SNP (Single Nucleotide Polymorphism) molecular marker related to drought resistance of corn and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker related to drought resistance of corn and application of the SNP molecular marker, and relates to the field of agricultural molecular biology. The SNP molecular marker comprises at least one of a first SNP molecular marker and a second SNP molecular marker; the first SNP molecular marker is located at a basic group at a 97605381 site of a chromosome 9 of a corn reference genome Zeamas.AGPv4, and the polymorphism of the first SNP molecular marker is C / T; the second SNP molecular marker is located at the basic group of the 97604011 site of the No.9 chromosome of the corn reference genome Zeamas.AGPv4, and the polymorphism is C / T. The SNP molecular marker disclosed by the invention has the characteristics of high specificity, high sensitivity, high resolution and high typing quality; the drought resistance of the corn material can be quickly judged according to the genotype, and the method has wide application universality and can be used for corn breeding.

Description

Technical Field

[0001] The invention belongs to the field of agricultural molecular biology, and in particular relates to a SNP molecular marker related to corn drought resistance and an application thereof. Background Art

[0002] Corn is an important food crop, and also one of the raw materials for animal feed, industrial raw materials and biomass energy. It is sensitive to water stress. Once corn is subjected to drought stress during its growth period, it will seriously affect the normal growth and development of corn, resulting in reduced photosynthesis, wilting leaves, reduced plant height, delayed silking, increased flowering interval (ASI) between male and female ears, asynchronous pollination and silking, and reduced pollination fruiting rate, thereby reducing corn yields.

[0003] At present, the drought-resistant breeding of corn mainly relies on conventional breeding based on phenotypic selection. The investigation and identification of phenotypes mainly rely on the investigation of breeding samples after they show phenotypes during the growth process. This method depends on the visual recognition of morphological characteristics and biological characteristics. The judgment criteria are often difficult to quantify accurately, are highly subjective, and have low detection sensitivity and resolution. They are easily affected by environmental and cultivation conditions, and have poor accuracy and stability. They are time-consuming and have poor timeliness. They require a lot of manpower and material resources and are costly. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a SNP molecular marker related to drought resistance of corn and its application.

[0005] The present invention also provides a primer set for detecting the above SNP molecular markers.

[0006] The present invention also provides a kit.

[0007] The invention also provides a gene chip.

[0008] The present invention also proposes the application of the above-mentioned SNP molecular marker, primer set, kit and / or gene chip.

[0009] The present invention also provides a method for comparing the drought resistance of tested corn, screening or assisting in screening corn strains or varieties with relatively strong drought resistance, screening or assisting in screening corn strains or varieties with relatively weak drought resistance, or corn breeding.

[0010] According to a first aspect of the present invention, a SNP molecular marker associated with drought resistance of maize is provided, comprising at least one of a first SNP molecular marker and a second SNP molecular marker;

[0011] The first SNP molecular marker is located at base 97605381 of chromosome 9 of the maize reference genome Zea_mays.AGPv4, and the polymorphism is C / T;

[0012] The second SNP molecular marker is located at base 97604011 of chromosome 9 of the maize reference genome Zea_mays.AGPv4, and the polymorphism is C / T.

[0013] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular marker is proposed.

[0014] In some embodiments of the present invention, the primer set for amplifying the first SNP molecular marker includes a first specific primer; the first specific primer includes a first Primer X with a nucleotide sequence as shown in SEQ ID NO:1 and a first Primer Y with a nucleotide sequence as shown in SEQ ID NO:2.

[0015] In some embodiments of the present invention, the primer set for amplifying the second SNP molecular marker includes a second specific primer; the second specific primer includes a second Primer X with a nucleotide sequence as shown in SEQ ID NO:4 and a second Primer Y with a nucleotide sequence as shown in SEQ ID NO:5.

[0016] In some embodiments of the present invention, the primer set for amplifying the first SNP molecular marker further includes a first Common Primer having a nucleotide sequence as shown in SEQ ID NO:3.

[0017] In some embodiments of the present invention, the primer set for amplifying the second SNP molecular marker further includes a second Common Primer whose nucleotide sequence is shown as SEQ ID NO:6.

