KASP markers, primer sets for identifying maize haploids and applications thereof

Abstract

The present application relates to the field of biotechnology, in particular to KASP markers for identifying maize haploid, primer sets and application thereof. The present application utilizes maize Maize 60K chip to screen 8 groups of haploid (10 haploids are induced as repeats in each group) in the offspring F1 of 8 selfing lines (Mo17, B73, Qi319, HZ4, Z58, W64A, W22, TX) respectively crossed with inducer line CAU5, and 145 sites are obtained which are all stably completed paternal chromosome elimination at 100% in the respective 10 haploids of the 8 F1 combinations. For the 145 sites, 94 stable KASP markers are developed, which provide a new way for accurate screening of haploid.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to KASP markers for identifying maize haploid, primer sets and application thereof. BACKGROUND

[0002] Maize is a model crop using heterosis. The most critical step in the use of heterosis is the breeding of pure lines. There are two common methods: the first is pedigree method, which requires 5-7 years to obtain pure lines through continuous multiple selfing or backcrossing; the other is haploid breeding technology, which uses haploid induced by haploid inducer to become homozygous diploid after doubling, and pure lines can be obtained within 1-2 years. DH breeding plays an increasingly important role in the process of commercial maize breeding, and has become a core technology of modern maize breeding together with molecular breeding technology and transgenic technology.

[0003] Haploid refers to an individual with gamete chromosome number in cells. The process of using haploid to rapidly produce pure lines is called double haploid breeding, i.e. DH breeding. Haploid has only one set of chromosomes, and recessive genes and dominant genes can be expressed in the same generation, so early generation can be screened for excellent traits, and undesirable traits can be eliminated in time, which is beneficial to the rapid aggregation of beneficial alleles such as yield and resistance.

[0004] Because haploid technology can produce homozygous inbred lines in a short time, it has become one of the main technologies of maize genetic breeding for many breeding institutions, units or companies at home and abroad in the past few years. One of the important steps of haploid engineering breeding is to simply and quickly select haploid grains from a small proportion of haploid grains in a large number of heterozygous diploid grains, and the accuracy and speed of screening directly affect the large-scale application of haploid breeding. There are many methods for haploid identification, including plant morphology identification, cytogenetic identification, seed morphology identification, radioactive method and fluorescent identification, etc. The most effective and basic method for identifying haploid seeds is chromosome number counting method, but this method often uses dividing vigorous somatic cells such as root tip meristem and other tissues, and needs to use fluorescence in situ hybridization technology, which is complicated and inefficient. The relatively simple method of selecting haploid seeds by R1-nj color marker often produces many false positive haploids, which interferes with subsequent experiments.

[0005] With the rapid development of molecular biology, molecular markers are also an effective method to identify plant ploidy. The molecular marker identification method based on SSR, SNP and other markers mainly uses the DNA of parents to screen the polymorphism of the offspring. The band type of haploid genotype is consistent with the maternal material, and the band type of diploid gene is heterozygous with the genotype of both parents, so as to accurately distinguish haploid grain and diploid grain. Although this method is extremely accurate, it is limited by high-cost chips and the need for high-throughput extraction of mature grain DNA, which has too high detection cost and a relatively complicated operation process, thus greatly limiting the popularization and application of this technology.

[0006] Kompetitive allele specific PCR (KASP) is a new genotyping technology based on single nucleotide polymorphisms (SNP), which can accurately genotype SNP and insertion-deletions (InDels) at the genome level, and can meet the requirements of low, medium and high throughput genotyping based on ordinary laboratory operations, has certain flexibility, and is suitable for experimental design with great changes in target sites and sample quantity. Compared with SNP genotyping platform TaqMan, KASP uses universal probes that can be used with various different gene-specific primers, without the need for probe synthesis for each specific site, which greatly reduces the reagent cost of the experiment by about 80%. At the same time, the genotyping based on KASP technology is a simple method that does not depend on gel electrophoresis detection, which does not require special equipment, and researchers can use conventional qPCR instruments for SNP genotyping, which has good compatibility. By designing two primers to amplify the allele-specific single nucleotide polymorphism site in different directions, the single nucleotide polymorphism can be converted into length polymorphism. In addition, KASP achieves a higher success rate of analysis design and a higher rate of successful work detection. In addition, compared with SNP genotyping platform Illumina GoldenGate, the average genotyping error of positive control DNA samples is lower than that observed using GoldenGate, which has higher accuracy. We can also order KASP reagents and use qRT-PCR instruments for result analysis, which can greatly reduce experimental costs and improve experimental accuracy, so it is widely used in the field of life science research.

