An InDel marker primer for identifying F1 generation of cherry blossom hybrids and its application

Abstract

This application belongs to the field of molecular biology technology, specifically disclosing an InDel marker primer for identifying the F1 generation of cherry blossom hybrids and its application. Based on cherry blossom whole-genome resequencing data, this application develops insertion / deletion InDel polymorphic sites suitable for identifying cherry blossom hybrid offspring. Furthermore, candidate InDel markers capable of effectively distinguishing between 'Yae Hanhizakura' and 'Xiangyun' hybrid offspring are screened. The selected markers are experimentally verified using the genomic DNA of the two parents, 'Yae Hanhizakura' and 'Xiangyun', and their F1 individuals. From this, the highly specific and high-resolution InDel marker primer IDG2 is identified, thus establishing a rapid, accurate, and efficient F1 generation identification technology system. This marker exhibits outstanding advantages such as high stability, high specificity, and ease of operation in identifying genuine 'Yae Hanhizakura' and 'Xiangyun' hybrid F1 generations.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, specifically to an InDel marker primer for identifying F1 generation of cherry blossom hybrids and its application. Background Technology

[0002] Cherry blossoms belong to the subgenus *Prunus* of the genus *Prunus* in the family Rosaceae. They are a group of distinctive woody plants with extremely high landscape value, and have become one of the most widely used core ornamental tree species in urban green space systems and garden landscape design. In terms of natural distribution, the native distribution area of ​​the *Prunus* subgenus is highly concentrated in the temperate to subtropical climate zones of the Northern Hemisphere. Globally, approximately 150 native species of the *Prunus* subgenus have been formally recorded, of which 52 native species and varieties are distributed in my country, accounting for more than one-third of the total known germplasm resources worldwide. my country's native *Prunus* subgenus plants not only have abundant germplasm resources, but also exhibit extremely diverse intraspecific phenotypic variations, possessing immense potential for new variety breeding and industrial development. In particular, wild cherry blossom groups such as *Prunus cerasifera*, *Prunus cerasifera var. caudatus*, and *Prunus campanulata* have wide distribution ranges in their natural habitats, and possess outstanding ornamental traits, strong environmental adaptability, and rich genetic diversity, making them core basic genetic materials for the creation of superior new germplasm and the breeding of distinctive new varieties.

[0003] In recent years, the number of cherry blossom varieties with independent intellectual property rights authorized in my country has exceeded 200. Faced with this vast resource of cultivated varieties, existing classification systems typically focus on morphological indicators such as tree shape, floral organ morphology, fruit characteristics, leaf morphology, and winter bud structure, constructing three- and five-level classification standards, supplemented by auxiliary rules based on flowering time and flower color intensity. However, in practical application, significant subjective differences exist in the thresholds for morphological traits among researchers. Furthermore, the widespread phenomenon of overlapping traits and characteristics among many varieties, coupled with the strong phenotypic plasticity of most morphological traits due to changes in the cultivation environment, has led to numerous insurmountable technical bottlenecks in traditional morphological variety identification. Consequently, industry irregularities such as non-standard variety naming, synonymy, and homonymy persist. In addition, most cherry blossom varieties exhibit the typical growth characteristic of flowering before leafing out. The biological characteristics of different stages of flower and leaf development greatly increase the probability of natural hybridization between species, further aggravating the richness of morphological variation within the group. At the same time, cherry blossoms are long-life-history trees with a relatively short flowering period per tree. Currently, the industry has not yet developed a set of rapid and accurate standardized identification methods for varieties.