[0018] In some embodiments of the present invention, the first Primer X and the first Primer Y are respectively connected to different fluorescent reporter groups.

[0019] In some embodiments of the present invention, the second Primer X and the second Primer Y are respectively connected to different fluorescent reporter groups.

[0020] In some embodiments of the present invention, the fluorescent reporter group is selected from one of FAM, HEX, FITC, RED, TET, JOE, and R110.

[0021] According to a third aspect of the present invention, a kit is provided, the kit comprising the above-mentioned primer set.

[0022] According to a fourth aspect of the present invention, a gene chip is provided, wherein the gene chip comprises the above-mentioned primer set.

[0023] According to a fifth aspect of the present invention, the use of the above-mentioned SNP molecular marker, primer set, kit or gene chip in any one of A1) to A6) is proposed:

[0024] A1) Identify or assist in identifying drought resistance of maize;

[0025] A2) screening or assisting in screening maize lines or varieties with relatively strong drought resistance;

[0026] A3) Screening or assisting in the screening of maize lines or varieties with relatively weak drought resistance;

[0027] A4) Compare the drought resistance of the tested corn;

[0028] A5) Maize molecular marker-assisted breeding;

[0029] A6) Corn breeding.

[0030] In some embodiments of the present invention, the corn molecular marker-assisted breeding or the corn breeding includes improvement of corn drought resistance traits, etc.

[0031] According to the sixth aspect of the present invention, the use of the above-mentioned SNP molecular marker or primer set in the preparation of a product having any one of the functions B1) to B6) is proposed:

[0032] B1) Identify or assist in identifying drought resistance of maize;

[0033] B2) Screening or assisting in the screening of maize lines or varieties with relatively strong drought resistance;

[0034] B3) Screening or assisting in the screening of maize lines or varieties with relatively weak drought resistance;

[0035] B4) Compare the drought resistance of the tested corn;

[0036] B5) Maize molecular marker-assisted breeding;

[0037] B6) Corn breeding.

[0038] In some embodiments of the present invention, the product is selected from a kit, a chip or a system.

[0039] According to a seventh aspect of the present invention, a method for identifying or assisting in identifying drought resistance of corn, comparing the drought resistance of corn to be tested, screening or assisting in screening corn lines or varieties with relatively strong drought resistance, screening or assisting in screening corn lines or varieties with relatively weak drought resistance, corn molecular marker-assisted breeding or corn breeding is provided, comprising the following steps:

[0040] Detecting the genotype of the SNP molecular marker in the genome of the tested corn, and determining the drought resistance level of the tested corn according to the genotype;

[0041] Among them, for the first SNP molecular marker, the drought resistance of TT genotype and CT genotype is better than that of CC genotype;

[0042] Regarding the second SNP molecular marker, the drought resistance of CC genotype and CT genotype is better than that of TT genotype.

[0043] In some embodiments of the present invention, if the first SNP molecular marker in the genome of the corn to be tested is a TT genotype or a CT genotype, and the second SNP molecular marker is a CC genotype or a CT genotype, the drought resistance of the corn to be tested is relatively strong; otherwise, the drought resistance of the corn to be tested is relatively poor.

[0044] In some embodiments of the present invention, the genome of the corn to be tested is extracted by CTAB method or phenol / chloroform extraction method.

[0045] In some embodiments of the present invention, the drought resistance-related traits include yield under drought stress. For example, for the first SNP molecular marker, the drought resistance of TT genotype and CT genotype is better than that of CC genotype, including: the yield of corn of TT genotype and CT genotype under drought stress is higher than that of corn of CC genotype. For the second SNP molecular marker, the drought resistance of CC genotype and CT genotype is better than that of TT genotype, including: the yield of corn of CC genotype and CT genotype under drought stress is higher than that of corn of TT genotype.

[0046] In some embodiments of the present invention, the genotype of the above SNP molecular markers in the genome of the tested corn is detected by KASP (competitive allele-specific PCR) technology.

