A method for screening nitrogen-efficient wheat varieties

Abstract

The invention discloses a method for screening nitrogen-efficient wheat varieties, comprising the following steps: step 1, selecting several wheat varieties for testing, conducting field tests, and setting fertilization treatments with two nitrogen supply levels: no nitrogen fertilizer treatment N0, and normal nitrogen fertilizer treatment N1; step 2, determining the root activity and rhizosphere soil enzyme activity of the tested wheat varieties under the N0 and N1 treatments, and performing high-throughput sequencing of soil microorganisms; step 3, analyzing the measurement data in step 2, screening out the tested wheat varieties with high root activity, high rhizosphere soil enzyme activity, high richness index and high diversity index of rhizosphere microorganisms, i.e., nitrogen-efficient wheat varieties. The method for screening nitrogen-efficient wheat varieties of the present invention, the screened nitrogen-efficient wheat varieties with high root activity, high rhizosphere soil enzyme activity, high richness index and high diversity index of rhizosphere microorganisms, provide a theoretical basis for screening nitrogen-efficient wheat varieties from the perspective of rhizosphere micro-domain environmental differences.

Description

Technical Field

[0001] The invention relates to the technical field of seed selection and breeding, and in particular to a method for screening nitrogen-efficient wheat varieties. Background Art

[0002] Soil enzymes and soil microorganisms are important participants in a variety of biochemical reactions, material cycles and energy metabolism in the soil. They play a key role in nutrient cycling, decomposition of organic matter and degradation of pollutants, and can characterize soil fertility and soil health. The rhizosphere is an important connecting platform for plants, soil, microorganisms and their growth environment, and is a microdomain at the junction of the root system and soil. Analyzing and comparing the microecological characteristics of rhizosphere soil enzyme activity and microbial community structure of different crops is conducive to providing a theoretical basis for low-fertilizer and high-efficiency crop cultivation from a microecological perspective.

[0003] Wheat is one of China's important food crops, and the development of the wheat industry is directly related to China's food security and social stability. In recent years, China's wheat production has increased year by year, but the increase in production is accompanied by excessive application of nitrogen fertilizers and decreased fertilizer efficiency, which affects the healthy development of the ecological environment. For example, the large-scale application of chemical fertilizers has led to a series of ecological and environmental problems such as soil acidification, greenhouse effect and loss of biodiversity. Based on the dual requirements of economic benefits and ecological and environmental protection, relevant scholars have conducted research from different angles on the issue of "double reduction of fertilizers and pesticides" in wheat. Yang Xiaoka et al. explored a high-yield and high-efficiency model with the lowest apparent nitrogen loss from the perspectives of cultivation measures and rotation systems. Zhao Ben et al. constructed a wheat aboveground nitrogen deficiency model based on critical nitrogen concentration for precise fertilization. Zhang Juanjuan et al. conducted a nitrogen nutrition diagnosis study on wheat varieties with different nitrogen efficiencies and found that different varieties had different nitrogen absorption and utilization efficiencies, root vitality and tolerance to low nitrogen stress. However, there are few studies on the mechanism of efficient nitrogen utilization. In recent years, the relationship between sustainable productivity of grain-soil and plant rhizosphere microbial flora has gradually attracted the attention of scientific researchers. Dong Hangyu studied the relationship between soil microorganisms, soil enzyme activity and efficient nitrogen utilization of japonica rice from the perspective of rhizosphere microecology. Yang Zhen and others explored the mechanism of plant disease occurrence based on the rhizosphere microdomain of crops and explored potential microbial resources; conversely, the differences between different crops or different varieties of the same crop can also be reflected through the soil enzyme activity and microbial diversity in the rhizosphere microdomain. At present, the research on the microecological flora of wheat rhizosphere soil is mostly focused on the absorption, utilization and tillage methods of nitrogen, and there are few studies on the differences in rhizosphere microorganisms and soil enzyme activity of wheat varieties with different nitrogen efficiencies. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for screening nitrogen-efficient wheat varieties in view of the deficiencies in the prior art. The screened wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index are nitrogen-efficient wheat varieties, providing a theoretical basis for screening nitrogen-efficient wheat varieties from the perspective of rhizosphere micro-domain environmental differences.

