Molecular marker for identifying navel orange and primer and application thereof

Abstract

This invention discloses a molecular marker and primers for identifying navel oranges and their applications, belonging to the field of plant breeding technology, aiming to solve the identification and screening problem of the Longhui Sweet variety in the prior art. It includes a molecular marker for identifying navel oranges, characterized in that a sequence 34332695-37319781 on chromosome 34332695-37319781 of the navel orange to be identified is deleted, and a DNA transposon is inserted at the breakpoint to form the molecular marker CsChr5-LHT. The nucleotide sequence of the molecular marker CsChr5-LHT is shown in SEQ ID NO.1. This invention is applicable to the screening and identification of navel orange varieties.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a molecular marker CsChr5-LHT and primers for identifying navel oranges and their applications, which are suitable for identifying high-quality navel oranges of the Longhui Sweet variety. Background Technology

[0002] The Gannan navel orange industry suffers from an imperfect variety structure, and there is a need to select and promote more superior varieties. The existing market has selected a new high-quality navel orange variety, Longhui Sweet (LHT), from Newhall (NHE) navel oranges.

[0003] Longhui sweet navel orange trees have a moderate vigor and a slightly drooping shape. The leaves are relatively slender and willow-shaped, with thinner branches. The fruit ripens in early to mid-November, reaching full orange-red color 7-10 days later than Newhall navel oranges. The fruit has a smooth, orange-red surface with fine, bright orange-red oil vesicles and an attractive shape. The peel is approximately 4.37 mm thick, thinner than Newhall navel oranges. The fruit shape index is approximately 1.14, making it more oval than Newhall navel oranges. The average single fruit weight is approximately 223 grams, slightly lighter than Newhall navel oranges. Longhui sweet navel oranges are of high quality, with a rich, orangey aroma, high solids content, and a sweet, intense flavor. Samples taken from the citrus orchards in Qiling Village, Longhui Town, in 2017 and 2018 were sent to the Jiangxi Provincial Pollution-Free Agricultural Products Quality Supervision and Inspection Station for testing. The solids content of Longhui sweet navel oranges was 13.5% and 15% respectively, higher than that of Newhall navel oranges during the same period, which had solids contents of 12.5% ​​and 14% respectively. Longhui sweet navel oranges have good resistance to citrus canker. Observations over many years have shown that the incidence of citrus canker is less than that of Newhall navel oranges. The average yield of mature Longhui sweet navel oranges is 4,500-5,000 jin per mu, with no significant alternate bearing, resulting in high and stable yields.

[0004] The existing high-quality navel orange variety Longhui Sweet has excellent quality, but there is a lack of molecular markers that are closely linked to candidate genes and have a stable screening effect, which can be used to screen for high-quality Longhui Sweet. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a molecular marker and primer for identifying navel oranges and their application, which facilitates rapid identification of high-quality Longhui sweet navel orange seedlings.

[0006] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0007] In a first aspect, the present invention provides a molecular marker for identifying navel oranges. The molecular marker is a molecular marker CsChr5-LHT formed by inserting a DNA transposon at the breakpoint of a deletion in the 34332695-37319781 sequence of a Chr5 chromosome of the navel orange to be identified. The nucleotide sequence of the molecular marker CsChr5-LHT is shown in SEQ ID NO.1. When the sequence shown in SEQ ID NO.1 is present, it is a Longhui sweet navel orange; when the sequence shown in SEQ ID NO.1 is missing, it is a non-Huilong sweet navel orange.

[0008] In a second aspect, the present invention provides a primer for amplifying the molecular marker described in the first aspect.

[0009] Furthermore, the primers include CsChr5-LHT-F and CsChr5-LHT-R;

[0010] The sequence of CsChr5-LHT-F is shown in SEQ ID NO.2, and the sequence of CsChr5-LHT-R is shown in SEQ ID NO.3.

[0011] Furthermore, one primer was located on the Chr5 chromosome of the navel orange, and the other primer was located on the inserted DNA transposon.

[0012] Thirdly, the present invention provides a kit comprising primers for amplifying the molecular marker CsChr5-LHT as described in any of the second aspects.

[0013] Fourthly, the present invention provides a method for identifying navel oranges, which detects the molecular markers described in the first aspect in the genome of the navel orange to be tested.

[0014] Furthermore, the genomic DNA of the navel orange to be tested is amplified by PCR using the primers described in any of the second aspects, and the PCR products are analyzed by electrophoresis to screen out the target navel oranges.

[0015] Furthermore, the test result showing a single 7067 bp band was from Longhui sweet navel oranges.

[0016] Furthermore, the target navel orange is the Longhui sweet navel orange.

[0017] Fifthly, the present invention provides the use of the molecular marker described in the first aspect, or the primer described in any of the second aspects, or the kit described in the third aspect, or the method described in any of the fourth aspects, in the identification of navel orange germplasm.

