InDel molecular marker for mandarin orange, primer pair, application and identification method

Abstract

The invention relates to the technical field of molecular marker assisted breeding, in particular to a Hua sweet orange InDel molecular marker, a primer pair, application and an identification method. The mandarin orange InDel molecular marker is located in a Nanfeng mandarin orange genome Chr3: 2131843, and the polymorphism of the mandarin orange InDel molecular marker is a deletion / insertion nucleic acid sequence; the deletion nucleic acid sequence is shown as SEQ ID NO.1, and the basic group of the insertion nucleic acid sequence is cytosine. A primer pair for amplifying the InDel molecular marker of the mandarin orange in the claim 1 is shown as SEQ ID NO.2 and SEQ ID NO.3. The invention also discloses a kit for detecting the InDel molecular marker of the mandarin orange in the mandarin orange in the mandarin The method can be used for rapidly and accurately identifying the Nanfeng tangerine slag melting superior line Hua sweet tangerine in a short time, so that the variety identification cost is reduced, and a foundation is laid for Nanfeng tangerine germplasm resource identification and breeding utilization.

Description

Technical Field

[0001] The invention relates to the technical field of molecular marker-assisted breeding, and in particular to Huagan mandarin orange InDel molecular markers and primer pairs, applications and identification methods. Background Art

[0002] Citrus belongs to the Rutaceae family and is an important fruit species in the world, grown in more than 140 countries and regions. There are many types of citrus, and production mainly involves three genera and eight categories. The three genera refer to Citrus, Fortunella and Trifoliate Orange. The eight categories include Citrus, sweet orange, sour orange, pomelo and grapefruit, citron, lemon, as well as fortunella and trifoliate orange.

[0003] Most common citrus fruits belong to the genus Citrus, which not only have bright colors and beautiful shapes, but are also rich in nutrients, containing a variety of vitamins, carotenoids and aromatic substances. The genus Citrus is often used as a rootstock due to its strong cold and disease resistance. The fruits of the genus Fortunella are smaller, and some varieties have been cultivated into ornamental plants for use in gardens and potted plants. Some wild species have been developed into model plants for studying the functional genome of citrus and used as genetic transformation materials.

[0004] Nanfeng mandarin oranges, produced in Nanfeng County, Jiangxi Province, are the oldest varieties of wide-skinned citrus. They are selected from milk oranges and have a 1,300-year cultivation history. Today, they are grown in Jiangxi, Zhejiang, Fujian, Hunan, Hubei, Sichuan, Guangxi and other places. This variety is juicy, fragrant, high-yielding, of excellent quality, cold-resistant, and ulcer-resistant, but with a slightly thicker husk and poor slag-forming properties. Through breeding methods such as bud mutation and natural hybridization, Nanfeng mandarin oranges have gradually formed two variety groups, the Nanfeng mandarin orange variety group and the Nanfeng Guangju variety group. The former includes early-maturing series, large-fruit series, small-fruit series and osmanthus-pedicle series, and the latter includes Mi Guang and Hong Guang. Mi Guang is the hybrid offspring of Nanfeng mandarin oranges and fire oranges. Although it has a thin skin and juicy flesh, and its flesh is tender and slag-forming, its aroma is not as good as that of Nanfeng mandarin oranges.

[0005] In recent years, several bud mutations with excellent pulp-forming properties have been discovered in Xiagang Village, Nanfeng County, Jiangxi Province. Not only are their fruits significantly better at pulp-forming properties than ordinary Nanfeng mandarin oranges, but they also have a strong aroma and good fruit quality. They are expected to become new citrus varieties. The development of this new variety can not only improve the variety structure of Nanfeng mandarin oranges, but also provide new germplasm resources for the research on pulp-forming properties of Nanfeng mandarin oranges.

[0006] Citrus varieties are rich in resources, with diverse breeding methods. The affinity between species is very strong, and the various mutations that occur are preserved by asexual reproduction, resulting in a complex genetic background between varieties. In order to analyze the genetic diversity and phylogenetic relationship of citrus, a variety of molecular marker technologies have been developed. Molecular markers are based on the differences in nucleotide sequences of genetic material between individuals. They directly reflect the genetic diversity between varieties at the DNA level, so that new varieties can be identified by constructing variety-specific fingerprint maps. This method is stable and efficient, and can effectively exclude environmentally induced changes. It has gradually become an important means of identifying bud mutations. Based on the differences in the means of detecting DNA polymorphisms, molecular markers can be divided into three types. The first type is DNA markers based on molecular hybridization technology, such as RFLP; the second type is DNA markers based on PCR technology, such as AFLP and SSR; the third type is DNA markers based on single nucleotide polymorphisms, such as SNP.

