An InDel marker primer combination and detection method for identifying Xiangyan and Xiangyun cherry hybrid F1 generation

Abstract

The application provides an InDel marker primer combination and a detection method for identifying Xiangyan (a paternal parent) and Xiangyun (a maternal parent) cherry hybrid F1 generation, and belongs to the field of molecular biology; the InDel molecular marker developed by the application comprises an IDC10 marker primer group and an IDG8 marker primer group, and can be completed through PCR amplification and agarose gel electrophoresis detection, thereby solving problems such as a long phenotype identification period, relatively low accuracy and efficiency; as long as the amplification product of the hybrid F1 generation to be detected has a specific band of the paternal parent, the F1 generation is a true hybrid offspring; the InDel marker method is used to identify the authenticity of the Xiangyan and Xiangyun cherry hybrid F1 generation, and the application has low requirements on instruments and equipment, can be completed through conventional PCR and agarose gel electrophoresis, has simple operation steps, is short in period, high in efficiency, and accurate and reliable in result.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, and in particular to an InDel marker primer combination for identifying F1 generation of Prunus serrulata cv. Xiangyan and Prunus serrulata cv. Xiangyun and a detection method. BACKGROUND

[0002] Prunus serrulata is a collective term for plants of the Rosaceae Prunus subg. Cerasus, which has excellent ornamental value and is widely used in landscaping and urban beautification. Prunus plants are mainly distributed in the temperate and subtropical regions of the Northern Hemisphere. There are more than 150 species of Prunus worldwide, 52 species and varieties in China, and more than one-third of them are native to China. Prunus plants in China not only have a wide variety of species, but also have a wide variety of variations, with a broad prospect for development and utilization. Wild Prunus resources such as Prunus serrulata, P. cerasoides, P. dielsiana, and P. campanulata have good ornamental value, strong adaptability, and rich genetic diversity, making them valuable materials for creating excellent Prunus germplasm and breeding new varieties.

[0003] In the past century, more than 800 Prunus horticultural varieties have been obtained through natural variation screening and hybrid breeding. In response to these horticultural varieties, taxonomists have proposed three- and five-level classification standards based on morphological characteristics such as tree shape, flower, fruit, leaf, and winter bud, as well as flowering period and flower color classification standards. Due to differences in judgment standards for morphological characteristics among different scholars, the overlap of characteristics between varieties, and the strong plasticity of characteristics in different environments, morphological identification is difficult, leading to confusion of variety names, homonymy, and synonymy. Moreover, most Prunus plants have flowers before leaves, with different flowering and leafing periods, making them easy to hybridize and have rich and diverse morphological variations. They have a long life cycle and a relatively short flowering period, lacking a rapid and accurate identification method.

[0004] ‘Xiangyan’ and ‘Xiangyun’ are new Prunus varieties selected from natural variation individuals of P. campanulata and P. dielsiana, respectively. They have bright flower colors and tall tree bodies, with high ornamental value and broad application prospects, and are also important parents for hybrid breeding. Practice has shown that the offspring obtained by crossing ‘Xiangyan’ as the father and ‘Xiangyun’ as the mother have rich genetic variations. Some individuals have both paternal and maternal characteristics in their leaf blades and other vegetative organs, while some individuals have morphological characteristics similar to the mother. Therefore, traditional morphological methods cannot be used to identify them, and the long cycle time and the influence of growth and development and the environment make it impossible to meet the needs of identifying hybrid offspring. Therefore, it is urgent to establish a rapid, accurate, and efficient molecular identification technology system for Prunus hybrid offspring, shorten the breeding cycle, speed up the breeding process, and promote the development of Prunus variety breeding, popularization, and high-quality seed industry. SUMMARY

[0005] In order to solve the above technical problems, the present application is based on genome resequencing technology, excavates insertion and deletion mutation (InDel) sites suitable for genotype analysis and hybrid F1 generation identification of cherry, screens InDel markers that can be used to identify hybrid offspring of Xiangyan and Xiangyun, and then uses the genomic DNA of Xiangyan, Xiangyun and their hybrid F1 generation to verify the candidate markers, so as to obtain markers with good specificity and high resolution, thereby achieving the purpose of rapid, accurate and efficient identification of F1 generation.

