Indel molecular marker for identifying color and luster of citrus peel in seedling stage and use method of indel molecular marker

Abstract

The invention discloses an indel molecular marker for identifying color and luster of citrus peel in a seedling stage and application of the indel molecular marker. The corresponding sites of the indel molecular marker of the citrus are positioned in chr7: 18, 398, 798-18, 399 and 289 intervals of a chromosome 7 of an SWO3.0 reference genome; the genome DNA of the citrus seedling stage plant is used as a template, the Indel molecular marker is amplified through PCR, the citrus seedling stage plant can be divided into a homozygous insertion type, a homozygous deletion type and a heterozygous type according to a PCR amplification result, and the three types are associated with the color of the citrus fruit for identifying the color of the citrus peel, so that the early screening of the seedling stage target traits of the citrus breeding population is realized, and the early screening of the seedling stage target traits of the citrus breeding population is realized. The breeding period is remarkably shortened, the breeding cost is reduced, and important technical support is provided for citrus fruit color improvement breeding.

Description

Technical Field

[0001] This invention relates to the field of citrus molecular breeding, specifically to an indel molecular marker for identifying the color of citrus peel during the seedling stage and its application method. Background Technology

[0002] Citrus is one of the world's most important economic fruit trees, and the appearance quality of its fruit (especially fruit color) directly affects consumers' willingness to buy, making it one of the core target traits for high-quality citrus breeding. Traditional citrus breeding mainly relies on hybridization and bud mutation selection, but this has significant limitations: citrus is a perennial woody plant, and it takes 3-5 years from hybridization and pollination to fruit maturity, resulting in a long breeding cycle; at the same time, the hybrid offspring population is large, requiring long-term field observation, which increases breeding costs and severely restricts the efficiency of fruit color improvement.

[0003] Molecular marker-assisted selection (MAS) technology can screen breeding materials at the seedling stage by detecting molecular markers linked to target traits, without waiting for plant maturity, which can significantly shorten the breeding cycle and reduce costs. Currently reported molecular markers related to citrus fruit color mainly include SNPs and SSRs: SNP markers require a real-time quantitative PCR platform (such as KASP technology), which has high equipment costs and is not suitable for small and medium-sized breeding units; SSR markers are mostly polymorphic and complex, and some markers have low linkage to traits, resulting in poor detection stability.

[0004] InDel (insertion / deletion) markers are molecular markers based on the insertion or deletion of short base fragments in the genome. They have advantages such as codominant inheritance, simple detection methods (requiring only routine PCR + agarose gel electrophoresis), low cost, and high stability, making them easier to promote and apply in grassroots breeding units. However, there are currently very few functional InDel markers for citrus fruit color, and no InDel markers that can be directly used to identify fruit color at the seedling stage have been reported. There is an urgent need to develop efficient and low-cost InDel molecular markers to meet the needs of citrus breeding. Summary of the Invention

[0005] To address the aforementioned technical problems, the main objective of this invention is to provide an indel molecular marker for identifying citrus peel color during the seedling stage and its application method, enabling rapid, accurate, and convenient prediction of citrus peel color, assisting in the early screening of target traits in citrus breeding, shortening breeding time, and reducing breeding costs.

[0006] To achieve the objectives of this invention, the following technical solutions are provided: In one aspect, the present invention provides an indel molecular marker for identifying the peel color of citrus fruits during the seedling stage. The indel molecular marker is located at the chr7: 18,398,798-18,399,289 region on chromosome 7 of the SWO3.0 reference genome. The indel molecular marker has three genotypes: homozygous insertion, homozygous deletion, and heterozygous. Using the genomic DNA of citrus seedlings as a template, the indel molecular marker is amplified by PCR. A 744bp band is amplified, and the genotype is defined as homozygous insertion. Two bands, 744bp and 216bp, are amplified, and the genotype is defined as heterozygous. A 216bp band is amplified, and the genotype is defined as homozygous deletion. The mature citrus fruits corresponding to the homozygous insertion and heterozygous types are orange, while the mature citrus fruits corresponding to the homozygous deletion type are light yellow.

[0007] Secondly, the present invention also provides amplification primers for the indel molecular marker for identifying the color of citrus peel as described in the above technical solution, wherein the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0008] Furthermore, the present invention also provides the application of the amplification primers described in the above technical solution in the preparation of citrus peel color detection reagents, kits, genomic chips or liquid phase probes.

