A SNP molecular marker associated with drought resistance in maize and its application

Abstract

This invention discloses a SNP molecular marker related to drought resistance in maize and its application, relating to the field of agricultural molecular biology. The SNP molecular marker includes at least one of a first SNP molecular marker and a second SNP molecular marker; the first SNP molecular marker is located at position 97605381 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a C / T polymorphism; the second SNP molecular marker is located at position 97604011 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a C / T polymorphism. The SNP molecular marker of this invention features high specificity, high sensitivity, high resolution, and high genotyping quality; it can rapidly determine the drought resistance of maize materials based on genotype, has broad applicability, and can be used in maize breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural molecular biology, specifically relating to a SNP molecular marker related to drought resistance in maize and its application. Background Technology

[0002] Corn is an important food crop and also a source of animal feed, industrial raw materials, and biomass energy. It is relatively sensitive to water stress. Drought stress during the corn's growth period can severely affect its normal growth and development, leading to reduced photosynthesis, wilting leaves, decreased plant height, delayed silking, increased inter-tapering interval (ASI), asynchronous pollen shedding and silking, and decreased pollination and seed setting rate, thereby reducing corn yield.

[0003] Currently, drought-resistant maize breeding mainly relies on conventional breeding methods that depend on phenotypic selection. Phenotypic investigation and identification primarily rely on investigating the phenotypes exhibited by the breeding samples during growth. This method depends on visual recognition of morphological characteristics and biological traits, and the judgment criteria are often difficult to quantify precisely, resulting in high subjectivity, low detection sensitivity and resolution; it is easily affected by environmental and cultivation conditions, leading to poor accuracy and stability; it is time-consuming and lacks timeliness; and it requires a large investment of manpower and resources, resulting in high costs. Summary of the Invention

[0004] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a SNP molecular marker related to drought resistance in maize and its application.

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

[0006] The present invention also proposes a reagent kit.

[0007] This invention also proposes a gene chip.

[0008] This invention also proposes the application of the above-mentioned SNP molecular markers, primer sets, kits and / or gene chips.

[0009] The present invention also proposes a method for comparing the drought resistance of maize samples, screening or assisting in the screening of maize lines or varieties with relatively strong drought resistance, screening or assisting in the screening of maize lines or varieties with relatively weak drought resistance, or for maize breeding.

[0010] According to a first aspect of the present invention, SNP molecular markers related to drought resistance in maize are proposed, including at least one of a first SNP molecular marker and a second SNP molecular marker;

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

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

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

[0014] In some embodiments of the present invention, the primer set used to amplify 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 used to amplify 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 used to amplify the first SNP molecular marker further includes a first Common Primer with a nucleotide sequence as shown in SEQ ID NO:3.

[0017] In some embodiments of the present invention, the primer set used to amplify the second SNP molecular marker further includes a second Common Primer with a nucleotide sequence as shown in 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-described primer set.

[0022] According to a fourth aspect of the present invention, a gene chip is provided, the gene chip comprising the aforementioned primer set.

[0023] According to a fifth aspect of the present invention, the application of the above-described SNP molecular markers, primer sets, kits, or gene chips in any one of A1) to A6) is proposed:

[0024] A1) To identify or assist in the identification of corn drought resistance;

[0025] A2) Screening or assisting in the screening of maize strains 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 corn samples being tested;

[0028] A5) Marker-assisted breeding of maize;

[0029] A6) Maize breeding.

[0030] In some embodiments of the present invention, maize molecular marker-assisted breeding or said maize breeding includes methods for improving maize drought resistance traits, etc.

[0031] According to a sixth aspect of the present invention, the application of the above-described SNP molecular markers or primer sets in the preparation of products having any one of the functions of B1) to B6) is proposed:

[0032] B1) To identify or assist in the identification of corn drought resistance;

[0033] B2) Screening or assisting in the screening of maize strains 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 corn samples being tested;

[0036] B5) Marker-assisted breeding of maize;

[0037] B6) Maize breeding.

[0038] In some embodiments of the present invention, the product is selected from reagent kits, chips, or systems.

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

[0040] The genotypes of the above-mentioned SNP molecular markers in the genome of the maize to be tested are detected, and the drought resistance level of the maize to be tested is determined based on the genotypes;

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

[0042] For the second SNP molecular marker, the CC and CT genotypes showed better drought resistance than the TT genotype.

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

[0044] In some embodiments of the present invention, the genome of the maize 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, regarding the first SNP molecular marker, the drought resistance of the TT and CT genotypes is superior to that of the CC genotype, meaning that maize with the TT and CT genotypes yields more under drought stress than maize with the CC genotype. Regarding the second SNP molecular marker, the drought resistance of the CC and CT genotypes is superior to that of the TT genotype, meaning that maize with the CC and CT genotypes yields more under drought stress than maize with the TT genotype.

