Primer and method for identifying authenticity of distant hybridization offspring of clausena lansium and citrus

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of citrus hybrid identification, and particularly relates to a primer and a method for identifying the authenticity of distant hybrid offspring of wampee and citrus. Background Art

[0002] Citrus fruits, including oranges, lemons, grapefruits, and tangerines, are rich in vitamin C, dietary fiber, and antioxidants. They not only play an important role in our daily diet but are also widely used in juice production, food processing, and the pharmaceutical industry. However, citrus fruits are vulnerable to pests and diseases, such as citrus greening disease and root rot. These diseases not only affect fruit quality and yield, but in severe cases can lead to large-scale production losses or even the death of fruit trees.

[0003] The kumquat tree is highly disease-resistant, resisting common citrus diseases such as Huanglongbing and root rot. This makes it relatively easy to manage during cultivation, reducing reliance on chemical pesticides. Although both citrus and kumquat are Rutaceae crops, they belong to different genera, making hybridization with citrus resources that exhibit superior resistance very difficult.

[0004] Distant hybridization refers to the mating between different individuals of taxonomic units above the species and genus in taxonomy. Distant hybridization is an important way to create new plant varieties. Through distant hybridization, the isolation between species and genera can be broken, and the excellent traits specific to species and genera can be aggregated and recombined under artificial hybridization conditions to form new varieties. Compared with normal hybridization, distant hybridization faces difficulties such as incompatibility before fertilization, chromosome pairing obstacles after fertilization, and fertilization and hybrid embryo abortion. Huangpi belongs to the Huangpi genus of the Rutaceae family. Hybridization with Huangpi as the male parent and different genera of the Rutaceae family is of great significance for the breeding of new varieties of Rutaceae plants.

[0005] During distant hybridization, some species in the Rutaceae family exhibit self-compatibility. The resulting progeny from distant hybridization are not necessarily true hybrids; they may be the result of self-pollination of the maternal parent, without incorporation of paternal genes. Therefore, identifying whether the progeny from distant hybridization are "true hybrids" is an essential technique for distant hybridization.

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

[0007] In order to solve the above technical problems, the main purpose of the present invention is to provide a primer for identifying the authenticity of the offspring of distant hybridization between wampee and citrus, so as to quickly, accurately and simply identify the authenticity of the offspring of distant hybridization between wampee and citrus.

[0008] In order to achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0009] In the first aspect, a primer for identifying the authenticity of distant hybridization progeny between wampee and citrus comprises a wampee-specific primer, wherein the wampee-specific primer is a primer combination comprising nucleotide sequence 1F and sequence 1R, or a primer combination comprising nucleotide sequence 2F and sequence 2R, or a primer combination comprising nucleotide sequence 3F and sequence 3R, and the nucleotide sequence is as shown in SEQ ID.1-6.

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

[0011] Furthermore, the detection reagent, kit or chip also includes other reagents for PCR amplification.

[0012] In a third aspect, the detection reagent, kit or chip of the present invention can be used in any of the following:

[0013] a) To identify the authenticity of the progeny of distant hybridization between Wampee and Citrus;

[0014] b) Identifying the presence of the paternal genome in the progeny of distant hybridization between Wampee and Citrus;

[0015] c) screening for true hybrids among the distant hybridization progenies of Wampee and Citrus;

[0016] d) Distant hybridization of wampee and tangerine;

[0017] e) preparing products for authenticating the authenticity of distant hybrid progeny of wampee and citrus;

[0018] f) preparing a product for identifying the presence of the paternal genome in the progeny of distant hybridization between wampee and citrus;

[0019] g) preparing a product for screening true hybrids among distant hybrid progenies of wampee and citrus;

[0020] h) preparing products of distant hybridization breeding between wampee and citrus.

[0021] In a fourth aspect, the present invention further provides a method for identifying the authenticity of distant hybridization progeny of wampee and citrus, comprising the following steps:

[0022] A1. Extract genomic DNA from the progeny of distant hybridization between wampee and citrus;

[0023] A2. Using the genomic DNA extracted in step A1 as a template, PCR amplification is performed using a primer combination, wherein the primer combination comprises a Wampee-specific primer, wherein the Wampee-specific primer is a primer combination comprising nucleotide sequence 1F and sequence 1R, or a primer combination comprising nucleotide sequence 2F and sequence 2R, or a primer combination comprising nucleotide sequence 3F and sequence 3R, and the nucleotide sequences of the primer combination are shown in SEQ ID. 1-6;

[0024] A3. Typing the PCR amplification product obtained in step A2 and performing band identification on the typing results: If a product of 270 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 1F and the sequence 1R, or a product of 296 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 2F and the sequence 2R, or a product of 235 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 3F and the sequence 3R, then the hybrid offspring is a true hybrid; if the primer combination comprising the nucleotide sequence 1F and the sequence 1R does not amplify a product of 270 bp, the primer combination comprising the nucleotide sequence 2F and the sequence 2R does not amplify a product of 296 bp, and the primer combination comprising the nucleotide sequence 3F and the sequence 3R does not amplify a product of 235 bp, then the hybrid offspring is not a true hybrid.

