CN114606334A - Development and Application of SNP Molecular Markers for Maize Flowering Stage Gene - Google Patents

Abstract

The invention discloses development and application of SNP molecular markers of maize flowering phase genes, and belongs to the technical field of crucibles. Detecting the genotype of the corn to be detected, and identifying or assisting in identifying the flowering phase of the corn according to the genotype of the corn to be detected; the genotype is the genotype of KASP _ D8 locus in the corn genome; the KASP _ D8 site is an SNP site in the genome of corn and is located at the 272694744 th site of the No.1 chromosome of corn, and the SNP site is C or G which is the 101 th nucleotide of SEQ ID No. 4. A KASP marker KASP _ D8 closely linked to a maize flowering gene D8, which is developed based on KASP technology and can detect the 272694744 th base of chromosome 1 of maize genome at high throughput. The invention applies KASP technology to carry out genotype identification on the maize flowering phase gene D8, has the advantages of simple and convenient operation, low cost, short detection period, stable marking, environmental protection and the like, can accurately detect the maize flowering phase gene D8, and has important significance for promoting maize flowering phase breeding.

Description

Development and application of SNP molecular markers for maize flowering genes

technical field

The invention relates to the field of biotechnology, in particular to the development and application of SNP molecular markers related to maize flowering stage genes.

Background technique

Flowering is an important physiological transition of plants from the vegetative growth stage to reproductive growth. Entering the flowering stage earlier and shortening the crop cultivation period can reduce the cost of field management and increase the output value, which is of great significance to improving the income of the people. Breeding maize varieties at early flowering stage is the most efficient, economical and environmentally friendly method to shorten the cultivation period of maize (Wang Di. Research on QTL mapping and genetic basis of maize flowering and tassel-related traits [D]. Chinese Academy of Agricultural Sciences, 2011.).

At present, some maize flowering stage genes have been cloned, such as ZCN8 gene and D8 gene (Tian Feng, Guo Li, Wang Xuehan. Two specific molecular markers of maize flowering stage gene ZCN8 and its application: CN108866227A[P].2018.; Ou Yanghong, Wu Wenwen, Shao Yuanjian, et al. CAPS marker development and its application in the development of D8 gene functional markers [J]. Molecular Plant Breeding, 2016(10):2716-2721.). At the same time, molecular markers linked to genes have also been developed, and the improved breeding of maize at flowering stage is carried out by molecular marker-assisted selection (MAS), which makes up for many shortcomings in traditional breeding and becomes a solution to the difficulty of variety selection. effective approach to the problem.

However, although the linked markers of maize flowering stage-related genes can be used for molecular marker-assisted selection, most of them are CAPS markers, SSR markers, InDel markers or enzyme cleavage markers, which have low detection efficiency and may also produce aerosols to pollute the environment, which is not applicable. On a high-throughput molecular detection platform (Tian Feng, Guo Li, Wang Xuehan. Two specific molecular markers of maize flowering stage gene ZCN8 and its application: CN108866227A[P]. 2018; Ou Yanghong, Wu Wenwen, Shao Yuanjian, et al. .CAPS marker development and its application in the development of D8 gene functional markers [J]. Molecular Plant Breeding, 2016(10):2716-2721.). Therefore, the development of low-cost, high-throughput molecular markers is an urgent need to promote gene identification at early flowering stage of maize, increase breeding accuracy and improve breeding efficiency at early flowering stage.

KASP (Kompetitive Allele-Specific PCR, competitive allele-specific PCR) achieves the effect of genotyping by specifically identifying gene loci with fluorescent probes, and can quickly detect SNP loci. Compared with molecular markers such as SSR, RFLP, and InDel, KASP markers have the characteristics of rapid detection, low cost, and easy large-scale application. KASP markers do not need to be typed according to the size of DNA fragments, and can get rid of the relative steps of traditional gel electrophoresis. The cumbersome, low-throughput, and expensive detection method is more suitable for the rapidly developing high-throughput molecular detection platform at this stage. Therefore, the development of low-cost, high-throughput molecular detection platform KASP molecular markers for maize flowering genes is of great significance for popularizing the application of molecular marker technology and improving the breeding efficiency and breeding level of maize early flowering in my country.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a SNP marker closely linked with the maize flowering stage gene D8 and its application method.

