InDel molecular marker closely linked to citrus fruit peel color and application thereof

Abstract

The application discloses an InDel molecular marker closely linked with the fruit peel color of Citrus sinensis and application thereof, and a nucleotide sequence of the InDel molecular marker is shown as SEQ ID No. 1. The application obtains a kit for identifying the fruit peel color of Citrus sinensis, and the kit is used for identifying red-skinned Citrus sinensis and yellow-skinned Citrus sinensis, and can overcome problems of long period and low efficiency caused by dependence on field fruit phenotype observation in traditional methods. By using the molecular marker diagnosis technology at the seedling stage, early screening of the fruit peel color trait can be realized at the seedling stage of the Citrus sinensis, the dependence on the fruit mature season is broken, and the breeding period is significantly shortened. The kit is simple in operation and low in cost, can be used for rapid screening and identification of target single plants in large-scale different Citrus sinensis germplasms or Citrus sinensis hybrid offspring populations, and greatly accelerates the breeding process of excellent fruit peel color Citrus sinensis germplasms.

Description

Technical Field

[0001] This invention relates to the field of molecular marker development technology related to fruit peel color, specifically to an InDel molecular marker closely linked to the peel color of oranges and its application. Background Technology

[0002] Orange ( Citrus junos This is a hybrid variety with significant utilization value and an important local citrus germplasm resource in my country. Under natural conditions, the peel of ordinary sweet oranges is yellow or orange-yellow when ripe. In recent years, a bud mutation variety of red-skinned sweet orange with a bright red peel has been discovered. Its red appearance significantly improves the commercial quality of the fruit, giving the red-skinned sweet orange higher commercial value and market competitiveness.

[0003] Currently, in production and breeding practices, the identification of the red peel trait in sweet oranges mainly relies on phenotypic observation during fruit maturity. However, as a perennial woody fruit tree, citrus has a juvenile period of 6–8 years, and the red peel trait only stabilizes after fruit maturity, making early screening during the seedling stage impossible. This limits the efficiency of this trait in early selection during breeding.

[0004] In recent years, with the development of molecular biology, molecular marker-assisted breeding has become an important technical means in modern breeding due to its advantages such as simple operation, accurate results, and good reproducibility. This technology can identify and screen target traits at an early stage in the seedling stage, significantly shorten the breeding cycle, improve the screening efficiency of new red-skinned orange germplasm, and provide important support for cultivating varieties with differentiated peel color that are competitive in the market.

[0005] Currently, there is a lack of molecular markers that can effectively identify the color of orange peel. Therefore, developing a molecular marker that can rapidly identify peel color and is applicable to the screening of different orange varieties is of great significance for promoting the screening and application of orange varieties with different peel colors. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an InDel molecular marker closely linked to the color of orange peel and its application. Based on this molecular marker, this invention has developed a corresponding detection kit, which can rapidly identify the color of orange peel at the seedling stage, thereby improving the screening efficiency of orange varieties with superior peel color.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides an InDel molecular marker that is closely linked to the color of orange peel, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0008] This invention also provides a primer pair for amplifying the above-mentioned InDel molecular marker, wherein the primer pair is: Upstream primer F: 5'-TAGGGCTGTTGCTACGCT-3', as shown in SEQ ID No. 2. Downstream primer R: 5'-TTGCCTGTCCTCCGTAAT-3', as shown in SEQ ID No. 3.

[0009] The present invention also provides an application of the above-mentioned primer pair in identifying the color of orange peel.

[0010] The present invention also provides the application of the above-mentioned primer pair in the preparation of a kit for identifying the color of orange peel.

[0011] The present invention also provides a kit for identifying the color of orange peel, the kit comprising the primer pair described above.

[0012] This invention also provides a method for identifying the color of orange peel, comprising the following steps: 1) Extract DNA from the tissue of the orange seedlings to be tested; 2) Perform PCR using the primer pairs or kits described above; 3) Electrophoresis of the amplification products. The color of the orange peel to be tested is determined based on the band characteristics of the amplification products. The judgment criteria are as follows: When the electrophoresis lane contains only one short band, it indicates that the orange peel being tested is red. Alternatively, if the electrophoresis lane contains two bands of different lengths or one long band, it indicates that the orange peel being tested is yellow.

