Primers and methods for identifying authenticity of phyllium and citrus outcross progeny

Abstract

The application discloses a primer for identifying the authenticity of a distant hybrid offspring of phellodendron amurense and citrus, which can amplify target bands of 270bp, 296bp and 235bp in the distant hybrid offspring of phellodendron amurense and citrus, and the hybrid offspring with the target bands of 270bp, 296bp and 235bp is the real hybrid offspring. The method provided by the application can quickly and accurately identify the authenticity of the distant hybrid offspring of phellodendron amurense and citrus, has the characteristics of short identification time, high accuracy and strong specificity, provides an effective basis for identification research of the distant hybrid of phellodendron amurense and citrus, and is beneficial to genetic improvement of citrus varieties.

Description

Technical Field

[0001] This invention belongs to the field of citrus hybrid identification technology, specifically relating to a primer and method for identifying the authenticity of offspring from distant hybrids of yellow peel and citrus. Background Technology

[0002] Citrus fruits are diverse, including oranges, lemons, grapefruits, and tangerines. Rich in vitamin C, dietary fiber, and antioxidants, they not only occupy an important place in our daily diet but are also widely used in juice, food processing, and the pharmaceutical industry. However, citrus fruits are susceptible to pests and diseases, such as citrus Huanglongbing (HLB) and root rot. These diseases not only affect fruit quality and yield but can also lead to large-scale yield reduction or even death of the trees in severe cases.

[0003] The wampee tree has strong disease resistance, able to resist common citrus diseases such as Huanglongbing (HLB) and root rot, making it relatively easy to manage during cultivation and reducing reliance on chemical pesticides. Although citrus and wampee are both Rutaceae crops, they belong to different genera, making it very difficult to obtain citrus resources with excellent resistance through hybridization.

[0004] Distant hybridization refers to mating between individuals from different taxonomic units at the species or genus level. It is an important method for creating new plant varieties. Through distant hybridization, the isolation between species and genera can be broken down, allowing the aggregation of species- and genus-specific superior traits, which can then be recombinated under artificial hybridization conditions to form new varieties. Compared with normal hybridization, distant hybridization faces challenges such as incompatibility before fertilization, chromosome pairing obstacles after fertilization, and fertilization and hybrid embryo abortion. *Phellodendron amurense* (also known as yellow-skinned vine) belongs to the genus *Phellodendron* in the Rutaceae family. Hybridization with different genera within the Rutaceae family using *Phellodendron amurense* as the male parent is of great significance for the cultivation of new varieties of Rutaceae plants.

[0005] During distant hybridization, some genera and species in the Rutaceae family exhibit self-compatibility. The offspring obtained from distant hybridization are not necessarily true hybrids; they may be derived from self-pollination of the maternal parent without the incorporation of paternal genes. Therefore, identifying whether the offspring of distant hybridization are "true hybrids" is a necessary technique in distant hybridization work.

[0006] Molecular markers are specific sequences that exhibit identifiable genetic differences at the DNA level. They are used to differentiate the genetic backgrounds of different parents based on specific DNA sequence variations in the genome, thereby inferring whether offspring from distant hybridization are "true hybrids." Currently, there is a lack of unified and efficient molecular marker tools for identifying these interspecies genetic differences. Therefore, developing a highly polymorphic, easy-to-use, and parent-specific molecular marker is crucial. Summary of the Invention

[0007] To address the aforementioned technical problems, the main objective of this invention is to provide a primer for identifying the authenticity of offspring from distant hybrids of wampee and citrus, enabling rapid, accurate, and convenient identification of the authenticity of offspring from distant hybrids of wampee and citrus.

[0008] To achieve the objectives of this invention, the following technical solutions are provided:

[0009] In a first aspect, a primer for identifying the authenticity of offspring from distant hybridization of yellow peel and citrus includes yellow peel-specific primers, wherein the yellow peel-specific primers are primer combinations containing nucleotide sequences 1F and 1R, or primer combinations containing nucleotide sequences 2F and 2R, or primer combinations containing nucleotide sequences 3F and 3R, wherein the nucleotide sequences are shown in SEQ ID. 1-6.