[0047] In some embodiments of the present invention, the composition of the KASP reaction mixture for detecting SNP molecular markers using the KASP technology is as follows:

[0048]

[0049] In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using KASP technology is: 94°C for 15 min; 94°C for 20 s, 65°C-57°C (annealing temperature decreases by 0.8°C each cycle) for 60 s, 10 cycles; 94°C for 20 s, 57°C for 60 s, 33 cycles.

[0050] The present invention has at least the following beneficial effects:

[0051] The SNP molecular markers ZM900039 and ZM900040 linked to the drought resistance trait of corn provided by the present invention are significantly correlated with the drought resistance of corn independently, and the selection result of drought resistance is basically consistent with the field identification, and can be used as functional molecular markers to screen corn resources and varieties with strong drought resistance. It is beneficial to reduce the field planting scale of the breeding population, shorten the breeding period, and accelerate the breeding process, and has a good application prospect in breeding related to the drought resistance trait of corn. The two marker sites are high in quality, single-copy, and highly polymorphic, and have a wide range of application universality.

[0052] The primer set developed by the present invention can detect the genotype of the SNP molecular marker of corn quickly, with high throughput and low cost, so as to determine its drought resistance level. The detection method realized by the primer set is simple, fast, and low in detection cost, and is applicable to different detection instruments and equipment. The detection method realized based on the Douglas Array Tape platform is simple, with high automation, high throughput, high speed, small amount of reagents, low detection cost, accurate detection results, good repeatability and stability, and data results from different detection laboratories can be compared and verified with each other, and the data has universal comparability.

[0053] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0055] Figure 1 Developing a flow chart for the molecular markers of the present invention;

[0056] Figure 2 This is a diagram showing the typing results of the molecular marker ZM900039 in Example 1 of the present invention;

[0057] Figure 3 This is a diagram of the typing results of the molecular marker ZM900040 in Example 1 of the present invention. DETAILED DESCRIPTION

[0058] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0059] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials and reagents used, unless otherwise specified, are all reagents and materials available from commercial sources.

[0060] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0061] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0062] In the phenotypic identification of the present invention, the method is to set up two treatments, a water stress area (DRT) and a normal irrigation area (W), each treatment is repeated 3 times, the plot is a single-row area, the row length is 2.2m, the row spacing is 0.5m, the plant spacing is 0.2m, and 11 plants / plot. A 3m water isolation belt is set between the DRT area and the W area. After the corn in the DRT area is sown, it is no longer irrigated. In case of severe drought (1 / 3 of the leaves of the plants are curled all day, and the lower leaves are dried up), water is supplemented with 1 / 3 of the normal irrigation amount in the W area during the same period until harvest. If drought occurs during the whole growth period of corn in the W area, irrigation is carried out in time. Except for the different water treatment conditions in the two treatment areas, other field management measures are the same as the local field level. After harvesting, the yield of the water stress area (DRT) and the normal irrigation area (W) is compared and counted. If the corn yield is reduced by less than 20%, the plant is judged to be a drought-resistant material; if the corn yield is reduced by more than 20%, the plant is judged to be a non-drought-resistant material.

[0063] Example 1: SNP molecular markers associated with drought resistance in maize

[0064] 1. Acquisition of SNP molecular markers:

[0065] The design process of molecular markers is as follows Figure 1 shown.

[0066] Based on the publicly available germplasm resources (from Dunhuang Seed Group Co., Ltd., Gansu Province), 62 core germplasm resources that have been phenotypically verified were selected for resequencing data to construct a maize drought resistance gene germplasm resource database. Through a large number of literature searches and data analysis, multiple SNP sites were obtained and about 150bp of their flanking sequences were obtained. For the obtained SNP sites, KASP primer markers were designed based on the maize reference genome Zea_mays.AGPv4 using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). Each group of markers has three primers (specific primer Primer X, specific primer Primer Y, and common primer Common Primer), and the 5' ends of the two specific primers are connected to FAM and HEX fluorescent sequences, respectively. After the design is completed, the primer sequence is further analyzed for the whole genome copy number, and the high-quality single-copy KASP marker site design marker information is finally obtained as shown in Table 1 below. The primers were commissioned to be synthesized by Bioengineering.

