermal profile was as follows: 94 °C (2 min), 30 cycles of 94 °C (30 s), 50 °C (40 s), 68 °C (1 min) and 68 °C (5 min). PCR products were separated in 1 % agarose gels, three products of expected size and all amplicons larger than expected were cut out, purified, cloned, and sequenced. Subsequently, PCR under the same conditions as above was carried out using DNAs of 141 randomly chosen BAC clones. Amplicons were separated in 1 % agarose gels and scored. We estimated the copy number of DcSto1 elements in the carrot genome taking into account that the average BAC clone size was 0.121 Mbp (Cavagnaro et al. 2009) and that the 2n carrot genome was approximately 980 Mbp (Bennett and Leith 1995).
Inverse PCR, Design, and Validation of Site-Specific Primers
Inverse PCR was set up as described by Collins and Weissman (1984). Ca. 100 ng of genomic DNA was digested with 1 U TaqI (5′T^CGA3′), MspI (5′C^CGG3′), EcoRI (5′G^AATTC3′), or NdeI (5′CA^TATG3′) (Fermentas) at 37 °C for 3 h in the total volume of 10 μl and incubated for 20 min at 65 °C for thermal inactivation of the enzyme. Within DcSto sequence, restriction sites of the applied enzymes were not present. To achieve intramolecular circularization, 5 μl of each digestion mixture was incubated overnight at 4 °C with 5 U of T4 DNA ligase (Fermantas).
Inverse PCR products were amplified with primers anchored in opposite directions at both ends of the DcSto1 element (DcSiPCR-F 5′ CCA CTT CAC CCA CTT TTC CT 3′ and DcSiPCR-R 5′TTT TAG GAA AGT TTT GTA ATG TAA AGA 3′). Primers were designed with Primer3 (v. 0.4.0) (Rozen and Skaletsky 2000) based on the sequence of identified DcSto elements. Each 20 μl reaction mixture contained 10 ng self-ligated DNA, 1 mM each primer, 0.25 mM dNTPs (Fermentas), 1 U Taq DNA polymerase (Fermentas) and 1× Taq buffer. The reactions were carried out as follows: 94 °C (1 min), 30 cycles of 94 °C (30 s), 50 °C (45 s), 68 °C (4 min), and a final extension step of 68 °C (10 min). To obtain flanking sequences long enough to design site-specific primers, only products longer than 400 bp were cloned and sequenced.
Local BLAST search was used to mine BAC-end sequences (BES) database with DcSto1 sequence as a query (e-value cutoff was 0.01). Identified BES sequences with insertions of DcSto elements carrying characteristic TSD, TIR sequence, and for which enough flanking sequence was available, were used for further analysis. Boundary sequences of insertion sites obtained following iPCR or in silico analysis of BES, were used to design site-specific primers with Primer3 (v. 0.4.0) using default parameters.
Site-specific PCR was carried out in 10 μl containing around 20 ng genomic DNA, 1 mM forward and reverse primer, 0.25 mM dNTPs (Fermentas), 0.5 U Taq DNA polymerase (Fermentas), and 1× Taq buffer supplied with MgCl2 (Fermentas). Amplification profile was as follows: 94 °C (1 min), 30 cycles of 94 °C (30 s), variable annealing temperature from 55 to 58 °C (depending on the primer combination) (30 s), 68 °C (variable time depending on the primer combination, from 1 to 3 min), and 68 °C (6 min). All primer sequences, the corresponding annealing temperatures and times of elongation are provided in supplementary Table 2. Products were separated on 1 % agarose gels and selected amplicons were sequenced.
Sequence evaluation and analysis
DcSto sequences were aligned using ClustalX (Thompson et al. 1997) and manually edited in BioEdit (Hall 1999). Genetic distances were calculated with Dnadist in PHYLIP (Felsenstein 1996) using Kimura two-parameter model of nucleotide substitution, Neighbor Joining (NJ) tree was produced with Neighbor and plotted with TreeView (Page 1996). To represent relationships among DcSto1 elements amplified from different sources, NJ tree was generated using MEGA 5.05 (Tamura et al. 2011). Consensus sequences of DcSto1 to DcSto9 were used to predict secondary structures in RNAfold (Hofacker 2003), to search for putative promoter regions using TSSP (Softberry), 3′-end cleavage and polyadenylation sites using POLYAH (Softberry), regulatory DNA sequences in RegSite database using NSITE-PL (Softberry), and to identify transposons inserted in/close to coding regions in the sequences deposited in GenBank using blastn algorithm (Altschul et al. 1997).
Fluorescence in situ hybridization
Localization of DcSto1 elements on chromosomes of cv. Amsterdam 3 (AS33) was carried out by means of fluorescence in situ hybridization (FISH). The DcSto1 probe was amplified with DcS-TIR primer and cloned into pGEM-T vector. All steps of multi color FISH were carried out as described by Nowicka et al. (2012).
Digestion of 10 μg genomic DNA was performed in 400 μl with 100 U of EcoRI (Fermentas) in 1× of EcoRI buffer for 3 h at 37 °C followed by 20 min at 65 °C. Digested DNAs were separated in 1 % TAE agarose gel for 16 h at 1 V/cm. The gel was washed for 8 min with 0.25 M HCl, twice for 15 min with denaturation buffer (0.5 M NaOH, 1.5 M NaCl), 1 min with distilled water, twice for 15 min with neutralization buffer (0.5 M Tris–HCl, pH 7.5, 1.5 M NaCl). After washing with 20× SSC (3 M NaCl, 0.3 M sodium citrate, pH7) for 15 min, the DNA was transferred onto Immobilion™-P transfer membrane (Millipore) at room temperature. The membrane was then exposed to UV light (120 mJ/cm2) using CL-100 Ultraviolet Crosslinker (UVP), washed for 2 min with 2× SSC, and dried. To prepare the probe, PCR was set up in 30 μl using 15 ng of template (a DcSto1 element cloned into pGEM-T) with 1 mM DcS-TIR primer, 0.1 mM digoxigenin-11-dUTP alkali-labile (Roche), 0.25 mM dNTPs (Fermentas), 1.5 U Long PCR Enzyme Mix (Fermentas) and 1× long PCR buffer. The temperature profile was as follows: 94 °C (1 min), 30 cycles of 94 °C (30 s), 50 °C (40 s), 68 °C (1 min) and 68 °C (4 min). Overnight hybridization at 65 °C was performed in 10 ml of hybridization buffer (7 % SDS, 50 % deionized formamide, 5× SSC, 0.1 % N-lauroyl sarcosine, 2 % blocking solution, 50 mM sodium phosphate, pH 7.0) with 30 μl of the denatured probe. Detection was carried out using DIG luminescent detection kit (Roche) following instructions provided by the manufacturer.