A SNP molecular marker for corn variety identification and its application

Abstract

The present invention relates to a SNP molecular marker for corn variety identification and its application. This study screened out a SNP marker for distinguishing the sweet and sticky traits of corn through whole genome sequencing analysis. The molecular marker is located at the polymorphic C / T site at position 1029462 of chromosome 3 of corn chromosome (GenBank: LR618876.1). The site can effectively distinguish between sticky corn and sweet corn varieties, and no false positive results were found in common corn varieties. It is confirmed that the molecular marker developed by this patent fills the gap of no SNP molecular marker for the sweet and sticky traits of corn.

Description

Technical Field

[0001] The invention relates to the technical field of corn varieties, and in particular to a SNP molecular marker for corn variety identification and application thereof. Background Art

[0002] Corn is a food that many people love to eat. Common corn varieties on the market can be divided into glutinous corn, sweet corn, and glutinous sweet corn according to their taste. Among them, the endosperm characteristics of sweet corn are different from those of ordinary corn. It is sweet, fragrant, crispy, and tender, and is known as "fruit corn" and "vegetable corn". Waxy corn is also called waxy corn or sticky corn. The endosperm of the grains is almost entirely composed of amylopectin, which is easier to digest than ordinary corn. The digestibility of glutinous corn is about 85%, while that of ordinary corn is about 69%. The unique flavor and rich grain color of glutinous corn are more popular among people. In the market, consumers' demands for glutinous corn and sweet corn are not completely consistent. Some consumers like corn with a soft and glutinous taste, while some consumers prefer corn with a sweet and crisp taste, and some consumers like corn that is both sweet and glutinous. How to effectively distinguish the glutinous and sweet characteristics of different corns is a technical problem that needs to be solved urgently.

[0003] Single nucleotide polymorphism (SNP) refers to the change of a single base in the DNA sequence of a population genome, including the insertion, deletion and substitution of a single base. SNP variation sites have the following advantages: large number, wide distribution, and representativeness. They can be converted into molecular markers through technologies such as CAPs (restriction site polymorphism markers), dCAPs (interventional restriction site polymorphism markers), KASP (competitive allele-specific PCR) and AS-PCR (allele-specific PCR technology), which is convenient for molecular marker detection. Molecular markers based on SNPs will not separate the marker and the target gene, which can greatly improve the accuracy and stability of the marker. However, there is no report on SNP molecular markers related to the sweet and sticky traits of corn. Summary of the invention

[0004] The present invention aims at the technical problems existing in the prior art and provides a SNP molecular marker for corn variety identification and application thereof.

[0005] In one aspect, the present invention provides a SNP molecular marker for corn variety identification, characterized in that the molecular marker is a SNP marker, whose sequence is shown in SEQ ID NO.1, and the base at the 26th position from the 5' end of the sequence is C or T.

[0006] In one aspect, the present invention provides a KASP molecular marker for corn variety identification, characterized in that the specific amplification primer sequence of the KASP molecular marker is shown in SEQ ID NO.2-5.

[0007] Preferably, the 5' ends of SEQ ID NO. 2 and SEQ ID NO. 3 carry different fluorescent gene markers respectively.

[0008] More preferably, the 5' end of SEQ ID NO. 2 carries a FAM tag, and the 5' end of SEQ ID NO. 3 carries a ROX tag.

[0009] In one aspect, the present invention provides a method for screening SNP molecular markers for corn variety identification, characterized in that the screening method comprises the following steps:

[0010] 1) Extracting genomic DNA of waxy corn, sweet corn and sweet waxy corn respectively;

[0011] 2) constructing Illumina sequencing libraries for genomic DNA and performing high-throughput sequencing;

[0012] 3) Annotate and compare the sequencing data to screen for polymorphic sites, and screen for sites with population marker polymorphism (MAF) greater than 0.01 and less than 0.05;

[0013] 4) Conduct corn trait association analysis on candidate loci and screen candidate regions associated with sweet and waxy traits of corn;

[0014] 5) Perform sequence comparison analysis on the candidate regions to screen for polymorphic sites;

[0015] 6) Perform KASP technical analysis and verification on the polymorphic sites, use KASP platform primers to evaluate and analyze the candidate sites, and select SNP sites that effectively distinguish between waxy corn and sweet corn.