[0067] Table 1

[0068]

[0069] By using the above two molecular markers designed based on the KASP reaction principle, the drought resistance mutation type and drought resistance of corn materials can be detected with high throughput.

[0070] When the genotype detected by the ZM900039 molecular marker is CC, it indicates that the corn material is not drought-resistant; when the genotype detected is TT, it indicates that the corn material is drought-resistant; when the genotype detected is TC, it is a heterozygous genotype, indicating that the corn material is drought-resistant.

[0071] When the genotype obtained by the ZM900040 molecular marker detection is CC, it indicates that the corn material has drought resistance; when the genotype obtained by the detection is TT, it indicates that the corn material does not have drought resistance; when the genotype obtained by the detection is TC, it is a heterozygous genotype, indicating that the corn material has drought resistance.

[0072] 2. KASP marker genotyping quality verification:

[0073] (1) DNA extraction: The CTAB method was used to extract genomic DNA from the above 62 corn samples.

[0074] Those skilled in the art may also use other well-known methods (such as phenol / chloroform extraction method, etc.) to extract DNA, as long as the genomic DNA of corn can be extracted.

[0075] (2) KASP markers were validated and detected using the Douglas Scientific Array Tape system. The ArrayTape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for signal scanning, and INTELLICS for data analysis.

[0076] 2.1) PCR system preparation: NEXAR was used to automatically assemble the PCR amplification system. The PCR amplification system is shown in Table 2 below.

[0077] Table 2

[0078] Components Final concentration Actual usage 100μM Primer_C 0.42μM 0.0033μL 100μM Primer_X 0.17μM 0.0013μL 100μM Primer_Y 0.17μM 0.0013μL 2×KASP Master Mix 1× 0.3945μL Ultrapure water 0.3995μL DNA (add DNA to tape membrane and dry) 20 ng~50 ng Total volume 0.8μL

[0079] 2.2) PCR amplification: PCR amplification was performed using SOELLEX.

[0080] The amplification conditions were as follows: 94°C for 15 min; 94°C for 20 sec, 65°C-57°C (annealing temperature decreased by 0.8°C each cycle) for 60 sec, 10 cycles; 94°C for 20 sec, 57°C for 60 sec, 33 cycles.

[0081] 2.3) Signal scanning and genotyping: After the PCR reaction is completed, ARAYA is used to scan the fluorescence signal of the reaction system; then INTELLICS is used for genotyping and data analysis.

[0082] In the KASP marker genotyping test, the genotypes of the samples are divided into 3 clusters. Among them, the X cluster indicates that the sample contains a homozygous X allele at this KASP marker site (marked in red in the genotyping diagram, located in the upper left corner of the graph), the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker site (marked in blue in the genotyping diagram, located in the lower right corner of the graph), and the blank control is marked in black in the genotyping diagram.

[0083] The typing results of 62 samples are as follows Figure 2 and Figure 3 shown.

[0084] The homozygous clusters of KASP markers ZM900039 and ZM900040 were well-typed and compact, with single-copy loci, and the detection rates were higher than 98%, with high consistency with the phenotype. Therefore, the genotyping quality of KASP markers ZM900039 and ZM900040 can fully meet the precise detection of drought resistance traits in maize.

[0085] Example 2: Specificity and practicality testing

[0086] In order to detect the specificity and practicality of the markers ZM900039 and ZM900040 in the present invention, a population of 41 improved strains was selected for field germplasm experiments and genotype detection according to the above detection method.

[0087] The test results are shown in Table 3.

[0088] Table 3

[0089]

[0090]

[0091]

[0092] The genotypes and phenotypes of the 41 samples were basically consistent. The markers ZM900039 and ZM900040 of the present invention have high specificity in detecting drought resistance of corn and can quickly and accurately identify whether the test material is drought-resistant.