[0009] The present invention also provides a citrus peel color detection product, which includes the amplification primers described in the above technical solution.

[0010] Thirdly, the present invention also provides the application of the indel molecular marker for identifying the color of citrus peel, the amplification primers described in the above-mentioned technical solutions, or the citrus peel color detection products described in the above-mentioned technical solutions in the identification or differentiation of citrus peel color.

[0011] Fourthly, the present invention also provides a method for using the above-mentioned technical solution, comprising the following steps: Step 1: Extract genomic DNA from the citrus plants to be tested. Step 2: Using the genomic DNA extracted in Step 1 as a template, perform PCR amplification using amplification primers. The nucleotide sequences of the forward and reverse primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Step 3: Perform gel electrophoresis on the PCR amplification products to obtain gel electrophoretic amplification bands and determine the results based on the size of the gel electrophoretic amplification bands. If a 744bp band is amplified, the genotype of the citrus sample being tested is homozygous insertion; if two bands, 744bp and 216bp, are amplified, the genotype of the citrus sample being tested is heterozygous; if a 216bp band is amplified, the genotype of the citrus sample being tested is homozygous deletion. The citrus fruits corresponding to homozygous insertion and heterozygous types are orange at maturity, while the citrus fruits corresponding to homozygous deletion types are light yellow at maturity.

[0012] Furthermore, the total volume of the PCR amplification reaction system in step 2 is 10 μL, containing 1.0 μL of genomic DNA at a concentration of 100-200 ng / μL, 0.25 μL of forward primer at a concentration of 10 μmol / L, 0.25 μL of reverse primer at a concentration of 10 μmol / L, 5.0 μL of 2× Hieff Canace® AdvanceFast PCR Master Mix, and 3.5 μL of ddH2O.

[0013] Furthermore, the PCR amplification reaction conditions are as follows: initial pre-denaturation at 98℃ for 5 minutes; followed by 40 cycles, each cycle including denaturation at 98℃ for 30 seconds, annealing at 52℃ for 30 seconds, and extension at 72℃ for 90 seconds; after the cycle, final extension at 72℃ for 10 minutes; and finally, incubation at 12℃ for 10 minutes.

[0014] The above scheme was used for seedling screening of citrus hybrid breeding populations, eliminating homozygous missing seedlings and retaining homozygous inserted or heterozygous seedlings.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to utilize the Indel molecular marker method for seedling screening of citrus hybrid breeding populations. Compared to the SNP and SSR markers commonly used in citrus breeding, the Indel molecular markers provided by this invention are more suitable for actual breeding needs. The Indel molecular marker method eliminates the need for a fluorescence quantitative PCR instrument dependent on SNPs, and also eliminates the need for polyacrylamide gel electrophoresis equipment required for SSRs; only a regular PCR instrument and agarose gel electrophoresis apparatus are required, resulting in lower equipment requirements. In terms of cost, the single-sample testing fee for the Indel molecular marker method is less than 5 yuan, far lower than the 20-50 yuan for SNPs and 10-15 yuan for SSRs, with no special consumables required. Furthermore, the Indel molecular marker method is simpler to operate; interpretation can be performed directly by electrophoresis after PCR amplification, without the need for software analysis for SNPs or silver staining and destaining steps for SSRs. Personnel can operate it after basic training. In addition, InDels are based on fragment insertion / deletion, exhibiting strong genetic conservation and superior detection stability compared to SSRs. They are better suited for field seedling samples, efficiently supporting large-scale breeding screening, and significantly lowering the threshold for industrialization. Attached Figure Description

[0016] The disclosure of this invention will become more readily understood with reference to the accompanying drawings. It will be readily understood by those skilled in the art that these drawings are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention.

