SNP (Single Nucleotide Polymorphism) marker for identifying purity of pepper seeds and application of SNP marker

Abstract

The invention relates to the technical field of biological agents, in particular to an SNP marker for identifying the purity of pepper seeds and application of the SNP marker. The SNP marker comprises the following components: Chr0118489307, Chr0285569078, and Chr05116774799, and the SNP marker comprises the following components: Chr0118489307, the SNP marker of a sample is detected to determine the genotyping of the sample, so that whether the sample is a false hybrid or a true hybrid can be determined, and the purity of the pepper seeds is identified.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological agents, and particularly relates to a SNP marker for identifying the purity of pepper seeds and application thereof. BACKGROUND

[0002] Pepper is a widely planted vegetable and spice crop in the world, and the main producing countries include China, India, Mexico, Thailand, etc. According to the statistics of the Food and Agriculture Organization (FAO) of the United Nations, the annual output of pepper in the world exceeds 40 million tons, which is an important source of income for farmers in many developing countries. Pepper is the core component of diet in Latin America, Asia, Africa, etc., and its pungent property endows food with unique flavor and promotes appetite. However, the single pepper variety and degeneration lead to problems such as concentrated harvest period and quality decline, which have become one of the problems urgently needed to be solved by the industry. The selection and popularization of new varieties is an effective means to adjust the variety structure and improve the quality and efficiency of the industry. At present, artificial hybridization is the main method for the selection of new pepper varieties. However, the breeding cycle is long, and false hybrids are easily produced due to open pollination. In addition, the identification of hybrid authenticity according to the field phenotypic traits of hybrid offspring is time-consuming and labor-intensive, and is easily affected by environment and human factors. With the centralized use of parents, the morphological differences of hybrid offspring are becoming smaller and smaller, and it is more and more difficult to identify true and false hybrids by morphological characteristics.

[0003] DNA molecular markers are used to identify the variety identity, which has the advantages of accuracy, reliability, simplicity, rapidity, easy operation and good timeliness. They are widely used in the identification of true and false varieties of citrus, apple, grape, cherry, peach, kiwi fruit, etc. The International Union for the Protection of New Varieties of Plants (UPOV) has recommended SSR marker technology and single nucleotide polymorphism technology (SNP) as two technologies for variety identification or seed purity identification in the BMT molecular test guide. However, the SSR technology has defects such as fewer detection sites, low throughput, poor representativeness of results and difficulty in realizing data sharing. SNP markers are uniformly distributed in the genome with high density, which can realize high-throughput detection and are more suitable for the integration and sharing of databases. Therefore, SNP markers are considered to be the most promising molecular marker technology.

[0004] At present, the identification of pepper hybrid offspring is mainly carried out by observing the morphological characteristics of the varieties. SUMMARY

[0005] In view of the above problems of the prior art, the present application aims to provide a SNP marker for identifying the purity of pepper seeds and application thereof, and to provide a new way for the identification of pepper hybrid offspring.

[0006] In order to solve the above problems, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a SNP marker for identifying the purity of pepper seeds, which comprises Chr01_18489307, Chr02_85569078 or Chr05_116774799.

[0008] Further, the pepper variety is Boyue 2306.

[0009] Further, the SNP marker pepper reference genome is CM334.

[0010] In a second aspect, the present application provides a primer for detecting the SNP marker for identifying the purity of pepper seeds, which comprises SEQ ID NO. 1-SEQ ID NO. 9.

[0011] In a third aspect, the present application provides a method for identifying the purity of pepper seeds, which comprises detecting the genotyping of the SNP marker of a pepper sample, and if the genotyping of the pepper sample is homozygous genotype, it is false hybrid; if the genotyping of the pepper sample is heterozygous genotype, it is true hybrid.

[0012] Further, the PCR amplification procedure for detecting the genotyping of the SNP marker of the pepper sample according to claim 1 is 94℃, 15min; 94℃, 20s, 65℃, 1min and reducing 0.7℃ for each cycle, for ten cycles; 94℃, 20s; 57℃, 1min.

[0013] In a fourth aspect, the present application provides the application of the SNP marker in pepper screening breeding.

[0014] In a fifth aspect, the present application provides the application of the primer in preparing a reagent for pepper screening breeding.

[0015] The present application has the beneficial effects that the present application determines the SNP marker for identifying the purity of pepper seeds, which comprises Chr01_18489307, Chr02_85569078 and Chr05_116774799; by detecting the SNP marker of the sample, the genotyping is determined, and it is determined whether the sample is false hybrid or true hybrid, and the purity of Boyue 2306 pepper seeds is identified. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The figure is the genotyping result of part of the primer.

[0017] Figure 2 The figure is the genotyping result of Chr01_18489307.

[0018] Figure 3 The figure is the genotyping result of Chr02_85569078.

[0019] Figure 4 Figure for typing result of Chr05_116774799. DETAILED DESCRIPTION

[0020] The application will be further described below in connection with specific embodiments.

[0021] It should be noted that the examples are only used to illustrate the application, but not to limit the application, and simple improvements of the method under the concept of the application are within the scope of the application.

[0022] Example 1

[0023] 1.1 Materials

[0024] High-generation inbred line 803 was used as the male parent, and high-generation inbred line 802 was used as the female parent. After artificial emasculation, the two were crossed to produce F1 seeds (“Bo Yue 2306”). Ninety-three seeds were randomly selected for seedling raising for seed purity identification.

