Chlorophyll analysis method for rapidly identifying column type apples

Abstract

The invention discloses a chlorophyll analysis method for rapidly identifying columnar apples, and belongs to the technical field of apple breeding. According to the method disclosed by the invention, the cylindrical apples are rapidly identified by utilizing the content ratio (Chla / Chlb) of chlorophyll a to chlorophyll b in apple leaves for the first time. According to the invention, 125 parts of materials from 17 apple varieties and 20 apple hybrid progenies are taken as test materials, the chlorophyll analysis method is experimented, and the experiment result shows that the accuracy rate of identifying the columnar material and the non-columnar material by using the chlorophyll analysis method provided by the invention can reach 97.6%, and the accuracy rate of identifying the columnar material is 100%. The invention provides an efficient solution for rapid identification of columnar apple hybrid progeny in the seedling stage and breeding and popularization of columnar apples. The identification method is low in cost, rapid, easy to implement and high in accuracy, the breeding efficiency of the columnar apples can be remarkably improved, and the method has actual application value and wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of apple breeding, in particular to a chlorophyll analysis method for rapidly identifying columnar apples. Background Art

[0002] Tree structure is a crucial factor in orchard intensive management, influencing aspects of fruit tree shaping, pruning, pollination, and fruit yield, making it a crucial agronomic trait. Dwarfing and dense planting is the current trend in modern apple production in my country, and the future development of the apple industry cannot be separated from tree forms suitable for simplified and mechanized management. The Colmnar apple, a short-internode bud mutation found on the common apple, features compact growth, short internodes, numerous short branches from axillary buds, few or no long branches, and high photosynthetic efficiency. This tree form requires minimal pruning and is amenable to standardized mechanized management, making it an important germplasm resource for the selection and breeding of simplified apple cultivars.

[0003] The inheritance of the columnar trait in apples is a quality trait controlled by a dominant single gene, Co, and is genetically stable. Current research has shown that columnar apples can be identified using molecular markers. However, molecular identification methods require complex experimental methods, high requirements for instruments and reagents, and certain toxicity. This places high demands on the professionalism of the experimental personnel and the standardization of the experimental site, making it unsuitable for rapid identification of columnar apple materials in the field. Therefore, there is an urgent need to develop a simple method for rapid identification of columnar apple materials based on their physiological characteristics to facilitate the selection and promotion of columnar apple materials. Summary of the Invention

[0004] The purpose of the present invention is to provide a chlorophyll analysis method for quickly identifying columnar apples to solve the problems existing in the above-mentioned prior art. The present invention proposes for the first time to calculate the ratio of chlorophyll a to chlorophyll b content (Chla / Chlb) in apple leaves to identify columnar apples, providing an efficient solution for quickly identifying hybrid offspring of columnar apples in the seedling stage and for the breeding and promotion of columnar apples.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for identifying columnar apples, comprising the following steps:

[0007] Taking leaves of apples to be tested, preparing a test solution, and detecting the absorbance of the test solution;

[0008] According to the absorbance, the chlorophyll a content is calculated and recorded as Chla, the chlorophyll b content is calculated and recorded as Chlb, and the ratio of Chla to Chlb is calculated and recorded as Chla / Chlb;

[0009] Identify whether the apple to be tested is a columnar apple.

[0010] Furthermore, if Chla / Chlb of the leaf is less than 2, the apple to be tested is identified as a columnar apple;

[0011] If the Chla / Chlb of the leaf is ≥2, the apple to be tested is identified as a non-columnar apple.

[0012] Furthermore, the method of taking the leaves of the apple to be tested comprises the following steps:

[0013] Counting from top to bottom, the first leaf of the normally growing branch of the apple to be tested with a length greater than 0.5 cm is recorded as the first leaf, and the eighth leaf is taken.

[0014] Furthermore, the preparation of the test liquid comprises the following steps:

[0015] The leaf is deveined, the leaf is chopped, an ethanol solution is added, and the leaf is ground to obtain a tissue homogenate;

[0016] Add ethanol solution to the tissue homogenate, grind until the tissue turns white, use ethanol solution to make up to volume, filter, and obtain the test solution.

[0017] Furthermore, the volume fraction of ethanol in the ethanol solution is 95%; and the volume after constant volume is 15 mL.

[0018] Furthermore, the detecting the absorbance of the test liquid comprises the following steps:

[0019] The absorbance of the test solution at a wavelength of 665 nm was measured with a 95% ethanol solution as a blank, and recorded as A. 665 , measure the absorbance of the test solution at a wavelength of 649 nm, recorded as A 649 .