[0007] Since InDel markers are mainly developed based on the insertion and deletion of nucleotide fragments in homologous sequences of the genomes of different individuals, the acquisition of homologous sequences is a prerequisite for their development. At present, with the development of next-generation sequencing technology, the cost of whole genome resequencing has been reduced, the time cycle has been shortened, and the reference genome sequences of various citrus fruits have been made public, which has greatly promoted the development of InDel markers. InDel markers are co-dominant markers with the advantages of rich sites, high polymorphism, stable variation, and simple operation. They are often used for citrus variety identification or identification of kinship between species. Based on the previous research of our research group, the use of InDel markers to quickly identify the superior line of Huazhaya variant 'Hua Gan Miju' is efficient and accurate, and is a molecular marker technology worthy of widespread promotion. Summary of the invention

[0008] The purpose of the present invention is to solve the shortcomings of the prior art and to propose InDel molecular markers and primer pairs, applications and identification methods for Huagan mandarin oranges.

[0009] One of the purposes of the present invention is to provide an InDel molecular marker of Huagan mandarin orange, which is located at Chr3:2131843 of the Nanfeng mandarin orange genome, and its polymorphism is a deletion / insertion nucleic acid sequence; the deletion nucleic acid sequence is shown in SEQ ID NO.1, and the base of the insertion nucleic acid sequence is cytosine.

[0010] The second object of the present invention is to provide a primer pair for amplifying the above-mentioned InDel molecular marker of Wagami mandarin orange, including: InDel-7-F and InDel-7-R; the nucleic acid sequence of InDel-7-F is shown in SEQ ID NO.2, and the nucleic acid sequence of InDel-7-R is shown in SEQ ID NO.3.

[0011] A third object of the present invention is to provide a kit comprising the above primer pair.

[0012] Preferably, the above kit also includes reagents required for PCR reaction and / or electrophoresis.

[0013] A fourth object of the present invention is to provide the use of the above-mentioned Huagan mandarin InDel molecular marker, or the above-mentioned primer pair, or the above-mentioned kit in identifying Nanfeng mandarin varieties or strains, or in Huagan mandarin breeding.

[0014] A fifth object of the present invention is to provide a method for identifying the variety or strain of Nanfeng mandarin oranges, comprising the following steps:

[0015] (1) extracting genomic DNA of the Nanfeng mandarin orange to be tested;

[0016] (2) using the DNA of the sample to be tested in step (1) as a template, and performing PCR amplification using the above primer pairs;

[0017] (3) Performing electrophoresis or sequencing on the amplified product obtained in step (2).

[0018] Preferably, in step (3), sequencing is used for detection. If the base at genome Chr3:2131843 is cytosine and lacks the nucleic acid sequence of SEQ ID NO.1, then the Nanfeng mandarin to be tested is Huagan mandarin, otherwise it is other Nanfeng mandarin varieties or strains.

[0019] Preferably, gel electrophoresis is used in step (3), and the specific operation is as follows: the amplification product obtained in step (2) is placed in an agarose gel for electrophoresis separation for 40 minutes.

[0020] If two bands appear in the above gel electrophoresis separation, located at less than 500 bp and 500-750 bp respectively, the Nanfeng mandarin orange to be tested is Huagan mandarin orange.

[0021] If no bands appear in the above gel electrophoresis separation, the Nanfeng mandarin orange to be tested is one of the large-leaf red mandarin orange, small-leaf Guang mandarin orange, and small-leaf red mandarin orange.

[0022] If only one band appears in the above gel electrophoresis separation, and the band is located at 500-750bp, then the Nanfeng mandarin orange to be tested is one of the other Nanfeng mandarin orange varieties or strains.

[0023] Preferably, capillary electrophoresis is used for detection in step (3), and the specific operation is as follows: the amplification product obtained in step (2) is placed in capillary electrophoresis for electrophoresis separation for 20 minutes.

[0024] If two bands appear in the capillary electrophoresis separation, which are 482 bp and 598 bp respectively, then the Nanfeng mandarin orange to be tested is Huagan mandarin orange;

[0025] If no bands appear in the capillary electrophoresis separation, the Nanfeng mandarin orange to be tested is one of the large-leaf red orange, small-leaf Guang orange, and small-leaf red orange;

[0026] If only one 578 bp band appears in the above capillary electrophoresis separation, the Nanfeng mandarin orange to be tested is one of the other Nanfeng mandarin orange varieties or strains.

