SV marker combination for identifying Hongmeiren hybrid oranges and application of SV marker combination

Abstract

The invention belongs to the technical field of plant breeding, and discloses an SV molecular marker combination for identifying Hongmeiren hybrid citrus, which is characterized by comprising the following two structural variation sites: (1) Chr1: 29803181 site, the sequence of which is as shown in SEQ ID NO: 1 and SEQ ID NO: 2; and (2) a Chr2: 1013046 site, wherein the sequence of the Chr2: 1013046 site is as shown in SEQ ID NO: 3 and SEQ ID NO: 4. The invention further discloses a primer pair for detecting the SV molecular marker combination, a kit comprising the primer pair and application of the kit. The invention provides the SV molecular marker combination for identifying the variety of the citrus reticulata Blanco, the authenticity of the citrus reticulata Blanco can be rapidly identified in the seedling stage, the market counterfeit behavior is restrained, molecular evidence is provided for intellectual property protection, and the application of the molecular marker can assist in filial generation screening and shorten the breeding period.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant breeding, and particularly relates to a SV molecular marker for identifying red beauty hybrid mandarin, a primer pair, a kit and application thereof. BACKGROUND

[0002] Red beauty hybrid mandarin (Citrus. unshiu x C. clementina) x ((C. sinensis x C. unshiu) x C. unshiu) x C. reticulata) (Citrus. unshiu x C. clementina) x ((C. sinensis x C. unshiu) x C. unshiu) x C. reticulata) Red beauty hybrid mandarin is a Citrus genus plant, also known as 'Yamaguchi 2', which is a fine single plant of a hybrid offspring of 'Nanxiang' (female parent) and 'Tianguo' (male parent). Red beauty hybrid mandarin has stable horticultural properties, a jelly-like pulp taste, a lot of juice, a delicious taste, a rich flavor and excellent quality, and is a highly competitive category in the citrus market.

[0003] In China, red beauty hybrid mandarin is mainly planted and popularized on a large scale in Zhejiang, Chongqing, Hubei and other places, and is also a backbone parent in citrus hybrid breeding. However, with the expansion of the consumer market, there is a risk of economic loss of the planters due to the mixing of fake and inferior seedlings, resulting in a reduction in yield. In the protection of seed industry intellectual property rights, the development of variety-specific molecular markers can quickly identify the seedlings through leaf DNA at the seedling stage, which is a core technical means to protect the safety of seedlings, standardize the market and prevent fake and inferior germplasm from entering the market, and is also a legal basis and urgent need for the protection and rights maintenance of high-value varieties.

[0004] At present, the identification of existing citrus varieties mainly depends on morphological characteristics and simple sequence repeat (SSR) markers, but red beauty hybrid mandarin is a ternary hybrid offspring of tangerines, pummelos and oranges, and the genome is highly hybridized, and similar properties are prone to occur in related varieties, resulting in a false positive risk in identification. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background, and to provide a SV molecular marker for identifying red beauty hybrid mandarin, a primer pair, a kit and application thereof.

[0006] To solve the above technical problems, the technical solution provided by the present application is as follows: A SV molecular marker combination for identifying red beauty hybrid mandarin comprises the following two structural variation sites: (1) Chr1:29803181 site, the sequence of which is shown in SEQ ID NO:1 and SEQ ID NO:2; (2) Chr2:1013046 site, the sequence of which is shown in SEQ ID NO:3 and SEQ ID NO:4; The Chr1:29803181 site is located at position 29803181 of chromosome 1 of the sweet orange reference genome SWO.v.3.0, and the Chr2:1013046 site is located at position 1013046 of chromosome 2.

[0007] The SV molecular marker combination is used for identifying red beautiful person Citrus sinensis. The 350 bp and 250 bp bands are amplified at the Chr1:29803181 site, and only the 250 bp band is amplified at the Chr2:1013046 site.

[0008] Based on a general inventive concept, the present application further provides a primer pair for detecting the SV molecular marker combination, comprising: (a) The primer pair sequence for the Chr1:29803181 site is: Forward primer: 5'-AGTGGGATCGAATGTCACTCT-3' (SEQ ID NO: 5), Reverse primer: 5'-ACAGAATTCACCAACGAGCTG-3' (SEQ ID NO: 6); (b) The primer pair sequence for the Chr2:1013046 site is: Forward primer: 5'-AACAGCCAGCACAGACCT-3' (SEQ ID NO: 7), Reverse primer: 5'-TCACACCTGACGCCTCCT-3' (SEQ ID NO: 8).

[0009] Based on a general inventive concept, the present application further provides a kit for identifying red beautiful person Citrus sinensis, comprising the primer pair.

