WO2018107852A1 - 高类黄酮优异种质'csr6r6-777'在功能型苹果育种中的应用 - Google Patents

高类黄酮优异种质'csr6r6-777'在功能型苹果育种中的应用 Download PDF

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WO2018107852A1
WO2018107852A1 PCT/CN2017/103348 CN2017103348W WO2018107852A1 WO 2018107852 A1 WO2018107852 A1 WO 2018107852A1 CN 2017103348 W CN2017103348 W CN 2017103348W WO 2018107852 A1 WO2018107852 A1 WO 2018107852A1
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apple
genotype
plant
csr6r6
pulp
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French (fr)
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陈学森
王楠
姜生辉
许海峰
王意程
毛志泉
姜远茂
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山东农业大学
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Priority to US15/761,069 priority Critical patent/US11109548B2/en
Priority to ZA2018/01651A priority patent/ZA201801651B/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
    • A01H6/7418—Malus domestica, i.e. apples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the application of the high flavonoid excellent germplasm 'CSR6R6-777' in functional apple breeding.
  • Functional apple breeding is the effective integration and balance of multiple quality traits (genes).
  • the research team has taken three measures: First, the hybrid generation of Xinjiang red meat apple and apple varieties Based on the study of genetic variation of traits such as total phenolic content, the apple breeding method (Patent No. ZL 2013 1 0205419.6) was proposed and implemented, and the breeding efficiency was significantly improved. Second, the genetic background was complicated. Apple breeds such as ' ⁇ ', 'American No.
  • the invention provides an application of a high flavonoid excellent germplasm 'CSR6R6-777' in apple breeding.
  • the excellent germplasm of high flavonoids provided by the present invention is 'CSR6R6-777', also known as apple (Malus domestica) CSR6R6-777, which was deposited on December 8, 2016 in the General Microbiology Center of China Microbial Culture Collection Management Committee (abbreviated as CGMCC, address: North of Chaoyang District, Beijing No. 3, No. 1 Chenxi Road, Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is CGMCC NO.12468.
  • Apple (Malus domestica) CSR6R6-777CGMCC NO.12468 is referred to as Apple CSR6R6-777.
  • the invention claims the application of apple CSR6R6-777 in apple breeding.
  • Apple CSR6R6-777 is one of the parents.
  • Apple CSR6R6-777 is used as a parent.
  • the purpose of the breeding is to obtain a plant full of red flesh.
  • the purpose of the breeding is to obtain a plant having the R6R1 genotype and full red flesh.
  • the purpose of the breeding is to obtain an apple germplasm having excellent traits.
  • the apple germplasm excellent in the trait is an apple plant satisfying the following (a) and/or (b) and/or (c) and/or (d): (a) whole flesh of the pulp; (b) high flavonoids of pulp Content; (c) high anthocyanin content of pulp; (d) high antioxidant capacity of pulp.
  • the apple germplasm excellent in the trait is the R6R1 genotype.
  • the high flavonoid content of the pulp refers to a flavonoid content of more than 200 mg per kilogram of fresh weight pulp.
  • the high anthocyanin content of the pulp means that the anthocyanin content per kilogram of fresh weight pulp is 30 mg or more.
  • the high antioxidant capacity of the pulp means that the antioxidant capacity (also known as the antioxidant content) per kilogram of fresh weight pulp is 1 ⁇ mol or more.
  • the invention also protects an apple breeding method comprising the following steps:
  • step (3) The seedlings obtained in the step (2) are transplanted to the field, and after the fruits are grown, the fruits are not bagged, and the plants of interest are screened.
  • the method for hybridizing apple variety A and apple variety B is as follows: pollen of apple variety A is taken, and apple variety B after emasculation is pollinated.
  • the hybrid seed is first subjected to a 1-3 ° C layering treatment to break the dormancy.
  • the time of the stratification treatment may specifically be 60 days.
  • the conditions for the seedling are: 25 ° C, 12 hours of light per day, and an illumination intensity of 3000 lx, and the nutrient solution is poured every 7-10 days from the start of seed germination.
  • the conditions for the seedling raising are specifically as follows: the hybrid seed is sown in a nutrient meal containing 3-5 seed seeds per seedling substrate, and the seedling height is raised to a seedling height. The degree is 8-15cm and the root neck is lignified (usually for 2-3 months), and then the seedlings are transplanted to a new nutrient meal containing the seedling substrate (1 strain per transplanted seedling) for cultivation.
  • the culture conditions are: 25 ° C, 12 hours of light per day (light intensity 3000 lx), nutrient solution is poured every 7-10 days from the start of seed germination (the large amount of element mother liquor of MS basic medium is diluted with water to 10 times volume) That is, nutrient solution), 40-50ml per nutrient solution.
  • the cultivation management method in the field is as follows: 6000 kg of organic fertilizer is applied per acre in the field where the planting seedlings are to be planted and watered and solidified, and the seedlings are planted in the field in the field. Then apply 6000kg of organic fertilizer and water it in time.
  • the organic fertilizer may specifically be a fully decomposed cow dung.
  • the plant of interest is a plant with red flesh.
  • the plant of interest is a plant of the R6R1 genotype and having a red flesh.
  • the plant of interest is an apple plant that satisfies the following (a) and / or (b) and / or (c) and / or (d): (a) full flesh of the pulp; (b) high flavonoid content of the pulp; ) high anthocyanin content in pulp; (d) high antioxidant capacity of pulp.
  • the apple germplasm excellent in the trait is the R6R1 genotype.
  • the high flavonoid content of the pulp refers to a flavonoid content of more than 200 mg per kilogram of fresh weight pulp.
  • the high anthocyanin content of the pulp means that the anthocyanin content per kilogram of fresh weight pulp is 30 mg or more.
  • the high antioxidant capacity of the pulp means that the antioxidant capacity (also known as the antioxidant content) per kilogram of fresh weight pulp is 1 ⁇ mol or more.
  • the trunk of the seedling is subjected to ring stripping at a distance of 20 cm from the ground, and the stripping width is 0.5-1.0 cm, which is deep to the xylem.
  • the plant of interest is obtained in the fifth year of transplanting.
  • the apple variety B is an apple variety of the R1R1 genotype.
  • the apple variety B is specifically a ' ⁇ ' apple or a 'red Fuji' apple.
  • the method for identifying the R1R1 genotype is as follows: taking apple from the apple plant to be tested, extracting the genomic DNA of the apple pulp, using the genomic DNA as a template, using a specific primer pair for PCR amplification, and then reading the genotype according to the following criteria: if PCR amplification The amplified product has a 386 bp DNA fragment and does not have a 497 bp DNA fragment, and the apple plant to be tested is the R1R1 genotype.