[0005] To achieve the above object, the present invention adopts the following technical scheme: A method for screening nitrogen efficient wheat varieties, comprising the following steps: Step 1, selecting several wheat varieties for test, conducting field tests, and setting two nitrogen supply level fertilization treatments: no nitrogen fertilizer treatment N0 (0kg N / hm 2 ), normal nitrogen fertilizer treatment N1;

[0006] Step 2, measuring the root activity and rhizosphere soil enzyme activity of the tested wheat varieties under N0 and N1 treatments, and performing high-throughput sequencing of soil microorganisms;

[0007] Step 3, analyzing the measured data in step 2, and screening out the test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, that is, nitrogen-efficient wheat varieties.

[0008] Furthermore, in step 1, the nitrogen fertilizer application rate in the normal nitrogen fertilizer treatment N1 is 165 kgN / hm 2 The ratio of base fertilizer to topdressing nitrogen fertilizer is 5:5, and topdressing is applied by digging furrows during the greening period.

[0009] Furthermore, the experimental plots in the field test in step 1 are randomly arranged, and the area of ​​each experimental plot is 6*9=54m 2 , row spacing 20cm, repeated 3 times, sowing rate 150kg / hm2, sowing in mid-October; phosphorus fertilizer and potassium fertilizer were applied as base fertilizer before sowing, of which nitrogen fertilizer was urea and phosphorus fertilizer was calcium dihydrogen phosphate 687.5kg / hm2 2 Potassium fertilizer is potassium sulfate, the application rate is 144.74kg / hm 2 .

[0010] Furthermore, the method for determining root activity in step 2 is: flushing the roots at the greening stage, jointing stage, booting stage, filling stage, and maturity stage, taking 5 cm of the roots at the root tip, and using the improved TTC reduction method to determine the root activity.

[0011] Furthermore, the soil enzymes in the rhizosphere soil enzyme activity determined in step 2 include: β-glucosidase, leucine aminopeptidase and polyphenol oxidase; the rhizosphere soil of the test wheat varieties under the N0 and N1 treatments at the booting stage were selected, and the soil BG enzyme and LAP enzyme activities were determined by microplate fluorescence analysis, and the soil POX enzyme activity was determined by microplate colorimetry.

[0012] Furthermore, the soil microbial high-throughput sequencing method in step 2 is: using the standard operating procedures of the Illumina MiSeq platform, the soil microbial community of the rhizosphere soil of the test wheat varieties under the N0 and N1 treatments at the booting stage is sequenced; the V3-V4 hypervariable region and the ITS region of the 16S rRNA gene are PCR amplified, and for each DNA sample, three replicates are independently PCR amplified using TransStart Fastpfu DNA polymerase on the ABI GeneAmp 9700PCR system, purified using the AxyPrepPCR purification kit, and double-end sequencing is performed using the Illumina MiSeq platform.

[0013] Furthermore, the method for analyzing the measured data of root activity in step 2 in step 3 is: analyzing the changing trends of root activity of different test wheat varieties along with the growth process; comparing the root activity values ​​of different test wheat varieties under N0 and N1 treatments.

[0014] Furthermore, in step 3, the method for analyzing the measured data of rhizosphere soil enzyme activity in step 2 is: selecting the rhizosphere soil enzyme activity values ​​under the N0 and N1 treatments at the booting stage, and comparing the rhizosphere soil enzyme activity values ​​between different tested wheat varieties.