[0013] Furthermore, in step 3), the judgment criteria are as follows: When the electrophoresis lane contains only one short band of 2,354 bp, it indicates that the peel color of the orange being tested is red. Alternatively, if the electrophoresis lane contains a long band of 3,237 bp and a short band of 2,354 bp, or only a long band of 3,237 bp, then the orange peel being tested is yellow.

[0014] Furthermore, in step 2), the PCR amplification system, in 20 μL, includes: 10 μL PCR Mix, 100 ng template, 1 μM each of upstream and downstream primers, and the remainder ddH2O.

[0015] Furthermore, in step 2), the PCR amplification program includes: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 3 min, 35 cycles; 72 °C further extension for 5 min, and storage at 12 °C.

[0016] The present invention also provides an application of the above-mentioned kit in molecular marker-assisted breeding of oranges.

[0017] The present invention also provides an application of the above-described kit in screening orange varieties with different peel colors.

[0018] The beneficial effects of this invention are: 1. This invention provides an InDel molecular marker closely linked to the color of orange peel. The invention designs a forward primer F upstream of the InDel molecular marker fragment and a reverse primer R downstream, forming a primer pair F / R. This invention allows for molecular marker detection using the aforementioned primer pair F / R, and the genotype of various traits at that locus can be determined solely from the gel image of the amplified products.

[0019] 2. This invention provides a kit for identifying the color of orange peel. Using this kit can overcome the dependence of traditional methods on the fruit ripening period, thereby shortening the breeding cycle.

[0020] 3. By utilizing molecular marker diagnostic technology at the seedling stage, the dependence of orange peel color identification on fruit maturity can be broken. Applying this kit for orange peel color identification offers advantages such as early screening and low cost, significantly improving the screening efficiency for orange varieties with superior peel color. Attached Figure Description

[0021] Figure 1 This is a diagram showing the amplification results of primer pairs in nine different orange germplasms. In the figure, M represents GL DNA marker 5000, and the bands from top to bottom are 5,000 bp, 3,000 bp, 2,000 bp, 1,500 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. Lanes 1-9 correspond to 9 different orange varieties: 1 is a red-skinned orange (seedling); 2 is a red-skinned orange (grafted seedling with trifoliate orange rootstock); 3 is a Japanese orange; 4 is a wood-shaped pomelo (Japanese orange); 5 is a small-fruited pomelo (Japanese orange); 6 is a tadanishi (Japanese orange); 7 is a jaila pomelo (Japanese orange); 8 is a Korean golden pomelo (Japanese orange); and 9 is a Ziyang orange. Among them, germplasm No. 1-2 is red-skinned, and germplasm No. 3-9 is yellow-skinned.

[0022] Figure 2 This is a sequence alignment diagram of molecular markers between yellow-skinned sweet orange (Japanese sweet orange) and red-skinned sweet orange (red-skinned sweet orange seedling), where the former amplifies a long band and the latter amplifies a short band; In the figure, the long strip is 3,237 bp in length; the short strip is 2,354 bp in length.

[0023] Figure 3 The amplification results of the kit for detecting four orange samples are shown in the figure. Among them, orange No. 1 is red-skinned, while oranges No. 2-4 are yellow-skinned. In the figure, M represents GL DNA marker 5000, and the bands from top to bottom are 5,000 bp, 3,000 bp, 2,000 bp, 1,500 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0025] Example 1 The specific steps for screening InDel molecular markers closely linked to orange peel color based on whole-genome sequencing are as follows: To identify genomic loci closely linked to the color of orange peel, Shanghai Paisennuo Biotechnology Co., Ltd. was commissioned to complete whole-genome sequencing (sequencing depth greater than 30×) of 9 different orange varieties, obtaining a total of more than 130 G of data.

[0026] Using BWA (v0.7.18) software, sequencing reads from nine different orange germplasms were aligned to the sweet orange v2.0 reference genome (http: / / citrus.hzau.edu.cn / data / Genome_info / SWO.v2.0 / SWO.v2.0.genome.fa) to obtain a BAM file containing information on all variant sites. IGV software was used to visualize and analyze the variant sites among different orange germplasms, comparing the variant information among germplasms with different peel colors, calculating the ratio of different bases / haplotypes at each variant site, and manually selecting the polymorphic sequence with the highest association with the peel color phenotype as candidate sites for molecular marker development. The results showed that a 902 bp insertion / deletion (InDel) exhibited significant differences between red and yellow-skinned oranges. This InDel is a molecular marker linked to orange peel color predicted based on whole-genome resequencing results, and its nucleotide sequence is shown in SEQ ID No. 1.