[0010] Based on a general inventive concept, the present application further provides a method for identifying red beautiful person Citrus sinensis, comprising the following steps: (1) Extracting genomic DNA of a citrus sample to be tested; (2) Performing PCR amplification on the genomic DNA using the primer pair; (3) Performing agarose gel electrophoresis detection on the PCR amplification product: when the sample simultaneously appears 350 bp and 250 bp bands at the Chr1 site, and only the 250 bp band appears at the Chr2 site, it is determined as red beautiful person Citrus sinensis.

[0011] The method, preferably, in step (2), the PCR amplification program is: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 57 ℃ annealing for 40 s, 72 ℃ extension for 40 s, cycle 35 times; 72 ℃ extension for 10 min.

[0012] The method, preferably, the agarose gel electrophoresis condition is: 2% agarose gel, 110 V voltage, electrophoresis for 1 hour.

[0013] Based on one overall inventive concept, the application also provides application of the SV molecular marker combination, the primer pair or the kit in true identification of red beauty mandarin variety, screening of counterfeit varieties in citrus seedling market or screening of hybrid breeding offspring.

[0014] Compared with the prior art, the application has the following beneficial effects: (1) The application provides a SV molecular marker combination for identifying beauty mandarin varieties, which can quickly identify the authenticity of red beauty mandarin at the seedling stage, curb market counterfeiting behavior, provide molecular evidence for intellectual property protection, and the application of the molecular marker can assist in screening of hybrid offspring, thereby shortening the breeding cycle.

[0015] (2) The application is based on analysis of citrus multi-variety resequencing data, which can significantly improve the mining of hybrid variety identification sites; combined with electronic PCR pre-screening, the number of experimental verification is reduced, and the cost of primer development is significantly reduced; in addition, the application uses agarose gel electrophoresis detection, the detection method is simple, the cost is low, no expensive equipment is needed, and the detection is convenient and can be popularized. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, a brief introduction will be given below to the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0017] Figure 1 is a gel electrophoresis map after specific primer amplification in the embodiments of the application: A. amplification result of the primer designed according to the chr1:29803181 site; B. amplification result of the primer designed according to the chr2:1013046 site; M represents DNA Marker, and 1-16 lanes respectively represent different citrus varieties, and 12 lane is red beauty mandarin.

[0018] Figure 2 is two differential bands amplified by the 1-181 primer in samples (red beauty mandarin, tiancao and nankang). The upper red bar corresponds to Figure 1 the lower gel band in A, and the white rectangle represents SV; the lower black bar represents Figure 1 the upper gel band in A.

[0019] Figure 3 is two differential bands amplified by the 2-046 primer in samples (red beauty mandarin, tiancao and nankang). The upper red bar corresponds to Figure 1The lower gel band in B, the white rectangle represents SV; the black bar below represents Figure 1 The upper gel band in B.

[0020] Figure 4 is the sequence amplified by 1-181 primers at 250 bp.

[0021] Figure 5 is the sequence amplified by 1-181 primers at 350 bp.

[0022] Figure 6 is the sequence amplified by 2-046 primers at 250 bp.

[0023] Figure 7 is the sequence amplified by 2-047 primers at 350 bp.

[0024] Figure 8 is the gel electrophoresis diagram based on specific primer amplification in the verification process of the application. DETAILED DESCRIPTION

[0025] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present application is not limited to the following specific embodiments.

[0026] Unless otherwise defined, all the professional terms used in the following have the same meaning as generally understood by those skilled in the art. The professional terms used in this paper are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present application.

[0027] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0028] Example 1 This embodiment is the development and application of the SV molecular marker combination, and the specific process is as follows: (1) Taking sweet orange genome (SWO.v.3.0) as reference, 21 pieces of citrus genome data released by NCBI were collected, and the resequencing data was mapped and aligned to the reference genome by using BWA software to generate BAM file; then the software tool SVIM-asm was used for detection and identification of structural variation sites (SV, insertion / deletion > 50 bp), and the parameters were set as minimum SV length 50 bp, confidence threshold ≥ 90%, and SV sites with polymorphism in hybrid citrus were screened; (2) Using IGV to load the genome and the aligned BAM file, manually verifying the SV site polymorphism, retaining only the SV sites that are specific to Hongmeiren and different from other Citrus varieties, and eliminating false positive sites; the following two SV sites were retained after manual verification, and the nucleotide sequences thereof are shown as SEQ ID NO: 1-4, and Figures 4-7 The yellow-marked in the figure is the different SV sequence, and the red-marked base is the SNP of the two sequences.