[0010] Secondly, the present invention also provides a detection reagent, kit, or chip containing the above-mentioned yellow peel-specific primers.

[0011] Furthermore, the detection reagents, kits, or chips may also include other reagents for PCR amplification.

[0012] Thirdly, the detection reagents, kits, or chips described in this invention can be used in any of the following ways:

[0013] a) Identify the authenticity of offspring from distant hybrids of wampee and citrus;

[0014] b) To identify whether the paternal genome exists in the offspring of distant hybrids of wampee and citrus;

[0015] c) Screening for true hybrids from distant hybrids of yellow-skinned fruit and citrus;

[0016] d) Distant hybridization breeding of wampee and citrus;

[0017] e) Prepare products to identify the authenticity of offspring from distant hybrids of wampee and citrus;

[0018] f) Prepare products to identify the presence of the paternal genome in offspring of distant hybrids of wampee and citrus;

[0019] g) Prepare products for screening true hybrids from distant hybrids of yellow peel and citrus;

[0020] h) Prepare products from distant hybridization breeding of yellow peel and citrus.

[0021] Fourthly, the present invention also provides a method for identifying the authenticity of offspring from distant hybridization of yellow peel and citrus, comprising the following steps:

[0022] A1. Extract genomic DNA from the offspring of distant hybrids of wampee and citrus;

[0023] A2. Using the genomic DNA extracted in step A1 as a template, PCR amplification is performed using a primer combination. The primer combination includes yellow-skin-specific primers. The yellow-skin-specific primers are primer combinations containing nucleotide sequences 1F and 1R, or primer combinations containing nucleotide sequences 2F and 2R, or primer combinations containing nucleotide sequences 3F and 3R. The nucleotide sequences of the primer combinations are shown in SEQ ID.1-6.

[0024] A3. Genuine the PCR amplification products obtained in step A2, and perform band discrimination on the genotyping results: If a primer combination containing nucleotide sequences 1F and 1R yields a product of 270 bp, or a primer combination containing nucleotide sequences 2F and 2R yields a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R yields a product of 235 bp, then the offspring is a true hybrid. If a primer combination containing nucleotide sequences 1F and 1R fails to yield a product of 270 bp, a primer combination containing nucleotide sequences 2F and 2R fails to yield a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R fails to yield a product of 235 bp, then the offspring is a non-true hybrid.

[0025] Fifthly, the present invention also provides a method for identifying the presence of the paternal genome in offspring of distant hybrids of yellow peel and citrus, comprising the following steps:

[0026] B1. Genomic DNA of the hybrid offspring obtained by crossing yellow-skinned fruit as the male parent and citrus fruit as the female parent was extracted.

[0027] B2. Using the genomic DNA extracted in step B1 as a template, PCR amplification is performed using a primer combination. The primer combination includes yellow-skin-specific primers. The yellow-skin-specific primers are primer combinations containing nucleotide sequences 1F and 1R, or primer combinations containing nucleotide sequences 2F and 2R, or primer combinations containing nucleotide sequences 3F and 3R. The nucleotide sequences of the primer combinations are shown in SEQ ID.1-6.

[0028] B3. Genuine the PCR amplification products obtained in step B2, and perform band analysis on the genotyping results: If a primer combination containing nucleotide sequences 1F and 1R yields a product of 270 bp, or a primer combination containing nucleotide sequences 2F and 2R yields a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R yields a product of 235 bp, then the offspring of this hybrid possesses the paternal genome. If a primer combination containing nucleotide sequences 1F and 1R fails to yield a product of 270 bp, a primer combination containing nucleotide sequences 2F and 2R fails to yield a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R fails to yield a product of 235 bp, then the offspring of this hybrid lacks the paternal genome.

