An InDel marker primer combination and detection method for identifying offspring of a cross between cherry varieties xiangyan and jingweng

Abstract

The application provides an InDel marker primer combination and a detection method for identifying offspring of a cross between a cherry variety Xiangyan (a paternal parent) and Jingong (a maternal parent), and belongs to the field of molecular biology. The InDel marker primer combination comprises an IDA2 marker primer group, an IDA13 marker primer group and an IDB17 marker primer group. The total DNA of Xiangyan, Jingong and a hybrid F1 generation to be detected is amplified by using the PCR amplification primers. If the amplification product of the cherry hybrid F1 generation to be detected contains a specific band of the paternal parent Xiangyan after electrophoresis, the hybrid F1 generation to be detected is a true offspring of Xiangyan and Jingong. The application detects 12 hybrid F1 generations of Xiangyan and Jingong. Through electrophoresis analysis of PCR products, the results show that the combination of the IDA2, IDA13 and IDB17 markers can identify 9 hybrid F1 generations, and the identification rate is 75%. Through experimental repetition, the accuracy rate can reach 100%.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to an InDel marker primer combination and detection method for identifying the hybrid offspring of the cherry blossom varieties Xiangyan and Jingweng. Background Technology

[0002] Cherry blossoms are a collective term for plants in the genus *Prunus*, subgenus *Cerasus*, of the family Rosaceae. Due to their excellent ornamental value, they are widely used in landscaping and urban beautification. *Prunus* species are mainly distributed in temperate and subtropical regions of the Northern Hemisphere. There are over 150 species of *Prunus* worldwide, with 52 species and varieties found in China, meaning more than one-third of *Prunus* species are native to China. my country not only has a large number of *Prunus* species but also a rich variety of variations, offering broad prospects for development and utilization. Wild cherry blossom resources such as *Prunus serrulata*, *P. cerasoides*, *P. dielsiana*, and *P. campanulata* are highly ornamental, adaptable, and genetically diverse, making them valuable materials for the creation of superior cherry blossom germplasm and the breeding of new varieties.

[0003] Over the past century, more than 800 cherry blossom horticultural varieties have been obtained through natural variation selection and hybridization. Taxonomists have proposed three- and five-level classification standards for these varieties based on morphological characteristics such as tree shape, flowers, fruits, leaves, and winter buds, as well as classification standards based on flowering period and flower color. However, due to differences in the criteria used by different scholars to judge morphological traits, the overlap and interrelation of traits among varieties, and their strong plasticity in different environments, morphological identification is difficult, leading to confusion in variety names, and frequent occurrences of synonyms and homonyms. Furthermore, most cherry blossoms bloom before their leaves appear, resulting in different flowering and leafing periods, facilitating hybridization, exhibiting rich and diverse morphological variations, long life cycles, and relatively short flowering periods, making rapid and accurate identification methods lacking.

[0004] 'Xiangyan' is a new cherry blossom variety bred from a natural mutation of *Prunus campanulata*. It boasts vibrant flower colors, a tall stature, and high ornamental value with broad application prospects. It is also an important parent variety for hybridization breeding. 'Jingweng' is a Japanese cherry blossom variety, possibly a combination of *Prunus pseudodocerasus* and *Prunus campanulata*, but this requires further verification. Practice has shown that offspring from a hybrid of 'Xiangyan' (male) and 'Jingweng' (female) exhibit rich genetic variation. Some individuals possess characteristics of both parents in their leaves and other vegetative organs, while others show morphological characteristics almost identical to the female parent, making morphological identification impossible. Furthermore, the long breeding cycle and susceptibility to growth, development, and environmental factors make it unsuitable for identifying hybrid offspring. Therefore, there is an urgent need to establish a rapid, accurate, and efficient molecular identification technology system for cherry blossom hybrids to shorten the breeding cycle, accelerate the breeding process, and promote the breeding, promotion, and high-quality development of my country's cherry blossom variety breeding and seed industry. Summary of the Invention

[0005] To address the aforementioned technical challenges, this approach utilizes genome resequencing technology to identify insertion-deletion (InDel) mutation sites suitable for cherry blossom genotype analysis and F1 hybrid identification. InDel markers are then screened for identification of the hybrid offspring of Xiangyan and Jingweng. Finally, the genomic DNA of Xiangyan, Jingweng, and their F1 hybrids is used to validate the candidate markers, resulting in markers with high specificity and resolution. This achieves the goal of rapid, accurate, and efficient identification of the F1 hybrids.

