An ssr molecular marker linked to osmanthus fragrans fertility trait, primer set and application

Abstract

This invention discloses an SSR molecular marker linked to the fruit-bearing trait of Osmanthus fragrans, a primer set, and its applications, belonging to the field of Osmanthus fragrans molecular marker technology. This invention provides an SSR molecular marker linked to the fruit-bearing trait of Osmanthus fragrans, located in the base region from position 23983153 to 23983435 on chromosome 3 of Osmanthus fragrans. Furthermore, by detecting the size of the amplified fragment containing this SSR molecular marker, the fruit-bearing trait of Osmanthus fragrans can be rapidly and accurately determined. Therefore, this SSR molecular marker has good application prospects in identifying the fruit-bearing trait of Osmanthus fragrans or in Osmanthus fragrans assisted breeding. In addition, this SSR molecular marker can be directly applied to the selection of Osmanthus fragrans hybrid parents, early screening of seedlings, identification of fruit-bearing traits in germplasm resource banks, and protection of new varieties.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology for Osmanthus fragrans, specifically relating to an SSR molecular marker, primer set, and application linked to the fruit-bearing trait of Osmanthus fragrans. Background Technology

[0002] Traditional methods for identifying the fruit-bearing characteristics of Osmanthus fragrans rely on long-term field observations, which have significant technical shortcomings. These shortcomings include: 1) A long fruit-bearing cycle: It takes 6-8 years for plants to consistently exhibit fruit-bearing characteristics after planting, significantly lagging behind the needs of breeding generations. 2) High environmental sensitivity: Factors such as rainfall during flowering, pollinating insects, and soil fertility can all cause fluctuations in fruit-bearing rates (e.g., the fruit-bearing rate of "Early Silver Osmanthus" varies significantly in different regions), easily leading to misjudgments of breeding materials. 3) High identification costs: It requires continuous tracking and statistical analysis of fruit set rates for 3-5 years, consuming substantial human and material resources.

[0003] Although SSR markers have been used for the identification of Osmanthus varieties, no specific molecular markers have been found that are directly related to fruit-bearing traits, making early and accurate identification impossible.

[0004] In view of this, it is necessary to develop an SSR molecular marker closely linked to the fruit-bearing trait of Osmanthus fragrans, so as to achieve rapid, accurate and low-cost identification of fruit-bearing traits in Osmanthus fragrans during the seedling stage, and provide technical support for efficient breeding and germplasm resource screening of Osmanthus fragrans. Summary of the Invention

[0005] The purpose of this invention is to provide an SSR molecular marker, primer set, and application linked to the fruit-bearing trait of Osmanthus fragrans. This addresses the problem in the prior art of lacking an SSR molecular marker that can rapidly, accurately, and cost-effectively identify the fruit-bearing trait of Osmanthus fragrans.

[0006] In a first aspect, the present invention provides the application of SSR molecular markers in identifying the fruit-bearing trait of Osmanthus fragrans or in Osmanthus fragrans assisted breeding. The SSR molecular markers are located in the base range of position 23983153-23983435 on chromosome 3 of Osmanthus fragrans.

[0007] In this invention, the inventors used bioinformatics software to scan the simple repetitive sequences in the Osmanthus genome to obtain an SSR molecular marker linked to the fruit-bearing trait of Osmanthus, located in the base region from position 23983153 to 23983435 on chromosome 3 of Osmanthus. Furthermore, by detecting the size of the amplified fragment containing this SSR molecular marker, the fruit-bearing trait of Osmanthus can be rapidly and accurately determined. Therefore, this SSR molecular marker has good application prospects in identifying the fruit-bearing trait of Osmanthus or in Osmanthus-assisted breeding.

[0008] In a second aspect, the present invention provides a primer set for identifying the fruit-bearing trait of Osmanthus fragrans, the nucleotide sequence of which is shown in SEQ ID NO.1-2.

[0009] In a third aspect, the present invention provides a kit for identifying the fruit-bearing characteristics of osmanthus, comprising the above-mentioned primer set.

[0010] In some implementations, the kit also includes at least one of the reagents required for DNA extraction and the reagents required for PCR amplification.

[0011] In a fourth aspect, the present invention provides the application of the primer set described above or any of the kits described above in identifying the fruit-bearing trait of Osmanthus fragrans and / or in Osmanthus fragrans assisted breeding.