[0029] Furthermore, the PCR amplification reaction system in step A2 or B2 consists of 2 μL of DNA template, 10 μL of PCR premix, 0.5 μL of forward primer, 0.5 μL of reverse primer, and ddH2O.

[0030] Furthermore, the PCR amplification reaction conditions are as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62℃ annealing for 30 s, 72℃ extension for 30 s, 75℃ final extension for 5 min, for a total of 30 cycles, and finally stored at 4℃.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] The primers and identification methods provided by this invention enable rapid and accurate identification of the authenticity and purity of distant hybrids between wampee and citrus. The molecular markers of this invention can quickly and accurately identify the true hybrids in distant hybrids of wampee and citrus, exhibiting highly specific amplified fragment characteristic peaks, significantly reducing the workload of hybrid selection; compared to traditional morphological identification methods, the error rate is lower. The method of identifying the authenticity of hybrids using the molecular markers of this invention has advantages such as low cost, safe experimental operation, and time and labor saving. This invention helps to quickly and accurately identify the presence of the target paternal genome in distant hybrids of wampee and citrus, which is of great significance for the efficient development of distant hybridization breeding between wampee and citrus. Attached Figure Description

[0033] 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.

[0034] Figure 1This is a graph showing the parental specificity of molecular markers; m represents the molecular weight marker, from top to bottom: 500bp, 250bp, and 100bp; the numbers correspond to different varieties of yellow-skinned mandarin orange, loose-skinned mandarin orange, pomelo, orange, citron, and lemon; A shows the PCR amplification results of specific primer pair 1 in 72 yellow-skinned mandarin orange varieties; B shows the PCR amplification results of specific primer pair 1 in 13 orange varieties; C shows the PCR amplification results of specific primer pair 1 in 5 citron and lemon varieties; D shows the PCR amplification results of specific primer pair 1 in 28 loose-skinned mandarin orange varieties; E shows the PCR amplification results of specific primer pair 1 in 47 pomelo varieties; F shows the PCR amplification results of specific primer pair 2 in 72 yellow-skinned mandarin orange varieties. G represents the PCR amplification results of specific primer pair 2 in 13 orange varieties; H represents the PCR amplification results of specific primer pair 2 in 5 citron and lemon varieties; I represents the PCR amplification results of specific primer pair 2 in 28 mandarin orange varieties; J represents the PCR amplification results of specific primer pair 2 in 47 pomelo varieties; K represents the PCR amplification results of specific primer pair 3 in 72 yellow-skinned varieties; L represents the PCR amplification results of specific primer pair 3 in 13 orange varieties; M represents the PCR amplification results of specific primer pair 3 in 5 citron and lemon varieties; N represents the PCR amplification results of specific primer pair 3 in 28 mandarin orange varieties; and O represents the PCR amplification results of specific primer pair 3 in 47 pomelo varieties.

[0035] Figure 2 This is a molecular marker verification diagram for the offspring of the hybrids of yellow peel and citrus; m represents the molecular weight marker, from top to bottom: 500bp, 250bp, and 100bp; Z1-Z10 represent the distant hybrid offspring to be identified; MB represents citrus (maternal parent); FB represents yellow peel (paternal parent); PCR amplification results of primer pair 1 (sequence 1F / sequence 1R) in the hybrid offspring; PCR amplification results of primer pair 2 (sequence 2F / sequence 2R) in the hybrid offspring; PCR amplification results of primer pair 3 (sequence 3F / sequence 3R) in the hybrid offspring. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] Example 1: Validation of Molecular Marker Specificity for Identifying the Authenticity of Progeny from Distant Hybrids of Yellow Peel and Citrus

[0039] I. Selection of Material Types

[0040] Seventy-two varieties of yellow-skinned fruit, 28 varieties of mandarin orange, 47 varieties of pomelo, 13 varieties of orange, and 5 varieties of citron and lemon were selected. The specific varieties are as follows:

[0041] The 72 varieties of wampee are: 1. Late-maturing sweet wampee, 2. Yingzui wampee, 3. Huangpu wampee, 4. Congcheng wampee, 5. Xinjia white sugar wampee, 6. Qijian wampee, 7. Fengshun No. 4, 8. Lutian single-kernel wampee, 9. Haizhu No. 7, 10. Panyu sweet wampee, 11. Jiexi sweet wampee, 12. Yakou No. 2, 13. Qugen sweet wampee, 14. Luzhuang No. 1, 15. Shixing 032, 16. Luoding sweet wampee, 17. Small-fruited sweet wampee, 18. Yang 19. Longshan Seedless Chicken, 20. Xingning No. 4, 21. Large Chicken Heart, 22. Late-ripening Chicken Heart, 23. Golden Chicken Heart, 24. Small Chicken Heart No. 2, 25. Yangshan Chicken Heart, 26. Grade 1 Chicken Heart, 27. Jieyang Chicken Heart, 28. Qishan Juedu, 29. Juedu No. 4, 30. Taxia Sweet Skin, 31. Taxia Sour Skin, 32. Small Round Head, 33. Long Round Yellow Skin, 34. Large Round Head, 35. Purple Skin, 36. Beef Heart, 37. Long-fleshed rock sugar, 38. Yunan seedless, 39. Yunan pearl, 40. Liannan No. 2, 41. Huangpu No. 1, 42. Yunan No. 1, 43. Dapu No. 3, 44. Lianjiang 069, 45. Chicken heart No. 2, 46. Chicken heart No. 3, 47. Chicken heart No. 5, 48. Zhanjiang No. 1, 49. Zhanjiang No. 2, 50. Yangxi No. 1, 51. Yangxi No. 2, 52. Chaozhou No. 1, 53. Chaozhou No. 2, 54. Chaozhou No. 5, 55. Chaozhou No. 6, 56. Chaozhou No. 7, 57. Chaozhou No. 8, 58. Chaozhou No. 10, 59. Chaozhou No. 11, 60. Fengshao No. 2, 61. Fengshao No. 3, 62. Fengshao No. 4, 63. Fengshao No. 5, 64. Fengshao No. 6, 65. Longmen No. 1, 66. Longmen No. 3, 67. Longmen No. 4, 68. Longmen No. 5, 69. Longmen No. 6, 70. Longmen No. 7, 71. Longmen No. 8, 72. Longmen No. 9.

[0042] The 28 varieties of loose-skinned mandarin oranges are: 1. Sugar Mandarin Orange, 2. Guizhou Seedless Zhu Mandarin Orange, 3. Datian Ponkan Orange, 4. Early-maturing Clementine, 5. Huangyan Local Early Mandarin Orange, 6. Guinong Local Early Mandarin Orange, 7. Seedless Mang Mandarin Orange, 8. Mashui Mandarin Orange, 9. Early Honey Ponkan Orange, 10. Xinnv Ponkan Orange, 11. Local Mandarin Orange, 12. Nanfeng Honey Mandarin Orange, 13. Qianyang Seedless Ponkan Orange, 14. Huazhong Agricultural University Local Early Mandarin Orange, 15. Huanglingmiao Mandarin Orange, 16. Mangshan Wild Mandarin Orange, 17. Daoxian Wild Mandarin Orange, 18. Wenzhou Honey Mandarin Orange, 19. Qiuhui, 20. Red Mandarin Orange, 21. Daoxian Smooth-skinned Mandarin Orange, 22. Rihui, 23. Clementine, 24. Local Guang Mandarin Orange, 25. Murcott, 26. Jiangyong Wild Mandarin Orange, 27. Dekopon, 28. Four Seasons Mandarin Orange.