[0046] In some embodiments of the present invention, the genotypes of the above-mentioned SNP molecular markers in the genome of the maize to be tested are 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 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 as follows: 94℃ for 15 min; 94℃ for 20 s, 65℃-57℃ (annealing temperature decreases by 0.8℃ per cycle) for 60 s, 10 cycles; 94℃ for 20 s, 57℃ 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 drought resistance traits in maize, provided by this invention, are independently and significantly associated with drought resistance in maize. The selection results for drought resistance are largely consistent with field identification, and they can be used as functional molecular markers to screen for drought-resistant maize resources and varieties. This is beneficial for reducing the field planting scale of breeding populations, shortening breeding cycles, and accelerating the breeding process, showing great application potential in breeding for drought resistance traits in maize. These two marker sites are of high quality, single-copy, and highly polymorphic, possessing broad applicability.

[0052] The primer set developed in this invention enables rapid, high-throughput, and low-cost detection of genotypes for maize SNP molecular markers, thereby determining their drought resistance level. The detection method implemented using this primer set is simple, fast, and cost-effective, and is applicable to various testing instruments and equipment. The detection method based on the Douglas Array Tape platform is simple, highly automated, high-throughput, fast, requires less reagent, has low cost, and provides accurate, repeatable, and stable results. Data from different testing laboratories can be compared and verified, demonstrating universal comparability.

[0053] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

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

[0055] Figure 1 This is a flowchart of the molecular marker development process of the present invention;

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

[0057] Figure 3 This is a genotyping result diagram of the molecular marker ZM900040 in Example 1 of the present invention. Detailed Implementation

[0058] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

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

[0060] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0061] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0062] In the phenotypic identification of this invention, the method involves establishing two treatments: a water stress zone (DRT) and a normal irrigation zone (W). Each treatment is repeated three times. Each plot is a single-row plot with a row length of 2.2m, a row spacing of 0.5m, a plant spacing of 0.2m, and 11 plants per plot. A 3m water isolation strip is set between the DRT and W plots. In the DRT plot, maize is not irrigated after sowing once the soil is sufficiently moist. If severe drought occurs (one-third of the plants have curled leaves throughout the day, and the lower leaves are withered), supplemental irrigation is provided at one-third of the normal irrigation amount in the W plot until harvest. In the W plot, maize is irrigated promptly if drought occurs throughout its growth period. Except for the water treatment conditions, all other field management practices are the same as in local fields. After harvest, the yields of the DRT and W plots are compared. If the maize yield reduction is less than 20%, the plant is considered drought-resistant; if the yield reduction is more than 20%, the plant is considered non-drought-resistant.

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

[0064] 1. Obtaining SNP molecular markers:

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

[0066] Based on publicly available germplasm resources (from Gansu Dunhuang Seed Industry Group Co., Ltd.), a maize drought resistance gene germplasm resource database was constructed using resequencing data from 62 core germplasm resources that had undergone phenotypic validation. Through extensive literature searches and data analysis, multiple SNP loci were obtained, and their flanking sequences (approximately 150 bp before and after the loci) were acquired. For the obtained SNP loci, KASP primer markers were designed using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) based on the maize reference genome Zea_mays.AGPv4. Each marker group consisted of three primers (specific primer Primer X, specific primer Primer Y, and universal primer Common Primer), with the 5' ends of the two specific primers linked to FAM and HEX fluorescent sequences, respectively. After design, genome-wide copy number analysis was performed on the primer sequences, resulting in high-quality single-copy KASP marker loci design information, as shown in Table 1 below. Primers were synthesized by Sangon Biotech.

[0067] Table 1

[0068]

[0069] Using the two molecular markers designed based on the KASP reaction principle, high-throughput detection of drought resistance mutants and drought resistance in maize materials can be performed.

[0070] When the genotype obtained by the ZM900039 molecular marker is CC, it indicates that the maize material does not have drought resistance; when the genotype obtained is TT, it indicates that the maize material has drought resistance; when the genotype obtained is TC, it is a heterozygous genotype, indicating that the maize material has drought resistance.

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

[0072] 2. Validation of KASP marker genotyping quality:

[0073] (1) DNA extraction: Genomic DNA was extracted from the above 62 maize samples using the CTAB method.

[0074] Those skilled in the art may also choose other known methods (such as phenol / chloroform extraction) for DNA extraction, as long as the genomic DNA of maize can be extracted.

[0075] (2) KASP marker validation and detection were performed 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: The PCR amplification system was automatically assembled using NEXAR. 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 (DNA is added to a tape membrane and then dried) 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℃ for 15 minutes; 94℃ for 20 seconds, 65℃~57℃ (annealing temperature decreased by 0.8℃ per cycle) for 60 seconds, 10 cycles; 94℃ for 20 seconds, 57℃ for 60 seconds, 33 cycles.

[0081] 2.3) Signal scanning and genotyping: After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then genotyping and data analysis were performed using INTELLICS.

