A method for breeding a light and simple machine apple variety
Patent Information
- Application Number
- CN202610118070.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-28
- Publication Date
- 2026-09-22
AI Technical Summary
但是，目前我国苹果产业受一家一户碎片化、果园基础地力差、机械化水平低及劳动力和生产成本上涨等多因素叠加影响，中国东部环渤海湾和西部黄土高原两个苹果主产区的果农收益空间大幅缩小，果园投入不足，栽培面积缩减
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fruit tree breeding and relates to a simplified and mechanizable method for cultivating apple varieties. Background Technology
[0002] Apples are one of the world's four major fruits. Through the collective efforts of generations of Chinese apple growers over a century, apples have become the largest deciduous fruit tree species in my country, accounting for over 50% of the world's cultivation area and output, with an annual output value exceeding 200 billion yuan. It is one of the pillar industries for increasing farmers' income and rural revitalization. However, currently, my country's apple industry is affected by a combination of factors, including fragmented production by individual households, poor soil fertility in orchards, low levels of mechanization, and rising labor and production costs. This has significantly reduced the profit margins for fruit farmers in the two main apple-producing areas of eastern China (the Bohai Rim region) and western China (the Loess Plateau region), leading to insufficient investment in orchards and a reduction in cultivated area. Therefore, in response to the increasing production costs of apple orchards and the need for large-scale modern cultivation models, a simplified and mechanized apple breeding objective has been proposed.
[0003] Simplified Production: Currently, labor costs for bagging and unbagging account for over 60% of the production cost in China's apple industry, necessitating the development of bag-free apple varieties. Implementing green and ecological development concepts and achieving cost reduction and efficiency improvement also requires cultivating disease-resistant apple varieties. Storage tolerance is not only a necessary characteristic for fruit to become a marketable product but also a crucial trait for simplified production. Therefore, bagging-free production, disease resistance, and storage tolerance are the breeding goals for simplified apple varieties.
[0004] Mechanization: Analysis of successful cases of modern cultivation models in China reveals that there are currently 22,000 hectares of mechanized agriculture in Weihai City, Shandong Province. 2 In modern apple orchards, 'Venus Gold' accounts for 85% of the production. This variety is a seedling descendant of 'Golden Delicious' and is considered a "triple-good" variety. It not only boasts a rich aroma, making it delicious and marketable, but also exhibits low sensitivity to pruning, making it suitable for mechanical pruning. It tolerates extensive pruning management, flowers easily, bears fruit early and yields abundantly, uses fruit to suppress vegetative growth, resulting in a compact canopy. It is also easy to manage and suitable for mechanized operations, quickly generating high profits for relevant enterprises. Therefore, easy flowering, early fruiting, high yield, and suitability for mechanized management are the breeding goals for mechanized apple orchards.
[0005] Developing new apple varieties that are easy to simplify and suitable for mechanization is of great significance for promoting the sustainable and high-quality development of China's apple industry and the modernization of the Chinese-style apple industry. Summary of the Invention
[0006] The purpose of this invention is to provide a simplified and machine-friendly method for cultivating apple varieties.
[0007] This invention provides primer pairs, including primer pair I, primer pair II, and primer pair III;
[0008] Primer pair I consists of primer BBX24F and primer BBX24R; primer BBX24F is the single-stranded DNA molecule shown in SEQ ID NO: 1, and primer BBX24R is the single-stranded DNA molecule shown in SEQ ID NO: 2; Primer pair II consists of primer MYB62F and primer MYB62R; primer MYB62F is the single-stranded DNA molecule shown in SEQ ID NO: 5, and primer MYB62R is the single-stranded DNA molecule shown in SEQ ID NO: 6; The primer pair III consists of primer NAC29F and primer NAC29R; primer NAC29F is the single-stranded DNA molecule shown in SEQ ID NO: 7, and primer NAC29R is the single-stranded DNA molecule shown in SEQ ID NO: 8.
[0009] Specifically, the primer combination also includes primer pair IV. Primer pair IV consists of primer ActinF and primer ActinR; primer ActinF is the single-stranded DNA molecule shown in SEQ ID NO: 3, and primer ActinR is the single-stranded DNA molecule shown in SEQ ID NO: 4.
[0010] Specifically, the primer pair consists of primer pair I, primer pair II, and primer pair III.
[0011] Specifically, the primer pair consists of primer pair I, primer pair II, primer pair III, and primer pair IV.
[0012] The present invention also protects the application of the primer combination as follows (a1) or (a2) or (a3) or (a4): (a1) Identification or auxiliary identification of simplified and mechanizable apple germplasm; (a2) Selecting or assisting in the selection of simplified and mechanizable apple germplasm; (a3) Prepare a kit for identifying or assisting in the identification of simplified and easily mechanized apple germplasm; (a2) Prepare a kit for breeding or assisting in the breeding of lightweight, easy-to-organize apple germplasm.
[0013] This invention also protects a method for breeding lightweight, simple, and biocompatible apple germplasm, comprising the following steps: (1) The male parent and the female parent are crossed to harvest hybrid seeds; the male parent is a red-fleshed apple; the female parent is a white-fleshed apple; (2) Cultivate the hybrid seeds obtained in step (1) into plants; (3) The plants obtained in step (2) are used as plants to be screened. Plants that meet standard 1, standard 2 and standard 3 are selected, which are the simplified and easy-to-organize apple germplasm. The method for screening plants that meet criterion 1 includes the following steps: Collect the pericarp of the fruit from the test plant and the pericarp of the fruit from the parent plant, extract total RNA and reverse transcribe it to obtain cDNA; use the cDNA as a template, and... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdBBX24 The relative expression levels of genes; then, plants meeting the following criterion 1 are screened: the pericarp of the fruit of the tested plant... MdBBX24 The relative expression level of the gene is the same as that of the fruit peel of the maternal plant. MdBBX24 The relative expression level of the gene is less than 1 / 5; The method for screening plants that meet criterion 2 includes the following steps: The pulp of the fruit from the test plant and the pulp of the fruit from the parent plant are taken respectively; total RNA is extracted and reverse transcribed to obtain cDNA; using the cDNA as a template, ... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdMYB62 The relative expression level of genes; then, plants that meet the following criterion 2 are screened: the pulp of the fruit of the tested plant... MdMYB62 The relative expression level of the gene is the same as that of the pulp of the maternal plant's fruit. MdMYB62 More than 5 times the relative expression level of the gene; The method for screening plants that meet criterion 3 includes the following steps: buds from the test plants and buds from the parent plants are taken respectively; total RNA is extracted and reverse transcribed to obtain cDNA; using the cDNA as a template, ... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdNAC29 The relative expression level of genes; then, plants that meet the following criterion 3 are screened: the buds of the tested plants... MdNAC29 The relative expression level of the gene is that of the bud of the maternal plant. MdNAC29 More than five times the relative expression level of the gene.
