A SNP locus for identifying potato tuber flesh color and its application

Abstract

The present invention provides a SNP site for identifying the color of potato tuber flesh and its application, wherein 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. The present invention mines the SNP marker site closely linked to the color trait of potato tuber flesh through the third-generation molecular marker technology, which is of great significance to the molecular marker-assisted breeding of tetraploid potato tuber flesh color.

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 an annual herb in the Solanum subgenus of the Solanaceae family. During the domestication and cultivation of potatoes, on the one hand, due to external environmental stimuli such as high altitude and high latitude areas, plants produce a large number of secondary metabolites under adverse conditions, such as anthocyanins and phenolic substances; on the other hand, human intervention in the domestication process has 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 levels, people's demand for potato products has become more and more diversified. Therefore, in addition to the development of potatoes as staple foods, 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 most 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).

[0004] At present, the research patents on colored potatoes are concentrated 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, use BWA to align the quality-controlled sequence to the tetraploid potato Qingshu 9 genome sequence, use Samtools to sort, remove PCR duplications through 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 conducted based on the obtained population SNP information. The cMLM model of GAPIT was used to conduct a correlation analysis of 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 time, and all materials are planted at the Qinghai Alpine Potato Experimental Station at 36º680N, 101º260E.

[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 genotyping 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 the Qinghai Academy of Agriculture and Forestry Sciences for a long time. All materials were planted at the Qinghai Alpine Potato Experimental Station (36º680N, 101º260E). This application planted the experimental materials in the form of tubers, with a tuber diameter of 35 mm~50 mm, no sprouts, healthy appearance, and no pests and diseases. The field trial was divided into 3 plots, with 6-10 potato plants planted for each variety, with a plant spacing of 30 cm and a row spacing of 90 cm. A protective row was 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, Uniformity 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 correlation analysis on potato tuber flesh color. 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 used the tetraploid 'Qingshu 9' genome as a reference to resequence 769 tetraploid potato natural populations, conducted GWAS research and analysis on potato flesh color, and found that the control of potato flesh color is mainly on chromosome 3 A2 haplotype. Through this discovery, the corresponding SNP marker site (snp: 38178848) was obtained, such as 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] 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. Application of detecting SNP site genotype in assisting identification of potato tuber flesh color, taking the tetraploid 'Qingshu 9' genome as a reference, the SNP site is the SNP single nucleotide polymorphism site Chr03A2: 38178848 of the A2 haplotype of chromosome 3 of the tetraploid 'Qingshu 9' potato genome, and the nucleotide base of the site is C or T; when the genotype is TC or CC, the tuber flesh color of the potato sample is significantly darker than that of the sample with the genotype of TT; when the genotype is TT, the potato tuber flesh color is white, milky white or light yellow.

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