The present invention provides a SNP molecular marker KASP_D8 closely linked with the maize florescence gene D8, the molecular marker KASP_D8 is a marker developed based on a SNP site in the maize florescence gene D8, and the SNP site is the nucleotide type of C Or G, is the 101st nucleotide of SEQ ID No. 4 in the sequence listing.

The application of the KASP molecular marker closely linked to the maize flowering stage gene D8 in C1, C2, C3 or C4 should also fall within the protection scope of the present invention:

C1, the application of detecting the polymorphism or genotype of the KASP_D8 site in the corn genome in identifying or assisting in identifying the flowering period of corn;

C2, the application of detecting the polymorphism or genotype of the KASP_D8 site in the corn genome in the preparation of identification or auxiliary identification of the product of the flowering period of corn;

C3, the application of detecting the polymorphism or genotype of the KASP_D8 site in the maize genome in maize assisted breeding or preparing maize assisted breeding products;

C4. The application of detecting the polymorphism or genotype of the KASP_D8 site in the maize genome in the selection of maize resources at the early flowering stage.

The present invention also provides a method for identifying or assisting in identifying the flowering period of corn, comprising detecting the genotype of the corn to be tested, and identifying or assisting in identifying the flowering period of the corn according to the genotype of the corn to be tested; the genotype is the genotype of KASP_D8 in the corn genome ; The KASP_D8 site is that the nucleotide type is C or G, which is the 101st nucleotide of SEQ ID No.4 in the sequence listing.

Wherein, when the genotype of the KASP_D8 site is the GG genotype or the CG genotype, the maize is in the early flowering stage or the candidate is the early flowering stage; when the genotype of the KASP_D8 site is the CC genotype, the maize is in the late flowering stage or the candidate is late flowering; wherein, the GG genotype indicates that the nucleotide type of the KASP_D8 site in the maize genome is a homozygous type of G; the CC genotype represents the nucleotide of the KASP_D8 site in the maize genome The species is homozygous for C; the GC genotype indicates that the nucleotide species at the KASP_D8 site in the maize genome are heterozygous for G and C.

The application of the material for detecting the polymorphism or genotype of the KASP_D8 site in the maize genome in A1, A2, A3 or A4 should also fall within the protection scope of the present invention:

A1, the application of detecting the polymorphism or genotype of the KASP_D8 site in the corn genome in identifying or assisting in identifying the flowering period of corn;

A2, the application of detecting the polymorphism or genotype of the KASP_D8 site in the maize genome in the preparation of identification or auxiliary identification of the product of maize flowering;

A3, the application of detecting the polymorphism or genotype of the KASP_D8 site in the maize genome in maize assisted breeding or preparing maize assisted breeding products;

A4. The application of detecting the polymorphism or genotype of the KASP_D8 locus in the maize genome in the selection and breeding of maize resources at the early flowering stage.

The present invention also provides a method for breeding maize resources at early flowering stage, comprising selecting maize whose genotype at the KASP_D8 site is GG genotype or GC for breeding, wherein the genotype is the KASP_D8 site of D8 in the maize genome The KASP_D8 site is the nucleotide type of G or C, which is the 101st nucleotide of SEQ ID No. 4 in the sequence listing; the GG genotype represents the KASP_D8 site in the maize genome The nucleotide species of KASP_D8 is homozygous for G; the GC genotype indicates that the nucleotide species at the KASP_D8 site in the maize genome are heterozygous for G and C.