[0014] In the method described, detection MdBBX24 The primer pair used for the gene is primer pair I; In the method described, detection MdMYB62 The primer pair used for the gene is primer pair II; In the method described, detection MdNAC29 The primer pair used for the gene is primer pair III.
[0015] In the method described, detection MdActin The primer pair for the gene is primer pair IV.
[0016] This invention also protects the lightweight, simple, and biocompatible apple germplasm obtained by the methods described above.
[0017] The simplified and machine-friendly apple germplasm mentioned above refers to apple germplasm with an anthocyanin content in the peel of the fruit ≥300mg / kg and an flowering rate ≥60%.
[0018] The simplified and biocompatible apple germplasm mentioned above refers to apple germplasm with an anthocyanin content in the peel ≥300mg / kg, a flowering rate ≥60%, and a soluble sugar content of ≥14%.
[0019] The simplified and biocompatible apple germplasm mentioned above refers to apple germplasm with an anthocyanin content in the peel ≥300mg / kg and apple germplasm with ring rot lesions ≤5cm². 2 Furthermore, the flowering rate of this apple variety is ≥60%.
[0020] The simplified and biocompatible apple germplasm mentioned above refers to apple germplasm with an anthocyanin content in the peel ≥300mg / kg and apple germplasm with ring rot lesions ≤5cm². 2 Furthermore, the flowering rate of this apple variety is ≥60%, and the soluble sugar content of the fruit of this apple variety is ≥14%.
[0021] The method for taking buds includes the following steps: the main stem of the test plant, starting from the top of the main stem and ending at the part of the branch closest to the ground, is divided into three equal parts according to length, namely the top, middle and bottom, and the branches growing on it are respectively the top branch, the middle branch and the bottom branch; three branches are randomly selected from the top branch, three branches are randomly selected from the middle branch and three branches are randomly selected from the bottom branch; one terminal bud and two random lateral buds are taken from each of the branches.
[0022] This invention also protects the application of Mei Hong 5 red-fleshed apples; (a) Application in apple production; (b) Application in apple breeding; The red-fleshed apple, Mei Hong 5, has the preservation registration number CGMCC No. 46989.
[0023] The apples produced are those that meet the following standards: anthocyanin content in the peel ≥ 300 mg / kg and flowering rate ≥ 60%. Alternatively, the apples produced can meet the following standards: anthocyanin content in the peel ≥ 400 mg / kg and flowering rate ≥ 70%. Finally, the apples produced can meet the following standards: anthocyanin content in the peel 430-440 mg / kg and flowering rate 71%-75%.
[0024] The apples produced are those that meet the following standards: the anthocyanin content in the fruit peel is ≥300mg / kg and the area of ring rot lesions on the fruit is ≤5cm². 2 Furthermore, the flowering rate is ≥60%. The apples produced are those meeting the following standards: anthocyanin content in the peel ≥400 mg / kg and area of ring rot lesions ≤3 cm². 2 Furthermore, the flowering rate must be ≥70%. The apple fruit produced must meet the following standards: the anthocyanin content in the fruit peel must be 430-440 mg / kg, and the area of ring rot lesions on the fruit must be 2.8-3.0 cm². 2 The flowering rate is 71%-75%.
[0025] The apples produced are those that meet the following standards: anthocyanin content in the peel ≥300 mg / kg, flowering rate ≥60%, and soluble sugar content ≥14%. Alternatively, the apples produced can meet the following standards: anthocyanin content in the peel ≥400 mg / kg, flowering rate ≥70%, and soluble sugar content ≥14%. Finally, the apples produced can meet the following standards: anthocyanin content in the peel 430-440 mg / kg, flowering rate 71%-75%, and soluble sugar content 15%-16%.
[0026] The apples produced are those that meet the following standards: the anthocyanin content in the fruit peel is ≥300mg / kg and the area of ring rot lesions on the fruit is ≤5cm². 2 Furthermore, the flowering rate must be ≥60% and the soluble sugar content of the fruit must be ≥14%. The apple fruit produced must meet the following standards: anthocyanin content in the fruit peel ≥400 mg / kg and the area of ring rot lesions ≤3 cm². 2 Furthermore, the flowering rate must be ≥70% and the soluble sugar content of the fruit must be ≥14%. The apple fruit produced must meet the following standards: anthocyanin content in the fruit peel must be 430-440 mg / kg and the area of ring rot lesions on the fruit must be 2.8-3.0 cm². 2 Furthermore, the flowering rate is 71%-75%, and the soluble sugar content of the fruit is 15%-16%.
[0027] The apples produced are those that meet the following standards: The anthocyanin content in the fruit peel is ≥300 mg / kg. Alternatively, the apples produced can meet the following standards: The anthocyanin content in the fruit peel is ≥400 mg / kg. Finally, the apples produced can meet the following standards: The anthocyanin content in the fruit peel is 430-440 mg / kg.
[0028] The apples produced are those that meet the following standards: the anthocyanin content in the fruit peel is ≥300mg / kg and the area of ring rot lesions on the fruit is ≤5cm². 2 The apples produced are those that meet the following standards: anthocyanin content in the peel ≥ 400 mg / kg and area of ring rot lesions ≤ 3 cm². 2 The apples produced are those that meet the following standards: the anthocyanin content in the fruit peel is 430-440 mg / kg, and the area of ring rot lesions on the fruit is 2.8-3.0 cm². 2 .
[0029] The apples produced are those that meet the following standards: anthocyanin content in the peel ≥ 300 mg / kg and soluble sugar content ≥ 14%. Alternatively, the apples produced can meet the following standards: anthocyanin content in the peel ≥ 400 mg / kg and soluble sugar content ≥ 14%. Finally, the apples produced can meet the following standards: anthocyanin content in the peel 430-440 mg / kg and soluble sugar content 15%-16%.
[0030] The apples produced are those that meet the following standards: the anthocyanin content in the fruit peel is ≥300mg / kg and the area of ring rot lesions on the fruit is ≤5cm². 2 Furthermore, the soluble sugar content of the fruit is above 14%. The apple fruit produced refers to apple fruit that meets the following standards: anthocyanin content in the fruit peel ≥ 400 mg / kg and the area of ring rot lesions ≤ 3 cm². 2 Furthermore, the soluble sugar content of the fruit is above 14%. The apple fruit produced meets the following standards: the anthocyanin content in the fruit peel is 430-440 mg / kg, and the area of ring rot lesions on the fruit is 2.8-3.0 cm². 2 Furthermore, the soluble sugar content of the fruit is 15%-16%.
