Hulless barley (Hordeum vulgare), also known as naked barley and Yuanmai, belongs to the Hordeum genus in the family of Poaceae. It is a variety of cultivated barley called naked barley because its inner and outer glumes of the seeds are separated from the glumes at maturity and the seeds are exposed (Wang et al., 2019). In Yunnan, the perennial sowing area of hulless barley is 1.40×10⁴ hm², 70% of which is planted in Diqing Tibetan Autonomous Prefecture and Lijiang City in the northwestern of Yunnan, which is the food crop that the Tibetan people depend on for survival (Bai et al., 2018; Xia et al., 2020, Private communication). At the same time, straw and seeds are also good feed for poultry and livestock (Qiang et al., 2008, Tibet’s Science and Technology, (3): 11-17). In recent years, barley food such as barley cookies, barley noodles, barley flower cake and barley wine have gradually emerged. Meanwhile, with the improvement of people's living standard and the research on the unique physiological effects of functional components such as β-glucan, flavonoids, alkaloids and resistant starch rich in barley in the prevention of cardiovascular diseases and diabetes (Zhang et al., 2002; Nirupama et al., 2015), the nutritional and health value of barley is gradually attracting attention (Yang et al., 2017), and barley is gradually developing from a regional food and feed crop to a global medicinal and food crop. The imperfect cultivation technology support related to barley is one of the factors restricting the development of barley industry. Therefore, strengthening the research on barley cultivation technology to improve the barley yield has a positive effect on the national poverty alleviation and the development of rural revitalization.

Density and nitrogen fertilizer are key factors affecting crop growth and development and yield formation, and research on nitrogen fertilizer and density for specific varieties is an important theoretical support for supporting good seeds and methods and promoting variety promotion (Jing et al., 2018). 'Yundamai 12' is a variety registered by the Food Crop Research Institute of Yunnan Academy of Agricultural Sciences as a non-major crop in Yunnan Province and the Ministry of Agriculture and Rural Areas in 2014 and 2020 respectively. It is a two-rowed hulless barley variety whose area in Yunnan Province has increased year by year, refreshed and maintained the national highland barley high-yield record for many times in recent years. Among them, the high-yield acceptance yield in 2019 reached 9 412.50 kg/hm² (Wang et al., 2020). This study intends to study the effects of different density and nitrogen application on the yield, agronomic characters and photosynthetic characteristics of 'Yundamai 12', in order to provide a basis for the standardization and popularization of 'Yundamai 12'.

1 Results and Analysis

1.1 Effects of density and nitrogen application rate on agronomic traits and yield

The results showed that density had a highly significant effect on yield and single plant tillers, but no significant effect on other agronomic traits. Nitrogen application had no significant effect on yield and only a highly significant effect on single plant tillers. The interaction of density and nitrogen had no significant effect on yield and agronomic traits. Both density and nitrogen application rate had a very significant effect on the single plant tillers of 'Yundamai 12' (Table 1). It indicates that density has a greater effect on yield and single tillering, and the amount of nitrogen applied is not the main factor affecting yield, and reasonable density is the main factor for high yield of 'Yundamai 12'.

1.2 Effect of density and nitrogen application rate on photosynthetic parameters and chlorophyll content

Density and nitrogen application had no significant effects on the four photosynthetic parameters of net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, transpiration rate and chlorophyll content (Table 2), while density and nitrogen application interacted to have significant effects on net photosynthetic rate, but not on stomatal conductance, intercellular CO₂ concentration, transpiration rate and chlorophyll. It indicates that different densities and nitrogen applications have little effect on photosynthetic parameters and chlorophyll content in general.

1.3 Effect of density and nitrogen application on yield and agronomic traits

With the increase of density, the yield first decreased and then increased (Table 3). The yield of A1 was the highest, up to 6 737.50 kg/hm², which was significantly higher than that of other treatments. Different nitrogen application rates had no significant effect on yield, and there was no significant difference among treatments. It showed that density was the main factor affecting the yield of 'Yundamai 12'. Single plant tillers decreased with the increase of density, there were significant differences among different densities, and increased with the increase of nitrogen application. Different nitrogen application rates had no effect on the number of grains per ear and 1000-grain weight. 1000-grain weight decreased with the increase of density, and A1 (51.92 g) was significantly different from A3 (49.02 g). The ear forming rate, ear length, plant height and unit weight were relatively stable, and density and nitrogen application rate had no significant effect on them, indicating that these four traits were not easily affected by cultivation methods.

