InDel molecular marker for distinguishing ziyang orange and japanese orange and application thereof

Abstract

The application discloses an InDel molecular marker for distinguishing Ziyang orange and Japanese orange 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 distinguishing Ziyang orange and Japanese orange, and the kit can be used to identify Ziyang orange and Japanese orange at a seedling stage, and can overcome problems of long period and low efficiency caused by dependence on field fruit phenotype observation in traditional methods. By using a seedling stage molecular marker diagnosis technology, seedling stage screening of Ziyang orange and Japanese orange can be realized, dependence on a fruit ripening season is broken, and a breeding period is significantly shortened. The kit is simple in operation, low in cost, and can be used for large-scale rapid screening and identification of Ziyang orange and Japanese orange.

Description

Technical Field

[0001] This invention relates to the field of molecular marker development technology related to citrus variety identification, specifically to an InDel molecular marker for distinguishing Ziyang oranges from Japanese oranges and its application. Background Technology

[0002] Orange ( Citrus junos It is a hybrid variety with important utilization value, and it is generally believed to have originated from the loose-skinned orange ( Citrus reticulata ) and Yichang orange ( Citrus ichangensis Natural hybridization. Although Ziyang oranges and Japanese oranges originated from similar parental combinations and have similar genetic backgrounds, they have undergone significant phenotypic differentiation in terms of botanical traits, functional components, ecological adaptability, and production and utilization methods during long-term natural evolution and artificial selection, thus forming germplasm types with different applications.

[0003] Ziyang fragrant orange is an important local citrus germplasm resource in my country. This germplasm boasts vigorous growth, a well-developed root system, and wide adaptability, maintaining good growth even in alkaline soil conditions. Its alkali resistance is significantly superior to commonly used rootstocks such as trifoliate orange. Furthermore, Ziyang fragrant orange exhibits characteristics such as polyembryony, good grafting compatibility, and early fruiting and high yield, and has been widely used as a citrus rootstock in production, playing a crucial role in improving plant adaptability and yield stability. In contrast, Japanese fragrant oranges differ significantly in morphological characteristics and utilization methods. Their fruit has a rich aroma and a distinctive volatile composition, making them more suitable for processing, such as flavor products or juice production.

[0004] However, since sweet oranges are of hybrid origin and exhibit polyembryony, it is difficult to accurately distinguish Ziyang sweet oranges from Japanese sweet oranges based solely on morphological or physiological indicators, which is particularly limiting in the seedling stage and germplasm resource identification. Currently, there are no molecular markers that can effectively distinguish between Ziyang sweet oranges and Japanese sweet oranges. Therefore, developing a method that can rapidly identify these two types of sweet oranges and is applicable to material screening is of great significance for promoting the screening and application of sweet orange germplasm resources. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an InDel molecular marker for distinguishing Ziyang oranges from Japanese oranges and its application. This invention develops a detection kit based on the InDel molecular marker for distinguishing Ziyang oranges from Japanese oranges. This kit can quickly distinguish between Ziyang oranges and Japanese oranges in the seedling stage, thereby improving the germplasm screening efficiency of Ziyang oranges and Japanese oranges.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides an InDel molecular marker for distinguishing Ziyang oranges from Japanese oranges, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0007] This invention also provides a primer pair for amplifying the above-mentioned InDel molecular marker, wherein the primer pair is: Upstream primer F: 5'-CATGGTCCTAAGCCTATT-3', as shown in SEQ ID No. 2. Downstream primer R: 5'-CTACAAAGGATCGGACTA-3', as shown in SEQ ID No. 3.

[0008] The present invention also provides an application of the above-mentioned primer pair in distinguishing between Ziyang oranges and Japanese oranges.

[0009] The present invention also provides the application of the above-mentioned primer pair in the preparation of a kit for distinguishing between Ziyang oranges and Japanese oranges.

[0010] The present invention also provides a kit for distinguishing between Ziyang oranges and Japanese oranges, the kit comprising the primer pairs described above.