[0006] To achieve the above objectives, this solution first provides an InDel marker primer set for identifying the hybrid offspring of the cherry blossom varieties Xiangyan and Jingweng. The InDel marker primer set is characterized by comprising the IDA2 marker primer set, the IDA13 marker primer set, and the IDB17 marker primer set.

[0007] The nucleotide sequences of the IDA2-labeled primer set are shown in SEQ ID NO.1 to SEQ ID NO.2;

[0008] The nucleotide sequences of the IDA13-labeled primer set are shown in SEQ ID NO.3 to SEQ ID NO.4;

[0009] The nucleotide sequences of the IDB17 labeled primer set are shown in SEQ ID NO.5 to SEQ ID NO.6.

[0010] Based on a general inventive concept, this solution also provides a method for detecting the authenticity of the F1 generation of the cross between the cherry blossom varieties Xiangyan and Jingweng using InDel-labeled primer combinations, characterized by the following steps:

[0011] S1. Total DNA was extracted from the leaves of Xiangyan, Jingweng and the F1 generation of hybrid samples to be tested using the modified CTAB method.

[0012] S2. Using the total DNA from step S1 as an amplification template, PCR amplification is performed using the three marker primer sets described in claim 1 as amplification primers.

[0013] S3. Perform agarose gel electrophoresis on the amplification products of step S2. If the F1 generation sample to be tested has a specific band of the father in the amplification products labeled with IDA2, IDA13 or IDB17, then the F1 generation hybrid sample to be tested is a true offspring of Xiangyan and Jingweng hybridization.

[0014] In step S1, Xiangyan is the father and Jingweng is the mother.

[0015] Preferably, the PCR amplification reaction system in step S2 is as follows: 12.5 μL of 2×Taq Master Mix, 1 μL each of 10 μmol / L upstream and downstream primers, 9.5 μL of ddH2O, and 1 μL of 50 ng / μL of DNA from Xiangyan, Jingweng, or the F1 generation material to be tested.

[0016] Preferably, the PCR amplification reaction program in step S2 is as follows: For IDA2-labeled PCR: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, storage at 4℃; For IDA13-labeled PCR: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 52℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, storage at 4℃; For IDB17-labeled PCR: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 49℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, storage at 4℃.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The detection method provided by the present invention reduces the dependence on morphological identification. The designed InDel marker is a third-generation molecular marker with good stability, strong specificity, and simple operation. It is not affected by the plant development period, growth stage and environment. Only a small amount of fresh tissue or silica gel-dried tissue sample is needed to accurately and quickly identify the sample to be tested. Under the premise of complete instruments and equipment, the detection results can be obtained in about 3 hours.

[0019] (2) The three InDel markers provided by this invention were used to detect 12 F1 hybrids of Xiangyan and Jingweng. The results of PCR product electrophoresis analysis showed that 9 F1 hybrids could be identified by combining IDA2, IDA13 and IDB17 markers, with an identification rate of 75%. Through experimental replication, the accuracy rate can reach 100%. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results of the IDA2 marker identification of the F1 generation in Experiment Example 1;

[0022] Figure 2 The results of the IDA13 marker identification of the F1 generation in Experiment Example 1;

[0023] Figure 3 The results of the IDB17 marker identification of the F1 generation of the hybrid in Experiment Example 1 are shown. Detailed Implementation

[0024] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0025] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0026] 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.

