Tea tree nitrogen efficient phenotype identification method and application

Abstract

The invention provides a tea tree nitrogen efficient phenotype identification method and application, and discloses a calculation method for quantifying the tea tree nitrogen efficient phenotype, stable isotope 15N marking time capable of indicating a woody plant tea tree nitrogen storage migration rule, a marking method and the tea tree nitrogen storage migration rule for the first time. The phenotypes of the nitrogen utilization efficiency of different tea tree varieties can be rapidly and efficiently evaluated on the basis of not destroying the assimilation level of background nitrogen of the tea tree and the normal growth and development state of the tea tree by applying the method to the tea tree varieties with different genotypes. The invention provides a rapid and efficient nitrogen high-efficiency phenotype identification method for the field of tea tree breeding, has a wide application prospect in the current seed industry science and technology period, and can accelerate the breeding process especially for tea trees with a long breeding period.

Description

Technical Field

[0001] This invention relates to the field of woody plant cultivation technology and the field of nitrogen-efficient variety breeding and phenotypic identification, and in particular to a method and application for identifying nitrogen-efficient phenotypic traits in tea trees. Background Technology

[0002] Nitrogen is one of the essential macro-mineral elements for the growth and development of tea trees, and it is also a limiting factor for yield and quality. The biosynthesis of quality components in tea, such as amino acids and caffeine, requires nitrogen. Tea trees are perennial woody plants, and their growth is seasonal. Before bud break in early spring, root activity is low, and their ability to absorb nitrogen from the soil is weak. Therefore, nitrogen stored in the tree plays a crucial role in the germination of dormant buds and the growth and development of new shoots. Obtaining the transport patterns and reuse efficiency of stored nitrogen in tea trees without disrupting their normal growth and development is an economical and effective method for identifying nitrogen-efficient phenotypic characteristics in tea trees.

[0003] Studies have reported that plant nitrogen use efficiency is closely related to its nitrogen reuse efficiency. Although some studies have reported differences in nitrogen absorption and utilization among different tea varieties, there are few studies on the reuse of stored nitrogen in tea trees, and in particular, there is currently no evaluation method for the reuse of stored nitrogen.

[0004] To address the aforementioned issues, this invention presents a method for identifying nitrogen-efficient phenotypes in tea trees. This method can rapidly and efficiently evaluate the nitrogen use efficiency of tea trees, which is of great significance for cultivating nitrogen-efficient tea varieties. Summary of the Invention

[0005] This invention provides a method for identifying nitrogen-efficient phenotypes in tea plants, comprising the following steps: Step 1: In early January, top the tea tree canopy by removing one bud and two leaves that are not fully developed at the top. Step 2: In late January or early February, select the first mature leaf corresponding to the dormant bud at the top of the canopy, mark it, and apply... 15 N-urea; Step 3: Collect the marked leaves one month later and measure them. 15 N content; Step 4: The sample was dried using a freeze dryer, and the biomass of the dried sample was determined. Then, it was pulverized using a micro-ultrafine ball mill. The nitrogen content was determined using an elemental analyzer combined with a stable isotope mass spectrometer. 15 N abundance.

[0006] Furthermore, in step one, the tea tree varieties are selected from 5-year-old tea tree cultivars grown under field planting conditions, including Zhongcha 108, Tieguanyin, Fuding Dabai, Dangui, Wuniuzao, Baiye No. 1, Longjing 43, Huangjinya, Zhenong 117, and Zijuan.

[0007] Further, the labeling process in step two is as follows: Select an even number of new tea shoots of each variety with a diameter of 0.2-0.4 cm from the tea canopy, measure and record the shoot diameter as D0, and the height of the canopy surface from the ground as H0. Then, apply 46 μg of... 15 N-urea was evenly applied to the first mature leaf corresponding to the dormant bud, and the leaf area was measured and recorded as LA0, and the leaf height from the ground was H1.

[0008] Furthermore, in step three, half of the marked leaves from each tea variety were collected, measured, and recorded: the area of ​​the mature leaf one month after application was recorded as LA1, and the dry weight per unit leaf area was recorded as UW1. 15 The proportion of N is 15 N dffM The amount of nitrogen (N0) assimilated into the tea plant was detected as a baseline sample. For the other half of the branches, when the first flush of new shoots from the tea plant develops to one bud and two leaves, collect samples of one bud and two leaves along with their corresponding mature leaves, and measure and record the following: leaf area as LA2, branch diameter as D1, new shoot dry weight as W1, and nitrogen concentration as N1. 15 The proportion of N is 15 N dffN ; Calculation transferred to new shoots 15 N content, divided by the baseline mature leaf sample 15 The nitrogen content is considered as the nitrogen reuse efficiency of tea plants.

