SNP combination marker for potato germplasm identification and application

Abstract

The application discloses a SNP molecular marker combination for DNA fingerprinting of tetraploid potato varieties, application and method. Through sequencing comparison of 135 potato germplasm samples, 3397137 SNPs are identified, and the SNPs are filtered and screened to obtain an SNP set for identifying tetraploid potato germplasm. The 21 SNP molecular markers screened by the application have high specificity, sensitivity and resolution. The application uses 187 potato breeding varieties to predict the distinguishing efficiency of the developed SNP set, and the result shows that the distinguishing rate of the application to the breeding varieties can reach 100%. It is indicated that the detection of the single nucleotide marker site of the application has the advantages of simplicity, rapidness and low cost, and can realize large-scale application in production.

Description

Technical Field

[0001] This invention belongs to the field of potato germplasm resource identification and breeding, and specifically relates to an SNP combination marker for potato germplasm resource identification. Background Technology

[0002] potato( Solanum tuberosum L) belongs to the Solanaceae family ( Solanaceae Solanum genus ( Solanum L) Important crop. Traditional phenotypic methods for analyzing the genetic diversity of germplasm resources have been widely used in potatoes. However, phenotypic identification is easily affected by environmental and subjective human factors, which can influence the research results. In contrast, molecular marker technologies based on DNA sequence diversity are less affected by environmental conditions and subjective human factors. Currently, molecular markers used in the analysis of genetic diversity of potato germplasm resources include AFLP (Nunziata et al., 2010; Wang et al., 2017), SSR (Simple Sequence Repeats) (Carputo et al., 2013; Kolechet et al., 2016; Nunziata et al., 2010; Wang et al., 2017), and SNP (Chiang et al., 2021; Igarashi et al., 2018; Jo et al., 2021; Kolech et al., 2016; Voset et al., 2015). Since single nucleotide polymorphisms (SNPs) are the most abundant variants in plant genomes and can be detected at low cost and high throughput, SNP markers are a highly promising molecular marker technology (Semagn et al., SNP microarray technology (2014) has been widely applied in various crops. In potatoes, SNP microarray technology has been used to study potato genetic diversity, QTL mapping, and breeding history (Chiang et al., 2021; Stich et al., 2013; Uitdewilligen et al., 2013; Vos et al., 2015; Zhang et al., 2022). This has laid a good foundation for molecular breeding of potatoes. However, as potatoes are asexually propagated materials, problems such as contamination or labeling errors are prone to occur during preservation, propagation, and germplasm resource exchange. Therefore, identifying the authenticity of potato germplasm resources and whether they belong to the same variety is of great significance for breeding work.

[0003] Problems with existing technologies: Phenotypic identification of potato germplasm resources is time-consuming, labor-intensive, and easily affected by environmental conditions and human subjectivity. Existing molecular markers such as ISSR, SRAP, and SSR are complex to operate, have long experimental cycles, and low throughput. Existing SNP marker technologies are all based on GBS and SNP microarray technology, requiring specific equipment and bioinformatics expertise, making them unsuitable for large-scale population studies. This application provides a set of SNP marker primers and uses ABI QuantStudio6 QS6 real-time quantitative PCR reaction. After the reaction, the instrument's built-in program collects FAM, HEX, and ROS fluorescence values ​​and performs genotyping analysis. Based on the genotyping results, varieties are identified. Summary of the Invention

[0004] The key technical problem this invention aims to solve is to develop SNP marker primers capable of classifying 187 potato germplasm accessions by performing genome resequencing on a large number of potato materials with different genetic backgrounds. To solve the above technical problem, this invention adopts the following technical solution:

[0005] 1. A potato SNP combination marker, wherein the SNP combination marker is identified using primers shown in SEQ NO. ID 1-63 of the sequence listing.

[0006] 2. A screening method for potato SNP combinatorial markers, including the following steps: (1) SLAF-seq genome sequencing, (2) SNP detection, (3) Perfect SNP filtering, (4) primer design and synthesis, (5) primer screening, (6) PCR reaction, and (7) gene analysis of the obtained fluorescence signal values ​​using the fitploy R package.

[0007] 3. Application of potato SNP combination markers in variety identification, the application is achieved by the following method: (1) extracting potato DNA to be identified, (2) synthesizing primers as shown in SEQ NO. ID 1-63 (also recorded in Table 1), (3) establishing a PCR reaction system, (4) performing real-time quantitative PCR, and (5) genotype comparison identification: comparing the experimental results with known varieties (see attached table). Figure 2-4 If the gene analysis results are completely identical, the potato variety is the same. When testing two unknown potato materials, identical results indicate the same potato variety, while different results indicate different potato varieties.