[0018] Beneficial effects

[0019] To achieve a leapfrog development and variety improvement in the navel orange industry, this invention designs a related molecular marker based on a mutation site provided by the Chr5 chromosome of the new variety Longhui Sweet. This marker specifically targets the mutation site provided by this invention, enabling the rapid identification of high-quality Longhui Sweet navel oranges in seedlings or other propagation materials. Thanks to this site for identifying navel orange varieties, the workload of selection can be effectively reduced, the breeding scale can be scaled down, and the breeding cycle can be shortened.

[0020] This invention provides primer pairs for the molecular marker CsChr5-LHT for the detection of LHT navel oranges, which are convenient for rapid identification of LHT navel orange seedlings. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the differences between the Chr5 chromosome sequence of LHT navel orange germplasm and the Chr5 chromosome sequence of Newhall navel orange provided by this invention;

[0023] Figure 2 This is a schematic diagram showing the results of detecting LHT navel orange and Newhall navel orange using the molecular marker CsChr5-LHT provided by this invention;

[0024] Figure 3 This is a schematic diagram showing the results of detecting LHT with other main navel orange varieties in southern Jiangxi using the molecular marker CsChr5-LHT provided by this invention. Detailed Implementation

[0025] The above content is further illustrated below with specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments. All technologies implemented based on the above content of this invention fall within the scope of this invention.

[0026] It should be understood that all experimental procedures not detailed in the experiment are routine experimental procedures well known to those skilled in the art.

[0027] The sequence list for the following embodiments is shown in Table 1.

[0028] Example 1

[0029] This embodiment focuses on the identification of differentially expressed sites and the development of molecular markers. The specific process is as follows:

[0030] (1) Identification of differential sites

[0031] The samples used for identification, including Newhall and bud mutation LHT navel orange materials, were obtained from the nursery of the National Navel Orange Engineering Technology Research Center. Three biological replicates were selected from different sample materials. DNA was extracted from navel orange leaves using the CTAB method, and a library was constructed, followed by next-generation sequencing at a depth of 30×. This invention uses the sweet orange genome as a reference (http: / / citrus.hzau.edu.cn) and analyzes the sequencing data on a Linux server. Comparison revealed a deletion of the 34332695-37319781 sequence on chromosome 5 of the LHT navel orange.

[0032] (2) Differential site sequence cloning analysis

[0033] To further clarify the sequence variation characteristics, primers were designed based on the sequences anterior to site 34332695 and posterior to site 37319781 on chromosome 5 of the reference genome. Using LHT and Newhall navel orange DNA as templates, the mutated sequences were cloned. The results showed that the corresponding sequence could not be cloned from Newhall navel orange, while the corresponding sequence could be cloned from high-quality navel orange LHT. Bioinformatics analysis indicated that an insertion sequence, a 6.93 kb DNA transposon, existed at this deletion site. After insertion, the molecular marker CsChr5-LHT was formed, and its sequence is shown in SEQ ID NO.1. A schematic diagram illustrating the differences between the Chr5 chromosome sequence of high-quality navel orange LHT and the Chr5 chromosome sequence of Newhall navel orange provided by this invention is shown below. Figure 1 As shown.

[0034] (3) Molecular marker design

[0035] Based on the characteristics of the inserted sequence, this invention designs a molecular marker primer pair. One amplification primer is located on the Chr5 chromosome of navel orange, and the other primer is located on the inserted transposon sequence. The insertion process is as follows: Figure 1 As shown. This molecular marker primer pair is named the CsChr5-LHT primer pair, which includes CsChr5-LHT-F and CsChr5-LHT-R. The sequence of CsChr5-LHT-F is shown in SEQ ID NO.2, and the sequence of CsChr5-LHT-R is shown in SEQ ID NO.3.

[0036] (4) Identification of molecular markers for LHT navel orange and Newhall navel orange materials

[0037] DNA was extracted from the leaves of LHT and Newhall navel oranges using the CTAB method for molecular marker identification of LHT and Newhall navel orange materials.

[0038] The extracted leaf DNA was amplified in a PCR reaction system of 10 μl, which contained 0.5 μl DNA template, 0.25 μl each of upstream and downstream primers at a concentration of 1 mmol / L, 5 μl Mix, and 4 μl ddH2O.

[0039] PCR reaction procedure: denaturation at 95°C for 5 min; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at Tm (dissolution temperature, 56°C in this example) for 30 s, extension at 72°C for 2 min; extension at 72°C for 5 min; and finally storage at 10°C.

[0040] The PCR amplification products of this invention were subjected to 1% agarose gel electrophoresis, and the gel was scanned and analyzed in the ChemiScope imaging system.