[0043] The 47 pomelo varieties are as follows: 1. Taiwan Dwarf Late-ripening Pomelo, 2. Xiangfeng Green Pomelo, 3. Yuhuan No. 1, 4. Yunnan Dongshi Early-ripening Pomelo, 5. Gongshui Red Pomelo, 6. Weisi Green Pomelo, 7. Quanzhou Pomelo, 8. June Early-ripening Pomelo, 9. Huaju Red Pomelo, 10. Japanese Red-fleshed Pomelo, 11. Guanxi Red-fleshed Pomelo, 12. Crispy Sweet Pomelo, 13. Guoqiang Madou, 14. Xishi Pomelo, 15. Taiwan Golden Silk Pomelo, 16. Juxiang Pomelo, 17. Qingtian Pomelo, 18. Thai Ruby Pomelo, 19. Nangnei Pomelo, 20. Shatin Pomelo, 21. Dianjiang White Pomelo, 22. Hainan Crystal Pomelo, 23. Guanxi Honey Pomelo, 24. Annong No. 1. 25. Cili Chopping Board Pomelo, 26. Glutinous Rice Pomelo, 27. Changsha Shatang Pomelo, 28. Ox Leg Pomelo, 29. Zhangjiajie Local Red Heart Pomelo, 30. White Jade Frost Pomelo, 31. Cili Golden Pomelo, 32. Early-maturing High-Sugar Fragrant Pomelo No. 3, 33. Cili Dwarf Pomelo, 34. Three-Stem Pomelo, 35. False Pomegranate Pomelo, 36. Hollow Tower Pomelo, 37. Late White Pomelo, 38. Local Pomelo Variation, 39. Early-maturing High-Sugar Fragrant Pomelo No. 1, 40. Cili Fragrant Pomelo, 41. Cili Sweet Pomelo No. 3, 42. Wendan, 43. Qiangdele Pomelo, 44. Jinlan Pomelo, 45. Chrysanthemum Heart Pomelo, 46. Early-maturing High-Sugar Fragrant Pomelo No. 2, 47. Large Fruit Red Flesh Pomelo.

[0044] The 13 orange varieties are: 1. Red Dark Willow Navel Orange, 2. Early Golden Sweet Orange, 3. Peach Leaf Orange, 4. Delta Sweet Orange, 5. Bud Blood Orange, 6. Newhall Sweet Orange, 7. Hamlin Sweet Orange, 8. Early Golden Sweet Orange, 9. Big Red Sweet Orange, 10. Fengjie 72-1, 11. Summer Orange, 12. Chenggu Sour Orange, and 13. Zigui Sour Orange.

[0045] The five citron and lemon varieties were: 1. Tibetan citron, 2. Yunnan citron, 3. Fragrant lemon, 4. Fujian sweet lemon, and 5. Eukaryotic lemon.

[0046] II. DNA Extraction from the Supply Material

[0047] Genomic DNA was extracted from the leaves of the test materials using the Adley novel plant genomic DNA rapid extraction kit.

[0048] 1. Take an appropriate amount of plant tissue (100mg of fresh tissue or 20mg of dry tissue; you can take more samples to make up for the loss that sticks to the mortar) and grind it into a fine powder in the mortar with liquid nitrogen.

[0049] 2. Prepare a 1.5 mL centrifuge tube, add 400 μL of buffer AP1 and 4 μL of RNase A (RNase A concentration is 10 mg / mL), take 100 mg of fresh tissue powder or 20 mg of dry tissue powder and put it into the centrifuge tube, vortex to mix thoroughly to help with lysis.

[0050] 3. Place the centrifuge tube in a 65℃ water bath for 10 minutes. During the water bath, the centrifuge tube can be inverted 2-3 times to mix the sample.

[0051] 4. Add 130 μL of buffer AP2, vortex to mix for 1 min, place on ice for 5 min, centrifuge at 13000 rpm for 5-10 min, and carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, being careful not to aspirate any interfacial material.

[0052] 5. Calculate the volume of supernatant, add 1.5 times the volume of AP3 / E, and immediately shake to mix.

