Peanut cultivar Zhonghua 8 is an erect growth genotype with short growth period (105 to 120 days from sowing to maturity), leaf spot and stunt viruses resistances, drought tolerance, high oil content (56.13%), big pod (100-pod weight 192.0 g), big seed (100-seed weight 84.2 g), high shelling percentage (75%), desirable pod and kernel features. Zhonghua 8 plants were grown under weak light in the greenhouse at the Institute of Oil Crops, Chinese Academy of Agricultural Sciences. The young etiolated leaves were washed with fresh water, then airdried at room temperature and frozen with liquid nitrogen, quickly stored at -80°C for use.
BAC vector preparation
The CopyControl™ BAC Cloning Kit (Epicentre company) was used for construction of the library. The BAC vector was digested with Hin d III, dephosphorylated, and stored at -20°C in 25 ng/ul aliquots until needed.
Preparation and digestion of high-molecular weight (HMW) DNA
The isolation of peanut nuclei was performed according to the protocols reported by Zhang et al. (1995) and Yüksel and Paterson (2005) with some modifications. 20 g of frozen leaves were ground in liquid nitrogen until fine powder was obtained, then dissolved in 200 ml of fresh extraction buffer [0.005 M citric acid, 0.5 M glucose, 0.01 M Na2EDTA, 2.0% (w/v) polyvinylpyrrolidone-40 (PVP-40), 5% (v/v) Triton X-100, 0.25% (w/v) spermidine, 0.1% (w/v) ascorbic acid, 0.2% (v/v) 2-mercaptoethanol, 0.1% (w/v) disodium diethylthiocarbamate (Na2Et2dtc) and 0.4% (w/v) NaHSO3, titrated to pH 6.5 with NaOH] on ice for about 15 min. The samples were filtered twice through two layers of cheese clothes and two layers of miraclothes. The filtrates were centrifuged for 15 min at 1600 g after a centrifuge for 15 min at 60 g. The precipitated nuclei were dissolved in extraction buffer and centrifuged at the same speed three times until the nuclei looked clean, the final wash of the nuclei was in the same extraction buffer without Triton X-100. The following steps were only followed for HMW DNA extraction: the plugs were incubated at 50°C for 24 h in lysis buffer [0.005 M citric acid, 0.14 M NaCl, 0.05 M Na2EDTA, 2% (w/v) PVP-40, 1% (w/v) sodium dodecyl sulfate (SDS), 1% sodium lauryl sarcosine titrated to pH 6.5 with NaOH, and autoclaved] and the same antioxidants at similar proportions as in the extraction buffer, and 0.2 mg/ml Proteinase K was added. The buffer was replaced, and plugs were incubated at the same temperature for another 24 h. The plugs were incubated at room temperature for at least 4 h in 70% ethanol then stored at -20°C until use.
The plugs were placed into T10E1 (PH = 8.0) containing 0.1 mM PMSF (Phenylmethanesulfonyl fluoride) and vortexed on ice for 1 h, repeated three times, then treated three times in T10E1 (PH = 8.0) without PMSF under the same conditions. The plugs were then digested with Hin d III enzyme as the method reported by Yüksel and Paterson (2005). Briefly, serial dilutions of Hin d III (NEB) (0, 0.1, 0.15, 0.2, 0.3, 0.5, 0.75, 1, and 2 U per milligram of plug) were added to the samples and the samples incubated at 4°C for 4 h. Partial digestion was carried out at 37°C for 7 min, then 0.5 M EDTA was added to the tubes to stop the reactions. The partially digested samples were resolved on 1% agarose gels run in 0.5 × TBE buffer by pulsed field gel electrophoresis (PFGE) at 6 V/cm, with 1- to 40-s switch times and a linear ramp, for 18 h. Optimum enzyme concentration was determined by visualizing maximum fragment concentration in the 100- to 300-kb range.
