WO2023208205A1 - Method of screening for high nitrogen use efficiency wheat cultivars - Google Patents
Method of screening for high nitrogen use efficiency wheat cultivars Download PDFInfo
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- WO2023208205A1 WO2023208205A1 PCT/CN2023/091643 CN2023091643W WO2023208205A1 WO 2023208205 A1 WO2023208205 A1 WO 2023208205A1 CN 2023091643 W CN2023091643 W CN 2023091643W WO 2023208205 A1 WO2023208205 A1 WO 2023208205A1
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- nitrogen
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- wheat varieties
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Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Definitions
- the present invention relates to the technical field of selective breeding, and in particular to a method for screening nitrogen-efficient wheat varieties.
- Soil enzymes and soil microorganisms are important participants in a variety of biochemical reactions, material cycles and energy metabolism in soil. They play a key role in nutrient cycling, decomposing organic matter and degrading pollutants, and can characterize soil fertility and soil health.
- the rhizosphere is an important connection platform for plants, soil, microorganisms and their growth environment. It is a micro domain at the interface between the root system and soil. Analyzing and comparing microecological characteristics such as rhizosphere soil enzyme activity and microbiome structure of different crops will help provide theoretical basis for low-fertilizer and high-efficiency cultivation of crops from a microecological perspective.
- Zhao Ben et al. constructed a wheat aboveground nitrogen deficit model based on critical nitrogen concentration for precise fertilization.
- Zhang Juanjuan et al. conducted nitrogen nutrition diagnostic studies on wheat varieties with different nitrogen efficiencies and found that different varieties have different nitrogen absorption and utilization efficiency, root activity and low nitrogen stress tolerance. However, there are few studies on the mechanism of efficient nitrogen utilization. In recent years, the relationship between grain-soil sustainable productivity and plant rhizosphere microflora has gradually attracted the attention of scientific researchers.
- the purpose of the present invention is to provide a method for screening nitrogen-efficient wheat varieties in view of the shortcomings of the existing technology.
- the screened out wheat varieties have high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and Test wheat varieties with high diversity index are nitrogen-efficient wheat varieties, which provides a theoretical basis for screening nitrogen-efficient wheat varieties from the perspective of rhizosphere micro-environment differences.
- the present invention adopts the following technical solution: a method for screening nitrogen-efficient wheat varieties, including the following steps: Step 1, select several test wheat varieties, conduct field trials, and set up two nitrogen supply levels of fertilization treatments : No nitrogen fertilizer treatment N0 (0kg N/hm 2 ), normal nitrogen fertilizer treatment N1;
- Step 2 Determine the root activity and rhizosphere soil enzyme activity of the test wheat varieties under N0 and N1 treatments, and conduct high-throughput sequencing of soil microorganisms;
- Step 3 Analyze the measurement data in Step 2 and select test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, which are nitrogen-efficient wheat varieties.
- step 1 the amount of nitrogen fertilizer applied in the normal nitrogen fertilizer treatment N1 is 165kgN/hm 2 , the nitrogen fertilizer base topdressing ratio is 5:5, and the topdressing fertilizer is applied in ditches during the greening stage.
- step 1 the test plots in the field test were randomly arranged.
- the seeding rate was 150kg/hm, sowing in mid-October; phosphate fertilizer and potassium fertilizer were It is applied once as a base fertilizer before sowing.
- the nitrogen fertilizer is urea
- the phosphorus fertilizer is calcium dihydrogen phosphate 687.5kg/hm 2
- the potassium fertilizer is potassium sulfate at an application rate of 144.74kg/hm 2 .
- the method for measuring root activity in step 2 is: punch the roots at the greening stage, jointing stage, booting stage, grain filling stage, and maturity stage, take 5cm of the root system from the root tip, and use the modified TTC reduction method to measure the root activity.
- the soil enzymes measured in rhizosphere soil enzyme activity in step 2 include: ⁇ -glucosidase, leucine aminopeptidase and polyphenol oxidase; select the rhizosphere soil of test wheat varieties under N0 and N1 treatments at the booting stage.
- soil BG enzyme and LAP enzyme activities were measured by microplate fluorescence analysis method
- soil POX enzyme activity was measured by microplate colorimetric method.
- the high-throughput sequencing method of soil microorganisms in step 2 is: using the standard operating procedures of the Illumina MiSeq platform to sequence the soil microbial communities in the rhizosphere soil of the test wheat varieties under the N0 and N1 treatments at the booting stage; PCR amplification of the 16S rRNA gene For each DNA sample, three replicates were independently PCR amplified using TransStart Fastpfu DNA Polymerase on the ABI GeneAmp 9700 PCR System and purified using the AxyPrep PCR Purification Kit. And use the Illumina MiSeq platform for paired-end sequencing.
- step 3 the method for analyzing the measurement data of root activity in step 2 is: analyze the changing trend of root activity of different test wheat varieties with the growth process; compare the root activity of different test wheat varieties under N0 and N1 treatments numerical value.
- step 3 the method for analyzing the measurement data of rhizosphere soil enzyme activity in step 2 is: select the rhizosphere soil enzyme activity values under the N0 and N1 treatments at the booting stage, and compare the rhizosphere soil enzyme activity values between different tested wheat varieties. .
- the method for analyzing the soil microbial high-throughput sequencing data in step 2 in step 3 is: 1) The sequencing results first use QIIME to splice, filter, and remove chimeras on the original data; select the sequence length greater than 200 bp, Barcode and primers High-quality sequences with no error bases and an average quality score Q ⁇ 25; use USEARCH software to divide classification operation units under the 97% threshold, according to the Silva number According to the database, OTU representative sequences are compared and classified, and Mothur software is used to calculate Shannon, Simpson diversity index and Chao1, ACE richness index to evaluate bacterial alpha-diversity; 2) According to the rhizosphere soil bacterial community at the phylum level and the relative abundance of dominant bacterial genera in the rhizosphere soil to analyze the bacterial community structure of the rhizosphere soil of the tested wheat varieties.
- the method of screening nitrogen-efficient wheat varieties of the present invention comprehensively analyzes the differences in wheat root activity of different nitrogen-efficient varieties and their impact on rhizosphere soil enzyme activity and microbial community diversity, and concludes that nitrogen-efficient wheat varieties can enhance soil enzyme activity and increase bacterial communities. Diversity and improved bacterial community composition.
- the test wheat varieties screened by the present invention have high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, which can be mutually confirmed with known wheat varieties with different nitrogen efficiencies. It provides a theoretical basis for screening nitrogen-efficient wheat varieties from the perspective of rhizosphere micro-environment differences.
- Figure 1 shows the difference in root activity of wheat varieties with different nitrogen efficiencies of the present invention (average data from 2018 to 2020);
- Figure 2 shows the relative abundance of rhizosphere soil bacterial communities at the phylum level of wheat varieties with different nitrogen efficiencies of the present invention
- Figure 3 shows the relative abundance of dominant bacterial genera in the rhizosphere soil of wheat varieties with different nitrogen efficiencies of the present invention.
- a method for screening nitrogen-efficient wheat varieties including the following steps: Step 1, select several wheat varieties for testing, conduct field trials, and set two nitrogen supply levels of fertilization treatments: no nitrogen fertilizer treatment N0 (0kg N/hm 2 ), normal nitrogen fertilizer treatment N1;
- Step 2 Determine the root activity and rhizosphere soil enzyme activity of the test wheat varieties under N0 and N1 treatments, and conduct high-throughput sequencing of soil microorganisms;
- Step 3 Analyze the measurement data in Step 2 and select test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, which are nitrogen-efficient wheat varieties.
- step 1 the nitrogen fertilizer application amount in the normal nitrogen fertilizer treatment N1 is 165kgN/hm 2 , the nitrogen fertilizer base topdressing ratio is 5:5, and the topdressing fertilizer is applied in ditches during the greening stage.
- Nitrogen fertilizer is urea (containing N46%).
- Phosphate fertilizer and potassium fertilizer are applied once as base fertilizer before sowing.
- the phosphate fertilizer is calcium dihydrogen phosphate (containing P 2 O 5 12%) 687.5kg/hm 2
- the potassium fertilizer is potassium sulfate (containing K 2 O 57%) 144.74kg/hm. 2.
- Other cultivation measures are the same as general high-yield field management.
- step 2 punch the roots at the greening stage, jointing stage, booting stage, grain filling stage, and maturity stage, take 5cm of the root system from the root tip, and use Modified TTC reduction method was used to measure root activity.