[0016] In one aspect, the present invention further provides a method for identifying corn varieties, characterized in that the method comprises the following steps:

[0017] (1) extracting total DNA from the corn sample to be tested;

[0018] (2) using the total DNA of the corn to be tested as a template, and performing a PCR amplification reaction using the primer pair of the KASP molecular marker shown in SEQ ID NO. 2-5;

[0019] (3) Read the fluorescence data of the PCR amplification product and judge the result based on the fluorescence color.

[0020] Preferably, the PCR reaction system in step (2) has a total volume of 25 μL, specifically including 8 μL of 10X Easy Taq buffer, 2 μL of dNTP (10 mM), 1 μL of forward and reverse primers (10 mM), 2 μL of DNA template (100-200 ng / μL), 1 μL of Easy Taq enzyme (5 U / μL), and 10 μL of ddH2O.

[0021] Preferably, the PCR reaction program in step (2) is: 94°C pre-denaturation for 3 min, 94°C denaturation for 45 s, 55°C annealing for 30 s, 72°C extension for 45 s, 40 cycles, 72°C extension for 10 min, and 4°C storage for 10 min.

[0022] Preferably, the result determination method in step (3) is: if the fluorescent staining is green, the sample to be tested is a glutinous corn variety; if the fluorescent staining is red, the sample to be tested is a sweet corn variety; if the fluorescent staining is yellow, the sample to be tested is a sweet and glutinous corn variety.

[0023] Preferably, the result determination method in step (3) is: if the sample to be tested is a CC genotype, then the sample to be tested is a glutinous corn variety; if the sample to be tested is a TT genotype, then the sample to be tested is a sweet corn variety; if the sample to be tested is a CT genotype, then the sample to be tested is a sweet and glutinous corn variety.

[0024] In one aspect, the present invention also provides a kit for identifying corn varieties, characterized in that the kit comprises a primer pair shown in SEQ ID NO.2-5.

[0025] On the other hand, the present invention also provides the use of the molecular marker or the kit in screening or identifying sweet and sticky corn varieties or identifying sweet and sticky corn genotypes or assisting in sweet and sticky corn breeding;

[0026] The beneficial effect of the present invention is that: this study screened out SNP markers that distinguish the sweet and sticky traits of corn through whole genome sequencing analysis. The molecular marker is located at the polymorphic C / T site at position 1029462 of chromosome 3 of corn (GenBank: LR618876.1). The site can effectively distinguish between waxy corn and sweet corn varieties, wherein the CC genotype corresponds to the waxy corn variety, the TT genotype corresponds to the sweet corn variety, and the CT genotype corresponds to the sweet and sticky corn variety. No false positive results were found in the test of common corn varieties, confirming that the molecular marker developed by this patent fills the gap of no SNP molecular markers for the sweet and sticky traits of corn. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below in conjunction with specific embodiments. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein belong to the meanings commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0028] Example 1: Screening of SNP molecular markers for corn variety identification

[0029] 1) Extract genomic DNA from waxy corn (Sukenuo 1701), sweet corn (Jingtian 3) and sweet waxy corn (Sukenuo 1902) respectively;

[0030] 2) constructing Illumina sequencing libraries for genomic DNA and performing high-throughput sequencing;

[0031] 3) Annotate and compare the sequencing data to screen for polymorphic sites, and screen for sites with population marker polymorphism (MAF) greater than 0.01 and less than 0.05;

[0032] 4) Conduct corn trait association analysis on candidate loci and screen candidate regions associated with sweet and waxy traits of corn;

[0033] 5) Perform sequence comparison analysis on the candidate regions to screen for polymorphic sites;

[0034] 6) Perform KASP technical analysis and verification on polymorphic sites, use KASP platform primers to evaluate and analyze candidate sites, and select SNP sites that effectively distinguish waxy corn from sweet corn;

[0035] 7) The polymorphic C / T locus located at position 1029462 of chromosome 3 of maize (GenBank: LR618876.1) was finally obtained through identification and analysis. This locus can effectively distinguish between waxy corn and sweet corn varieties. The CC genotype corresponds to the waxy corn variety, the TT genotype corresponds to the sweet corn variety, and the CT genotype corresponds to the sweet and waxy corn variety.