[0011] This invention also provides a method for distinguishing Ziyang oranges from Japanese oranges, comprising the following steps: 1) Extract DNA from the tissue of the orange variety to be tested; 2) Perform PCR using the primer pairs or kits described above; 3) Electrophoresis of amplified products: Ziyang oranges and Japanese oranges are distinguished based on the band characteristics of the amplified products. The judgment criteria are as follows: When the electrophoresis lane contains only one long band, it indicates that the orange variety being tested is a Japanese orange. Alternatively, if the electrophoresis lane contains two bands, one long and one short, it indicates that the orange variety being tested is Ziyang orange.

[0012] Furthermore, in step 3), the judgment criteria are as follows: When the electrophoresis lane contains only one long band of 549 bp, it indicates that the orange variety being tested is a Japanese orange. Alternatively, if the electrophoresis lane contains a long band of 549 bp and a short band of 360 bp, it indicates that the orange variety being tested is Ziyang orange.

[0013] 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.

[0014] Furthermore, in step 2), the PCR amplification program includes: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 10 sec, annealing at 55 °C for 20 sec, extension at 72 °C for 45 sec, 35 cycles; further extension at 72 °C for 5 min, and storage at 12 °C.

[0015] The present invention also provides an application of the above-mentioned kit in molecular marker-assisted breeding of oranges.

[0016] The present invention also provides an application of the above-described reagent kit in distinguishing between Ziyang oranges and Japanese oranges.

[0017] The beneficial effects of this invention are: 1. This invention provides an InDel molecular marker to distinguish between Ziyang sweet oranges and Japanese sweet oranges. The invention designs a forward primer F upstream of the InDel molecular marker fragment and a reverse primer R downstream, forming primer pair F and R. This invention allows for molecular marker detection using the aforementioned primer pair F and R, and the genotype of each material at that locus can be determined solely based on the gel image of the amplified product.

[0018] 2. This invention provides a kit that can distinguish between Ziyang oranges and Japanese oranges. Using this kit for identification can avoid the uncertainty of traditional reliance on biological traits such as fruit after fruit set for variety identification, and at the same time effectively shorten the identification cycle.

[0019] 3. By utilizing seedling-stage molecular marker diagnostic technology, the dependence of orange type identification on post-fruiting fruit traits can be broken. This kit can be used to distinguish Ziyang oranges from Japanese oranges, offering the advantage of early screening and significantly accelerating the screening efficiency of Ziyang oranges and Japanese oranges. Attached Figure Description

[0020] Figure 1 A schematic diagram of InDel molecular markers used to distinguish Ziyang oranges from Japanese oranges; Figure 2 This is a diagram showing the amplification results of primer pairs in eight different orange germplasms. In the figure, M represents GL DNA marker 1,000, and the bands from top to bottom are 1,000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp. Lanes 1-8 correspond to 8 different sweet orange germplasms: 1 is Ziyang sweet orange (Quzhou); 2 is Ziyang sweet orange (Wuhan); 3 is Japanese sweet orange; 4 is Mutouyu (Japanese sweet orange); 5 is Xiaoguoyu (Japanese sweet orange); 6 is Tada Nishiki (Japanese sweet orange); 7 is Zailaiyu (Japanese sweet orange); and 8 is Korean Golden Yuzu (Japanese sweet orange).

[0021] Figure 3Sequence alignment diagram of the long band amplified from Ziyang orange (Quzhou) and the short band amplified from Japanese orange; The long band represents SEQ ID No. 4, which has the sequence SEQ ID No. 1, and the short band represents SEQ ID No. 5, which does not have the sequence SEQ ID No. 1.

[0022] Figure 4 Amplification results of seven orange samples tested with the kit; In the figure, M represents GL DNA marker 1,000, and the bands from top to bottom are 1,000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp. Lanes 1-7 correspond to 7 different orange ingredients: 1, 2, and 3 are Ziyang oranges; 4, 5, 6, and 7 are Japanese oranges. Detailed Implementation

[0023] 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.

[0024] Example 1 The InDel molecular markers that can distinguish Ziyang oranges from Japanese oranges were identified based on whole-genome sequencing. The specific steps are as follows: To identify genomic loci that can distinguish Ziyang oranges from Japanese oranges, Shanghai Paisennuo Biotechnology Co., Ltd. was commissioned to complete whole-genome sequencing (sequencing depth greater than 30×) of 6 Japanese orange varieties and 2 Ziyang orange varieties, obtaining a total of more than 120G of data.