- the method for measuring rhizosphere soil enzyme activity in step 2 is as follows: the rhizosphere soil is at the wheat booting stage, 3 points are randomly selected in each test plot, and the plants are dug up and down. out, use the soil shaking method to collect the rhizosphere soil.
- the rhizosphere soil is 0 to 5mm away from the root system. Store it at 4°C and bring it back to the laboratory as soon as possible. After passing through a 2mm sieve, remove the root residue and divide it into two parts.
- Soil enzymes include: ⁇ -glucosidase (B-glucosidase, BG for short), leucine amiopeptidase (LAP for short) and polyphenol oxidase (POX for short).
- Soil BG enzyme and LAP enzyme activities are analyzed by microplate fluorescence method.
- the measurement principle 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. , the latter has a maximum absorption peak at 405nm.
- the soil POX enzyme activity adopts the microplate colorimetric method.
- the measurement principle is that POX enzyme can catalyze the substrate to produce colored products, and the chromogenic material has a maximum absorption peak at 460nm.
- the substrates of the three enzymes BG, LAP and POX are shown in Table 1.
- nmol/H/g is the amount of substances that decompose the corresponding substrate to produce specific products per gram of fresh soil per hour.
- the soil microorganism high-throughput sequencing method in step 2 is: using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) to label Standard operating procedures for sequencing soil microbial communities.
- the V3-V4 hypervariable regions of the 16S rRNA gene as well as the ITS region were PCR amplified.
- three Independent PCR amplification was performed in duplicate, purified using the AxyPrep PCR Purification Kit (Axygen Biosciences, Union City, CA, USA), and paired-end sequencing was performed using the Illumina MiSeq platform.
- step 3 the method for analyzing the measurement data of root activity in step 2 is: analyzing the changing trend of root activity of different wheat varieties tested with the growth process; comparing the results between N0 and N1 Root activity values of different tested wheat varieties under treatment.
- Xuke 168 and Zhengpinmai 8 were selected as the test wheat varieties. The measured root activity of the two wheat varieties is shown in Figure 1. The root activity of Xuke 168 and Zhengpinmai 8 both increased with the growth process. The tendency is to increase and then decrease, with the root activity being the strongest at the booting stage.
- the root system is an important organ of the plant. It has the function of fixing and supporting the plant, affecting the plant's absorption of nutrients, water and minerals. It is also an important place for the synthesis of polyions, organic acids, amino acids, etc.
- Wheat is a fibrous root crop, and the root activity reflects the metabolic activity of the root system to a certain extent. Domestic and foreign researchers 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 capabilities of crop roots, and objectively reflects the strong metabolism of the root system. weak. Studies by Xiong Shuping and others believe that nitrogen-efficient genotype wheat has higher root activity and root metabolic capacity. The results of this study also confirmed this.
- the root activity of Xuke 168 was higher than that of Zhengpinmai 8, with average increases of 16.76-70.14% and 14.44-37.04% respectively; among them, under N0 treatment, the root activity of Xuke 168 in the jointing stage to maturity stage was The root activity was significantly higher than that of Zhengpinmai No. 8. Under N1 treatment, except for the jointing stage and booting stage, there was no significant difference in root activity between the two varieties in other growth stages. 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 8 increased by 0.97-42.63% on average.
- step 3 the method for analyzing the measurement data of rhizosphere soil enzyme activity in step 2 is: selecting the results of Xuke 168 and Zhengpinmai 8 under the N0 and N1 treatments at the booting stage.
- the results of rhizosphere soil enzyme activity changes showed that compared with Zhengpinmai 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% and 34.43% respectively. and 30.50%, and reached a significant level of difference; the increase under N1 treatment was not obvious, which were 9.31%, 18.88% and 9.05% respectively.
- 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. It is not only an important participant in the material cycle and energy conversion in the soil, but also the catalyst for all biochemical reactions in the soil. Research shows that BG, LAP and POX participate in the catalytic reactions at the end of the carbon cycle and nitrogen cycle, and can excellently reflect the metabolic level of soil nutrients.
- the level of rhizosphere soil enzyme activity is closely related to the mineralization amount of soil nutrients and the degree of decomposition of organic matter. The increase in enzyme activity promotes the absorption of nutrients by crops.
- the rhizosphere ⁇ -glucosidase, leucine aminopeptidase and polyphenol oxidase activities of the nitrogen-efficient wheat variety Xuke 168 were 59.38% higher than those of the nitrogen-inefficient wheat variety Zhengpinmai No. 8, respectively. 34.43% and 30.50%. It can be seen that no matter under low nitrogen N0 or high nitrogen N1, the activities of BG, POX and LAP enzymes in the rhizosphere soil of the nitrogen-efficient wheat variety Xuke 168 are higher than those of the nitrogen-inefficient wheat variety Zhengpinmai 8.
- Example 8 The difference between this example and Example 8 is that the method for analyzing the soil microbial high-throughput sequencing data in step 2 in step 3 is: 1) The sequencing results are first analyzed using QIIME (Quantitative Insights Into Microbial Ecology) (v1.2.1) Raw data were spliced, filtered, and chimeras removed. Select high-quality sequences with a sequence length greater than 200 bp, Barcode and primer sequences without error bases, and an average quality score Q ⁇ 25. Use USEARCH software to divide classification operation units (OTUs for short) at the 97% threshold. According to the Silva database, OTU representative sequences were compared and classified.
- QIIME Quality of Into Microbial Ecology
- 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 8.
- the increases were 7.22%, 7.35% and 12.95% respectively, among which the Shannon index reached a significant difference level of 5%.
- the richness index (Chao1 index and ACE index) of Xuke 168 It is significantly higher than Zhengpinmai No. 8.
- the Shannon index and Simpson index of Xu Ke 168 diversity index are both higher than Zhengpinmai No. 8. Under N1 treatment, the difference in richness index and diversity index between Xuke 168 and Zhengpinmai 8 was not obvious.
- the relative abundance of Acidobacteria and Verrucomicrobia in the wheat variety Xuke 168 under the N1 treatment was significantly reduced by 38.68% and 28.16%, respectively.
- the relative abundance of phylum increased significantly by 17.68%, 66.50% and 59.68% respectively; while the Acidobacteria, Planctomycetes and Verrucomicrobia in Zhengpinmai 8 increased significantly by 62.36%, 40.79% and 50.18%, and the green Bay fungi were significantly reduced by 32.84%.
- 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. Microbial diversity and community richness are considered important indicators of soil fertility status. There is a certain correlation between plant species and their rhizosphere microbial community structure and abundance. Research by Nong Zemei and others concluded that there are significant differences in the abundance of the main bacterial groups in the root microorganisms among different varieties. The results of this study also confirmed this view.
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Abstract
Provided is a method of screening for high nitrogen use efficiency wheat cultivars, comprising the following steps: step 1, selecting a plurality of wheat cultivars for testing, carrying out a field test, and setting two fertilization treatment nitrogen supply levels: fertilization treatment without application of nitrogen (N0), and fertilization treatment with normal application of nitrogen (N1); step 2, taking measurements to determine the root system activity and rhizosphere soil enzyme activity of said wheat cultivars under the N0 treatment and the N1 treatment, and carrying out high-throughput sequencing on the soil microorganisms; step 3, analyzing the measurement data of step 2, and screening for wheat cultivars having high root system activity, high rhizosphere soil enzyme activity, a high rhizosphere microorganism richness index and a high diversity index, such cultivars being considered high nitrogen use efficiency wheat cultivars. In the method of screening for high nitrogen use efficiency wheat cultivars, the identified high nitrogen use efficiency wheat cultivars having high root system activity, high rhizosphere soil enzyme activity, a high rhizosphere microorganism richness index, and a high diversity index provide a theoretical basis for screening for high nitrogen use efficiency wheat cultivars in different rhizosphere micro-domain environments.
Description
本发明涉及选种育种技术领域，尤其是涉及一种筛选氮高效小麦品种的方法。The present invention relates to the technical field of selective breeding, and in particular to a method for screening nitrogen-efficient wheat varieties.
土壤酶和土壤微生物是土壤中多种生化反应、物质循环和能量代谢的重要参与者，在养分循环、分解有机质及降解污染物方面发挥关键作用，可以表征土壤肥力和土壤健康状况。根际是植物、土壤、微生物及其生长环境的一个重要衔接平台，是根系与土壤交界的微域。分析和比较不同作物的根际土壤酶活性及微生物群结构等微生态特性，有利于从微生态学角度提供作物低肥高效栽培理论依据。Soil enzymes and soil microorganisms are important participants in a variety of biochemical reactions, material cycles and energy metabolism in soil. They play a key role in nutrient cycling, decomposing organic matter and degrading pollutants, and can characterize soil fertility and soil health. The rhizosphere is an important connection platform for plants, soil, microorganisms and their growth environment. It is a micro domain at the interface between the root system and soil. Analyzing and comparing microecological characteristics such as rhizosphere soil enzyme activity and microbiome structure of different crops will help provide theoretical basis for low-fertilizer and high-efficiency cultivation of crops from a microecological perspective.