[0053] 6. Add the mixture obtained in the previous step (including any precipitate that may appear) to an adsorption column AC. Place the adsorption column in a collection tube and centrifuge at 13000 rpm for 30-60 seconds. Discard the waste liquid in the collection tube. In this process, first add 650 μL, centrifuge, discard the waste liquid, then add the remaining solution and centrifuge again.

[0054] 7. Add 600 μL of rinsing buffer (WB), centrifuge at 13000 rpm for 30 seconds, and discard the waste liquid.

[0055] 8. Repeat step 7 once.

[0056] 9. Place the adsorption column AC back into the empty collection tube, centrifuge at 13000 rpm for 2 min, and remove as much of the washing solution as possible to avoid residual ethanol in the washing solution inhibiting the downstream reaction.

[0057] 10. Remove the adsorption column AC and place it in a clean centrifuge tube. Add 50-100 μL of elution buffer EB, incubate at room temperature for 3-5 minutes, and centrifuge at 13000 rpm for 1 minute. Add the resulting solution back into the adsorption column, incubate at room temperature for 2 minutes, and centrifuge at 13000 rpm for 1 minute to obtain the genomic DNA of the sample.

[0058] 11. Genomic DNA from the sample can be stored at 2-8℃. If it needs to be stored for a long time, it can be placed at -20℃.

[0059] III. PCR Amplification

[0060] Using the genomic DNA extracted in step two as a template, PCR amplification was performed using a yellow-skin-specific primer pair to obtain the PCR product. The primer sequences of the yellow-skin-specific primer pair are as follows:

[0061] HP1-F (forward primer): 5'-ACAATGGTGCTCGAGTGCAA-3' (sequence 1F, SEQ ID NO.1),

[0062] HP1-R (reverse primer): 5'-TCAAATCCACGACTCTGGGA-3' (sequence 1R, SEQ ID NO.2).

[0063] The PCR amplification system consisted of 20 μL: 2 μL DNA template, 10 μL PCR premix, 0.5 μL forward primer, 0.5 μL reverse primer, and 7 μL ddH2O. The PCR premix was selected from Yisheng Biotechnology (Shanghai) Co., Ltd. PCRMaster Mix.

[0064] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 62℃ annealing for 30 s, 72℃ extension for 30 s, 75℃ final extension for 5 min, for a total of 30 cycles, and finally stored at 4℃.

[0065] IV. Detection of PCR products by agarose gel electrophoresis

[0066] After the PCR reaction was completed, 8 μL of the PCR product was taken and placed in a 1.0% agarose gel. After electrophoresis at 150V for 20 min, the gel was observed and photographed using an automated gel imaging system.

[0067] Example 2

[0068] The same material selection and DNA extraction methods as in Example 1 were used. The PCR amplification method was basically the same as in Example 1, except that the primer sequences of the yellow-skin-specific primer pair are as follows:

[0069] HP2-F (forward primer): 5'-TCAACACAGCACGACCCATG-3' (sequence 2F, SEQ ID NO.3),

[0070] HP2-R (reverse primer): 5'-CCCAATCAACTTCAGCCATC-3' (sequence 2R, SEQ ID NO.4).

[0071] The method for detecting PCR products by agarose gel electrophoresis is the same as in Example 1.

[0072] Example 3

[0073] The same material selection and DNA extraction methods as in Example 1 were used. The PCR amplification method was basically the same as in Example 1, except that the primer sequences of the yellow-skin-specific primer pair are as follows:

[0074] HP3-F (forward primer): 5'-CACGGCATCACTCTCAATTC-3' (sequence 3F, SEQ ID NO.5),

[0075] HP3-R (reverse primer): 5'-ACTCAAGTTGGCAACAACCA-3' (sequence 3R, SEQ ID NO.6).

[0076] The method for detecting PCR products by agarose gel electrophoresis is the same as in Example 1.

