Method for identifying red-flesh trait of apple fruit and specific primer pair

Abstract

The application discloses a method for identifying a red-flesh trait of an apple fruit and a specific primer pair. The application provides a primer pair, named specific primer pair B, which is composed of a single-stranded DNA molecule shown in sequence 5 of a sequence listing and a single-stranded DNA molecule shown in sequence 6 of the sequence listing. The application also protects the application of the specific primer pair B in identifying the anthocyanin content of apple flesh. The application also protects the application of the specific primer pair B in identifying or breeding red-flesh apples. The anthocyanin content of apple plants can be identified by using the primer combination and the method provided by the application, early identification and early exclusion can be achieved, targeted breeding objectives can be selected, breeding costs can be reduced, breeding efficiency can be improved, and important theoretical basis is provided for cultivating high-anthocyanin-content red-flesh apple varieties.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to a method for identifying the red flesh characteristics of apple fruit and a specific primer pair. Background Technology

[0002] Apples are a widely cultivated deciduous fruit tree worldwide, and apple fruit is a major source of vitamins and polyphenols for the human body. Flavonoids, as easily absorbed free polyphenols, possess antioxidant, anti-aging, and cardiovascular disease prevention properties, offering high nutritional and health benefits. my country ranks first in the world in both apple cultivation area and annual production. However, recent breeding efforts in my country have overemphasized fruit appearance and storage durability, resulting in approximately 70% of cultivated varieties being Fuji apples with low flavonoid content and poor nutritional quality. Specialty varieties with high nutritional quality are scarce, leading to an unbalanced varietal structure. Therefore, developing a variety with high flavonoid content is a new breeding goal for breeders.

[0003] In recent years, significant progress has been made both domestically and internationally in effectively utilizing modern molecular biology techniques to actively explore the formation and regulation mechanisms of quality traits in specialty fruit tree resources or new varieties. An increasing number of functional genes related to apple yield, resistance, and quality have been cloned and identified. The high content of flavonoids in red-fleshed apples has also attracted widespread attention from breeders worldwide in recent years. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying the red flesh characteristics of apple fruits and a specific primer pair.

[0005] This invention provides a primer pair, named specific primer pair B, which consists of a single-stranded DNA molecule shown in sequence 5 of the sequence listing and a single-stranded DNA molecule shown in sequence 6 of the sequence listing.

[0006] This invention also protects the application of the specific primer pair B in identifying the anthocyanin content in apple pulp.

[0007] The determination of anthocyanin content in apple pulp refers to the determination of anthocyanin content in the pulp of the apple plant.

[0008] The purpose of this application is to screen apple plants with high anthocyanin content in the pulp.

[0009] This invention also protects the use of the specific primer pair B in the identification or selection of red-fleshed apples.

[0010] The identification or selection of red-fleshed apples refers to the identification or selection of apple plants that produce red-fleshed apples.

[0011] The purpose of this application is to screen apple plants that produce red-fleshed apples.

[0012] In this application, the apple plants used for screening are: the hybrid offspring obtained by crossing apple varieties with the R1R1 genotype and apple varieties with the R6R6 genotype.

[0013] This invention also protects a method for identifying the anthocyanin content in the pulp of apple plants, comprising the following steps:

[0014] Using the genomic DNA of the tested apple plant as a template, PCR amplification was performed on the B sample using the specific primers described above;

[0015] If the PCR amplification product is of one type and its size is 385bp, the tested apple plant is of the M1M1 genotype; if the PCR amplification product is of two types and its sizes are 385bp and 222bp respectively, the tested apple plant is of the M1M2 genotype.

[0016] The anthocyanin content in the pulp of apple plants with the M1M1 genotype is higher than that in apple plants with the M1M2 genotype.

[0017] This invention also protects a method for identifying or breeding apple plants that produce red-fleshed apples, comprising the following steps:

[0018] Using the genomic DNA of the tested apple plant as a template, PCR amplification was performed on the B sample using the specific primers described above;

[0019] If the PCR amplification product is of one type and its size is 385bp, the tested apple plant is of the M1M1 genotype; if the PCR amplification product is of two types and its sizes are 385bp and 222bp respectively, the tested apple plant is of the M1M2 genotype.

[0020] M1M1 genotype apple plants are candidate apple plants for producing red-fleshed apples.

[0021] The tested apple plants were hybrid offspring obtained by crossing apple varieties with the R1R1 genotype and apple varieties with the R6R6 genotype.