[0031] The apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥300 mg / kg and flowering rate ≥60%. Alternatively, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥400 mg / kg and flowering rate ≥70%. Finally, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel 430-440 mg / kg and flowering rate 71%-75%.
[0032] The apple breeding program aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥300mg / kg and area of ring rot lesions ≤5cm². 2Furthermore, the flowering rate is ≥60%. The apple breeding program aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥400 mg / kg and the area of ring rot lesions on the fruit ≤3 cm². 2 Furthermore, the flowering rate is ≥70%. The apple breeding program aims to cultivate apple germplasm that meets the following criteria: the anthocyanin content in the fruit peel is 430-440 mg / kg, and the area of ring rot lesions on the fruit is 2.8-3.0 cm². 2 The flowering rate is 71%-75%.
[0033] The apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥300 mg / kg, flowering rate ≥60%, and soluble sugar content ≥14%. Alternatively, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥400 mg / kg, flowering rate ≥70%, and soluble sugar content ≥14%. Finally, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel 430-440 mg / kg, flowering rate 71%-75%, and soluble sugar content 15%-16%.
[0034] The apple breeding program aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥300mg / kg and area of ring rot lesions ≤5cm². 2 Furthermore, the flowering rate should be ≥60% and the soluble sugar content of the fruit should be ≥14%. The apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥400 mg / kg and the area of ring rot lesions on the fruit ≤3 cm². 2 Furthermore, the flowering rate should be ≥70% and the soluble sugar content of the fruit should be ≥14%. The apple breeding aims to cultivate apple germplasm that meets the following criteria: the anthocyanin content in the fruit peel is 430-440 mg / kg and the area of ring rot lesions on the fruit is 2.8-3.0 cm². 2 Furthermore, the flowering rate is 71%-75%, and the soluble sugar content of the fruit is 15%-16%.
[0035] The apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥ 300 mg / kg. Alternatively, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥ 400 mg / kg. Finally, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel 430-440 mg / kg.
[0036] The apple breeding program aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥300mg / kg and area of ring rot lesions ≤5cm². 2The apple breeding program aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥ 400 mg / kg and area of ring rot lesions ≤ 3 cm². 2 The apple breeding program aims to cultivate apple germplasm that meets the following criteria: the anthocyanin content in the fruit peel is 430-440 mg / kg, and the area of ring rot lesions on the fruit is 2.8-3.0 cm². 2 .
[0037] The apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥ 300 mg / kg and soluble sugar content ≥ 14%. Alternatively, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥ 400 mg / kg and soluble sugar content ≥ 14%. Finally, the apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel 430-440 mg / kg and soluble sugar content 15%-16%.
[0038] The apple breeding program aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥300mg / kg and area of ring rot lesions ≤5cm². 2 Furthermore, the soluble sugar content of the fruit is above 14%. The apple breeding aims to cultivate apple germplasm that meets the following criteria: anthocyanin content in the fruit peel ≥ 400 mg / kg and the area of ring rot lesions on the fruit ≤ 3 cm². 2 Furthermore, the soluble sugar content of the fruit is above 14%. The apple breeding aims to cultivate apple germplasm that meets the following criteria: the anthocyanin content in the fruit peel is 430-440 mg / kg, and the area of ring rot lesions on the fruit is 2.8-3.0 cm². 2 Furthermore, the soluble sugar content of the fruit is 15%-16%.
[0039] The area of ring rot lesions mentioned above refers to the area of ring rot lesions on apple fruits caused by the pathogenic fungus *Botryosphaeria dothidea*. Specifically, the pathogenic fungus is *Botryosphaeria dothidea*.
[0040] The area of the ring spot disease lesions refers to the area of the ring spot disease lesions detected in the ring spot disease resistance test.
[0041] For example, the ring spot disease resistance test includes the following steps: ① Inoculate the pathogenic fungus of apple ring rot onto PDA medium and incubate at 25°C until the colony diameter is about 5cm. Then, use a sterile punch with a diameter of 7 mm to collect mycelial blocks, which are the pathogenic inoculum. ② Take the fruit of the tested apple germplasm, disinfect the surface with 70% ethanol aqueous solution, then rinse with sterile water and air dry naturally. Then make a needle puncture wound with a depth of 2 mm with a sterile needle and place the pathogenic inoculum on the needle puncture wound. ③ Five days after vaccination, the area of ring spot disease lesions was counted.
[0042] Specifically, as described in apple cDNA MdNAC29 The gene is shown in SEQ ID NO: 11.
[0043] Specifically, as described in apple cDNA MdBBX24 The gene is shown in SEQ ID NO: 9.
[0044] Specifically, as described in apple cDNA MdMYB62 The gene is shown in SEQ ID NO: 10.
[0045] Specifically, the parent variety is the 'Meihong' apple.
[0046] Specifically, the parent variety is the 'Changfu 2' apple.
[0047] The present invention also provides a protein, named MdNAC29 protein, as shown in SEQ ID NO: 12.
[0048] This invention also provides MdNAC29 protein-related biomaterials, for MdNAC29 Genes, possess MdNAC29 Gene expression cassettes, with MdNAC29 Recombinant plasmids of genes, possessing MdNAC29 Recombinant microorganisms or those with genes MdNAC29 Plants with genetically modified organisms; the aforementioned MdNAC29 The gene is the gene that encodes the MdNAC29 protein.
[0049] Specifically, the aforementioned MdNAC29 The gene is shown in SEQ ID NO: 11.
[0050] Specifically, the aforementioned MdBBX24 The gene is shown in SEQ ID NO: 9.
[0051] Specifically, the aforementioned MdMYB62 The gene is shown in SEQ ID NO: 10.
[0052] This invention also provides the application of MdNAC29 protein or MdNAC29 protein-related biomaterials in improving the flowering rate of plants.
[0053] The present invention also provides a method for improving the flowering rate of plants, comprising the following steps: introducing MdNAC29 protein-related biological materials into plants to obtain transgenic plants with improved flowering rate.
[0054] Any of the plants mentioned above are either monocotyledons or dicotyledons.
[0055] Specifically, any of the plants mentioned above are either Arabidopsis or Tomato.
[0056] For example, any of the plants mentioned above are Arabidopsis thaliana ecotype or Micro Tom tomato.
[0057] The apples mentioned above are ripe apples.
[0058] The simplified indicators refer to: coloring traits (specifically reflected in the anthocyanin content of the peel) and disease resistance traits (specifically reflected in the area of apple ring rot lesions).