[0004] 'Xiangyun' is a distinctive new cherry blossom variety selected from a seedling mutation of the natural population of *Prunus cerasifera* var. *truncata*, while 'Yaezakura Hanhizakura' is a double-flowered natural mutation of *Prunus campanulata* var. *bellflower*. Both varieties possess advantages such as vibrant flower color, outstanding resistance to adverse conditions, and a wide range of environmental adaptability. They have extremely high value for landscaping applications and broad market prospects, and are also indispensable core parent materials in the current cherry blossom directional hybridization breeding program. Previous research results have shown that the offspring population obtained by artificially hybridizing 'Xiangyun' as the female parent and 'Yaezakura Hanhizakura' as the male parent exhibits extremely rich types of phenotypic segregation and genetic variation: some hybrid offspring show superimposed morphological characteristics of both parents in their leaves and other vegetative organs, and a considerable proportion of offspring do not show observable significant differences in phenotype from the female parent 'Xiangyun'. In such complex germplasm identification scenarios, traditional morphological identification methods not only fail to accurately trace the parentage of offspring, but also suffer from excessively long identification cycles. Furthermore, the identification results are highly susceptible to interference from plant growth and development stages and external environmental factors, completely failing to meet the practical production needs of early screening and rapid identification of offspring in hybridization breeding. Therefore, the industry urgently needs to construct a rapid, accurate, and efficient molecular marker identification technology system for the hybrid offspring of 'Xiangyun' and 'Yae no Hanhizakura', thereby significantly shortening the cherry blossom breeding cycle, accelerating the breeding process, and promoting the high-quality development of my country's independent cherry blossom variety selection, promotion, and application, as well as the entire cherry blossom seed industry. Summary of the Invention

[0005] Molecular marker technology has the significant advantage of being unaffected by plant developmental stages and external environmental conditions. It requires only a small amount of fresh tissue or samples rapidly dried with silica gel to achieve efficient and accurate identification of specific plant varieties and their F1 hybrids. This application, based on whole-genome resequencing data, identifies insertion-deletion (InDel) polymorphic sites suitable for identifying cherry blossom hybrids. Furthermore, it screens candidate InDel markers that can effectively distinguish between hybrids of 'Yaehana' and 'Xiangyun'. The selected markers are experimentally validated using genomic DNA from two parents ('Yaehana' and 'Xiangyun') and their F1 individuals. This process identifies highly specific and high-resolution molecular markers, thereby establishing a rapid, accurate, and efficient F1 generation identification technology system.

[0006] To achieve the above objectives, this application first provides an InDel-marked primer for identifying the F1 generation of cherry blossom hybrids. The InDel-marked primer includes the IDG2 primer set, the nucleotide sequences of which are shown in SEQ ID NO.1~SEQ ID NO.2.

[0007] As a preferred option, the male parent variety of the F1 generation of the cherry blossom hybrid is Yaezakura, and the female parent variety is Xiangyun.

[0008] Based on a general inventive concept, this application also provides the application of the InDel marker primer in identifying the F1 generation of the hybrid cherry blossom variety Yaezakura and Xiangyun.

[0009] Preferably, the detection method of the InDel-marked primers in identifying the F1 generation of the hybrid cherry blossom variety Yaezakura and Xiangyun includes the following steps:

[0010] S1. Extract total DNA from the Yae-hime, Xiangyun and F1 generation samples of the plants to be tested respectively;

[0011] S2. Using the total DNA extracted in step S1 from the double-flowered cherry blossom, Xiangyun and its hybrid F1 plants as amplification templates, and using the InDel-labeled primers as amplification primers, PCR amplification was performed respectively.

[0012] S3. Perform agarose gel electrophoresis on the PCR amplification products obtained in step S2. If the F1 generation sample to be tested has both the Yaehana cherry blossom and Xiangyun parental bands, it is identified as a true hybrid F1 generation.

[0013] Preferably, the total DNA extraction in step 1 is performed using a modified CTAB method.

[0014] Preferably, the PCR amplification system in step S2 is: 12.5 μL of 2×Taq Master Mix, 1 μL each of the upstream and downstream primers of the InDel-labeled primers, 9.5 μL of ddH2O, and 1 μL of amplification template DNA.

[0015] Preferably, the PCR reaction program in step S2 is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 20 s, annealing at 55.8℃ for 20 s, extension at 72℃ for 10 s, 35 cycles; extension at 72℃ for 5 min, and storage at 4℃.

