InDel marker for identifying authenticity of fraxinus velutina hybrid and application thereof

Abstract

The application belongs to the technical field of identification of Fraxinus velutina hybrid, and particularly relates to an InDel marker for identifying the authenticity of Fraxinus velutina hybrid and application thereof. The InDel marker is located at positions 63-150 of the sequence shown in SEQ ID NO:2, and the sequence of the InDel marker is shown in SEQ ID NO:1. Meanwhile, based on the InDel marker, the application obtains a group of primers with stable polymorphism, high amplification efficiency and clear bands, and establishes a technical system for identifying the authenticity of hybrid offspring at the seedling stage without relying on expensive equipment, thereby providing an efficient and low-cost molecular tool for Fraxinus velutina new variety breeding, variety right protection and market supervision, and having good application value.

Description

Technical Field

[0001] This invention belongs to the field of identification technology for velvet ash hybrids, specifically involving an InDel marker for identifying the authenticity of velvet ash hybrids and its application. Background Technology

[0002] Velvet wax ( Fraxinus velutina With its rapid growth, cold resistance, strong stress resistance, and golden autumn foliage, *Fraxinus velutipes* is widely used in ecological restoration and landscape construction. Artificially controlled pollination to obtain hybrid offspring with target traits has become an important method for breeding new varieties of *Fraxinus velutipes*. However, the morphological differences between different varieties of *Fraxinus velutipes* are small, and traditional field identification methods suffer from drawbacks such as long cycles, low accuracy, and susceptibility to environmental interference. Furthermore, the F1 generation of hybrids lacks stable and quantifiable morphological markers in the early stages (seedling stage), affecting the timely application and protection of new variety rights for *Fraxinus velutipes*.

[0003] Insertion / deletion (InDel) variants are codominant markers widely distributed in the genome, with readily apparent length differences and convenient PCR detection. They combine the high stability of SNPs with the high polymorphism of SSRs, making them particularly suitable for rapid identification of the authenticity of hybrid offspring. Compared to SSR markers, InDel markers do not require complex capillary electrophoresis platforms; visualization and interpretation can be achieved solely through conventional PCR combined with agarose gel electrophoresis, significantly reducing the detection threshold and cost. Furthermore, compared to SNPs, InDels exhibit more pronounced length differences and can be directly distinguished by electrophoresis, making them more suitable for grassroots breeding units and resource-limited scenarios.

[0004] Therefore, developing specific InDel sites for different parents of *Fraxinus velutipes* is of great significance for the breeding of new *Fraxinus velutipes* varieties, the protection of variety rights, and market supervision. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to provide an InDel marker for identifying the authenticity of velvety ash hybrids and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an InDel marker for identifying the authenticity of a velvety ash hybrid, the InDel marker being located at positions 63 to 150 of the sequence shown in SEQ ID NO:2, the sequence of the InDel marker being shown in SEQ ID NO:1.

[0007] A second aspect of the invention provides the use of detecting the InDel-labeled substance described in the first aspect in the identification of velvet ash hybrids.

[0008] Furthermore, the substance contains PCR primers for amplifying genomic DNA fragments of velvet wax containing the InDel molecular marker.

[0009] Furthermore, the PCR primers consist of a forward primer and a reverse primer, wherein the forward primer is a single-stranded DNA that specifically binds upstream of the InDel molecular marker in the genomic DNA of *Cephalotaxus fortunei*, and the reverse primer is a single-stranded DNA that specifically binds downstream of the InDel molecular marker in the genomic DNA of *Cephalotaxus fortunei*.

[0010] Furthermore, the forward primer sequence is shown in SEQ ID NO:8, and the reverse primer sequence is shown in SEQ ID NO:9.

[0011] Furthermore, the velvety ash hybrid is a velvety ash hybrid with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent.

[0012] A third aspect of the present invention provides a method for identifying a hybrid of velvety white wax, wherein the method uses a primer set with a forward primer sequence as shown in SEQ ID NO:8 and a reverse primer sequence as shown in SEQ ID NO:9 to perform PCR amplification on the sample to be tested, and identifies the hybrid based on the characteristics of the amplified fragments. The aforementioned velvety ash hybrid is a velvety ash hybrid with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent.

[0013] Furthermore, the PCR amplification reaction system includes: DNA polymerase, reaction buffer, amplification primers, DNA of the sample to be tested, and sterile water.

[0014] Furthermore, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 39 cycles; 72℃ extension for 10 min.

[0015] Furthermore, the identification methods include agarose gel electrophoresis or first-generation sequencing.