The present invention provides a product containing a substance for detecting the polymorphism or genotype of the KASP_D8 site in the corn genome, which is any product in C1)-C4):

C1) Products that detect SNP polymorphisms or genotypes associated with corn flowering;

C2) Identify or assist in identifying the products at the flowering stage of corn;

C3) Products for corn-assisted breeding;

C4) Products used for breeding corn resources in early flowering stage.

The application of the product containing the substance for detecting the polymorphism or genotype of the KASP_D8 site in the corn genome in B1, B2, B3 or B4 should also fall within the protection scope of the present invention:

B1, the application of detecting the polymorphism or genotype of the KASP_D8 site in the corn genome in identifying or assisting in identifying the flowering period of corn;

B2, the application of detecting the polymorphism or genotype of the KASP_D8 site in the corn genome in the preparation of identification or auxiliary identification of the product of the flowering period of corn;

B3, the application of detecting the polymorphism or genotype of the KASP_D8 site in the maize genome in maize assisted breeding or preparing maize assisted breeding products;

B4. The application of detecting the polymorphism or genotype of the KASP_D8 site in the maize genome in breeding maize resources at the early flowering stage.

The substance for detecting the polymorphism or genotype of the KASP_D8 site in the maize genome is the following D1), D2) or D3):

D1) The substance for detecting the polymorphism or genotype of the KASP_D8 site in the corn genome contains PCR primers for amplifying the DNA fragment of the corn genome including the KASP_D8 site;

D2) the substance that detects the polymorphism or genotype of the KASP_D8 site in the corn genome is a PCR reagent containing the PCR primer;

D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

The PCR primers are P1 or P2:

P1. The PCR primer is a single-stranded DNA whose nucleotide sequence is the 22-40th position of SEQ ID No. 1 in the sequence table, and the nucleotide sequence is the 22-40th position of SEQ ID No. 2 in the sequence table. The single-stranded DNA and nucleotide sequence of the bit is the primer set composed of the single-stranded DNA of SEQ ID No.3 in the sequence listing;

P2. The PCR primers are the single-stranded DNA shown in SEQ ID No.1 in the sequence listing, the single-stranded DNA shown in SEQ ID No.2 in the sequence listing, and the single-stranded DNA shown in SEQ ID No.3 in the sequence listing. Primer sets for strand DNA.

The present invention provides the development and application of a KASP marker closely linked to the maize florescence gene D8, and the molecular marker is the KASP marker KASP_D8 closely linked to the maize florescence gene D8. The marker was developed based on KASP technology and can detect the 272694744th base of chromosome 1 in the maize genome with high throughput. The invention uses KASP technology to identify the genotype of the maize flowering stage gene D8, and has the advantages of simple operation, low cost, short detection period, stable marking, green environmental protection and the like, can accurately detect the maize flowering stage gene D8, and has important significance for promoting maize flowering stage breeding .

Description of drawings

Figure 1 is a genotyping diagram of using the molecular marker KASP_D8 to detect 8 tested maize variety materials in Example 1, wherein FAM indicates that the genotype is CC, VIC indicates that the genotype is GG, and H indicates that the genotype is GC.

Figure 2 is a genotype diagram of 48 F2 test populations detected by molecular marker KASP_D8 in Example 2, wherein FAM indicates that the genotype is CC, VIC indicates that the genotype is GG, and H indicates that the genotype is GC.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The corn varieties used in the following examples are all commercially available.

The inventor of the present invention discovered a SNP locus related to maize flowering through literature (Ou Yanghong, Wu Wenwen, Shao Yuanjian, et al. Development of CAPS marker and its application in the development of D8 gene functional marker [J]. Molecular Plant Breeding , 2016(10):2716-2721.), which is anchored at position 272694744 of maize chromosome 1 (https://www.ncbi.nlm.nih.gov/assembly/GCF_902167145.1/, Genome Version Zm-B73-REFERENCE-NAM-5.0), the SNP flanking sequence is shown in SEQ ID No.4. KASP_D8 is located at position 101 of SEQ ID No. 4, where the nucleotide sequence is G or C.