[0008] 4. Application of potato SNP combination markers in constructing potato SNP fingerprinting, the application is achieved by the following methods: (1) extracting potato DNA to be identified, (2) synthesizing primers as shown in SEQ NO. ID 1-63, (3) establishing a PCR reaction system, (4) performing real-time fluorescence quantitative PCR, and (5) genotype comparison identification.

[0009] 5. Application of potato SNP combination markers in distinguishing genetic distance in hybrid offspring, the application is achieved by the following methods: (1) extracting potato DNA to be identified, (2) synthesizing primers as shown in SEQ NO. ID 1-63, (3) establishing a PCR reaction system, (4) performing real-time quantitative PCR, and (5) genotype comparison identification: the greater the difference in individual scores in the hybrid population, the greater the genetic distance between the two, and the closer the scores, the closer the genetic distance.

[0010] Beneficial effects: This invention develops 21 sets of SNP molecular markers. Using this combination of SNP molecular markers, the influence of environmental conditions and subjective factors can be avoided, enabling accurate variety identification. Real-time quantitative PCR analysis is highly efficient and convenient, and more convenient than traditional electrophoresis detection. Attached Figure Description

[0011] Figure 1 The experimental procedure for SNP screening.

[0012] Figure 2 187 potato SNP fingerprints were generated using 21 primer sets (Part 1).

[0013] Figure 3 187 potato SNP fingerprints were generated using 21 primer sets (Part II).

[0014] Figure 4 187 potato SNP fingerprints were generated using 21 primer sets (Part 3). Specific implementation methods Unless otherwise specified, the methods and apparatus used in the following embodiments of this invention are conventional methods and apparatus; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. To make the objectives, technical solutions, and advantages of this invention clearer, the specific implementation methods of this invention are described in detail below with reference to specific embodiments. Examples of these preferred embodiments are illustrated in the specific embodiments. It should also be noted that, in order to avoid obscuring the technical solution of this invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solution according to this invention are shown in the embodiments, while other details that are not closely related are omitted.

[0015] Example 1 This embodiment provides an SNP combination marker for potato germplasm resource identification, comprising 21 sets of SNP primers as shown in Table 1 below. The sequences are also recorded in the sequence listing SEQ NO.ID 1-63, where the sequences in the sequence listing contain the same set of sequences: Pimer-X, Pimer-Y, and Primer-C.

[0016] Table 1. Potato SNP sites and their primers

[0017] Example 2 This embodiment provides a method for screening SNP combination markers for potato germplasm resource identification, such as... Figure 1 As shown, it includes the following steps: 1. Simplified genome sequencing of potato 135 using SLAF-seq simplified genome technology. Approximately 0.1 g of each of 135 young potato leaves was randomly selected. Total DNA was extracted from the leaves using the CTAB method. DNA integrity was assessed by agarose gel electrophoresis, and the leaves were double-digested using HaeIII-Hpy166II enzymes. The 135 potato DNA samples were then used for library construction and sequencing, following the procedures outlined in Sun et al. (Sun X, 2013). The resulting reads were then obtained.

[0018] 2. SNP detection Sequencing reads obtained using the SLAF-seq simplified genome sequencing method were aligned to the potato reference genome using bwa software (Zhou, Q. 2020). SNPs were then detected using Smatools software, yielding a total of 3,397,137 high-quality SNPs.

[0019] 3. Perfect SNP Filtering According to the criteria of Liu et al, perfect SNPs were screened, and the screening criteria were as follows: (1) Minimum allele frequency (MAF) > 0.4; (2) Mismatch rate < 0.25 (the mismatch rate in the literature is < 0.2); (3) Heterozygosity < 0.5 (the heterozygosity rate in the literature is < 0.4); (4) No other mutations within 50 bp to the left and right of the SNP site (100 bp in the literature).

[0020] 4. Primer design and synthesis Primers were designed using Primer3, and a linker for fluorescence matching amplification of FAM was added to primer X (GAGGTGACCAAGTTCATGCT); a linker for fluorescence matching with HEX was added to primer Y (GAGGTCGGAGTCAACGGATT). The synthesized primers were then sent to Shanghai Sangon Biotech for further synthesis.

[0021] 5. Primer screening.