  • the method for identifying the R1R1 genotype is as follows: taking an apple from the apple plant to be tested, and extracting genomic DNA of the apple pulp, Using genomic DNA as a template, PCR amplification was performed using a specific primer pair, and then the genotype was judged according to the following criteria: If the PCR amplification product is one band and 386 bp, the apple plant to be tested is the R1R1 genotype.
  • the method for identifying the R6R1 genotype is as follows: taking apple from the apple plant to be tested, extracting the genomic DNA of the apple pulp, using the genomic DNA as a template, using a specific primer pair for PCR amplification, and then reading the genotype according to the following criteria: if PCR amplification The product has a 497 bp DNA fragment and a 386 bp DNA fragment, and the apple plant to be tested is the R6R1 genotype.
  • the method for identifying the R6R1 genotype is as follows: taking apple from the apple plant to be tested, extracting the genomic DNA of the apple pulp, using the genomic DNA as a template, using a specific primer pair for PCR amplification, and then reading the genotype according to the following criteria: if PCR amplification The product was increased into two bands and was 497 bp and 386 bp, respectively, and the apple plant to be tested was the R6R1 genotype.
  • the specific primer pair is composed of F1 and R1; the F1 is a single-stranded DNA molecule represented by SEQ ID NO: 1 of the Sequence Listing; and R1 is a single-stranded DNA molecule represented by SEQ ID NO: 2 of the Sequence Listing.
  • the invention also protects the application of apple CSR6R6-777 in the production of foods based on apple fruit.
  • the foods based on apple fruits include fresh apples, dried apples, applesauce, and the like.
  • the invention also protects the application of apple CSR6R6-777 in the production of food processed products using apple fruit as raw material.
  • the food processed products using the apple fruit as raw materials include cider, apple cider vinegar and the like.
  • Figure 1 shows the phenotypic comparison of the fruits of Xinjiang Red Meat Apple, 'Red Fuji' and 'CSR6R6-777'.
  • Figure 2 shows the relative expression levels of individual genes.
  • Figure 3 is the content of flavanol and leucocyanidin, the content of proanthocyanidin, the content of flavanone, the content of chalcone and dihydrochalcone, the content of flavonol, cyanine in apple pulp.
  • Figure 4 shows the results of accurate identification of flavonols in apple pulp.
  • Figure 5 is a photograph of an apple fruit of a part of a progeny plant.
  • Figure 6 is a photograph of an apple fruit of a part of a progeny plant.
  • Figure 7 is a photograph of an apple fruit of a part of a progeny plant.
  • Fig. 8 shows the results of genotype identification of "Red Heart No. 11", “Red Heart No. 16", “Zi Xia No. 1", “Zi Xia No. 2", “Zi Xia No. 3" and " ⁇ ".
  • 80% acetone solution 4 parts by volume of acetone was mixed with 1 part by volume of water.
  • the method for identifying apple plants as R1R1 genotype, R6R6 genotype or R6R1 genotype is as follows: taking apple from the apple plant to be tested, extracting genomic DNA of apple pulp, using genomic DNA as a template, using primer pairs composed of F1 and R1 PCR amplification, and then read the genotype according to the following criteria: If the PCR amplification product is a band and is 497 bp, the apple plant to be tested is the R6R6 genotype; if the PCR amplification product is a band and is 386 bp, the apple plant to be tested is R1R1 genotype; if the PCR amplification product is two bands and is 497 bp and 386 bp, respectively, the apple plant to be tested is the R6R1 genotype.
  • F1 sequence 1 of the sequence listing: 5'-GGTGGTCAAAGATGTGTGTTGT-3';
  • Xinjiang red meat apples are crossed with white meat-cultivated apple varieties such as 'Red Fuji'.
  • white meat-cultivated apple varieties such as 'Red Fuji' (R1R1 genotype)
  • the plant was found in genotype 3 R6R6 of F 1 hybrid population (868) in the.
  • Each of the three plants had the following phenotypes: the leaves, flowers, peels, and flesh were all purplish red at each stage of development.
  • the stem of a single plant is also purple-red, which is named 'CSR6R6-777'.
  • the stems of the other two plants were light red, and were named as control strain A and control strain B, respectively.
  • the 'CSR6R6-777' was expanded by grafting grafts or tissue culture.
  • CSR6R6-777' also known as Apple (Malus domestica) CSR6R6-777, was deposited on December 8, 2016 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Beichen West, Chaoyang District, Beijing) No. 3, No. 1 Road, Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is CGMCC NO.12468.
  • control strain A and control strain B were used as the plants to be tested, the apples on the plants were taken, the total RNA of apple pulp was extracted, and the key enzymes in the flavonoid synthesis pathway were detected by real-time quantitative RT-PCR.
  • Gene CHS gene, F3H gene, ANS gene, LAR gene, FLS base
  • MYB10 gene the expression level of the key transcription factor gene
  • the primer pairs used to detect the CHS gene are as follows:
  • the primer pairs used to detect the F3H gene are as follows:
  • F3H-F 5'-TGGAAGCTTGTGAGGACTGGGGT-3';
  • F3H-R 5'-CTCCTCCGATGGCAAATCAAAGA-3'.
  • the primer pairs used to detect the ANS gene are as follows:
  • the primer pairs used to detect the LAR gene are as follows:
  • the primer pairs used to detect the FLS gene are as follows:
  • FLS-F 5'-AACCACTGTGAACAAGGATA-3';
  • FLS-R 5'-CATAGTCGCCGTACTTCTT-3'.
  • the primer pairs used to detect the MYB10 gene are as follows:
  • MYB10-F 5'-TGCCTGGACTCGAGAGGAAGACA-3';
  • MYB10-R 5'-CCTGTTTCCCAAAAGCCTGTGAA-3'.
  • control strain A and control strain B were used as the plants to be tested, respectively.
  • step 2 Take the pulp obtained in step 1 and grind it in liquid nitrogen to obtain a powder.
  • step 3 Weigh 2g of the powder obtained in step 2, add 5mL of 0.5% hydrochloric acid methanol solution, and let it stand for 2h at 4°C, then centrifuge at 8000rpm for 20min, and collect the supernatant and residue separately.
  • step 4 Take the residue obtained in the step 3, add 5 mL of a 0.5% hydrochloric acid methanol solution, and let stand for 1 hour at 4 ° C, then centrifuge at 8000 rpm for 20 min, and collect the supernatant.
  • the supernatant obtained in the step 3 and the supernatant obtained in the step 4 are mixed to obtain a mixed solution.
