Characteristic sequence, marker primer and identification method of fig variety Hongyan

Abstract

The invention relates to a characteristic sequence and a molecular specific marker primer of a fig variety Hongyan, and a method capable of rapidly identifying the fig variety Hongyan. The molecular specific marker primer has the following sequences: (1) a 16793104 primer group: 16793104 LF: ATTTTTAGAACTTTCACCAGCCG16793104 RR: GTTGACCACCACAAATGTCTG16793104 SF: GCCCTAAAATTTCAAATTCTGATTTGAG4526148RR: GTTAATACTCTCGCAATGCAG4526148SF: TCAATGAACCGGTCACATAACACC, and the molecular specific marker primer disclosed by the invention can be used for carrying out rapid early identification on the fig variety Hongyan, and the method is simple, rapid and convenient.

Description

Technical Field

[0001] The invention relates to a characteristic sequence of a fig variety Hongyan, a molecular specific marker primer combination, and a method for specifically identifying the fig variety Hongyan by utilizing the molecular specific marker primer combination. Background Art

[0002] Fig Ficus carica Ficus genus, Moraceae Ficus A deciduous shrub or small tree native to the Mediterranean coast, it was first cultivated throughout Xinjiang, my country, and is now being introduced and cultivated in many locations throughout my country. It boasts high yields and rapid returns. Currently, over 20 varieties have performed well in my country, including 'Bojihong,' 'Blanrick,' and 'Masyi Taufen.' The fruit of the fig contains a variety of active ingredients, including polysaccharides, flavonoids, and psoralens, with benefits such as treating sore throats, fighting tumors, lowering blood sugar and lipids, and boosting immunity. Consequently, market demand for figs is growing. While the fruit shape, size, color, and flavor of different fig cultivars vary significantly, the differences between individual plants are minimal. Furthermore, the naming of hybrids between some fig cultivars can be confusing, hindering cultivar identification, promotion, communication, and the development of new varieties. Therefore, efforts are underway to develop stable and specific DNA markers at the molecular level, offering a scientific approach to accurately and rapidly identifying fig varieties. Summary of the Invention

[0003] The purpose of the present invention is to provide a characteristic sequence of the fig variety Hongyan, a molecular specific marker primer combination, and a method for specifically identifying the fig variety Hongyan using the molecular specific marker primer combination; The technical solution adopted in the present invention is: The characteristic sequence group of the fig variety Hongyan is as follows: 1) 16793_104: 5'-CCGACCTTCAATTAGTGCCAAATCCCCGATTTGCCCTCATCTCCCACCCGCCTAAAAGTCACTATCCCTCGAATGCCCTAAATACCCTAAAAAAATTTCAAACTATCTTCTACAGTGTTTAG AGANNNNNNNNNNACCGAATTTTTAGAACTTTCACCAGCCGATGACCTTTTGGATGACTTAGCCGTAACTCGGACTGACCTCTTCTCGGGACGAGTGTTTTTCTCAGACATTTTAGTGGGTGTCAACATGGGTAGTTTT -3' 2) 45261_48: 5'-ACCTTTCGACAGGATACATCCACCTCAAATGAACCGGTCCACATAACCTAACTTCCCGAACTAAGTGCACCGTTAGATGGATCATTATATCAAAAAAGGCTGGTAGGAAATACTTCTCAAGCATGCNNN NNNNNNNACTCTTCCAACTTCAAAAGTTTGGTGTCGATGAAATATTTGAGGCTCGTTGGAATGAAAACACATGATTGTCTTATTTTGATGCAACAATTATTACCTCTTGCATTGCGGAGAGTATTAACGAAAG -3' Where NNNNNNNNNN is an unknown sequence within 300bp.

[0004] The present invention also relates to a molecular specific marker primer set for the fig variety Hongyan, wherein the primer sequence is: 1) Primer set 16793_104: 16793_104LFATTTTTAGAACTTTCACCAGCCG 16793_104RRGTTGACACCCACTAAAATGTCTG 16793_104SF GCCCTAAATACCCTAAAAAAATTTCAAACT 2) 45261_48 primer set: 45261_48LF GTCGATGAAATATTTGAGGGCTCG 45261_48RR GTTAATACTCTCCGCAATGCAAG 45261_48SF TCAAATGAACCGGTCCACATAACC The two primer sets described above were derived from molecular marker development using SLAF-seq (Specific-Locus Amplified Fragment Sequencing) technology on 23 accessions, generating genome-wide molecular markers. An average of 118,891 SLAF tags were developed per sample in this project, with an average sequencing depth of 10.12x. A total of 72.00 Mb reads were generated. Bioinformatics analysis identified 166,254 SLAF tags, including 6,575 polymorphic SLAF tags, for a total of 72,678 population SNPs. Based on these results, over 1,000 primer pairs were designed and screened and validated in 23 samples. DNA fragments specific to the fig variety Hongyan were identified; no specific fragments were obtained for the selected primer combinations in other fig varieties. It should be noted that the molecular marker primer combinations described in this invention are limited to fig variety identification, meaning that the samples tested are limited to figs.