[0007] In maize, Jagtap et al. developed 100 pairs of KASP markers for genes related to high-temperature stress response. They found that 71% of these markers were polymorphic, 21% produced only one gene or only a heterozygous gene, and the remaining 8% failed to produce useful amplification signals. Comparative analysis showed that the KASP marker development efficiency in this study was higher than that in wheat and rice. Lu et al. identified a series of SNP loci using whole-genome resequencing data from maize inbred lines from different sources and developed 700 pairs of KASP markers. From these, 202 representative KASP marker pairs were selected for further phylogenetic tree construction and population structure analysis. The results showed that the cluster analysis results based on KASP marker loci were highly consistent with those based on total SNP loci, with a genetic distance similarity coefficient as high as 89.5%, successfully distinguishing maize heterotic groups. Therefore, KASP markers can also play an important role in maize germplasm resource analysis and heterotic group classification.

[0008] However, research on the use of KASP markers in haploid screening of maize kernels is relatively limited. In 2011, Huang Min et al. used 47 SSR markers to conduct genetic analysis on 10 maize haploid plants, finding that 29 pairs of markers were polymorphic between Zheng 58 and B73, and 16 pairs were polymorphic between Xi 502 and Chang 7-2. However, these polymorphic markers, on average, numbered less than three per chromosome in maize, failing to evenly cover most locus information and easily leading to biased experimental results. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a KASP marker, primer set, and its application for identifying maize haploids.

[0010] The present invention specifically adopts the following technical solution:

[0011] In a first aspect, the present invention provides a KASP marker for identifying maize haploids, said KASP marker being any one of KA01 to KA94:

[0012]

[0013]

[0014]

[0015] The KASP marker was determined based on the whole genome sequence alignment of maize variety B73, and the version number of the whole genome sequence of maize variety B73 is B73 RefGen V3.

[0016] Applicant uses maize Maize 60K chip to screen 145 sites which are all stably completed paternal chromosome elimination at 100% in 8 groups of 10 haploids of each F1 combination of 8 selfing lines (Mo17, B73, Qi319, HZ4, Z58, W64A, W22, TX) respectively crossed with inducing line CAU5, which can be used for accurate identification of haploids, and the distribution on corn chromosomes 1-10 is: 23, 37, 10, 4, 9, 8, 11, 22, 9, 12, which can fully ensure the reasonable density of markers in the identification process. For the 145 sites, 94 stable KASP markers are developed, compared with less than 3 markers on each chromosome before, the marker sites of the application greatly improve the coverage of markers on each chromosome, which can fully ensure the reasonable density of markers in the identification process, and provide a new way for accurate screening of haploids.

[0017] In a second aspect of the application, a primer set for amplifying the KASP marker is provided.

[0018] Further, the primer set is composed of ninety-four primer pairs, which correspond to KASP markers numbered KA01 to KA94 in sequence, each primer pair consisting of a specific primer 1, a specific primer 2 and a reverse primer targeting the corresponding KASP marker, the common sequence of all specific primers 1 being as shown in SEQ ID NO. 1, and the common sequence of all specific primers 2 being as shown in SEQ ID NO. 2.

[0019] Still further, the tail of the specific primer 1 is added with a specific sequence capable of binding with FAM fluorescence and labeled with a FAM fluorescent group, and the tail of the specific primer 2 is added with a specific sequence capable of binding with HEX fluorescence and labeled with a HEX fluorescent group.

[0020] In a third aspect of the application, a kit containing the primer set is provided.

[0021] In a fourth aspect of the application, the KASP marker, the primer set or the kit is used for identifying corn haploids.

[0022] In a fifth aspect of the application, a method for identifying corn ploidy is provided, comprising the following steps:

[0023] 1) Using the genomic DNA of the corn to be tested and its parents as a template, the genotype data of the corn to be tested and its parents at at least one SNP site in KA01 to KA94 of the KASP marker is obtained;

[0024] 2) According to the genotype data determination result, if the to-be-tested corn is the same as the genotype of the female parent, the to-be-tested corn is a haploid plant; if the genotype of the to-be-tested corn is a heterozygote of the genotype of the male parent and the female parent, the to-be-tested corn is a diploid plant.