[0022] This invention also protects a primer pair consisting of specific primer pair A and specific primer pair B;

[0023] The specific primer pair A consists of a single-stranded DNA molecule shown in Sequence 1 of the sequence listing and a single-stranded DNA molecule shown in Sequence 2 of the sequence listing;

[0024] The specific primer pair B is the specific primer pair B described above.

[0025] This invention also protects the use of the primer combination in identifying the anthocyanin content in apple pulp.

[0026] The determination of anthocyanin content in apple pulp refers to the determination of anthocyanin content in the pulp of the apple plant.

[0027] The purpose of this application is to screen apple plants with high anthocyanin content in the pulp.

[0028] This invention also protects the use of the primer combination in the identification or selection of red-fleshed apples.

[0029] The identification or selection of red-fleshed apples refers to the identification or selection of apple plants that produce red-fleshed apples.

[0030] The purpose of this application is to screen apple plants that produce red-fleshed apples.

[0031] This invention also protects an apple breeding method, comprising the following steps:

[0032] (1) Cross apple varieties with the R1R1 genotype and apple varieties with the R6R6 genotype to obtain hybrid offspring;

[0033] (2) Select plants with the M1M1 genotype from the hybrid offspring of the R1R6 genotype;

[0034] The methods for determining the R1R1, R6R6, and R1R6 genotypes are as follows: Using the genomic DNA of the tested apple plant as a template, PCR amplification is performed using the specific primers. If the PCR amplification product shows a single band of 493 bp, the tested apple plant has the R6R6 genotype; if the PCR amplification product shows a single band of 392 bp, the tested apple plant has the R1R1 genotype; if the PCR amplification product shows two bands of 493 bp and 392 bp, respectively, the tested apple plant has the R1R6 genotype.

[0035] The method for determining the M1M1 genotype is as follows: Using the genomic DNA of the tested apple plant as a template, PCR amplification is performed on the tested apple plant using the specific primers; if the PCR amplification product is of the same type and its size is 385bp, the tested apple plant is of the M1M1 genotype.

[0036] The goal of apple breeding is to select apples with high anthocyanin content in the pulp.

[0037] The goal of apple breeding is to select apple plants with high anthocyanin content in the pulp.

[0038] The goal of the apple breeding program is to select red-fleshed apples.

[0039] The goal of the apple breeding program is to select apple plants that produce red-fleshed apples.

[0040] The apple variety with the R1R1 genotype can specifically be the 'Gala' apple.

[0041] The apple variety with the R6R6 genotype can specifically be the 'Purple Red No. 1' apple.

[0042] The specific steps for obtaining hybrid offspring are as follows: collect pollen from 'Zihong No. 1' apple plants, perform hybrid pollination on 'Gala' apple plants with stamens removed to obtain hybrid seeds; stratify the obtained hybrid seeds in sand at low temperature; after the seeds germinate, sow and cultivate seedlings to obtain seedlings.

[0043] The inventors of this invention discovered a molecular marker through extensive experiments and developed a specific primer pair B based on this marker. Specific primer pair B can be used to determine the anthocyanin content in the pulp of hybrid offspring of Purple Red 1 and Gala apples. Using specific primer pair A and specific primer pair B in combination can identify the anthocyanin content in the pulp of tested apple plants, thus enabling the breeding of apples with high anthocyanin content. This invention is of great significance for those skilled in the art to analyze hybrid parents and offspring populations through molecular biology-assisted breeding, and to selectively choose target offspring, thereby improving breeding efficiency, shortening breeding time, and reducing breeding costs. Using the primer combinations and methods provided by this invention to identify the anthocyanin content in the pulp of apple plants allows for early identification and exclusion, targeted selection of breeding targets, and simultaneously reduces breeding costs and improves breeding efficiency, providing an important theoretical basis for cultivating red-fleshed apple varieties with high anthocyanin content. Attached Figure Description

[0044] Figure 1 The images shown are cross-sectional photographs of apple fruits from Example 1 and electrophoresis diagrams used for genotyping.

[0045] Figure 2 This is an exemplary result of detecting the anthocyanin content in the test apples in Example 3.

[0046] Figure 3 Examples of apple fruit cross-section photographs and electrophoresis images used for genotyping in Example 3 are shown.

[0047] Figure 4 These are cross-sectional photographs of the apple fruits tested in Example 4 and electrophoresis images used for genotyping.