[0012] In a fifth aspect, the present invention provides a method for identifying the fruit-bearing trait of Osmanthus fragrans, comprising the following steps: extracting genomic DNA from the Osmanthus fragrans sample to be tested; performing PCR amplification on the genomic DNA of the Osmanthus fragrans sample to be tested using the above-mentioned primer set or any of the above-mentioned kits to obtain amplification products; performing electrophoresis on the amplification products to obtain the band size of the amplification products in the genomic DNA of the Osmanthus fragrans sample to be tested, and determining the fruit-bearing trait of Osmanthus fragrans based on the band size.

[0013] In some implementations, in the step of PCR amplification of the genomic DNA of the Osmanthus fragrans sample to be tested, the PCR amplification reaction system includes: 2-4 μL of 4×VAHTS Multi-PCR Mix, 0.5-0.7 μL of upstream primer, 0.5-0.7 μL of downstream primer, 1-2 μL of genomic DNA, and 6-6.5 μL of ddH2O.

[0014] In some implementations, the PCR amplification procedure for the genomic DNA of the Osmanthus fragrans sample to be tested includes: pre-denaturation at 98-100℃ for 1-3 minutes; denaturation at 98-100℃ for 13-17 seconds, annealing at 60-65℃ for 3-5 minutes, for 30-34 cycles; extension at 70-75℃ for 8-15 minutes; and storage of the product at 0-4℃.

[0015] In some implementations, the electrophoresis step of amplifying the products includes agarose gel electrophoresis.

[0016] In some implementations, the step of obtaining the band size of the amplified product in the genomic DNA of the Osmanthus sample to be tested and determining the fruit-bearing trait of the Osmanthus based on the band size specifically includes: when the band size of the amplified product in the genomic DNA of the Osmanthus sample to be tested is 282bp or 282 / 392bp, the Osmanthus sample to be tested is non-fruit-bearing; when the band size of the amplified product in the genomic DNA of the Osmanthus sample to be tested is 392bp, the Osmanthus sample to be tested is fruit-bearing.

[0017] The beneficial effects of this invention are as follows: Unlike the prior art, this invention provides an SSR molecular marker linked to the fruit-bearing trait of Osmanthus fragrans, located in the base region from position 23983153 to 23983435 on chromosome 3 of Osmanthus fragrans; furthermore, by detecting the size of the amplified fragment containing this SSR molecular marker, the fruit-bearing trait of Osmanthus fragrans can be rapidly and accurately determined. Therefore, this SSR molecular marker has good application prospects in identifying the fruit-bearing trait of Osmanthus fragrans or in Osmanthus fragrans assisted breeding; in addition, this SSR molecular marker can be directly applied to the fields of Osmanthus fragrans hybrid parent selection, early screening of seedlings, identification of fruit-bearing traits in germplasm resource banks, and protection of new varieties. Attached Figure Description

[0018] Figure 1 The agarose gel electrophoresis results of the amplified products in Example 1 of this invention are shown. Lane M is Maker, and its molecular weight from bottom to top is 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. Lanes 1-16 are samples 12, 8, 1, 4, 9, 10, 13, 22, 28, 31, 40, 45, 51, 56, 59, and 62, respectively. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0021] Currently, there is a lack of SSR molecular markers in existing technologies that can quickly, accurately, and at low cost identify the fruit-bearing traits of Osmanthus fragrans.

[0022] To address the lack of SSR molecular markers in existing technologies that can rapidly, accurately, and cost-effectively identify the fruit-bearing trait of Osmanthus fragrans, this invention provides an SSR molecular marker linked to the fruit-bearing trait of Osmanthus fragrans, a primer set, and its application.

[0023] In a first aspect, the present invention provides the application of SSR molecular markers in identifying the fruit-bearing trait of Osmanthus fragrans or in Osmanthus fragrans assisted breeding. The SSR molecular markers are located in the base range of position 23983153-23983435 on chromosome 3 of Osmanthus fragrans.