[0025] 1.2 Method

[0026] 1.2.1 Design and screening of primers

[0027] DNA was extracted from the leaves of high-generation inbred lines 803 and 802 using the CTAB method and sent to Berry Genomics for whole-genome resequencing. The obtained reads were then aligned to the pepper reference genome CM334 (V1.55), and SNP detection was performed using GATK. Eleven SNP sites that were homozygous between 802 and 803 and had differences were randomly selected, and primers were designed using Primers5 software (Table 1). All primers were synthesized by Shanghai Generay Biotech. 802, 803, and five random “Bo Yue 2306” seeds were used as materials for primer screening, and the SNP typing reagent PARMS was purchased from Wuhan Jingsai Biological. The reaction system was as follows:

[0028] Table 1 PCR reaction system

[0029] Ingredients Volume 2x mix 2.5ul Primer X 0.075ul Primer Y 0.075ul Primer C 0.2ul DNA template 3ul Double distilled water 2.15ul

[0030] PCR was performed on an ABI QuantStudio6 QS6 instrument, and genotyping was performed on the instrument. The reaction program is shown in Table 2. Seven primers that could genotype the materials including the parents were selected, and a total of three groups of SNP primers that could be used for hybrid authenticity identification were obtained.

[0031] Table 2 PCR thermal cycling program

[0032]

[0033] 1.3 Hybrid purity molecular identification

[0034] The selected primers Chr01_18489307, Chr02_85569078 and Chr05_116774799 were used to genotype 93 single plants of 'Boluo 2306' and 802, 803. The genotype of each sample was counted, and when the genotype of the sample was homozygous, it was a false hybrid; when the genotype of the sample was heterozygous, it was a true hybrid.

[0035] 1.4 Results and analysis

[0036] 1.4.1 Primer screening

[0037] From the resequencing data of advanced inbred lines 802 and 803, SNP homozygous sites with differences between parents were selected. 11 groups of primers (Table 3) were designed to screen primers using 802, 803 and 5 single plants of 'Boluo 2306' as materials by PARMS-SNP marker method. The results showed that 11 groups of primers were distributed on Chr01, Chr02, Chr03, Chr04, Chr05 and Chr08, a total of 6 chromosomes. After PCR amplification, 9 groups of primers could not distinguish 8 materials, only Chr01_18489307, Chr02_85569078 and Chr05_116774799 could distinguish 8 materials into 3 types, and met the theoretical genotype of parents. It is shown that Chr01_18489307, Chr02_85569078 and Chr05_116774799 can be used for purity identification of 'Boluo 2306' hybrid varieties. Part of the genotyping results of primers are shown in Figure 1 (blue represents the genotype of '802', red represents the genotype of '803', and green represents the genotype of hybrid F1 generation).

[0038] Table 3 SNP markers for purity identification of 'Boluo 2306'

[0039]

[0040]

[0041] 1.4.2 Hybrid purity identification

[0042] DNA of single plant leaves of the parents 802, 803 and 93 strains of "Boyue 2306" were extracted by alkali cracking method, and then SNP typing of the single plants of the parents 802, 803 and 93 strains of "Boyue 2306" was performed by using CHROl_18489307, CHRO2_85569078 and CHRO5_116774799. The results showed that the 95 single plants could be divided into three genotypes by the three markers. Among them, 2 strains of single plants were identified to be consistent with the genotype of 802 and 803 by using CHROl_18489307, CHRO2_85569078 and CHRO5_116774799 respectively (the genotyping results of CHROl_18489307, CHRO2_85569078 and CHRO5_116774799 are shown in Figures Figures 2-4 ) respectively). Only one SNP genotyping result consistent with the genetic rule can determine whether it is true or false hybrid. By comprehensively using the three markers, 90 true hybrids, 2 false hybrids and 1 undetected hybrid were identified in the 93 strains of "Boyue 2306" seedlings, and the seed purity was 96.77% (Table 4).

[0043] Table 4 Statistical results of hybrid purity identification of "Boyue 2306"

[0044]

[0045]

[0046]

[0047] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described by referring to the preferred embodiments of the present application, it should be understood by those skilled in the art that various changes can be made in form and details without departing from the spirit and scope of the present application as defined in the appended claims.

Claims

1. An SNP marker for identifying the purity of chili pepper seeds, characterized in that, The SNP tags include Chr01_18489307, Chr02_85569078, or Chr05_116774799.

2. The SNP marker for identifying the purity of chili seeds according to claim 1, characterized in that, The chili pepper variety mentioned is Boyue 2306.

3. The SNP marker for identifying the purity of chili seeds according to claim 1, characterized in that, The reference genome for the SNP marker in chili peppers is CM334.

4. A primer for detecting the SNP marker used in claim 1 for identifying the purity of chili seeds, characterized in that, The primers include those such as SEQ ID NO.1-SEQ ID NO.

9.

5. A method for identifying the purity of chili pepper seeds, characterized in that, The method includes genotyping of chili pepper samples using the SNP markers described in claim 1. If the genotyping of the chili pepper sample is homozygous, it is a pseudohybrid; if the genotyping of the chili pepper sample is heterozygous, it is a true hybrid.

6. The method for identifying the purity of chili seeds according to claim 5, characterized in that, The PCR amplification program for detecting the SNP marker genotyping of the chili pepper sample as described in claim 1 is as follows: 94℃, 15min; 94℃, 20s; 65℃, 1min, with the temperature decreasing by 0.7℃ for each cycle, for ten cycles; 94℃, 20s; 57℃, 1min.

7. The application of the SNP marker in pepper screening and breeding as described in claim 1.

8. The application of the primers according to claim 4 in the preparation of pepper screening breeding reagents.

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