[0015] Furthermore, in step 3, the method for analyzing the soil microbial high-throughput sequencing data in step 2 is as follows: 1) the sequencing results are first spliced, filtered, and chimeras are removed using QIIME; high-quality sequences with a sequence length greater than 200 bp, no erroneous bases in the Barcode and primer sequences, and an average quality score of Q≥25 are selected; the USEARCH software is used to divide the classification operation units at a threshold of 97%, and the OTU representative sequences are aligned and classified according to the Silva database, and the Shannon and Simpson diversity indices and the Chao1 and ACE richness indices are calculated using Mothur software to evaluate bacterial α-diversity; 2) the rhizosphere soil bacterial community structure of the tested wheat varieties is analyzed based on the relative abundance of the rhizosphere soil bacterial community at the phylum level and the relative abundance of the dominant bacterial genera in the rhizosphere soil.

[0016] The beneficial effects of the present invention are:

[0017] The method for screening nitrogen-efficient wheat varieties of the present invention comprehensively analyzes the differences in root activity of wheat of different nitrogen-efficient varieties and their effects on rhizosphere soil enzyme activity and microbial community diversity, and concludes that nitrogen-efficient wheat varieties can enhance soil enzyme activity, increase bacterial community diversity, and improve bacterial community composition. The test wheat varieties screened by the present invention with high root activity, high rhizosphere soil enzyme activity, high richness index of rhizosphere microorganisms, and high diversity index can be mutually verified with known different nitrogen-efficient wheat varieties, providing a theoretical basis for screening nitrogen-efficient wheat varieties from the perspective of rhizosphere microdomain environmental differences. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Attached Figure 1 The difference in root activity of wheat varieties with different nitrogen efficiencies of the present invention (average data from 2018 to 2020);

[0019] Attached Figure 2 is the relative abundance of rhizosphere soil bacterial community at the phylum level of wheat varieties with different nitrogen efficiency in the present invention;

[0020] Attached Figure 3 The relative abundance of dominant bacterial genera in the rhizosphere soil of wheat varieties with different nitrogen efficiency in the present invention. DETAILED DESCRIPTION

[0021] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0022] Example 1

[0023] A method for screening nitrogen-efficient wheat varieties comprises the following steps: Step 1, selecting several wheat varieties for testing, conducting field trials, and setting two fertilization treatments with nitrogen supply levels: no nitrogen fertilizer treatment N0 (0 kg N / hm2) and 2 ), normal nitrogen fertilizer treatment N1;

[0024] Step 2, measuring the root activity and rhizosphere soil enzyme activity of the tested wheat varieties under N0 and N1 treatments, and performing high-throughput sequencing of soil microorganisms;

[0025] Step 3, analyzing the measured data in step 2, and screening out the test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, that is, nitrogen-efficient wheat varieties.

[0026] Example 2

[0027] The difference between this embodiment and embodiment 1 is that the nitrogen fertilizer application rate in the normal nitrogen fertilizer treatment N1 in step 1 is 165 kgN / hm 2 The ratio of nitrogen fertilizer to topdressing is 5:5, and the topdressing is applied in furrows during the green period. The nitrogen fertilizer is urea (containing 46% N).

[0028] Example 3

[0029] The difference between this embodiment and embodiment 2 is that: in step 1, the experimental plots in the field test are randomly arranged, and the area of ​​each experimental plot is 6*9=54m 2 , row spacing 20cm, repeated 3 times, sowing rate 150kg / hm2, sowing in mid-October. Phosphate fertilizer and potash fertilizer were applied as basal fertilizer before sowing. Phosphate fertilizer was calcium dihydrogen phosphate (containing P2O5 12%) 687.5kg / hm2 2 Potassium fertilizer is potassium sulfate (containing K2O 57%) 144.74kg / hm 2 Other cultivation measures are the same as general high-yield field management.

[0030] Example 4

[0031] The difference between this embodiment and embodiment 1 is that the method for determining the root activity in step 2 is: flushing the roots at the greening stage, jointing stage, booting stage, filling stage, and maturity stage, taking 5 cm of the root tip, and using the improved TTC reduction method to determine the root activity.