The following GG genotype indicates that the nucleotide sequence of KASP_D8 is homozygous for G; CC genotype indicates that the nucleotide sequence of KASP_D8 is homozygous for C; GC genotype indicates that the nucleotide types of KASP_D8 are G and C the hybrid type.

Example 1 Development of KASP marker closely linked to maize flowering stage-related gene D8

1. Design of primers: According to the KASP_D8 site, extract flanking sequences of 100 bp on the left and right sides, and preferably use Primer5.0 software to design 3 sets of KASP primers. After detection by Douglas Scientific's ArrayTape platform, a set of KASP primers with good polymorphism was selected for subsequent verification.

The specific KASP primers used to detect KASP_D8 are as follows:

Primer X: 5'-gaaggtcggagtcaacggattCATCCTCCACAGGCTCACG-3' (SEQ ID No. 1, the lowercase part is the specific fluorescent tag sequence VIC);

Primer Y: 5'-gaaggtgaccaagttcatgctCATCCTCCACAGGCTCACC-3' (SEQ ID No. 2, the lowercase part is the specific fluorescent tag sequence FAM);

Primer R: 5'-TGTATTCCAGCACAAGGGTTTC-3' (SEQ ID No. 3).

2. DNA extraction: Genomic DNA was extracted from maize leaves by conventional CTAB method.

3. KASP Response Test

KASP marker amplification and reaction system:

(1) Fluorescence quantitative PCR instrument AB-Q6 Flex detection:

5μL PCR fluorescence quantitative instrument detection reaction system includes: genomic DNA 50ng, primer mixture 0.07μL (preferable primer mixture ratio: forward primer Primer X, Primer Y 100pmol·L-1 each 12μL, reverse primer Primer R100pmol·L-1 30μL, ddH2O 46μL, other reasonable primer mixture ratio can also achieve the same detection purpose), LGC company 2×KASP Mix (Low Rox) 2.5μL, according to the fluorescence quantitative PCR instrument AB-Q6 instrument operation manual, edit the sample table , execute the running program and save the data.

The above reaction system is the preferred reaction system of AB-Q6 Flex, and other reasonable reaction systems can also achieve the same detection purpose.

(2) Select Douglas Scientific's ArrayTape platform for detection

1.6μL PCR ArrayTape platform detection reaction system includes: 0.8μL containing genomic DNA 50ng/μL, primer mixture 0.03μL (preferable primer mixture ratio: forward primer Primer X, Primer Y 100pmol·L-1 each 12μL, reverse primer Primer R 100pmol·L-1 30μL, ddH2O 46μL, other reasonable primer mixture ratio can also achieve the same detection purpose), LGC company 2×KASP Mix (Std Rox) 0.8μL. According to the ArrayTape platform instrument operation manual, write the sample table, run the program, and read the data.

The above reaction system is the preferred reaction system of Douglas Scientific's ArrayTape platform, and other reasonable reaction systems can also achieve the same detection purpose.

Note: The above are the recommended detection methods. Other detection methods that can achieve the same detection purpose can also be applied to the molecular marker-assisted breeding process of the above markers.

Among them, 2×KASP Mix consists of fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B, as well as high-fidelity Taq enzyme, dNTP, Mg2+, etc. The nucleotide sequence of fluorescent probe A is: 5'-GAAGGTCGGAGTCAACGGATT-3', and a VIC fluorescent group is connected to the 5' end; the nucleotide sequence of fluorescent probe B is: 5'-GAAGGTGACCAAGTTCATGCT-3', its 5 A FAM fluorescent group is connected to the ' end; the nucleotide sequence of quenching probe A is: 5'-AATCCGTTGACTCCGACCTTC-3', and its 3' end is connected to a quenching group BHQ; the nucleotide sequence of quenching probe B is: The sequence is: 5'-AGCATGAACTTGGTCACCTTC-3', and its 3' end is connected with a quenching group BHQ.