[0025] The sequencing reads from eight materials were aligned to the sweet orange v2.0 genome (http: / / citrus.hzau.edu.cn / data / Genome_info / SWO.v2.0 / SWO.v2.0.genome.fa) using BWA (v0.7.18) software to obtain a BAM file containing all variation information. The variation sites between Ziyang sweet orange and Japanese sweet orange were visualized using iGV software. Variation information between Ziyang sweet orange and Japanese sweet orange was compared and examined one by one. The ratios of various bases / haplotypes at the variation sites were statistically analyzed. Polymorphic sequences that could distinguish Ziyang sweet orange from Japanese sweet orange were manually examined and screened as candidate sites for molecular marker development. A 189 bp InDel was found to be significantly different between Ziyang sweet orange and Japanese sweet orange. When this 189 bp was present, it indicated that the sweet orange was from the Japanese sweet orange. Figure 1 This result is based on the prediction of a linkage marker (InDel molecular marker) that can distinguish between Ziyang orange and Japanese orange based on whole genome resequencing results. The nucleotide sequence of this 189 bp InDel molecular marker is shown in SEQ ID No. 1.

[0026] Example 2 I. Primer pairs F / R were designed based on the InDel molecular markers described above, as follows: Upstream primer F: 5'-CATGGTCCTAAGCCTATT-3', as shown in SEQ ID No. 2. Downstream primer R: 5'-CTACAAAGGATCGGACTA-3', as shown in SEQ ID No. 3.

[0027] II. A reagent kit for distinguishing between Ziyang oranges and Japanese oranges. The kit includes the primer pair F / R described above.

[0028] The method for distinguishing Ziyang oranges from Japanese oranges using a reagent 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.

[0029] 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 45 sec, 35 cycles; 72 °C further extension for 5 min, and storage at 12 °C.

[0030] 3) Electrophoresis of the amplification products: Based on the band characteristics of the amplification products, determine whether it is Ziyang fragrant orange or Japanese fragrant orange. When the electrophoresis lane contains only a single 549 bp band (as shown in SEQ ID No. 4), it indicates that the orange being tested is a Japanese orange. Alternatively, if the electrophoresis lane contains a long band of 549 bp (sequence shown in SEQ ID No. 4) and a short band of 360 bp (sequence shown in SEQ ID No. 5), it indicates that the orange being tested is a Ziyang orange.

[0031] Example 3 The above-mentioned kit and identification method were used to identify 6 Japanese orange samples and 2 Ziyang orange samples: 1) Genomic DNA was extracted from eight orange samples 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.

[0032] The thermal cycling parameters were: 95 °C for 3 min; 95 °C for 10 sec, 55 °C for 20 sec, 72 °C extension for 45 sec, 35 cycles; 72 °C extension for another 5 min, and storage at 12 °C. The reaction was performed on a BIOCENER instrument.

[0033] 3) The amplified products were detected by 2.0% agarose gel electrophoresis on a horizontal electrophoresis tank of the Beijing 61 Electrophoresis System. The electrophoresis buffer was 1xTAE buffer (0.04M Tris-acetate, 0.001M 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).

[0034] like Figure 2 As shown, the amplification results of primer pair F / R in eight orange germplasms showed that lanes 1 and 2 (Ziyang orange) amplified a long band of 549 bp and a short band of 360 bp, exhibiting a double band pattern; lanes 3-8 (Japanese orange) amplified only a single band of 549 bp. The band types of the marker in the eight materials were completely consistent with their actual orange types, with a consistency of 100% (8 / 8), indicating that the InDel molecular marker is a co-dominant marker, and Ziyang orange (double band) and Japanese orange (single band) can be directly distinguished based on the electrophoretic bands.

[0035] The results are as follows Figure 3 As shown: the long band (549 bp, SEQ ID No. 4) contains the 189 bp InDel sequence shown in SEQ ID No. 1, while the short band (360 bp, SEQ ID No. 5) lacks this sequence. The sequence difference between the two is precisely the 189 bp fragment shown in SEQ ID No. 1. This result confirms at the sequence level that the difference between the long and short bands lies in the presence or absence of the InDel sequence shown in SEQ ID No. 1. Ziyang oranges exhibit a double band because they possess both the long allele containing the InDel and the short allele not containing it, while Japanese oranges exhibit a single band because they only possess the long allele containing the InDel.