小麦是中国重要的粮食作物之一，小麦产业发展直接关系到中国的粮食安全和社会稳定。近年来，中国小麦连年增产，但增产的同时伴随着氮肥的过量施用及肥效下降，从而影响生态环境的健康发展。例如，大量施用化肥导致了土壤酸化、温室效应及生物多样性丧失等一系列的生态环境问题。基于经济效益和生态环境保护的双重要求，相关学者已就小麦“肥药双减”问题开展了不同角度的研究。杨晓卡等从栽培措施、轮作体系上探讨出氮素表观损失量最低的高产高效模式。赵犇等构建了基于临界氮浓度的小麦地上部氮亏缺模型进行精准施肥。张娟娟等通过不同氮效率小麦品种的氮素营养诊断研究，发现不同品种的氮吸收利用效率、根系活力及耐低氮胁迫能力不同。但有关氮素高效利用机制的研究较少。近年来，粮食-土壤可持续生产力与植物根际微生物区系的关系逐渐被科研工作者关注。董航宇从根际微生态角度研究了土壤微生物、土壤酶活性与粳稻高效利用氮的关系。杨珍等基于作物根际微域探究植物病害发
生机理，挖掘具有潜力的微生物资源；相反，通过根际微域中的土壤酶活性和微生物多样性也能体现出不同作物或者同一作物不同品种间的差异。目前关于小麦根际土壤微生态区系的研究多集中在氮素的吸收、利用及耕作方式方面，关于不同氮效率小麦品种的根际微生物和土壤酶活性差异的研究较少。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 after year, but the increase in production has been accompanied by excessive application of nitrogen fertilizers and reduced fertilizer efficiency, which has affected 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 environment protection, relevant scholars have conducted research on the issue of "double reduction of fertilizers and pesticides" in wheat from different angles. Yang Xiaoka and others explored a high-yield and high-efficiency model with the lowest apparent nitrogen loss based on cultivation measures and crop rotation systems. Zhao Ben et al. constructed a wheat aboveground nitrogen deficit model based on critical nitrogen concentration for precise fertilization. Zhang Juanjuan et al. conducted nitrogen nutrition diagnostic studies on wheat varieties with different nitrogen efficiencies and found that different varieties have different nitrogen absorption and utilization efficiency, root activity and low nitrogen stress tolerance. However, there are few studies on the mechanism of efficient nitrogen utilization. In recent years, the relationship between grain-soil sustainable productivity and plant rhizosphere microflora has gradually attracted the attention of scientific researchers. Dong Hangyu studied the relationship between soil microorganisms, soil enzyme activity and the efficient use of nitrogen by japonica rice from the perspective of rhizosphere microecology. Yang Zhen et al. studied the occurrence of plant diseases based on crop rhizosphere micro-regions. Physiology, and explore potential microbial resources; on the contrary, 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 micro-domain. At present, most research on wheat rhizosphere soil microecological flora focuses on nitrogen absorption, utilization and farming methods. There are few studies on the differences in rhizosphere microorganisms and soil enzyme activities of wheat varieties with different nitrogen efficiencies.
发明内容Contents of the invention
有鉴于此，本发明的目的是针对现有技术的不足，提供一种筛选氮高效小麦品种的方法，筛选出的根系活力高、根际土壤酶活性高、根际微生物的丰富度指数高和多样性指数高的供试小麦品种即为氮高效小麦品种，为从根际微域环境差异角度筛选氮高效小麦品种提供理论依据。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 shortcomings of the existing technology. The screened out wheat varieties have high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and Test wheat varieties with high diversity index are nitrogen-efficient wheat varieties, which provides a theoretical basis for screening nitrogen-efficient wheat varieties from the perspective of rhizosphere micro-environment differences.
为达到上述目的，本发明采用以下技术方案：一种筛选氮高效小麦品种的方法，包括如下步骤：步骤1，选取若干个供试小麦品种，进行大田试验，设置2个供氮水平的施肥处理：不施氮肥处理N0(0kg N/hm2)、正常施氮肥处理N1；In order to achieve the above purpose, the present invention adopts the following technical solution: a method for screening nitrogen-efficient wheat varieties, including the following steps: Step 1, select several test wheat varieties, conduct field trials, and set up two nitrogen supply levels of fertilization treatments : No nitrogen fertilizer treatment N0 (0kg N/hm 2 ), normal nitrogen fertilizer treatment N1;
步骤2，测定N0和N1处理下供试小麦品种的根系活力、根际土壤酶活性，以及进行土壤微生物进行高通量测序；Step 2: Determine the root activity and rhizosphere soil enzyme activity of the test wheat varieties under N0 and N1 treatments, and conduct high-throughput sequencing of soil microorganisms;
步骤3，分析步骤2中的测定数据，筛选出根系活力高、根际土壤酶活性高、根际微生物的丰富度指数高和多样性指数高的供试小麦品种，即为氮高效小麦品种。Step 3: Analyze the measurement data in Step 2 and select test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, which are nitrogen-efficient wheat varieties.
进一步地，步骤1中正常施氮肥处理N1中氮肥施用量为165kgN/hm2，氮肥基追比为5∶5，追肥于返青期开沟施入。Further, in step 1, the amount of nitrogen fertilizer applied in the normal nitrogen fertilizer treatment N1 is 165kgN/hm 2 , the nitrogen fertilizer base topdressing ratio is 5:5, and the topdressing fertilizer is applied in ditches during the greening stage.
进一步地，步骤1中大田试验中试验小区随机排列，每个试验小区面积为6*9＝54m2，行距20cm，重复3次，播种量为150kg/hm，10月中旬播种；磷肥和钾肥在播前作为基肥一次性施入，其中氮肥为尿素，磷肥为磷酸二氢钙687.5kg/hm2，钾肥为硫酸钾施用量144.74kg/hm2。
Further, in step 1, the test plots in the field test were randomly arranged. The area of each test plot was 6*9=54m 2 and the row spacing was 20cm. Repeated 3 times, the seeding rate was 150kg/hm, sowing in mid-October; phosphate fertilizer and potassium fertilizer were It is applied once as a base fertilizer before sowing. The nitrogen fertilizer is urea, the phosphorus fertilizer is calcium dihydrogen phosphate 687.5kg/hm 2 , and the potassium fertilizer is potassium sulfate at an application rate of 144.74kg/hm 2 .
进一步地，步骤2中测定根系活力的方法为：分别于返青期、拔节期、孕穗期、灌浆期、成熟期冲根，取根尖处5cm根系，采用改良TTC还原法测定根系活力。Further, the method for measuring root activity in step 2 is: punch the roots at the greening stage, jointing stage, booting stage, grain filling stage, and maturity stage, take 5cm of the root system from the root tip, and use the modified TTC reduction method to measure the root activity.
进一步地，步骤2中测定根际土壤酶活性中土壤酶包括：β-葡萄糖苷酶、亮氨酸氨基肽酶和多酚氧化酶；选取孕穗期N0和N1处理下供试小麦品种根际土，土壤BG酶和LAP酶活性采用微孔板荧光法分析法测定，土壤POX酶活性采用微孔板比色法测定。Further, the soil enzymes measured in rhizosphere soil enzyme activity in step 2 include: β-glucosidase, leucine aminopeptidase and polyphenol oxidase; select the rhizosphere soil of test wheat varieties under N0 and N1 treatments at the booting stage. , soil BG enzyme and LAP enzyme activities were measured by microplate fluorescence analysis method, and soil POX enzyme activity was measured by microplate colorimetric method.
