Artificial hybridization, peg counts and sampling
The high oleic peanut cultivar Huayu 665 (HY665) was used as the female parent, and the compatible wild species A. monticola PⅠ 219824 from section Arachis and the incompatible wild species A. appressipilla PⅠ 289637 from section Procumbentes were used as the male parents. The cross Huayu 665 × A. appressipilla was the experimental group, whereas the cross Huayu 665 ×A. monticola was used as the control.
Parents were sown in May 2021 at the Laixi Experimental Station, Shandong Peanut Research Institute. Artificial hybridization including emasculation at the afternoon and pollination at the following morning during full flowering phase were performed as previously described. To minimize sampling errors, only flowers on the first nodes of cotyledonary branches of the female parent were used for hand crossing. Pollinated flowers and the resultant pegs were marked by ropes with different colors to distinguish the date at which pollination was made. Pegs formed were counted 7 days after pollination. Pegging percentage was calculated using the following formula: Pegging percentage=number of pegs formed/number of pollinations.
For each cross, 0.23 g of flower bases, 1.40 g of pegs (whole pegs from bases to tips), and 2.71 g of peg/pods (whole pegs/pods from bases to tips) were collected with sterilized razor blades 3 days after pollination, and 5 or 10 days after soil penetration of pegs, respectively. All samples were quickly frozen in liquid nitrogen, and then stored at -80°C before use.
Sample preparation and phytohormone extraction
Peanut samples were ground into powder (30 Hz, 1 min). 50 mg of ground samples were placed into a 2 ml plastic microtube and dissolved in 1 ml of methanol/water/formic acid extractant (15:4:1, V/V/V). 10 μl of internal standard mixed solution (100 ng/ml) was added into the extract as internal standards (IS) for the quantification. The mixture was vortexed for 10 min., and then centrifugated at 4°C at 12000 r/min for 5 min. The supernatant was transferred to a clean plastic microtube, followed by evaporation to dryness and dissolved in 100 μl of 80% methanol (V/V), and passes through a 0.22 μm filter membrane and placed in a feed bottle for further LC-MS/MS analysis (Li et al. 2016)(Floková et al. 2014).
UPLC conditions
Sample extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC™ AD ; MS,Applied Biosystems 6500 Triple Quadrupole). The analytical conditions were listed below. LC: column, Waters ACQUITY UPLC HSS T3 C18 (100 mm×2.1 mm i.d.,1.8 µm); solvent system: water with 0.04% acetic acid (A), acetonitrile with 0.04% acetic acid (B); gradient program, started at 5% B (0~1 min), increased to 95% B (1~8 min), 95% B (8~9 min), finally ramped back to 5% B (9.1~12 min); flow rate: 0.35 ml/min; temperature: 40°C; injection volume: 2 μl (Cai et al. 2014; Niu et al. 2014; Xiao et al. 2018).
ESI-MS/MS conditions
Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® 6500+ LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in both positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows. Ion source: ESI+/-; source temperature: 550 ℃; ion spray voltage (IS): 5500 V(Positive), -4500 V(Negative); curtain gas (CUR): set at 35 psi. Phytohormones were analyzed using scheduled multiple reaction monitoring (MRM). Data acquisitions were performed using Analyst 1.6.3 software (Sciex). Multiquant 3.0.3 software (Sciex) was used to quantify all metabolites. Mass spectrometer parameters including the declustering potentials (DP) and collision energies (CE) for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period (Pan et al. 2010; Šimura et al. 2018; Cui et al. 2015).
Detection of phytohormones
Phytohormones contents were detected by MetWare(http://www.metware.cn/) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. A total of 75 phytohormones belonging to 5 categories, namely auxins, cytokinins (CK), ethylene (ETH), gibberellins (GA) and strigolactones (SL), were analyzed in the present study.
Screening for differential phytohormones
Phytohormones were considered significantly different for fold change ≥ 2 or fold change ≤ 0.5 in experimental groups as compared with the control groups. Where,fold change=(Value in experimental group)/(Value in control group).