A SNP-CAPS Molecular Marker Related to Anthocyanin in Colored Potatoes and Its Identification

Abstract

The present invention discloses an SNP-CAPS molecular marker related to anthocyanin in colored potatoes and its identification, which relates to the technical fields of potato breeding and molecular genetics. Through transcriptome sequencing and single nucleotide polymorphism (SNP) site analysis of purple flesh (Zicai No. 3) and yellow flesh (Longshu No. 7) at different tuber development stages, the present invention discovers that there is a non-synonymous single nucleotide mutation in the CDS region of the potato anthocyanin synthesis gene StCHS . Based on the difference of this single nucleotide, a CAPS molecular marker CAPS9-325 is developed. This CAPS molecular marker has high accuracy and good repeatability, and can quickly and effectively detect whether different potato genomes contain StCHS functional genes and their homozygous and heterozygous states, and then screen out potato materials with anthocyanin, so as to conduct molecular marker-assisted selection breeding in the early stage, which is beneficial to accelerating the breeding process of new colored potato varieties with high anthocyanin content.

Description

Technical Field

[0001] The present invention relates to the technical fields of potato breeding and molecular genetics, and particularly relates to an SNP-CAPS molecular marker related to anthocyanin in colored potatoes and its identification. Background Art

[0002] Potato ( Solanum tuberosum Solanum tuberosum L.), also known as potato and sweet potato, belongs to the Solanaceae family and the Solanum genus, and is an annual herbaceous plant. It is the world's largest non-cereal food crop and one of the important cash crops in China. Potatoes originated in the areas of Peru and Chile in the Andes Mountains of South America and were spread to all over the world by colonists after being domesticated and cultivated by the Indians. At first, potatoes were only used as ornamental plants. As people's understanding and knowledge of potatoes continued to increase, potatoes began to be widely planted. Due to the strong adaptability and versatility of potatoes, they are currently planted in more than 150 countries and regions around the world and are used as staple food crops in many countries. China ranks first in the world in potato production. With the implementation of the "Potato Staple Foodization" strategy, its planting area is still continuously increasing, playing an important role in food security.

[0003] Potatoes are rich in nutrients. As a vegetable, the tubers of potatoes contain a large amount of carbohydrates, which are mainly stored in the form of starch, accounting for more than 20%. Therefore, potatoes are classified as "starchy vegetables". It is reported that a type of "resistant starch" in potatoes has been proven to promote the growth of intestinal flora, improve intestinal health, and thus reduce the incidence of intestinal diseases. The protein content in fresh potato tubers is about 1.7%-2.1%. Although it is not as good as the crops that are the main sources of protein, it is relatively higher than other tuber and rhizome crops. In terms of the nutritional value of protein, potatoes are superior to other plant proteins and are a "complete" protein. In addition, potatoes contain a complete range of vitamins, including carotene and vitamin C that are not present in cereal grain crops, and the Vc content is 10 times that of apples. Therefore, potatoes are known as the "underground apple". The mineral content in potatoes is about 4.38%, among which the phosphorus and potassium contents are relatively high, and the potassium content is much higher than that of bananas. According to research reports, a low-sodium and high-potassium diet can reduce the risks of hypertension and stroke, and potatoes are an ideal food that meets this dietary requirement and can be included in the diet pattern for controlling hypertension. In addition to its rich nutritional value, potatoes also play an important role in feed and industrial uses and are of great significance in ensuring national food security.

[0004] Colored potatoes, also known as colored potatoes, are a special type of cultivated potato germplasm resources. Because their tubers are rich in anthocyanins, the potato skin and potato flesh appear in a variety of different colors such as red, blue, purple, and pink. Compared with ordinary white or yellow-fleshed potato varieties, colored potatoes are not only colorful, but also rich in anthocyanins, and their antioxidant capacity is 2.5-3 times higher than that of ordinary potatoes. As a natural water-soluble pigment commonly found in the vacuoles of plant cells, anthocyanins are brightly colored and are an important substance for the coloring of fruit, vegetable, and flower plants. Studies have shown that anthocyanins can not only make plants appear colorful, but also resist ultraviolet rays, diseases, and pests. In addition, in terms of nutrition and health care, anthocyanins have a variety of physiological functions such as reducing the growth rate of cancer cells, regulating blood sugar, anti-aging, anti-tumor, and enhancing vision, which are beneficial to human health. As a source crop of natural pigments, colored potatoes have a lower anthocyanin content compared to anthocyanin-rich crops such as blueberries and black currants, but their pigment yield per unit area is high, and the pigments they contain are stable, easy to process and storable, making them one of the ideal sources of high-quality anthocyanins.