[0017] Figure 1 Agarose gel electrophoresis images of the InDel loci in 40 natural citrus populations are shown. M in the image represents the GL DNA marker 5000. Lanes 1-40 correspond to representative materials from the 40 natural populations: 1. Mangshan wild orange; 2. Daoxian wild orange; 3. Nanfeng mandarin orange; 4. Wenzhou mandarin orange; 5. Clementine; 6. Red mandarin orange; 7. Phoenix pomelo; 8. Acid-free pomelo; 9. Late-blooming white pomelo; 10. Gaoban pomelo; 11. Shatang pomelo; 12. HB pomelo; 13. Buddha's hand; 14. Citron 1; 15. Citron 2; 16. Citron 3; 17. Citron 4; 18. Citron 5; 19. Yichang orange 1; 2 0. Yichang Orange 2; 21. Yichang Orange 3; 22. Yichang Orange 4; 23. Yichang Orange 5; 24. Yichang Orange 6; 25. Newhall; 26. Hamlin; 27. Fulingxia Orange; 28. Red Summer Orange; 29. ​​Royal Grapefruit; 30. Starbucks; 31. Cocktail Grapefruit; 32. Chunxiang Tangerine; 33. Beijing Lemon; 34. Coarse Lemon; 35. Eureka Lemon; 36. Green Lemon; 37. Zigui Sour Orange; 38. Chengdu Sour Orange; 39. Brazilian Sour Orange; 40. Daidai Sour Orange.

[0018] Figure 2 This is an agarose gel electrophoresis image of the InDel loci of Chengdu sour orange and its self-crossed progeny. M in the image represents GL DNA marker 5000. The bands, from top to bottom, are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. Lanes 1-22 correspond to the parents and 21 self-crossed progeny: 1. CDLB-M; 2. CDLB3; 3. CDLB231; 4. CDLB618; 5. CDLB712; 6. CDLB905; 7. CDLB40; 8. CDLB... B62; 9.CDLB355; 10.CDLB378; 11.CDLB486; 12.CDLB616; 13.CDLB625; 14.CDLB647; 15.CDLB65 6; 16. CDLB670; 17. CDLB249; 18. CDLB610; 19. CDLB619; 20. CDLB651; 21. CDLB658; 22. CDLB675. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0020] The separating agent and usage method provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] This invention collects materials from different natural citrus populations, covering orange-peeled varieties (such as Newhall, Hamlin, and Nanfeng mandarin oranges) and non-orange-peeled varieties (such as Shatin pomelo, citron, and Yichang oranges). All materials are from the National Citrus Germplasm Resource Center and have a clear phenotype—the mature fruit color is orange or light yellow. By performing whole-genome resequencing on different natural citrus population materials, using the SWO3.0 genome as a reference, an InDel locus located in the chr7: 18,398,798-18,399,289 interval was screened through comparative analysis. It was found that there is a 528bp fragment insertion at this locus, and its genotype is strictly associated with the fruit color.

[0022] Homozygous insertion type (both chromosomes contain a 528bp insertion fragment): the mature fruit is orange; Heterozygous (one chromosome contains the insertion segment, and the other does not): The fruit is orange at maturity; Homozygous deletion type (neither chromosome contains the insertion segment): The mature fruit is light yellow.

[0023] Validated using natural population data, the genotype of the InDel locus corresponds to fruit color with 100% accuracy, with no false positives or false negatives, demonstrating a stable association between the locus and the orange fruit trait.

[0024] Based on the conserved genomic sequences flanking the InDel site (to avoid non-specific amplification), a dedicated primer pair (forward primer F and reverse primer R) was designed to ensure that the amplification product contains the complete InDel region—the homozygous insertion amplification product is "conserved region + 528bp insert" (744bp), the homozygous deletion amplification product is "conserved region only" (216bp), and the heterozygous amplification product produces both products simultaneously. The primer sequences are as follows: Forward primer F: 5'-ACCTAAGTAAGGAGAAACATTACCAC-3' (SEQ ID NO.1) Reverse primer R: 5'-AATTTTATGTGCGTGTCAATTAATTG-3' (SEQ ID NO.2) Example 1: Detection of indel molecular markers in natural population samples I. Plant Sample Collection Forty citrus materials were selected, all from the National Citrus Germplasm Resource Center, including mandarin orange, sweet orange, pomelo, Yichang orange, sour orange, grapefruit, and tangerine pomelo. Specifically, they are: 1. Mangshan wild mandarin orange; 2. Daoxian wild mandarin orange; 3. Nanfeng mandarin orange; 4. Wenzhou mandarin orange; 5. Clementine; 6. Red mandarin orange; 7. Phoenix pomelo; 8. Acid-free pomelo; 9. Late white pomelo; 10. Gaoban pomelo; 11. Shatang pomelo; 12. HB pomelo; 13. Buddha's hand; 14. Citron 1; 15. Citron 2; 16. Citron 3; 17. Citron 4; 18. Citron 5; 19. Yichang Orange 1; 20. Yichang Orange 2; 21. Yichang Orange 3; 22. Yichang Orange 4; 23. Yichang Orange 5; 24. Yichang Orange 6; 25. Newhall; 26. Hamlin; 27. Fulingxia Orange; 28. Red Summer Orange; 29. ​​Royal Grapefruit; 30. Starbucks; 31. Cocktail Grapefruit; 32. Chunxiang Tangerine; 33. Beijing Lemon; 34. Coarse Lemon; 35. Eureka Lemon; 36. Green Lemon; 37. Zigui Sour Orange; 38. Chengdu Sour Orange; 39. Brazilian Sour Orange; 40. Daidai Sour Orange.