进一步地，步骤2中土壤微生物高通量测序方法为：利用Illumina MiSeq平台标准操作规程，对孕穗期N0和N1处理下供试小麦品种根际土土壤微生物群落进行测序；PCR扩增16S rRNA基因的V3-V4高变区以及ITS区域，对于每个DNA样品，在ABI GeneAmp 9700 PCR系统上使用TransStart Fastpfu DNA聚合酶对三个重复进行独立的PCR扩增，使用AxyPrep PCR纯化试剂盒进行纯化，并使用Illumina MiSeq平台进行双末端测序。Furthermore, the high-throughput sequencing method of soil microorganisms in step 2 is: using the standard operating procedures of the Illumina MiSeq platform to sequence the soil microbial communities in the rhizosphere soil of the test wheat varieties under the N0 and N1 treatments at the booting stage; PCR amplification of the 16S rRNA gene For each DNA sample, three replicates were independently PCR amplified using TransStart Fastpfu DNA Polymerase on the ABI GeneAmp 9700 PCR System and purified using the AxyPrep PCR Purification Kit. And use the Illumina MiSeq platform for paired-end sequencing.
进一步地，步骤3中分析步骤2中根系活力的测定数据方法为：分析不同供试小麦品种根系活力均随着生育进程的变化趋势；对比在N0和N1处理下不同供试小麦品种的根系活力数值。Further, in step 3, the method for analyzing the measurement data of root activity in step 2 is: analyze the changing trend of root activity of different test wheat varieties with the growth process; compare the root activity of different test wheat varieties under N0 and N1 treatments numerical value.
进一步地，步骤3中分析步骤2中根际土壤酶活性的测定数据方法为：选取孕穗期N0和N1处理下根际土壤酶活性数值，对比不同供试小麦品种之间的根际土壤酶活性数值。Further, in step 3, the method for analyzing the measurement data of rhizosphere soil enzyme activity in step 2 is: select the rhizosphere soil enzyme activity values under the N0 and N1 treatments at the booting stage, and compare the rhizosphere soil enzyme activity values between different tested wheat varieties. .
进一步地，步骤3中分析步骤2中土壤微生物高通量测序数据方法为：1)测序结果首先使用QIIME对原始数据进行拼接，过滤，并去除嵌合体；挑出序列长度大于200bp，Barcode和引物序列无错误碱基，平均质量得分Q≥25的高质量序列；采用USEARCH软件在97％的阈值下划分分类操作单元，根据Silva数
据库，比对OTU代表序列并进行分类，利用Mothur软件计算Shannon、Simpson多样性指数及Chao1、ACE丰富度指数，用于评价细菌α-多样性；2)根据根际土壤细菌群落在门水平上的相对丰度，以及根际土壤优势细菌属的相对丰度，分析供试小麦品种根际土壤细菌群落结构。Further, the method for analyzing the soil microbial high-throughput sequencing data in step 2 in step 3 is: 1) The sequencing results first use QIIME to splice, filter, and remove chimeras on the original data; select the sequence length greater than 200 bp, Barcode and primers High-quality sequences with no error bases and an average quality score Q ≥ 25; use USEARCH software to divide classification operation units under the 97% threshold, according to the Silva number According to the database, OTU representative sequences are compared and classified, and Mothur software is used to calculate Shannon, Simpson diversity index and Chao1, ACE richness index to evaluate bacterial alpha-diversity; 2) According to the rhizosphere soil bacterial community at the phylum level and the relative abundance of dominant bacterial genera in the rhizosphere soil to analyze the bacterial community structure of the rhizosphere soil of the tested wheat varieties.
本发明的有益效果是：The beneficial effects of the present invention are:
本发明筛选氮高效小麦品种的方法综合分析不同氮效率品种小麦根系活力差异及其对根际土壤酶活性和微生物群落多样性的影响，得出氮高效小麦品种能够增强土壤酶活性、提高细菌群落多样性及改善细菌群落组成。本发明筛选出的根系活力高、根际土壤酶活性高、根际微生物的丰富度指数高和多样性指数高的供试小麦品种，可以与已知的不同氮效率小麦品种相互印证，为从根际微域环境差异角度筛选氮高效小麦品种提供理论依据。The method of screening nitrogen-efficient wheat varieties of the present invention comprehensively analyzes the differences in wheat root activity of different nitrogen-efficient varieties and their impact on rhizosphere soil enzyme activity and microbial community diversity, and concludes that nitrogen-efficient wheat varieties can enhance soil enzyme activity and increase bacterial communities. Diversity and improved bacterial community composition. The test wheat varieties screened by the present invention have high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, which can be mutually confirmed with known wheat varieties with different nitrogen efficiencies. It provides a theoretical basis for screening nitrogen-efficient wheat varieties from the perspective of rhizosphere micro-environment differences.
附图1为本发明不同氮效率小麦品种根系活力的差异(2018-2020两年的平均数据)；Figure 1 shows the difference in root activity of wheat varieties with different nitrogen efficiencies of the present invention (average data from 2018 to 2020);
附图2为本发明不同氮效率小麦品种的根际土壤细菌群落在门水平上的相对丰度；Figure 2 shows the relative abundance of rhizosphere soil bacterial communities at the phylum level of wheat varieties with different nitrogen efficiencies of the present invention;
附图3为本发明不同氮效率小麦品种的根际土壤优势细菌属的相对丰度。Figure 3 shows the relative abundance of dominant bacterial genera in the rhizosphere soil of wheat varieties with different nitrogen efficiencies of the present invention.
以下是本发明的具体实施例，对本发明的技术方案作进一步的描述，但本发明并不限于这些实施例。The following are specific examples of the present invention to further describe the technical solution of the present invention, but the present invention is not limited to these examples.
实施例1Example 1
一种筛选氮高效小麦品种的方法，包括如下步骤：步骤1，选取若干个供试小麦品种，进行大田试验，设置2个供氮水平的施肥处理：不施氮肥处理N0(0kg
N/hm2)、正常施氮肥处理N1；A method for screening nitrogen-efficient wheat varieties, including the following steps: Step 1, select several wheat varieties for testing, conduct field trials, and set two nitrogen supply levels of fertilization treatments: no nitrogen fertilizer treatment N0 (0kg N/hm 2 ), normal nitrogen fertilizer treatment N1;
步骤2，测定N0和N1处理下供试小麦品种的根系活力、根际土壤酶活性，以及进行土壤微生物进行高通量测序；Step 2: Determine the root activity and rhizosphere soil enzyme activity of the test wheat varieties under N0 and N1 treatments, and conduct high-throughput sequencing of soil microorganisms;
步骤3，分析步骤2中的测定数据，筛选出根系活力高、根际土壤酶活性高、根际微生物的丰富度指数高和多样性指数高的供试小麦品种，即为氮高效小麦品种。Step 3: Analyze the measurement data in Step 2 and select test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, which are nitrogen-efficient wheat varieties.
实施例2Example 2
本实施例与实施例1的不同之处在于：步骤1中正常施氮肥处理N1中氮肥施用量为165kgN/hm2，氮肥基追比为5∶5，追肥于返青期开沟施入。氮肥为尿素(含N46％)。The difference between this embodiment and Example 1 is that in step 1, the nitrogen fertilizer application amount in the normal nitrogen fertilizer treatment N1 is 165kgN/hm 2 , the nitrogen fertilizer base topdressing ratio is 5:5, and the topdressing fertilizer is applied in ditches during the greening stage. Nitrogen fertilizer is urea (containing N46%).
实施例3Example 3
本实施例与实施例2的不同之处在于：步骤1中大田试验中试验小区随机排列，每个试验小区面积为6*9＝54m2，行距20cm，重复3次，播种量为150kg/hm，10月中旬播种。磷肥和钾肥在播前作为基肥一次性施入，磷肥为磷酸二氢钙(含P2O512％)687.5kg/hm2，钾肥为硫酸钾(含K2O 57％)144.74kg/hm2，其他栽培措施同一般高产田管理。The difference between this embodiment and Example 2 is that in the field test in step 1, the test plots are randomly arranged, the area of each test plot is 6*9=54m 2 , the row spacing is 20cm, repeated three times, and the seeding rate is 150kg/hm , sown in mid-October. Phosphate fertilizer and potassium fertilizer are applied once as base fertilizer before sowing. The phosphate fertilizer is calcium dihydrogen phosphate (containing P 2 O 5 12%) 687.5kg/hm 2 , and the potassium fertilizer is potassium sulfate (containing K 2 O 57%) 144.74kg/hm. 2. Other cultivation measures are the same as general high-yield field management.
实施例4Example 4
本实施例与实施例1的不同之处在于：步骤2中测定根系活力的方法为：分别于返青期、拔节期、孕穗期、灌浆期、成熟期冲根，取根尖处5cm根系，采用改良TTC还原法测定根系活力。The difference between this embodiment and Example 1 is that the method for measuring root activity in step 2 is: punch the roots at the greening stage, jointing stage, booting stage, grain filling stage, and maturity stage, take 5cm of the root system from the root tip, and use Modified TTC reduction method was used to measure root activity.