[0009] Furthermore, the formula for calculating the nitrogen reuse efficiency of tea plants is as follows: Nitrogen efficiency (NRE) = (LA2 ​​- LA1) ÷ LA0 × (H1 - H0) ÷ 46 + ( 15 N dffN (× W1 × N1 × D1) ÷ ( 15 N dffM (× UW1 × N0 × D0) × 100%.

[0010] Furthermore, in step four, the basic leaves of 10 varieties were detected... 15 N dff The abundance was all below 0.1%, indicating that the labeling method did not disrupt the baseline nitrogen metabolism level of tea plants and can be used as a method to trace the reuse of stored nitrogen in plants.

[0011] Furthermore, the transport patterns of stored nitrogen in tea plants were derived: (1) During the dormancy period, i.e. one month after the marking was applied, four different tissue parts of the tea tree were collected: the first mature leaf, the second mature leaf, the third mature leaf, and the stem corresponding to the dormant bud at the top of the 10 tea tree varieties. (2) Test each tissue according to the method given in step four.15 The nitrogen content was measured, and the recovered nitrogen content was determined. 15 The proportion of nitrogen distributed in the four tissues was consistent, with more than 80% of the stored nitrogen stored in the first mature leaf corresponding to the terminal dormant bud, while the proportion distributed in the second and third mature leaves was small. This method identified Longjing 43 as having the highest reuse rate. (3) The transport pattern of stored nitrogen in tea trees was found to be consistent among different tea varieties, and the reuse rate of stored nitrogen among different varieties was significantly different.

[0012] This invention also discloses the application of a method for identifying nitrogen-efficient phenotypic characteristics in tea trees, which is used to regulate the growth and development of tea trees and improve tea quality through nitrogen nutrition management measures, while providing an auxiliary method for identifying phenotypic characteristics of nitrogen-efficient tea tree varieties. Attached Figure Description

[0013] Figure 1 Stable isotopes 15 N marks the location.

[0014] Figure 2 for 15 N-labeled samples were detected in the basal blades one month later. 15 N abundance.

[0015] Figure 3 Nitrogen transport patterns during storage in different tea varieties.

[0016] Figure 4 Identification of nitrogen reuse phenotypes in different tea varieties. Detailed Implementation

[0017] The present invention will be further described and illustrated below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the reagents, consumables, etc., used are all commercially available.

[0018] Please see Figure 1-4 This invention provides a method for identifying nitrogen-efficient phenotypes in tea plants, comprising the following steps: Step 1: In early January, top the tea tree canopy by removing one bud and two leaves that are not fully developed at the top. Step 2: In late January or early February, select the first mature leaf corresponding to the dormant bud at the top of the canopy, mark it, and apply... 15 N-urea; Step 3: Collect the marked leaves one month later and measure them. 15 N content; Step 4: The sample was dried using a freeze dryer, and the biomass of the dried sample was determined. Then, it was pulverized using a micro-ultrafine ball mill. The nitrogen content was determined using an elemental analyzer combined with a stable isotope mass spectrometer. 15 N abundance.

[0019] Example 1 Stable Isotopes 15 N-labeling method Experimental materials: Five-year-old tea trees planted in the field under the following cultivars: Zhongcha 108 (ZC108), Tieguanyin (TGY), Fuding Dabai (FDDB), Dangui (DG), Wuniuzao (WNZ), Baiye No.1 (BY1), Longjing 43 (LJ43), Huangjinya (HJY), Zhenong 117 (ZN117), and Zijuan (ZJ).

[0020] Experimental methods: 1) In early January, top the tea tree canopy, removing the one bud and two leaves that are not fully developed at the top. Figure 1 ).

[0021] 2) At the end of January, select the first mature leaf corresponding to the dormant bud at the top of the canopy and apply 100 μL of a 2% solution prepared with ddH2O. 15 N-urea.

[0022] 3) Collect the marked leaves one month later and measure them. 15 N content.

[0023] 4) The samples were dried using a freeze dryer, and the biomass of the dried samples was determined. They were then pulverized using a micro-ultrafine ball mill. The nitrogen content was determined using an elemental analyzer combined with a stable isotope mass spectrometer. 15 N abundance.

[0024] 5) Detected in the basic leaves of 10 varieties 15 N dff The abundance was all below 0.1%, and the content was extremely low (Table 1). Figure 2 This indicates that the labeling method did not disrupt the baseline nitrogen metabolism level of tea plants and can be used as a method to trace the reuse of stored nitrogen in plants.

[0025] Table 1.