[0027] Example 2 I. Primer pairs F / R were designed based on the InDel molecular markers described above, as follows: Upstream primer F: 5'-TAGGGCTGTTGCTACGCT-3', as shown in SEQ ID No. 2. Downstream primer R: 5'-TTGCCTGTCCTCCGTAAT-3', as shown in SEQ ID No. 3.

[0028] II. Kit for Identifying the Color of Orange Peel The kit includes the primer pair F / R described above.

[0029] The method for identifying the color of orange peel using a kit includes the following steps: 1) Extract DNA from the tissue of the orange seedlings to be tested; 2) Perform PCR using the kit described above; where, The PCR amplification system, in 20 μL increments, includes: 10 μL PCR Mix, 100 ng template, 1 μM each of upstream and downstream primers, and the remainder ddH2O.

[0030] The PCR amplification program included: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 3 min, 35 cycles; 72 °C further extension for 5 min, and storage at 12 °C.

[0031] 3) Electrophoresis of the amplification products; the color of the orange peel to be tested is determined based on the band characteristics of the amplification products. When the electrophoresis lane contains only a short band of 2,354 bp (as shown in SEQ ID No. 5), it indicates that the peel color of the orange being tested is red. Alternatively, if the electrophoresis lane contains a long band of 3,237 bp (as shown in SEQ ID No. 4) and a short band of 2,354 bp (as shown in SEQ ID No. 5), or only a long band of 3,237 bp, then the orange peel color to be tested is yellow.

[0032] Example 3 The above-mentioned kits and methods were used to identify nine orange germplasm accessions: 1) Genomic DNA was extracted from nine orange germplasm accessions using a modified CTAB method. 2) Using each genomic DNA as a template, PCR was performed using a kit to obtain amplification products, among which, The PCR reaction system is as follows: 10 μL PCR Mix (purchased from Yisheng Company), 100 ng DNA, 1 μM each of forward and reverse primers (synthesized by Tianyi Company), and ddH2O added to a final volume of 20 μL.

[0033] The thermal cycling parameters were: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 3 min, 35 cycles; 72 °C extension for 5 min, storage at 12 °C. The reaction was performed on a BIOCENER instrument.

[0034] 3) The amplified products were detected by 1.0% agarose gel electrophoresis on a horizontal electrophoresis tank of the Beijing 61 Electrophoresis System. The electrophoresis buffer was 1xTAE buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0), voltage 120 V / cm, current 400 mA, and electrophoresis time 20 min. After electrophoresis, images were taken and stored using a BIO-RAD gel imaging system (UVP).

[0035] like Figure 1 and Figure 2 As shown, among the nine orange germplasm samples, the molecular marker identification results were completely consistent with the peel color phenotype: two red-skinned orange samples amplified only a short band of 2,354 bp in length; while seven yellow-skinned orange samples amplified a long band of 3,237 bp in length, or simultaneously amplified a long band of 3,237 bp in length and a short band of 2,354 bp in length. Figure 2 Sequence alignment results further confirmed that this site exhibits a genuine insertion / deletion difference between red and yellow-skinned oranges, and... Figure 1 The PCR amplification results corroborated each other. Statistical analysis of the amplification results from nine orange germplasms showed a 100% concordance rate for the marker, indicating that it is a co-dominant InDel molecular marker for orange peel color. This marker can be used for seedling screening of red-skinned oranges, overcoming the limitations of traditional methods that rely on phenotypic observation at fruit maturity, and has significant practical value for accelerating red-skinned orange breeding.

[0036] Therefore, the use of the above-mentioned kit provides a simple, rapid and effective screening method for the early diagnosis of orange peel color, which can improve the screening efficiency of orange varieties with different peel colors.

[0037] Table 1. Validation of molecular markers for nine orange germplasm samples with different peel colors. Note: "√" indicates that it meets the requirements.