[0025] In a fifth aspect, the present invention also provides a method for identifying whether the paternal genome exists in the offspring of distant hybridization between wampee and citrus, comprising the following steps:

[0026] B1. Extracting genomic DNA from hybrid offspring obtained by hybridizing wampee as the male parent and citrus as the female parent;

[0027] B2. Using the genomic DNA extracted in step B1 as a template, PCR amplification is performed using a primer combination, wherein the primer combination comprises a Wampee-specific primer, wherein the Wampee-specific primer is a primer combination comprising nucleotide sequence 1F and sequence 1R, or a primer combination comprising nucleotide sequence 2F and sequence 2R, or a primer combination comprising nucleotide sequence 3F and sequence 3R, and the nucleotide sequences of the primer combination are shown in SEQ ID. 1-6;

[0028] B3. Typing the PCR amplification product obtained in step B2 and performing band identification on the typing results: If a product of 270 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 1F and sequence 1R, or a product of 296 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 2F and sequence 2R, or a product of 235 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 3F and sequence 3R, then the paternal genome is present in the hybrid offspring; if the primer combination comprising the nucleotide sequence 1F and sequence 1R does not amplify a product of 270 bp, the primer combination comprising the nucleotide sequence 2F and sequence 2R does not amplify a product of 296 bp, and the primer combination comprising the nucleotide sequence 3F and sequence 3R does not amplify a product of 235 bp, then the paternal genome is not present in the hybrid offspring.

[0029] Furthermore, the PCR amplification reaction system of 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 reaction conditions of the PCR amplification were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, final extension at 75°C for 5 min, for a total of 30 cycles, and finally storage at 4°C.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The primers and identification method provided by the present invention can be used to quickly and accurately identify the authenticity and purity of the offspring of distant hybridization between Wampee and Citrus. The molecular markers of the present invention can quickly and accurately determine the true hybrids in the offspring of distant hybridization between Wampee and Citrus, and have characteristic peaks of amplified fragments with good specificity, which greatly reduces the workload of hybrid screening; compared with traditional morphological identification methods, the error rate is lower. The method of using the molecular markers of the present invention to identify the authenticity of hybridization has the advantages of low cost, safe experimental operation, and time and labor saving. The present invention helps to quickly and accurately identify whether the target paternal genome exists in the offspring of distant hybridization between Wampee and Citrus, and is of great significance to the efficient development of distant hybridization breeding between Wampee and Citrus. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The disclosure of the present invention will become more easily understood with reference to the accompanying drawings. Those skilled in the art will readily appreciate that these drawings are merely used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

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

[0035] Figure 2 This is a molecular marker verification diagram for the hybrid offspring of wampee and citrus; m is a molecular weight marker, which is 500bp, 250bp, and 100bp from top to bottom respectively; Z1-Z10 are the distant hybrid offspring to be identified respectively; MB is citrus (maternal parent); FB is wampee (paternal parent); PCR amplification results of the line-specific primer pair 1 where sequence 1F / sequence 1R is located in the hybrid offspring; PCR amplification results of the line-specific primer pair 2 where sequence 2F / sequence 2R is located in the hybrid offspring; PCR amplification results of the line-specific primer pair 3 where sequence 3F / sequence 3R is located in the hybrid offspring. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of this application.

[0037] The separating agent and the method of use provided by the present invention are 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 authenticity identification of distant hybrid progeny of wampee and citrus

[0039] 1. Selection of material varieties

[0040] 72 yellow-peel varieties, 28 wide-peel mandarin varieties, 47 grapefruit varieties, 13 orange varieties, and 5 citron and lemon varieties were selected. The specific varieties are as follows:

[0041] The 72 varieties of kumquats are: 1. Late-ripening sweet kumquat, 2. Yingzui kumquat, 3. Huangpu kumquat, 4. Congcheng kumquat, 5. Xinjiao white sugar, 6. Qijian kumquat, 7. Fengshun No. 4, 8. Lutian single core, 9. Haizhu No. 7, 10. Panyu sweet kumquat, 11. Jiexi sweet kumquat, 12. Yakou No. 2, 13. Qugen sweet kumquat, 14. Luzhuang No. 1, 15. Shixing 032, 16. Luoding sweet kumquat, 17. Small fruit sweet kumquat, 18. Yang Shan Daye, 19. Longshan Seedless, 20. Xingning No. 4, 21. Large Chicken Heart, 22. Late Ripening Chicken Heart, 23. Golden Chicken Heart, 24. Fine Chicken Heart No. 2, 25. Yangshan Chicken Heart, 26. First-Class Chicken Heart, 27. Jieyang Chicken Heart, 28. Qishan Juedu, 29. Juedu No. 4, 30. Taxia Sweet Peel, 31. Taxia Sour Peel, 32. Small Round Head, 33. Long Round Yellow Peel, 34. Large Round Head, 35. Purple Peel, 36. Ox Heart, 37. Long fruit 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. Jixin No. 2, 46. Jixin No. 3, 47. Jixin 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 wide-skinned tangerines are: 1. Sugar tangerine, 2. Guizhou seedless tangerine, 3. Datian tangerine, 4. Early-maturing Clementine, 5. Huangyan local early tangerine, 6. Guinong local early tangerine, 7. Seedless Mangtangerine, 8. Mashui tangerine, 9. Early honey tangerine, 10. Xinnu tangerine, 11. Local tangerine, 12. Nanfeng mandarin orange, 13. Qianyang seedless tangerine, 14. Huanong local early tangerine, 15. Huangling temple tangerine, 16. Mangshan wild tangerine, 17. Daoxian wild tangerine, 18. Wenzhou mandarin orange, 19. Qiuhui, 20. Red tangerine, 21. Daoxian slippery tangerine, 22. Rihui, 23. Clementine, 24. Local Guang tangerine, 25. Murcott, 26. Jiangyong wild tangerine, 27. Unknown fire, 28. Four-season tangerine.

[0043] The 47 varieties of pomelo are: 1. Taiwan Dwarf Late Pomelo, 2. Xiangfeng Green Pomelo, 3. Yuhuan No. 1, 4. Yunnan Dongshi Early Pomelo, 5. Gongshui Red Pomelo, 6. Weisi Green Pomelo, 7. Quanzhou Pomelo, 8. June Early Pomelo, 9. Huaju Red Pomelo, 10. Japanese Red Pomelo, 11. Guanxi Red Pomelo, 12. Crisp and 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. Shatian Pomelo, 21. Dianjiang White Pomelo, 22. Hainan Crystal Pomelo, 23. Guanxi Honey Pomelo, 24. Annong No. 1, 25. Cili cutting board pomelo, 26. Glutinous rice pomelo, 27. Changsha Shatian pomelo, 28. Niutui pomelo, 29. Zhangjiajie local red-heart pomelo, 30. Baiyu frost pomelo, 31. Cili golden pomelo, 32. Early-maturing high-sugar fragrant pomelo No. 3, 33. Cili dwarf pomelo, 34. Three-and-a-half 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-fruited red-fleshed pomelo.

[0044] The 13 orange varieties are: 1. Red Dark Willow Navel Orange, 2. Early Golden Sweet Orange, 3. Peach Leaf Orange, 4. Delta Orange, 5. Bud Blood Orange, 6. Newhall Orange, 7. Hamlin 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 5 varieties of citron and lemon are: 1. Tibetan citron, 2. Yunnan citron, 3. Perfume lemon, 4. Fujian sweet lemon, 5. Ukulele lemon.

[0046] 2. DNA Extraction from Materials

[0047] The genomic DNA of the leaves of the test materials was extracted using the new plant genomic DNA rapid extraction kit.

[0048] 1. Take an appropriate amount of plant tissue (100 mg of fresh tissue or 20 mg of dry weight tissue, and take more sample to make up for the loss of sample sticking to the mortar) and add liquid nitrogen to the mortar and grind it into fine powder.

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

[0050] 3. Place the centrifuge tube in a 65°C water bath for 10 minutes. Invert the tube 2-3 times during the water bath to mix the sample.

[0051] 4. Add 130 μL of buffer AP2, vortex and mix for 1 min, place on ice for 5 min, centrifuge at 13,000 rpm for 5-10 min, and carefully pipette the supernatant into a new 1.5 mL centrifuge tube, taking care not to aspirate the interface material.

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

[0053] 6. Add the mixture from the previous step (including any precipitate) to an adsorption column AC. Place the column in a collection tube and centrifuge at 13,000 rpm for 30-60 seconds. Discard the waste liquid from the collection tube. First, add 650 μL of the solution and centrifuge, discard the waste liquid, then add the remaining solution and centrifuge again.