[0005] The results of a random sampling of 5 plants of this variety showed that the characteristics of the Hongyan variety fig are shown in the table: The present invention also relates to a method for rapidly identifying the fig variety Hongyan by using the molecular specific marker primer combination. The method comprises the following steps: extracting genomic DNA from leaves of the fig variety to be tested as a template, using the molecular specific marker primer set as amplification primers, and performing PCR amplification. The primers correspond to the binding sites on the characteristic sequences, such as Figure 1 , Figure 2 As shown; The key to the method of the present invention lies in the selection of amplification primer combinations, while DNA extraction, PCR reaction system and reaction conditions, and electrophoresis detection can all be performed according to conventional methods in the art. Compared with existing molecular marker methods for fig varieties, such as SSR markers, the method of the present invention has the following advantages: (1) Because the primers used have been sequenced and repeatedly verified, their reliability has been greatly improved; (2) The detection is convenient and intuitive. The presence or absence of band combinations can be directly determined by ordinary electrophoresis. However, if the SSR marker method is used, further analysis or sequencing by high-resolution electrophoresis is required after amplification. (3) The sample requirements are relatively low, and DNA samples from tissues such as leaves can meet the needs of variety identification; Preferably, the PCR amplification system of the present invention is composed as follows: The final concentration of PCR Buffer is 1× dNTPs 1 mmol / L MgCl2 2.5 mmol / L Taq enzyme 1.0 U / reaction 0.2 μM each of upstream and downstream primers Template DNA 60 ng / reaction The balance is ddH2O; The PCR amplification conditions were as follows: pre-denaturation at 94°C for 300 s, denaturation at 95°C for 10 s, annealing at 56°C for 50 s, extension at 72°C for 40 s, for a total of 30 cycles, and a final fill-in at 72°C for 300 s; the termination temperature was 4°C; The final concentration of PCR Buffer is 1×, which means that the concentrations of the components in the reaction system are the same as those of 1× PCR Buffer. 10× PCR Buffer is usually used, with a volume of 1 / 10 of the reaction system volume. 10× PCR Buffer consists of: 100 mM Tris-HCl (pH 8.5), 500 mM KCl, 25 mM MgCl2, and 1.0% Triton-X-100, with ddH2O as the solvent. Specifically, the method is as follows: (1) Take the fig leaves to be tested, grind them with liquid nitrogen, and extract the genomic DNA of the fig leaves to be tested using the CTAB method; (2) Using the genomic DNA extracted in step (1) as a template and the molecular specific labeled primers as amplification primers, PCR amplification is performed: The PCR reaction system per 15 μL is composed as follows: 2×TsingKE master mix 7.5μL 0.2 μL each of 10 μM upstream and downstream primers 20 ng / μL template DNA 2 μL dd H2O 5.1 μL; PCR reaction conditions are as follows: After pre-denaturation at 94°C for 300 s, the reaction was cycled for 30 cycles: denaturation at 95°C for 10 s, annealing at 56°C for 50 s, and extension at 72°C for 40 s. The final cycle was completed at 72°C for 300 s. The termination temperature was 4°C. (3) Take 3 μL of the amplified product from step (2), mix it with 1 μL of 0.25% bromophenol blue buffer, spot it on a 1.5% agarose gel, and electrophorese it in 1×TAE buffer at 5 V / cm. After the electrophoresis, stain it with EB and photograph it on an automatic gel image analyzer. If the electrophoresis results show that the DNA sample can be PCR amplified into two bands in the 16793_104 primer set and two bands in the 45261_48 primer set, then the fig variety to be tested from which the DNA sample comes is Hongyan; if it does not have this characteristic, then it is not. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 Schematic diagram of the relative positions of the primers in the 16793_104 primer set within the fig DNA signature sequence fragment 16793_104; Figure 2 Schematic diagram of the relative positions of the primers in the 45261_48 primer set within the fig DNA signature sequence fragment 45261_48; Figure 3 PCR amplification results of DNA from 23 fig varieties using the 16793_104 primer set (variety numbers 1-23 represent: 1. Brooke Red; 2. Grace; 3. Brunswick; 4. Early Yellow; 5. Mas-yi-Taufin; 6. Strawberry; 7. B1011; 8. Huishankou; 9. Jinaofen; 10. Qinghua; 11. Hongyan; 12. Papa John's; 13. BNR; 14. Hardy; 15. BBR; 16. Baishan; 17. Sala; 18. Jiashan; 19. Lukang 1; 20. Dela; 21. Baiya; 22. California Black; 23. Baraunay). Takara DL2000 marker was used as the marker. Two specific DNA bands were amplified from the fig variety numbered 11, Hongyan, using the 16793_104 primer set. Figure 4 PCR amplification results of DNA from 23 fig varieties using the 45261_48 primer set (variety numbers 1-23 represent: 1. Brooke Red; 2. Grace; 3. Brunswick; 4. Early Yellow; 5. Mas-Yitaufen; 6. Strawberry; 7. B1011; 8. Huishankou; 9. Jinaofen; 10. Qinghua; 11. Hongyan; 12. Papa John's; 13. BNR; 14. Hardy; 15. BBR; 16. Baishan; 17. Sala; 18. Rockery; 19. Green Anti-1; 20. Dela; 21. Baiya; 22. California Black; 23. Baraunay). Takara DL2000 marker was used as the marker. Two specific DNA bands were amplified from the fig variety 11, Hongyan, using the 45261_48 primer set. DETAILED DESCRIPTION

