SNP (Single Nucleotide Polymorphism) site for identifying potato pulp color of potato tuber and application

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site for identifying the color of potato tuber pulp and application, the SNP site is an SNP single nucleotide polymorphism site Chr03A2: 38178848 of a haplotype of a potato genome chromosome 3 A2, and the nucleotide base of the site is C or T. The invention further provides a method for identifying the color of potato tuber pulp. According to the invention, excavation of SNP marker sites closely linked to the color character of the potato pulp of the potato tuber by a third-generation molecular marker technology is of great significance to development of molecular marker-assisted breeding work of the color of the potato pulp of the potato tuber of the potato tuber of the tetraploid potato.

Description

Technical Field

[0001] The invention belongs to the technical field of potato germplasm resource identification, and relates to a SNP site for identifying the color of potato tuber flesh and an application thereof. Background Art

[0002] Potato is the world's largest tuber crop and the fourth most important food crop after rice, wheat and corn. It has gradually developed into the world's third largest food crop (Sun Huisheng, 2003, 45-47). Potato belongs to the Solanum subgenus of the Solanaceae family. It is an annual herb that originated in Peru and Chile in South America, around Lake Titicaca in the Andes Mountains. Primitive people discovered and began to domesticate it. In the 16th century, potatoes went out of South America and first entered Europe, and then spread to all parts of the world. They were introduced to China during the Wanli period of the Ming Dynasty. Although China has only a cultivation history of more than 400 years, it has already played an important strategic role in food security. During the domestication and cultivation of potatoes, on the one hand, they were stimulated by the environment of high altitude and high latitude areas, and under adverse conditions, plants produced a large number of secondary metabolites, such as anthocyanins and phenolic substances; on the other hand, human intervention in the domestication process caused potatoes of different colors to be selected, preserving the potato varieties of different colors that can be seen today. With the improvement of global consumption level, people's demand for potato products is becoming more and more diversified. Therefore, in addition to developing potatoes as staple food, how to increase the added value of potato processing products to meet people's consumption needs, especially the needs of the fast food and food industries has become one of the hot spots in potato research.

[0003] The color of potato tuber skin is one of the important quality traits of potato, which affects the commodity attributes of potato and the choice of consumers. The color of potato tubers is usually white or yellow, while purple, red, blue or black potatoes are called "colored potatoes" (Kyoungwon, 2016, 67(5):1519-1533; Liu, 2015, 10(6):e0129148). In addition to various antioxidants contained in ordinary potatoes, such as polyphenols, carotenoids, flavonoids and vitamin C, colored potatoes also contain anthocyanins. A large number of studies have shown that the higher the anthocyanin content of colored potatoes, the stronger the antioxidant activity (Brown, 2005, 130 (2): 174-180; Hamouz, 2011, 57 (10): 478-485; Nayak, 2011, 35: 571-580; Yin, 2016, 22 (2): 219-226; Galani, 2017, 3 (2): 73-89). Because of the important research value of colored potatoes in the development of health foods and natural antioxidants, they are gradually accepted and favored by the market.

[0004] At present, the research patents on colored potatoes focus on methods for quickly identifying colored potatoes from seed populations (CN117129428A), quickly judging the color of potato skin and flesh in the field (CN107806930B), and preparing and evaluating colorimetric cards for evaluating the color of potato skin and flesh (CN113484250A). However, there is still a lack of research on methods for quickly screening potato flesh color based on SNP molecular markers at the DNA level. Summary of the invention

[0005] Based on the above, the purpose of this application is to provide a SNP site and application for identifying the color of potato tuber flesh, which can quickly and accurately screen potato flesh color in large quantities, and is of great significance for the development of molecular marker-assisted breeding of tetraploid potato tuber flesh color.

[0006] The invention provides a SNP site for identifying the color of potato tuber flesh. The SNP site is a SNP single nucleotide polymorphism site Chr03A2:38178848 of the A2 haplotype of chromosome 3 of the potato genome, and the nucleotide base of the site is C or T.

[0007] Preferably, when the genotype is TC / CC, the potato flesh color of the potato sample is significantly higher than that of the sample with the genotype of TT; when the genotype is TT, the potato flesh color is white or milky white.