[0036] Example 2: Application of SNP molecular markers for corn variety identification

[0037] Ten samples of common glutinous corn (Jinnuo 1607, Wannuo 2000, Zhenuoyu 14, Sidanuo 49), sweet corn varieties (Zhetian 11, Guangliangtian 27, Jinguan 218, Huameitian 8) and sweet glutinous corn varieties (Meiyunuo 16, Zhentiannuo 108, Caitiannuo 6, Mihuatiannuo 3) were selected for efficiency identification analysis of SNP molecular markers. The analysis method includes the following steps:

[0038] (1) extracting total DNA from the corn sample to be tested;

[0039] (2) Using the total DNA of the corn to be tested as a template, a PCR amplification reaction was performed using the primer pairs shown in SEQ ID NO.1-3; the PCR reaction system had a total volume of 25 μL, specifically including 8 μL of 10X Easy Taq buffer, 2 μL of dNTP (10 mM), 1 μL of each forward and reverse primer (10 mM), 2 μL of DNA template (100-200 ng / μL), 1 μL of Easy Taq enzyme (5 U / μL), and 10 μL of ddH2O.

[0040] PCR reaction program: 94°C pre-denaturation for 3 min, 94°C denaturation for 45 s, 55°C annealing for 30 s, 72°C extension for 45 s, 40 cycles, 72°C extension for 10 min, and 4°C storage for 10 min.

[0041] (3) The PCR product is placed in an ELISA instrument to read the fluorescence data, and the result is judged according to the fluorescence color. The results are shown in Table 1 below. The results show that the 1029462 position of chromosome 3 in all waxy corn varieties presents a CC genotype, the sweet corn is a TT genotype, and the sweet waxy corn variety presents a CT genotype. That is, the SNP molecular marker of the present invention can achieve the purpose of 100% effective differentiation of sweet waxy corn varieties.

[0042] Table 1. SNP genotype analysis of sweet and waxy corn

[0043]

[0044]

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying corn varieties, characterized in that: The method comprises the following steps: (1) Extracting total DNA from the corn sample to be tested; (2) Using the total DNA of the corn to be tested as a template, a PCR amplification reaction is performed using the primer pair of the KASP molecular marker shown in SEQ ID NO.2-5; the 5' end of the primer of the KASP molecular marker shown in SEQ ID NO.2 is labeled with FAM, and the 5' end of the primer of the KASP molecular marker shown in SEQ ID NO.3 is labeled with ROX; (3) Reading the fluorescence data of the PCR amplification product and judging the result according to the fluorescence color; the result judgment method is as follows: if the base of the sample to be tested at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 is CC genotype, the sample to be tested is a waxy corn variety; if the base of the sample to be tested at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 is TT genotype, the sample to be tested is a sweet corn variety; if the base of the sample to be tested at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 is CT genotype, the sample to be tested is a sweet and waxy corn variety.

2. The method according to claim 1, characterized in that The PCR reaction system in step (2) has a total volume of 25 μL, specifically including 8 μL of 10X Easy Taq buffer, 2 μL of dNTP, 1 μL of each forward and reverse primer, 2 μL of DNA template, 1 μL of Easy Taq enzyme, and 10 μL of ddH2O; the PCR reaction program in step (2) is: 94°C pre-denaturation for 3 min, 94°C denaturation for 45 s, 55°C annealing for 30 s, 72°C extension for 45 s, 40 cycles, 72°C extension for 10 min, and storage at 4°C for 10 min.

3. Application of a primer set for detecting KASP molecular markers in screening or identifying sweet and sticky corn varieties, characterized in that: The KASP molecular marker is a SNP marker, and the sequence of the KASP molecular marker is shown in SEQ ID NO.1, in which the base at the 26th position from the 5' end is C or T; if the base at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 of the sample to be tested is a CC genotype, then the sample to be tested is a waxy corn variety; if the base at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 of the sample to be tested is a TT genotype, then the sample to be tested is a sweet corn variety; if the base at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 of the sample to be tested is a CT genotype, then the sample to be tested is a sweet and waxy corn variety.

4. The application of detecting KASP molecular markers in identifying sweet and sticky corn genotypes is characterized in that: The KASP molecular marker is a SNP marker, and its sequence is shown in SEQ ID NO.

1. In the sequence, the base at the 26th position from the 5' end is C or T; if the base at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 of the sample to be tested is a CC genotype, then the sample to be tested is a waxy corn variety; if the base at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 of the sample to be tested is a TT genotype, then the sample to be tested is a sweet corn variety; if the base at the 26th position from the 5' end of the sequence shown in SEQ ID NO.1 of the sample to be tested is a CT genotype, then the sample to be tested is a sweet and waxy corn variety.

Images