[0029] SEQ ID NO: 1: (276 bp) AGTGGGATCGAATGTCACTCTTACTGATGATTCCAATAGGATAGAGGTATCTATATCCTTTAAATTTCATTTTTTTTTTCTCCTTTATTGGGGACCTGTTTCATTAAAATTTAAATGTTTAAGTTTTTTCATTGTATTTATTTGTGTTTTTTTTTTAATCAAGTAAAAGCACCTAAAAGCAGTTATCTTAAAAAAAAAGTAGATATTAATTAAAGTGCTTTTACCATTTTGAAAGCGCAAGTTATAATTGCTTTCAGCTCGTTGGTGAATTCTGT SEQ ID NO: 2: (362 bp) AGTGGGATCGAATGTCACTCTTACTGATGATTCCAATAGGATAGAGGTATCTATAGCTTTTAAATTTCATTTATTTTTTCTCCTTTATTGGGGACCTGTTTCATTAAAATTTAAATGTTTAAGTTTGTTCATTGTATTTATTTGTGTTTTTTTTTTAATCAAGTAAAAGCACCTAAAAGCAGTTATCATAAAAAAAAAGTAGCAAAACATATATCGATTGCTTCTTTGCCCTCCCCCCCGCAGGGCCGCCCAAAAAAAAAATCTATTTTTGCCGAATTTATAAATTAGATATTAATTAAAGTGCTTTTACCATTTTGAAATCACAAGTTATAATTGCTTTCAGCTCGTTGGTGAATTCTGT SEQ ID NO: 3: (256 bp) AACAGCCAGCACAGACCTAATGAGTTGCAGATTGACTTCGCCAATCCAGGCTTGTGATAATAAACAAATAAGCTTATTGCACTGAGTGAAACAACTTAAAAGCACTACTTAAACTAAATCAATTAACATAAATATACGAATAATCAATAGACTCTACTTATAATCTGGATGCATATATGCCTACTGCACAAGAATACAGTATAAATATGGACAGGAATCACAGTAGGTTTAGAGAATAAGGAGGCGTCAGGTGTGA SEQ ID NO: 4: (342 bp) AACAGCCAGCACAGACCTAATGAATTGCAGATTGACTTCGCCAATCCAGGCTTGTGATAATAAACAAATAAGCTTATTGCAGTGAGTGAAACAACTTAAAAGCACTACTTAAACTAAATCAATTAACATAAATATACGAATAATCAATAGACTTTACTATAAGAAGAAGTACACAGATATACATGGATCATAAAAGAATATAAGTAGAAGCAGTTCAAACCTAGAAGCAGTTCAAACCAAGTATTATAATCTGGATGCATATATGCCTACTGCACAAGAATACAGTATAAATATGGACAGGAATCACAGTAGGTTTAGAGAATAAGGAGGCGTCAGGTGTGA (3) Based on the above-identified and verified structural variation sites, extract the SV site variation sequence, use Primer 3 to design amplification primers in the upstream and downstream of the site in batches, and the parameter settings are GC content 40-60%, Tm value 55-65℃. And use In-Silico PCR to simulate electronic PCR amplification, and screen primers that meet the following conditions: ① The primer amplification specificity is strong, and only one or two bands can be amplified in different samples, meeting the characteristics of citrus diploid heterozygosity; ② The primer amplification product length is 200-1000 bp, which is convenient for reducing the extension time required during PCR amplification; ③ The primer amplification band difference length is within 200 bp, which is convenient for gel detection after amplification.

[0030] PCR amplification and gel electrophoresis verification: DNA was extracted from *Citrus 'Red Beauty'* and 15 other citrus varieties. PCR amplification was performed using screening primers in a 10 μL reaction system (containing 1× buffer, 0.2 mM dNTPs, 0.5 μM primers, and 1 U Taq enzyme). The PCR amplification program was: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 57 ℃ annealing for 40 s, 72 ℃ extension for 40 s, 35 cycles; 72 ℃ extension for 10 min. After amplification, electrophoresis was performed on a 2% agarose gel (110 V, 1 h). Variety identification was performed based on differences in amplified bands, and primers with clear and stable amplified bands were retained. The two primer pairs obtained through agarose gel electrophoresis screening, as shown in Table 1 and SEQ ID NO: 5–8, can be used to identify primer combinations for the authenticity identification of *Citrus 'Red Beauty'*.

[0031] Table 1 Primer pairs for detecting SV molecular marker combinations

[0032] Figure 2 These are two differential bands amplified by primers 1-181 in the samples (Red Beauty, Amakusa, and Nanxiang). The red bar at the top corresponds to... Figure 1 The lower gel band in A, SEQ ID NO:1 276bp, is represented by a white rectangle indicating SV; the black bar below represents... Figure 1 The upper gel band in A, SEQ ID NO:2 362bp.

[0033] Figure 3 These are two differentially amplified bands in the samples (Red Beauty, Amakusa, and Nanxiang) using primer 2-046. The red bar at the top corresponds to... Figure 1 The lower gel band in B, SEQ ID NO:3 256bp, is represented by a white rectangle indicating SV; the black bar below it represents... Figure 1 The upper gel band in B, SEQ ID NO:4 342bp.