[0006] In order to achieve the above purpose, the present application first provides an InDel marker primer combination for identifying the hybrid F1 generation of Xiangyan and Xiangyun cherry, which comprises an IDC10 marker primer combination and an IDG8 marker primer combination.

[0007] The nucleotide sequence of the IDC10 marker primer combination is shown in SEQ ID NO. 1-2.

[0008] The nucleotide sequence of the IDG8 marker primer combination is shown in SEQ ID NO. 3-4.

[0009] Based on a general inventive concept, the present application also provides a detection method for identifying the authenticity of the hybrid F1 generation of Xiangyan and Xiangyun cherry using an InDel marker primer combination, comprising the following steps:

[0010] S1, using a modified CTAB method to extract total DNA from the leaves of Xiangyan, Xiangyun and the F1 generation sample to be tested;

[0011] S2, using the total DNA of step S1 as an amplification template, using the IDC10 marker primer combination and the IDG8 marker primer combination as amplification primers, and performing PCR amplification;

[0012] S3, performing agarose gel electrophoresis detection on the amplification product of step S2, if the F1 generation sample to be tested has a specific band of the father in the amplification product of the IDC10 marker or the IDG8 marker, then the F1 generation sample to be tested is a true hybrid offspring of Xiangyan and Xiangyun;

[0013] In the S1 step, Xiangyan is the father and Xiangyun is the mother.

[0014] As a preferred embodiment, the reaction system of PCR in step S2 is as follows: 2×Taq Master Mix 12.5 μL, 10 μmol / L of each of the upper and lower primers 1 μL, ddH2O 9.5 μL, 50 ng / μL of DNA of Xiangyan, Xiangyun or the F1 generation material to be tested 1 μL, and the total reaction system is 25 μL.

[0015] As preferred, the reaction procedure of PCR in the S2 step is: the IDC10 labeled PCR reaction procedure: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 57.6℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, 4℃ storage; the IDG8 labeled PCR reaction procedure: 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, 4℃ storage.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] (1) The InDel molecular marker primer set developed in the application solves the problem that the true F1 generation cannot be completely identified by morphology, overcomes the defects of long period, and is limited by time and plant growth and development stage, and can quickly and accurately identify the demand of hybrid F1 generation, and the true F1 generation plant can amplify the paternal band type, so that the true F1 generation can be easily identified.

[0018] (2) The two InDel markers in the application are applied to detect 79 hybrid F1 generations of Xiangyan and Xiangyun, and through PCR product electrophoresis analysis, combined with IDC10 and IDG82 markers, 68 hybrid F1 generations can be identified, the identification rate is 86.08%, and the accuracy can reach 100%.

[0019] (3) In summary, the InDel marker method is used to identify the true F1 generation of Xiangyan and Xiangyun, the InDel marker has good stability, strong specificity, simple operation, and is not affected by plant development period, growth stage and environment, under the premise of complete instruments and equipment, the detection result can be obtained in about 3 hours, the detection process has low requirement on instruments and equipment, and the operation steps are simple, and the result is accurate and reliable. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0021] Figure 1 IDC10 marker hybrid F1 generation identification results in experimental example 1;

[0022] Figure 2 IDG8 marker hybrid F1 generation identification results in experimental example 1. DETAILED DESCRIPTION

[0023] To make the technical problems, technical solutions and advantages of the present application clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0024] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0025] If not specifically indicated, the technical means used in the examples are the conventional means known to those skilled in the art; if not specifically indicated, the reagents used in the examples are commercially available.