[0038] Example 4 The above-described kit and method were used to identify four orange samples: 1) Genomic DNA was extracted from four orange seedlings using a modified CTAB method. 2) Using each genomic DNA as a template, PCR was performed using a kit to obtain amplification products, among which, The PCR reaction system is as follows: 10 μL PCR Mix (purchased from Yisheng Company), 100 ng DNA, 1 μM each of forward and reverse primers (synthesized by Tianyi Company), and ddH2O added to a final volume of 20 μL.

[0039] The thermal cycling parameters were: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 3 min, 35 cycles; 72 °C extension for 5 min, storage at 12 °C. The reaction was performed on a BIOCENER instrument.

[0040] 3) The amplified products were detected by 1.0% agarose gel electrophoresis on a horizontal electrophoresis tank of the Beijing 61 Electrophoresis System. The electrophoresis buffer was 1xTAE buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0), voltage 120 V / cm, current 400 mA, and electrophoresis time 20 min. After electrophoresis, images were taken and stored using a BIO-RAD gel imaging system (UVP).

[0041] Table 2. Molecular marker analysis results of four orange samples. Depend on Figure 3 As shown in Table 2, among the four samples, one was a red-skinned orange and three were yellow-skinned oranges (among which, the double band indicates that the orange was heterozygous at this locus but was still classified as yellow-skinned); this fully demonstrates that the InDel marker is a codominant molecular marker and can also distinguish between homozygous and heterozygous individuals.

[0042] The kit identification results in this embodiment were clear and well-defined, with no non-specific amplification, further verifying the specificity and stability of the primer pair F / R. This marker can distinguish color and genotype at the seedling stage, which is of great significance for the early and accurate screening of hybrid offspring populations. In particular, the identification of heterozygous individuals can provide a basis for parent selection in further hybridization breeding.

[0043] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An InDel molecular marker closely linked to the color of orange peel, characterized in that: The nucleotide sequence of the InDel molecular marker is shown in SEQ ID No.

1.

2. The application of a primer pair for amplifying the InDel molecular marker as described in claim 1 in distinguishing between red and yellow peels of oranges, characterized in that: The primer pair is Upstream primer F: 5'-TAGGGCTGTTGCTACGCT-3', as shown in SEQ ID No.

2. Downstream primer R: 5'-TTGCCTGTCCTCCGTAAT-3', as shown in SEQ ID No.

3.

3. The use of a primer pair for amplifying the InDel molecular marker as described in claim 1 in the preparation of a kit for distinguishing between red and yellow peels of oranges.

4. A method for identifying the color of orange peel, characterized in that: Includes the following steps: 1) Extract DNA from the tissue of the orange seedlings to be tested; 2) Perform PCR using primer pairs; the primer pairs are: Upstream primer F: 5'-TAGGGCTGTTGCTACGCT-3', as shown in SEQ ID No.

2. Downstream primer R: 5'-TTGCCTGTCCTCCGTAAT-3', as shown in SEQ ID No. 3; 3) Electrophoresis of the amplification products: The corresponding peel color of the orange to be tested is determined based on the band characteristics of the amplification products; the judgment criteria are as follows: When the electrophoresis lane contains only one short band of 2,354 bp, it indicates that the peel color of the orange being tested is red. Alternatively, if the electrophoresis lane contains a long band of 3,237 bp and a short band of 2,354 bp, or only a long band of 3,237 bp, then the orange peel being tested is yellow.

5. The method according to claim 4, characterized in that: In step 2), The PCR amplification system, in 20 μL increments, includes: 10 μL PCR Mix, 100 ng template, 1 μM each of upstream and downstream primers, and the remainder ddH2O. In step 2), the PCR amplification program includes: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 3 min, 35 cycles; 72 °C further extension for 5 min, and storage at 12 °C.

6. An application of a reagent kit in screening red-peel and yellow-peel orange germplasm, characterized in that: The kit includes primer pairs, and the primer pairs are... Upstream primer F: 5'-TAGGGCTGTTGCTACGCT-3', as shown in SEQ ID No.

2. Downstream primer R: 5'-TTGCCTGTCCTCCGTAAT-3', as shown in SEQ ID No. 3.

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