  • the mixed solution obtained in the step 5 was removed by steaming at 37 ° C, and the residue was dissolved in 2-3 ml of methanol, and then centrifuged at 8000 rpm for 20 min, and the supernatant was collected.
  • the supernatant obtained in the step 6 was taken up to 5 ml with methanol, and then filtered through a 0.45 ⁇ m filter to collect the filtrate.
  • the column was a BEH C18 column (100 mm ⁇ 2.1 mm), the particle size of the filler was 1.7 ⁇ m; the column temperature was 45 ° C; the injection volume was 1 ⁇ L;
  • the mobile phase is a mixture of liquid A and liquid B, the flow rate is 0.3mL/min; liquid A is acetonitrile, liquid B is aqueous solution containing 0.2% (volume fraction) formic acid; 0-0.1min, liquid A accounts for the volume of mobile phase
  • the fraction is 5%; 0.1-20min, the volume fraction of liquid A in the mobile phase increases linearly from 5% to 20%; in 20-22min, the volume fraction of liquid A in the mobile phase increases linearly from 20% to 80%; 22-22.1 Min, the volume fraction of liquid A in the mobile phase decreased linearly from 80% to 5%; 22.1-25 min, the volume fraction of liquid A in the mobile phase was 5%.
  • the mass spectrometer was WATERS MALDI SYNAPT Q-TOF MS, ESI ionization source, electrospray ionization positive ion acquisition mode (ESI+); scanning range 100-1500 m/z; capillary voltage 3.5 kV, cone voltage 30 V; source temperature 100 ° C, The desolvation temperature was 300 ° C; the desolvation gas flow rate was 500 L / h.
  • the apple pulp of 'CSR6R6-777' contains 41 kinds of flavonol substances, 9 of which are unique, and are not contained in the apple pulp of the control strain A and the control strain B.
  • the flavonols specific to the apple pulp of 'CSR6R6-777' are shown in Table 1.
  • the detection methods of the nine unique substances in Table 1 belong to the flavonoid component and the content detection method. References (Chen Xuesen, Zhang Jing, Liu Daliang, et al. Xinjiang Red Meat Apple Hybrid Generational Genetic Variation and Evaluation of Functional Apples[J]. ⁇ ,2014,47(11):2193-2204.
  • Example 2 using 'CSR6R6-777' as a parental hybrid to obtain a full red plant
  • the BC 1 hybrid seed obtained in step 1 was sown in a nutrient meal containing 3-5 seed seeds per seedling substrate (the seedlings were seeded with 3-5 seeds per seedling) until the seedling height was 8-15 cm and the root neck was lignified. (usually for 2-3 months of sowing), then transplant the seedlings to a new nutrient meal containing the seedling substrate (1 strain per transplanted seedling) for cultivation; the culture conditions in this step are: 25 °C 12 hours of light per day (light intensity 3000lx), every 5-10 days from the start of seed germination, nutrient solution (diluted a large amount of element mother liquor of MS basic medium to 10 times volume is used as nutrient solution), each time The nutrition is poured 40-50ml.
  • Hybrid seedlings are planted (in Guan County, Shandong City)
  • the genotypes of the five plants are as follows:
  • Zixia 1 is the R6R1 genotype
  • Zixia 2 is the R6R1 genotype
  • 'Zixia No. 3' is the R6R1 genotype.
  • the identification map is shown in Figure 8.
  • a standard curve was prepared using rutin (rutin, Sigma chemical, ST, Loiuis, USA) as a standard.
  • ⁇ A (A 510 nm - A 700 nm ) Under the conditions of pH 1.0 - (A 510 nm - A 700 nm ) Under pH 4.5 conditions .
  • Acetic acid buffer pH 3.6, 300 mM: 16.8 g of glacial acetic acid and 0.8 g of sodium hydroxide were mixed with water to make a 1 liter solution.
  • TPTZ solution an aqueous solution containing 10 mM 2,4,6-tripyridyltriazine and 40 mM hydrochloric acid.
  • step (6) After completion of the step (6), 4 mL of the solution was taken, mixed with 30 ⁇ L of the sample to be tested, and allowed to stand at 37 ° C for 120 min, and then the absorbance was measured at 593 nm. A standard curve was prepared using Trolox as a standard.
  • control strain A was used instead of 'CSR6R6-777', and the procedure of the first step of Example 2 was followed. operating. In August 2016, no fleshy red plants were obtained from the backcross progeny, and only one plant with red and white flesh (detected as the R6R1 genotype) was obtained, and it was named 'Hongza No.1'.
  • the control strain B was used instead of 'CSR6R6-777', and the procedure of the first step of Example 2 was followed. In August 2016, no fleshy red plants were obtained from the backcross progeny, and only one plant with red and white flesh (detected as the R6R1 genotype) was obtained, and it was named 'Hongza No. 2'.
  • Example 3 using 'CSR6R6-777' as a parental hybrid to obtain a full red plant
  • the operation of the first step of the second embodiment is carried out by replacing the ' ⁇ ' apple with the 'Red Fuji' apple.
  • 'Zixia No. 4', 'Zixia No. 5', 'Zixia No. 6', and 'Red Heart No. 35' were all R6R1 genotypes.
  • the apple CSR6R6-777 provided by the invention has high content of flavonoids and anthocyanins in the pulp of the apple fruit, and has strong antioxidant capacity, and increases the functional health effect above the edible value of ordinary apples, and the long-term consumption will be taken The health of the person has a beneficial effect.
  • Will Apple CSR6R6-777, The apple fruit produced as a raw material to produce cider/apple vinegar, etc., can have a higher content of flavonoids and anthocyanins in cider and apple vinegar, thereby having a health care effect.
  • the apple CSR6R6-777 provided by the invention can also be used as a parent to cross with other varieties, thereby obtaining more new high flavonoid apple germplasm, which is of great value for high flavonoid apple breeding.