Construction of BAC library
For ligation, a constant 25 ng of vector was used, and varying amounts of insert ranging from 60 to 120 ng were tested for each size selection. The vector/insert (V/I) ratio, which gave the best efficiency and average insert size, was chosen. Ligation reactions were performed in 60 μl volumes and incubated at 16°C for 10 h. After desalting, 2–3 μl of reactions were transformed into Escherichia coli EP1300 (Epicentre) competent cells by electroporation (BioRad Gene Pulser® II Electroporation System). For electroporation, 1.25 kV and 200× of resistance were used. The electroporated cells were immediately mixed with 1 ml of SOC media and grown for 1 h at 37°C before separation on selective medium (LB medium) with 12.5 μg chloroamphenicol, 0.55 mM IPTG, and 80 μg/ml X-gal. After 18 h of incubation at 37°C, a sampling of 10–20 colonies were picked and tested. Randomly selected white colonies were inoculated into 1 ml LB CM liquid medium and grown for 16 h at 37°C. The liquid cultures were subjected to minipreps by an alkaline lysis protocol, and the DNA was digested with 10 U of Not I (NEB) for 4 h at 37°C. The digested samples were resolved on 1%, 0.5 × TBE agarose by PFGE with the following parameters: 3- to 20-s linear ramp, 6 V/cm, and a 16-h run time. The ligation reactions with average insert size of 100 kb or more were mass-transformed, plated, and directly picked with toothpicks into 384-well plates. The clones were replicated three times with Replicator and stored in FM medium [LB + 36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM sodium citrate, 0.4 mM MgSO4, 6.8 mM (NH4)2SO4, and 4.4% glycerol] at -80°C.
200 clones were randomly picked out for insert-size characterization. The Not I-digested clones were detected on 1%, 1 × TAE agarose by PFGE with the following parameters: 5- to 15-s linear ramp, 6 V/cm, and a 16-h run time.
Detection of organelle DNAs contamination
The BAC clones were dotted on N+ nylon filter with Replicator, and then conversely cultured on LB medium containing chloramphenicol at 37°C overnight. The clone carrying empty BAC vector was used as a negative control, the clones containing the psbA gene of barley chloroplast and the atp6 gene of rice mitochondrion, respectively, were used as positive controls. The filters were treated under following sequential conditions: 10% SDS, denaturing solution, neutralizing solution, neutralizing solution, 2xSSC, 0.1% SDS, 2xSSC, 5 min, respectively, then 0.4 N NaOH 20 min. The sequential washing of filters was performed with 0.1% SDS (in 5xSSC) for 20 min twice and 2xSSC for 10 min twice, then the filters were dried at 80°C for 2 h.
1 μL (25 ng) probe DNA mixed with 2 μL primers and 11 μL water was denatured at 95°C for 3 min, placed on ice for at least 10 min, then 2.5 μL 10 × Buffer, 2.5 μL dNTP Mixture, 5 μL α-32P dCTP and 1 μL Klenow enzyme were added into the probe solution, the labeling was performed at 37°C for 1–2 h, subsequently the mixture was denatured again at 95°C for 3 min, placed on ice until for use. The filter was pre-hybridized at 68°C for 3 h, then 5 μL labeled probes each were added into the pre-hybridization solution (6 × SSC, 0.05 × blotto, 0.5% SDS, 100 μg/ml ssDNA), the hybridization was performed at 68°C for 16 h. Finally, the hybridized filters were washed twice with solution I (2xSSC, 0.1% SDS) at room temperature for 5–10 min, then washed twice with solution II (1xSSC, 0.1% SDS) at 68°C for 2 h, and imaged and quantitatively analyzed using a Biorad FX molecular imager.
Detection of BAC clone stability
Three BAC clones were randomly picked out for successive cultures; the clones of the 100th generation could be obtained on the fifth day since E. coli propagated 20 generations each day (Sambrook and Russell 2001). The BAC clones of the first day and the fifth day were digested with Not I to analyze their stability.