[0048] Figure 5 The images shown are illustrative cross-sectional photographs of the fruit from the tested apple plants in Example 4, as well as electrophoresis images used for genotyping. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. 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 present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are all commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0050] The apple variety 'Zihong No. 1' is described in the following literature: Wang Yan et al., Analysis of antioxidant properties and anthocyanins in the flesh of 'Zihong No. 1' red-fleshed apples.

[0051] The apple variety 'Mei Hong' is described in the following literature: Wang Nan et al., A new apple variety 'Mei Hong' with high flavonoid content.

[0052] The specific steps for detecting anthocyanin content are as follows:

[0053] 1. Weigh 0.5g of fresh apple pulp, add liquid nitrogen and grind it into powder. Then add 5ml of 1% hydrochloric acid methanol solution pre-cooled at 4℃, and then let it stand at 4℃ in the dark for 24h to extract. Then centrifuge at 8000r / min for 10min and collect the supernatant.

[0054] 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°C in the dark for 15 min, then centrifuge at 8000 r / min for 10 min and collect the supernatant.

[0055] 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°C in the dark for 15 min, then centrifuge at 8000 r / min for 10 min and collect the supernatant.

[0056] 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.

[0057] Anthocyanin content (mg / kg) = △A × 5 × 0.005 × 1000 × 449.2 × 1000 / (26900 × 0.5);

[0058] △A=(A 510nm -A 700nm ) (pH＝1.0) -(A 510nm -A 700nm ) (pH＝4.5) .

[0059] The "kg" in the anthocyanin content unit "mg / kg" refers to the fresh weight of the apple pulp.

[0060] 1% hydrochloric acid-methanol solution: Mix 97.2 ml of methanol with 2.8 ml of concentrated hydrochloric acid. Concentrated hydrochloric acid is commercially available 12 mol / L hydrochloric acid.

[0061] KCl buffer (pH=1, 0.025M): Dissolve 1.86g KCl in 980ml of distilled water, adjust the pH to 1.0 with concentrated hydrochloric acid, transfer to a 1L volumetric flask, and dilute to volume with distilled water.

[0062] NaAC buffer (pH=4.5, 0.4M): Dissolve 54.43g NaAC in 960ml of distilled water, adjust the pH to 4.5 with concentrated hydrochloric acid, transfer to a 1L volumetric flask, and dilute to volume with distilled water.

[0063] Example 1: Identification of apple genotype and anthocyanin content using primer pair A

[0064] The apple fruits tested were: apple variety 'Zihong No. 1', apple variety 'Gala', and apple variety 'Meihong'.

[0065] I. Determination of anthocyanin content in the tested apples

[0066] The pulp of the tested apple fruit (ripe fruit) was extracted and the anthocyanin content was determined.

[0067] Take 10 apples of each type and average the results.

[0068] The average anthocyanin content in the pulp of 'Zihong No. 1' is 195.63±12.5mg / kg.

[0069] The average anthocyanin content of Gala apple pulp is 1.14 ± 0.4 mg / kg.

[0070] The average anthocyanin content in the pulp of 'Mei Hong' is 76.37±5.5 mg / kg.

[0071] II. Identification of apple genotypes using primer pair A

[0072] Primer pair A consists of primer R6F and primer R6R.

[0073] Primer R6F (Sequence 1 in the sequence listing): 5'-GGAGGGGAATGAAGAAGAGG-3';

[0074] Primer R6R (Sequence 2 in the sequence listing): 5'-TCCACAGAAGCAAACACTGAC-3'.

[0075] Extract genomic DNA from the pulp of the tested apple fruit. Using the genomic DNA as a template, perform PCR amplification with primer pair A, followed by 1.5% agarose gel electrophoresis. The genotype is then determined according to the following criteria: If the PCR amplification product shows a single band of 493 bp (exemplary sequencing results are shown in sequence 3 of the sequence listing), the apple pulp is of genotype R6R6; if the PCR amplification product shows a single band of 392 bp (exemplary sequencing results are shown in sequence 4 of the sequence listing), the apple pulp is of genotype R1R1; if the PCR amplification product shows two bands of 493 bp and 392 bp respectively, the apple pulp is of genotype R1R6.

[0076] The genotypes of apple flesh were identified using primer pair A. 'Zihong No. 1' was identified as having the R6R6 genotype, 'Gala' as having the R1R1 genotype, and 'Meihong' as having the R1R6 genotype.