[0025] II. DNA Extraction from Sample Plants Genomic DNA was extracted from control sample plants using the CTAB method. The CTAB extraction buffer contained 2% CTAB, 1% β-mercaptoethanol, 100 mmol / L Tris-HCl, 20 mmol / L EDTA, 1.4 mol / L NaCl, and the buffer pH was 8.0.

[0026] III. PCR Amplification Using the genomic DNA extracted in step two as a template, PCR amplification was performed using the indel molecular marker primers designed in this invention for identifying the color of citrus peel during the seedling stage, and the PCR product was obtained.

[0027] The total volume of the PCR amplification reaction system was 10 μL, containing 1.0 μL of genomic DNA at a concentration of 100-200 ng / μL, 0.25 μL of forward primer at a concentration of 10 μmol / L, 0.25 μL of reverse primer at a concentration of 10 μmol / L, 5.0 μL of 2× Hieff Canace® AdvanceFast PCR Master Mix (Shanghai Yisheng Biotechnology), and 3.5 μL of ddH2O. The PCR amplification program was as follows: initial pre-denaturation at 98℃ for 5 minutes; followed by 40 cycles, each cycle consisting of denaturation at 98℃ for 30 seconds, annealing at 52℃ for 30 seconds, and extension at 72℃ for 90 seconds; a final extension at 72℃ for 10 minutes after the cycles; and finally, incubation at 12℃ for 10 minutes.

[0028] IV. Detection of PCR products by agarose gel electrophoresis After the PCR reaction, the PCR amplification products were collected and electrophoresed on a 1.5% agarose gel at a constant voltage of 130V for 10 minutes. The results were then observed using a gel imaging system. Under these conditions, the 744bp and 216bp bands can be clearly separated, and the electrophoresis time is short, allowing for rapid results.

[0029] Agarose gel electrophoresis results of InDel sites in 40 natural populations of citrus fruits are as follows: Figure 1 As shown.

[0030] V. Result Determination The results were determined by observing the size of the amplified bands using a gel imaging system: a 744bp band was amplified, and its genotype was defined as homozygous insertion; two bands, 744bp and 216bp, were amplified, and their genotypes were defined as heterozygous; a 216bp band was amplified, and its genotype was defined as homozygous deletion. The citrus fruits corresponding to the homozygous insertion and heterozygous types were orange at maturity, while the citrus fruits corresponding to the homozygous deletion type were light yellow at maturity.

[0031] Of the 40 materials, 18 orange phenotype materials were either homozygous insertions (AA, 6 materials) or heterozygous (Aa, 12 materials); of the 22 light yellow phenotype materials, 21 were homozygous deletions (aa) and 1 was heterozygous. A comparison of the genotype and phenotype with the phenotype predicted by this invention shows that the Indel molecular marker designed in this invention can effectively identify the color of citrus peel with an accuracy of 97%. The specific comparison results are shown in Table 1.

[0032] Table 1. Validation results of natural populations of 40 citrus species

[0033] Example 2: Detection of indel molecular markers in self-crossed population samples In this example, 21 Chengdu sour orange plants and their self-crossed progeny were used as samples for indel molecular marker detection. Of these, 15 plants were homozygous insertions or heterozygous, and 6 plants were homozygous deletions. The sample DNA extraction method, PCR amplification method, and detection method used were the same as in Example 1. The final agarose gel electrophoresis results of the InDel sites of the Chengdu sour orange plants and their self-crossed progeny are shown below. Figure 2 As shown.