实施例5Example 5
本实施例与实施例1的不同之处在于：步骤2中测定根际土壤酶活性的方法为：根际土于小麦孕穗期，每个试验小区内随机选取3个点，将植株连根挖
出，采用抖土法收集根际土，根际土距离根系周围0～5mm，于4℃条件下保存并尽快带回试验室，过2mm筛后，除去根系残体，分成两部分，一部分自然风干过0.25mm筛，用于测定土壤养分含量；一部分鲜样保存在-80℃冰箱，用于测定土壤细菌群落和根际土壤酶活性，土壤酶包括：β-葡萄糖苷酶(B-glocusidase，简称BG)、亮氨酸氨基肽酶(leucine amiopeptidase，简称LAP)和多酚氧化酶(简称POX)。The difference between this embodiment and Example 1 is that the method for measuring rhizosphere soil enzyme activity in step 2 is as follows: the rhizosphere soil is at the wheat booting stage, 3 points are randomly selected in each test plot, and the plants are dug up and down. out, use the soil shaking method to collect the rhizosphere soil. The rhizosphere soil is 0 to 5mm away from the root system. Store it at 4°C and bring it back to the laboratory as soon as possible. After passing through a 2mm sieve, remove the root residue and divide it into two parts. One part is naturally Air-dried and passed through a 0.25mm sieve for determination of soil nutrient content; some fresh samples were stored in a -80°C refrigerator for determination of soil bacterial communities and rhizosphere soil enzyme activities. Soil enzymes include: β-glucosidase (B-glucosidase, BG for short), leucine amiopeptidase (LAP for short) and polyphenol oxidase (POX for short).
土壤BG酶和LAP酶活性采用微孔板荧光法分析法，测定原理是BG酶分解底物生成对-硝基苯酚，后者在400nm有最大吸收峰；LAP酶分解底物生成对硝基苯胺，后者在405nm有最大吸收峰。Soil BG enzyme and LAP enzyme activities are analyzed by microplate fluorescence method. The measurement principle 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. , the latter has a maximum absorption peak at 405nm.
土壤POX酶活性采用微孔板比色法，测定原理是POX酶可催化底物生成有色产物，其显色物质在460nm处有最大吸收峰。BG、LAP和POX酶三种酶的底物见表1。The soil POX enzyme activity adopts the microplate colorimetric method. The measurement principle is that POX enzyme can catalyze the substrate to produce colored products, and the chromogenic material has a maximum absorption peak at 460nm. The substrates of the three enzymes BG, LAP and POX are shown in Table 1.
表1 土壤酶名称、缩写、编号和底物
Table 1 Soil enzyme names, abbreviations, numbers and substrates
Table 1 Soil enzyme names, abbreviations, numbers and substrates
根据测定原理，使用酶标仪(Labsystems Multiskan MS，芬兰)在特定波长下读取吸光值，通过测定吸光值升高速率来计算各种土壤酶活性。所得结果统一单位为nmol/H/g，即每小时每克鲜土分解相应底物产生特定产物的物质的量。According to the measurement principle, a microplate reader (Labsystems Multiskan MS, Finland) was used to read the absorbance value at a specific wavelength, and various soil enzyme activities were calculated by measuring the increase rate of the absorbance value. The unified unit of the obtained results is nmol/H/g, which is the amount of substances that decompose the corresponding substrate to produce specific products per gram of fresh soil per hour.
实施例6Example 6
本实施例与实施例5的不同之处在于：步骤2中土壤微生物高通量测序方法为：利用Illumina MiSeq平台(Illumina Inc.，San Diego，CA，USA)标
准操作规程，对土壤微生物群落进行测序。PCR扩增16S rRNA基因的V3-V4高变区以及ITS区域，对于每个DNA样品，在ABI GeneAmp 9700 PCR系统(Applied Biosystems，Foster City，CA，USA)上使用TransStart Fastpfu DNA聚合酶对三个重复进行独立的PCR扩增，使用AxyPrep PCR纯化试剂盒(Axygen Biosciences，Union City，CA，USA)进行纯化，并使用Illumina MiSeq平台进行双末端测序。The difference between this example and Example 5 is that the soil microorganism high-throughput sequencing method in step 2 is: using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) to label Standard operating procedures for sequencing soil microbial communities. The V3-V4 hypervariable regions of the 16S rRNA gene as well as the ITS region were PCR amplified. For each DNA sample, three Independent PCR amplification was performed in duplicate, purified using the AxyPrep PCR Purification Kit (Axygen Biosciences, Union City, CA, USA), and paired-end sequencing was performed using the Illumina MiSeq platform.
实施例7Example 7
本实施例与实施例6的不同之处在于：步骤3中分析步骤2中根系活力的测定数据方法为：分析不同供试小麦品种根系活力均随着生育进程的变化趋势；对比在N0和N1处理下不同供试小麦品种的根系活力数值。The difference between this embodiment and Example 6 is that: in step 3, the method for analyzing the measurement data of root activity in step 2 is: analyzing the changing trend of root activity of different wheat varieties tested with the growth process; comparing the results between N0 and N1 Root activity values of different tested wheat varieties under treatment.
选取许科168和郑品麦8号作为供试小麦品种，测得两个小麦品种的根系活力如图1所示，许科168和郑品麦8号的根系活力均随着生育进程呈先增加后降低的趋势，孕穗期的根系活力最强。Xuke 168 and Zhengpinmai 8 were selected as the test wheat varieties. The measured root activity of the two wheat varieties is shown in Figure 1. The root activity of Xuke 168 and Zhengpinmai 8 both increased with the growth process. The tendency is to increase and then decrease, with the root activity being the strongest at the booting stage.
根系是植物的重要作用器官，具有固定和支撑植物的功能，影响植物对养分、水分和矿物质等的吸收，同时又是多离种离子、有机酸、氨基酸等合成的重要场所。小麦属于须根系作物，根系活力的大小一定程度反映了根系新陈代谢活动的强弱。国内外学者研究发现，根系活力与作物的品种、土壤类型、肥水措施及自身基因遗传性状等密切相关；它反映了作物根系吸收、合成、呼吸和氧化能力等，客观体现了根系新陈代谢能力的强弱。熊淑萍等研究认为，氮高效基因型小麦具有较高的根系活力及根系代谢能力。本研究结果也证实了这一点。The root system is an important organ of the plant. It has the function of fixing and supporting the plant, affecting the plant's absorption of nutrients, water and minerals. It is also an important place for the synthesis of polyions, organic acids, amino acids, etc. Wheat is a fibrous root crop, and the root activity reflects the metabolic activity of the root system 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 capabilities of crop roots, and objectively reflects the strong metabolism of the root system. weak. Studies by Xiong Shuping and others believe that nitrogen-efficient genotype wheat has higher root activity and root metabolic capacity. The results of this study also confirmed this.
N0和N1处理下，许科168根系活力均高于郑品麦8号，平均增加幅度分别为16.76-70.14％和14.44-37.04％；其中N0处理下，拔节期-成熟期许科168的
根系活力显著高于郑品麦8号，N1处理下，除了拔节期和孕穗期，其它生育期两品种间的根系活力差异不显著。与N0处理相比，N1处理下许科168的根系活力平均增加了16.75-44.56％，郑品麦8号平均增加了0.97-42.63％。可知不论在低氮N0或高氮N1下，氮高效小麦品种许科168的根系活力均高于氮低效小麦品种郑品麦8号，低氮处理下差异明显，达到5％的显著水平。Under N0 and N1 treatments, the root activity of Xuke 168 was higher than that of Zhengpinmai 8, with average increases of 16.76-70.14% and 14.44-37.04% respectively; among them, under N0 treatment, the root activity of Xuke 168 in the jointing stage to maturity stage was The root activity was significantly higher than that of Zhengpinmai No. 8. Under N1 treatment, except for the jointing stage and booting stage, there was no significant difference in root activity between the two varieties in other growth stages. 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 8 increased by 0.97-42.63% on average. It can be seen that regardless of low nitrogen N0 or high nitrogen N1, the root activity of the nitrogen-efficient wheat variety Xuke 168 is higher than that of the nitrogen-inefficient wheat variety Zhengpinmai 8. The difference is obvious under the low-nitrogen treatment, reaching a significance level of 5%.