Abstract

苹果(Malus domestica)CSR6R6-777在苹果育种中的应用。苹果(Malus domestica)CSR6R6-777的保藏编号为CGMCC NO.12468。育种的目的为获得性状优异的苹果种质。性状优异的苹果种质为满足如下(a)和/或(b)和/或(c)和/或(d)的苹果植株：(a)果肉全红；(b)果肉高类黄酮含量；(c)果肉高花青苷含量；(d)果肉高抗氧化能力。该方法对于功能型苹果育种具有重大价值。

Description

高类黄酮优异种质‘CSR6R6-777’在功能型苹果育种中的应用 技术领域
本发明涉及高类黄酮优异种质‘CSR6R6-777’在功能型苹果育种中的应用。
背景技术
“医食同源”是发展方向，“吃营养，吃健康”已成为人们的共识。为此，开展功能型苹果育种对改进人类健康具有重要意义。
功能型苹果育种是多个品质性状(基因)的有效集成与平衡，为完善新品种选育方案，提高育种效率，课题组采取了三项措施：一是在新疆红肉苹果与苹果品种杂种一代果实总酚含量等性状遗传变异研究的基础上，提出并实施了“三选两早一促”的苹果育种法(专利号ZL 2013 1 0205419.6)，育种效率显著提高；二是利用遗传背景复杂的‘嘎啦’、‘美国八号’、‘寒富’及‘富士’等苹果品种与新疆红肉苹果(M.sieversii f.niedzwetzkyana)进行多亲本杂交与反复回交，旨在进行品质育种，目前已构建回交一、二代分离群体40个，定植杂种实生苗4万株，并申报了“果树多种源品质育种法(专利号ZL 2015 10428448.8)”及“易着色苹果品种培育法(专利号ZL 201510890141.X)”2个育种技术发明专利；三是及时地以性状基本稳定的后代株系为试材，进行品质性状评价及发育机理的研究，并已取得了诸多重要进展。目前，已创建了常规杂交与生物技术有机结合的苹果高效育种技术体系，创制了一批新品种及优异种质，研发了苹果新品种配套高效栽培技术体系。授权和申报发明专利10余项，育成新品种(系)16个；发表相关研究论文120篇，其中SCI论文20余篇，这些研究成果总体处在国际同类研究的领先水平。
发明公开
本发明提供了一种高类黄酮优异种质‘CSR6R6-777’在苹果育种中的应用。
本发明提供的高类黄酮优异种质为‘CSR6R6-777’，又称苹果(Malus domestica)CSR6R6-777，已于2016年12月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址为：北京市朝阳区北 辰西路1号院3号，中国科学院微生物研究所)，保藏登记号为CGMCC NO.12468。苹果(Malus domestica)CSR6R6-777CGMCC NO.12468简称苹果CSR6R6-777。
本发明要求保护苹果CSR6R6-777在苹果育种中的应用。所述应用中，苹果CSR6R6-777作为亲本之一。所述应用中，苹果CSR6R6-777作为父本。所述育种的目的为获得果肉全红的植株。
所述育种的目的为获得R6R1基因型且果肉全红的植株。
所述育种的目的为获得性状优异的苹果种质。所述性状优异的苹果种质为满足如下(a)和/或(b)和/或(c)和/或(d)的苹果植株：(a)果肉全红；(b)果肉高类黄酮含量；(c)果肉高花青苷含量；(d)果肉高抗氧化能力。所述性状优异的苹果种质为R6R1基因型。所述果肉高类黄酮含量指的是每千克鲜重的果肉中的类黄酮的含量为200mg以上。所述果肉高花青苷含量指的是每千克鲜重的果肉中的花青苷含量为30mg以上。果肉高抗氧化能力指的是每千克鲜重的果肉中的抗氧化能力(又称抗氧化物含量)为1μmol以上。
本发明还保护一种苹果育种方法，包括如下步骤：
(1)将苹果品种A和苹果品种B进行杂交，获得杂交种子；所述苹果品种A为苹果CSR6R6-777；所述苹果品种B为除苹果品种A以外的苹果品种；
(2)将步骤(1)得到的杂交种子播种并育苗，得到实生苗；
(3)将步骤(2)得到的实生苗移栽至大田，待其长出果实后不对果实进行套袋，筛选得到目的植株。
所述将苹果品种A和苹果品种B进行杂交的方法如下：取苹果品种A的花粉，对去雄后的苹果品种B进行授粉。
所述步骤(2)中，进行所述播种前，先将所述杂交种子进行1-3℃层积处理以打破休眠。所述层积处理的时间具体可为60天。
所述步骤(2)中,进行所述育苗的条件为：25℃、每天光照12小时、光照强度3000lx，从种子萌发开始每7-10天浇一次营养液。
所述步骤(2)中,进行所述育苗的条件具体为：将所述杂交种子播种于装有育苗基质的营养钵(每个营养钵播种3-5粒种子)培养至实生苗高 度为8-15cm且根颈处木质化(通常为播种2-3个月)，然后将实生苗移栽至新的装有育苗基质的营养钵(每个营养钵移栽1株)进行培养；所述培养的条件为：25℃、每天光照12小时(光照强度3000lx)，从种子萌发开始每7-10天浇一次营养液(将MS基本培养基的大量元素母液用水稀释至10倍体积即为营养液)、每次每个营养钵浇40-50ml。
所述步骤(3)中,在所述大田中进行栽培管理的方法如下：在拟定植实生苗的大田中每亩施入有机肥6000kg并浇水沉实，定植实生苗后在大田中每亩再施入有机肥6000kg并及时浇水。所述有机肥具体可为充分腐熟的奶牛粪。
所述目的植株为果肉全红的植株。
所述目的植株为R6R1基因型且果肉全红的植株。
所述目的植株为满足如下(a)和/或(b)和/或(c)和/或(d)的苹果植株：(a)果肉全红；(b)果肉高类黄酮含量；(c)果肉高花青苷含量；(d)果肉高抗氧化能力。所述性状优异的苹果种质为R6R1基因型。所述果肉高类黄酮含量指的是每千克鲜重的果肉中的类黄酮的含量为200mg以上。所述果肉高花青苷含量指的是每千克鲜重的果肉中的花青苷含量为30mg以上。果肉高抗氧化能力指的是每千克鲜重的果肉中的抗氧化能力(又称抗氧化物含量)为1μmol以上。
所述方法中，移栽第4年，在离地面20cm处对实生苗的主干进行环剥，环剥宽度为0.5-1.0cm，深达木质部。
所述方法中，移栽第5年，在实生苗的盛花期进行如下操作：每个花序的5朵花仅保留中心花，其他全部疏除。
所述方法中，移栽第5年得到所述目的植株。
所述苹果品种B为R1R1基因型的苹果品种。
所述苹果品种B具体为‘嘎啦’苹果或‘红富士’苹果。
鉴定R1R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA，以基因组DNA为模板，采用特异引物对进行PCR扩增，然后按如下标准判读基因型：如果PCR扩增产物中具有386bp的DNA片段且不具有497bp的DNA片段，待测苹果植株为R1R1基因型。鉴定R1R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA， 以基因组DNA为模板，采用特异引物对进行PCR扩增，然后按如下标准判读基因型：如果PCR扩增产物为一条带且为386bp，待测苹果植株为R1R1基因型。