[0007] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto: Example

[0008] (1) Extraction of genomic DNA from fig varieties: 0.05 g of young leaves of the tested fig variety were taken and ground thoroughly with liquid nitrogen. Genomic DNA from the leaves was extracted using the CTAB method. After multiple extractions, a genomic DNA extract of the fig variety was obtained. The DNA extract was then subjected to 1.5% agarose gel electrophoresis to test integrity, purity, and concentration. Band brightness was determined for subsequent PCR amplification. The DNA extract was stored at -20°C until use. (2) Design specific PCR amplification primers. The sequences of the primer pairs are: 1) Primer set 16793_104: 16793_104LFATTTTTAGAACTTTCACCAGCCG 16793_104RRGTTGACACCCACTAAAATGTCTG 16793_104SF GCCCTAAATACCCTAAAAAAATTTCAAACT 2) 45261_48 primer set: 45261_48LF GTCGATGAAATATTTGAGGGCTCG 45261_48RR GTTAATACTCTCCGCAATGCAAG 45261_48SF TCAAATGAACCGGTCCACATAACC Synthesized by Shanghai Bioengineering Technology Co., Ltd.; (3) PCR amplification: PCR reaction solution composition (15 μL): 2×TsingKE master mix (Qingke Biotechnology, Beijing) 7.5μL 0.2 μL each of 10 μM upstream and downstream primers 20 ng / μL template DNA 2 μL dd H2O 5.1 μL; The amplification reaction was performed on a TC-XP thermal amplification instrument. The amplification conditions were as follows: 94°C pre-denaturation for 300 s, 95°C denaturation for 10 s, 56°C annealing for 50 s, and 72°C extension for 40 s, for a total of 30 cycles, followed by a final fill-in at 72°C for 300 s. The termination temperature was 4°C. (4) Electrophoresis detection: Take 3 μL of the PCR amplification product from step (3), mix it with 1 μL of 0.25% bromophenol blue buffer, apply it to a 1.5% agarose gel, and run electrophoresis in 1×TAE buffer at 5 V / cm. After the electrophoresis, stain it in an aqueous solution containing 0.5 μg / ml EB for 30 minutes, and then photograph it on the Bio-rad gel imaging system Gel Doc; According to the above method, 24 fig varieties (numbered 1-23, representing fig varieties, are as follows: 1, Brooke Red; 2, Grace; 3, Brunswick; 4, Early Yellow; 5, Masyi Dauphin; 6, Strawberry; 7, B1011; 8, Huishankou; 9, Jinaofen; 10, Qinghua; 11, Hongyan; 12, Papa John's; 13, BNR; 14, Hardy; 15, BBR; 16, Baishan; 17, Sala; 18, Rockery; 19, Green Anti-1; 20, Dela; 21, Baiya; 22, California Black; 23, Balaonai) were detected by electrophoresis. The marker used was Takara DL2000. The results of electrophoresis are shown in the table. Figure 3 , Figure 4 ; Among them, only the fig variety numbered 11, Hongyan, amplified two specific DNA bands using the primer set 16793_104 ( Figure 3 ), and two specific DNA bands were also amplified in the 45261_48 primer set ( Figure 4 The number of bands for the remaining numbered fig varieties was not identical. This indicates that the molecular-specific marker primer pair developed by the present invention is highly stable and specific for early identification of the fig variety Hongyan.