[0082] In KASP marker genotyping, the genotypes of the samples were divided into three clusters. The X cluster indicates that the sample contains a homozygous X allele at this KASP marker locus (marked in red in the upper left corner of the genotyping graph), the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker locus (marked in blue in the lower right corner of the genotyping graph), and the blank control is marked in black in the genotyping graph.

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

[0084] The homozygous clusters of KASP markers ZM900039 and ZM900040 exhibit good and compact genotyping, with single-copy loci and detection rates exceeding 98%, and high consistency with phenotypes. Therefore, the genotyping quality of KASP markers ZM900039 and ZM900040 fully meets the requirements for accurate detection of drought resistance traits in maize.

[0085] Example 2: Specificity and Practicality Testing

[0086] To test the specificity and practicality of markers ZM900039 and ZM900040 in this invention, 41 improved strains were selected for field germplasm experiments and genotyping according to the above detection methods.

[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 this invention have high specificity in detecting drought resistance in maize and can quickly and accurately identify whether the test materials are drought resistant.

[0093] The reagents and consumables used in this invention with the Douglas Arraytape genotyping platform were all purchased from LGC Ltd., UK. Advantages of KASP marker detection based on the Douglas Arraytape platform: KASP marker detection based on the Douglas Arraytape platform achieves 90% automation, significantly reducing laboratory manpower and human error. High throughput: 122,880 data points can be obtained in 8 hours, 10 times that of traditional 96-well plate SNP genotyping methods. Low reaction volume (only 0.8 μL / reaction): Compared with traditional 96-well plate SNP genotyping methods, reagent and consumable costs are reduced by 70%-90%.

[0094] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The use of a primer set for amplifying SNP molecular markers, a kit comprising said primer set, or a gene chip comprising said primer set in any of A1) to A6): A1) To identify or assist in the identification of drought resistance in maize; A2) Screening or assisting in the screening of 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 corn samples being tested; A5) Marker-assisted breeding of maize; A6) Maize breeding; The SNP molecular marker includes at least one of a first SNP molecular marker and a second SNP molecular marker; The first SNP molecular marker is located at position 97605381 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a polymorphism of C / T; The second SNP molecular marker is located at position 97604011 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a polymorphism of C / T; The primer set used to amplify the first SNP molecular marker includes a first specific primer and a first common primer with a nucleotide sequence as shown in SEQ ID NO: 3; 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; the first Primer X and the first Primer Y are respectively linked to different fluorescent reporter groups; The primer set used to amplify the second SNP molecular marker includes a second specific primer and a second common primer with a nucleotide sequence as shown in SEQ ID NO: 6; 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 linked to different fluorescent reporter groups.

2. The application according to claim 1, characterized in that, The fluorescent reporter group is selected from one of FAM, HEX, FITC, RED, TET, JOE, and R110.

3. Application of primer sets for amplifying SNP molecular markers in the preparation of products with any of the functions of B1) to B6): B1) To identify or assist in the identification of corn drought resistance; B2) Screening or assisting in the screening of maize strains 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 varieties; B5) Marker-assisted breeding of maize; B6) Maize breeding; The SNP molecular marker includes at least one of a first SNP molecular marker and a second SNP molecular marker; The first SNP molecular marker is located at position 97605381 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a polymorphism of C / T; The second SNP molecular marker is located at position 97604011 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a polymorphism of C / T; The primer set used to amplify the first SNP molecular marker includes a first specific primer and a first common primer with a nucleotide sequence as shown in SEQ ID NO: 3; 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; the first Primer X and the first Primer Y are respectively linked to different fluorescent reporter groups; The primer set used to amplify the second SNP molecular marker includes a second specific primer and a second common primer with a nucleotide sequence as shown in SEQ ID NO: 6; 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 linked to different fluorescent reporter groups.

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

5. A method for identifying or assisting in the identification of drought resistance in maize, comparing the drought resistance of maize samples, screening or assisting in the screening of maize lines or varieties with relatively strong drought resistance, screening or assisting in the screening of maize lines or varieties with relatively weak drought resistance, and for maize molecular marker-assisted breeding or maize breeding, characterized in that, The method includes the following steps: The genotypes of SNP molecular markers in the genome of a maize sample are detected, and the drought resistance level of the maize sample is determined based on the genotypes. The SNP molecular markers include at least one of a first SNP molecular marker and a second SNP molecular marker. The first SNP molecular marker is located at position 97605381 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a polymorphism of C / T. The second SNP molecular marker is located at position 97604011 on chromosome 9 of the maize reference genome Zea_mays.AGPv4, with a polymorphism of C / T. Among them, for the first SNP molecular marker, the drought resistance of the TT genotype and CT genotype is better than that of the CC genotype; For the second SNP molecular marker, the CC and CT genotypes showed better drought resistance than the TT genotype.

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