鉴定R6R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA，以基因组DNA为模板，采用特异引物对进行PCR扩增，然后按如下标准判读基因型：如果PCR扩增产物中具有497bp的DNA片段和386bp的DNA片段，待测苹果植株为R6R1基因型。鉴定R6R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA，以基因组DNA为模板，采用特异引物对进行PCR扩增，然后按如下标准判读基因型：如果PCR扩增产物为两条带且分别为497bp和386bp，待测苹果植株为R6R1基因型。
所述特异引物对由F1和R1组成；所述F1为序列表的序列1所示的单链DNA分子；所述R1为序列表的序列2所示的单链DNA分子。
本发明还保护苹果CSR6R6-777在生产以苹果果实为原料的食品中的应用。所述以苹果果实为原料的食品包括鲜食苹果、苹果干、苹果酱等。
本发明还保护苹果CSR6R6-777在生产以苹果果实为原料的食品加工品中的应用。所述以苹果果实为原料的食品加工品包括苹果酒、苹果醋等。
附图说明
图1为新疆红肉苹果、‘红富士’以及‘CSR6R6-777’的果实的表型比较。
图2为各个基因的相对表达量。
图3为苹果果肉中，黄烷醇和无色花青素的含量、原花青苷的含量、黄烷酮的含量、查尔酮和二氢查耳酮的含量、黄酮醇的含量、花青苷的含量。
图4为苹果果肉中的黄酮醇进行精确鉴定的结果。
图5为部分后代植株的苹果果实的照片。
图6为部分后代植株的苹果果实的照片。
图7为部分后代植株的苹果果实的照片。
图8为‘红心11号’、‘红心16号’、‘紫霞1号’‘紫霞2号’‘紫霞3号’和‘嘎啦’的基因型鉴定结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
芦丁的结构式如下：
Figure PCTCN2017103348-appb-000001
Trolox的结构式如下：
Figure PCTCN2017103348-appb-000002
0.5％盐酸甲醇溶液的制备方法：将0.5体积份35％浓盐酸与99.5体积份甲醇混合。
1％盐酸甲醇溶液的制备方法：将1体积份35％浓盐酸与99体积份甲醇混合。
80％丙酮溶液：将4体积份丙酮与1体积份水混合。
鉴定苹果植株为R1R1基因型、R6R6基因型还是R6R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA，以基因组DNA为模板，采用F1和R1组成的引物对进行PCR扩增，然后按如下标准判读基因型：如果PCR扩增产物为一条带且为497bp，待测苹果植株为R6R6基因型；如果PCR扩增产物为一条带且为386bp，待测苹果植株为R1R1基因型；如果PCR扩增产物为两条带且分别为497bp和386bp，待测苹果植株为R6R1基因型。
F1(序列表的序列1)：5’-GGTGGTCAAAGATGTGTGTTGT-3’；
R1(序列表的序列2)：5’-TTTGCCTGCTACCCACTTCA-3’。
提及‘红富士’苹果和‘嘎啦’苹果的参考文献：陈学森，辛培刚等，元帅和金帅在苹果新品种选育中的作用，山东农业大学学报，1994，25(2)：236—248。经检测，‘红富士’苹果和‘嘎啦’苹果均为R1R1基因型。
提及‘红色之爱’苹果的文献：郑吉文、姚少群、刘淑贞，红肉苹果新品种“红色之爱”，西北园艺，201304。
实施例1、苹果优异种质‘CSR6R6-777’的鉴定
一、‘CSR6R6-777’的获得
新疆红肉苹果作为亲本，与‘红富士’等白肉栽培苹果品种杂交。按照孟德尔遗传定律，新疆红肉苹果(R6R1基因型)与‘红富士’(R1R1基因型)等白肉栽培苹果品种杂交,其后代群体应为红肉表型(R6R1基因型):白肉表型(R1R1基因型)＝1:1。但是，在杂交F1代群体(868株)中发现3株R6R6基因型的单株。
3株单株均具有如下表型：叶、花、果皮和果肉等各部分及各个发育阶段均为紫红色。一株单株的茎也为紫红色，将其命名为‘CSR6R6-777’。另外两株单株的茎为淡红色，分别命名为对照株甲和对照株乙。
在杂交F1代群体随机选定一个R1R1基因型的单株(命名为对照株A)和一个R6R1基因型的单株(命名为对照株B)。
新疆红肉苹果、‘红富士’以及‘CSR6R6-777’的果实的表型比较见图1。
通过嫁接枝条或组培的方式，将‘CSR6R6-777’扩繁。
二、‘CSR6R6-777’的保藏
‘CSR6R6-777’，又称苹果(Malus domestica)CSR6R6-777，已于2016年12月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏登记号为CGMCC NO.12468。
三、类黄酮生物合成相关基因表达分析
分别将‘CSR6R6-777’、对照株A和对照株B作为待测植株，取待测植株上的苹果，提取苹果果肉的总RNA，通过实时定量RT-PCR检测类黄酮合成途径上的关键酶基因(CHS基因、F3H基因、ANS基因、LAR基因、FLS基 因)以及关键转录因子基因(MYB10基因)的表达量。
用于检测CHS基因的引物对如下：
CHS-F：5’-GGAGACAACTGGAGAAGGACTGGAA-3’；
CHS-R：5’-CGACATTGATACTGGTGTCTTC-3’。
用于检测F3H基因的引物对如下：
F3H-F：5’-TGGAAGCTTGTGAGGACTGGGGT-3’；
F3H-R：5’-CTCCTCCGATGGCAAATCAAAGA-3’。
用于检测ANS基因的引物对如下：
ANS-F：5’-CCAAGTGAAGCGGGTTGTGCT-3’；
ANS-R：5’-CAAAGCAGGCGGACAGGAGTAGC-3’。
用于检测LAR基因的引物对如下：
LAR-F：5’-CACCGTCAAGTCCTTCAA-3’；
LAR-R：5’-ACCTCTTAACTGTACCAACTG-3’。
用于检测FLS基因的引物对如下：
FLS-F：5’-AACCACTGTGAACAAGGATA-3’；
FLS-R：5’-CATAGTCGCCGTACTTCTT-3’。
用于检测MYB10基因的引物对如下：
MYB10-F：5’-TGCCTGGACTCGAGAGGAAGACA-3’；
MYB10-R：5’-CCTGTTTCCCAAAAGCCTGTGAA-3’。
以MdActin为内参，数据分析采用2-ΔΔCT方法(Livak&Schmittgen，2001)计算。
各个基因的相对表达量见图2。‘CSR6R6-777’中各个基因的表达量均极为显著的高于对照株A和对照株B中相应基因的表达量。结果表明，‘CSR6R6-777’具有很强的类黄酮合成能力。
四、类黄酮组分含量分析
分别将‘CSR6R6-777’、对照株A和对照株B作为待测植株。
1、取待测植株上的苹果，取苹果果肉。
2、取步骤1得到的果肉，在液氮中研磨得到粉末。