[0022] Twenty-four potato samples were randomly selected, and DNA was extracted using the CTAB method. These 24 potato DNA samples were then used as templates for primer screening. The SNP genotyping reagent PARMS was purchased from Wuhan Jingtai Biotechnology. The reaction system is as follows: Table 2 PCR reaction system

[0023] PCR reactions were performed using an ABI QuantStudio 6 QS6 real-time quantitative PCR instrument. The reaction procedure is shown in Table 3. After the reaction, FAM, HEX, and ROS fluorescence values ​​were collected using the instrument's built-in program, and genotyping analysis was performed. If a primer could distinguish 24 potato samples, it was considered usable and could be used for genotyping of 187 potato samples. If a primer could not distinguish the 24 samples, it was considered unusable. After screening, a total of 69 usable SNP primers were obtained. Based on the genotyping results, the top 21 SNP primers with good genotyping results were selected as core primers.

[0024] Genotyping was performed using the Fitploy R software package. PCR reactions were conducted on 187 high-generation potato lines using the 21 sets of SNP primers described above, and FAM, HEX, and ROS fluorescence values ​​were collected. Genotyping was then performed using the Fitploy R software package based on the FAM, HEX, and ROS fluorescence values. The genotyping results are shown below. Figure 2-4 As shown. The final results are represented by 0, 1, 2, 3, and 4, indicating the final typing result for each sample.

[0025] Table 3 PCR thermal cycling program

[0026] Example 2 This embodiment provides an application of SNP combination markers for potato germplasm resource identification, as detailed below: 1. Application of SNP markers in potato DNA fingerprinting Using the genotyping results of the above 21 sets of SNP primers on 187 potato samples, fingerprint patterns were constructed as shown in the attached figure. Figure 2-4As shown.

[0027] 2. Identification of potato varieties (1) Extracting DNA from potatoes to be identified: Take about 0.1g of young potato leaves, extract total DNA from the leaves using the CTAB method, and detect the integrity of the DNA using agarose electrophoresis.

[0028] (2) Primer synthesis: 21 sets of primers as shown in Table 1 were synthesized at Shanghai Sangon Biotech.

[0029] (3) Establish PCR reaction system: As shown in Table 2, establish PCR reaction system.

[0030] (4) Real-time quantitative PCR: PCR was performed using an ABI QuantStudio6 QS6 real-time quantitative PCR instrument. The reaction procedure is shown in Table 3. After the reaction, the FAM, HEX, and ROS fluorescence values ​​were collected and analyzed using the instrument's built-in program. Genotyping was then performed using the Fitploy R software package based on the FAM, HEX, and ROS fluorescence values. The final results are represented by 0, 1, 2, 3, and 4 to indicate the final genotyping result for each sample.

[0031] (5) Genotype comparison and identification: attach the experimental results Figure 2-4 By comparing the gene analysis tables, those with completely identical results belong to the same variety. When testing two unknown potato materials, identical results indicate they are the same potato variety, while different results indicate they are different potato varieties.

[0032] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

[0033] References: Sun X, Liu D, Zhang X, et al. SLAF-seq: an efficient method of large-scale De novo SNP discovery and genotyping using high-throughput sequencing[J]. PloS one, 2013, 8(3): e58700 Zhou, Q.; Tang, D.; Huang, W.; Yang, Z.; Zhang, Y.; Hamilton, J. P.;Visser, R. G. F.; Bachem, C. W. B.; Robin Buell, C.; Zhang, Z.; Zhang, C.;Huang, S., Haplotype-resolved genome analyses of a heterozygous diploidpotato. Nat Genet 2020, 52, (10), 1018-1023. Stacklies, W.; Redestig, H.; Scholz, M.; Walther, D.; Selbig, J.,pcaMethods—a bioconductor package providing PCA methods for incomplete data.Bioinformatics 2007, 23, (9), 1164-1167 Liu, W.; Qian, Z.; Zhang, J.; Yang, J.; Wu, M.; Barchi, L.; Zhao, H.;Sun, H.; Cui, Y.; Wen, C., Impact of fruit shape selection on geneticstructure and diversity uncovered from genome-wide perfect SNPs genotyping ineggplant. Molecular Breeding 2019, 39, (10-11).

Claims

1. The application of primers for identifying potato SNP combination markers in variety identification, characterized in that... The application is implemented by the following methods: (1) extracting the DNA of the potato to be identified, (2) synthesizing primers as shown in SEQ ID NO. 1-63, (3) establishing a PCR reaction system, (4) performing real-time fluorescence quantitative PCR, and (5) genotype comparison identification: testing two unknown potato materials, if the results are consistent, it indicates that they are the same potato variety, and if the results are different, they are different potato varieties.

Images