[0016] The main benefits of this application are as follows:

[0017] 1. Existing methods for identifying F1 generation cherry blossom hybrids primarily rely on morphological methods, distinguishing them by comparing morphological characteristics of leaves, branches, floral organs, and fruits. This method is not only time-consuming but also severely limited by the plant's growth and development stage and seasonal variations, making it difficult to meet the practical needs for rapid and accurate identification. The InDel marker used in this application belongs to third-generation molecular marker technology, possessing outstanding advantages such as high stability, strong specificity, and ease of operation. It is unaffected by plant development stage, growth phase, or external environmental conditions. Only a small amount of fresh tissue or a sample rapidly dried with silica gel is required for accurate and rapid identification of the sample, and the entire detection process can be completed within 5 hours.

[0018] 2. The InDel marker primers provided in this application were used to detect the F1 generation of hybrids of 'Yae Hanhizakura' and 'Xiangyun'. The electrophoretic analysis of the PCR products showed that the IDG2 marker had a 100% accuracy rate in identifying the true F1 generation of hybrids of 'Yae Hanhizakura' and 'Xiangyun'. The screening results of the F1 generation were accurate and reliable, significantly shortening the breeding cycle, reducing breeding costs, and enabling earlier screening of breeding target offspring. This effectively improved the identification efficiency of cherry blossom hybrids and provided a rapid, accurate, and efficient F1 generation identification technology system for accurately identifying the authenticity of interspecific hybrid offspring of cherry blossoms. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a gel electrophoresis result of the IDG2 primer set used in Experiment Example 1 of this application to identify the F1 generation of cherry blossom hybrids. Detailed Implementation

[0021] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0022] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0023] Unless otherwise specified, the percentage sign "%" in this application refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0024] The weight units mentioned in this application may be well-known weight units in the art, such as μg, mg, g, kg, etc., or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0025] Experimental materials: Twenty young leaves were collected from cherry blossom varieties 'Yae Hanhizakura' and 'Xiangyun' and their F1 hybrids, respectively, for screening and specificity verification of InDel markers.

[0026] Example 1: Whole genome resequencing of Prunus campanulata

[0027] (1) Experimental materials: The experimental materials in this embodiment are 17 healthy young leaves of Prunus campanulata collected from three core original distribution areas in Hunan, Fujian and Taiwan. All samples were collected from the fully unfolded young leaves at the top of the current year's new branches that were free from pests and diseases. After collection, they were immediately placed in cryovials containing liquid nitrogen for quick freezing, and then transferred to an ultra-low temperature freezer at -80℃ for storage to avoid DNA degradation affecting subsequent experimental results.

[0028] (2) Cherry Blossom Genome Resequencing Analysis: In this embodiment, total genomic DNA was extracted from 17 young leaf samples of Cherry Blossom using a modified CTAB method. The specific operation steps are as follows: about 0.2g of young leaf sample was placed in a pre-cooled mortar, and sufficient liquid nitrogen was added to quickly grind it into a uniform powder. The powder was immediately transferred to a 2mL centrifuge tube preheated with 65℃ CTAB extraction buffer. After thorough mixing by inverting, the sample was placed in a 65℃ water bath for lysis for 30min, and inverted and mixed once every 10min. Then, an equal volume of liquid nitrogen was added to the centrifuge tube. The chloroform-isoamyl alcohol mixture (volume ratio 24:1) was gently inverted and centrifuged at 12,000 rpm for 10 min. The upper aqueous phase was carefully transferred to a new centrifuge tube. After repeating the extraction once, 0.8 volumes of pre-chilled isopropanol were added, and the mixture was gently mixed and placed at -20°C for 30 min to precipitate. The DNA extraction was then completed by centrifugation, washing the precipitate twice with 75% ethanol, air-drying at room temperature, and dissolving it in enzyme-free water. The final total DNA was temporarily stored at -20°C.

[0029] After DNA extraction, the integrity and degradation of the DNA were detected by 1% agarose gel electrophoresis. The DNA concentration was accurately determined using a Qubit 3.0 fluorescence quantitative quantitation instrument. Only qualified samples with clear electrophoretic bands without obvious tailing, DNA concentration ≥50ng / μL, and total DNA amount ≥2μg were retained for subsequent library construction.