3、称取2g步骤2得到的粉末，加入5mL 0.5％盐酸甲醇溶液，4℃静置提取2h，然后8000rpm离心20min，分别收集上清液和残渣。
4、取步骤3得到的残渣，加入5mL 0.5％盐酸甲醇溶液，4℃静置提取1h，然后8000rpm离心20min，收集上清液。
5、将步骤3得到的上清液和步骤4得到的上清液混合，得到混合液。
6、取步骤5得到的混合液，37℃旋蒸除去甲醇，残留物用2-3ml甲醇溶解，然后8000rpm离心20min，收集上清液。
7、取步骤6得到的上清液，用甲醇定容至5ml，然后用0.45μm滤膜过滤，收集滤液。
8、将步骤7得到的滤液进行HPLC-MS分析。
液相色谱条件：
采用WATERS ACQUITY UPLC色谱仪，色谱柱为BEH C18柱(100mm×2.1mm)，填料粒径1.7μm；柱温45℃；进样体积1μL；
流动相为A液和B液的混合液，流速为0.3mL/min；A液为乙腈，B液为含0.2％(体积分数)甲酸的水溶液；0-0.1min，A液占流动相的体积分数为5％；0.1-20min，A液占流动相的体积分数由5％线性上升至20％；20-22min，A液占流动相的体积分数由20％线性上升至80％；22-22.1min，A液占流动相的体积分数由80％线性下降至5％；22.1-25min，A液占流动相的体积分数为5％。
质谱条件：
质谱仪为WATERS MALDI SYNAPT Q-TOF MS，ESI电离源，电喷雾离子化正离子采集模式(ESI+)；扫描范围100-1500m/z；毛细管电压3.5kV，锥孔电压30V；源温度100℃，脱溶温度300℃；脱溶剂气流量500L/h。
每克鲜重的苹果果肉中，黄烷醇和无色花青素的含量、原花青苷的含量、黄烷酮的含量、查尔酮和二氢查耳酮的含量、黄酮醇的含量、花青苷的含量见图3。‘CSR6R6-777’的苹果果肉中，原花青苷的含量、查尔酮和二氢查耳酮的含量、黄酮醇的含量、花青苷的含量均显著高于对照株A和对照株B中的相应物质含量。
对苹果果肉中的黄酮醇进行精确鉴定，结果见图4。‘CSR6R6-777’的苹果果肉中含有41种黄酮醇物质，其中9种为其特有的，在对照株A和对照株B的苹果果肉中都不含有。‘CSR6R6-777’的苹果果肉特有的9种黄酮醇物质见表1。表1中的9种特有物质的检测方法均属于类黄酮组分及含量检测方法，参考文献(陈学森,张晶,刘大亮,等.新疆红肉苹果杂种一 代的遗传变异及功能型苹果优株评价[J].中国农业科学,2014,47(11):2193-2204.。
表1
Figure PCTCN2017103348-appb-000003
以上结果表明，‘CSR6R6-777’具有很强的类黄酮合成能力，果肉富含类黄酮，并且含有特殊的黄酮醇类组分，是培育功能型苹果品种的优异种质。
实施例2、应用‘CSR6R6-777’作为亲本杂交获得全红植株
一、从杂交后代中选育全红植株
1、回交(在山东烟台市牟平区进行)
2011年4月，取‘CSR6R6-777’的花粉，对去雄后的‘嘎啦’苹果进行授粉，收获BC1杂交种子，将BC1杂交种子洗净后放到1-3℃冰箱保鲜室层积处理60d左右(目的是通过满足需冷量来解除种子休眠)。
2、温室育苗(在山东泰安进行)
2011年12月，将步骤1得到的BC1杂交种子播种于装有育苗基质的营养钵(每个营养钵播种3-5粒种子)培养至实生苗高度为8-15cm且根颈处木质化(通常为播种2-3个月)，然后将实生苗移栽至新的装有育苗基质的营养钵(每个营养钵移栽1株)进行培养；本步骤中的培养条件为：25℃、每天光照12小时(光照强度3000lx)，从种子萌发开始每7-10天浇一次营养液(将MS基本培养基的大量元素母液用水稀释至10倍体积即为营养液)、每次每个营养钵浇40-50ml。
3、杂种实生苗定植(在山东冠县进行)
2012年2月，在拟定植实生苗的选种圃(大田)中每亩施入有机肥(本实施例中采用的为充分腐熟的奶牛粪)6000kg并浇水沉实；2012年4月，将3600株步骤2得到的实生苗定植于选种圃，2012年4月在定植有实生苗的选种圃中每亩再施入有机肥(本实施例中采用充分腐熟的奶牛粪)6000kg并及时浇水。
4、杂种实生苗环剥处理
2015年5月，在离地面20cm处对各实生苗的主干进行环剥，环剥宽度为0.5-1.0cm，深达木质部。此操作的目的是促进营养物质的积累及花芽分化，以缩短童期、提早结果。
5、花果管理
2016年4-5月，在各实生苗的盛花期进行疏花(每个花序的5朵花仅保留中心花，其他全部疏除)。幼果不套袋，常规管理。
6、全红植株的获得
2016年8月，从回交后代群体中选育出5个果肉全红植株：‘紫霞1号’、‘紫霞2号’、‘紫霞3号’、‘红心11号’和‘红心16号’。
二、表型特征
5个植株上生长的苹果的果实特征见图5、图6和图7。
三、基因型
5个植株的基因型如下：
‘红心11号’为R6R1基因型；
‘红心16号’为R6R1基因型；
‘紫霞1号’为R6R1基因型；
‘紫霞2号’为R6R1基因型；
‘紫霞3号’为R6R1基因型。
鉴定图谱见图8。
结果表明，从‘CSR6R6-777’的杂种后代分离群体中，能够选育出R6R1基因型且果肉全红的植株。
四、类黄酮含量、花青苷含量及抗氧化能力分析
待测材料：‘红心11号’上生长的苹果、‘红心16号’上生长的苹 果、‘紫霞1号’上生长的苹果、‘紫霞2号’上生长的苹果、‘紫霞3号’上生长的苹果以及‘CSR6R6-777’上生长的苹果和‘嘎啦’上生长的苹果。
1、类黄酮含量测定
(1)取1g果肉，液氮研磨，然后加入10ml 4℃预冷的65％(体积百分含量)乙醇水溶液并混匀，4℃避光静置提取4h，然后12000g离心20min，收集上清液。
(2)取试管，加入0.5ml步骤(1)得到的上清液，然后依次加入1mL5g/100ml NaNO2水溶液、1ml 10g/100ml AL(NO3)3水溶液、4mL 2M NaOH水溶液，混匀后静置15min，8000rpm离心10min，取上清，然后在510nm下测定吸光值。
以芦丁(rutin,Sigma chemical,ST,Loiuis,USA)为标样做标准曲线。
2、花青苷含量测定
(1)取0.5g果肉，液氮研磨，加入5ml 4℃预冷的1％盐酸甲醇溶液，4℃避光静置提取24h。
(2)取1ml步骤(1)得到的提取液，加入4ml KCl缓冲液或加入4ml NaAC缓冲液，混匀后4℃避光静置提取15min，然后8000r/min离心10min，收集上清液。
KCl缓冲液(pH＝1、0.025M)：1.86g KCl用980ml蒸馏水溶解，用浓盐酸调pH为1.0，转移到1L容量瓶中，用蒸馏水定容。
NaAC缓冲液(pH＝4.5、0.4M)：54.43g NaAC用960ml蒸馏水溶解，用浓盐酸调pH为4.5，转移到1L容量瓶中，用蒸馏水定容。
(3)取步骤(2)得到的上清液，分别测510nm和700nm下的吸光值。
花青苷含量(mg/g)＝△A*5*0.005*1000*449.2/(26900*0.5)；