[0032] Example 5

[0033] The difference between this embodiment and embodiment 1 is that: the method for determining the rhizosphere soil enzyme activity in step 2 is as follows: at the wheat heading stage, 3 points are randomly selected in each test plot, the plants are dug out with roots, and the rhizosphere soil is collected by shaking the soil method. The rhizosphere soil is 0 to 5 mm away from the root system, stored at 4°C and brought back to the laboratory as soon as possible. After passing through a 2 mm sieve, the root residues are removed and divided into two parts, one part is naturally air-dried and passed through a 0.25 mm sieve for determining the soil nutrient content; the other part is freshly stored in a -80°C refrigerator for determining the soil bacterial community and rhizosphere soil enzyme activity, and the soil enzymes include: β-glucosidase (B-glocusidase, referred to as BG), leucine aminopeptidase (leucine amiopeptidase, referred to as LAP) and polyphenol oxidase (referred to as POX).

[0034] The activities of soil BG enzyme and LAP enzyme were analyzed by microplate fluorescence method. The principle of the determination is that BG enzyme decomposes the substrate to generate p-nitrophenol, which has a maximum absorption peak at 400nm; LAP enzyme decomposes the substrate to generate p-nitroaniline, which has a maximum absorption peak at 405nm.

[0035] The soil POX enzyme activity was measured using a microplate colorimetric method. The principle of the assay is that POX enzyme can catalyze the substrate to generate a colored product, and its color-developing substance has a maximum absorption peak at 460 nm. The substrates of the three enzymes, BG, LAP and POX enzyme, are shown in Table 1.

[0036] Table 1 Soil enzyme names, abbreviations, numbers and substrates

[0037]

[0038] According to the determination principle, the absorbance value was read at a specific wavelength using a microplate reader (Labsystems Multiskan MS, Finland), and the various soil enzyme activities were calculated by measuring the rate of increase of the absorbance value. The results were uniformly expressed in nmol / H / g, i.e., the amount of substances that decompose the corresponding substrate to produce specific products per gram of fresh soil per hour.

[0039] Example 6

[0040] The difference between this embodiment and embodiment 5 is that the soil microbial high-throughput sequencing method in step 2 is: the soil microbial community is sequenced using the standard operating procedures of the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). The V3-V4 hypervariable region and the ITS region of the 16S rRNA gene were PCR amplified, and for each DNA sample, three replicates were independently PCR amplified using TransStart Fastpfu DNA polymerase on an ABI GeneAmp 9700PCR system (Applied Biosystems, Foster City, CA, USA), purified using an AxyPrep PCR purification kit (Axygen Biosciences, Union City, CA, USA), and double-end sequencing was performed using the Illumina MiSeq platform.

[0041] Example 7

[0042] The difference between this embodiment and embodiment 6 is that: the method for analyzing the measured data of the root activity in step 2 in step 3 is: analyzing the changing trend of the root activity of different test wheat varieties along the growth process; comparing the root activity values ​​of different test wheat varieties under N0 and N1 treatments.

[0043] Xuke 168 and Zhengpinmai 8 were selected as the tested wheat varieties. The root activity of the two wheat varieties was measured. Figure 1 As shown in the figure, the root activity of Xuke 168 and Zhengpinmai 8 increased first and then decreased with the growth process, and the root activity was the strongest in the booting stage.

[0044] The root system is an important organ of plants. It has the function of fixing and supporting plants, affecting the absorption of nutrients, water and minerals by plants. It is also an important place for the synthesis of polyvalent ions, organic acids, amino acids, etc. Wheat is a fibrous root crop. The size of the root activity reflects the strength of the root metabolic activity to a certain extent. Domestic and foreign scholars have found that root activity is closely related to crop varieties, soil types, fertilizer and water measures, and their own genetic traits; it reflects the absorption, synthesis, respiration and oxidation capacity of crop roots, and objectively reflects the strength of the root metabolic capacity. Xiong Shuping et al. believe that nitrogen-efficient genotype wheat has higher root activity and root metabolic capacity. The results of this study also confirmed this.