[0024] In the above-mentioned applications provided by this invention, the inventors scanned simple repetitive sequences in the Osmanthus genome using bioinformatics software to obtain an SSR molecular marker linked to the fruit-bearing trait of Osmanthus, located in the base region from position 23983153 to 23983435 on chromosome 3 of Osmanthus. Furthermore, by detecting the size of the amplified fragment containing this SSR molecular marker, the fruit-bearing trait of Osmanthus can be quickly and accurately determined. Therefore, this SSR molecular marker has good application prospects in identifying the fruit-bearing trait of Osmanthus or in Osmanthus-assisted breeding. In addition, this SSR molecular marker can be directly applied to the selection of Osmanthus hybrid parents, early screening of seedlings, identification of fruit-bearing traits in germplasm resource banks, and protection of new varieties. At the same time, identification can be carried out during the seedling stage of Osmanthus (2 months after planting), shortening the observation cycle of 6-8 years to a few hours, greatly accelerating the breeding process.

[0025] Specifically, the physical location of the aforementioned SSR molecular markers was determined based on the OFL v1.0 genome sequence of Osmanthus fragrans.

[0026] In a second aspect, the present invention provides a primer set for identifying the fruit-bearing trait of Osmanthus fragrans, the nucleotide sequence of which is shown in SEQ ID NO.1-2.

[0027] It is understood that primer sets can be designed based on the flanking sequences of SSR molecular markers using conventional primer design software (such as Primer Premier 5), as long as they can efficiently amplify the target fragment containing the SSR molecular marker. For example, in this invention, the preferred nucleotide sequence of the primer set is shown in SEQ ID NO. 1-2.

[0028] In a third aspect, the present invention provides a kit for identifying the fruit-bearing characteristics of osmanthus, comprising the above-mentioned primer set.

[0029] In this invention, the above-mentioned kit can be used to quickly, accurately and cost-effectively identify the fruit-bearing characteristics of Osmanthus fragrans. Furthermore, the kit has the technical advantages of high specificity and high accuracy in identifying the fruit-bearing characteristics of Osmanthus fragrans.

[0030] In some implementations, the kit also includes at least one of the reagents required for DNA extraction and the reagents required for PCR amplification.

[0031] Understandably, the reagents required for DNA extraction can be selected from conventional DNA extraction reagents in existing technologies, as long as they can efficiently and quickly extract genomic DNA with high purity. Similarly, the reagents required for PCR amplification can be selected from conventional PCR amplification reagents in existing technologies, as long as they can efficiently and quickly amplify products with high purity.

[0032] In a fourth aspect, the present invention provides the application of the primer set described above or any of the kits described above in identifying the fruit-bearing trait of Osmanthus fragrans and / or in Osmanthus fragrans assisted breeding.

[0033] In this invention, the target fragment containing the SSR molecular marker can be efficiently amplified using the above primer set or the above kit. Then, based on the size of the amplified target fragment, the fruit-bearing characteristics of Osmanthus can be determined quickly, accurately, and at low cost.

[0034] In a fifth aspect, the present invention provides a method for identifying the fruit-bearing trait of Osmanthus fragrans, comprising the following steps: extracting genomic DNA from the Osmanthus fragrans sample to be tested; performing PCR amplification on the genomic DNA of the Osmanthus fragrans sample to be tested using the above-mentioned primer set or any of the above-mentioned kits to obtain amplification products; performing electrophoresis on the amplification products to obtain the band size of the amplification products in the genomic DNA of the Osmanthus fragrans sample to be tested, and determining the fruit-bearing trait of Osmanthus fragrans based on the band size.

[0035] The method for identifying the fruit-bearing traits of Osmanthus provided by this invention is simple, uses inexpensive and readily available raw materials, and is suitable for large-scale testing. Therefore, this method can be used to identify Osmanthus seedlings (2 months after planting), shortening the observation cycle of 6-8 years to a few hours, greatly accelerating the breeding process. At the same time, it can be directly applied to the selection of Osmanthus hybrid parents, early screening of seedlings, identification of fruit-bearing traits in germplasm resource banks, and protection of new varieties.