[0016] Furthermore, the identification method is agarose gel electrophoresis. If the sample shows a 176 bp electrophoretic band, the sample is the female parent of "Lu La No. 2"; if the sample shows a 264 bp electrophoretic band, the sample is the male parent of "Lu Rong"; if the sample shows both 176 bp and 264 bp electrophoretic bands, the sample is a hybrid of velvety white wax with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent.

[0017] Furthermore, the identification method is first-generation sequencing. If the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO:3, then the sample to be tested is the female parent "Lu La No. 2"; if the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO:2, then the sample to be tested is the male parent "Lu Rong"; if the sequence of the electrophoretic recovered fragment contains both the sequences shown in SEQ ID NO:2 and SEQ ID NO:3, then the sample to be tested is a hybrid of velvety white wax with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention uses two excellent velvety ash varieties, "Lu La 2" and "Lu Rong," as the maternal and paternal parents, respectively. Through high-depth whole-genome resequencing, specific InDel sites between the parents were identified. InDel primer pairs with stable polymorphism, high amplification efficiency, and clear bands were designed and screened. A technical system was established that can identify the authenticity of hybrid offspring without relying on expensive equipment during the seedling stage. This provides an efficient and low-cost molecular tool for the breeding of new velvety ash varieties, variety rights protection, and market supervision, and has good application value. Attached Figure Description

[0019] Figure 1 The figures show the amplification results of the core primers 2-4, 14-4, 15-2, 15-4, 18-4, and 21-3 in the two parental lines “Lu La No. 2” and “Lu Rong” in the embodiments of the present invention. In the figure, M represents the DNA molecular weight standard, L2 represents the maternal parent “Lu La No. 2”, and LR represents the paternal parent “Lu Rong”.

[0020] Figure 2 The figure shows the amplification band results of primer 15-2 in the two parents, “Lu La No. 2” and “Lu Rong”, and their real hybrids in the embodiments of the present invention. In the figure, M represents the DNA molecular weight standard, L2 represents the maternal parent “Lu La No. 2”, LR represents the paternal parent “Lu Rong”, and 1 to 29 are the real hybrids with “Lu La No. 2” as the maternal parent and “Lu Rong” as the paternal parent. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0023] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0024] The experimental material “Lu La No. 2” was collected from Qingdao, Shandong, and “Lu Rong” was collected from Dongying, Shandong.

[0025] Example 1: InDel marker discovery and core primer screening (1) Genomic DNA extraction and quality testing: Two superior velvety ash varieties, "Lu La No. 2" and "Lu Rong," were used as the female and male parents, respectively. High-quality genomic DNA was extracted from the leaves of the two parents and 29 true hybrids identified in the field using a modified CTAB method. The DNA concentration and purity (OD260 / 280 required to be between 1.8 and 2.0) were determined using an ultra-micro spectrophotometer for later use.

[0026] (2) Whole genome resequencing and InDel marker discovery: Libraries were constructed from DNA of "Lu La 2" (maternal parent) and "Lu Rong" (paternal parent), and sequenced using an Illumina NovaSeq6000 PE150 at a sequencing depth ≥15×. Raw data underwent quality control and alignment to the *Cephalotaxus fortunei* reference genome using BWA-MEM, and InDel detection was performed. High-confidence, specific loci with a length difference ≥30 bp between parents were selected for subsequent primer design. PCR primers were designed using Primer3 software in conserved regions flanking the differing loci.

[0027] (3) Validation of InDel markers and screening of core primers: Using parental DNA as a template, all candidate primers were validated by PCR amplification. Reaction mixture (20 μL): 10 μL 2 x Taq Plus Master Mix, 1 μL each of forward and reverse primers (10 μM), 1 μL template DNA, and ddH2O to make up the volume. Reaction program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, annealing at 45–60℃ for 30 s (optimized according to primer Tm values), extension at 72℃ for 45 s, for a total of 39 cycles; final extension at 72℃ for 10 min.

[0028] PCR products were separated by electrophoresis on a 3% agarose gel (containing safe nucleic acid dye) at 110 V for 40–60 min. The gel imaging system showed that stable, clear core primers with expected length differences could be amplified between the two parents.

[0029] Six pairs of core primers (2-4, 14-4, 15-2, 15-4, 18-4, and 21-3) exhibiting polymorphism between the two parents were obtained through screening. Primer information is shown in Table 1, and amplification results are as follows: Figure 1 As shown.

[0030] Table 1 Primer Information

[0031] Example 2: Verification of Core Primer Accuracy Using genomic DNA from the two parental varieties, "Lu La 2" and "Lu Rong," and a real hybrid as templates, PCR amplification was performed using the core identification primers screened in Example 1. The reaction mixture (20 μL) consisted of 10 μL of 2 x Taq Plus Master Mix, 1 μL each of forward and reverse primers (10 μM), 1 μL of template DNA, and ddH2O to make up the volume. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 39 cycles; and a final extension at 72℃ for 10 min. The PCR products were analyzed by electrophoresis on a high-resolution agarose gel (3%).