[0077] The PCR product detection results of Examples 1-3 are as follows: Figure 1 As shown, the results indicate that the specific molecular markers for wampee could amplify the target bands in all 72 wampee varieties, such as... Figure 1 A, Figure 1 F, Figure 1 K. Specifically, primer combinations containing nucleotide sequences 1F and 1R yielded a 270 bp product, primer combinations containing nucleotide sequences 2F and 2R yielded a 296 bp product, and primer combinations containing nucleotide sequences 3F and 3R yielded a 235 bp product. The target band was not amplified in other citrus varieties, such as... Figure 1 B. Figure 1 C Figure 1 D、 Figure 1 G, Figure 1 H, Figure 1 I, Figure 1 J、 Figure 1 L, Figure 1 M, Figure 1 N、 Figure 1 O.

[0078] Example 4: Identification of the authenticity of distant hybrids between wampee and citrus using primer combinations containing nucleotide sequences 1F and 1R.

[0079] I. Selection of Material Types

[0080] The offspring Z1-Z10 were obtained by crossing citrus as the female parent and wampee as the male parent.

[0081] II. Authenticity Verification of Hybrid Offspring

[0082] Genomic DNA was extracted from the parents and hybrid offspring Z1-Z10 in this example. The yellow-skin-specific primers, DNA extraction method, PCR amplification, and amplification product detection method used were the same as in Example 1. The authenticity of the hybrid offspring of the varieties in Example 4 was verified.

[0083] Example 5: Identification of the authenticity of distant hybrids between wampee and citrus using primer combinations containing nucleotide sequences 2F and 2R.

[0084] I. Selection of Material Types

[0085] The offspring Z1-Z10 were obtained by crossing citrus as the female parent and wampee as the male parent.

[0086] II. Authenticity Verification of Hybrid Offspring

[0087] Genomic DNA was extracted from the parents and hybrid offspring Z1-Z10 in this example. The yellow-skin-specific primers, DNA extraction method, PCR amplification, and amplification product detection method used were the same as in Example 2. The authenticity of the hybrid offspring of the varieties in Example 5 was verified.

[0088] Example 6: Identification of the authenticity of distant hybrids between wampee and citrus using primer combinations containing nucleotide sequences 3F and 3R.

[0089] I. Selection of Material Types

[0090] The offspring Z1-Z10 were obtained by crossing citrus as the female parent and wampee as the male parent.

[0091] II. Authenticity Verification of Hybrid Offspring

[0092] Genomic DNA was extracted from the parents and hybrid offspring Z1-Z10 in this example. The yellow-skin-specific primers, DNA extraction method, PCR amplification, and amplification product detection method used were the same as in Example 3. The authenticity of the hybrid offspring of the varieties in Example 6 was verified.

[0093] The results of the identification are as follows Figure 2 As shown, the results indicate that primer combinations containing nucleotide sequences 1F and 1R, 2F and 2R, and 3F and 3R amplified bands of 270bp, 296bp, and 235bp respectively in Z3 of the Z1-Z10 hybrid offspring. This indicates that the genome contains paternal (yellow-skinned) specific sequences and yellow-skinned genomic information, confirming that it is a genuine distant hybrid offspring. The other nine materials did not amplify bands of 270bp, 296bp, and 235bp, and are not distant hybrid offspring.

[0094] In summary, the method of identifying the authenticity of distant hybrid offspring of wampee and citrus using molecular markers has the following advantages: the wampee-specific primers used in this invention can save time and space while identifying the authenticity of hybrid offspring, and have high accuracy. They can quickly and accurately identify the lines with paternal genetic information in the hybrid offspring of wampee and citrus, thereby determining the true distant hybrid offspring.

[0095] 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. A primer for identifying the authenticity of hybrid offspring produced by crossing a yellow-skinned mandarin orange as the male parent and a citrus mandarin orange as the female parent, characterized in that, The primers contain yellow-skin-specific primers, which are primer combinations containing nucleotide sequences 1F and 1R, or primer combinations containing nucleotide sequences 2F and 2R, or primer combinations containing nucleotide sequences 3F and 3R, as shown in SEQ ID.1-6.

