KASP marker and primer group for identifying corn haploid and application of KASP marker and primer group

Abstract

The invention relates to the technical field of biology, in particular to a KASP marker for identifying corn haploids, a primer group and application of the KASP marker and the primer group. According to the invention, a corn Maize 60K chip is used for respectively hybridizing eight inbred lines (Mo17, B73, Qi319, HZ4, Z58, W64A, W22 and TX) with an induction line CAU5 to screen out eight groups of haploids (10 haploids are respectively induced in each group as repetitions) from filial generations F1, 145 sites which are shown as 100% stable completion of male parent chromosome elimination in respective 10 haploids of the eight F1 combinations are obtained, and for the 145 sites, the male parent chromosome elimination is completely completed. 94 stable KASP markers are developed, and a new way is provided for accurate screening of haploids.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a KASP marker, a primer set and an application thereof for identifying maize haploid. Background Art

[0002] Corn is a model crop that utilizes hybrid vigor. The most critical link in the utilization of hybrid vigor is the selection of pure lines. There are two commonly used methods: the first is the pedigree method, which is to obtain pure lines through continuous multi-band selfing or backcrossing, which usually takes 5-7 years; the other is haploid breeding technology, which uses haploid induction lines to double the haploids to become homozygous diploids, and pure lines can be obtained within 1-2 years. DH breeding plays an increasingly important role in the commercial breeding process of corn, and has become the core technology of modern corn breeding together with molecular breeding technology and transgenic technology.

[0003] Haploid refers to an individual with the same number of gamete chromosomes in its cells. The process of rapidly producing pure lines by doubling haploids is called double haploid breeding, or DH breeding. Haploids have only one set of chromosomes, and both recessive and dominant genes can be manifested in the present generation. Therefore, excellent traits can be screened in the early generations, and undesirable traits can be eliminated in a timely manner, which is conducive to the rapid aggregation of favorable alleles such as yield and resistance.

[0004] Because haploid technology can produce homozygous inbred lines in a relatively short period of time, it has become one of the main technologies for corn genetic breeding in many breeding institutions, units or companies at home and abroad in the past few years. It is one of the important links in haploid engineering breeding to simply and quickly select haploid grains from a large number of heterozygous diploids, and the accuracy and speed of screening directly affect the large-scale application of haploid breeding. There are many methods for haploid identification, and the commonly used methods include: plant morphology identification, cytogenetic identification, seed morphology identification, and radioactive methods and fluorescence identification. Among them, the most effective and basic method for identifying haploid grains is the chromosome number counting method, but because the experimental materials required by this method are often somatic cells with vigorous division, such as root tips and stem tips, and the use of fluorescence in situ hybridization technology, the operation process is cumbersome and inefficient. The simpler result of selecting haploid grains by R1-nj color marking 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 for identifying plant ploidy. The molecular marker identification method based on markers such as SSR and SNP mainly uses the DNA of both parents to screen the polymorphisms of the molecular markers of the offspring. The band pattern of the haploid genotype is consistent with that of the female parent material, while the diploid gene band pattern has a heterozygous band pattern of both parental genotypes, thus being able to accurately distinguish haploid grains from diploid grains. Although this method is extremely accurate, it is limited by the high-cost chips and the need for high-throughput extraction of DNA from mature grains. The detection cost is too high and the operation process is relatively cumbersome, so it greatly limits the popularization and application of this technology.