[0020] Furthermore, the calculation formula for calculating the chlorophyll a content is:

[0021] Chlorophyll a content = 13.95 × A 665 -6.88×A 649 .

[0022] Furthermore, the calculation formula for calculating the chlorophyll b content is:

[0023] Chlorophyll b content = 24.96 × A 649 -7.32×A 665 .

[0024] The present invention also provides an application of the above method in the trait screening and / or genetic breeding of columnar apples.

[0025] The present invention also provides an application of the above method in the development and protection of columnar apple germplasm resources.

[0026] The present invention discloses the following technical effects:

[0027] This invention proposes, for the first time, the rapid identification of columnar apples using the ratio of chlorophyll a to chlorophyll b (Chla / Chlb) in apple leaves. The chlorophyll analysis method described above was tested using 125 samples from 17 apple varieties and 20 apple hybrid offspring. The experimental results demonstrated that the chlorophyll analysis method provided by the present invention achieved an accuracy rate of 97.6% for identifying columnar and non-columnar apples, with an accuracy rate of 100% for identifying columnar apples. This invention provides a highly efficient solution for the rapid identification of columnar apple hybrid offspring at the seedling stage and for the breeding and promotion of columnar apples. The identification method of the present invention is low-cost, rapid, easy, and highly accurate, significantly improving the breeding efficiency of columnar apples and possessing practical application value and broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 Schematic diagram of leaf phenotypes and chlorophyll extracts of different apple varieties; C indicates that the sample is a columnar apple material, and N indicates that the sample is a non-columnar apple material; the scale represents 1.5 cm;

[0030] Figure 2 This is a statistical chart of the chlorophyll analysis results of different apple varieties; **** indicates P < 0.0001;

[0031] Figure 3 This is the electrophoresis result diagram of molecular marker identification of different apple varieties; among them, M represents DNA Marker, C indicates that the sample is determined to be a columnar apple material, and N indicates that the sample is determined to be a non-columnar apple material. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The apple varieties used in the present invention include non-columnar apple varieties Golden Delicious, Granny Smith, New Red Star, Red Moon, Wanglin, Ruixue, Pink Lady, Fuli, Fumei and Xu, as well as columnar apple varieties Tuscan, Guifei, Fulei, Ruga 5, Ruga 4, Wumei and Wessex. Among them, Granny Smith and Pink Lady have been described in the literature "Jia Xiaohui, Jason Johnston, Nicolette Niemann, et al. Research report on production, market sales and quality of New Zealand apples [J]. Chinese Fruit Trees, 2024, 11: 149-154. "; Golden Crown, Fuli, New Red Star, Xu, Wesekxu, Telamon, Tuskan, Dounan, Guifei, Luga 4 and Luga 5 have been published in the literature "Liu Gengsen. Development of apple SSR and SNP markers and their application in genetic map construction and variety identification [D]. Hunan Agricultural University, 2018."; Fulei has been published in the literature "Jiao Jiale, Wang Yanbo, Song Chunyan, et al. Effects of different pollination varieties on the growth and development and fruit quality of 'Fuli' apple [J]. Shandong Agricultural Sciences, 2023, 55(01 ):46-54."; Wang Lin and Rui Xue have been disclosed in the document "Xue Xiaomin, Wang Jinzheng. Development history and prospects of apple industry in Shandong Province [J]. Deciduous Fruit Trees, 2024, 56(06):8-11."; Wu Mei has been disclosed in the document "Liu Hongyu, Sun Zihao, Li Baohua, et al. Detection and full sequence analysis of apple rust virus infecting 'Wu Mei' apples [J]. Plant Protection, 2019, 45(04):176-179."; Hong Yue has been disclosed in the document "Wu Yaoguo, Chen Shuo, Wang Yangyang. Research on the creation path of regional public brands of agricultural products in developing regions - taking Yanyuan apples as an example [J]. Macro Quality Research, 2025, 1-15". The chlorophyll extraction method is a common method for analyzing plant chlorophyll. Using the principle of like dissolves like, chlorophyll can be dissolved in organic solvents such as ethanol. Based on the absorption coefficients of the wavelengths at which chlorophyll a and chlorophyll b have their maximum absorption peaks in the organic solvent, the absorbance at those wavelengths is measured and analyzed to calculate characteristics such as the chlorophyll concentration ratio. In a chlorophyll solution in 95% ethanol, the maximum absorption peaks of chlorophyll a and chlorophyll b are 665 nm and 649 nm, respectively. Combining the absorption coefficients of chlorophyll a and chlorophyll b at these two wavelengths, the chlorophyll a and chlorophyll b concentrations, Chla and Chlb, can be calculated using the formula.