[0045] Under N0 and N1 treatments, the root activity of Xuke 168 was higher than that of Zhengpinmai No. 8, with an average increase of 16.76-70.14% and 14.44-37.04%, respectively. Under N0 treatment, the root activity of Xuke 168 was significantly higher than that of Zhengpinmai No. 8 in the jointing-maturity period. Under N1 treatment, except for the jointing and booting period, the root activity of the two varieties in other growth periods was not significantly different. Compared with the N0 treatment, the root activity of Xuke 168 increased by 16.75-44.56% on average under N1 treatment, and the root activity of Zhengpinmai No. 8 increased by 0.97-42.63% on average. It can be seen that the root activity of Xuke 168, a nitrogen-efficient wheat variety, was higher than that of Zhengpinmai No. 8, a nitrogen-inefficient wheat variety, under low nitrogen NO or high nitrogen N1, and the difference was obvious under low nitrogen treatment, reaching a significant level of 5%.

[0046] Example 8

[0047] The difference between this embodiment and embodiment 7 is that: the method for analyzing the determination data of rhizosphere soil enzyme activity in step 2 in step 3 is: the results of the changes in rhizosphere soil enzyme activity of Xuke 168 and Zhengpinmai No. 8 under the N0 and N1 treatments at the booting stage showed that compared with Zhengpinmai No. 8, the BG, LAP and POX enzyme activities of Xuke 168 increased under the two fertilization treatments; the increases under the N0 treatment were 59.38%, 34.43% and 30.50%, respectively, and reached a significant difference level; the increases under the N1 treatment were not obvious, which were 9.31%, 18.88% and 9.05%, respectively.

[0048] Table 2 Differences in rhizosphere soil enzyme activities between Xuke 168 and Zhengpinmai 8 (average data from 2018 to 2020)

[0049]

[0050] Soil enzymes are produced by animals, plants and microorganisms, and their enzyme activity is a key indicator of soil nutrient cycling and microbial metabolic activity. They are not only important participants in material cycling and energy conversion in the soil, but also catalysts for all biochemical reactions in the soil. Studies have shown that BG, LAP and POX participate in the catalytic reactions at the end of the carbon cycle and nitrogen cycle, and can well reflect the metabolic level of soil nutrients. The level of rhizosphere soil enzyme activity is closely related to the mineralization of soil nutrients and the degree of decomposition of organic matter. The increase in enzyme activity promotes the absorption of nutrients by crops. The results of this study showed that the application of nitrogen fertilizer increased soil enzyme activity, which may be due to the fact that microorganisms promote their own growth by assimilating and utilizing the applied nitrogen, resulting in a significant increase in the number and types of enzymes produced. Under normal nitrogen fertilizer N1 and no nitrogen fertilizer N0 conditions, the nitrogen efficient variety Xuke 168 showed higher soil enzyme activity, that is, under low nitrogen stress, BG, POX and LAP in the rhizosphere soil of the nitrogen efficient variety Xuke 168 still maintained high activity. Higher soil enzyme activity means that the material and energy conversion of the soil are very strong, thus promoting good plant growth. This shows that the nitrogen efficient wheat variety Xuke 168 has good adaptability under low nitrogen stress conditions.

[0051] Under low nitrogen conditions, the rhizosphere β-glucosidase, leucine aminopeptidase and polyphenol oxidase activities of the nitrogen-efficient wheat variety Xuke 168 were 59.38%, 34.43% and 30.50% higher than those of the nitrogen-inefficient wheat variety Zhengpinmai 8, respectively. It can be seen that the BG, POX and LAP enzyme activities in the rhizosphere soil of the nitrogen-efficient wheat variety Xuke 168 were higher than those of the nitrogen-inefficient wheat variety Zhengpinmai 8, regardless of low nitrogen NO or high nitrogen N1.