[0059] The indicator for adaptability to mechanization is: flowering rate.
[0060] This invention can be used to cultivate new apple varieties that are lightweight, simplified, and suitable for mechanization, which is of great significance for promoting the sustainable and high-quality development of China's apple industry and the modernization of China's apple industry.
[0061] Preservation Instructions Germplasm name: Mei Hong 5 Category Naming: Red-fleshed Apple Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Abbreviation of depositary institution: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: December 25, 2025 Registered with the China National Collection Center (CGMCC) No. 46989. Attached Figure Description
[0062] Figure 1 Photo of 'Meihong No. 5' fruit setting in the field.
[0063] Figure 2 Photos showing the resistance of 'Meihong 5' and 'Changfu 2' apples to ring spot disease.
[0064] Figure 3 This is a photograph of the flower phenotype identification in Example 5. Detailed Implementation
[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0066] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. Concentrated hydrochloric acid is commercially available 12mol / L hydrochloric acid. The organic fertilizer used in the examples is fully decomposed cow manure. The seed nursery (field) is located in Tai'an, Shandong Province, covering an area of 50 mu. The purpose of the stratification treatment in the examples is to break seed dormancy by meeting the chilling requirement. The premixed solution used in the examples is 2×TransStart® Tip Green qPCR SuperMix, Beijing TransGen Biotech Co., Ltd., product catalog number AQ141. Seedling substrate: Ward Agricultural Technology Co., Ltd. MS basal medium stock solution (Murashige & Skoog Basal Medium with Vitamins): www.phytotechlab.com, product catalog number M519. Dilute the stock solution of macroelements in MS basal medium with water to 10 times its volume to obtain the nutrient solution. Unless otherwise specified, light-dark alternation means 16 hours of light / 8 hours of darkness. The pPZP211 vector, a plant expression vector in the prior art, has its full sequence shown in GenBank: U10490.1 (28-JAN-1995).
[0067] The 'Meihong' apple variety is described in the following literature: A new high-flavonoid apple variety 'Meihong'. Journal of Horticulture, 2018, 45(S2):2705-2706.
[0068] The 'Changfu No. 2' apple variety is described in the following literature: Differences in the internal quality components of 'Changfu No. 2' apples grown with and without bagging. Journal of Northwest A&F University (Natural Science Edition), 2025, (12): 1-9.
[0069] The pathogen of apple ring rot used in this example is *Botryosphaeria dothidea*. Botryosphaeria dothidea The pathogenic function of the pectin lyase gene Bdpl1 of Botryosphaeria dothidea is described in the following literature: Acta Phytopathologica Sinica, 2019, 49(03):314-325.
[0070] Example 1: Preparation of a hybrid population of 'Meihong' apple and 'Changfu 2' apple 1. Preparation of hybrid seeds The 'Meihong' apple plant was used as the male parent and the 'Changfu 2' apple plant as the female parent. They were crossbred and cultivated under normal conditions. The apples from the female parent plant were harvested, and the seeds were collected to obtain the hybrid seeds. After washing, the hybrid seeds were stratified in a refrigerator at 1-3℃ for approximately 60 days.
[0071] 2. Greenhouse seedling cultivation Sow the seeds obtained from step 1 in seedling pots filled with seedling substrate (3-5 seeds per pot), and cultivate them until the seedlings are 8-15cm tall and the root collar is lignified (usually 2-3 months after sowing). Then, transplant the seedlings into new seedling pots filled with seedling substrate (1 seedling per pot) for further cultivation.
[0072] The cultivation conditions in this step are: 25℃, 12 hours of light / 12 hours of darkness, and a light intensity of 3000 lx. During the cultivation process, water each seedling pot with nutrient solution every 7-10 days from the start of seed germination, using 40-50 ml of solution each time.
[0073] 3. Transplanting of hybrid seedlings In February, apply organic fertilizer (6000 kg / mu) to the seed selection nursery and water to settle the soil. In April, transplant the 1400 seedlings obtained in step 2 to the seed selection nursery, apply organic fertilizer again (6000 kg / mu), and water promptly. Perform normal cultivation management. The year of seedling transplantation is considered the first year; fruiting begins in the third year.
[0074] Example 2: Discovering genes whose expression levels are correlated with target traits Study subjects: 100 seedlings were randomly selected from the hybrid population prepared in step one of Example 1.
[0075] I. Identifying genes whose expression levels are correlated with anthocyanin content 1. In the third year (the year of transplanting seedlings is considered the first year), five mature fruits were randomly selected from each plant. The peel was removed using a peeler, and the anthocyanin content was measured. The average value was then calculated. The differences in anthocyanin content in the peel of different plants were compared. Transcriptomic analysis was used to identify target genes whose expression levels are related to anthocyanin content in the peel.
[0076] 2. In the fourth year (the year of transplanting seedlings is considered the first year), 10 mature fruits were randomly harvested from each plant. The peel was removed using a peeler, and the anthocyanin content was measured. The average value was then calculated. The differences in anthocyanin content in the peel of different plants were compared. The correlation between the anthocyanin content in the peel and the expression level of the target gene identified in step 1 was verified. The target gene whose expression level was negatively correlated with the anthocyanin content in the peel was finally verified as follows: MdBBX24 Genes (in apple cDNA) MdBBX24 (The gene is shown in SEQ ID NO: 9).
[0077] II. Establish a system for testing MdBBX24 Methods for screening target plants based on gene expression levels The test fruit refers to the mature fruit on the test plant. 5-10 mature fruits are taken from each test plant.
[0078] Collect the test fruit, remove the pericarp with a peeler, extract total RNA, and reverse transcribe it to obtain cDNA. Use the cDNA as a template... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdBBX24 Relative gene expression levels (2) -△△CT (Method). Used for detection. MdBBX24 The primer pair for the gene consists of primer BBX24F and primer BBX24R. It is used for detection. MdActin A gene primer pair consists of primer ActinF and primer ActinR. F represents the upstream primer and R represents the downstream primer.
[0079] Primer BBX24F (SEQ ID NO: 1): 5'-TTCAGCCAATAGCCTCTC-3'; Primer BBX24R (SEQ ID NO: 2): 5'-GCAGTAAGTCATCAACACC-3'.
[0080] Primer ActinF (SEQ ID NO: 3): 5'-TGTTCTTAGTGGTGGTTCA-3'; Primer ActinR (SEQ ID NO: 4): 5'-CACATCTGCTGGAAGGTA-3'.