[0036] In some implementations, during the PCR amplification step of the genomic DNA from the Osmanthus fragrans sample to be tested, the PCR amplification reaction system includes: 2-4 μL (e.g., 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, or other values ​​within this range) of 4×VAHTS Multi-PCR. Mix, 0.5-0.7 μL (e.g., 0.5 μL, 0.55 μL, 0.6 μL, 0.65 μL, 0.7 μL or other values ​​within this range) of upstream primer, 0.5-0.7 μL (e.g., 0.5 μL, 0.55 μL, 0.6 μL, 0.65 μL, 0.7 μL or other values ​​within this range) of downstream primer, 1-2 μL (e.g., 1 μL, 1.2 μL, 1.5 μL, 1.8 μL, 2 μL or other values ​​within this range) of genomic DNA, and 6-6.5 μL (e.g., 6 μL, 6.1 μL, 6.2 μL, 6.3 μL, 6.4 μL, 6.5 μL or other values ​​within this range) of ddH2O.

[0037] In some preferred embodiments, the PCR amplification reaction system comprises: 3 μL of 4×VAHTS Multi-PCRMix, 0.6 μL of upstream primer, 0.6 μL of downstream primer, 1.5 μL of genomic DNA, and 6.3 μL of ddH2O.

[0038] In some implementations, the PCR amplification procedure for the genomic DNA of the Osmanthus fragrans sample includes: pre-denaturation at 98-100℃ (e.g., 98℃, 98.5℃, 99℃, 99.5℃, 100℃, or other values ​​within this range) for 1-3 minutes (e.g., 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, or other values ​​within this range); denaturation at 98-100℃ (e.g., 98℃, 98.5℃, 99℃, 99.5℃, 100℃, or other values ​​within this range) for 13-17 seconds (e.g., 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, or other values ​​within this range); and denaturation at 60-65℃ (e.g., 60℃, 61℃, 62 ... Anneal at 70-75°C (e.g., 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, or other values ​​within the range) for 3-5 minutes, for example, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, or other values ​​within the range, for 30-34 cycles, for example, 30 cycles, 31 cycles, 32 cycles, 33 cycles, 34 cycles, or other values ​​within the range; extend at 70-75°C (e.g., 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, or other values ​​within the range) for 8-15 minutes, for example, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, or other values ​​within the range; store the product at 0-4°C (e.g., 0°C, 1°C, 2°C, 3°C, 4°C, or other values ​​within the range).

[0039] In some preferred embodiments, the PCR amplification reaction procedure includes: 99°C pre-denaturation for 2 minutes; 99°C denaturation for 15 seconds, 63°C annealing for 4 minutes, 32 cycles; 72°C extension for 10 minutes, and product storage at 4°C.

[0040] In this invention, by optimizing the PCR amplification reaction system and reaction procedure, a target fragment containing SSR molecular markers with good purity can be obtained, which facilitates the subsequent determination of the size of the target fragment, thereby enabling rapid, accurate, and low-cost determination of the fruit-bearing characteristics of Osmanthus fragrans.

[0041] In some implementations, the electrophoresis step of amplifying the products includes agarose gel electrophoresis.

[0042] It is understandable that the electrophoresis method can be selected from conventional electrophoresis methods in the existing technology according to actual needs, as long as the size of the target fragment containing the SSR molecular marker can be obtained. For example, in this invention, the electrophoresis preferably includes agarose gel electrophoresis.

[0043] In some implementations, the step of obtaining the band size of the amplified product in the genomic DNA of the Osmanthus sample to be tested and determining the fruit-bearing trait of the Osmanthus based on the band size specifically includes: when the band size of the amplified product in the genomic DNA of the Osmanthus sample to be tested is 282bp or 282 / 392bp, the Osmanthus sample to be tested is non-fruit-bearing; when the band size of the amplified product in the genomic DNA of the Osmanthus sample to be tested is 392bp, the Osmanthus sample to be tested is fruit-bearing.

[0044] In this invention, the phenotypic characteristics of fruit setting in osmanthus samples can be rapidly, accurately, and cost-effectively determined by measuring the band size of the amplified products in the genomic DNA of the osmanthus sample.

[0045] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0046] Example 1 In this embodiment, the aim is to develop SSR molecular markers linked to the fruit-bearing trait of Osmanthus fragrans samples.

[0047] Specifically, it includes the following steps: 1) Filter regions containing SSR repeat sequences From the genome transcriptome data of the target species Osmanthus fragrans, simple repeat sequences were scanned using the bioinformatics software MISA to screen out DNA fragments containing SSR repeats.