[0077] See the cross-sectional photos of the apple fruit and the electrophoresis images used for genotyping. Figure 1 .

[0078] Based on steps one and two, the conclusions are as follows: the anthocyanin content of the pulp of the R6R6 genotype is greater than that of the pulp of the R1R6 genotype, which is greater than that of the pulp of the R1R1 genotype.

[0079] Example 2: Preparation of hybrid offspring

[0080] Pollen from 'Zihong No. 1' apple plants was collected and used to cross-pollinate 'Gala' apple plants (with stamens removed) to obtain hybrid seeds. The hybrid seeds were then subjected to low-temperature sand stratification. After germination, the seeds were sown and seedlings were cultivated to obtain seedlings.

[0081] Example 3: Using primer pair A to identify the genotype of the hybrid offspring and to detect the anthocyanin content in the fruit pulp.

[0082] Apple plants tested: 100 plants grown from seedlings prepared in Example 2 (all plants were planted 6 years after planting and had started bearing fruit from the 3rd year after planting).

[0083] I. Determination of anthocyanin content in the tested apples

[0084] Take mature fruits from the tested apple plants (10 mature fruits per plant), extract the pulp, and test the anthocyanin content.

[0085] See the example results Figure 2 The average anthocyanin content of mature fruits from the five tested apple plants was 0.3±0.02 mg / kg, 7.1±0.4 mg / kg, 43.4±3.5 mg / kg, 82.4±4.3 mg / kg, and 157.6±12.3 mg / kg, respectively.

[0086] Of the 100 apple trees tested, 38 had anthocyanin content in the pulp below 10 mg / kg, 49 had anthocyanin content in the pulp between 11 and 50 mg / kg, 9 had anthocyanin content in the pulp between 50 and 80 mg / kg, and 4 had anthocyanin content in the pulp greater than 80 mg / kg.

[0087] The above results indicate that the anthocyanin content in the pulp of different tested plants varies considerably.

[0088] II. Identifying the genotype of hybrid offspring using primer pair A

[0089] Leaves were taken from the tested apple plants, genomic DNA was extracted, and the genotype of the plants was detected according to the method in step two of Example 1.

[0090] Examples of apple fruit cross-section photographs and electrophoresis images used for genotyping are shown below. Figure 3 .

[0091] All 100 apple plants tested were of the R1R6 genotype.

[0092] Based on steps one and two: the hybrid offspring of 'Zihong No. 1' and 'Gala' apples all share the R1R6 genotype, yet the anthocyanin content in the pulp of the fruits from different plants shows significant differences. The lowest anthocyanin content in the pulp was only 0.3±0.02 mg / kg, while the highest was 157.6±12.3 mg / kg. Therefore, using primer pair A to identify the apple genotype cannot fully explain the differences in anthocyanin accumulation among individuals with the same genotype.

[0093] Example 4: Using primer pair B to identify the genotypes of the parents and hybrid offspring, and to detect the anthocyanin content in the fruit pulp.

[0094] Apple plants tested: 100 plants grown from seedlings prepared in Example 2 (all plants were planted 6 years after planting and had started bearing fruit from the 3rd year after planting).

[0095] Apple fruits tested: apple variety 'Zihong No. 1' and apple variety 'Gala'.

[0096] I. Detection of anthocyanin content

[0097] Take mature fruits from the tested apple plants (10 mature fruits per plant), extract the pulp, and test the anthocyanin content.

[0098] II. Genotyping using primer pairs B

[0099] 1. Take the pulp of the apple fruit to be tested and extract genomic DNA.

[0100] 2. Take leaves from the tested apple plants and extract genomic DNA.

[0101] 3. Using the genomic DNA obtained in step 1 or step 2 as a template, perform PCR amplification with primer pair B, then perform 1.5% agarose gel electrophoresis and interpret the genotype according to the following criteria: If the PCR amplification product is of one type and its size is 385 bp (exemplary sequencing results are shown in sequence 7 of the sequence listing), then the apple plant to be tested is of genotype M1M1; if the PCR amplification product is of two types and its sizes are 385 bp and 222 bp, respectively, then the apple plant to be tested is of genotype M1M2 (exemplary sequencing results for the 385 bp amplification product are shown in sequence 7 of the sequence listing, and exemplary sequencing results for the 222 bp amplification product are shown in sequence 8 of the sequence listing).

[0102] Primer pair B consists of primer W10F and primer W10R.