1.4 Effect of density and nitrogen application rate on photosynthetic parameters and chlorophyll content

The effects of different densities and nitrogen application on photosynthetic parameters and chlorophyll content were generally small, only chlorophyll was affected by density, and chlorophyll content increased with increasing density, with A3 (50.61 mg/g) having a significantly higher content than A1 (48.05 mg/g) (Table 4).

1.5 Effects of different treatments on yield and agronomic traits

Analysis of variance (ANOVA) was conducted for different treatments and the results showed (Table 5) that the yield amounts ranged from 5 280.00 to 7 282.33 kg/hm², with the highest yield of 7 282.33 kg/hm² at A1B2, indicating that the maximum yield was achieved at a density of 850 000/hm² and a nitrogen application of 112.5 kg/hm², which was the optimal density and nitrogen fertilizer program for high yield. Single plant tiller reached the highest at A1B3 with 19.07 tillers/plant and the lowest at A3B1 with 8.84 tillers/plant, a highly significant difference, indicating that single plant tiller was the highest at the lowest density with the highest nitrogen application and the smallest at the lowest density with the lowest nitrogen fertilizer. Plant height was highest at A2B3 (71.61 cm) and shortest at A2B1 (58.31 cm). The maximum bulk density was 721.11 g at A2B3 and the minimum was 694.11 g at A2B1. The four agronomic traits of spike rate, spike length, spike grain number and 1000-grain weight were relatively stable and did not differ significantly among treatments.

1.6 Effects of different treatments on photosynthetic parameters and chlorophyll content

Net photosynthetic rate (12.21 μmol CO²/(m²·s)) and stomatal conductance (0.13 μmol H₂O/(m²·s)) reached their maximum and chlorophyll content (46.05 mg/g) was lowest under the A1B1 treatment. The net photosynthetic rate (7.44 μmol CO₂/(m²·s)) was the lowest, and the intercellular CO₂ concentration was the highest (239.52 μmol CO₂/mol) under the A3B2 treatment. The transpiration rate (2.98 mmol H₂O/(m²·s)) and chlorophyll content (51.85 mg/g) reached their maximum under the A3B3 treatment. Stomatal conductance (0.07 μmol H₂O/(m²·s)), intercellular CO₂ concentration (213.39 μmolCO²/mol) and transpiration rate (1.17 mmol H₂O/(m²·s)) were all minimal under the A2B1 treatment. The net photosynthetic rate (7.44 μmol CO²/(m²·s)) was the lowest under A3B2 treatment (Table 6).

2 Discussion

With the increase of density, the yield of 'Yundamai 12' first decreased and then increased, and the yield was the highest at A1 (850 000/hm²), which was significantly different from other treatments. At the same time, 'Yundamai 12' had the highest tillering per plant at A1, which was significantly different from other densities, and sown 43.33% less than the conventional variety with a sowing rate of about 1.5 million/hm² (Wang et al., 2015, Primary Agricultural Technology Extension, 3(10): 95-97), indicating that 'Yundamai 12' was a high-yield variety with strong tillering ability and suitable for sparse sowing.

In recent years, there are much research on planting density and fertilizer amount (Zhou, 1993, Tibet Journal of Agricultural Sciences, 15(2): 19-21; Li et al., 2017, Jiangsu Agricultural Sciences, 45(1): 76-79; Wang et al., 2019). Reasonable fertilizer and planting density are conducive to the improvement of hulless barley yield. Previous studies have shown that the increase of nitrogen application can significantly improve the yield of hulless barley (Feng et al., 2009; Wei and Ma, 2020). While the results of this study showed that the effect of nitrogen application rate on the yield of Yundamai 12' was not significant, which may be related to the fact that the material used in previous studies was a multi ribbed hulless barley variety with few tillers, and the population number mainly depended on the sowing rate. And in this study, both density and nitrogen application rate could significantly affect the tillers per plant of 'Yundamai 12'. The tillers per plant decreased with the increase of density and increased with the increase of nitrogen application rate. The average tillers per plant was 13.67. The tillers of 'Yundamai 12' were related to the strong ability to regulate at the same time. Under different treatments, the four agronomic characters of 'Yundamai 12' were relatively stable, including spike rate, spike length, grains and 1000-grain weight. There was no significant difference between different treatments, indicating that the four agronomic characters were relatively stable and not easy to be affected by the cultivation environment. The results of this study showed that 'Yundamai 12' had the highest yield under A1B2 treatment, indicating that the best cultivation system of 'Yundamai 12' was the sowing rate of 850 000/hm² and the nitrogen application rate of 112.5 kg/hm².

