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

Abstract

This invention discloses an InDel molecular marker closely linked to the peel color of pomelo and its application. The nucleotide sequence of the InDel molecular marker is shown in SEQ ID No. 1. This invention provides a kit for identifying the peel color of pomelo. Using this kit to identify red and yellow peels of pomelo overcomes the problems of long cycles and low efficiency associated with traditional methods that rely on field fruit phenotypic observation. Utilizing seedling-stage molecular marker diagnostic technology, early screening of peel color traits can be achieved during the pomelo seedling stage, breaking the dependence on the fruit ripening season and significantly shortening the breeding cycle. This kit is simple to operate, low in cost, and can be used for rapid screening and identification of target individual plants in large-scale hybrid progeny populations, greatly accelerating the breeding process of pomelo germplasm with superior peel color.

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 pomelo and its application. Background Technology

[0002] Under natural conditions, ordinary pomelos typically turn pale yellow or orange-yellow when ripe. In recent years, however, a red-skinned bud mutation of the pomelo has been discovered, producing a vibrant red peel that is far more appealing. In the fresh fruit market, this distinctive red color significantly enhances the fruit's appearance, increasing consumer acceptance and purchasing desire, thus giving the red-skinned pomelo higher commercial value and market competitiveness.

[0003] Traditional methods for identifying red-skinned pomelos primarily rely on visual phenotypic observation after fruit maturity. However, perennial crops like citrus have long seedling stages, typically requiring 6-8 years from planting to first fruiting. Furthermore, the peel color phenotypic of pomelos only becomes apparent at maturity. The inability to perform early screening for the red-skinned trait during the seedling stage limits the efficient selection of highly ornamental and economically valuable red-skinned pomelos.

[0004] In recent years, with the development of molecular biology, molecular marker-assisted breeding has gradually become an important tool in modern breeding due to its convenient operation, accurate results, and good reproducibility. This technology can identify and screen target traits at an early stage in seedlings, significantly shortening the breeding cycle and greatly improving the screening efficiency of new red-skinned pomelo germplasm, providing strong technical support for accelerating the cultivation of competitive varieties with differentiated peel colors.

[0005] Currently, there are no effective molecular markers for identifying the color of pomelo peel. Therefore, developing a molecular marker that can rapidly identify the color of pomelo peel and applying it to the screening of pomelos with different peel colors is of great significance for promoting the screening and dissemination of pomelo varieties with different peel colors. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an InDel molecular marker closely linked to the color of pomelo peel and its application. This invention develops a detection kit based on the InDel molecular marker closely linked to the color of pomelo peel. This kit can quickly identify the color of pomelo peel at the seedling stage, thereby improving the efficiency of screening pomelo germplasm 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 closely linked to the color of pomelo 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'-ATCTTCATTGTCGTGCCA-3', as shown in SEQ ID No. 2. Downstream primer R: 5'-GATTGTGGCTGATGGAAA-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 pomelo 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 pomelo peel.

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

[0012] This invention also provides a method for identifying the color of pomelo peel, comprising the following steps: 1) Extract DNA from the pomelo seedling tissue to be tested; 2) Perform PCR using the primer pairs or kits described above; 3) Electrophoresis of the amplification products. The color of the grapefruit 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 long stripe, it indicates that the peel color of the pomelo being tested is red. Alternatively, if the electrophoresis lane contains two bands, one long and one short, or one short band, it indicates that the peel color of the pomelo being tested is yellow.

[0013] Furthermore, in step 3), the judgment criteria are as follows: When the electrophoresis lane contains only one long band of 2,049 bp, it indicates that the peel color of the grapefruit being tested is red. Alternatively, if the electrophoresis lane contains a long band of 2,049 bp and a short band of 1,747 bp, or only a short band of 1,747 bp, then the peel color of the grapefruit 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: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 1 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 pomelo.

[0017] The present invention also provides an application of the above-mentioned kit in screening pomelo 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 pomelo 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 each material at that locus can be determined solely from the gel image of the amplification product.

[0019] 2. This invention provides a kit for identifying the color of pomelo peel. Using this kit to identify the color of pomelo peel can overcome the traditional dependence on the fruit ripening season and significantly shorten the breeding cycle.