[0093] The reagents and consumables of the Douglas Arraytape genotyping platform used in the present invention are all purchased from LGC, UK. Advantages of KASP marker detection based on the Douglas Array Tape platform: The automation level of KASP markers based on the Douglas Array Tape platform reaches 90%, which greatly reduces the manpower and human errors in the laboratory. The detection throughput is high, and 122,880 data points can be obtained in 8 hours, which is 10 times that of the traditional 96-well plate SNP genotyping method. The detection reaction volume is low (only 0.8 μL / reaction), and the cost of reagents and consumables is reduced by 70%-90% compared with the traditional 96-well plate SNP genotyping method.

[0094] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A SNP molecular marker, characterized in that: Including at least one of a first SNP molecular marker and a second SNP molecular marker; The first SNP molecular marker is located at base 97605381 of chromosome 9 of the maize reference genome Zea_mays.AGPv4, and the polymorphism is C / T; The second SNP molecular marker is located at base 97604011 of chromosome 9 of the maize reference genome Zea_mays.AGPv4, and the polymorphism is C / T.

2. A primer set for amplifying the SNP molecular marker as described in claim 1.

3. The primer set according to claim 2, characterized in that The primer set for amplifying the first SNP molecular marker includes a first specific primer; the first specific primer includes a first Primer X having a nucleotide sequence as shown in SEQ ID NO: 1 and a first Primer Y having a nucleotide sequence as shown in SEQ ID NO: 2; the first Primer X and the first Primer Y are respectively connected to different fluorescent reporter groups; Preferably, the primer set for amplifying the first SNP molecular marker also includes a first Common Primer whose nucleotide sequence is shown as SEQ ID NO:

3.

4. The primer set according to claim 2, characterized in that: The primer set for amplifying the second SNP molecular marker includes a second specific primer; the second specific primer includes a second Primer X with a nucleotide sequence as shown in SEQ ID NO:4 and a second Primer Y with a nucleotide sequence as shown in SEQ ID NO:5; the second Primer X and the second Primer Y are respectively connected to different fluorescent reporter groups; Preferably, the primer set for amplifying the second SNP molecular marker further includes a second Common Primer whose nucleotide sequence is shown as SEQ ID NO:

6.

5. The primer set according to claim 3 or 4, characterized in that: The fluorescent reporter group is selected from one of FAM, HEX, FITC, RED, TET, JOE, and R110.

6. A kit, characterized in that: The kit comprises the primer set according to any one of claims 2 to 5.

7. A gene chip, characterized in that: The gene chip comprises the primer set according to any one of claims 2 to 5.

8. Use of the SNP molecular marker according to claim 1, the primer set according to any one of claims 2 to 5, the kit according to claim 6 or the gene chip according to claim 7 in any one of A1) to A6): A1) Identify or assist in identifying drought resistance of maize; A2) screening or assisting in screening maize lines or varieties with relatively strong drought resistance; A3) Screening or assisting in the screening of maize lines or varieties with relatively weak drought resistance; A4) Compare the drought resistance of the tested corns; A5) Maize molecular marker-assisted breeding; A6) Corn breeding.

9. Use of the SNP molecular marker according to claim 1 or the primer set according to any one of claims 2 to 5 in preparing a product having any one of the functions B1) to B6): B1) Identify or assist in identifying drought resistance of maize; B2) Screening or assisting in the screening of maize lines or varieties with relatively strong drought resistance; B3) Screening or assisting in the screening of maize lines or varieties with relatively weak drought resistance; B4) Compare the drought resistance of the tested corn; B5) Maize molecular marker-assisted breeding; B6) Corn breeding.

10. A method for identifying or assisting in identifying drought resistance of corn, comparing the drought resistance of corn to be tested, screening or assisting in screening corn strains or varieties with relatively strong drought resistance, screening or assisting in screening corn strains or varieties with relatively weak drought resistance, corn molecular marker-assisted breeding or corn breeding, characterized in that: The method comprises the following steps: Detecting the genotype of the SNP molecular marker in the genome of the tested corn, and determining the drought resistance level of the tested corn according to the genotype; Among them, for the first SNP molecular marker, the drought resistance of TT genotype and CT genotype is better than that of CC genotype; Regarding the second SNP molecular marker, the drought resistance of CC genotype and CT genotype is better than that of TT genotype.

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