[0038] The chlorophyll extraction method in the embodiment of the present invention is:

[0039] Reagent: 95% ethanol solution (95 mL of anhydrous ethanol diluted to 100 mL with water)

[0040] Method: Remove the veins of the leaves to be tested and cut them into small pieces. After weighing, add 2.5 mL of 95% ethanol solution and grind into a tissue homogenate. Add 10 mL of 95% ethanol and continue grinding until the tissue turns white. Let it stand for 3 minutes, then transfer the residue into a 15 mL volumetric flask and dilute to volume with 95% ethanol. Dilute 1 mL of the test solution with 2 mL of 95% ethanol solution and transfer it to a 1 cm optical path cuvette. Using 95% ethanol as a blank, measure the absorbance at wavelengths of 665 nm and 649 nm using a spectrophotometer. Calculate the ratio of chlorophyll a content to chlorophyll b content using the formula.

[0041] The inventor's team's preliminary research found that the surface of the leaves of transgenic poplars heterologously expressing the apple columnar gene MdCoL was wrinkled and difficult to flatten, but the leaf color was dark green and the chlorophyll content was significantly increased, with chlorophyll a and chlorophyll b increasing by 1.1 and 1.5 times respectively, and the chlorophyll a content / chlorophyll b content significantly decreased; in transgenic apples overexpressing MdCoL, the total chlorophyll content also increased, and the chlorophyll a content / chlorophyll b content significantly decreased.

[0042] Example 1 Rapid Identification of Columnar and Non-columnar Apple Varieties Using Chlorophyll Analysis

[0043] Based on the functional characteristics of the apple columnar gene MdCoL, verified in previous genetic transformation experiments, and combined with leaf tissue chlorophyll analysis, the inventors have developed a method for analyzing the ratio of chlorophyll a to chlorophyll b in apple leaf tissue to rapidly identify columnar and non-columnar apples. Columnar apples have a chlorophyll a to chlorophyll b ratio less than 2, designated as Chla / Chlb < 2, while non-columnar apples have a Chla / Chlb ratio ≥ 2.

[0044] 1. Leaf location selection and chlorophyll extraction method

[0045] In this embodiment, non-columnar apple varieties Golden Delicious, Granny Smith, New Red Star, Red Moon, Wanglin, Ruixue, Pink Lady, Fuli, Fumei, and Xu, as well as columnar apple varieties Tuscan, Guifei, Fulei, Ruga No. 5, Ruga No. 4, Wumei, and Wessex Xu were selected, totaling 17 varieties. 3-5 leaves were taken from each variety, totaling 63 materials, as identification test materials.

[0046] Take a leaf with normal growth, count the first leaf with a length greater than 0.5 cm as the first leaf, select the eighth leaf, remove the veins of the leaf to be tested and cut it into pieces, weigh 0.5 g of leaf tissue, add 2.5 mL of 95% ethanol solution and grind it into a tissue homogenate, then add 10 mL of 95% ethanol and continue grinding until the tissue turns white, let it stand for 3 minutes, transfer it together with the residue into a 15 mL volumetric flask and dilute it with 95% ethanol to obtain the test solution. Figure 1 shown.

[0047] 2. Measurement, calculation and analysis methods

[0048] Filter 1 mL of the test solution and transfer it into a cuvette with a light path of 1 cm. Then add 2 mL of 95% alcohol to dilute it. Use 95% ethanol solution as a blank and measure the absorbance at wavelengths of 665 nm and 649 nm using a spectrophotometer. Perform four technical replicates.

[0049] The chlorophyll content was calculated according to the following formula:

[0050] Chlorophyll content (mg / g) = C × V × N / ω;

[0051] Where: C represents the chlorophyll concentration, mg / L; V represents the volume of the test solution, mL; N represents the measurement dilution factor; ω represents the fresh weight of plant leaf tissue, g.

[0052] In 95% ethanol solution, according to the Lambert-Beer law and the absorption coefficients of chlorophyll a and chlorophyll b at their maximum absorption peaks of 665 nm and 649 nm, the concentrations of chlorophyll a and chlorophyll b can be calculated according to the following formulas:

[0053] Chla=13.95×A 665 -6.88×A 649 ;

[0054] Chlb=24.96×A 649 -7.32×A 665 .

[0055] Where: Chla represents the concentration of chlorophyll a, mg / L; Chlb represents the concentration of chlorophyll b, mg / L; A 665 Indicates the absorbance of the test solution at 665 nm; A 649 Indicates the absorbance of the test solution at 649 nm.