[0052] Example 9

[0053] The difference between this embodiment and embodiment 8 is that: the method for analyzing the high-throughput sequencing data of soil microorganisms in step 2 in step 3 is: 1) The sequencing results are first spliced, filtered, and chimeras are removed using QIIME (Quantitative Insights Into MicrobialEcology) (v1.2.1). High-quality sequences with a sequence length greater than 200bp, no incorrect bases in the Barcode and primer sequences, and an average quality score of Q≥25 are selected. The USEARCH software is used to divide the classification operation unit (OTU for short) at a threshold of 97%. According to the Silva database, the OTU representative sequences are aligned and classified. The Shannon and Simpson diversity indices and the Chao1 and ACE richness indices are calculated using Mothur software to evaluate bacterial α-diversity; 2) According to the relative abundance of rhizosphere soil bacterial communities at the phylum level and the relative abundance of dominant bacterial genera in rhizosphere soil, the rhizosphere soil bacterial community structure of Xuke 168 and Zhengpinmai No. 8 is analyzed.

[0054] From the characteristics of soil bacterial α-diversity under different nitrogen treatments in Table 3, it can be seen that the richness index (Chao1 index and ACE index) of the nitrogen-efficient wheat variety Xuke 168 is significantly higher than that of the nitrogen-inefficient wheat variety Zhengpinmai No. 8, with increases of 7.22%, 7.35% and 12.95% respectively, among which the Shannon index reaches a significant difference level of 5%. It can be seen that under the N0 treatment, the richness index (Chao1 index and ACE index) of Xuke 168 is significantly higher than that of Zhengpinmai No. 8, and the Shannon index and Simpson index of the diversity index of Xuke 168 are both higher than those of Zhengpinmai No. 8. Under the N1 treatment, there is no significant difference in the richness index and diversity index between Xuke 168 and Zhengpinmai No. 8.

[0055] Table 3 α-diversity characteristics of rhizospheric soil bacteria of Xuke 168 and Zhengpinmai 8

[0056]

[0057] like Figure 2 As shown in the figure, by classifying the soil bacterial communities of Xuke 168 and Zhengpinmai 8 at the phylum level, the two treatments contained a total of 28 phyla, of which 11 phyla had a relative abundance greater than 1%, among which Acidobacteria, Actinobacteria, Bacteroidetes, Proteobacteria and Thaumarchaea were dominant phyla, with a relative abundance of 84.36%-88.20%.

[0058] Compared with the N0 treatment, the relative abundance of Acidobacteria and Verrucomicrobia in the wheat variety Xuke 168 under N1 treatment decreased significantly by 38.68% and 28.16%, respectively, while the relative abundance of Bacteroidetes, Chlorobacterium and Firmicutes increased significantly by 17.68%, 66.50% and 59.68%, respectively; while the relative abundance of Acidobacteria, Planctomyces and Verrucomicrobia in Zhengpinmai No. 8 increased significantly by 62.36%, 40.79% and 50.18%, and Chlorobacterium decreased significantly by 32.84%.

[0059] Under the same treatment, the abundance of dominant phyla in the two varieties was also different. Under the N0 treatment, the Acidiobacillaceae of Xuke 168 increased by 66.21% compared with Zhengpinmai No. 8, and the Thaumarchaeota decreased by 11.74%. Under the N1 treatment, the Acidiobacillaceae of Xuke 168 decreased by 37.23% compared with Zhengpinmai No. 8, and the Thaumarchaeota increased by 13.30%. Other differences were not obvious.

[0060] Further comparative analysis showed that the two treatments contained a total of 857 genera, of which GP6, Nitrosococconia, and Unclassified Bacteria were dominant genera, e.g. Figure 3 shown.

[0061] Compared with Zhengmaipin 8, under N1 treatment, the genus Nitrosococconia in the rhizosphere soil of Xuke 168 increased significantly by 21.79%, the genus Unclassified Bacteria decreased significantly by 27.63%, and the genus GP6 decreased by 9.49%; under N0 treatment, the genus Nitrosococconia in the rhizosphere soil of Xuke 168 increased significantly by 38.06%, the genus Unclassified Bacteria increased by 12.92%, and the genus GP6 decreased significantly by 21.33%.