[0041] (5) Results and Analysis

[0042] The molecular marker CsChr5-LHT can distinguish LHT navel oranges from their bud mutation, Newhall navel oranges. In the gel electrophoresis results, CsChr5-LHT is a linear marker with two band patterns: a band size of 7067 bp and no band. The orange with the 7067 bp band is the LHT navel orange, while the orange without the band is the Newhall navel orange.

[0043] like Figure 2 As shown, the molecular marker CsChr5-LHT provided by this invention is used to detect LHT navel orange and Newhall navel orange; M in the figure is DL-15000 Marker, channel 1 is the band pattern of CsChr5-LHT for detecting LHT navel orange, and channel 2 is the band pattern of CsChr5-LHT for detecting Newhall navel orange.

[0044] Example 2

[0045] In this embodiment, the molecular marker CsChr5-LHT was used to identify LHT and other navel orange germplasms.

[0046] (1) Molecular marker detection of LHT and other navel orange germplasm.

[0047] DNA was extracted from the leaves of LHT and other domestically cultivated navel orange materials using the CTAB method for molecular marker identification of LHT and other domestic navel orange materials.

[0048] The extracted DNA was amplified by PCR. The specific PCR reaction system volume was 10 μl, which contained 0.5 μl DNA template, 0.25 μl each of upstream and downstream primers at a concentration of 1 mmol / L, 5 μl Mix, and 4 μl ddH2O.

[0049] PCR reaction procedure: denaturation at 95℃ for 5 min; followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at Tm (56℃) for 30 s, extension at 72℃ for 2 min; further extension at 72℃ for 5 min; and final storage at 10℃. PCR amplification products were analyzed by 1% agarose gel electrophoresis. The films were scanned and analyzed using a ChemiScope imaging system.

[0050] (2) Results and analysis.

[0051] The molecular marker CsChr5-LHT can distinguish LHT navel oranges from other major domestic navel orange germplasms. In the gel electrophoresis results, CsChr5-LHT is a linear marker with two band patterns: a band size of 7067 bp and no band. The 7067 bp band indicates LHT navel oranges, while other navel orange germplasms show no band.

[0052] like Figure 3 As shown, the molecular marker CsChr5-LHT provided by this invention is used to detect LHT in navel orange varieties in southern Jiangxi. M is the DL-15000 Marker, channels 1-2 are the band patterns of LHT-detected navel oranges by CsChr5-LHT, and channels 3-24 are the band patterns of other navel orange varieties by CsChr5-LHT.

[0053] The results above show that the molecular marker CsChr5-LHT designed in this invention can be used to screen out the target Longhui Sweet variety and shorten the breeding time.

[0054] SEQ ID NO. 1 (Molecular marker CsChr5-LHT sequence):

[0055]

[0056] Table 1: Sequence List.

[0057]

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular marker for identifying navel oranges, characterized in that, The molecular marker is a molecular marker CsChr5-LHT formed by inserting a DNA transposon into a breakpoint position of a sequence deletion of a Chr5 chromosome 34332695-37319781 of navel orange to be identified, a nucleotide sequence of the molecular marker CsChr5-LHT is shown as SEQ ID NO. 1; when the sequence shown in SEQ ID NO. 1 exists, it is Longhu sweet navel orange, and when the sequence shown in SEQ ID NO. 1 is deleted, it is non-Longhu sweet navel orange.

2. A primer, characterized in that, The primer is used for amplifying the molecular marker in claim 1.

3. The primer of claim 2, wherein, The primer comprises CsChr5-LHT-F and CsChr5-LHT-R. The sequence of the CsChr5-LHT-F is shown as SEQ ID NO. 2, and the sequence of the CsChr5-LHT-R is shown as SEQ ID NO.

3.

4. The primer of claim 3, wherein One of the primers is located on a Chr5 chromosome of navel orange, and the other primer is located on the inserted DNA transposon.

5. A kit comprising the primer for amplifying the molecular marker CsChr5-LHT according to any one of claims 2-4.

6. A method of identifying navel oranges, characterized by, Detecting the molecular marker in claim 1 in a genome of a navel orange to be tested.

7. The method of authenticating navel oranges according to claim 6, characterized in that, The genome DNA of the navel orange to be tested is subjected to PCR amplification by using the primer according to any one of claims 2-4, and the PCR product is subjected to electrophoresis analysis, so as to screen a target navel orange.

8. The method of authenticating navel oranges according to claim 6, characterized in that, The Longhu sweet navel orange is detected by a single 7067 bp band.

9. The method of claim 6, wherein, The target navel orange is the Longhu sweet navel orange.

10. Use of the molecular marker in claim 1, the primer according to any one of claims 2-4, the kit in claim 5, or the method according to any one of claims 6-9 in identifying navel orange germplasm.

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