[0025] Further, the method for acquiring genotype data includes a fluorescent quantitative PCR detection method and a high-throughput SNP detection analysis method.

[0026] The present application has the following beneficial effects:

[0027] The markers listed in the present application are sites that are 100% stable in completing the paternal chromosome elimination in 8 inbred lines, have wide adaptability and are more stable. Instead of identifying the polymorphism of the parent by naked eye in the artificial rubber band identification process, the machine is directly used to read the band, and the results can be batched, which is more convenient. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a phenotype diagram of the hybrid F1 generation of the haploid and diploid grains of 8 inbred lines and the inducing line CAU5.

[0029] Figure 2 It is a genotype diagram of the DNA of the haploid, diploid and hybrid amplified by the developed KASP markers. The green color is the HEX fluorescence, and the green scatter diagram position represents the consistency of the parent genotype connected with the HEX connector (for example, AA). The blue marker is the FAM fluorescence, and the blue scatter diagram position represents the consistency of the parent connected with the FAM connector (for example, TT), and the red color represents the hybrid (for example, AT).

[0030] Figure 3 It is a distribution diagram of the 94 KASP markers developed on the 10 chromosomes of corn.

[0031] Figure 4 It is a field performance diagram of the haploid and diploid single plants screened according to the KASP markers. The left diagram is the field diploid plant verified by the KASP markers, and the right diagram is the field haploid.

[0032] Figure 5 It is a genotype result of the plants with haploid and diploid performance in the field identified by the KASP markers KA01 to KA47.

[0033] Figure 6 It is a genotype result of the plants with haploid and diploid performance in the field identified by the KASP markers KA48 to KA94.

[0034] Figure 5 and Figure 6In the table, A to N are B73 inbred, haploid in F1 of B73 x CAU5, Mo17 inbred, haploid in F1 of Mo17 x CAU5, Z58 inbred, haploid in F1 of Z58 x CAU5, HZ4 inbred, haploid in F1 of HZ4 x CAU5, W22 inbred, haploid in F1 of W22 x CAU5, Qi319 inbred, haploid in F1 of Qi319 x CAU5, TX inbred, haploid in F1 of TX x CAU5. DETAILED DESCRIPTION

[0035] The application will be described in detail below with reference to the drawings and specific examples, but should not be understood as limiting the application. If not specifically stated, the technical means used in the following examples are conventional means known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0036] Example 1

[0037] 1. Construction of materials and method for screening haploid

[0038] The grains on the F1 plants obtained by crossing 8 maize inbreds (Mo17, B73, Qi319, HZ4, Z58, W64A, W22, TX) with the common inducer CAU5 were first screened for seeds of pseudo-haploid by R1-nj color, and then the pseudo-haploid seeds were germinated, the DNA of each pseudo-haploid seed was extracted, and the haploid was screened using 12 KASP markers on chromosome 7 of maize with all known polymorphisms between parents (Table 1). The single plants that showed the maternal genotype in all 12 markers were kept as true haploids for the next screening experiment with Maize 60K chip. 10 haploid seeds of each inbred combination were selected as repeats, and the DNA was extracted for the next round of screening. Figure 1

[0039] 2. Experimental method of Maize 60K Chip

[0040] 1) Transfer 8.7 μl of haploid DNA of each combination described above into a 96-well PCR plate, centrifuge at 250xg for a short time at 20°C, and reserve.

[0041] 2) Prepare the denaturation solution (DMM) according to Table 2, vortex to mix, centrifuge at 250xg for a short time at room temperature. Add 8.7 μl of denaturation solution to each well, centrifuge at 1000xg for a short time, vortex to mix for 10 min, add 56.6 μl of neutralization solution to each well to terminate denaturation, centrifuge at 250xg for a short time, vortex to mix, and stand.

[0042] ​3) Prepare the amplification solution according to Table 3, invert the plate and vortex mix again, add 100.1 μΐ amplification solution to each well, 250 x g, short spin, vortex mix, centrifuge at 1000 rpm for 1 min at room temperature. Then put the AMP Plate into the 37°C hybridization oven for 22-24 hours.

[0043] 4) After amplification, perform the fragmentation experiment.