[0056] If the absorbance of the test solution at 665 nm and 649 nm is greater than 3, dilute the test solution by the corresponding multiple and re-measure.

[0057] The above method was used to calculate the average Chla / Chlb value and the original Chla / Chlb value of each test material. The results showed that the average Chla / Chlb value of columnar apples was 1.79, and the average Chla / Chlb value of non-columnar apples was 2.33. The Chla / Chlb ratios of columnar apples and non-columnar apples were clearly separated, and the analysis found that the Chla / Chlb ratio was distributed around 2.00 (such as Figure 2As shown in the figure). Therefore, the Chla / Chlb ratio was used as the evaluation content, and the dividing line between columnar apples and non-columnar apples was 2.00. If Chla / Chlb < 2.00, the material was columnar apple, and if Chla / Chlb ≥ 2.00, the sample was non-columnar apple.

[0058] The results in Table 1 demonstrate that the chlorophyll analysis method is highly effective in identifying columnar apple material. Using a Chla / Chlb ratio of 2.00 as the dividing line, columnar and non-columnar apple material can be clearly distinguished. The accuracy rate for identifying columnar and non-columnar materials reached 98.4%, with a 100% accuracy rate for identifying columnar material. This demonstrates the feasibility and effectiveness of the method. This method is low-cost, rapid, efficient, and has a low error rate. It provides a simple and effective approach for the breeding and promotion of columnar apples, significantly improving production efficiency.

[0059] Table 1 Statistics of identification results of different apple varieties using chlorophyll analysis method

[0060]

[0061] Note: The "C" or "N" before the variety name in the variety column and the "C" or "N" in the result column indicate that the sample is a "columnar" or "non-columnar" apple material; the numbers after the variety name indicate the biological replicates of different trees; Chla / Chlb in the table is the average of 4 Chla / Chlb data.

[0062] Example 2 Identification of hybrid offspring of different columnar and non-columnar apples by chlorophyll analysis and molecular markers

[0063] To test the accuracy of chlorophyll analysis in identifying columnar apples, 20 hybrids of columnar and non-columnar apples were randomly selected from the hybrid nursery of Qingdao Agricultural University. Three to five leaves from each plant, totaling 62 samples, were used as identification materials. Chlorophyll analysis was used to measure and calculate the Chla / Chlb ratio, and columnar and non-columnar materials were identified. DNA extraction and molecular marker identification were also performed.

[0064] 1. Chlorophyll analysis method

[0065] From the 62 test materials, normally growing leaves were collected. The first leaf greater than 0.5 cm in length was designated leaf 1. The eighth leaf was selected, the veins of the leaf to be tested removed, and the leaf was chopped into small pieces. After weighing, the leaf was added with 2.5 mL of 95% ethanol solution and ground into a homogenate. 10 mL of 95% ethanol was then added and ground until the tissue turned white. The mixture was allowed to stand for 3 minutes, and the residue was transferred to a 15 mL volumetric flask and brought to volume with 95% ethanol. 1 mL of the test solution was diluted with 2 mL of 95% ethanol solution and transferred to a 1 cm optical path cuvette. Using 95% ethanol as a blank, the absorbance at wavelengths of 665 nm and 649 nm was measured using a spectrophotometer. The ratio of chlorophyll a content to chlorophyll b content was calculated according to the formula.

[0066] 2. Molecular marker identification method

[0067] A young leaf was collected from each of the 20 apple hybrid progeny samples. DNA was extracted using a plant genomic DNA extraction kit (Extron). DNA sample quality was assessed using a UV imager after electrophoresis on a 1% agarose gel.

[0068] The DNA concentration should be 10-30 ng / μL. 260 / 280 =1.8-2.0, A 260 / 230 =1.8-2.0.

[0069] Primers used for molecular markers:

[0070] AF1: 5'-GGTTTTGCGTCAAGTCTTATGTTAG-3' (SEQ ID NO. 1);

[0071] AR1: 5'-CCCTCTAGCTAGTCCGTCATTTATC-3' (SEQ ID NO. 2);

[0072] AR2: 5'-AAGGTTTTGGGCTACGTCCATACTA-3' (SEQ ID NO. 3).

[0073] The PCR amplification system contained 1.4 μL AF1, 0.8 μL AR1 and 0.8 μL AR2, 1 μL DNA sample, 10 μL 2× Taq Mix, and 6 μL sterile water. The final concentration of each primer was 10 μM.

[0074] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; 30 cycles of denaturation (95°C for 30 s), annealing (56.5°C for 30 s), and extension (72°C for 30 s); finally, extension at 72°C for 2 min and storage at 4°C.