Amplification program: pre-denaturation at 95°C for 10 min, 1 cycle; denaturation at 95°C for 20s, annealing at 55-62°C (preferably 55°C) for 60s, and set for 40 cycles.

At the same time, a blank control (NTC) without template DNA in the reaction system was set in the experiment, and one or more blank controls were set in each plate.

The scanning data is analyzed, then the genotype of the KASP_D8 site in the maize genome to be tested is determined as follows (that is, the 272694744th base of chromosome 1 of the maize genome is detected as G or C), if the amplification of the maize to be tested The fluorescence signal data of the product is analyzed by Douglas genotyping software near the X axis (VIC signal), and the genotype of the KASP_D8 site in the maize genome to be tested is GG homozygous (that is, the 272694744th base of chromosome 1 in the maize genome). is G homozygous type); if the fluorescence signal data of the amplified product of maize to be tested is close to the Y axis (FAM signal) through Douglas genotyping software analysis, then the genotype of the KASP_D8 site in the maize genome to be tested is CC homozygous (that is, the 272694744th base of chromosome 1 of the maize genome is C homozygous); if the fluorescence signal data of the amplified product of the maize to be tested is analyzed by Douglas genotyping software, it is located in the middle of the X-axis and the Y-axis (VIC and FAM signal), then the genotype of the KASP_D8 site in the maize genome to be tested is GC heterozygous (that is, the 272694744th base of chromosome 1 in the maize genome is GC heterozygous). The samples shown in black in the lower left corner are blank controls.

5. Marker typing data analysis

In order to verify the reliability of KASP_D8 marker, 8 maize materials were identified in the field, and the field flowering period data of the materials were obtained. The genotype of the material was detected using the ArrayTape platform of Douglas Scientific Company, and the detection method, reaction system and amplification procedure were implemented according to the above preferred scheme.

The amplification results showed that KASP_D8 marker could obtain stable PCR products in 8 materials, and two alleles G and C could be detected (Figure 1). Two early-flowering maize materials were GG genotype, and six late-flowering maize materials were CC genotype. Thus, it can be seen that the KASP_D8 marker can be used for the selection of maize at different flowering stages.

Therefore, the KASP_D8 marker described in the present invention can be used for molecular marker-assisted breeding of the gene D8 related to the flowering stage of maize.

Table 1. Flowering date and genotype information of 8 tested maize materials

Note: GG, homozygous for genotype G; CC, homozygous for genotype C; GC, heterozygous for genotype G and C; '*' means no detection signal.

Example 2 Application of KASP marker closely linked to maize flowering stage-related gene D8 in molecular marker-assisted selection of maize flowering stage plants

In order to detect the practicability of the KASP_D8 marker of the present invention, the corn early flowering stage material Ji 495 (as the female parent) described in Table 1 was crossed with the corn late flowering stage material Zheng 22 (as the male parent) to prepare an F2 segregated population, and the F2 segregated population was carried out. KASP marker detection and field flowering statistics (see Figure 2 and Table 2 for the detection results), the implementation method of marker detection and flowering identification refers to Example 1. Through statistical analysis of the flowering period and genotype of the segregating population, the average flowering period of the segregating population with genotypes of GG and GC was 70.25 days, the average flowering period of the segregated population with genotype CC was 78.33 days, and the average flowering period of the segregated population with genotypes of GG or GC was 78.33 days. The average flowering time of segregating population was significantly earlier than that of segregating population with genotype CC. The above results show that the marker KASP_D8 has high practicability in the screening of maize flowering plants.