[0005] Anthocyanin, as an important quality trait of colored potatoes, has become the research focus and one of the important breeding goals of breeders in recent years. Traditional breeding methods have the disadvantages of being time-consuming, labor-intensive, and highly dependent on the environment. The emergence of molecular marker-assisted breeding has broken the limitations of traditional breeding and accelerated the process of crop breeding. Since the anthocyanin content trait in colored potatoes is a quantitative trait controlled by multiple genes, this trait is not only controlled by major effect genes, but also some minor effect genes will indirectly affect the correlation of markers. In addition, tetraploid potatoes have high genetic heterozygosity, and the number of molecular markers that can be effectively used for colored potatoes is extremely limited. SNP markers, as the latest generation of molecular markers, are currently widely used in various biological research fields. However, SNP-CAPS molecular markers related to colored potato anthocyanins have not yet been reported. Therefore, the development of molecular markers related to colored potato anthocyanins will be of great significance for molecular-assisted breeding of new colored potato varieties. Summary of the invention

[0006] The purpose of the present invention is to provide a SNP-CAPS molecular marker and identification related to colored potato anthocyanins to solve the following technical problems:

[0007] The existing common cultivated potatoes are autologous tetraploids with highly heterozygous genomes, which makes genetic analysis complex, and the number of molecular markers that can be effectively used for colored potatoes is extremely limited.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A SNP-CAPS molecular marker related to anthocyanins in colored potatoes, wherein the CAPS molecular marker is CAPS9-325, and the molecular marker is based on potatoStCHS A CAPS primer developed based on a SNP locus on the CDS region of the gene chr9_2.77858834, with polymorphisms of A or G.

[0010] As a further aspect of the present invention: CAPS9-325 is A in purple potato flesh and G in yellow potato flesh, and can distinguish potato materials with homozygous purple potato flesh, heterozygous purple potato flesh, homozygous yellow potato flesh, and heterozygous yellow potato flesh.

[0011] As a further aspect of the present invention: A method for identifying SNP-CAPS molecular markers, comprising the following steps:

[0012] (1) Using primer sequences to perform PCR amplification on the extracted genomic DNA to obtain a PCR amplification product;

[0013] (2) Using the PCR amplification product to prepare a digestion system and perform a digestion reaction;

[0014] (3) Using 3% agarose gel electrophoresis to detect the digested product, with the electrophoresis buffer being 1% TAE solution, and performing electrophoresis at 150 V for 25 min.

[0015] As a further aspect of the present invention: The PCR amplification system and its components are as follows:

[0016]

[0017] As a further aspect of the present invention: The upstream primer NOPur-F: 5'-GCTATCATTATGGGTTCG-3'; the downstream primer NOPur-R: 5'-CTCTTGTAGCCCTAAGTTTC-3'.

[0018] As a further aspect of the present invention: The PCR amplification program is as follows:

[0019]

[0020] As a further aspect of the present invention: The digestion system and its components are as follows:

[0021]

[0022] As a further aspect of the present invention: The specific steps of the digestion reaction are: reacting at 65°C for 15 min.

[0023] Advantages of the present invention:

[0024] By performing transcriptome sequencing and single nucleotide polymorphism (SNP) locus analysis on purple potato flesh (Zicai No. 3) and yellow potato flesh (Longshu No. 7) at different tuber development stages, the present invention discovers that there are differences in potato anthocyanin synthesis genes between the two.StCHS There is a non-synonymous single nucleotide mutation in the CDS region of StCHS . According to the difference of this single nucleotide, the CAPS molecular marker CAPS9-325 was developed. This CAPS molecular marker has high accuracy and good repeatability, and can quickly and effectively detect whether different potato genomes contain BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below with reference to the accompanying drawings.

[0026] Figure 1 shows the PCR amplification of primers for potato varieties 1-20 in M1, M2 and Table 4; where M1 is DL2000 Marker and M2 is 50 bp DNA Ladder;

[0027] Figure 2 shows the PCR amplification of primers for potato varieties (lines) 1, 2, 21-38 in M1, M2 and Table 4; where M1 is DL2000 Marker and M2 is 50 bp DNA Ladder. DETAILED DESCRIPTION OF THE INVENTION

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1

[0030] The identification method of the SNP-CAPS molecular marker includes the following steps:

[0031] (1) DNA extraction: Using the TSINGKE plant DNA extraction kit (general type), genomic DNA was extracted; the test materials were 29 different potato varieties and 9 new colored potato lines selected by our research group, and the specific information is shown in Table 4;

[0032] (2) The molecular marker is located in tetraploid potato StCHSThe CDS region of the gene (chr9_2.77858834) is A in purple potato flesh and G in yellow potato flesh. (Transcriptome sequencing was performed on the purple potato variety Zicai 3 and the yellow potato variety Longshu 7 at different tuber development stages, and the results of single nucleotide polymorphism (SNP) sites obtained from the transcriptome sequencing were analyzed. It was found that StCHS there is a non-synonymous mutation SNP in the CDS region (chr9_2,77858834) of the gene);

[0033] Forward and reverse primer sequences of this molecular marker: Extract 500 bp sequences upstream and downstream of the SNP mutation site, and use Primer5.0 software to design primers. The product size is 325 bp, and the amplified product contains the above non-synonymous mutation SNP site. Use SnapGene to detect the effective restriction enzyme sites on the expected amplified product sequence (the SNP mutation site is located on the restriction enzyme site), and select the restriction enzyme BstBI for the digestion reaction.