2. Detection reagents, kits, or chips containing the primers described in claim 1.

3. The use of the detection reagent, kit, or chip according to claim 2 in any of the following: a) To verify the authenticity of hybrid offspring produced by crossing yellow-skinned fruit as the male parent and citrus as the female parent; b) To determine whether the paternal genome exists in the offspring of a cross between a yellow-skinned male parent and a citrus female parent; c) Screen for true hybrids from the offspring of crosses where yellow-skinned mandarin orange is the male parent and citrus is the female parent; d) To produce authentic hybrid offspring from crosses between yellow-skinned mandarin oranges and citrus fruits; e) To determine whether the offspring of a cross between yellow-skinned mandarin orange and citrus mandarin orange contain the paternal genome; f) Prepare products from the true hybrid offspring of hybrids produced by crossing yellow-skinned fruit as the male parent and citrus fruit as the female parent.

4. A method for identifying the authenticity of offspring from distant hybridization of wampee and citrus, characterized in that, Includes the following steps: A1. Extract genomic DNA from the offspring of hybrids that were crossed with yellow-skinned mandarin orange as the male parent and citrus as the female parent; A2. Using the genomic DNA extracted in step A1 as a template, PCR amplification is performed using a primer combination. The primer combination includes yellow-skin-specific primers. The yellow-skin-specific primers are primer combinations containing nucleotide sequences 1F and 1R, or primer combinations containing nucleotide sequences 2F and 2R, or primer combinations containing nucleotide sequences 3F and 3R. The nucleotide sequences of the primer combinations are shown in SEQ ID.1-6. A3. Genuine the PCR amplification products obtained in step A2, and perform band discrimination on the genotyping results: If a primer combination containing nucleotide sequences 1F and 1R yields a product of 270 bp, or a primer combination containing nucleotide sequences 2F and 2R yields a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R yields a product of 235 bp, then the offspring is a true hybrid. If a primer combination containing nucleotide sequences 1F and 1R fails to yield a product of 270 bp, a primer combination containing nucleotide sequences 2F and 2R fails to yield a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R fails to yield a product of 235 bp, then the offspring is a non-true hybrid.

5. A method for identifying the presence of the paternal genome in offspring of distant hybrids of yellow-skinned fruit and citrus, characterized in that, Includes the following steps: B1. Genomic DNA of the hybrid offspring obtained by crossing yellow-skinned fruit as the male parent and citrus fruit as the female parent was extracted. B2. Using the genomic DNA extracted in step B1 as a template, PCR amplification is performed using a primer combination. The primer combination includes yellow-skin-specific primers. The yellow-skin-specific primers are primer combinations containing nucleotide sequences 1F and 1R, or primer combinations containing nucleotide sequences 2F and 2R, or primer combinations containing nucleotide sequences 3F and 3R. The nucleotide sequences of the primer combinations are shown in SEQ ID.1-6. B3. Genuine the PCR amplification products obtained in step B2, and perform band analysis on the genotyping results: If a primer combination containing nucleotide sequences 1F and 1R yields a product of 270 bp, or a primer combination containing nucleotide sequences 2F and 2R yields a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R yields a product of 235 bp, then the offspring possesses the paternal genome. If a primer combination containing nucleotide sequences 1F and 1R fails to yield a product of 270 bp, a primer combination containing nucleotide sequences 2F and 2R fails to yield a product of 296 bp, or a primer combination containing nucleotide sequences 3F and 3R fails to yield a product of 235 bp, then the offspring lacks the paternal genome.

6. The method according to claim 4 or 5, characterized in that, The PCR amplification reaction system consists of 2 μL of DNA template, 10 μL of PCR premix, 0.5 μL of forward primer, 0.5 μL of reverse primer, and ddH2O.

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