[0020] 3. By utilizing molecular marker diagnostic technology at the seedling stage, the dependence of pomelo peel color identification on the maturity stage can be broken. This kit can be used to identify pomelo peel color, offering advantages such as early screening and low cost, significantly accelerating the screening efficiency for pomelo varieties with superior peel color. Attached Figure Description

[0021] Figure 1 This is a diagram showing the amplification results of primer pairs in 20 different pomelo samples. In the figure, M represents GL DNA marker 2000, and the bands from top to bottom are 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. Lanes 1-20 correspond to 20 portions of pomelo material: 1 is Crispy Red Pomelo; 2 is Changshan Pomelo Orange No. 9; 3 is Pomelo No. 2; 4 is Crispy Treasure Pomelo; 5 is Pear-shaped Pomelo; 6 is Dong'an Red Skin Pomelo; 7 is Low-acid Large Fruit Pomelo; 8 is 83 Series Pomelo; 9 is Ancestral Tree Pomelo; 10 is Yellow Skin Pomelo; 11 is Chentang Reservoir Pomelo; 12 is Taigongshan (Aijia) Pomelo; 13 is Zhongyou Base Pomelo; 14 is Seawater Base Pomelo; 15 is Zhou Jinhua Base Pomelo; 16 is Sanjian Base Pomelo; 17 is Tongmensi Base Pomelo; 18 is Fengjiashan Base Pomelo; 19 is Zheng Zhongxuan Base Pomelo; 20 is Xie Yugen Base Pomelo.

[0022] Figure 2 Alignment diagram of molecular marker sequences for long bands amplified from red-skinned pomelo (i.e., Dong'an red-skinned pomelo) and short bands amplified from yellow-skinned pomelo (Xie Yugen base pomelo); In the figure, A represents the long band amplified from the red-skinned pomelo, and the length of the long band is 2,049 bp; 'a' indicates the short band amplified from the yellow-skinned pomelo, with a length of 1,747 bp.

[0023] Figure 3 PCR amplification results for identifying 8 sample materials using the kit; In the figure, M represents GL DNA marker 2000, and the bands from top to bottom are 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. Lanes 1-6 contain 6 portions of pomelo, and lanes 7-8 contain 2 portions of lemon. 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 the color of pomelo peel based on whole-genome sequencing are as follows: To identify genomic loci closely linked to the color of pomelo peel, Shanghai Paisennuo Biotechnology Co., Ltd. was commissioned to complete whole-genome sequencing (sequencing depth greater than 30×) of 20 different pomelo samples, obtaining a total of more than 300 G of data.

[0026] Using BWA (v0.7.18) software, sequencing reads from 20 different pomelo 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) to obtain a BAM file containing all variation information. IGV software was used to visualize variation sites among different pomelo materials. Variation information for pomelo resources with different peel colors was compared and examined individually. Based on the peel color of the material, the ratios of various bases / haplotypes at variation sites were statistically analyzed. Sequence polymorphisms with the highest association with the peel color phenotype were manually examined and screened as candidate sites for molecular marker development. A 288 bp InDel was found to be significantly different between red and yellow pomelo. This result is a linkage marker (i.e., the InDel molecular marker) predicted based on whole-genome resequencing results and associated with pomelo peel color. The nucleotide sequence of this marker 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'-ATCTTCATTGTCGTGCCA-3', as shown in SEQ ID No. 2. Downstream primer R: 5'-GATTGTGGCTGATGGAAA-3', as shown in SEQ ID No. 3.

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

[0029] The method for identifying the color of pomelo peel using a reagent kit includes the following steps: 1) Extract DNA from the pomelo seedling tissue 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: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 1 min, 35 cycles; 72 °C further extension for 5 min, and storage at 12 °C.

[0031] 3) Electrophoresis of the amplified products; the color of the grapefruit peel to be tested is determined based on the band characteristics of the amplified products. When the electrophoresis lane contains only one long band of 2,049 bp (the long band sequence is shown in SEQ ID No. 4), it indicates that the peel color of the pomelo being tested is red. Alternatively, if the electrophoretic lane contains a long band of 2,049 bp and a short band of 1,747 bp, or only a short band of 1,747 bp (the short band sequence is shown in SEQ ID No. 5), it indicates that the peel color of the grapefruit being tested is yellow.

[0032] Example 3 The above-mentioned kit and method were used to identify 20 pomelo samples: 1) Genomic DNA was extracted from 20 pomelo 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.

[0033] The thermal cycling parameters were: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 1 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 1.5% agarose gel electrophoresis on a horizontal electrophoresis tank of the Beijing 61 Electrophoresis System. The electrophoresis was performed using 1xTAE buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0), at a voltage of 120 V / cm and a current of 400 mA for 20 min. After electrophoresis, images were taken and stored using a BIO-RAD gel imaging system (UVP).