[0081] The reaction system consisted of: 1 μL template (50 ng DNA), 1 μL upstream primer solution, 1 μL downstream primer solution, 10 μL premix, and 7 μL ddH₂O. The upstream primer solution provided the active ingredient of the upstream primer (primer BBX24F or primer ActinF), and the downstream primer solution provided the active ingredient of the downstream primer (primer BBX24R or primer ActinR). The concentrations of the upstream and downstream primers in the reaction system were both 0.2 μM.
[0082] The reaction was performed using a Bio-Rad (USA) iCycler iQ5 real-time quantitative PCR instrument. The reaction program was as follows: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, for 40 cycles; 72℃ extension for 5 min.
[0083] The result for each plant is the average of all the fruits tested from that plant.
[0084] The parent plant (i.e., the 'Changfu 2' apple plant) was used as the reference plant for the test plants, and the above steps were followed for testing. MdBBX24 The relative expression level of genes, defined as reference data 1. If the tested plants'... MdBBX24 The relative expression level of the gene was less than 1 / 5 (inclusive) of that of the reference data 1, and the tested plant was the target plant.
[0085] III. Identifying genes whose expression levels are correlated with disease resistance 1. In the third year (the year of transplanting seedlings is considered the first year), five mature fruits were randomly selected from each plant to test their resistance to ring rot disease, and the average value was taken. Differences in ring rot resistance among different plants were compared. Target genes were identified through transcriptomic analysis. Target genes are those whose expression levels are related to ring rot resistance.
[0086] 2. In the fourth year (the year of transplanting seedlings is considered the first year), 10 mature fruits were randomly selected from each plant to test their resistance to ring rot disease, and the average value was taken. The differences in ring rot resistance among different plants were compared. The correlation between ring rot resistance and the expression level of the target gene identified in step 1 was verified. The target gene whose expression level was positively correlated with ring rot resistance was finally verified as follows: MdMYB62 Genes (in apple cDNA) MdMYB62 (The gene is shown in SEQ ID NO: 10).
[0087] IV. Establishing a testing system MdMYB62 Methods for screening target plants based on gene expression levels The test fruit refers to the mature fruit on the test plant. 5-10 mature fruits are taken from each test plant.
[0088] The pulp of the tested fruit was collected, total RNA was extracted, and cDNA was obtained through reverse transcription. Using the cDNA as a template, ... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdMYB62 Relative gene expression levels (2) -△△CT (Method). Used for detection. MdMYB62 The primer pair for the gene consists of primer MYB62F and primer MYB62R. It is used for detection. MdActin A gene primer pair consists of primer ActinF and primer ActinR. F represents the upstream primer and R represents the downstream primer.
[0089] Primer MYB62F (SEQ ID NO: 5): 5'-ATCAAGAACTACTGGAGAACA-3'; Primer MYB62R (SEQ ID NO: 6): 5'-CTGCGAAATGGTATGGTTT-3'.
[0090] Primer ActinF (SEQ ID NO: 3): 5'-TGTTCTTAGTGGTGGTTCA-3'; Primer ActinR (SEQ ID NO: 4): 5'-CACATCTGCTGGAAGGTA-3'.
[0091] The reaction system consisted of: 1 μL template (50 ng DNA), 1 μL upstream primer solution, 1 μL downstream primer solution, 10 μL premix, and 7 μL ddH₂O. The upstream primer solution provided the active ingredient of the upstream primer (MYB62F or ActinF), and the downstream primer solution provided the active ingredient of the downstream primer (MYB62R or ActinR). The concentrations of the upstream and downstream primers in the reaction system were both 0.2 μM.
[0092] The reaction was performed using a Bio-Rad (USA) iCycler iQ5 real-time quantitative PCR instrument. The reaction program was as follows: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, for 40 cycles; 72℃ extension for 5 min.
[0093] The result for each plant is the average of all the fruits tested from that plant.
[0094] The parent plant (i.e., the 'Changfu 2' apple plant) was used as the reference plant for the test plants, and the above steps were followed for testing. MdMYB62 The relative expression level of the gene, defined as reference data 2. If the tested plants' MdMYB62 The relative expression level of the gene was more than 5 times (including 5 times) that of the reference data 2, and the tested plant was the target plant.
[0095] V. Identifying genes whose expression levels are correlated with flowering rate 1. In the second year (the year of transplanting seedlings is considered the first year), buds from the plants are taken in June (during the physiological differentiation period of flower buds) for transcriptomic analysis (all buds taken from each plant are mixed before transcriptomic analysis). In the third year (the year of transplanting seedlings is considered the first year), the flowering rate of each plant is measured. Target genes are identified. Target genes are those whose expression levels are related to the flowering rate.
[0096] 2. In the third year (the year of seedling transplanting is considered the first year), buds from the plants were collected in June (during the physiological differentiation period of flower buds) for transcriptomic analysis. In the fourth year (the year of seedling transplanting is considered the first year), the flowering rate of each plant was measured. The correlation between the flowering rate and the expression level of the target gene identified in step 1 was validated. The target gene whose expression level was positively correlated with the flowering rate was finally verified to be... MdNAC29 Genes (in apple cDNA) MdNAC29 (The gene is shown in SEQ ID NO: 11).
[0097] VI. Establish a testing system MdNAC29 Methods for screening target plants based on gene expression levels During the physiological differentiation stage of flower buds, buds were collected from the test plants. Total RNA was extracted from all buds from a single plant and reverse transcribed to obtain cDNA. Using the cDNA as a template, ... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdNAC29 Relative gene expression levels (2) -△△CT (Method). Used for detection. MdNAC29 The primer pair for the gene consists of primer NAC29F and primer NAC29R. It is used for detection. MdActin A gene primer pair consists of primer ActinF and primer ActinR. F represents the upstream primer and R represents the downstream primer.
[0098] Primer NAC29F (SEQ ID NO: 7): 5'-CTGAGGGACTTTGTTCTTTAC-3'; Primer NAC29R (SEQ ID NO: 8): 5'-GCAACTACCATTACCAGGA-3'.
[0099] Primer ActinF (SEQ ID NO: 3): 5'-TGTTCTTAGTGGTGGTTCA-3'; Primer ActinR (SEQ ID NO: 4): 5'-CACATCTGCTGGAAGGTA-3'.
[0100] The reaction system consisted of: 1 μL template (50 ng DNA), 1 μL upstream primer solution, 1 μL downstream primer solution, 10 μL premix, and 7 μL ddH₂O. The upstream primer solution provided the active ingredient of the upstream primer (primer NAC29F or primer ActinF), and the downstream primer solution provided the active ingredient of the downstream primer (primer NAC29R or primer ActinR). The concentrations of the upstream and downstream primers in the reaction system were both 0.2 μM.