△A＝(A510nm-A700nm)pH1.0条件下-(A510nm-A700nm)pH4.5条件下。
3、抗氧化能力测定
(1)取果肉，液氮研磨，得到粉末。
(2)称取10g步骤(1)得到的粉末，加入50mL 80％丙酮溶液，4℃静置提取2h，8000rpm离心10min，收集上清。
(3)取完成步骤(2)的残渣，加入50mL 80％丙酮溶液，4℃静置提取1h，8000rpm离心10min，收集上清。
(4)将步骤(2)得到的上清和步骤(3)得到的上清合并，得到混合液。
(5)取步骤(4)得到的混合液，40℃旋蒸除去丙酮，残留部分5000rpm离心，取上清液，用去离子水定容至20mL，得到待测样品。
(6)10体积份乙酸缓冲液、1体积份20mM FeCl3·6H2O水溶液和1体积份TPTZ溶液混合，37℃水浴5min。
乙酸缓冲液(pH3.6、300mM)：16.8克冰醋酸和0.8克氢氧化钠与水配制成1升溶液。
TPTZ溶液：含10mM 2,4,6–三吡啶基三嗪和40mM盐酸的水溶液。
(7)完成步骤(6)后，取4mL溶液，与30μL待测样品混合，37℃静置反应120min，然后593nm测其吸光度。以Trolox为标样做标准曲线。
各个待测材料的类黄酮含量、花青苷含量以及抗氧化能力(又称抗氧化物含量)检测结果见表2。结果表明，以‘CSR6R6-777’为亲本杂交育成的全红植株的果肉的类黄酮含量、花青苷含量以及抗氧化能力均显著的高于其亲本嘎啦。‘红心11号’和‘红心16号’酸甜适口，细脆多汁，鲜食品质优良。‘紫霞1号’、‘紫霞2号’和‘紫霞3号’可作为深加工的原料。因此，‘CSR6R6-777’是功能苹果育种的珍贵种质。
表2
Figure PCTCN2017103348-appb-000004
对比例、
用对照株甲代替‘CSR6R6-777’，按照实施例2的步骤一的方案进行 操作。2016年8月，从回交后代中未得到果肉全红植株，仅得到一株果肉红白参杂的植株(经检测，为R6R1基因型)，将其命名为‘红杂1号’。
用对照株乙代替‘CSR6R6-777’，按照实施例2的步骤一的方案进行操作。2016年8月，从回交后代中未得到果肉全红植株，仅得到一株果肉红白参杂的植株(经检测，为R6R1基因型)，将其命名为‘红杂2号’。
按照实施例2的步骤四的方法检测‘红杂1号’和‘红杂2号’的类黄酮含量、花青苷含量以及抗氧化能力，结果见表3。
实施例3、应用‘CSR6R6-777’作为亲本杂交获得全红植株
用‘红富士’苹果代替‘嘎啦’苹果，按照实施例2的步骤一的方案进行操作。
2016年8月，从回交后代群体中选育出4个果肉全红植株：‘紫霞4号’、‘紫霞5号’、‘紫霞6号’、‘红心35号’。
经检测，‘紫霞4号’、‘紫霞5号’、‘紫霞6号’、‘红心35号’均为R6R1基因型。
按照实施例2的步骤四的方法检测‘紫霞4号’、‘紫霞5号’、‘紫霞6号’、和‘红心35号’的类黄酮含量、花青苷含量以及抗氧化能力，结果见表3。
表3
Figure PCTCN2017103348-appb-000005
工业应用
本发明提供的苹果CSR6R6-777，生产的苹果果实的果肉中，类黄酮和花青苷含量高，抗氧化能力强，在普通苹果的食用价值以上又增加了功能保健作用，长期食用将对服用者的健康产生有益效果。将苹果CSR6R6-777， 生产的苹果果实作为原料生产苹果酒/苹果醋等，可以使苹果酒、苹果醋中具有较高含量的类黄酮和花青苷，从而具有保健效果。本发明提供的苹果CSR6R6-777，还可以作为亲本与其它品种杂交，从而获得更多的新的高类黄酮苹果种质，对于高类黄酮苹果育种具有重大价值。

Claims (12)

  1. 苹果(Malus domestica)CSR6R6-777在苹果育种中的应用；苹果(Malus domestica)CSR6R6-777的保藏编号为CGMCC NO.12468。
  2. 一种苹果育种方法，包括如下步骤：
    (1)将苹果品种A和苹果品种B进行杂交，获得杂交种子；所述苹果品种A为苹果(Malus domestica)CSR6R6-777；苹果(Malus domestica)CSR6R6-777的保藏编号为CGMCC NO.12468；所述苹果品种B为除苹果品种A以外的苹果品种；
    (2)将步骤(1)得到的杂交种子播种并育苗，得到实生苗；
    (3)将步骤(2)得到的实生苗移栽至大田，待其长出果实后不对果实进行套袋，筛选得到目的植株。
  3. 如权利要求2所述的方法，其特征在于：移栽第4年，在离地面20cm处对实生苗的主干进行环剥，环剥宽度为0.5-1.0cm，深达木质部。
  4. 如权利要2所述的方法，其特征在于：移栽第5年，在实生苗的盛花期进行如下操作：每个花序的5朵花仅保留中心花，其他全部疏除。
  5. 如权利要求2所述的方法，其特征在于：所述苹果品种B为‘嘎啦’苹果或‘红富士’苹果。
  6. 如权利要求2所述的方法，其特征在于：所述苹果品种B为R1R1基因型的苹果品种；
    鉴定R1R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA，以基因组DNA为模板，采用特异引物对进行PCR扩增，然后按如下标准判读基因型：如果PCR扩增产物中具有386bp的DNA片段且不具有497bp的DNA片段，待测苹果植株为R1R1基因型；
    所述特异引物对由F1和R1组成；所述F1为序列表的序列1所示的单链DNA分子；所述R1为序列表的序列2所示的单链DNA分子。
  7. 如权利要求2所述的方法，其特征在于：所述苹果品种B为R1R1基因型的苹果品种；
    鉴定R1R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA，以基因组DNA为模板，采用特异引物对进行PCR扩增， 然后按如下标准判读基因型：如果PCR扩增产物为一条带且为386bp，待测苹果植株为R1R1基因型；
    所述特异引物对由F1和R1组成；所述F1为序列表的序列1所示的单链DNA分子；所述R1为序列表的序列2所示的单链DNA分子。
  8. 如权利要求2至7中任一所述的方法，其特征在于：所述目的植株为果肉全红的植株。
  9. 如权利要求2至7中任一所述的方法，其特征在于：所述目的植株为R6R1基因型且果肉全红的植株；
    鉴定R6R1基因型的方法如下：从待测苹果植株上取苹果，提取苹果果肉的基因组DNA，以基因组DNA为模板，采用特异引物对进行PCR扩增，然后按如下标准判读基因型：如果PCR扩增产物中具有497bp的DNA片段和386bp的DNA片段，待测苹果植株为R6R1基因型；
    所述特异引物对由F1和R1组成；所述F1为序列表的序列1所示的单链DNA分子；所述R1为序列表的序列2所示的单链DNA分子。