[0027] Example 1

[0028] InDel marker filtering

[0029] 1. Genome resequencing analysis of Prunus campanulata germplasm resources

[0030] Seventeen young leaf samples of *Prunus campanulata* were collected from Hunan, Fujian, and Taiwan. Total DNA was extracted using a modified CTAB method. After DNA testing, the DNA was mechanically fragmented (using ultrasound). The fragmented DNA was then purified, end-repaired, 3′-A-added, and ligated with sequencing adapters. Fragment size selection was performed using agarose gel electrophoresis, followed by PCR amplification to form sequencing libraries. The constructed libraries underwent quality control, and those that passed were sequenced using an Illumina NovaSeq 6000 platform. The sequencing depth was greater than 30× (data greater than 10Gb). The obtained sequencing reads were remapped to the *Prunus campanulata* reference genome. By comparing the positions of clean reads on the reference genome, the sequencing depth and genome coverage of each sample were statistically analyzed to facilitate subsequent variant detection.

[0031] 2. InDel tag development

[0032] Based on the localization results of the clean reads from the *Prunus campanulata* genome resequencing in the *Prunus campanulata* reference genome, insertion / deletion mutations (InDel) were detected using GATK v4.1.4.1 software. The obtained variant sites were then filtered to select highly reliable variants, resulting in the final InDel site set. InDel marker statistics were then performed. Two hundred sites with insertions or deletions of 30 bp or more were randomly selected from the InDel site set for subsequent validation experiments.

[0033] 3. InDel-labeled PCR primer design

[0034] Using the *Prunus campanulata* genome sequence as a template, PCR primers for 200 InDel sites were designed using Primer Premier 6.0 software. Key primer design parameters were: length 18–26 bp; GC content 40%–60%; melting temperature (Tm) 54–61℃; amplified product size 100–400 bp; primers themselves should not have four consecutive complementary bases to avoid hairpin structures; primers should not have four consecutive complementary bases to avoid primer dimers; and primer specificity and high amplification efficiency were ensured.

[0035] 4. Polymorphism InDel marker filtering

[0036] Total DNA was extracted from the cherry blossom varieties Xiangyan and Xiangyun using a modified CTAB method, and PCR amplification was performed using the DNA as a template. Polymorphic markers were screened from 200 candidate InDel markers. The markers that showed significant differences in the size of the PCR amplification products, as detected by 2% agarose gel electrophoresis, could effectively distinguish the two cherry blossom varieties Xiangyan and Xiangyun.

[0037] Experimental Example 1

[0038] Identification of F1 offspring of Xiangyan and Jingweng

[0039] 1. Experimental Materials

[0040] Tender leaves from cherry blossom varieties 'Xiangyan' and 'Jingweng', as well as the F1 generation to be tested, were collected in a total of 14 samples for screening and specificity verification of InDel markers.

[0041] 2. InDel marker detection

[0042] 2.1 DNA Extraction

[0043] Total DNA was extracted from 14 samples, including Xiangyan, Jingweng, and the F1 generation to be tested, using a modified CTAB method.

[0044] 2.2 PCR amplification

[0045] Using the DNA extracted in step 2.1 from 'Xiangyan', 'Jingweng' and their F1 hybrids as amplification templates, PCR amplification was performed using three InDel-labeled specific primers (see Table 1).

[0046] Table 1 Information on Specific Marker Primers

[0047]

[0048]

[0049] 2.3 PCR reaction system

[0050] The PCR reaction system is 25 μL, including 12.5 μL of 2×Taq Master Mix, 1 μL each of 10 μmol / L upstream and downstream primers, 9.5 μL of ddH2O, and 1 μL of 50 ng / μL DNA from Xiangyan, Jingweng, or the material to be tested.

[0051] 2.4 PCR reaction procedure

[0052] The IDA2-labeled PCR reaction procedure was as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, and storage at 4℃.

[0053] The PCR reaction procedure for IDA13 labeling was as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 52℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, and storage at 4℃.