[0101] The reaction was performed using a Bio-Rad (USA) iCycler iQ5 real-time quantitative PCR instrument. The reaction program was as follows: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, for 40 cycles; 72℃ extension for 5 min.
[0102] The parent plant (i.e., the 'Changfu 2' apple plant) was used as the reference plant for the test plants, and the above steps were followed for testing. MdNAC29 The relative expression level of genes, defined as reference data 3. If the tested plants... MdNAC29 The relative expression level of the gene was more than 5 times (including 5 times) that of the reference data 2, and the tested plant was the target plant.
[0103] Example 3: Method Verification Study subjects: Twenty seedlings were randomly selected from the hybrid population prepared in Example 1 (not overlapping with the 100 seedlings in Example 2). The 20 seedlings, along with the male parent ('Meihong' apple plant) and the female parent ('Changfu 2' apple plant), were used as test plants.
[0104] In the fourth year (the year of transplanting seedlings is considered the first year), the procedure in step two of Example 2 is followed.
[0105] In the fourth year (the year of transplanting seedlings is considered the first year), the procedure in step four of Example 2 is followed.
[0106] In the fourth year (the year of transplanting seedlings is considered the first year), the procedure in step six of Example 2 is followed.
[0107] In the fourth year, the anthocyanin content in the fruit peel and its resistance to ring rot were tested.
[0108] In the 5th year, the flowering rate was measured.
[0109] The results are shown in Table 1. Standard 1: The tested plants MdBBX24The relative expression level of a gene is less than 1 / 5 (inclusive) of that in reference data 1. Mark with √ if satisfied, and × if not. Standard 2: The expression level of the tested plants... MdMYB62 The relative expression level of the gene is 5 times or more (including 5 times) that of reference data 2. Mark with √ if satisfied, and × if not. Standard 3: The expression level of the tested plants... MdNAC29 If the relative expression level of a gene is 5 times or more (including 5 times) that of reference data 3, it is indicated by √ and not by ×.
[0110] Table 1
[0111] Example 4: Breeding simplified and mechanizable apple varieties The population to be screened was the hybrid population (1400 seedlings) prepared in Example 1.
[0112] 1. Select target plants from the population to be screened.
[0113] The tested plants were each plant in the population to be screened, as well as the male parent plant ('Meihong' apple plant) and the female parent plant ('Changfu 2' apple plant).
[0114] In the fourth year (the year of transplanting seedlings is considered the first year), the procedure in step two of Example 2 is followed.
[0115] In the fourth year (the year of transplanting seedlings is considered the first year), the procedure in step four of Example 2 is followed.
[0116] In the fourth year (the year of transplanting seedlings is considered the first year), the procedure in step six of Example 2 is followed.
[0117] Plants that meet criteria 1, 2, and 3 are selected as target plants. Criterion 1: The tested plants... MdBBX24 The relative expression level of a gene is less than 1 / 5 (inclusive) of that in reference data 1. Mark with √ if satisfied, and × if not. Standard 2: The expression level of the tested plants... MdMYB62 The relative expression level of the gene is 5 times or more (including 5 times) that of reference data 2. Mark with √ if satisfied, and × if not. Standard 3: The expression level of the tested plants... MdNAC29 If the relative expression level of a gene is 5 times or more (including 5 times) that of reference data 3, it is indicated by √ and not by ×.
[0118] Ten target plants were selected from 1400 seedlings and named 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9# or 10# respectively.
[0119] 2. Detection characteristics The tested plants were the 10 plants selected in step 1, as well as the male parent plant ('Meihong' apple plant) and the female parent plant ('Changfu 2' apple plant).
[0120] In the fourth year, the anthocyanin content in the peel, resistance to ring rot disease, and soluble sugar content in the fruit were tested.
[0121] In the 5th year, the flowering rate was measured.
[0122] The results are shown in Table 2.
[0123] Table 2
[0124] Simplified and mechanizable apple plants refer to those with an anthocyanin content in the peel ≥300mg / kg and lesion area ≤5cm² in the ring rot resistance test. 2 And the flowering rate is ≥60%.
[0125] Plant #5 was named Mei Hong 5 (also known as 'Mei Hong No. 5'), a red-fleshed apple. Mei Hong 5 was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 46989. Field photos of Mei Hong 5 fruit setting can be found... Figure 1 Photos of the resistance tests for ring rot disease conducted on 'Meihong 5' and 'Changfu 2' apple varieties can be found here. Figure 2 .
[0126] Example 5: Cloning of MdNAC29 and identification of flowering phenotypes in its transgenic plants. I. Preparation of Infection Solution 1. Replace the small fragment between the SalI and SpeI restriction enzyme recognition sequences in the pPZP211 vector with the double-stranded DNA molecule shown in SEQ ID NO: 11 (encoding the protein shown in SEQ ID NO: 12), keeping the other sequences unchanged, to obtain the recombinant plasmid (which has been verified by sequencing), which is used for overexpression. MdNAC29 The recombinant plasmid of the gene was named recombinant plasmid 35S::MdNAC29.
[0127] 2. The recombinant plasmid 35S::MdNAC29 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.
[0128] 3. Resuspend the recombinant Agrobacterium in ddH2O to increase OD. 600nm The value is 0.6-0.8. Then, acetylsyl syringone is added to make the concentration in the system 150 μM, which is the inoculation solution.
[0129] II. Preparation of transgenic Arabidopsis The infection solution was used to genetically transform Columbia ecotype Arabidopsis thaliana plants using the flower immersion method. Seeds from the plants were then harvested, yielding T1 generation seeds. These T1 seeds were cultured into plants, resulting in T1 generation plants. Transgenic plants were selected from the T1 generation plants. The T1 generation transgenic plants were self-pollinated, and their seeds were harvested, yielding T2 generation seeds. These T2 generation seeds were cultured into plants, resulting in T2 generation plants. Transgenic plants were selected from the T2 generation plants. The T2 generation transgenic plants were self-pollinated, and their seeds were harvested, yielding T3 generation seeds. These T3 generation seeds were cultured into plants, resulting in T3 generation plants. Transgenic plants were selected from the T3 generation plants. For a given T2 generation plant, if all T3 generation plants obtained from its self-pollination are transgenic plants, then the T2 generation plant is a homozygous transgenic plant, and all its self-pollinated offspring will also be homozygous transgenic plants.
[0130] Method for screening transgenic plants: Take leaves from the plant, extract genomic DNA, and perform PCR amplification using primer pair composed of NAC29F0 and NAC29R0. If an amplification product of 912 bp is obtained, the plant is a transgenic plant.
[0131] NAC29F0: 5'-ATGGGAAACATCAGCAGCAG-3'; NAC29R0: 5'-TCAGGAGCATTTCCAAGCTG-3'.