[0026] Example 1

[0027] Screening of InDel markers

[0028] 1. Genome resequencing analysis of Prunus subhirtella var. pendula germplasm resources

[0029] Seventeen Prunus subhirtella var. pendula tender leaf samples from Hunan, Fujian, Taiwan and other places were collected, and the total DNA was extracted by using the improved CTAB method. After the DNA detection was qualified, the DNA was fragmented by mechanical breaking (ultrasonic wave), and then the fragmented DNA was subjected to fragment purification, end repair, 3' end A addition, ligation of sequencing adapters, and then subjected to fragment size selection by agarose gel electrophoresis, PCR amplification to form a sequencing library, and the library was first subjected to library quality inspection. The qualified library was subjected to sequencing by using the Illumina NovaSeq6000 platform. The sequencing depth was more than 30 times (data more than 10 Gb). The obtained sequencing reads were repositioned to the Prunus subhirtella reference genome, the positions of the Clean reads on the reference genome were aligned and positioned, and the sequencing depth and genome coverage of each sample were counted, so as to facilitate the next step of variation detection.

[0030] 2. Development of InDel markers

[0031] According to the positioning results of the Prunus subhirtella genome resequencing Clean reads on the Prunus subhirtella reference genome, the GATK v4.1.4.1 software was used to detect insertion / deletion mutations (Insertion / deletion mutation, InDel), and then the obtained variation sites were filtered to screen high-reliability variation results, to obtain the final InDel site set, and the InDel marker was counted. 200 sites with insertion or deletion of more than 30 bp of bases were randomly selected from the InDel site set for subsequent verification experiments.

[0032] 3. Design of InDel marker PCR primers

[0033] The genomic sequence of the clock flower is used as a template, and the primer premier 6.0 software is used to design 200 InDel site PCR amplification primers. The main parameters of primer design: length 18-26 bp; GC content 40%-60%, melting temperature (Tm) 57-61℃, amplification product size 100-400 bp; The primer itself cannot have continuous 4 bases complementary to avoid forming a hairpin structure; The primers cannot have continuous 4 bases complementary to avoid forming primer dimers; Ensure the specificity and high amplification efficiency of the primers.

[0034] 4, screening of polymorphic InDel markers

[0035] The total DNA of cherry varieties Xiangyan and Xiangyun is extracted by the improved CTAB method, and is used as a template for PCR amplification. From 200 candidate InDel markers, the markers with polymorphism are screened, that is, through 2% agarose gel electrophoresis detection, the PCR amplification product size has obvious difference, which can well distinguish the two cherry varieties Xiangyan and Xiangyun.

[0036] Experimental example 1

[0037] Identification of Xiangyan and Xiangyun hybrid F1 generation

[0038] 1, experimental materials

[0039] Collect the tender leaves of cherry varieties Xiangyan, Xiangyun and the hybrid F1 generation to be tested, a total of 81 materials, for screening and specificity verification of InDel markers.

[0040] 2, InDel marker detection

[0041] 2.1 DNA extraction

[0042] The total DNA of the above Xiangyan, Xiangyun and the hybrid F1 generation to be tested is extracted by the improved CTAB method.

[0043] 2.2 PCR amplification

[0044] The DNA of Xiangyan, Xiangyun and their hybrid F1 generation extracted in step 2.1 is used as the amplification template, and 2 pairs of specific InDel marker primers are used as amplification primers for PCR amplification. The InDel marker primers are shown in Table 1:

[0045] Table 1 specific marker primer information

[0046]

[0047]

[0048] 2.3 PCR reaction system

[0049] The PCR reaction system was 25 μL, including 2x Taq Master Mix 12.5 μL, 10 μmol / L of upstream and downstream primers 1 μL each, ddH2O 9.5 μL, 50 ng / μL of Xiangyan, Xiangyun or DNA of hybrid F1 material to be tested 1 μL.

[0050] 2.4 PCR reaction program

[0051] The PCR reaction program of IDC10 marker was 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 57.6℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, 4℃ preservation.

[0052] The PCR reaction program of IDG8 marker was 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 57.6℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, 4℃ preservation.