Table 2. Phenotypic and Genotypic Information of Maize Segregating Populations

Note: GG, homozygous for genotype G; CC, homozygous for genotype C; GC, heterozygous for genotype G and C; '*' means no detection signal.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. It should be understood by those skilled in the art that the present invention is not limited by the above-mentioned embodiments, and without departing from the spirit and scope of the present invention, the present invention will also have various changes and improvements, and these changes and improvements all fall into the protection requirements within the scope of the present invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

sequence listing

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Claims (10)

1. the development method of the SNP molecular marker of maize florescence gene, it is characterized in that: comprise the genotype that detects the maize to be tested, identify according to the genotype of maize to be tested or assist the identification of maize florescence; Described genotype is the KASP_D8 position in the maize genome The genotype of the point; the KASP_D8 site is a SNP site in the maize genome, located at position 272694744 of maize chromosome 1, and the SNP site is a nucleotide type of C or G, which is SEQ ID No.4 Nucleotide at position 101; The genotype of the KASP_D8 site is the GG genotype or the GC genotype. The flowering period of the corn to be tested is earlier than or the candidate is earlier than the genotype of the KASP_D8 site. The tested corn of the CC genotype; the KASP_D8 site The genotype of GC genotype and the genotype of the KASP_D8 site is the GG genotype of corn to be tested, there is no significant difference in flowering; wherein, the GG genotype represents the KASP_D8 position in the corn genome. The nucleotide species of the point is the homozygous type of G; the CC genotype represents that the nucleotide species of the KASP_D8 site in the maize genome is the homozygous type of C; the GC genotype represents the maize genome described in The nucleotide species at the KASP_D8 site is a hybrid of G and C. 2. method according to claim 1 is characterized in that, is applied in corn breeding. 3. according to the development method of the SNP molecular marker of maize florescence gene in claim 1, it is characterized in that, the application of the polymorphism of described KASP_D8 site or genotype material in identifying or assisting in identifying maize florescence. 4. according to the development method of the SNP molecular marker of maize florescence gene in claim 1, it is characterized in that, the application of the polymorphism of described KASP_D8 site or genotype material in preparation identification or auxiliary identification of the product of maize florescence. 5. according to the development method of the SNP molecular marker of maize florescence gene in claim 1, it is characterized in that, the application of the polymorphism or genotype material of described KASP_D8 site in maize assisted breeding or preparation of maize assisted breeding products. 6. The method for corn breeding, characterized in that, comprising selecting the corn whose genotype of the KASP_D8 site described in claim 1 is GG or the genotype of GC as a parent for breeding, and the GG genotype represents the corn genome described in the corn genome. The nucleotide species at the KASP_D8 site is homozygous for G; the GC genotype indicates that the nucleotide species at the KASP_D8 site in the maize genome are heterozygous for G and C; the maize breeding may be breeding Early flowering corn. 7. A product containing a substance for detecting the polymorphism or genotype of the KASP_D8 site described in claim 1, which is any product in C1)-C3): C1) Products that detect SNP polymorphisms or genotypes associated with maize flowering; C2) Identify or assist in the identification of corn blooming products; C3) Products for corn-assisted breeding. 8. The application according to any one of claims 3-5 or the product according to claim 7, wherein the substance for detecting the polymorphism or genotype of the KASP_D8 site in claim 1 is the following D1) , D2) or D3): D1) The substance for detecting the polymorphism or genotype of the KASP_D8 site in claim 1 contains PCR primers for amplifying the maize genomic DNA fragment including the KASP_D8 site; D2) The substance for detecting the polymorphism or genotype of the KASP_D8 site in claim 1 is a PCR reagent containing the PCR primer; D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2). 9. The application or product according to claim 8, wherein the PCR primer is a single-stranded DNA, nucleotide whose nucleotide sequence is the 22-40th position of SEQ ID No.1 in the sequence table The sequence is a primer set consisting of the single-stranded DNA at positions 22-40 of SEQ ID No. 2 in the sequence listing and the nucleotide sequence is the single-stranded DNA of SEQ ID No. 3 in the sequence listing. 10. The application or product according to any one of claims 8 or 9, wherein the PCR primer is the single-stranded DNA shown in SEQ ID No. 1 in the sequence listing, and SEQ ID No. 1 in the sequence listing. The primer set of the single-stranded DNA shown in 2 and the single-stranded DNA shown by SEQ ID No. 3 in the Sequence Listing.

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