[0032] Result determination: Hybrid (F1) type: has heterozygous bands or characteristic bands of the paternal parent. This is sufficient evidence for identifying hybrids. The accuracy of the primers is verified using real hybrid plants.

[0033] Accuracy rate = (Number of individuals with heterozygous bands or characteristic bands of the paternal parent) / (Total number of hybrid seedlings) * 100%.

[0034] One pair of InDel marker primers with 100% accuracy was obtained: 15-2F / R. The primer sequence is shown in Table 1. The corresponding site of the primer is located at the 7674371st base of chromosome 15 (reference genome is the velvet ash genome JAZGXY000000000).

[0035] No mutation was found at this site in “Lu La No. 2”, and the amplification product sequence is: GTGACCATCATGCCCAAGGACATCCAGCTAGCTCGTCGGATTAGGGGCGAGCGTGCTTAGACATTGTTAAACTCATTAGCTATAATTTTAATTCTAGTTGGTTGTGTGGACAATGATTTTCAATGGCCGGTGGTGGTGGTGTGCTGTAATGAATTTGGCGTTTTTGTTGTAGGATG (SEQ ID NO:3).

[0036] The "Lu Rong" gene has an 88 bp base insertion at this site, and its amplified product sequence is: GTGACCATCATGCCCAAGGACATCCAGCTAGCTCGTCGGATTAGGGGCGAGCGTGCTTAGACGACGACCCCTGATCATCTTGCTTACATGTGATTTTAGTAAACTATGGTGTTCTTTAGTCTATCTTTGTTAAGGAATATATAGTTAATCATTGTTAAACTCATTAGCTATAATTTTAATTCTAGTTGGTTGTGTGGACAATGATTTTCAATGGCCGGTGGTGGTGGTGTGCTGTAATGAATTTGGCGTTTTTGTTGTAGGATG (SEQ ID NO:2). The inserted sequence is: GACGACCCCTGATCATCTTGCTTACATGTGATTTTAGTAAACTATGGTGTTCTTTAGTCTATCTTTGTTAAGGAATATATAGTTAATC (SEQ ID NO:1), and the two parents differ by 88 bp at this site. Figure 2 As shown, the primers amplified heterozygous bands with both paternal and maternal specific bands in the offspring of genuine hybrids with a 100% accuracy rate. The method of this invention can quickly and accurately identify the authenticity of the hybrid of "Lu La No. 2" (maternal parent) and "Lu Rong" (paternal parent), laying a good foundation for the identification of hybrids of different parental combinations of velvet ash.

[0037] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An InDel marker for identifying the authenticity of velvety ash hybrids, characterized in that, The InDel marker is located at positions 63 to 150 of the sequence shown in SEQ ID NO:2, and the sequence of the InDel marker is shown in SEQ ID NO:1; the velvety ash hybrid is a velvety ash hybrid with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent.

2. The application of the InDel-labeled substance as described in claim 1 in the identification of velvet ash hybrids; wherein the velvet ash hybrid is a velvet ash hybrid with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent.

3. The application as described in claim 2, characterized in that, The substance contains PCR primers for amplifying genomic DNA fragments of velvet wax containing the InDel molecular marker.

4. The application as described in claim 3, characterized in that, The PCR primers consist of a forward primer and a reverse primer. The forward primer is a single-stranded DNA that specifically binds upstream of the InDel molecular marker in the genomic DNA of *Euonymus alatus*, and the reverse primer is a single-stranded DNA that specifically binds downstream of the InDel molecular marker in the genomic DNA of *Euonymus alatus*.

5. The application as described in claim 4, characterized in that, The forward primer sequence is shown in SEQ ID NO:8, and the reverse primer sequence is shown in SEQ ID NO:

9.

6. A method for identifying hybrid varieties of velvety ash, characterized in that, The method uses a primer set with a forward primer sequence as shown in SEQ ID NO:8 and a reverse primer sequence as shown in SEQ ID NO:9 to perform PCR amplification on the test sample, and identifies the amplified fragments based on their characteristics. The aforementioned velvety ash hybrid is a velvety ash hybrid with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent; The identification method is agarose gel electrophoresis. If the sample to be tested shows electrophoretic bands of 176 bp and 264 bp, then the sample to be tested is a hybrid of velvet ash with "Lu La No. 2" as the female parent and "Lu Rong" as the male parent.

7. The method as described in claim 6, characterized in that, The PCR amplification reaction system includes: DNA polymerase, reaction buffer, amplification primers, DNA from the sample to be tested, and sterile water.

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