实施例8Example 8
本实施例与实施例7的不同之处在于：步骤3中分析步骤2中根际土壤酶活性的测定数据方法为：挑选出孕穗期N0和N1处理下，许科168和郑品麦8号的根际土壤酶活性变化结果表明，与郑品麦8号相比，两种施肥处理下，许科168的BG、LAP和POX酶活性均增加；N0处理下增加幅度分别为59.38％、34.43％和30.50％，而且达到差异显著水平；N1处理下增加幅度不明显，分别为9.31％、18.88％和9.05％。The difference between this example and Example 7 is that: in step 3, the method for analyzing the measurement data of rhizosphere soil enzyme activity in step 2 is: selecting the results of Xuke 168 and Zhengpinmai 8 under the N0 and N1 treatments at the booting stage. The results of rhizosphere soil enzyme activity changes showed that compared with Zhengpinmai 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% and 34.43% respectively. and 30.50%, and reached a significant level of difference; the increase under N1 treatment was not obvious, which were 9.31%, 18.88% and 9.05% respectively.
表2 许科168和郑品麦8号根际土壤酶活性的差异(2018-2020两年的平均数据)
Table 2 Differences in rhizosphere soil enzyme activities between Xu Ke 168 and Zheng Pinmai 8 (average data from 2018 to 2020)
Table 2 Differences in rhizosphere soil enzyme activities between Xu Ke 168 and Zheng Pinmai 8 (average data from 2018 to 2020)
土壤酶是由动物、植物和微生物产生的，其酶活性是表征土壤养分循环及微生物代谢活性的关键指标。不仅是土壤中物质循环和能量转换的重要参与者，还是土壤中进行一切生化反应的催化剂。研究表明，BG、LAP和POX参与碳循环和氮循环末端的催化反应，能够极好的反映土壤养分的代谢水平。根际土壤酶活性的高低与土壤养分的矿化量及有机质的分解程度密切相关，酶活性的提高促进作物对养分的吸收。本研究结果表明，施用氮肥提高了土壤酶活性，这可能是由于微生物通过同化利用这些施入的氮素来促进自身的生长，导致产生的
酶数量和种类均显著增加所致。正常施氮肥N1和不施氮肥N0条件下，氮高效品种许科168均表现出了较高的土壤酶活性，即低氮胁迫下，氮高效品种许科168根际土壤中BG、POX和LAP依然保持较高的活性，较高的土壤酶活性代表土壤的物质和能量转化都很强盛，从而促进了植株的良好生长。这说明氮高效小麦品种许科168在低氮胁迫条件下具有良好的适应能力。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. It is not only an important participant in the material cycle and energy conversion in the soil, but also the catalyst for all biochemical reactions in the soil. Research shows that BG, LAP and POX participate in the catalytic reactions at the end of the carbon cycle and nitrogen cycle, and can excellently reflect the metabolic level of soil nutrients. The level of rhizosphere soil enzyme activity is closely related to the mineralization amount 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 show that the application of nitrogen fertilizer increases 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 the production of The number and types of enzymes increased significantly. Under normal application of nitrogen fertilizer N1 and no application of nitrogen fertilizer N0, the nitrogen-efficient variety Xuke 168 showed higher soil enzyme activities, that is, under low nitrogen stress, BG, POX and LAP in the rhizosphere soil of the nitrogen-efficient variety Xuke 168 Still maintaining high activity, high soil enzyme activity means that the material and energy transformation of the soil are strong, thus promoting good plant growth. This shows that the nitrogen-efficient wheat variety Xuke 168 has good adaptability under low nitrogen stress conditions.
低氮条件下，氮高效小麦品种许科168的根际β-葡萄糖苷酶、亮氨酸氨基肽酶和多酚氧化酶活性分别比氮低效小麦品种郑品麦8号高出59.38％、34.43％和30.50％。可知，不论在低氮N0或高氮N1下，氮高效小麦品种许科168的根际土壤中BG、POX和LAP酶活性均高于氮低效小麦品种郑品麦8号。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% higher than those of the nitrogen-inefficient wheat variety Zhengpinmai No. 8, respectively. 34.43% and 30.50%. It can be seen that no matter under low nitrogen N0 or high nitrogen N1, the activities of BG, POX and LAP enzymes in the rhizosphere soil of the nitrogen-efficient wheat variety Xuke 168 are higher than those of the nitrogen-inefficient wheat variety Zhengpinmai 8.
实施例9Example 9
本实施例与实施例8的不同之处在于：步骤3中分析步骤2中土壤微生物高通量测序数据方法为：1)测序结果首先使用QIIME(Quantitative Insights Into Microbial Ecology)(v1.2.1)对原始数据进行拼接，过滤，并去除嵌合体。挑出序列长度大于200bp，Barcode和引物序列无错误碱基，平均质量得分Q≥25的高质量序列。采用USEARCH软件在97％的阈值下划分分类操作单元(简称OTU)。根据Silva数据库，比对OTU代表序列并进行分类。利用Mothur软件计算Shannon、Simpson多样性指数及Chao1、ACE丰富度指数，用于评价细菌α-多样性；2)根据根际土壤细菌群落在门水平上的相对丰度，以及根际土壤优势细菌属的相对丰度，分析许科168和郑品麦8号根际土壤细菌群落结构。The difference between this example and Example 8 is that the method for analyzing the soil microbial high-throughput sequencing data in step 2 in step 3 is: 1) The sequencing results are first analyzed using QIIME (Quantitative Insights Into Microbial Ecology) (v1.2.1) Raw data were spliced, filtered, and chimeras removed. Select high-quality sequences with a sequence length greater than 200 bp, Barcode and primer sequences without error bases, and an average quality score Q ≥ 25. Use USEARCH software to divide classification operation units (OTUs for short) at the 97% threshold. According to the Silva database, OTU representative sequences were compared and classified. Use Mothur software to calculate Shannon and Simpson diversity indexes and Chao1 and ACE richness indices to evaluate bacterial α-diversity; 2) According to the relative abundance of rhizosphere soil bacterial communities at the phylum level, and the dominant bacteria in rhizosphere soil The relative abundance of genera and the bacterial community structure in the rhizosphere soil of Xuke 168 and Zhengpinmai 8 were analyzed.
从表3不同氮处理下土壤细菌α-多样性特征可以看出，氮高效小麦品种许科168的丰富度指数(Chao1指数和ACE指数)显著高于氮低效小麦品种郑品麦8号，增加幅度分别为7.22％、7.35％和12.95％，其中Shannon指数达到5％的差异显著水平。可知在N0处理下，许科168的丰富度指数(Chao1指数和ACE指数)
显著高于郑品麦8号，许科168多样性指数的Shannon指数和Simpson指数均高于郑品麦8号。在N1处理下，许科168和郑品麦8号的丰富度指数和多样性指数差异不明显。As can be seen from Table 3, the α-diversity characteristics of soil bacteria under different nitrogen treatments, 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 8. The increases were 7.22%, 7.35% and 12.95% respectively, among which the Shannon index reached a significant difference level of 5%. It can be seen that under N0 treatment, the richness index (Chao1 index and ACE index) of Xuke 168 It is significantly higher than Zhengpinmai No. 8. The Shannon index and Simpson index of Xu Ke 168 diversity index are both higher than Zhengpinmai No. 8. Under N1 treatment, the difference in richness index and diversity index between Xuke 168 and Zhengpinmai 8 was not obvious.
表3 许科168和郑品麦8号根际土壤细菌α-多样性特征
Table 3 Alpha-diversity characteristics of rhizosphere soil bacteria of Xu Ke 168 and Zheng Pinmai 8
Table 3 Alpha-diversity characteristics of rhizosphere soil bacteria of Xu Ke 168 and Zheng Pinmai 8
如图2所示，通过对许科168和郑品麦8号土壤细菌群落在门水平上进行分类，2个处理共含有28个门，相对丰度大于1％的门有11个，其中酸杆菌门、放线菌门、拟杆菌门、变形菌门和奇古菌门为优势门，相对丰度为84.36％-88.20％。As shown in Figure 2, 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, and 11 phyla had a relative abundance greater than 1%, among which acid Bacilli, Actinobacteria, Bacteroidetes, Proteobacteria and Thaumarchaeota are the dominant phyla, with relative abundances ranging from 84.36% to 88.20%.