[0075] After 1.8% agarose gel electrophoresis, the molecular marker identification results were observed by UV imaging (e.g. Figure 3 shown).

[0076] 3. Experimental Results

[0077] The average values ​​of Chla / Chlb of 62 samples of 20 apple hybrid offspring were statistically analyzed. The results are shown in Table 2. Among all 24 samples of 8 hybrid offspring, the Chla / Chlb was less than 2.0, and they were identified as columnar apples. Among all 38 samples of the remaining 12 hybrid offspring, 36 samples had Chla / Chlb ≥ 2.0, and they were identified as non-columnar apples.

[0078] The molecular marker identification results are as follows Figure 3 As shown, the PCR products of 8 hybrid offsprings among the test materials had two bands, the same as the columnar control materials "Guifei" and "Fulei", and were determined to be columnar apples; the PCR products of 12 hybrid offsprings had only one non-columnar band, which was consistent with the non-columnar control materials "Fuli" and "Xinhongxing", and were determined to be non-columnar apples. The result numbers were consistent with the identification result numbers of the above-mentioned chlorophyll analysis method.

[0079] Based on the results of molecular marker identification, the overlap between the identification results using the Chla / Chlb method of chlorophyll analysis and the molecular marker identification results was as high as 96.8%, of which the overlap for identifying columnar apples reached 100%. A comprehensive analysis of the identification results of 63 test materials from 10 known non-columnar apple varieties and 7 known columnar apple varieties in Example 1, as well as 62 test materials from 20 hybrid offspring of this example, showed that using the chlorophyll analysis method of the present invention, the accuracy rate for identifying columnar and non-columnar materials reached 97.6%, of which the accuracy rate for identifying columnar materials was 100%. This proves that the method of chlorophyll analysis Chla / Chlb for rapidly identifying columnar apples is effective.

[0080] Table 2 Statistics of identification results of apple materials with different columnar and non-columnar shapes in hybrid lines

[0081]

[0082] Note: In the hybrid number column, the number after “-” indicates biological replicates of different trees; Chla / Chlb in the table is the average of 4 Chla / Chlb data; “C” or “N” in the “Molecular Marker Result” column indicates that the sample is a “columnar” or “non-columnar” apple material; “Y” in the “Are the results consistent” column indicates that the Chla / Chlb identification results are consistent with the molecular marker results, and “N” indicates inconsistency.

[0083] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for identifying columnar apples, characterized in that: The following steps are involved: Taking leaves of apples to be tested, preparing a test solution, and detecting the absorbance of the test solution; According to the absorbance, the chlorophyll a content is calculated and recorded as Chla, the chlorophyll b content is calculated and recorded as Chlb, and the ratio of Chla to Chlb is calculated and recorded as Chla / Chlb; Identify whether the apple to be tested is a columnar apple.

2. The method according to claim 1, wherein If the Chla / Chlb ratio of the leaf is less than 2, the apple to be tested is identified as a columnar apple; If the Chla / Chlb of the leaf is ≥2, the apple to be tested is identified as a non-columnar apple.

3. The method according to claim 1, wherein The method of taking the leaves of the apple to be tested comprises the following steps: Counting from top to bottom, the first leaf of the normally growing branch of the apple to be tested with a length greater than 0.5 cm is recorded as the first leaf, and the eighth leaf is taken.

4. The method according to claim 1, wherein The preparation of the test solution comprises the following steps: The leaf is deveined, the leaf is chopped, an ethanol solution is added, and the leaf is ground to obtain a tissue homogenate; Add ethanol solution to the tissue homogenate, grind until the tissue turns white, use ethanol solution to make up to volume, filter, and obtain the test solution.

5. The method according to claim 4, wherein The volume fraction of ethanol in the ethanol solution is 95%; the volume after the constant volume is 15 mL.

6. The method according to claim 1, wherein The method of detecting the absorbance of the liquid to be tested comprises the following steps: With a volume fraction of 95% ethanol solution as blank, the absorbance of the test solution at a wavelength of 665nm was measured and recorded as A 665 , measure the absorbance of the test solution at a wavelength of 649 nm, recorded as A 649 .

7. The method according to claim 6, wherein The calculation formula for calculating the chlorophyll a content is: Chlorophyll a content = 13.95 × A 665 -6.88×A 649 .

8. The method according to claim 6, wherein The calculation formula for calculating the chlorophyll b content is: Chlorophyll b content = 24.96 × A 649 -7.32×A 665 .

9. Use of the method according to any one of claims 1 to 8 in trait screening and / or genetic breeding of columnar apples.

10. Use of the method according to any one of claims 1 to 8 in the development and protection of columnar apple germplasm resources.

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