[0026] variety <![CDATA[ 15 N dff (%)]]> N content (%) <![CDATA[ 15 Total N (mg per leaf) China Tea 108 0.0027±0.0003 3.21±0.15 0.1723±0.0162 Tieguanyin 0.0026±0.0008 2.73±0.12 0.1593±0.0489 Fuding Big White 0.0026±0.0004 2.60±0.10 0.1343±0.0222 Osmanthus fragrans 0.0064±0.0021 2.67±0.12 0.3469±0.1071 Wu Niu Zao 0.0034±0.0006 3.04±0.08 0.1865±0.0355 Anji White Tea 0.0059±0.0013 2.84±0.36 0.3038±0.0774 Longjing 43 0.0022±0.0007 2.89±0.14 0.0990±0.0423 Golden Buds 0.0123±0.0074 3.16±0.27 0.2533±0.1408 Zhejiang Agricultural University 117 0.0018±0.0006 2.56±0.09 0.0917±0.0366 Purple Azalea 0.0073±0.0036 2.91±0.10 0.6883±0.3737 After implementing the marking method of this invention, tea leaves 15 The nitrogen content is extremely low and does not disrupt the background nitrogen level in the leaves.

[0027] Example 2: Transport patterns of stored nitrogen in tea plants Experimental methods: 1) In Example 1, for 10 tea tree varieties, during the dormant period (one month after marking), samples were collected from four different tissue parts of the tea tree: the first mature leaf (L1) corresponding to the terminal dormant bud, the second mature leaf (L2) from the bottom, the third mature leaf (L3), and the stem (S). Figure 3 A).

[0028] 2) Detect each tissue according to the method in Example 1. 15 N content.

[0029] 3) Recycled 15 The distribution of nitrogen (N) across the four tissues was consistent, with over 80% of stored nitrogen located in the first mature leaf corresponding to the terminal dormant bud, ranging from 82.61% to 90.38%. Smaller proportions were distributed in the second (0.48%-3.77%) and third mature leaves (0.18%-5.36%), while 8.35%-14.6% was allocated to the stem. Figure 3 B).

[0030] Example 3: Phenotypic Evaluation of Nitrogen Reuse Efficiency of Different Tea Varieties 1) Collect new shoots (NS) and the corresponding first mature leaf (L1) of 10 tea varieties in Example 1 at the one bud and two leaf stage. T2 The second mature leaf from the bottom (L2) T2 ), the third mature leaf (L3) T2 ) and stem (S T2 () Figure 4 A).

[0031] 2) Following the method in Example 1, the samples were freeze-dried, and the biomass was weighed and tested in new shoots and mature leaves. 15 N content.

[0032] 3) The new shoots 15 N content divided by the mature leaf in Example 1 15 Multiplying the nitrogen content by 100% gives the nitrogen reuse efficiency of tea trees.

[0033] 4) The method of this invention identified Longjing 43 as having the highest reuse rate. Figure 4 B, where a and b are letters used for significance analysis. If there are significant differences in nitrogen reuse among different varieties, they are arranged in descending order of value. First, the analysis is performed according to the highest value. If there is a significant difference from the highest value, it is marked as b). This is consistent with the earlier report that Longjing 43 is a national-level excellent variety.

[0034] This invention provides the transport patterns of stored nitrogen in different tea varieties. The reuse of stored nitrogen in tea trees is consistent across different varieties. More than 80% of the stored nitrogen is mainly stored in the first mature leaf corresponding to the dormant bud at the top of the tea tree canopy, with less transport to other parts.

[0035] This invention also provides the application of this method in evaluating the nitrogen reuse efficiency of different tea varieties. Significant differences exist in the nitrogen reuse phenotypes among different tea varieties.

[0036] Compared with the prior art, the advantages of this invention are: 1) It fills the gap in the method of nitrogen storage in tea trees, and for the first time proposes a method that, based on the original field environment, does not damage the normal growth and development of tea trees, and does not disturb the background nitrogen metabolism level of tea trees, by applying a small amount of nitrogen tracing agent to the first mature leaf corresponding to the dormant bud at the top of the tea tree canopy. 15 N-urea was used to trace the transport of stored nitrogen. 2) It was clarified that the transport of stored nitrogen is consistent among different tea varieties, and the reuse of stored nitrogen varies significantly among different varieties. Therefore, the method involved in this invention is related to plant nitrogen utilization and can be applied to regulate the growth and development of tea trees and improve tea quality through nitrogen nutrition management and other measures. At the same time, it provides an auxiliary method for identifying the phenotypic characteristics of nitrogen-efficient tea varieties, and has practical application value.

[0037] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is limited by the appended claims and their equivalents.