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

[0055] 8. Repeat step 7.

[0056] 9. Place the adsorption column AC back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove as much rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions.

[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 13,000 rpm for 1 minute. Add the resulting solution back to the adsorption column, incubate at room temperature for 2 minutes, and centrifuge at 13,000 rpm for 1 minute to obtain the genomic DNA of the feed.

[0058] 11. The genomic DNA of the material can be stored at 2-8℃. If it needs to be stored for a long time, it can be placed at -20℃.

[0059] 3. PCR Amplification

[0060] Using the genomic DNA extracted in step 2 as a template, PCR amplification was performed using a pair of primers specific for Wampee to obtain a PCR product. The primer sequences of the Wampee-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 was 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 prepared from Yisheng Biotechnology (Shanghai) Co., Ltd. PCRMaster Mix.

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

[0065] 4. Detection of PCR products by agarose gel electrophoresis

[0066] After the PCR reaction was completed, 8 μL of PCR product was taken and electrophoresed on 1.0% agarose gel at 150 V for 20 min, and then observed and photographed in an automatic gel imaging system.

[0067] Example 2

[0068] The same material variety selection and material DNA extraction method as in Example 1 were used, and the PCR amplification method was basically the same as in Example 1, except that the primer sequences of the yellow peel-specific primer pair were 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 of detecting PCR products by agarose gel electrophoresis is the same as that in Example 1.

[0072] Example 3

[0073] The same material variety selection and material DNA extraction method as in Example 1 were used, and the PCR amplification method was basically the same as in Example 1, except that the primer sequences of the yellow peel-specific primer pair were 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 of detecting PCR products by agarose gel electrophoresis is the same as that in Example 1.

[0077] The PCR product detection results of Example 1-3 are as follows Figure 1 As shown in the results, the specific molecular markers for yellow peel can amplify the target bands in all 72 yellow peel varieties, such as Figure 1 A. Figure 1 F. Figure 1 Specifically, a product of 270 bp was amplified using a primer combination containing nucleotide sequence 1F and sequence 1R, a product of 296 bp was amplified using a primer combination containing nucleotide sequence 2F and sequence 2R, and a product of 235 bp was amplified using a primer combination containing nucleotide sequence 3F and sequence 3R. However, no target bands were amplified in the remaining 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 authenticity of distant hybrid progeny between Wampee and Citrus using a primer combination comprising nucleotide sequence 1F and sequence 1R

[0079] 1. Selection of material varieties

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

[0081] 2. Authenticity Identification of Hybrid Progeny

[0082] The genomic DNA of the parents and hybrid offspring Z1-Z10 in this example was extracted, and the yellow peel-specific primers, DNA extraction method, PCR amplification and amplification product detection methods used were the same as those in Example 1. The authenticity of the hybrid offspring of the variety in Example 4 was identified.

[0083] Example 5 Identification of authenticity of distant hybrid progeny between Wampee and Citrus using a primer combination containing nucleotide sequence 2F and sequence 2R

[0084] 1. Selection of material varieties

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

[0086] 2. Authenticity Identification of Hybrid Progeny

[0087] The genomic DNA of the parents and hybrid offspring Z1-Z10 in this example was extracted, and the yellow peel-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 variety in Example 5 was identified.

[0088] Example 6 Identification of authenticity of distant hybrid progeny between Wampee and Citrus using a primer combination containing nucleotide sequence 3F and sequence 3R

[0089] 1. Selection of material varieties

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

[0091] 2. Authenticity Identification of Hybrid Progeny

[0092] The genomic DNA of the parents and hybrid offspring Z1-Z10 in this example was extracted, and the yellow peel-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 variety in Example 6 was identified.

[0093] Identification results such as Figure 2 As shown, the results showed that the primer combination containing the nucleotide sequence 1F and sequence 1R, the primer combination containing the nucleotide sequence 2F and sequence 2R, and the primer combination containing the nucleotide sequence 3F and sequence 3R could respectively amplify bands of 270bp, 296bp and 235bp in Z3 of the hybrid offspring Z1-Z10, indicating that its genome contained the specific sequence of the father (yellow peel) and the yellow peel genome information, confirming that it was a true distant hybridization offspring, while the other 9 materials did not amplify bands of 270bp, 296bp and 235bp, and were not distant hybridization offspring.

[0094] In summary, the method of the present invention for authenticity identification of distant hybridization progeny of wampee and citrus using molecular markers has the following advantages: the wampee-specific primers used in the present invention can save time and space while identifying the authenticity of the hybridization progeny, with high accuracy, and can quickly and accurately identify the strains with paternal genetic information in the hybridization progeny of wampee and citrus, thereby determining the true distant hybridization progeny.