[0132] III. Preparation of Transgenic Tomatoes Leaves from Micro Tom tomato plants were used as explants. The explants were placed in the infection solution and shaken at room temperature for 20-30 minutes, then blotted dry with filter paper. They were then transferred to differentiation medium (MS solid medium containing 2 mg / L TDZ and 0.2 mg / L IAA) and cultured in the dark at 24°C for 2-3 days. Next, they were transferred to differentiation medium containing 300 mg / L carbenicillin and cultured in the dark at 24°C for 7-10 days. Then, they were cultured in alternating light and dark at 24°C until resistant shoots appeared on the callus, and subcultured every 2-3 weeks. The resistant shoots were then excised and transferred to subculture medium (MS solid medium containing 0.5 mg / L 6-BA and 0.2 mg / L IAA) and cultured in alternating light and dark at 24°C until the shoots reached a length of 1 cm. Finally, they were transferred to selection medium (MS solid medium containing 0.5 mg / L 6-BA, 0.2 mg / L IAA, and 50 mg / L kanamycin) and cultured in alternating light and dark at 24°C for 30 days. Then, the plants with good growth are transferred to rooting medium and cultured until they root, which are then called T0 generation plants.
[0133] T0 generation plants are self-pollinated, and seeds are harvested to obtain T1 generation seeds. These T1 generation seeds are then cultured into plants to obtain T1 generation plants. Transgenic plants are selected from the T1 generation plants. T1 generation transgenic plants are then self-pollinated, and seeds are harvested to obtain T2 generation seeds. These T2 generation seeds are then cultured into plants to obtain T2 generation plants. Transgenic plants are selected from the T2 generation plants. For a given T1 generation plant, if all T2 generation plants obtained from its self-pollination are transgenic plants, then the T1 generation plant is a homozygous transgenic plant, and all its self-pollinated offspring will be homozygous transgenic plants.
[0134] Method for screening transgenic plants: Take leaves from the plant, extract genomic DNA, and perform PCR amplification using primer pair composed of NAC29F0 and NAC29R0. If an amplification product of 912 bp is obtained, the plant is a transgenic plant.
[0135] IV. Identification of flowering phenotypes in Arabidopsis thaliana The T3 generation homozygous transgenic plants or Columbia ecotype Arabidopsis plants obtained in step two.
[0136] Culture conditions: 24℃, 16h light / 8h dark, light intensity 10000 lux.
[0137] The counting starts from the time the seed germinates; see the photo on day 35. Figure 3 The image above shows the transgenic Arabidopsis plants. Only sporadic flowering was observed in the Columbia ecotype, while abundant flowering was observed in the transgenic plants. Compared to the Columbia ecotype, the transgenic plants exhibited a phenotype with more and earlier flowering.
[0138] V. Phenotypic Identification of Tomato Flowering Formation The T2 generation homozygous transgenic plants or tomato Micro Tom plants obtained in step three.
[0139] Culture conditions: 24℃, 16h light / 8h dark, light intensity 10000 lux.
[0140] The counting starts from the time the seed germinates; see the photo on day 40. Figure 3 The image below shows the results. No flowering was observed in the Micro Tom tomato plants, while abundant flowering was observed in the transgenic plants. Compared to the Micro Tom tomato plants, the transgenic plants exhibited a phenotype of producing more and earlier flowers.
[0141] In this embodiment, the method for detecting the anthocyanin content in the fruit peel includes the following steps: ① Weigh 0.5g of fresh fruit peel, add liquid nitrogen and grind into powder. Then add 5ml of 1% hydrochloric acid-methanol solution pre-cooled at 4℃, and let it stand at 4℃ in the dark for 24h for extraction. Then centrifuge at 8000r / min for 10min and collect the supernatant. 1% hydrochloric acid-methanol solution: 97.2ml methanol and 2.8ml concentrated hydrochloric acid are mixed.
[0142] ② Take 1 ml of the supernatant obtained in step ①, add 4 ml of KCl buffer (pH=1.0) and mix well. Then, incubate at 4℃ in the dark for 15 min, followed by centrifugation at 8000 r / min for 10 min, and collect the supernatant. KCl buffer (pH=1, 0.025M): Dissolve 1.86 g of KCl in 980 ml of distilled water, adjust the pH to 1.0 with concentrated hydrochloric acid, transfer to a 1 L volumetric flask, and dilute to volume with distilled water.
[0143] ③ Take 1 ml of the supernatant obtained in step ①, add 4 ml of NaAc buffer (pH=4.5) and mix well. Then, incubate at 4℃ in the dark for 15 min, followed by centrifugation at 8000 r / min for 10 min, and collect the supernatant. NaAC buffer (pH=4.5, 0.4M): Dissolve 54.43 g of NaAC in 960 ml of distilled water, adjust the pH to 4.5 with concentrated hydrochloric acid, transfer to a 1 L volumetric flask, and dilute to volume with distilled water.
[0144] ④ Take the supernatant obtained in step ② and the supernatant obtained in step ③ respectively, and measure the absorbance at 510 nm and 700 nm.
[0145] Anthocyanin content (mg / kg) = △A × 5 × 0.005 × 1000 × 449.2 × 1000 / (26900 × 0.5); △A = (A 510nm -A 700nm ) (pH=1.0) - (A 510nm -A 700nm ) (pH=4.5) .
[0146] The "kg" in the anthocyanin content unit "mg / kg" refers to the fresh weight of the fruit peel.
[0147] In this embodiment, the method for detecting resistance to ring spot disease (lesion area) includes the following steps: ① Inoculate the pathogenic fungus of apple ring rot onto PDA medium and incubate at 25°C until the colony diameter is approximately 5 cm (7-10 days). Then, use a sterile punch with a diameter of 7 mm to collect mycelial blocks, which are the pathogenic inoculum.
[0148] ② PDA culture medium, obtained by punching with a sterile puncher with a diameter of 7 mm, serves as the control inoculum.
[0149] ③ Take apple fruits, disinfect the surface with 70% ethanol aqueous solution, then rinse with sterile water and air dry naturally. Then, make needle puncture wounds with a depth of 2 mm using a sterile needle (3 needle puncture wounds are evenly set for each fruit). Place a pathogenic inoculum on the first and second needle puncture wounds, and place a control inoculum on the third needle puncture wound.
[0150] ④ Place the apples that have completed step ③ in a sealed container with spacing between them. Take photos daily to record the disease development process and the date of onset for each fruit. Five days after inoculation, take a photo and resize it to 1:1 size with the actual fruit. Then, use calipers (Beaverton, OR, USA) to measure the size of the lesions in the photo. Calculate the lesion area using ImageJ software by averaging the two vertical diameters (longitudinal and transverse) at each lesion.