Claims

1. A method for identifying nitrogen-efficient phenotypic characteristics in tea plants, characterized in that, Includes the following steps: Step 1: In early January, top the tea tree canopy by removing one bud and two leaves that are not fully developed at the top. Step 2: In late January or early February, select the first mature leaf corresponding to the dormant bud at the top of the canopy, mark it, and apply... 15 N-urea; Step 3: Collect the marked leaves one month later and measure them. 15 N content; Step 4: The sample was dried using a freeze dryer, and the biomass of the dried sample was determined. Then, it was pulverized using a micro-ultrafine ball mill. The nitrogen content was determined using an elemental analyzer combined with a stable isotope mass spectrometer. 15 N abundance.

2. The method for identifying nitrogen-efficient phenotypic characteristics of tea plants according to claim 1, characterized in that: In step one, the tea tree varieties selected are 5-year-old tea tree cultivars under field planting conditions, including Zhongcha 108, Tieguanyin, Fuding Dabai, Dangui, Wuniuzao, Baiye No. 1, Longjing 43, Huangjinya, Zhenong 117, and Zijuan.

3. The method for identifying nitrogen-efficient phenotypic characteristics of tea plants according to claim 2, characterized in that, The marking process in step two is as follows: Select an even number of new tea shoots of each variety with a diameter of 0.2-0.4 cm from the tea canopy, measure and record the shoot diameter as D0, and the height of the canopy surface from the ground as H0. Then, apply 46 μg of... 15 N-urea was evenly applied to the first mature leaf that grew from the dormant bud, and the leaf area was measured and recorded as LA0. The height of the leaf from the ground was H1.

4. The method for identifying nitrogen-efficient phenotypic characteristics of tea plants according to claim 3, characterized in that, In step three, half of the marked leaves from each tea variety are collected, measured, and recorded: the area of ​​the mature leaf one month after application is recorded as LA1, and the dry weight per unit leaf area is recorded as UW1. 15 The proportion of N is 15 N dffM The amount of nitrogen (N0) assimilated into the tea plant was detected as a baseline sample. For the other half of the branches, when the first flush of new shoots from the tea plant develops to one bud and two leaves, collect samples of one bud and two leaves along with their corresponding mature leaves, and measure and record the following: leaf area as LA2, branch diameter as D1, new shoot dry weight as W1, and nitrogen concentration as N1. 15 The proportion of N is 15 N dffN ; Calculation transferred to new shoots 15 N content, divided by the baseline mature leaf sample 15 The nitrogen content is considered as the nitrogen reuse efficiency of tea plants.

5. The method for identifying nitrogen-efficient phenotypic characteristics of tea plants according to claim 4, characterized in that, The formula for calculating the nitrogen reuse efficiency of tea plants is: Nitrogen efficiency (NRE) = (LA2 ​​- LA1) ÷ LA0 × (H1 - H0) ÷ 46 + ( 15 N dffN (× W1 × N1 × D1) ÷ ( 15 N dffM (× UW1 × N0 × D0) × 100%.

6. The method for identifying nitrogen-efficient phenotypic characteristics of tea plants according to claim 4, characterized in that, The basic leaves of 10 varieties were detected in step four. 15 N dff The abundance was all below 0.1%, indicating that the labeling method did not disrupt the baseline nitrogen metabolism level of tea plants and can be used as a method to trace the reuse of stored nitrogen in plants.

7. The method for identifying nitrogen-efficient phenotypic characteristics of tea plants according to claim 6, characterized in that: (1) During the dormancy period, i.e. one month after the marking was applied, four different tissue parts of the tea tree were collected: the first mature leaf, the second mature leaf, the third mature leaf, and the stem corresponding to the dormant bud at the top of the 10 tea tree varieties. (2) Test each tissue according to the method given in step four. 15 The nitrogen content was measured, and the recovered nitrogen content was determined. 15 The proportion of nitrogen distributed in the four tissues was consistent, with more than 80% of the stored nitrogen stored in the first mature leaf corresponding to the terminal dormant bud, while the proportion distributed in the second and third mature leaves was small. This method identified Longjing 43 as having the highest reuse rate. (3) The transport pattern of stored nitrogen in tea trees was found to be consistent among different tea varieties, and the reuse rate of stored nitrogen among different varieties was significantly different.

8. An application of a method for identifying nitrogen-efficient phenotypic characteristics of tea plants according to any one of claims 1-7, characterized in that, It is applied to regulate the growth and development of tea trees and improve tea quality through nitrogen nutrition management measures, and at the same time provides an auxiliary method for identifying the phenotypic characteristics of nitrogen-efficient tea tree varieties.

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