[0095] The above is a further detailed description of the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. Those skilled in the art of the art to which the present invention belongs may make several substitutions or modifications to the described embodiments without departing from the scope of the present invention, and such substitutions or modifications should be considered to fall within the scope of protection of the present invention.

Claims

1. A primer for identifying the authenticity of distant hybridization progeny of wampee and citrus, characterized in that: It comprises a Wampee-specific primer, which is a primer combination comprising a nucleotide sequence 1F and a sequence 1R, or a primer combination comprising a nucleotide sequence 2F and a sequence 2R, or a primer combination comprising a nucleotide sequence 3F and a sequence 3R, and the nucleotide sequence is shown in SEQ ID.1-6.

2. A detection reagent, kit or chip containing the primer according to claim 1.

3. Use of the detection reagent, kit or chip according to claim 2 in any of the following: a) To identify the authenticity of the progeny of distant hybridization between Wampee and Citrus; b) Identifying the presence of the paternal genome in the progeny of distant hybridization between Wampee and Citrus; c) screening for true hybrids among the distant hybridization progenies of Wampee and Citrus; d) Distant hybridization of wampee and tangerine; e) preparing products for authenticating the authenticity of distant hybrid progeny of wampee and citrus; f) preparing a product for identifying the presence of the paternal genome in the progeny of distant hybridization between wampee and citrus; g) preparing a product for screening true hybrids among distant hybrid progenies of wampee and citrus; h) preparing products of distant hybridization breeding between wampee and citrus.

4. A method for identifying the authenticity of distant hybridization progeny of wampee and citrus, characterized in that: The following steps are involved: A1. Extract genomic DNA from the progeny of distant hybridization between wampee and citrus; A2. Using the genomic DNA extracted in step A1 as a template, PCR amplification is performed using a primer combination, wherein the primer combination comprises a Wampee-specific primer, wherein the Wampee-specific primer is a primer combination comprising nucleotide sequence 1F and sequence 1R, or a primer combination comprising nucleotide sequence 2F and sequence 2R, or a primer combination comprising nucleotide sequence 3F and sequence 3R, and the nucleotide sequences of the primer combination are shown in SEQ ID. 1-6; A3. Typing the PCR amplification product obtained in step A2 and performing band identification on the typing results: If a product of 270 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 1F and the sequence 1R, or a product of 296 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 2F and the sequence 2R, or a product of 235 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 3F and the sequence 3R, then the hybrid offspring is a true hybrid; if the primer combination comprising the nucleotide sequence 1F and the sequence 1R does not amplify a product of 270 bp, the primer combination comprising the nucleotide sequence 2F and the sequence 2R does not amplify a product of 296 bp, and the primer combination comprising the nucleotide sequence 3F and the sequence 3R does not amplify a product of 235 bp, then the hybrid offspring is not a true hybrid.

5. A method for identifying the presence of the paternal genome in the progeny of distant hybridization between wampee and citrus, characterized in that: The following steps are involved: B1. Extracting genomic DNA from hybrid offspring obtained by hybridizing wampee as the male parent and citrus as the female parent; B2. Using the genomic DNA extracted in step B1 as a template, PCR amplification is performed using a primer combination, wherein the primer combination comprises a Wampee-specific primer, wherein the Wampee-specific primer is a primer combination comprising nucleotide sequence 1F and sequence 1R, or a primer combination comprising nucleotide sequence 2F and sequence 2R, or a primer combination comprising nucleotide sequence 3F and sequence 3R, and the nucleotide sequences of the primer combination are shown in SEQ ID. 1-6; B3. Typing the PCR amplification product obtained in step B2 and performing band identification on the typing results: If a product of 270 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 1F and sequence 1R, or a product of 296 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 2F and sequence 2R, or a product of 235 bp is obtained by amplification using the primer combination comprising the nucleotide sequence 3F and sequence 3R, then the paternal genome is present in the hybrid offspring; if the primer combination comprising the nucleotide sequence 1F and sequence 1R does not amplify a product of 270 bp, the primer combination comprising the nucleotide sequence 2F and sequence 2R does not amplify a product of 296 bp, and the primer combination comprising the nucleotide sequence 3F and sequence 3R does not amplify a product of 235 bp, then the paternal genome is not present in the hybrid offspring.

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

7. The method according to claim 4 or 5, characterized in that The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, and final extension at 75°C for 5 min, for a total of 30 cycles, and finally storage at 4°C.

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