[0062] Soil microorganisms are one of the most active and important components in farmland soil ecosystems. They are rich in species and numbers, and play an important role in the mineralization and decomposition of soil organic matter and the formation of humic acid. The diversity of microorganisms and the richness of communities are considered important indicators for measuring soil fertility. There is a certain correlation between plant species and the structure and abundance of their rhizosphere microbial communities. Nong Zemei et al. believed that there were significant differences in the abundance of the main root microbial communities among different varieties. The results of this study also confirmed this view.

[0063] Based on the results of high-throughput sequencing analysis, the relative abundance of Acidobacteria in the rhizosphere soil of Xuke 168 was significantly higher than that of Zhengpinmai No. 8 under the N0 treatment, while the opposite was true under the N1 treatment. This may be because Acidobacteria are oligotrophic bacteria with slow growth rates and are enriched in environments with low nutrient content. The interaction between the root system and the rhizosphere soil of the nitrogen-efficient variety Xuke 168 under the N0 treatment is conducive to the growth of rhizosphere microorganisms, thereby increasing the activity of rhizosphere soil microorganisms. Some scholars believe that Acidobacteria and Thaumarchaeota are positively correlated in dryland crop soils, and the results of this study show that the relative abundance of Thaumarchaeota in the rhizosphere soil of Xuke 168 under the N0 treatment is significantly lower than that of Zhengpinmai No. 8, which may be related to the selected growth period and soil environment, because wheat varieties with different nitrogen efficiencies absorb soil available nitrogen to different degrees, affecting the balance of the soil nitrogen pool, changing soil properties to varying degrees, and thus affecting the soil microbial community.

[0064] Further analysis revealed that under the two nitrogen treatments, the dominant genera GP6 and Nitrosococconia of Xuke 168 and Zhengpinmai No. 8 had different degrees of difference. In addition, based on the results of high-throughput sequencing analysis, the main bacterial community species were basically similar between different nitrogen fertilizer treatments, and the main difference was in the abundance of the bacterial community. Under low nitrogen treatment, there were significant differences in the richness index and diversity index of rhizosphere microorganisms of wheat varieties with different nitrogen efficiency. Under high nitrogen treatment, the difference was not obvious. The fundamental reason was that the differences in root vitality, rhizosphere soil enzyme activity, etc. of wheat varieties with different nitrogen efficiency affected the distribution of soil bacterial community structure.