[0053] 2.5 Electrophoretic detection of PCR amplification product

[0054] The PCR amplification product was electrophoresed on a 2% agarose gel (5 μL of GelRed 10000x stock solution was added to each 50 mL agarose solution), the electrophoresis buffer was 1x TAE buffer, the voltage was set to 110 V, and the electrophoresis time was 40 min. The electrophoresis result was photographed in the gel imaging system, and if the PCR product size was consistent with the expected and there was no non-specific band, it was considered that the target nucleic acid fragment amplification was successful.

[0055] 3, Xiangyan, Xiangyun hybrid F1 generation specific InDel marker and detection analysis

[0056] In theory, the true hybrid F1 generation should amplify the specific band of the father (Xiangyan), and the identification result of IDC10 marker is shown in Figure 1 , 1, 2, 4, 6, 7, 9, 10, 12, 14, 15, 19, 20, 22, 23, 24, 27, 29, 33, 34, 35, 38, 40, 42, 43, 44, 46, 47, 49, 50, 51, 52, 53, 54, 55, 59, 60, 61, 63, 64, 65, 66, 68, 69, 72, 74, 77 and 79, a total of 47 samples of PCR amplification product had the specific band of the father, which was the true hybrid F1 generation, and the identification rate was 59.49%.

[0057] The identification result of IDG8 marker is shown in Figure 2As shown, PCR amplification products of 2, 5, 7, 8, 11, 12, 16, 17, 20, 22, 23, 24, 25, 26, 27, 28, 31, 32, 35, 36, 37, 40, 42, 43, 44, 46, 47, 50, 52, 53, 54, 56, 57, 58, 60, 61, 62, 63, 64, 66, 67, 68, 69, 70, 71, 72, 74, 76, 77, 78, and 79, a total of 51 samples have the specific band of the father, which are true hybrid F1 generation, and the identification rate is 64.56%.

[0058] According to the identification results of the combination of the IDC10 and IDG8 markers, 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 43, 44, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 76, 77, 78, and 79, a total of 68 samples are true hybrid F1 generation, and the identification rate is 86.08%.

[0059] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above. Improvements and changes made by those skilled in the art without departing from the technical concept of the present application should also be considered as the protection scope of the present application.

Claims

1. An InDel marker primer combination for identifying Xiangyan and Xiangyun cherry hybrid F1 generation, characterized in that, The InDel marker primer combination comprises an IDC10 marker primer group and an IDG8 marker primer group; The nucleotide sequence of the IDC10 marker primer group is shown as SEQ ID NO. 1-2; The nucleotide sequence of the IDG8 marker primer group is shown as SEQ ID NO. 3-4.

2. The InDel marker primer combination of claim 1 in the detection method for identifying the authenticity of Xiangyan and Xiangyun cherry hybrid F1 generation, characterized in that, The method comprises the following steps: S1, using a modified CTAB method to extract total DNA of Xiangyan, Xiangyun and F1 generation samples to be tested from leaf blades; S2, using the total DNA of S1 step as an amplification template, using the IDC10 marker primer group and the IDG8 marker primer group as amplification primers, and performing PCR amplification; S3, performing agarose gel electrophoresis detection on the amplification product of S2 step, if the F1 generation sample to be tested has specific bands of the father and the mother in the amplification product of the IDC10 marker or the IDG8 marker, then the F1 generation sample to be tested is the true hybrid offspring of Xiangyan and Xiangyun; In the S1 step, Xiangyan is the father and Xiangyun is the mother.

3. The detection method according to claim 2, characterized in that, In the S2 step, the reaction system of PCR is: 2x Taq Master Mix 12.5 μL, 10 μmol / L of each 1 μL of upstream and downstream primers, 9.5 μL of ddH2O, 1 μL of 50 ng / μL of Xiangyan, Xiangyun or F1 generation material to be tested DNA, and the reaction system is 25 μL.

4. The detection method according to claim 2, characterized in that, In the S2 step, the reaction program of PCR is: the reaction program of IDC10 marker PCR: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 57.6℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min, 4℃ storage; the reaction program of IDG8 marker 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, 4℃ storage.

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