Claims

1. The characteristic sequence of the fig variety Hongyan, which is composed of two coexisting specific DNA fragments, has the following sequence: 1) 16793_104: 5'-CCGACCTTCAATTAGTGCCAAATCCCCGATTTGCCCTCATCTCCCACCCGCCTAAAAGTCACTATCCCTCGAATGCCCTAAATACCCTAAAAAAATTTCAAACCATCTTCTACAGTGTTTTAG AGANNNNNNNNNNACCGAATTTTTAGAACTTTCACCAGCCGATGACCTTTTGGATGACTTAGCCGTAACTCGGACTGACCTCTTCTCGGGACGAGTGTTTTTCTCAGACATTTTAGTGGGTGTCAACATGGGTAGTTTT -3' 2）45261_48： 5'-ACCTTTCGACAGGATACATCCACCTCAAATGAACCGGTCCACATAACTTAACTTCCCGAACTAAGTGCACCGTTAGATGGATCATTATATCAAAAAAGGCTGGTAGGAAATACTTCTCAAGCATGCNNN NNNNNNNACTCTTCCAACTTCAAAAGTTTGGTGTCGATGAAATATTTGAGGCTCGTTGGAATGAAAACACATGATTGTCTTATTTTGATGCAACAATTATTACCTCTTGCATTGCGGAGAGTATTAACGAAAG -3' Where NNNNNNNNNN is an unknown sequence within 300bp.

2. A primer set for detecting the characteristic sequence of the fig variety Hongyan according to claim 1, wherein the primer set is composed of the following primer sequences: 1) Primer set 16793_104: 16793_104LF:ATTTTTAGAACTTTCACCAGCCG 16793_104RR:GTTGACACCCACTAAAATGTCTG 16793_104SF:GCCCTAAATACCCTAAAAAAATTTCAAACT 2) 45261_48 primer set: 45261_48LF:GTCGATGAATATTTGAGGGCTCG 45261_48RR:GTTAATACTCTCCGCAATGCAAG 45261_48SF:TCAAATGAACCGGTCCACACATAACC.

3. A method for rapidly identifying the fig variety Hongyan using the primer set of claim 2, the method comprising: extracting genomic DNA from leaves of the fig variety Hongyan to be tested as a template, performing PCR amplification using the molecular-specific marker primers in groups, and performing electrophoresis detection on the amplified products. If the electrophoresis results show two specific DNA bands amplified by the 16793_104 primer set and two specific DNA bands also appear in the 45261_48 primer set, then the fig variety to be tested is the fig variety Hongyan; otherwise, it is not. The two primer sets are composed of the following complete primer sequences: 1) Primer set 16793_104: 16793_104LF:ATTTTTAGAACTTTCACCAGCCG 16793_104RR:GTTGACACCCACTAAAATGTCTG 16793_104SF:GCCCTAAATACCCTAAAAAAATTTCAAACT 2) 45261_48 primer set: 45261_48LF:GTCGATGAATATTTGAGGGCTCG 45261_48RR:GTTAATACTCTCCGCAATGCAAG 45261_48SF:TCAAATGAACCGGTCCACACATAACC.

4. The method according to claim 3, wherein The PCR amplification conditions were as follows: pre-denaturation at 94°C for 300 s; denaturation at 95°C for 30 s, annealing at 56°C for 60 s, and extension at 72°C for 50 s, for a total of 30-40 cycles, and a final fill-in at 72°C for 300 s; the termination temperature was 4°C.

5. The method according to claim 3, wherein The method is as follows: 1) Take the fig leaves to be tested, grind them in liquid nitrogen, and extract the genomic DNA of the fig leaves using the SDS-CTAB method; 2) performing PCR amplification using the genomic DNA extracted in step 1) as a template and the primers in the molecular-specific marker primer set as amplification primers; 3) 3 μL of the amplified product from step 2) was mixed with 1 μL of 0.25% bromophenol blue buffer and spotted on a 1.5% agarose gel. The gel was electrophoresed in 1×TAE buffer at 5 V / cm. After electrophoresis, the gel was stained with EB and photographed on an automated gel image analyzer. If the electrophoresis results showed the characteristic band described in claim 3, the fig variety under test was considered to be Hongyan; otherwise, it was considered to be a negative variety.

Images