The results from a previous study on 50 oilseed rape genotypes allowed us to categorize the tested genotypes into four groups, based on their NUtE values: Nt-responder, Nt-nonresponder, Nt-efficiency, and Nt-inefficiency. Nt-responder referred to genotypes with an NUtE value above the mean at high N, while genotypes with a NUtE value below the mean were categorized into the Nt-nonresponder group. At the low N supply, genotypes displayed a NUtE value above the mean were called Nt-efficient, and Nt-inefficient genotypes were those genotypes with a NUtE value below the mean . According to the rank of responses reported previously, we chose 18 genotypes with distinctive NUtE for this study. These included 5 Nt-responder, 5 Nt-nonresponder, 4 Nt-efficiency and 4 Nt-inefficiency and are described in Table 1.
This study contained a field trail and a controlled pot experiment, and conducted at Yangling district, Shaanxi province (34⁰ 24’ N, 108⁰ 08’ E) from 2016-2018. In the field experiment, the soil was alluvial texture with a pH of 7.65, containing 13.7 g kg-1 organic matter, 1.19 g of total N kg-1, 24.7 mg of available N kg-1, 15.7 mg of Olsen-P kg-1 and 76.9 mg of available K kg-1. In the pot experiment, the red loess soil had the following properties: 9.21 g kg-1 organic matter, 0.92 g of total N kg-1, 22.0 mg of available N kg-1, 10.7 mg of Olsen-P kg-1 and 73.6 mg of available K kg-1, with a pH of 7.5.
Our previous study indicated that the high NUtE of N-efficient genotypes expressed only under high N supply conditions, while the low NUtE of N-inefficient genotypes expressed under low N supply conditions. Therefore, the high N efficient genotypes were planted with high N level (150 kg N ha-1 in the field experiment and 0.30 g of N kg–1 dry soil in the pot experiment), while the 8 low N genotypes were tested with low N level (0 kg N ha-1 in field experiment and 0.10 g of N kg–1 dry soil in pot experiment). Each experiment was arranged in a randomized complete block design with four replicates. In both experiments, sufficient phosphorus as superphosphate (P2O5135 kg ha-1 and 0.20 g kg–1 dry soil) and potassium (K2O 150 kg ha-1 and 0.30 g kg–1 dry soil) were supplied.
Observations and measurements
In order to monitor the dynamics of silique development, flowering and fertilization process of the genotypes were categorized into five stages: pre-embryo (20-80 / °C, 1-4 growing degree days after flowering), globular (95-136 / °C, 5-8 growing degree days after flowering), heart (153-224 / °C, 9-14 growing degree days after flowering), torpedo (240-396 / °C, 15-22 growing degree days after flowering), and maturation (391-540 / °C, 23-30 growing degree days after flowering) stages. For accurate monitoring, fully-opened flowers on the main inflorescence of each plant were marked with color strings around the peduncles in each day. In the pot experiment, five marked siliques of each plant at each developmental stage were chosen for the determination of net photosynthetic rate, silique length, width, surface area, EON, biomass and RNA expression levels.
At the flowering stage, after the sampled buds were removed from the petals, we recorded the stamen and anther number. The number of pollens was counted under light microscope (Axioplan 2; Zeiss), pollen vigor was measured according to HY Zheng, HM Wu, XY Pan, WW Jin and XX Li . The initiation ovule number and the abortion rate were determined according to XJ Wang, A Mathieu, PH Cournede, JM Allirand, A Jullien, PD Reffye and BG Zhang . The pollen vigor was expressed as the total number of germinated pollen grains divided by the total number of pollen grains. The abortion rate was calculated as the aborted ovules over the total number of ovules.
Measurement of agronomic traits
At maturation stage, plant height was measured from the base of the stem to the tip of the main inflorescence . The point of measurement of stem diameter was set at 10 cm from basal of the main stem . First valid branch height was measured as the height from the base of the stem to the effective primary branches at the bottom of the main stem. The number of the first valid branches was measured according to JS Xu, X Song, Y Cheng, XL Zou, L Zeng, X Qiao, GY Lu, GP Fu, Z Qu and XK Zhang .
Measurement of yield and N use efficiency
Number of siliques per plant was measured as the number of effective pods on the main inflorescence, branch inflorescence and the whole plant, respectively . Seed weight of each plant was measured by weighing 500 fully developed seeds with four replications, and then was converted to 1,000-seed weight for easy comparison with other studies . Seed yield per plant was measured as the average dry weight of seeds of the 4 randomly selected plants from each genotype . According to HY He, R Yang, YJ Li, A Ma, LQ Cao, XM Wu, BY Chen, H Tian and YJ Gao , N utilization efficiency (NUtE) and N uptake efficiency (NUpE) were estimated with the following equations: NUtE = Seed yield / The N accumulation (shoot); NUpE = The N accumulation (shoot) / The N supply.
Measurement of siliques indexes
At each stage (pre-embryo, globular, heart, torpedo, and maturation), silique gas exchange rate was measured on the marked siliques between 09:00 and 11:00, using a Portable Photosynthesis System (Li-6400, LI-COR, Lincoln, NE, USA). Afterwards, the marked siliques were picked from the main inflorescence. Half of each of sampled siliques was rapidly frozen in liquid N, and stored at - 80 °C for later RNA transcript analysis, and the other half was used for the determination of surface area  and the normal ovule numbers, which was counted under a light microscope. The sampled siliques were oven-dried at 105 °C for 30 min and then at 70 °C until a constant dry weight was reached and weighed.
Quantitative RT-PCR analysis
Silique samples of the 18 diverse NUtE genotypes were frozen in liquid nitrogen immediately after collection and stored at -80℃. Approximately 100 mg of tissue was ground in liquid nitrogen and total RNA was extracted using an EZNATM Plant RNA Kit (Omega Bio-Tek, USA). The sample was used for cDNA synthesis using TransScript® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Quantitative PCR analyses were performed in an QuantStudio® Design and Analysis (QuantStudio 5, Life Technologies, USA), using an TransStart Tip Green qPCR SuperMix (TransGen Biotech, China). All the primers used in the analysis are listed in Table S1. The low NUtE genotypes (Nt-nonresponder and Nt-inefficiency) were used as references (Nt-nonresponder for Nt-responder and Nt-inefficiency for Nt-efficiency, respectively). Data were expressed as the mean of four biological replicates ± SD.
All the data from both the field and pot experiments, were subjected to analysis of variance. Estimates of correlation coefficients and tests of significance, were performed using the SPSS version 17.0. Principal component analysis was performed using the Canoco 5.0 software. When the analysis showed significant, mean comparisons were made according to the least-significant difference test at P < 0.05 (LSD0.05). All the figures were created using Origin 9.0 software, Microsoft Excel and PowerPoint 2016.