[0054] IDB17-labeled PCR reaction procedure: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 49℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, store at 4℃.

[0055] 2.5 Electrophoretic detection of PCR amplification products

[0056] PCR amplification products were electrophoresed on a 2% agarose gel (5 μL of GelRed 10000× stock solution was added to every 50 mL of agarose solution) using 1×TAE buffer. The electrophoresis voltage was set to 110 V, and the electrophoresis time was 40 min. The electrophoresis results were photographed and recorded using a gel imaging system. If the PCR product size was consistent with the expectation and there were no nonspecific bands, the target nucleic acid fragment was considered to have been successfully amplified.

[0057] 3. Specific InDel markers and detection analysis of F1 generation from the cross between Xiangyan and Jingweng

[0058] Theoretically, a true hybrid F1 generation should amplify a paternal-specific band, and the identification results of the IDA2 marker should be as follows: Figure 1 As shown, the PCR amplification products of seven samples, namely 1, 2, 3, 5, 6, 8 and 10, have specific bands of the father (Xiangyan), and are true F1 hybrids, with an identification rate of 58.33%.

[0059] The identification results of the IDA13 marker are as follows: Figure 2 As shown, the PCR amplification products of six samples, namely 1, 2, 5, 6, 8 and 10, have paternal-specific bands, indicating that they are true F1 hybrids with an identification rate of 50%.

[0060] The identification results of the IDB17 marker are as follows: Figure 2 As shown, the PCR amplification products of four samples, namely No. 2, No. 8, No. 9 and No. 11, have paternal-specific bands, indicating that they are true F1 hybrids with an identification rate of 33.33%.

[0061] Based on the identification results of IDA2, IDA13 and IDB17 markers, samples 1, 2, 3, 5, 6, 8, 9, 10 and 11, a total of 9 samples, are true F1 hybrids, with an identification rate of 75%.

[0062] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, any improvements and modifications obtained without departing from the technical concept of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A kind of InDel marker primer combination in the method for detecting the authenticity of cherry variety Xiangyan and Jingong hybrid F1 generation, characterized in that, Includes the following steps: S1. Total DNA was extracted from the leaves of Xiangyan, Jingweng and the F1 generation of hybrid samples to be tested using the modified CTAB method. S2. Using the total DNA from step S1 as a template, PCR amplification is performed using the InDel-labeled primer set as the amplification primers. The InDel-labeled primer set consists of the IDA2-labeled primer set, the IDA13-labeled primer set, and the IDB17-labeled primer set. The nucleotide sequences of the IDA2-labeled primer set are shown in SEQ ID NO. 1 to SEQ ID NO. 2; the nucleotide sequences of the IDA13-labeled primer set are shown in SEQ ID NO. 3 to SEQ ID NO. 4; and the nucleotide sequences of the IDB17-labeled primer set are shown in SEQ ID NO. 5 to SEQ ID NO.

6. S3. Perform agarose gel electrophoresis on the amplification products of step S2. If the F1 generation sample to be tested has a specific band of the father in the amplification products labeled with IDA2, IDA13 or IDB17, then the F1 generation hybrid sample to be tested is a true offspring of Xiangyan and Jingweng hybridization. In step S1, Xiangyan is the father and Jingweng is the mother.

2. The method according to claim 1, characterized in that, The PCR amplification reaction system in step S2 is as follows: 12.5 μL of 2×Taq Master Mix, 1 μL each of 10 μmol / L upstream and downstream primers, 9.5 μL of ddH2O, and 1 μL of 50 ng / μL DNA from Xiangyan, Jingweng, or the F1 generation material to be tested.

3. The method according to claim 1, characterized in that, The PCR amplification reaction program in step S2 is as follows: IDA2-labeled PCR reaction program: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, storage at 4℃. PCR reaction procedure for IDA13 labeling: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 52℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, storage at 4℃; IDB17-labeled PCR reaction procedure: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 49℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, storage at 4℃.

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