[0035] like Figures 1-2 As shown: 20 samples of pomelo were found. Molecular marker identification identified 3 samples as red-skinned pomelo (a long band of 2,049 bp, as shown in SEQ ID No. 4), of which only 1 sample (plant No. 7) did not match the phenotypic expression; 17 samples of yellow-skinned pomelo (a long band of 2,049 bp and a short band of 1,747 bp or containing only a short band of 1,747 bp, as shown in SEQ ID No. 5) matched the phenotypic expression. Figure 2 Sequence alignment confirmed at the molecular level that this site exhibits genuine insertion / deletion differences among grapefruits of different peel colors. Figure 1 The results of the group validation were mutually corroborated. Further statistical analysis of the amplification results of 20 pomelo materials showed that the concordance rate of the marker reached (19 / 20)×100%=95%, indicating that the marker has a high explanatory power for the color of pomelo peel and can be applied to early seedling screening. This breaks the limitation of traditional methods that rely on phenotypic observation during the fruit ripening season and has important practical value for accelerating the breeding of red-skinned pomelo.

[0036] Therefore, the use of the above-mentioned reagent kit provides a simple, rapid and effective screening method for the early diagnosis of pomelo peel color, and improves the screening efficiency of different pomelo peel colors.

[0037] Table 1. Validation of molecular markers for peel color in 20 different pomelos Note: "√" means it meets the requirements, and "×" means it does not meet the requirements.

[0038] Example 4 The above-described kit and method were used to identify eight sample materials (six grapefruits and two lemons).

[0039] 1) Eight sample materials (six grapefruit samples and two lemon samples) were selected, and genomic DNA was extracted from the eight sample materials using a modified CTAB method. 2) Using each genomic DNA as a template, PCR amplification was performed using a kit (refer to Example 2), and the results were analyzed by electrophoresis and actual fruit peel color.

[0040] like Figure 3 As shown in Table 2: Among the eight samples, samples 1 and 2 only amplified one 2,049 bp band, predicted to be red-skinned; samples 3 and 4 amplified two bands, 2,049 bp and 1,747 bp, respectively; samples 5 and 6 amplified one 1,747 bp band, and these four samples were predicted to be yellow-skinned. The predicted results perfectly matched the actual peel color of each sample, and the identification accuracy of the four pomelo samples was 100%.

[0041] Both markers 7 and 8 amplified only one 2,049 bp band, predicted as red-skinned according to the interpretation rules; however, both actually had yellow peels, indicating a discrepancy between the predicted and actual phenotypes. This result demonstrates that the polymorphism at this marker site cannot explain the variation in lemon peel color.

[0042] Table 2. Identification results of molecular markers for fruit peel color in 8 sample materials. Note: "√" means it meets the requirements, and "×" means it does not meet the requirements.

[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 pomelo peel, 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'-ATCTTCATTGTCGTGCCA-3', as shown in SEQ ID No.

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

3.

3. The application of the primer pair according to claim 2 in identifying the color of pomelo peel.

4. The use of the primer pair according to claim 2 in the preparation of a kit for identifying the color of pomelo peel.

5. A reagent kit for identifying the color of pomelo peel, characterized in that: The kit includes the primer pair as described in claim 2.

6. A method for identifying the color of pomelo peel, characterized in that: Includes the following steps: 1) Extract DNA from the pomelo seedling tissue 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: The corresponding peel color of the grapefruit 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 long stripe, it indicates that the peel color of the pomelo being tested is red. Alternatively, if the electrophoresis lane contains two bands, one long and one short, or one short band, it indicates that the peel color of the pomelo being tested is yellow.

7. The method according to claim 6, characterized in that: In step 3), the judgment criteria are as follows: When the electrophoresis lane contains only one long band of 2,049 bp, it indicates that the peel color of the grapefruit being tested is red. Alternatively, if the electrophoresis lane contains a long band of 2,049 bp and a short band of 1,747 bp, or only a short band of 1,747 bp, then the peel color of the grapefruit being tested is yellow.

8. 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. In step 2), the PCR amplification program includes: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 sec, 55 °C annealing for 20 sec, 72 °C extension for 1 min, 35 cycles; 72 °C further extension for 5 min, and storage at 12 °C.

9. An application of the kit described in claim 5 in molecular marker-assisted breeding of pomelo.

10. An application of the kit described in claim 5 in screening grapefruit germplasm with different peel colors.

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