  10. 如权利要求2至7中任一所述的方法，其特征在于：所述目的植株为满足如下(a)和/或(b)和/或(c)和/或(d)的苹果植株：(a)果肉全红；(b)果肉高类黄酮含量；(c)果肉高花青苷含量；(d)果肉高抗氧化能力。
  11. 苹果(Malus domestica)CSR6R6-777在生产以苹果果实为原料的食品中的应用；苹果(Malus domestica)CSR6R6-777的保藏编号为CGMCCNO.12468。
  12. 苹果(Malus domestica)CSR6R6-777在生产以苹果果实为原料的食品加工品中的应用；苹果(Malus domestica)CSR6R6-777的保藏编号为CGMCC NO.12468。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110491444A (zh) * 2019-08-13 2019-11-22 西北农林科技大学 一种快速筛选苹果红肉性状的分子标记的开发方法及应用

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106718835B (zh) * 2016-12-12 2018-04-24 山东农业大学 高类黄酮优异种质‘csr6r6-777’在功能型苹果育种中的应用
CN107118919B (zh) * 2017-06-30 2018-10-26 山东农业大学 干高型高类黄酮苹果酒及其制备方法
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CN107118900B (zh) * 2017-06-30 2018-07-06 山东农业大学 干柔型高类黄酮苹果酒及其制备方法
CN107099422B (zh) * 2017-06-30 2018-07-27 山东农业大学 干低型高类黄酮苹果酒及其制备方法
CN107118899B (zh) * 2017-06-30 2018-07-27 山东农业大学 甜柔型高类黄酮苹果酒及其制备方法
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CN112831520B (zh) * 2021-01-07 2022-07-12 山东农业大学 R1r6基因型苹果作为受体材料在制备红肉转基因苹果中的应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1029926A1 (ru) * 1981-02-26 1983-07-23 Московский Ордена Ленина,Ордена Октябрьской Революции И Ордена Трудового Красного Знамени Государственный Университет Им.М.В.Ломоносова Способ селекции блони
WO2008017842A1 (en) * 2006-08-09 2008-02-14 Coressence Limited Polyphenol extraction process
JP2012044935A (ja) * 2010-08-27 2012-03-08 Nagoya Univ 赤果肉リンゴ及びその育種方法
CN103314840A (zh) * 2013-05-29 2013-09-25 山东农业大学 “三选两早一促”的苹果育种法
CN105123497A (zh) * 2015-07-20 2015-12-09 山东农业大学 果树多种源品质育种法
CN105359963A (zh) * 2015-12-04 2016-03-02 山东农业大学 一种易着色苹果品种培育法
CN106718835A (zh) * 2016-12-12 2017-05-31 山东农业大学 高类黄酮优异种质‘csr6r6‑777’在功能型苹果育种中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105837671B (zh) * 2016-05-06 2019-02-12 山东农业大学 获自功能型苹果的黄酮醇调控蛋白MsMYB22及其编码基因和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1029926A1 (ru) * 1981-02-26 1983-07-23 Московский Ордена Ленина,Ордена Октябрьской Революции И Ордена Трудового Красного Знамени Государственный Университет Им.М.В.Ломоносова Способ селекции блони
WO2008017842A1 (en) * 2006-08-09 2008-02-14 Coressence Limited Polyphenol extraction process
JP2012044935A (ja) * 2010-08-27 2012-03-08 Nagoya Univ 赤果肉リンゴ及びその育種方法
CN103314840A (zh) * 2013-05-29 2013-09-25 山东农业大学 “三选两早一促”的苹果育种法
CN105123497A (zh) * 2015-07-20 2015-12-09 山东农业大学 果树多种源品质育种法
CN105359963A (zh) * 2015-12-04 2016-03-02 山东农业大学 一种易着色苹果品种培育法
CN106718835A (zh) * 2016-12-12 2017-05-31 山东农业大学 高类黄酮优异种质‘csr6r6‑777’在功能型苹果育种中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL: "Genetic Variation of F 1 Population Between Malus sieversii f. neidzwetzkyana and Apple Varieties and Evaluation on Fruit Characters of Functional Apple Excellent Strains", SCIENTIA AGRICULTURA SINICA, vol. 47, no. 11, 31 December 2014 (2014-12-31), pages 2193 - 2204, XP055605815 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110491444A (zh) * 2019-08-13 2019-11-22 西北农林科技大学 一种快速筛选苹果红肉性状的分子标记的开发方法及应用

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