与N0处理相比，N1处理下的小麦品种许科168的酸杆菌门和疣微菌门的相对丰度分别显著降低了38.68％和28.16％，拟杆菌门、绿湾菌门和厚壁菌门相对丰度分别显著增加了17.68％、66.50％和59.68％；而郑品麦8号的酸杆菌门、浮霉菌门和疣微菌门则显著增加了62.36％、40.79％和50.18％，绿湾菌门显著降低了32.84％。Compared with the N0 treatment, the relative abundance of Acidobacteria and Verrucomicrobia in the wheat variety Xuke 168 under the N1 treatment was significantly reduced by 38.68% and 28.16%, respectively. The relative abundance of phylum increased significantly by 17.68%, 66.50% and 59.68% respectively; while the Acidobacteria, Planctomycetes and Verrucomicrobia in Zhengpinmai 8 increased significantly by 62.36%, 40.79% and 50.18%, and the green Bay fungi were significantly reduced by 32.84%.
同一处理下，两品种优势门丰度也有差别，N0处理下，许科168的酸酐菌门比郑品麦8号增加了66.21％，奇古菌门降低了11.74％，N1处理下许科168的酸酐菌门比郑品麦8号降低了37.23％，奇古菌门则增加了13.30％，其它差别不明显。Under the same treatment, there are also differences in the abundance of the dominant phylum of the two varieties. Under the N0 treatment, the Anhydrobacteria in Xuke 168 increased by 66.21% compared with Zhengpinmai No. 8, and the Thaumarchaeota decreased by 11.74%. Under the N1 treatment, the Anhydrobacteria in Xuke 168 increased by 66.21% compared with Zhengpinmai 8. The Anhydrobacteria phylum decreased by 37.23% compared with Zhengpinmai No. 8, while the Thaumarchaeota increased by 13.30%. Other differences were not obvious.
进一步对比分析，2个处理共含有857个属，其中GP6属、亚硝基球藻和未分类细菌属为优势属，如图3所示。
Further comparative analysis showed that the two treatments contained a total of 857 genera, among which GP6, Nitrosococcus and unclassified bacterial genera were the dominant genera, as shown in Figure 3.
与郑麦品8号相比，在N1处理下，许科168根际土壤亚硝基球藻属显著增加了21.79％，未分类细菌属显著降低了27.63％，GP6属降低了9.49％；N0处理下，许科168根际土壤的亚硝基球藻显著增加了38.06％，未分类细菌属增加了12.92％，GP6属显著降低了21.33％。Compared with Zhengmaipin No. 8, under the N1 treatment, the genus Nitrosococcus in the rhizosphere soil of Xuke 168 significantly increased by 21.79%, the unclassified bacterial genera significantly decreased by 27.63%, and the genus GP6 decreased by 9.49%; under the N0 treatment , Nitrosococcus in the rhizosphere soil of Xuke 168 increased significantly by 38.06%, unclassified bacterial genera increased by 12.92%, and GP6 genera decreased significantly by 21.33%.
土壤微生物是农田土壤生态系统中最活跃的重要成分之一，其种类丰富、数量繁多，在土壤有机质矿化分解及腐殖酸形成中扮演重要角色。微生物的多样性和群落的丰富度被视为衡量土壤肥力状况的重要指标。植物种类与其根际微生物群落结构及丰度有一定相关性。农泽梅等研究认为不同品种间根系微生物主要菌群丰度存在显著差异。本次研究结果也证实了这个观点。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. Microbial diversity and community richness are considered important indicators of soil fertility status. There is a certain correlation between plant species and their rhizosphere microbial community structure and abundance. Research by Nong Zemei and others concluded that there are significant differences in the abundance of the main bacterial groups in the root microorganisms among different varieties. The results of this study also confirmed this view.
基于高通量测序分析结果，N0处理下，许科168根际土壤酸杆菌门相对丰度显著高于郑品麦8号，而N1处理下相反。这可能是因为酸杆菌门属于贫营养型菌，生长速率缓慢、富集在养分含量较低环境中，而N0处理下氮高效品种许科168的根系与根际土壤的相互作用有利于根际微生物的生长，从而提高了根际土壤微生物的活性。有学者认为，酸杆菌门和奇古菌门在旱地作物土壤中是正相关关系，而本研究结果显示，N0处理下许科168根际土壤奇古菌门的相对丰度明显低于郑品麦8号，这可能与选择的生育时期及土壤环境有关，因为不同氮效率小麦品种吸收土壤有效氮程度不同，影响了土壤氮库的平衡，不同程度的改变了土壤性质，从而影响土壤微生物群落。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 8 under N0 treatment, while the opposite was true under N1 treatment. This may be because Acidobacteria are oligotrophic bacteria that grow slowly and are concentrated in environments with low nutrient content. The interaction between the root system of the nitrogen-efficient variety Xuke 168 under N0 treatment and the rhizosphere soil is beneficial to the rhizosphere. The growth of microorganisms thereby increases the activity of rhizosphere soil microorganisms. Some scholars believe that Acidobacteria and Thaumarchaeota are positively correlated in dryland crop soil, and the results of this study show that the relative abundance of Thaumarchaeota in the rhizosphere soil of Xuke 168 under N0 treatment was significantly lower than that of Zheng Pinmai No. 8, this may be related to the selected growth period and soil environment, because wheat varieties with different nitrogen efficiencies absorb soil available nitrogen to different extents, which affects the balance of soil nitrogen pools, changes soil properties to varying degrees, and thus affects soil microbial communities.
进一步分析发现，两种氮处理下，许科168和郑品麦8号的优势属GP6属、亚硝基球藻属等都有不同程度的差异。另外，基于高通量测序分析结果，不同氮肥处理间主要菌群种落基本相似，其主要差异表现在菌群丰度上，在低氮处理下不同氮效率小麦品种根际微生物的丰富度指数和多样性指数存在明显差异，高氮处理下，差异不明显，其根本原因在于不同氮效率小麦品种的根系活
力、根际土壤酶活性等的差异影响了土壤细菌群落结构的分布。Further analysis found that under the two nitrogen treatments, the dominant genera of Xuke 168 and Zhengpinmai 8 were different to varying degrees, such as GP6 and Nitrosococcum. In addition, based on the results of high-throughput sequencing analysis, the main bacterial communities between different nitrogen fertilizer treatments are basically similar. The main differences are reflected in the abundance of bacterial communities. The richness index of rhizosphere microorganisms of different nitrogen efficiency wheat varieties under low nitrogen treatment There is a significant difference between the diversity index and the diversity index. Under the high nitrogen treatment, the difference is not obvious. The fundamental reason is that the root activity of wheat varieties with different nitrogen efficiencies is different. Differences in soil strength and rhizosphere soil enzyme activity affect the distribution of soil bacterial community structure.
高通量测序分析结果显示，2个不同品种根际土壤细菌种群结构存在一定差异，与郑品麦8号相比，许科168显著提高了细菌群落α-多样性。同一处理，两品种的优势门丰度有一定差别，N0处理下，许科168的酸酐菌门比郑品麦8号增加了66.21％，奇古菌门降低了11.74％，N1处理下许科168的酸酐菌门比郑品麦8号降低了37.23％，奇古菌门则增加了13.30％，其它差别不明显；GP6属、亚硝基球藻和未分类细菌属为优势属，与氮低效品种郑麦品8号相比，N1处理下，许科168根际土壤亚硝基球藻属显著增加了21.79％；N0处理下，显著增加了38.06％。因此，本次研究结果证实了氮高效小麦品种能够提高细菌群落多样性及改善细菌群落组成。The results of high-throughput sequencing analysis showed that there were certain differences in the bacterial population structure of the rhizosphere soil between the two different varieties. Compared with Zhengpinmai 8, Xuke 168 significantly increased the α-diversity of the bacterial community. In the same treatment, there is a certain difference in the abundance of dominant phyla between the two varieties. Under the N0 treatment, the Anhydrobacteria in Xuke 168 increased by 66.21% compared with Zhengpinmai 8, and the Thaumarchaeota decreased by 11.74%. Under the N1 treatment, the Anhydrobacteria in Xuke The number of Anhydrobacteria in 168 decreased by 37.23% compared with Zhengpinmai No. 8, while Thaumarchaeota increased by 13.30%. Other differences were not obvious; GP6, Nitrosococcus and unclassified bacterial genera were the dominant genera, and Nitrogen was the dominant genera. Compared with the low-efficiency variety Zhengmaipin No. 8, under the N1 treatment, the genus Nitrosococcus in the rhizosphere soil of Xuke 168 significantly increased by 21.79%; under the N0 treatment, it increased significantly by 38.06%. Therefore, the results of this study confirmed that nitrogen-efficient wheat varieties can increase bacterial community diversity and improve bacterial community composition.
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，本领域普通技术人员对本发明的技术方案所做的其他修改或者等同替换，只要不脱离本发明技术方案的精神和范围，均应涵盖在本发明的权利要求范围当中。
Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Those of ordinary skill in the art may make other modifications or equivalent substitutions to the technical solutions of the present invention, as long as they do not deviate from the spirit and scope of the technical solutions of the present invention. The scope should be covered by the claims of the present invention.