[0044] 3, Method for identifying paternal chromosome complete elimination sites

[0045] After genotyping the data from the 60K chip, the total number of genotypes is divided into four categories, "-" represents that the site is not detected, "AA / TT / GG / CC" represents that the site is a homozygous genotype, and "AT / AC / AG / TC / TG / GC" represents that the site is a heterozygote. Taking the hybrid combination of Mo17 and CAU5 as an example, first, select the sites that are homozygous in both parents and have polymorphism (polymorphism refers to the genotype of the two parents at the site is different, for example, AA and TT) among the 61224 markers; second, divide each chromosome into 10 equal parts, and mark them as 0-9.99%, 10-19.99%, 20-29.99%, 30-39.99%, 40-49.99%, 50-59.99%, 60-69.99%, 70-79.99%, 80-89.99%, and 90-100% from top to bottom. The number of paternal chromosome complete elimination sites in each interval (complete elimination refers to the genotype of a haploid individual at the same site is the same as the genotype of the maternal parent, and the specific genotype information is shown in Table 4) accounts for the total number of sites in the interval (Table 5). Then, the number of complete elimination sites on each chromosome of the eight combinations is counted (Table 6), and the intersection is taken to finally select 145 sites that are stably completed in the paternal chromosome elimination in the 10 haploids of each of the eight hybrid combinations F1 (Table 4). Then, according to the CR (> 96%) and FLD (> 3.6) values of the markers in the group, the sites that can be used to develop KASP markers are evaluated. Figure 2

[0046] 4, Development process of KASP markers

[0047] 1) Primer and probe design

[0048] ​Firstly, two forward PCR primers for specific SNPs were designed, each primer corresponding to one allele of a SNP by adjusting the 3' end; secondly, a tag sequence was added to the 5' end of each forward primer. In addition, a fluorescent probe corresponding to the tag sequence was designed. The common sequence of specific primer 1 was 5'-GAAGGTGACCAAGTTCATGCT-3', as shown in SEQ ID NO. 1, and a specific sequence capable of binding with FAM fluorescence was added to the tail; the common sequence of specific primer 2 was 5'-GAAGGTCGGAGTCAACGGATT-3', as shown in SEQ ID NO. 2, and a specific sequence capable of binding with HEX fluorescence was added to the tail, and both shared a reverse primer sequence. The primer sequences are shown in Table 7.

[0049] 2) General PCR amplification

[0050] In the first round of PCR amplification, the allele-specific primer can recognize the specific allele template, and complete the allele recognition. From the second round of PCR amplification, the template carrying the universal tag sequence appears in the product, and this step completes the introduction of the universal tag sequence into the PCR product corresponding to the SNP. Subsequently, in the PCR amplification process, the fluorescent probe is added to the PCR product by binding to the complementary DNA strand of the universal sequence, and after multiple rounds of PCR amplification, more fluorescent probes are annealed to the newly synthesized complementary strand without a quenching group, gradually enhancing the fluorescence intensity of the PCR product.

[0051] 5) Use method of newly developed KASP markers

[0052] We designed 145 pairs of KASP markers according to the above method, and finally 94 pairs of KASP markers could be amplified to obtain correct bands, which were recorded as successful KASP markers, and then fluorescence quantitative PCR method could be used for accurate identification of haploid (reaction program see Table 8), wherein 50 μL of KASP Primer mix was prepared by mixing 6 μL of specific primer 1 (100 μmol / L), 6 μL of specific primer 2 (100 μmol / L), 15 μL of common primer (100 μmol / L), and 23 μL of Tris-HCl (10 mmol / L). The PCR reaction conditions are as follows: the first round of 94℃, 15min; 94℃, 20s, 61-55℃, 60s, each cycle decreases by 0.6℃, a total of 10 cycles; the second round of 94℃, 20s, 55℃, 60s, a total of 26 cycles. The PCR amplification is completed on the qPCR instrument 7900 (Applied Biosystems, USA), and the fluorescence signal is collected by end-point method, and then the SNP viewer 2.0 software developed by LGC company is used to read the detection data. This method is suitable for individuals or small-scale laboratories that need to identify haploid, and can be operated with small sample size, and the result can be obtained in the fastest 3 hours. Also, the DOUGLUS platform can be used for batch precise double-allele determination, which has the advantages of short cycle, high verification rate, low cost, strong flexibility, etc., and can efficiently, conveniently and accurately complete the screening of haploid, and can combine with the field phenotype identification work to confirm that the detection result of the KASP marker is consistent with the field phenotype of the single / double haploid.