[0008] The present invention also provides a method for screening the above-mentioned SNP sites, comprising:

[0009] 1) Extract DNA from natural potato population materials;

[0010] 2) Perform whole genome resequencing on the obtained DNA, perform quality control and filtering on the generated sequencing data, align the quality-controlled sequence to the tetraploid potato Qingshu 9 genome sequence using BWA, sort it using Samtools, remove PCR duplicates using Picard, and then use GATK to obtain population SNP information;

[0011] 3) According to the specifications of the "Guidelines for Testing Potato Plant Varieties for Specificity, Consistency and Stability", the tuber flesh color of potato population materials was identified, and a GWAS analysis of potato tuber flesh color was performed in combination with the obtained population SNP information. The cMLM model of GAPIT was used to perform a correlation analysis on potato tuber flesh color, and the SNP site was screened.

[0012] Preferably, in step 1), the natural potato population materials are collected and preserved by Qinghai Academy of Agriculture and Forestry Sciences for a long term, and all materials are planted at the Qinghai Alpine Potato Experimental Station at 36°680N, 101°260E.

[0013] Preferably, in step 2), the raw sequencing data is filtered using FastQC software.

[0014] Preferably, in step 3), the MAF of the GWAS analysis is > 0.05, and HWE is > 0.001.

[0015] The present invention further provides the use of the above SNP site in identifying the color of potato tuber flesh.

[0016] Preferably, when the genotype is TC / CC, the potato flesh color of the potato sample is significantly higher than that of the sample with the genotype of TT; when the genotype is TT, the potato flesh color is white or milky white.

[0017] The beneficial effects of the present invention are:

[0018] 1. This application uses 769 natural population materials from a wide range of sources. The trait variation and genetic variation within the population are relatively rich, which can cover all phenotypic traits of potato flesh color, and accumulate a lot of recombination results and mutation information, which greatly improves the resolution of GWAS positioning.

[0019] 2. The sequencing technology used in this application is whole genome resequencing. Compared with other sequencing technologies, resequencing technology can detect mutation information most comprehensively at the whole genome level and can capture rich variations, providing favorable conditions for conducting GWAS research and more accurately positioning the association between potato flesh color.

[0020] 3. Compared to other mapping techniques, QTL mapping is highly dependent on the genetic diversity of the parents, and the effects of the detected QTLs may vary in different populations. The QTL region may also be quite large, containing too many genes to be studied as potential candidate genes. The use of GWAS studies can overcome the limitations of QTL analysis and can use natural populations to narrow down the candidate region. The identification of candidate genes by BSA-seq technology relies on the construction of its mixed pool population, which is smaller than GWAS analysis, but has low precision.

[0021] 4. The present invention uses the third-generation molecular marker technology to mine SNP marker sites that are closely linked to the potato tuber flesh color trait, which is of great significance for the development of molecular marker-assisted breeding of tetraploid potato tuber flesh color. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: The results of the whole-genome association analysis of potato tuber flesh color, where: Figure A is the positioning analysis result of 769 tetraploid potato tuber flesh color based on the whole genome; Figure B is the positioning analysis result of 769 tetraploid potato tuber flesh color on the A1, A2, A3, and A4 haplotypes of chromosome 3.

[0023] Figure 2 : QQ chart of potato tuber flesh color.

[0024] Figure 3 : SNP genotype analysis of potato tuber flesh color. Results are based on 3 replicates, and error bars represent standard deviation (SD). DETAILED DESCRIPTION

[0025] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0026] Example

[0027] 1. Planting of Experimental Materials

[0028] The 769 natural potato population materials used in this application were collected and preserved by Qinghai Academy of Agriculture and Forestry Sciences for a long time. All materials were planted at the Qinghai Alpine Potato Experimental Station (36°680N, 101°260E). This application planted the experimental materials in the form of tubers, with a tuber diameter of 35mm to 50mm, no sprouts, healthy appearance, and no pests and diseases. The field trial was divided into 3 plots, with 6-10 potato plants planted in each variety, with a spacing of 30cm between plants and 90cm between rows, and a protection row set up every 15 rows. The young leaves of each sample were taken and preserved during the budding period for subsequent DNA extraction, library construction, and sequencing.

[0029] 2. Identification of potato tuber flesh color

[0030] This application identified the color of 769 potato tubers according to the "Guidelines for Testing Potato Plant Variety Specificity, Consistency and Stability" (GB / T19557.28-2018; https: / / openstd.samr.gov.cn / bzgk / gb / ).

[0031] Table 1. Color of tuber flesh of different potato varieties

[0032]

[0033] 3. Potato DNA extraction and detection

[0034] Take about 100 mg of fresh plant tissue or about 30 mg of dry weight tissue, add liquid nitrogen and grind thoroughly. Use CTAB method to extract sample DNA. Agarose electrophoresis detection; 2% agarose gel (60V electrophoresis for 35 minutes) to detect DNA integrity.