[0103] Primer W10F (Sequence 5 in the sequence listing): 5'-CTCGTAGGGCAACAAAAGATT-3';

[0104] Primer W10R (Sequence 6 in the sequence listing): 5'-GAACTTCAAGTGGAAAGTCCA-3'.

[0105] The genotypes of the flesh of the tested apple fruits were identified using primer pair B. 'Zihong No. 1' was identified as having the M1M1 genotype, and 'Gala' as having the M1M2 genotype.

[0106] The genotypes of the tested apple plants were identified using primer pair B. Some plants were identified as having the M1M1 genotype, while the remaining plants were identified as having the M1M2 genotype.

[0107] III. Grouping by genotype and calculating the average anthocyanin content

[0108] Based on the genotypes identified using primer pair B, the tested apple plants were divided into two groups.

[0109] Forty-two plants were of the M1M1 genotype. The average anthocyanin content in the pulp was 37.4 ± 1.5 mg / kg. The lowest anthocyanin content in the pulp was 22.7 ± 1.2 mg / kg. The highest anthocyanin content in the pulp was 157.6 ± 12.3 mg / kg.

[0110] Fifty-eight plants were of the M1M2 genotype. The average anthocyanin content in the pulp was 5.4 ± 0.25 mg / kg. The lowest anthocyanin content in the pulp was 0.3 ± 0.02 mg / kg. The highest anthocyanin content in the pulp was 19.2 ± 1.6 mg / kg.

[0111] See the cross-sectional photographs of the tested apple fruits and the electrophoresis images used for genotyping. Figure 4 .

[0112] Examples of fruit cross-section photographs of the tested apple plants and electrophoresis images used for genotyping are shown below. Figure 5 .

[0113] Based on steps one, two, and three, the conclusions are as follows: the anthocyanin content in the pulp of apple plants with the M1M1 genotype is greater than that in apple plants with the M1M2 genotype.

[0114] 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. A method for identifying the anthocyanin content in the pulp of an apple plant, comprising the following steps: Using the genomic DNA of the tested apple plant as a template, PCR amplification was performed using specific primer pair B; the specific primer pair B consists of single-stranded DNA molecules shown in sequence 5 and sequence 6 of the sequence listing; if the PCR amplification product is of one type and its size is 385 bp, the tested apple plant is of genotype M1M1; if the PCR amplification product is of two types and its sizes are 385 bp and 222 bp, respectively, the tested apple plant is of genotype M1M2; The anthocyanin content in the pulp of apple plants with the M1M1 genotype is higher than that in apple plants with the M1M2 genotype.

2. A method for identifying or breeding apple plants for producing red-fleshed apples, comprising the following steps: Using the genomic DNA of the tested apple plant as a template, PCR amplification was performed using specific primer pair B; the specific primer pair B consists of single-stranded DNA molecules shown in sequence 5 and sequence 6 of the sequence listing; if the PCR amplification product is of the same type and its size is 385 bp, the tested apple plant is of the M1M1 genotype; the M1M1 genotype apple plant is a candidate apple plant for producing red-fleshed apples.

3. An apple breeding method, comprising the following steps: (1) Cross apple varieties with the R1R1 genotype and apple varieties with the R6R6 genotype to obtain hybrid offspring; (2) Select plants with the M1M1 genotype from the hybrid offspring of the R1R6 genotype; the methods for determining the R1R1, R6R6, and R1R6 genotypes are as follows: using the genomic DNA of the tested apple plant as a template, perform PCR amplification using specific primer pair A; the specific primer pair A consists of single-stranded DNA molecules shown in sequence 1 of the sequence listing and single-stranded DNA molecules shown in sequence 2 of the sequence listing; if the PCR amplification product shows a single band with a size of 493 bp, the tested apple plant is of the R6R6 genotype; if the PCR amplification product shows a single band with a size of 392 bp, the tested apple plant is of the R1R1 genotype; if the PCR amplification product shows two bands with sizes of 493 bp and 392 bp respectively, the tested apple plant is of the R1R6 genotype; The method for determining the M1M1 genotype is as follows: Using the genomic DNA of the tested apple plant as a template, PCR amplification is performed using specific primer pair B; the specific primer pair B consists of single-stranded DNA molecules shown in sequence 5 of the sequence listing and single-stranded DNA molecules shown in sequence 6 of the sequence listing; if the PCR amplification product is of the same type and its size is 385 bp, the tested apple plant is of the M1M1 genotype.

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