3 Materials and Methods

3.1 Experimental materials

The high-yield hulless barley variety 'Yundamai 12' bred by the Food Crops Research Institute, Yunnan Academy of Agricultural Sciences, whose parent source is 07YD-4/Yundamai 2, passed the non-major crop registration of Yunnan Province (Number: Yunzhongjianding 2015008) and the Ministry of Agriculture and Rural Areas (Number: GPD barley (hulless barley) (2020) 530021) in 2014 and 2020, respectively. The variety is two rowed, weak spring, half creeping seedlings, with compact plant type and dark green leaves. The spike layer is neat, the spike is low and drooping at maturity, and the mature phase is good; The plant height is 70 cm, which is suitable for planting in high fertility paddy field, with strong lodging resistance and tillering ability, and the growth period is 155 d; The grains are slender, with 26 grains and a 1000-grain weight of 45.80 g; It has strong lodging resistance, medium resistance to powdery mildew and wide adaptability.

3.2 Experimental design

Split-plot experiment design is adopted, with the density (A) as the main area and the nitrogen fertilizer application amount (B) as the sub area. Three gradients are set respectively, in which the density (A1: 850 000/hm², A2: 1 200 000/hm², A3: 1 550 000/hm²), pure nitrogen (B1: 0 kg/hm², B2: 112.5 kg/hm², B3: 225 kg/hm²) and pure nitrogen content are converted by urea (N content of 46.4%) and applied in the form of seed fertilizer at one time, a total of 9 treatments, 3 repetitions and 27 plots.

The test materials were planted in Songming Agricultural Research Station (25°21′N, 103°05′E) of Yunnan Academy of Agricultural Sciences from 2019 to 2020. The altitude of the test field is 1 900 m. The terrain of the test field is flat and the soil fertility is uniform. The soil of the test field is brown soil. The basic nutrient content of the 0~30 cm plough layer soil is 86.60 mg/kg of available nitrogen, 60.00 mg/kg of available phosphorus and 106.00 mg/kg of available potassium. The plot is 3.57 m long, 1.82 m wide, 6.67 m² in area, and the row spacing is 23 cm. According to the germination rate, the basic seedlings were weighed into the rows, and 10 kg of compound fertilizer/mu was applied as the base fertilizer, and the rest was managed in the same way as the general field.

3.3 Measurement of indicators

At the seedling emergence stage, investigate the number of seedlings in the first two rows and the last two rows, and convert them into basic seedlings. The tillering stage is investigated every 7 days, and the highest tiller is selected. The effective spikes were investigated at the wax ripening stage, and the tillering and Spike rate per plant were calculated. 10 plants were randomly selected from each plot at the completion stage to investigate spike length, plant height, grains and false spikelet. Harvest in the whole area, calculate the yield after air drying, and investigate the 1000-grain weight and bulk density.

In the middle stage of grouting, select 10 plants in each plot from 10:00 to 11:30 in sunny weather, select the top two leaves with the same growth trend to light, measure the chlorophyll content of the upper, middle and lower parts of each leaf with SPAD-502 handheld chlorophyll meter, and take the average value. The net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration and transpiration rate were measured by Li-6400 portable photosynthetic apparatus (USA). The open gas path was used for measurement, and the average CO₂ concentration was (382.6±2.5) μL/L, red/blue LED light source for leaf chamber, PAR of 1 500 μmol·m^-2·s^-1, wind speed<1 m/s, humidity of 53%~56%, gas flow rate of 5 mL/min, reading after each measurement is stable for 2 min (Qiao et al., 2019).

3.4 Data processing

Excel was used for data processing. SPSS 26.0 was used to analyze the variance between treatments and compare the significance of differences. LSD method was used to detect the significance.

Authors’ Contributions

WZL and YJH are the executors of the experimental design and research of this study. WZL completed the data analysis and wrote the first draft of the paper. YYX, QXM, CJS, WZW, and CZ participated in the experimental design and the analysis of the experimental results. YJH is the designer and leader of the project, guiding experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the Major Science and Technology Special Project on Key Technology Research and Industrialization Development of Barley Industry Chain of Yunnan Province (2019ZG004) and the Modern Agricultural Wheat Industry Technology System of Yunnan Province (Yunnongkeban 2020-40).

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