[0034] The PCR amplification system and its components are shown in Table 1:

[0035]

[0036] Among them, the forward primer NOPur-F: 5'-GCTATCATTATGGGTTCG-3'; the reverse primer NOPur-R: 5'-CTCTTGTAGCCCTAAGTTTC-3';

[0037] The PCR amplification program is shown in Table 2:

[0038]

[0039] (3) Prepare the digestion system using the PCR amplification product and react at 65°C for 15 min;

[0040] The digestion system and its components are shown in Table 3:

[0041]

[0042] (4) Use 3% agarose gel electrophoresis to detect the digestion products. The electrophoresis buffer is 1% TAE solution, and electrophoresis is carried out at 150 V for 25 min.

[0043] After PCR amplification, enzyme digestion, and electrophoresis of the functional marker, 3 bands were amplified in the heterozygous purple-fleshed potato material, with lengths of 325 bp, 273 bp, and 52 bp (the fragment is too short to be shown in the figure); 1 band was amplified in the homozygous purple-fleshed potato material, with a length of 325 bp; 2 bands were amplified in the homozygous yellow (or white)-fleshed potato, with lengths of 273 bp and 52 bp respectively; and 3 bands were amplified in the heterozygous yellow (or white)-fleshed potato, with lengths of 325 bp, 273 bp, and 52 bp respectively.

[0044] Using the SNP-CAPS molecular marker provided by the present invention, PCR verification was carried out on existing potato varieties (lines); please refer to Figure 1-2 , where M1 and M2 are both Markers, M1 is DL2000 Marker, and M2 is 50 bp DNA Ladder; 1-38 correspond to the serial numbers of potato varieties (lines) in Table 4; among them, 1-29 are potato varieties, and 30-38 are new colored potato lines.

[0045] Table 4: Information Table of Each Potato Variety (Line)

[0046]

[0047] The above has described an embodiment of the present invention in detail, but the content described is only a preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the application of the present invention should still fall within the scope covered by the patent of the present invention.

Claims

1. A method for identifying SNP-CAPS molecular markers related to anthocyanins in colored potatoes, characterized in that: The steps include: (1) Perform PCR amplification on the extracted genomic DNA using primer sequences to obtain PCR amplification products; (2) Using the PCR amplification product to prepare an enzyme digestion system and perform an enzyme digestion reaction; (3) Detect the enzyme digestion products by 3% agarose gel electrophoresis, the electrophoresis buffer is 1% TAE solution, and the electrophoresis is performed at 150V for 25min; Upstream primer NOPur-F: 5′-GCTATCATTATGGGTTCG-3′; downstream primer NOPur-R: 5′-CTCTTGTAGCCCTAAGTTTC-3′; The enzyme digestion system and its components are as follows: 2 μL 10×NEB buffer, 1 μL endonuclease BstBI, 10 μL PCR product, 7 μL Nuclease-free Water; After PCR amplification, enzyme digestion and electrophoresis, the SNP-CAPS molecular marker amplified three bands in the heterozygous purple potato flesh material, with lengths of 325bp, 273bp and 52bp respectively; one band in the homozygous purple potato flesh material was amplified, with a length of 325bp; two bands in the homozygous yellow or white potato flesh were amplified, with lengths of 273bp and 52bp respectively; and three bands in the heterozygous yellow or white potato flesh were amplified, with lengths of 325bp, 273bp and 52bp respectively.

2. The identification method according to claim 1, characterized in that: The PCR amplification system and its components are as follows: 2 μL 2.0 mmol / L Mg 2+ 10× Buffer, 1.5μL 0.225mmol / L dNTPs, 0.4μL 5U / μL Easy Taq enzyme, 0.9μL 10ng / μL upstream primer, 0.9μL 10ng / μL downstream primer, 2μL 50ng / μL DNA, 12.3μL Nuclease-free Water.

3. The identification method according to claim 1, characterized in that: The PCR amplification procedure is as follows: (1) Pre-denaturation: temperature 94°C, time 4 min, cycle number 1; (2) Denaturation: temperature 94°C, time 30 s, cycle number 1; (3) Annealing: temperature 52°C, time 30 s; extension: temperature 72°C, time 60 s; extension: temperature 72°C, time 8 min; number of cycles is 35; (4) Store at 4°C.

4. The identification method according to claim 1, characterized in that: The specific steps of the enzyme digestion reaction are: react at 65°C for 15 minutes.

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