[0030] Genomic DNA samples that have passed quality inspection are placed in an ice-water bath environment and mechanically broken down using an ultrasonic disruptor. The complete genomic DNA is randomly broken into DNA fragments ranging from 300 to 350 bp in length using preset parameters. Subsequently, the target length fragments are purified and recovered using magnetic beads, the ends of the DNA fragments are repaired using T4 DNA polymerase and Klenow fragments, a single adenosine (A) base is added to the 3' end of the DNA fragment, and ligation reaction is performed with Illumina sequencing adapters with specific tags to obtain a ligated DNA mixture.

[0031] The ligation products were separated by 1.8% agarose gel electrophoresis, and the gel band containing the target fragment of 400-450 bp was precisely excised. The target fragment was purified by a gel extraction kit. The purified fragment was used as a template for 8-12 cycles of PCR amplification to finally construct a paired-end sequencing genomic library.

[0032] The constructed sequencing libraries were sequentially subjected to library concentration determination, insert length verification, and precise quantification of effective library concentration. Only libraries with a concentration ≥10nM, insert length meeting expectations, and no adapter contamination were retained. Paired-end 150bp sequencing was performed using the Illumina NovaSeq 6000 high-throughput sequencing platform, with the sequencing depth of each sample controlled to be no less than 30× and the effective sequencing data volume of a single sample no less than 10Gb, to ensure the coverage and accuracy of subsequent variant detection.

[0033] The raw sequencing reads obtained after sequencing were filtered to remove low-quality and adapter contamination sequences, resulting in clean reads. BWA alignment software was used to precisely locate all clean reads onto the publicly available Prunus campestris reference genome sequence. The sequencing depth distribution, 1× coverage, 5× coverage, and 10× coverage of each sample were statistically analyzed to complete the quality control verification of the sequencing data, providing reliable basic data support for the accurate detection of variant sites such as InDel and SNP in the whole genome.

[0034] Example 2 InDel tag design screening

[0035] (1) InDel marker development: Based on the localization results of the clean reads from the *Prunus campanulata* genome resequencing in the *Prunus campanulata* reference genome, insertion / deletion mutations (InDels) were detected using GATK v4.1.4.1 software. The obtained variant sites were then filtered to select highly reliable variant results, resulting in the final InDel site set. From the InDel site set, 500 sites with insertion or deletion bases larger than 50 bp were randomly selected for subsequent validation experiments.

[0036] (2) InDel-labeled PCR primer design: Using the cherry blossom genome sequence as a template, PCR amplification primers with 500 InDel sites were designed using Primer Premier 6.0 software. The main parameters for primer design were: length 18~26 bp; GC content 40%~60%; melting temperature (Tm) 55~61℃; amplification product size 100~400 bp; primers themselves should not have 4 consecutive complementary bases to avoid hairpin structures; primers should not have 4 consecutive complementary bases to avoid primer dimers; and primers should be specific and have high amplification efficiency.

[0037] (3) Screening of polymorphic InDel markers: Total DNA of cherry blossom varieties 'Yae Hanhizakura' and 'Xiangyun' was extracted using the modified CTAB method and PCR amplification was performed using the DNA as a template. Polymorphic markers were screened from 500 candidate InDel markers, i.e., the PCR amplification products showed significant differences in size, which could effectively distinguish the markers of the two cherry blossom varieties 'Yae Hanhizakura' and 'Xiangyun'.

[0038] Experimental Example 1: Authenticity Identification of F1 Generation of True Hybrids of Yaehana Cherry and Xiangyun Cherry

[0039] (1) DNA extraction

[0040] Total DNA was extracted from 20 samples (samples 1-18) of 'Yae no Hanhizakura' (paternal parent), 'Xiangyun' (maternal parent) and their hybrid F1 generation using the modified CTAB method in Example 1.