[0053] Figure 4 For a randomly selected new KASP marker KA77, plant ploidy identification was carried out, and it was found that the haploid plant (the same as the genotype of the female parent) had narrow and small leaves, larger leaf angle, shorter plant, pollen sterility and other characteristics compared with the diploid plant (genotype of the father and mother parent genotype heterozygote), which was consistent with the field phenotype of the haploid plant.

[0054] Figure 5 And Figure 6 For plants showing haploid and diploid in the field, the genotypes of the plants were identified by KASP markers KA01-KA94.

[0055] The KASP marker of the application has the following advantages:

[0056] 1) High marker reliability

[0057] The data of Maize 60K Chip of F1 haploid of hybrid offspring of 8 representative inbred lines and inducer CAU5 is the most comprehensive detection means under the existing experimental conditions, and for each site in the induced F1, there are three different cases, a. The father is completely eliminated (the F1 genotype is consistent with the mother); b. The father is partially eliminated (the F1 genotype is a heterozygous type of the genotypes of the parents); and c. The mother is eliminated (the F1 genotype is consistent with the father).

[0058] When screening sites, we prefer to screen the sites where the father's chromosome is completely eliminated (type a), which can completely avoid the interference of false positive sites (types b and c) in the use of markers, and ensure the accuracy of the use of markers.

[0059] 2) Wide coverage and uniform distribution of markers

[0060] After the polymorphism screening of 8 parents, it can be found that the number of chip markers with polymorphism between 8 inbred lines (Mo17, B73, Qi319, HZ4, Z58, W64A, W22, TX) and the parent CAU5 is 21820, 12992, 23012, 21775, 23461, 18627, 15148, and 15993 respectively. Taking the combination of Mo17 and CAU5 as an example, the number of chip markers with polymorphism between the parents on each chromosome is 3347, 2950, 2534, 2543, 2331, 1745, 1734, 1559, 1473, and 1613 respectively. Among them, 94 sites are developed as KASP markers for identifying haploid. Compared with less than 3 markers on each chromosome before, the marker sites protected in the application greatly improve the coverage of markers on each chromosome. Figure 3 )。

[0061] 3) Simple identification method

[0062] The identification method of the KASP marker mentioned in the application can directly determine the double alleles accurately by using the Douglas 7900 and enzyme meter platform, has short cycle, high verification rate, low cost and strong flexibility, can efficiently, conveniently and accurately complete the screening of haploids, and can combine with the field phenotype identification work to confirm that the detection result of the KASP marker is consistent with the field phenotype of the single / double haploid (the haploid leaf is narrow and small, the leaf angle is large, the plant is short, the pollen is sterile, etc.). Compared with the traditional gel electrophoresis detection method which is limited to processing at most 96-hole samples per test cycle and has low efficiency, the Douglas 7900 platform mentioned in the application can process 720 384-hole plates at a time, which is high in efficiency and also avoids the error of manual detection in the traditional method.

[0063] Table 1 KASP marker number and physical position for preliminary identification of haploids (taking the combination of Mo17 and CAU5 as an example)

[0064] Position-V4 Mo17 CAU5 Chr 1.298519 CC TT 7 8.358129 CC TT 7 9.621652 GG AA 7 42.068584 TT CC 7 56.253218 CC TT 7 88.89284 AA GG 7 137.670828 GG TT 7 126.109862 AA CC 7 148.349344 GG AA 7 162.970898 CC AA 7 169.116076 AA GG 7 180.747932 AA GG 7

[0065] Table 2 DMM denaturation liquid reaction system

[0066] Component Volume 10X Denat Soln 244 μΐ Axiom Water 2.2ml Total 2.444ml

[0067] Table 3 Amp MM amplification liquid reaction system

[0068] Component Volume Amp Soln 12ml Amp Enzyme 267 μΐ Total 12.267ml

[0069] Table 4 Genotypes and corresponding gene numbers of 145 sites of paternal chromosome complete elimination in 60K chip data of inducer CAU5 and 8 selfing lines

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Note: Space represents no corresponding gene number under the current version.