[0065] The results of high-throughput sequencing analysis showed that there were certain differences in the bacterial population structure of the rhizosphere soil of the two different varieties. Compared with Zhengpinmai No. 8, Xuke 168 significantly improved the α-diversity of the bacterial community. Under the same treatment, the abundance of dominant phyla of the two varieties was different. Under the N0 treatment, the Acidobacteria of Xuke 168 increased by 66.21% compared with Zhengpinmai No. 8, and the Thaumarchaea decreased by 11.74%. Under the N1 treatment, the Acidobacteria of Xuke 168 decreased by 37.23% compared with Zhengpinmai No. 8, and the Thaumarchaea increased by 13.30%. Other differences were not obvious; GP6, Nitrosococcobacteria and Unclassified Bacteria were dominant genera. Compared with the nitrogen-inefficient variety Zhengmai No. 8, Nitrosococcobacteria in the rhizosphere soil of Xuke 168 increased significantly by 21.79% under the N1 treatment; under the N0 treatment, it increased significantly by 38.06%. Therefore, the results of this study confirmed that nitrogen-efficient wheat varieties can increase the diversity of bacterial communities and improve the composition of bacterial communities.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for screening nitrogen-efficient wheat varieties, characterized in that: The following steps are included: Step 1: Select several wheat varieties for field experiment and set two nitrogen fertilizer treatments: no nitrogen fertilizer treatment N0 (0 kg N / hm2) 2 )、N1, normal nitrogen fertilizer treatment; Step 2, measuring the root activity and rhizosphere soil enzyme activity of the tested wheat varieties under N0 and N1 treatments, and performing high-throughput sequencing of soil microorganisms; Step 3, analyzing the measured data in step 2, and screening out the test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, that is, nitrogen-efficient wheat varieties; In step 1, the nitrogen fertilizer application rate in the normal nitrogen fertilizer treatment N1 was 165 kg N / hm 2 The ratio of nitrogen fertilizer to base and topdressing is 5:5, and topdressing is applied in furrows during the green period; Phosphate fertilizer and potash fertilizer were applied as base fertilizer before sowing. Nitrogen fertilizer was urea and phosphorus fertilizer was calcium dihydrogen phosphate 687.5 kg / hm2. 2 Potassium fertilizer is potassium sulfate, the application rate is 144.74kg / hm 2 ; The soil enzymes in the rhizosphere soil enzyme activity determined in step 2 include: β-glucosidase, leucine aminopeptidase and polyphenol oxidase; the rhizosphere soil of the tested wheat varieties under the N0 and N1 treatments at the booting stage were selected, and the soil BG enzyme and LAP enzyme activities were determined by microplate fluorescence analysis, and the soil POX enzyme activity was determined by microplate colorimetry; The method for analyzing the soil microbial high-throughput sequencing data in step 2 in step 3 is as follows: 1) The sequencing results are first spliced, filtered, and chimeras are removed using QIIME; high-quality sequences with a sequence length greater than 200bp, no incorrect bases in the Barcode and primer sequences, and an average quality score of Q≥25 are selected; the USEARCH software is used to divide the classification operation units at a threshold of 97%, and the OTU representative sequences are aligned and classified according to the Silva database. The Shannon and Simpson diversity indices and the Chao1 and ACE richness indices are calculated using Mothur software to evaluate bacterial α-diversity; 2) The rhizosphere soil bacterial community structure of the tested wheat varieties is analyzed based on the relative abundance of the rhizosphere soil bacterial community at the phylum level and the relative abundance of the dominant bacterial genera in the rhizosphere soil.

2. The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: In step 1, the experimental plots in the field experiment were randomly arranged, and the area of ​​each experimental plot was 6*9=54 m 2 , row spacing 20cm, repeated 3 times, sowing rate 150kg / hm, sowing in mid-October.

3. The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: The method for determining root activity in step 2 is: flush the roots at the greening stage, jointing stage, booting stage, filling stage, and maturity stage, take 5 cm of the root tip, and use the improved TTC reduction method to determine the root activity.

4. The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: The soil microbial high-throughput sequencing method in step 2 is: using the standard operating procedures of the Illumina MiSeq platform, the soil microbial community of the rhizosphere soil of the tested wheat varieties under the N0 and N1 treatments at the booting stage was sequenced; The V3-V4 hypervariable region and the ITS region of the 16S rRNA gene were PCR amplified. For each DNA sample, three independent PCR amplifications were performed on the ABI GeneAmp 9700 PCR system using TransStart Fastpfu DNA polymerase, purified using the AxyPrep PCR purification kit, and sequenced using the Illumina MiSeq platform.

5. The method for screening nitrogen-efficient wheat varieties according to claim 3, characterized in that: The method for analyzing the measured data of root activity in step 2 in step 3 is: analyzing the changing trends of root activity of different test wheat varieties with the growth process; comparing the root activity values ​​of different test wheat varieties under N0 and N1 treatments.

6. The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: The method for analyzing the measured data of rhizosphere soil enzyme activity in step 2 in step 3 is: selecting the rhizosphere soil enzyme activity values ​​under the N0 and N1 treatments at the booting stage, and comparing the rhizosphere soil enzyme activity values ​​between different tested wheat varieties.

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