[0027] The sixth object of the present invention is to provide an application of the above-mentioned Huagan mandarin InDel molecular marker, or the above-mentioned primer pair, or the above-mentioned kit, or the above-mentioned method for identifying Nanfeng mandarin varieties or strains in constructing a Nanfeng mandarin fingerprint.

[0028] The beneficial effects of the present invention include but are not limited to:

[0029] (1) The InDel sites obtained by the present invention are used to design specific primers, so that the method for identifying the varieties or strains of Nanfeng mandarin oranges is accurate and efficient, and the results are stable.

[0030] (2) The primer pair InDel-7 obtained in the present invention was used to amplify the DNA fragments different from those of other varieties / lines in the superior line of Nanfeng mandarin orange Huazha 'Hua Gan Mandarin Orange', and can be used for the rapid identification of the variety.

[0031] (3) The InDel marker developed by the present invention is simple to operate and easy to detect. Only agarose gel electrophoresis is required to quickly identify the 'Hua Gan Mandarin Orange', which greatly reduces the cost;

[0032] (4) The molecular markers developed in the present invention further confirmed that 'Hua Gan Mandarin' is a new superior bud mutation line of Nanfeng Mandarin, enriched the variety structure of Nanfeng Mandarin, and laid a foundation for the subsequent identification and genetic breeding of Nanfeng Mandarin varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The figure is an agarose gel electrophoresis pattern of the fragments amplified by the InDel-labeled primer pair InDel-7 obtained in the present invention in 20 Nanfeng mandarin orange varieties / lines.

[0034] Figure 2 The capillary electrophoresis pattern of the fragments amplified by the InDel-labeled primer pair InDel-7 obtained in the present invention in 20 Nanfeng mandarin orange varieties / lines. DETAILED DESCRIPTION

[0035] The present invention will be further explained below in conjunction with specific embodiments.

[0036] Example 1 Obtaining Molecular Marker Primers for Identification of Nanfeng Mandarin Orange Variety 'Hua Gan Mandarin Orange'

[0037] Twenty Nanfeng mandarin orange varieties / lines (as shown in Table 1) were selected for whole genome resequencing, and the clean reads obtained by filtering were compared with the Nanfeng mandarin orange reference genome using bwa software.

[0038] Table 1 20 Nanfeng mandarin orange varieties / lines

[0039]

[0040]

[0041] The differential InDel sites between 'Hua Gan Mandarin' and other 19 Nanfeng Mandarin varieties / lines were further compared and analyzed to screen out the specific InDel sites of this variety. Based on the results, InDel sites with insertion / deletion fragments larger than 3 bp were selected, and InDel sites with other insertion / deletion fragments 50 bp upstream and downstream were eliminated, and finally a differential InDel site library was obtained.

[0042] Subsequently, based on the complete genome sequence of Nanfeng mandarin orange, the screened InDel site was located on the genome, and primers were designed with a base length of 300bp on each side of the InDel site, a total of 600bp fragment length.

[0043] A total of 11 pairs of primers were designed through the website https: / / www.primer3plus.com. All primers amplified the target bands, with a successful amplification rate of 100%; 5 pairs of primers showed polymorphism in two or more Nanfeng mandarin orange varieties, with a polymorphism rate of 45.46%. The sequences of the 11 pairs of primers are shown in Table 2.

[0044] Table 2 Names and sequences of 11 pairs of primers

[0045]

[0046]

[0047] The designed and synthesized InDel primers were further used to perform PCR amplification on 'Hua Gan Mandarin' and the other 19 Nanfeng Mandarin varieties / lines, and the core InDel marker InDel-7 was screened. The primers designed for it are as follows:

[0048] InDel-7-F: GAATTATTGAGATTTGAGAAGGATACG.

[0049] InDel-7-R: AGAACAAATCTATGAATGAGTGAGA.

[0050] Example 2 Identification and application of the Nanfeng tangerine residue superior strain "Hua Gan tangerine" variety

[0051] (1) The CTAB method was used to extract DNA from 20 Nanfeng mandarin orange varieties / lines (the specific sample names are shown in Table 1).

[0052] (2) Using the DNA samples of the 20 Nanfeng mandarin orange varieties / lines as templates, PCR amplification reactions were performed.

[0053] a. PCR reaction system

[0054] The configuration of the 25 μL reaction system is shown in Table 3.