[0034] The genotype and phenotype were combined and compared with the phenotype predicted by this invention. The comparison results are shown in Table 2.

[0035] Table 2. Verification results of 21 Chengdu sour orange plants and their self-crossed progeny

[0036] The Indel molecular marker method designed in this invention predicted that the fruit color of 15 homozygous insert or heterozygous samples would be orange, and the fruit color of 6 homozygous deletion samples would be light yellow (yellow-skinned). Actual fruit phenotype analysis showed that 14 homozygous insert / heterozygous plants had orange fruit, and 1 heterozygous plant had light yellow fruit (possibly due to genetic background); 5 homozygous deletion plants had light yellow fruit, and 1 homozygous deletion plant had orange fruit (possibly due to genetic background). The results show that the Indel molecular marker method designed in this invention achieves an overall accuracy rate of over 90%, demonstrating the good stability of the marker in self-crossed offspring.

[0037] The above description, in conjunction with specific / preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

Claims

1. An indel molecular marker for identifying the peel color of citrus fruits at the seedling stage, wherein the indel molecular marker is located in the chr7:18,398,798-18,399,289 region on chromosome 7 of the SWO3.0 reference genome; the indel molecular marker has three genotypes: homozygous insertion, homozygous deletion, and heterozygous; wherein, using genomic DNA of citrus seedlings as a template, the indel molecular marker is amplified by PCR, and a 744bp band is amplified, defining the genotype as homozygous insertion; two bands, 744bp and 216bp, are amplified, defining the genotype as heterozygous; and a 216bp band is amplified, defining the genotype as homozygous deletion; the citrus fruits corresponding to the homozygous insertion and heterozygous types are orange at maturity, while the citrus fruits corresponding to the homozygous deletion type are light yellow at maturity.

2. Amplification primers for identifying the indel molecular marker for identifying citrus peel color during the seedling stage as described in claim 1, characterized in that, The forward primer nucleotide sequence of the amplification primer is shown in SEQ ID NO.1, and the reverse primer nucleotide sequence is shown in SEQ ID NO.

2.

3. A detection product for identifying the color of citrus peel during the seedling stage, characterized in that, The detection product is a detection reagent, kit, genomic chip, or liquid probe containing the amplification primers described in claim 2.

4. The application of the indel molecular marker of claim 1, the amplification primer of claim 2, or the detection product of claim 3 in identifying the color of citrus peel during the seedling stage.

5. A method for using an indel molecular marker to identify the color of citrus peel during the seedling stage, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from the citrus plants to be tested; Step 2: Using the genomic DNA extracted in Step 1 as a template, perform PCR amplification using primers. The nucleotide sequences of the forward and reverse primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Step 3: Perform gel electrophoresis on the PCR amplification products obtained in Step 2 to obtain gel electrophoretic amplification bands, and interpret the bands based on their size. If a 744bp band is amplified, the genotype of the citrus sample being tested is homozygous insertion; if two bands, 744bp and 216bp, are amplified, the genotype of the citrus sample being tested is heterozygous; if a 216bp band is amplified, the genotype of the citrus sample being tested is homozygous deletion. The citrus fruits corresponding to homozygous insertion and heterozygous types are orange at maturity, while the citrus fruits corresponding to homozygous deletion types are light yellow at maturity.

6. The method for using the indel molecular marker for identifying the color of citrus peel during the seedling stage according to claim 5, characterized in that, The total volume of the PCR amplification reaction system in step 2 is 10 μL, containing 1.0 μL of genomic DNA at a concentration of 100-200 ng / μL, 0.25 μL of forward primer at a concentration of 10 μmol / L, 0.25 μL of reverse primer at a concentration of 10 μmol / L, 5.0 μL of 2× Hieff Canace® AdvanceFast PCR Master Mix, and 3.5 μL of ddH2O.

7. The method for using the indel molecular marker for identifying the color of citrus peel during the seedling stage according to claim 5, characterized in that, The PCR amplification reaction conditions were as follows: initial pre-denaturation at 98℃ for 5 minutes; followed by 40 cycles, each cycle consisting of denaturation at 98℃ for 30 seconds, annealing at 52℃ for 30 seconds, and extension at 72℃ for 90 seconds; final extension at 72℃ for 10 minutes after the cycle; and finally incubation at 12℃ for 10 minutes.

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