Plant materials and DNA extraction
Three flue-cured tobacco varieties, K326, Honghuadajinyuan (Hongda) and Yunyan87, one sun-cured tobacco variety, Fuquanliuye, three F1 hybrids derived from crosses between the three flue-cured tobacco varieties (as female parents) and Fuquanliuye (as the male parent), and an F2 population derived from a cross between K326 (female) and Fuquanliuye (male) were used. K326, Hongda and Yunyan87 are the major flue-cured tobacco varieties planted in China, while Fuquanliuye is a PVY-resistant sun-cured tobacco landrace from Guizhou Province, China. All plant materials were collected and identified by one of authors (Rengang Wang) and deposited at the Guizhou Academy of Tobacco Science, Guiyang, China.
Genomic DNAs of the varieties and their F1, F2 and BC1F1 derivatives were isolated from young leaves using the AxyprepTM Multisource Genomic DNA Miniprep Kit (Axygen Scientific, USA). DNA concentrations were determined with a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All DNA samples were diluted to 80 ng/μl in distilled-deionized water and stored at -20 °C.
A necrotic strain of PVY (PVY-NGZ) was isolated from a tobacco field in Guizhou Province, China, and maintained by the Guizhou Academy of Tobacco Sciences. Inoculations were carried out manually, using the sap of PVY-infected N. tabacum leaves as the inoculum.
Identification of the deletion mutation of eIF4E1.S in Fuquanliuye tobacco
To characterize the region of the Fuquanliuye genome associated with eIF4E1.S, six different sets of partial-overlapping primers (Table 1) spanning from −55-bp to +4423-bp with respect to the translational start site of eIF4E1.S were designed and used for PCR amplifications in both wild type K326 and mutant Fuquanliuye tobacco genotypes. Amplification reactions were performed in a final volume of 20 μl containing 1×PCR buffer (Mg2+ plus), 1.0 U Taq DNA polymerase, 0.2 mM dNTPs, 0.4 μM each of the forward and reverse primers, and 64 ng of genomic DNA. PCR reactions were conducted in a C1000 Touch Thermal Cycler (Bio-rad, Hercules, CA, USA) with an initial denaturation step at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, annealing temperature of 52-58°C for 30 s and extension 72°C for 1 min, terminating with the final extension step at 72°C for 10 min. The amplicons were purified using a PCR Purification Kit (Qiagen) and ligated into the pGEM-T easy vector (Promega) followed by transformation into competent E.coli DH5α cells (TIANGEN). Ten colonies were randomly selected for each PCR product and sequenced by Invitrogen (Shanghai, China). Sequence assembly and analysis were performed using DNAMAN software (Version 6.0). BLAST alignments were carried out using the China Tobacco Genome Database (Version 4.0).
Cloning and verification of the deletion junction in Fuquanliuye
To obtain the junction fragment originating in the 3' region of eIF4E1.S in Fuquanliuye tobacco, a genome walking approach was initiated using the Genome Walking Kit (TaKaRa, Japan). The junction fragment was amplified by three rounds of thermal asymmetric interlaced PCR (TAIL PCR) using genomic DNA from Fuquanliuye tobacco as the template. Specific primers SP1, SP2, and SP3 (Table 1) were designed for the first, second, and third rounds of genome walking, respectively, corresponding to sequences at the 5'-end of eIF4E1.S located close to the breakpoint. The amplification reactions were performed in 50 μl reaction volumes according to the manufacturers protocol, using the specific primers mentioned above and arbitrary degenerate primers provided by the Genome Walking Kit. The amplification products were cloned into the pGEM-T easy vector and sequenced.
The genomic sequence obtained by genome walking was assembled using DNAMAN software, and the specific primer SP4 was designed from the downstream region where the sequence diverged from the wild type genomic DNA. PCR reactions utilizing SP4 together with SP1 were used in PCR reactions to validate the existence of this genomic configuration in Fuquanliuye. The PCR conditions involved an initial denaturation at 94 for 3 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min with a final extension at 72°C for 10 min. PCR products of expected size was cloned into the pGEM-T vector and sequenced. The resulting fragment was compared with the reference genome of Nicotiana tabacum using the BLAST program in the China Tobacco Genome Database (Version 4.0) to identify and characterize the extent of the deletion originating from the eIF4E1.S gene region in the Fuquanliuye genome.
Designing and testing of allele-specific primers
The common forward primer (Table 1; FWm) was designed according to a sequence 5’ of the deletion region, namely, a portion of the eIF4E1.S gene that is still present in Fuquanliuye. One reverse primer (Table 1; Rm) spans the deletion region to enable amplification of the mutant eiF4E1.Fu allele, generating a PCR product of 572 bp; the other reverse primer (Table 1; RW) was designed against a sequence that had been deleted, amplifying a 763 bp product in wild type tobaccos when used with FWm. The amplification reactions were performed in a final volume of 20 μl containing 1×PCR buffer (Mg2+ plus), 1.0 U Taq DNA polymerase, 0.2 mM dNTPs, 0.4 μM each of the forward and reverse primers, and 64 ng of genomic DNA. The PCR cycler conditions included an initial denaturation step at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 58°C for 30 s and 72°C for 1 min, with a final extension step at 72°C for 10 min. The PCR products were cloned into the pGEM-T vector and and validated by DNA sequence analysis.
Tri-primer multiplex PCR assay
To simplify the PCR protocol for use in marker-assisted selection, a tri-primer multiplex PCR assay was designed. PCR amplifications were performed in a 20 μl reaction volumes containing 1× Premix Taq Version 2.0 plus dye (TaKaRa, Japan), 0.4 μM each of the oligonucleotide primers (FWm/Rm/RW), and 64 ng of template DNA. Thermocycler conditions involved an initial denaturation step at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 58°C for 30 s and 72°C for 1 min, and a final extension step at 72°C for 10 min. The PCR products were analyzed by electrophoresis using 2% agarose gels.