[0151] In this embodiment, the method for taking buds from the test plant is as follows: the main stem is divided into three equal parts according to length, starting from the top of the main stem and ending at the part of the branch closest to the ground, namely the top, middle and bottom. The branches growing on it are respectively the top branch, the middle branch and the bottom branch. Three branches are randomly selected from the top branch, three branches are randomly selected from the middle branch and three branches are randomly selected from the bottom branch. One terminal bud and two random lateral buds are taken from each of the branches.
[0152] In this embodiment, the method for detecting the flowering rate in the Nth year includes the following steps: The main trunk, starting from its tip and ending at the point where the branches grow closest to the ground, is divided into three equal parts by length: top, middle, and bottom. The branches growing from these sections are designated as top branches, middle branches, and bottom branches, respectively. Three branches are randomly selected from the top, middle, and bottom branches. In year N-1, all buds on these branches are marked. The flowering rate is calculated during the flowering period of year N. Flowering rate = (Number of flower buds + Number of leaf buds) × 100%. A flower bud is a bud that develops into a flower. A leaf bud is a bud that develops into a leaf.
[0153] In this example, the soluble sugar content of the fruit was detected using an Atago PAL-1 saccharimeter.
[0154] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. Primer combinations, including primer pair I, primer pair II, and primer pair III; Primer pair I consists of primer BBX24F and primer BBX24R; primer BBX24F is the single-stranded DNA molecule shown in SEQ ID NO: 1, and primer BBX24R is the single-stranded DNA molecule shown in SEQ ID NO: 2; Primer pair II consists of primer MYB62F and primer MYB62R; primer MYB62F is the single-stranded DNA molecule shown in SEQ ID NO: 5, and primer MYB62R is the single-stranded DNA molecule shown in SEQ ID NO: 6; The primer pair III consists of primer NAC29F and primer NAC29R; primer NAC29F is the single-stranded DNA molecule shown in SEQ ID NO: 7, and primer NAC29R is the single-stranded DNA molecule shown in SEQ ID NO:
8.
2. The application of the primer combination according to claim 1 is as follows (a1) or (a2) or (a3) or (a4): (a1) Identification or auxiliary identification of simplified and mechanizable apple germplasm; (a2) Selecting or assisting in the selection of simplified and mechanizable apple germplasm; (a3) Prepare a kit for identifying or assisting in the identification of simplified and easily mechanized apple germplasm; (a2) Prepare a kit for breeding or assisting in the breeding of lightweight, easy-to-organize apple germplasm; The simplified and biocompatible apple germplasm refers to apple germplasm with an anthocyanin content in the peel ≥300mg / kg and a flowering rate ≥60%.
3. A method for breeding lightweight, simple, and machine-friendly apple germplasm includes the following steps: (1) The male parent and the female parent are crossed to harvest hybrid seeds; the male parent is a red-fleshed apple; the female parent is a white-fleshed apple; (2) Cultivate the hybrid seeds obtained in step (1) into plants; (3) The plants obtained in step (2) are used as plants to be screened. Plants that meet standard 1, standard 2 and standard 3 are selected, which are the simplified and easy-to-organize apple germplasm. The method for screening plants that meet criterion 1 includes the following steps: Collect the pericarp of the fruit from the test plant and the pericarp of the fruit from the parent plant, extract total RNA and reverse transcribe it to obtain cDNA; use the cDNA as a template, and... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdBBX24 The relative expression levels of genes; then, plants meeting the following criterion 1 are screened: the pericarp of the fruit of the tested plant... MdBBX24 The relative expression level of the gene is the same as that of the fruit peel of the maternal plant. MdBBX24 The relative expression level of the gene is less than 1 / 5; The method for screening plants that meet criterion 2 includes the following steps: The pulp of the fruit from the test plant and the pulp of the fruit from the parent plant are taken respectively; total RNA is extracted and reverse transcribed to obtain cDNA; using the cDNA as a template, ... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdMYB62 The relative expression level of genes; then, plants that meet the following criterion 2 are screened: the pulp of the fruit of the tested plant... MdMYB62 The relative expression level of the gene is the same as that of the pulp of the maternal plant's fruit. MdMYB62 More than 5 times the relative expression level of the gene; The method for screening plants that meet criterion 3 includes the following steps: buds from the test plants and buds from the parent plants are taken respectively; total RNA is extracted and reverse transcribed to obtain cDNA; using the cDNA as a template, ... MdActin The gene is an internal reference gene, and it is detected by real-time quantitative PCR. MdNAC29 The relative expression level of genes; then, plants that meet the following criterion 3 are screened: the buds of the tested plants... MdNAC29 The relative expression level of the gene is that of the bud of the maternal plant. MdNAC29 More than 5 times the relative expression level of the gene; The simplified and biocompatible apple germplasm refers to apple germplasm with an anthocyanin content in the peel ≥300mg / kg and a flowering rate ≥60%.
4. The method as described in claim 3, characterized in that: In the method described, detection MdBBX24 The primer pair used for the gene is primer pair I as described in claim 1; In the method described, detection MdMYB62 The primer pair used for the gene is primer pair II as described in claim 1; In the method described, detection MdNAC29 The primer pair used for the gene is primer pair III as described in claim 1.
5. The lightweight, easy-to-organize apple germplasm obtained by the method described in claim 3 or 4.
6. Application of Mei Hong 5 red-fleshed apples; (a) Application in apple production; (b) Application in apple breeding; The red-fleshed apple, Mei Hong 5, has the preservation registration number CGMCC No. 46989.
7. A protein, named MdNAC29 protein, as shown in SEQ ID NO:
12.
8. MdNAC29 protein-related biomaterials, for MdNAC29 Genes, possess MdNAC29 Gene expression cassettes, with MdNAC29 Recombinant plasmids of genes, possessing MdNAC29 Recombinant microorganisms or those with genes MdNAC29 Plants with genetically modified organisms; the aforementioned MdNAC29 The gene is the gene encoding the MdNAC29 protein as described in claim 7.
9. The application of MdNAC29 protein or MdNAC29 protein-related biomaterials in improving the flowering rate of plants; wherein the MdNAC29 protein is as described in claim 7; and the MdNAC29 protein-related biomaterials are as described in claim 8.
10. A method for improving the flowering rate of plants, comprising the following steps: introducing MdNAC29 protein-related biomaterials into plants to obtain transgenic plants with improved flowering rate; wherein the MdNAC29 protein-related biomaterials are as described in claim 8.