[0048] 2) Design SSR-specific primers Specific PCR primers were designed using Primer Premier5 to target the conserved flanking regions of the aforementioned candidate SSR loci. Primer parameters were set as follows: length 18-22 bp, Tm value 55-60℃, GG content 40-60%, and no hairpin structure / dimer; a total of 25 primer pairs were designed. Several representative Osmanthus fragrans samples (both fruit-bearing and non-fruit-bearing) were selected as DNA templates, and the amplification efficiency and specificity of these 25 primer pairs were preliminarily determined. The reaction system consisted of 20 μL: template DNA 50 ng, 2X TaqPCRMasterMix 10 μL, forward and reverse primers 0.4 μmol / L each, and ddH2O added to 20 μL. The amplification program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 10 min. The results showed that 20 primer pairs amplified clear target bands.

[0049] 3) Primer validation and marker typing Using 20 primer pairs initially selected, polymorphism verification was performed in a small population comprising 5 fruit-bearing samples and 5 non-fruit-bearing samples. The results, detected by agarose gel electrophoresis, showed that only one primer pair amplified fragments with stable polymorphic differences in length between fruit-bearing and non-fruit-bearing samples. This differential locus was ultimately identified as the SSR molecular marker linked to the fruit-bearing trait of Osmanthus fragrans, located at base positions 23983153-23983435 on chromosome 3 of Osmanthus fragrans. This marker primarily amplified a large fragment (392 bp) in fruit-bearing samples, while in non-fruit-bearing samples, it primarily amplified small and medium fragments (282 / 392 bp).

[0050] The physical location of this SSR molecular marker was determined based on the Osmanthus fragrans genome sequence OFL v1.0.

[0051] Example 2 In this embodiment, based on the SSR molecular marker in Example 1, the aim is to develop a primer set for identifying the fruit-bearing trait of Osmanthus fragrans.

[0052] Specifically, based on the SSR molecular marker in Example 1, and combined with the flanking sequence of the SSR molecular marker, a primer set for identifying the fruit-bearing trait of Osmanthus fragrans was designed.

[0053] The primer sequence is as follows: Upstream primer F: 5′-TAAATACCACAATCAATCAAAGCAGT-3′ (SEQ ID NO.1); Downstream primer R: 5′-CAATCAAATCAAATACCAAGGAGC-3′ (SEQ ID NO.2).

[0054] Example 3 In this embodiment, a method for identifying the fruit-bearing characteristics of Osmanthus fragrans was established based on the primer set developed in Example 2, and the accuracy of the primer set detection was verified.

[0055] Specifically, it includes the following steps: 1) 110 samples of Osmanthus fragrans population with known fruiting characteristics were selected (14 fruiting samples and 96 non-fruiting samples).

[0056] 2) Genomic DNA extraction: Take about 0.2g of fresh leaves from each variety during the seedling stage in step 1), grind them with liquid nitrogen, and then use a nanomagnetic bead plant DNA extraction kit for high-throughput genomic DNA extraction.

[0057] 3) Using the genomic DNA of the osmanthus sample obtained in step 2) as a template, PCR amplification was performed to obtain the amplification product; The PCR amplification reaction system included: 3 μL of 4×VAHTS Multi-PCR Mix, 0.6 μL of upstream primer, 0.6 μL of downstream primer, 1.5 μL of genomic DNA, and 6.3 μL of ddH2O. The PCR amplification reaction program included: 99℃ pre-denaturation for 2 minutes; 99℃ denaturation for 15 seconds, 63℃ annealing for 4 minutes, 32 cycles; 72℃ extension for 10 minutes; and storage of the product at 4℃.

[0058] 4) The amplification products obtained in step 3) were subjected to agarose gel electrophoresis to obtain the fragment sizes of the genomic DNA amplification products of 110 Osmanthus samples to be tested. The results are shown in Table 1 below.

[0059] For example, the agarose gel electrophoresis results of samples 12, 8, 1, 4, 9, 10, 13, 22, 28, 31, 40, 45, 51, 56, 59, and 62 are as follows: Figure 1 As shown.

[0060] from Figure 1 As can be seen, the target fragment bands of the corresponding size can be amplified in samples 12, 8, 1, 4, 9, 10, 13, 22, 28, 31, 40, 45, 51, 56, 59, and 62.

[0061] Table 1. Results of Osmanthus fragrans sample name, fruit set phenotype, and amplified fragment size.