[0041] (2) PCR amplification

[0042] Using the DNA extracted in step (1) above from 'Yae no Hana Cherry', 'Xiangyun', and their F1 hybrids as amplification templates, and the specific InDel-labeled primers (IDG2) as amplification primers, the primer sequences (5′-3′) are as follows:

[0043] IDG2F: AGCGAATAGCTCTCCATTCTACAC (SEQ ID NO.1);

[0044] IDG2R: ACCTTATGGTTTGGGAGGTCTTT (SEQ ID NO. 2).

[0045] (3) PCR reaction system

[0046] The PCR reaction system consisted of 25 μL, including 12.5 μL of 2×Taq Master Mix, 1 μL each of upstream and downstream primers (10 μmol / L), 9.5 μL of ddH2O, and 1 μL (approximately 50 ng) of DNA from 'Yae Hanhizakura', 'Xiangyun', or the material to be tested.

[0047] (4) PCR reaction procedure

[0048] Pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 20 s, annealing at 55.8℃ for 20 s, extension at 72℃ for 10 s, 35 cycles; extension at 72℃ for 5 min, storage at 4℃.

[0049] (5) Electrophoretic detection of PCR amplification products

[0050] Take 5 μL of the PCR amplification product and perform electrophoresis on a 2% agarose gel (add 1 μL of GelRed 10000× stock solution to every 10 mL of agarose solution). Electrophoresis is performed in 1×TAE buffer at 130 V for 60 min. The electrophoresis results are recorded by photographing the gel using a gel imaging system. If the size of the PCR product is consistent with the expectation and there are no nonspecific bands, the target nucleic acid fragment is considered to have been successfully amplified.

[0051] (6) Specific InDel markers and detection analysis of 'Yaehana Cherry Blossom' and 'Xiangyun' hybrid F1 generation

[0052] The identification results of the IDG2 marker are as follows: Figure 1 As shown: 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18. Among the 18 samples, 17 samples had PCR amplification products with specific bands of both the male parent 'Yae Hanhizakura' and the female parent 'Xiangyun', which are true hybrid F1 generation. Sample 5 only had the female parent band, which may be due to self-fertilization of the female parent or other true F1 generation.

[0053] The true F1 generation identification rate was calculated using the formula: Identification rate = (Number of true F1 generations / Total number of tests) × 100%. In this experimental case, the true F1 generation identification rate was 94.4%.

[0054] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. An InDel marker primer for identifying F1 generation of cherry blossom hybrids, characterized in that, The InDel-labeled primers include the IDG2 primer set, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.

2.

2. The InDel-labeled primer according to claim 1, characterized in that, The male parent variety of the F1 generation of the cherry blossom hybrid is Yaezakura, and the female parent variety is Xiangyun.

3. The application of the InDel marker primer as described in any one of claims 1 to 2 in identifying the F1 generation of the hybrid cherry blossom variety Yaezakura and Xiangyun.

4. The application according to claim 3, characterized in that, The detection method of the InDel marker primer in identifying the F1 generation of the hybrid cherry blossom variety Yaezakura and Xiangyun includes the following steps: S1. Extract total DNA from the Yae-hime, Xiangyun and F1 generation samples of the plants to be tested respectively; S2. Using the total DNA extracted in step S1 from the double-flowered cherry blossom, Xiangyun and its hybrid F1 plants as amplification templates, and using the InDel-labeled primers as amplification primers, PCR amplification was performed respectively. S3. Perform agarose gel electrophoresis on the PCR amplification products obtained in step S2. If the F1 generation sample to be tested has both the Yaehana cherry blossom and Xiangyun parental bands, it is identified as a true hybrid F1 generation.

5. The application according to claim 4, characterized in that, The total DNA extraction in step 1 was performed using a modified CTAB method.

6. The application according to claim 4, characterized in that, The PCR amplification system in step S2 is as follows: 12.5 μL of 2×TaqMaster Mix, 1 μL each of the upstream and downstream primers of the InDel-labeled primers, 9.5 μL of ddH2O, and 1 μL of amplification template DNA.

7. The application according to claim 4, characterized in that, The PCR reaction program in step S2 is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 20 s, annealing at 55.8℃ for 20 s, extension at 72℃ for 10 s, 35 cycles; extension at 72℃ for 5 min, and storage at 4℃.

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