[0055] Table 3 PCR reaction system

[0056]

[0057] The upstream / downstream primers used are as follows:

[0058] InDel-7-F: GAATTATTGAGATTTGAGAAGGATACG.

[0059] InDel-7-R: AGAACAAATCTATGAATGAGTGAGA.

[0060] b. PCR reaction conditions

[0061] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C-50°C (-1°C / cycle) for 30 s, extension at 72°C for 30 s, 10 cycles; denaturation at 95°C for 30 s, annealing at 47-60°C (specific values ​​are set according to primer annealing temperature) for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 5 min, hold at 12°C.

[0062] (3) Separate the PCR amplification products using 3% agarose gel electrophoresis.

[0063] a. Preparation of 3% agarose gel

[0064] Use a measuring cylinder to measure 40 mL of 1×TAE buffer, use an electronic balance to weigh 1.2 g of agarose, place in a conical flask, stir evenly, and heat in a microwave to melt. Take out and stir evenly again. Repeat 2-3 times. After the agarose is completely dissolved, place in a ventilated place to cool for a while. When the agarose solution cools to about 60°C, add 1.8 μL of nucleic acid dye, mix the dye evenly, pour the solution onto a horizontal plate, and insert a 24-hole comb.

[0065] b. Gel electrophoresis

[0066] Take out the solidified gel, pull out the comb, use a pipette to draw 10μL of PCR product and add it to the sample wells in turn, add 10μL of 2000bp marker on the far left, record the smoothness of spotting and the amount of sample added, put the gel into the electrophoresis tank, pour 1×TAE buffer into the tank until it covers the agarose gel by about 1mm, set the electrophoresis parameters as voltage U=120V, current I=200mA, and electrophoresis time 40min.

[0067] c. Result analysis

[0068] After electrophoresis, use a gel imager to photograph, name and save the gel.

[0069] (4) Band analysis

[0070] The selected InDel-7 primers were used to amplify all the DNA samples of Nanfeng mandarin oranges. Only the "Hua Gan Mandarin Orange" could specifically obtain two polymorphic bands after amplification, one less than 500bp and the other between 500bp and 750bp. The results are as follows Figure 1 shown.

[0071] Example 3 Construction of accurate identification and fingerprint of Nanfeng mandarin orange residue superior line 'Hua Gan mandarin orange'

[0072] (1) The CTAB method was used to extract DNA from 20 Nanfeng mandarin orange varieties / lines (the specific sample names are shown in Table 1).

[0073] (2) Using the DNA samples of the 20 Nanfeng mandarin orange varieties / lines as templates, PCR amplification reactions were performed.

[0074] a. PCR reaction system

[0075] The configuration of the 25 μL reaction system is shown in Table 4.

[0076] Table 4 PCR reaction system

[0077]

[0078] The upstream / downstream primers used are as follows:

[0079] InDel-7-F: GAATTATTGAGATTTGAGAAGGATACG.

[0080] InDel-7-R: AGAACAAATCTATGAATGAGTGAGA.

[0081] b. PCR reaction conditions

[0082] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C-50°C (-1°C / cycle) for 30 s, extension at 72°C for 30 s, 10 cycles; denaturation at 95°C for 30 s, annealing at 47-60°C (specific values ​​are set according to primer annealing temperature) for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 5 min, hold at 12°C.

[0083] (3) A fully automated capillary electrophoresis system (QI Advanced, QIAGEN) was used to separate the PCR amplification products. The instrument operation procedures followed the QIAxcel Advanced manufacturer's instructions.

[0084] a. Sample operation

[0085] Prepare 12 strips of tubes containing 15μL of Alignment Marker and Size Marker diluents respectively, and make sure there are no vacuoles in the liquid. Place the prepared Alignment Marker in the MARKER 1 position in the buffer reagent tank. Open the sample door and place the sample and Size Marker in the 96-well rack. If there is a gap in the sample in a well of the 96-well rack, fill it with 15μL of DNA dilution buffer. Set the running parameters, Size Marker position, and set the sample row, Alignment Marker and other information in the sample selection interface. After the settings are completed, click the "Run" button to start the run process.

[0086] b. Result analysis

[0087] The absolute migration distances of each capillary lane are aligned according to the Alignment Marker, and the sample bands are obtained in conjunction with the assignment in the system. All results are processed and analyzed in the QIAxcelScreenGel software.

[0088] (4) All the DNA samples of Nanfeng mandarin oranges were amplified using the selected InDel-7 primers. Only the “Hua Gan Mandarin Orange” could specifically obtain two polymorphic bands of 482 bp and 598 bp after amplification. The results are as follows: Figure 2 shown.