[0062] As shown in Table 1, when the amplified band size of the genomic DNA of the tested Osmanthus sample was 282 bp or 282 / 392 bp, the tested Osmanthus sample exhibited the non-fruiting trait, which was 100% consistent with the actual phenotype. When the amplified band size of the genomic DNA of the tested Osmanthus sample was 392 bp, the tested Osmanthus sample exhibited the fruiting trait, which was also 100% consistent with the actual phenotype. Therefore, the primer set of this invention can specifically amplify the target fragment containing the SSR molecular marker, and then, based on the size of the target fragment, efficient detection of the fruiting trait of Osmanthus can be achieved. Thus, this SSR molecular marker has good application prospects in identifying the fruiting trait of Osmanthus or in Osmanthus-assisted breeding; in addition, this SSR molecular marker can be directly applied to the selection of Osmanthus hybrid parents, early screening of seedlings, identification of fruiting traits in germplasm resource banks, and protection of new varieties.

[0063] In summary, this invention provides an SSR molecular marker linked to the fruit-bearing trait of Osmanthus fragrans, located in the base region from position 23983153 to position 23983435 on chromosome 3 of Osmanthus fragrans. Furthermore, by detecting the size of the amplified fragment containing this SSR molecular marker, the fruit-bearing trait of Osmanthus fragrans can be rapidly and accurately determined.

[0064] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0065] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of SSR molecular markers in identifying the fruiting traits of Osmanthus fragrans or in the assisted breeding of Osmanthus fragrans, characterized in that, The SSR molecular marker is located in the base range of position 23983153-23983435 on chromosome 3 of Guihua.

2. A primer set for identifying the fruiting trait of Osmanthus fragrans, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO. 1-2.

3. A kit for identifying the fruiting trait of Osmanthus fragrans, characterized in that, Includes the primer set as described in claim 2.

4. The kit of claim 3, wherein The kit also includes at least one of the reagents required for DNA extraction and the reagents required for PCR amplification.

5. The application of the primer set as described in claim 2 or the kit as described in any one of claims 3-4 in identifying the fruit-bearing trait of Osmanthus fragrans and / or in Osmanthus fragrans assisted breeding.

6. A method for identifying the fruiting trait of Osmanthus fragrans, characterized in that, Includes the following steps: Genomic DNA was extracted from the osmanthus samples to be tested; The genomic DNA of the Osmanthus fragrans sample to be tested was amplified by PCR using the primer set described in claim 2 or the kit described in any one of claims 3-4 to obtain the amplification product. The amplified products were subjected to electrophoresis to obtain the band size of the amplified products in the genomic DNA of the Osmanthus sample to be tested, and the fruit-bearing trait of Osmanthus was determined based on the band size.

7. The method according to claim 6, characterized in that, In the step of PCR amplification of the genomic DNA of the Osmanthus sample to be tested, the PCR amplification reaction system includes: 2-4 μL of 4×VAHTS Multi-PCR Mix, 0.5-0.7 μL of upstream primer, 0.5-0.7 μL of downstream primer, 1-2 μL of genomic DNA, and 6-6.5 μL of ddH2O.

8. The method according to claim 6, characterized in that, In the step of PCR amplification of the genomic DNA of the Osmanthus fragrans sample to be tested, the PCR amplification reaction program includes: pre-denaturation at 98-100℃ for 1-3 minutes; denaturation at 98-100℃ for 13-17 seconds, annealing at 60-65℃ for 3-5 minutes, 30-34 cycles; extension at 70-75℃ for 8-15 minutes, and storage of the product at 0-4℃.

9. The method according to claim 6, characterized in that, In the step of electrophoresis of the amplified products, the electrophoresis includes agarose gel electrophoresis.

10. The method according to claim 6, characterized in that, In the step of obtaining the band size of the amplified product in the genomic DNA of the osmanthus sample to be tested, and determining the fruit-bearing trait of osmanthus based on the band size, the determination specifically includes: When the band size of the amplified product in the genomic DNA of the Osmanthus sample to be tested is 282bp or 282 / 392bp, the Osmanthus sample to be tested is non-fruiting. When the amplified band size of the genomic DNA of the osmanthus sample to be tested is 392 bp, the osmanthus sample to be tested is considered to have a fruit-bearing phenotype.

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