[0035] 4. Genome Sequencing and Analysis

[0036] The obtained DNA was resequenced for the whole genome, and the sequencing data was quality controlled and filtered: the raw sequencing data was filtered by FastQC (https: / / github.com / s-andrews / FastQC / ) software. The sequence after quality control was aligned to the tetraploid potato Qingshu 9 genome sequence using BWA, sorted using Samtools, PCR duplications were removed by Picard, and then GATK was used to obtain population SNP information.

[0037] 5. GWAS of potato tuber color

[0038] The population of the association study included 769 materials, and GWAS analysis was performed on the filtered mutation sites (MAF>0.05 and HWE>0.001). The cMLM model of GAPIT was used to conduct a correlation analysis on the color of potato tuber flesh. CMplot was used to draw Manhattan and QQ plots and screen significant SNP sites. The completion of the assembly of multiple potato genomes has laid a solid foundation for the study of potato genome structure and function.

[0039] The present invention uses the tetraploid 'Qingshu 9' genome as a reference to resequence 769 tetraploid potato natural populations, conducts GWAS research and analysis on potato flesh color, and finds that the control of potato flesh color is mainly on chromosome 3 A2 haplotype. Through this discovery, the corresponding SNP marker site (snp: 38178848) is obtained. Figure 1 shown.

[0040] In order to check the reliability of SNP markers, a QQ plot was drawn, as shown in Figure 2 As shown, it was found that the model fit was good, and the significant SNP sites were significantly higher than the fitting curve.

[0041] like Figure 3 As shown in the figure, the effect analysis of different genotypes of the SNP locus on regulating potato flesh color shows that the potato flesh color of the potato samples with genotype TC / CC is significantly higher than that of the samples with genotype TT. The mining of SNP marker loci closely linked to the potato flesh color trait of potato tubers through the third-generation molecular marker technology is of great significance for the molecular marker-assisted breeding of tetraploid potato tuber flesh color.

[0042] This SNP locus was used to verify the potato flesh color of 76 potato populations by further identification and DNA extraction. The genotypes of the tested plants and the potato flesh color phenotype results are shown in Table 2. The potato flesh color of the samples with genotype TC / CC is darker than that of the samples with genotype TT (the potato flesh color is white or milky white).

[0043] Table 2. Genotypes of tested plants and potato flesh color phenotype results

[0044]

[0045]

[0046] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A SNP site for identifying the color of potato tuber flesh, wherein the SNP site is the SNP single nucleotide polymorphism site Chr03A2:38178848 of the A2 haplotype of chromosome 3 of the potato genome, and the nucleotide base of the site is C or T.

2. The SNP site according to claim 1, characterized in that: When the genotype is TC / CC, the potato flesh color of the potato samples is significantly higher than that of the samples with the genotype of TT; when the genotype is TT, the potato flesh color is white or milky white.

3. The method for screening SNP sites according to claim 1, comprising: 1) Extract DNA from natural potato population materials; 2) Perform whole genome resequencing on the obtained DNA, perform quality control and filtering on the generated sequencing data, align the quality-controlled sequence to the tetraploid potato Qingshu 9 genome sequence using BWA, sort it using Samtools, remove PCR duplicates using Picard, and then use GATK to obtain population SNP information; 3) According to the specifications of the "Guidelines for Testing Potato Plant Varieties for Specificity, Consistency and Stability", the tuber flesh color of potato population materials was identified, and a GWAS analysis of potato tuber flesh color was performed in combination with the obtained population SNP information. The cMLM model of GAPIT was used to perform a correlation analysis on potato tuber flesh color, and the SNP site was screened.

4. The screening method according to claim 3, characterized in that In step 1), the natural potato population materials are collected and preserved by Qinghai Academy of Agriculture and Forestry Sciences for a long time, and all materials are planted at the Qinghai Alpine Potato Experimental Station at 36°680N, 101°260E.

5. The screening method according to claim 3, characterized in that In step 2), the raw sequencing data is filtered using FastQC software.

6. The screening method according to claim 3, characterized in that In step 3), the MAF of the GWAS analysis was > 0.05 and HWE was > 0.

001.

7. Use of the SNP site according to claim 1 in identifying the color of potato tuber flesh.

8. The use according to claim 7, characterized in that: When the genotype is TC / CC, the potato flesh color of the potato samples is significantly higher than that of the samples with the genotype of TT; when the genotype is TT, the potato flesh color is white or milky white.

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