[0089] At the same time, according to the number and size of the bands obtained by amplifying all materials with the selected primer pairs (InDel-7-F and InDel-7-R), the fingerprints of the 20 Nanfeng mandarin orange varieties / lines in Table 1 were drawn (as shown in Table 5). In Table 5, 598 bp is the InDel-7-1 allele, 578 bp is the InDel-7-2 allele, and 482 bp is the InDel-7-3 allele.

[0090] Table 5 Fingerprints of 20 Nanfeng mandarin orange varieties / lines

[0091]

[0092]

[0093] Note: At the same migration position, the presence or absence of amplified fragment polymorphism is marked with "1" or "0". If there is a band, it is marked with "1", and if there is no band, it is marked with "0".

[0094] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Huagan mandarin orange InDel molecular marker, characterized in that: The InDel molecular marker is located at Chr3:2131843 of the Nanfeng mandarin orange genome, and its polymorphism is a deletion / insertion nucleic acid sequence; The deleted nucleic acid sequence is shown as SEQ ID NO.1, and the base of the inserted nucleic acid sequence is cytosine.

2. A primer pair for amplifying the InDel molecular marker of Huagan mandarin orange as claimed in claim 1, characterized in that: include: InDel-7-F and InDel-7-R; the nucleic acid sequence of InDel-7-F is shown in SEQ ID NO.2, and the nucleic acid sequence of InDel-7-R is shown in SEQ ID NO.

3.

3. A kit comprising the primer pair as claimed in claim 2.

4. The kit according to claim 3, characterized in that: It also includes reagents required for performing PCR reactions and / or electrophoresis.

5. Use of the Huagan mandarin InDel molecular marker as claimed in claim 1, or the primer pair as claimed in claim 2, or the kit as claimed in claim 3 in identifying Nanfeng mandarin varieties or strains, or in Huagan mandarin breeding.

6. A method for identifying Nanfeng mandarin orange varieties or strains, characterized in that: The steps include: (1) extracting genomic DNA of the Nanfeng mandarin orange to be tested; (2) using the DNA of the sample to be tested in step (1) as a template, and performing PCR amplification using the primer pair described in claim 2; (3) Performing electrophoresis or sequencing on the amplified product obtained in step (2).

7. The method for identifying the variety or strain of Nanfeng mandarin orange according to claim 6, characterized in that: In step (3), sequencing is used for detection. If the base at genome Chr3:2131843 is cytosine and lacks the nucleic acid sequence of SEQ ID NO.1, then the Nanfeng mandarin to be tested is Huagan mandarin. Otherwise, it is other Nanfeng mandarin varieties or strains.

8. The method for identifying the variety or strain of Nanfeng mandarin orange according to claim 6, characterized in that: In step (3), gel electrophoresis is used for detection, and the specific operation is as follows: the amplified product obtained in step (2) is placed in an agarose gel for electrophoresis separation for 40 minutes; If two bands appear in the electrophoresis separation, located at less than 500 bp and 500-750 bp respectively, the Nanfeng mandarin orange to be tested is Huagan mandarin orange; If no bands appear during electrophoresis separation, the Nanfeng mandarin orange to be tested is one of the following: large-leaf red orange, small-leaf Guang orange, or small-leaf red orange; If only one band appears in the electrophoretic separation and the band is located at 500-750bp, the Nanfeng mandarin orange to be tested is one of the other Nanfeng mandarin orange varieties or strains.

9. The method for identifying the variety or strain of Nanfeng mandarin orange according to claim 6, characterized in that: In step (3), capillary electrophoresis detection is used, and the specific operation is as follows: the amplification product obtained in step (2) is placed in a capillary electrophoresis for electrophoresis separation for 20 minutes; If two bands appear in the electrophoresis separation, which are 482 bp and 598 bp respectively, the Nanfeng mandarin orange to be tested is Huagan mandarin orange; If no bands appear during electrophoresis separation, the Nanfeng mandarin orange to be tested is one of the following: large-leaf red orange, small-leaf Guang orange, or small-leaf red orange; If only one 578bp band appears in the electrophoretic separation, the Nanfeng mandarin orange to be tested is one of the other Nanfeng mandarin orange varieties or strains.

10. Use of the Huagan mandarin InDel molecular marker as claimed in claim 1, or the primer pair as claimed in claim 2, or the kit as claimed in claim 3, or the method for identifying Nanfeng mandarin varieties or strains as claimed in claim 6 in constructing a Nanfeng mandarin fingerprint.

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