[0036] Further statistical analysis of the amplification results of 8 orange materials revealed that the molecular marker bands of the 8 hybrid offspring matched the phenotypic expression (Table 1). Overall, the match rate of the marker reached (8 / 8)×100%=100%, and it was considered that the marker was a codominant molecular marker that distinguishes Ziyang orange from Japanese orange.

[0037] Table 1. Molecular marker validation results of 8 orange samples Note: "√" indicates that it meets the requirements.

[0038] Example 4 The above-mentioned kit and identification method were used to identify seven orange samples: 1) Genomic DNA was extracted from seven orange samples using a modified CTAB method. 2) Using each genomic DNA as a template, PCR was performed using a kit to obtain amplification products. The PCR reaction system was 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 was added to a final volume of 20 μL.

[0039] The thermal cycling parameters were: 95 °C for 3 min; 95 °C for 10 sec, 55 °C for 20 sec, 72 °C extension for 45 sec, 35 cycles; 72 °C extension for another 5 min, and storage at 12 °C. The reaction was performed on a BIOCENER instrument.

[0040] 3) The amplified products were detected by 2.0% agarose gel electrophoresis on a horizontal electrophoresis tank of the Beijing 61 Electrophoresis System. The electrophoresis buffer was 1xTAE buffer (0.04M Tris-acetate, 0.001M 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] according to Figure 4 The gel electrophoresis images show that a total of 7 orange samples (lanes 1–7) were analyzed, and the marker (M) position was used to determine the results. Table 2. Molecular marker validation results of 7 orange samples Table 2 shows that the actual types of the 7 materials completely match the predicted types of the molecular markers, with a verification accuracy rate of 100%.

[0042] As shown above, this embodiment uses seedling materials (blind testing of unknown samples), further demonstrating that the InDel marker can be accurately identified at the seedling stage, independent of fruit phenotype. The InDel molecular marker is an accurate, stable, and co-dominant marker suitable for rapid screening at the seedling stage. Using this kit to distinguish between Ziyang sweet oranges and Japanese sweet oranges offers the advantage of early screening, significantly accelerating the screening efficiency between Ziyang sweet oranges and Japanese sweet oranges.

[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 for distinguishing Ziyang oranges from Japanese oranges, characterized in that: The nucleotide sequence of the InDel molecular marker is shown in SEQ ID No.

1.

2. A primer pair for amplifying the InDel molecular marker of claim 1, characterized in that: The primer pair is Upstream primer F: 5'-CATGGTCCTAAGCCTATT-3', as shown in SEQ ID No.

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

3.

3. The application of the primer pair according to claim 2 in distinguishing between Ziyang oranges and Japanese oranges.

4. The use of the primer pair according to claim 2 in the preparation of a kit for distinguishing between Ziyang oranges and Japanese oranges.

5. A reagent kit for distinguishing between Ziyang oranges and Japanese oranges, characterized in that: The kit includes the primer pair as described in claim 2.

6. A method for distinguishing Ziyang oranges from Japanese oranges, characterized in that: Includes the following steps: 1) Extract DNA from the tissue of the orange variety to be tested; 2) Perform PCR using the primer pair described in claim 2 or the kit described in claim 5; 3) Electrophoresis of the amplification products: Ziyang oranges and Japanese oranges are distinguished based on the band characteristics of the amplification products; the judgment criteria are as follows: When the electrophoresis lane contains only one long band of 549 bp, it indicates that the orange variety being tested is a Japanese orange. Alternatively, if the electrophoresis lane contains a long band of 549 bp and a short band of 360 bp, it indicates that the orange variety being tested is Ziyang orange.

7. The method according to claim 6, 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; 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 45 sec, 35 cycles; 72 °C further extension for 5 min, and storage at 12 °C.

8. An application of the kit described in claim 5 in molecular marker-assisted breeding of oranges.

9. An application of the kit according to claim 5 in distinguishing between Ziyang oranges and Japanese oranges.

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