[0034] Amplification results using primers designed based on the chr1:29803181 site showed bands at both 350 bp and 250 bp for *Chr1*. Amplification results using primers designed based on the chr2:1013046 site showed a band only at 250 bp, with no band at 350 bp. (See [link to relevant documentation]). Figure 1 And Table 2. Figure 1 In the text, M stands for DNA molecular marker, and lanes 1 to 16 represent various citrus varieties, including Kiyomi, Amakusa, Nanko, Nishiko, W. Murcott, Murcott, Wogan, Dayagan, Haruka, Harumi, Kanpei, Benijin, Dekopon, Jinshusu Tangju, Asumi, and Sunshine No. 1.

[0035] By combining the band patterns of two SV loci (350bp+250bp+250bp), Hongmeiren can be distinguished from other 15 varieties with 100% accuracy, as shown in Table 2.

[0036] Table 2 SV marker amplification combination of 216 hybrid mandarin varieties

[0037] (4) 22 citrus seedlings (including 4 Hongmeiren seedlings) were randomly selected for blind test, and were detected by the two pairs of markers described above. As shown in Table 3, the amplification bands of lanes 13, 15, 18 and 21 were consistent with Hongmeiren, and were indeed Hongmeiren seedlings, i.e. the detection rate of Hongmeiren seedlings was 100%, and the markers were stable. Among them, lanes 1-22 samples: M. DNA marker; 1. Miyagawa 4; 2. Jaffa; 3. Xin Nv Penggan; 4. Oshu; 5. Zao Meng Penggan; 6. Dapu 5; 7. Nanfeng Honey Orange; 8. Anjiang Xiangyu; 9. Jinlan Pummelo; 10. Qiangdele; 11. Juxianghong; 12. Shatian Pummelo; 13. Hongmeiren-1; 14. Qiongxi Honey Orange; 15. Hongmeiren-3; 16. Ganning; 17. Shiranami; 18. Hongmeiren-4; 19. Jinqi Sandatang Orange; 20. Tiancao; 21. Hongmeiren-2; 22. Chunxiang. Figure 8 ​

Claims

1. A combination of SV molecular markers for identifying Citrus grandis (L.) Osbeck x Citrus sinensis (L.) Osbeck, characterized in that, Comprise two structural variation sites as follows: (1) Chr1:29803181 site, the sequence of which is shown in SEQ ID NO:1 and SEQ ID NO:2; (2) Chr2:1013046 site, the sequence of which is shown in SEQ ID NO:3 and SEQ ID NO:4; The Chr1:29803181 site is located at the position of 29803181 of chromosome 1 of sweet orange reference genome SWO.v.3.0, and the Chr2:1013046 site is located at the position of 1013046 of chromosome 2.

2. The combination of SV molecular markers according to claim 1, wherein, In identifying red beauty trifoliate orange: 350 bp and 250 bp bands are amplified at the Chr1:29803181 site, and only 250 bp band is amplified at the Chr2:1013046 site.

3. A primer pair for detecting the SV molecular marker combination of claim 1 or 2, characterized in that, Comprise: (a) The sequence of the primer pair for the Chr1:29803181 site is as follows: Forward primer: 5'-AGTGGGATCGAATGTCACTCT-3', Reverse primer: 5'-ACAGAATTCACCAACGAGCTG-3'; (b) The sequence of the primer pair for the Chr2:1013046 site is as follows: Forward primer: 5'-AACAGCCAGCACAGACCT-3', Reverse primer: 5'-TCACACCTGACGCCTCCT-3'.

4. A kit for identifying red beauty mandarin, characterized by, Comprise the primer pair of claim 3.

5. A method for identifying red fleshed trifoliate orange, characterized by, Comprise the following steps: (1) Extracting the genomic DNA of the citrus sample to be tested; (2) Using the primer pair of claim 3 to perform PCR amplification on the genomic DNA; (3) Performing agarose gel electrophoresis detection on the PCR amplification product: when the sample simultaneously appears 350 bp and 250 bp bands at the Chr1 site, and only 250 bp band appears at the Chr2 site, it is determined as red beauty trifoliate orange.

6. The method of claim 5, wherein, In step (2), the PCR amplification procedure is as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 57 ℃ annealing for 40 s, 72 ℃ extension for 40 s, cycling for 35 times; 72 ℃ extension for 10 min.

7. The method of claim 5, wherein, The agarose gel electrophoresis condition is as follows: 2% agarose gel, 110 V voltage, electrophoresis for 1 hour.

8. The use of the SV molecular marker combination of claim 1 or 2, the primer pair of claim 3, or the kit of claim 4 in identifying the authenticity of red beauty trifoliate orange variety, screening the counterfeit variety of citrus seedling market, or screening the offspring of citrus cross breeding.

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