Claims (9)
- 一种筛选氮高效小麦品种的方法，其特征在于：包括如下步骤：步骤1，选取若干个供试小麦品种，进行大田试验，设置2个供氮水平的施肥处理：不施氮肥处理N0(0kg N/hm2)、正常施氮肥处理N1；A method for screening nitrogen-efficient wheat varieties, characterized by: including the following steps: Step 1, select several wheat varieties for testing, conduct field trials, and set two nitrogen supply levels of fertilization treatments: no nitrogen fertilizer treatment N0 (0kg N/hm 2 ), normal nitrogen fertilizer treatment N1;步骤2，测定N0和N1处理下供试小麦品种的根系活力、根际土壤酶活性，以及进行土壤微生物进行高通量测序；Step 2: Determine the root activity and rhizosphere soil enzyme activity of the test wheat varieties under N0 and N1 treatments, and conduct high-throughput sequencing of soil microorganisms;步骤3，分析步骤2中的测定数据，筛选出根系活力高、根际土壤酶活性高、根际微生物的丰富度指数高和多样性指数高的供试小麦品种，即为氮高效小麦品种。Step 3: Analyze the measurement data in Step 2 and select test wheat varieties with high root activity, high rhizosphere soil enzyme activity, high rhizosphere microbial richness index and high diversity index, which are nitrogen-efficient wheat varieties.
- 根据权利要求1所述的筛选氮高效小麦品种的方法，其特征在于：步骤1中正常施氮肥处理N1中氮肥施用量为165kg N/hm2，氮肥基追比为5∶5，追肥于返青期开沟施入。The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: in step 1, the nitrogen fertilizer application amount in the normal nitrogen fertilizer treatment N1 is 165kg N/hm 2 , the nitrogen fertilizer base topdressing ratio is 5:5, and the topdressing fertilizer is used for turning green. Digging trenches will be carried out during the period.
- 根据权利要求2所述的筛选氮高效小麦品种的方法，其特征在于：步骤1中大田试验中试验小区随机排列，每个试验小区面积为6*9＝54m2，行距20cm，重复3次，播种量为150kg/hm，10月中旬播种；磷肥和钾肥在播前作为基肥一次性施入，其中氮肥为尿素，磷肥为磷酸二氢钙687.5kg/hm2，钾肥为硫酸钾施用量144.74kg/hm2。The method for screening nitrogen-efficient wheat varieties according to claim 2, characterized in that: in the field test in step 1, the test plots are randomly arranged, the area of each test plot is 6*9=54m2, and the row spacing is 20cm , repeated 3 times, The seeding rate is 150kg/hm, sowing in mid-October; phosphate fertilizer and potassium fertilizer are applied once as base fertilizer before sowing. The nitrogen fertilizer is urea, the phosphate fertilizer is calcium dihydrogen phosphate 687.5kg/hm 2 , and the potassium fertilizer is potassium sulfate. The application rate is 144.74kg. /hm 2 .
- 根据权利要求1所述的筛选氮高效小麦品种的方法，其特征在于：步骤2中测定根系活力的方法为：分别于返青期、拔节期、孕穗期、灌浆期、成熟期冲根，取根尖处5cm根系，采用改良TTC还原法测定根系活力。The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: the method for measuring root activity in step 2 is: punching the roots at the greening stage, jointing stage, booting stage, filling stage, and maturity stage, and taking the roots. The root system at the tip of 5cm was measured using a modified TTC reduction method to measure root activity.
- 根据权利要求1所述的筛选氮高效小麦品种的方法，其特征在于：步骤2中测定根际土壤酶活性中土壤酶包括：β-葡萄糖苷酶、亮氨酸氨基肽酶和多酚氧化酶；选孕穗期N0和N1处理下供试小麦品种根际土，土壤BG酶和LAP酶活性采用微孔板荧光法分析法测定，土壤POX酶活性采用微孔板比色法测定。 The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: the soil enzymes in the rhizosphere soil enzyme activity measured in step 2 include: β-glucosidase, leucine aminopeptidase and polyphenol oxidase. ; Select the rhizosphere soil of the test wheat varieties under N0 and N1 treatments at the booting stage. The soil BG enzyme and LAP enzyme activities were measured by microplate fluorescence analysis method, and the soil POX enzyme activity was measured by microplate colorimetric method.
- 根据权利要求1所述的筛选氮高效小麦品种的方法，其特征在于：步骤2中土壤微生物高通量测序方法为：利用Illumina MiSeq平台标准操作规程，对孕穗期N0和N1处理下供试小麦品种根际土土壤微生物群落进行测序；PCR扩增16S rRNA基因的V3-V4高变区以及ITS区域，对于每个DNA样品，在ABI GeneAmp 9700 PCR系统上使用TransStart Fastpfu DNA聚合酶对三个重复进行独立的PCR扩增，使用AxyPrep PCR纯化试剂盒进行纯化，并使用Illumina MiSeq平台进行双末端测序。The method for screening nitrogen-efficient wheat varieties according to claim 1, characterized in that: the soil microorganism high-throughput sequencing method in step 2 is: using the Illumina MiSeq platform standard operating procedures, the test wheat under the N0 and N1 treatments at the booting stage The soil microbial communities in the rhizosphere soil of the cultivar were sequenced; the V3-V4 hypervariable region and the ITS region of the 16S rRNA gene were PCR amplified. For each DNA sample, three replicates were used on the ABI GeneAmp 9700 PCR system using TransStart Fastpfu DNA polymerase. Independent PCR amplification was performed, purified using the AxyPrep PCR Purification Kit, and paired-end sequencing using the Illumina MiSeq platform.
- 根据权利要求4所述的筛选氮高效小麦品种的方法，其特征在于：步骤3中分析步骤2中根系活力的测定数据方法为：分析不同供试小麦品种根系活力均随着生育进程的变化趋势；对比在N0和N1处理下不同供试小麦品种的根系活力数值。The method for screening nitrogen-efficient wheat varieties according to claim 4, characterized in that: in step 3, the method for analyzing the measurement data of root activity in step 2 is: analyzing the changing trend of root activity of different test wheat varieties with the growth process. ; Compare the root activity values of different tested wheat varieties under N0 and N1 treatments.
- 根据权利要求5所述的筛选氮高效小麦品种的方法，其特征在于：步骤3中分析步骤2中根际土壤酶活性的测定数据方法为：选取孕穗期N0和N1处理下根际土壤酶活性数值，对比不同供试小麦品种之间的根际土壤酶活性数值。The method for screening nitrogen-efficient wheat varieties according to claim 5, characterized in that: in step 3, the method for analyzing the measurement 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 , comparing the rhizosphere soil enzyme activity values between different tested wheat varieties.
- 根据权利要求6所述的筛选氮高效小麦品种的方法，其特征在于：步骤3中分析步骤2中土壤微生物高通量测序数据方法为：1)测序结果首先使用QIIME对原始数据进行拼接，过滤，并去除嵌合体；挑出序列长度大于200bp，Barcode和引物序列无错误碱基，平均质量得分Q≥25的高质量序列；采用USEARCH软件在97％的阈值下划分分类操作单元，根据Silva数据库，比对OTU代表序列并进行分类，利用Mothur软件计算Shannon、Simpson多样性指数及Chao1、ACE丰富度指数，用于评价细菌α-多样性；2)根据根际土壤细菌群落在门水平上的相对丰度，以及根际土壤优势细菌属的相对丰度，分析供试小麦品种根际土壤细菌群落结构。 The method for screening nitrogen-efficient wheat varieties according to claim 6, characterized in that: in step 3, the method for analyzing soil microbial high-throughput sequencing data in step 2 is: 1) the sequencing results first use QIIME to splice the original data and filter , and remove chimeras; select high-quality sequences with sequence length greater than 200 bp, Barcode and primer sequences without error bases, and average quality score Q≥25; use USEARCH software to divide classification operation units under the 97% threshold, according to the Silva database , compare and classify OTU representative sequences, and use Mothur software to calculate Shannon, Simpson diversity index and Chao1, ACE richness index, which are used to evaluate bacterial α-diversity; 2) According to the rhizosphere soil bacterial community at the phylum level The relative abundance, as well as the relative abundance of dominant bacterial genera in the rhizosphere soil, were analyzed to analyze the bacterial community structure in the rhizosphere soil of the tested wheat varieties.
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