[0076] Table 5 Taking the combination of Mo17 and CAU5 as an example to show the proportion of sites of paternal chromosome complete elimination on each chromosome

[0077] Percentage Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr8 Chr9 Chr10 0.00-9.99 12.0％ 13.5％ 12.1％ 12.1％ 22.4％ 8.9％ 13.1％ 10.2％ 20.5％ 14.3％ 10.00-19.99 18.0％ 10.1％ 5.9％ 9.8％ 8.3％ 2.5％ 5.6％ 8.1％ 10.2％ 3.2％ 20.00-29.99 16.2％ 6.8％ 4.5％ 12.1％ 5.4％ 5.6％ 0.0％ 0.3％ 1.5％ 0.0％ 30.00-39.99 9.6％ 4.5％ 6.0％ 5.8％ 9.9％ 9.3％ 0.0％ 7.6％ 8.1％ 0.0％ 40.00-49.99 1.1％ 10.6％ 4.6％ 7.4％ 5.5％ 8.4％ 4.1％ 5.6％ 5.1％ 0.0％ 50.00-59.99 3.3％ 12.4％ 13.7％ 6.3％ 4.9％ 11.5％ 9.8％ 10.8％ 14.5％ 5.2％ 60.00-69.99 11.6％ 7.0％ 14.6％ 13.1％ 11.3％ 14.5％ 16.2％ 11.9％ 6.8％ 7.3％ 70.00-79.99 8.2％ 13.5％ 14.6％ 10.7％ 10.9％ 11.5％ 16.6％ 12.7％ 9.7％ 16.8％ 80.00-89.99 11.4％ 13.2％ 9.1％ 12.2％ 14.3％ 16.4％ 17.8％ 15.4％ 10.2％ 24.1％ 90.00-100.0 8.6％ 8.4％ 14.8％ 10.6％ 7.2％ 11.4％ 16.8％ 17.2％ 13.3％ 29.1％

[0078] Table 6 Number of paternal chromosome completely eliminated loci per selfing line combination

[0079] Chr Mo17 B73 Qi319 HZ4 Z58 W64A W22 TX 1 2500 1519 2738 2619 2612 2504 2257 1961 2 2172 1214 2130 2080 2385 1901 1529 1112 3 1859 895 2002 1938 1992 1690 1546 1516 4 1861 897 1820 231 1792 1745 1425 1350 5 1775 860 1829 1890 1763 1648 1158 1354 6 1269 605 1324 1358 1288 1178 765 1148 7 1248 806 1380 1387 1422 1263 1242 1037 8 1197 804 1420 1369 1422 1032 1049 751 9 1058 678 1289 1155 1333 1039 804 740 10 1167 514 1187 1222 1107 967 1016 997

[0080] Table 7 Primer sequences for 94 KASP markers developed for 145 paternal chromosome completely eliminated loci

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] Table 8 KASP marker PCR amplification reaction mix

[0092] Component Volume KASP Master mix 1.5 μΐ KASP Primer mix 0.042 μΐ Template DNA 1.5 μΐ (50 ng / μΐ) Total 3.042 μΐ

[0093] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A primer set for KASP markers for amplification of identification of maize haploids, characterized in that, The KASP markers are KA01-KA94, the primer set is composed of ninety-four primer pairs, corresponding to the KASP markers numbered KA01-KA94 in turn, each primer pair is composed of specific primer 1, specific primer 2 and reverse primer targeting the corresponding KASP marker, the common sequence of all specific primer 1 is shown as SEQ ID NO. 1, the common sequence of all specific primer 2 is shown as SEQ ID NO. 2, and the ninety-four primer pairs are as follows: The tail of the specific primer 1 is marked with FAM fluorescent group, and the tail of the specific primer 2 is marked with HEX fluorescent group.

2. A kit comprising the primer set of claim 1.

3. Use of the primer set of claim 1 or the kit of claim 2 in identifying maize haploid.

4. A method of identifying the ploidy of maize, characterized by, The method comprises the following steps: 1) Using the genomic DNA of the maize to be tested and its parents as templates, and using the primer set of claim 1 to perform PCR amplification to obtain the genotype data of at least one SNP site in the target sequence of the maize to be tested and its parents; 2) According to the genotype data determination result, if the maize to be tested is the same as the genotype of the female parent, then the maize to be tested is a haploid plant; if the genotype of the maize to be tested is a heterozygote of the genotype of the male and female parents, then the maize to be tested is a diploid plant.

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