[0006] Kompetitive allele specific PCR (KASP) is a new genotyping technology based on Single Nucleotide Polymorphisms (SNP). It can accurately genotype SNPs and insertion-deletions (InDels) at the genomic level, and can meet the requirements of low, medium, and high-throughput genotyping on the basis of ordinary laboratory operations. It has a certain degree of flexibility and is suitable for experimental designs with very large variations in target sites and sample numbers. Compared with the SNP genotyping platform TaqMan, KASP uses universal probes that can be used in combination with various different gene-specific primers, without the need to synthesize probes 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 rely on gel electrophoresis detection. It does not require special equipment, and researchers can use conventional qPCR instruments for SNP genotyping, with good compatibility. By designing two primer pairs to amplify allele-specific single nucleotide polymorphism sites in different directions, single nucleotide polymorphisms can be converted into length polymorphisms. In addition, KASP achieves a higher success rate of analysis design and a conversion rate to successful working detection rates. Moreover, compared with the SNP genotyping platform Illumina GoldenGate, the average genotyping error of KASP in positive control DNA samples is lower than the value observed using GoldenGate, with higher accuracy. We can also order KASP reagents by ourselves and analyze the results using a qRT-PCR instrument, which can greatly reduce the experimental cost and improve the 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 heat stress response, and found that 71% of these markers were polymorphic, 21% of the markers produced only one allele or were only heterozygous, and the remaining 8% failed to produce useful amplification signals. Comparative analysis showed that the efficiency of KASP marker development in this study was higher than that in wheat and rice. Lu et al. identified a series of SNPs using whole-genome resequencing data of maize inbred lines from different sources and developed 700 pairs of KASP markers. Among them, 202 pairs of representative KASP markers were further used for phylogenetic tree construction and population structure analysis. The results showed that the clustering analysis results based on KASP marker loci were highly consistent with those based on total SNPs loci, and the similarity coefficient of genetic distance between the two was as high as 89.5%, which could successfully distinguish the heterotic groups of maize. Therefore, KASP markers can also play an important role in the analysis of maize germplasm resources and the classification of heterotic groups.

[0008] However, there is relatively little research on the use of KASP markers for screening maize haploids. In 2011, Huang Min et al. performed genetic analysis on 10 maize haploids using 47 SSR markers and found that 29 pairs of them were polymorphic between Zheng 58 and B73, and 16 pairs were polymorphic between Xi 502 and Chang 7-2. However, the average number of these polymorphic markers on each chromosome of maize was less than 3, which could not evenly cover most of the locus information and was prone to causing deviation in experimental results. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides KASP markers, primer sets and their applications for identifying maize haploids.

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

[0011] In the first aspect of the present invention, there is provided a KASP marker for identifying maize haploids, and the KASP marker is any one of KA01 to KA94:

[0012]

[0013]

[0014]

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

[0016] The applicant used the Maize 60K chip to screen 145 loci that showed 100% stable paternal chromosome elimination in each of the 10 haploids in 8 groups of haploids (10 haploids were induced in each group as replicates) in the F1 hybrids of 8 inbred lines (Mo17, B73, Qi319, HZ4, Z58, W64A, W22, TX) crossed with the inducer line CAU5 respectively. These loci can be used for accurate identification of haploids, and their distribution on maize chromosomes 1-10 is as follows: 23, 37, 10, 4, 9, 8, 11, 22, 9, 12, which can fully ensure a reasonable density of markers during the identification process. For these 145 loci, 94 stable KASP markers were developed. Compared with less than 3 markers on each chromosome before, the marker loci of the present invention greatly improve the coverage of markers on each chromosome, can fully ensure a reasonable density of markers during the identification process, and provide a new way for accurate screening of haploids.

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

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

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

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

[0021] In the fourth aspect of the present invention, the application of the KASP marker, the primer set, or the kit in identifying maize haploids is provided.

[0022] In the fifth aspect of the present invention, a method for identifying the ploidy of maize is provided, including the following steps:

[0023] 1) Using the genomic DNA of the maize to be tested and its parents as templates, genotype data of at least one SNP locus among KA01-KA94 of the KASP marker for the maize to be tested and its parents is obtained;

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

[0025] Further, the methods for obtaining genotype data include fluorescence quantitative PCR detection method and high-throughput SNP testing and analysis method.

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

[0027] The markers listed in the present invention are sites where paternal chromosome elimination is stably completed at 100% in 8 inbred lines, which have wide adaptability and are more stable. It is no longer restricted by visually identifying the polymorphisms of parents during the manual gel running identification process, but directly uses a machine to read out the bands, and can produce results in batches, which is more convenient. Description of the Drawings

[0028] Figure 1 It is a phenotypic diagram of haploid and diploid grains of the F1 generation of the hybrids of 8 inbred lines and the inducer CAU5.

[0029] Figure 2 It is a genotype diagram of amplifying the DNA of haploids, diploids and heterozygotes using the developed KASP markers. Green is HEX fluorescence, and the position of the green scatter plot represents the same genotype as the parent connected to the HEX linker (for example, AA), the blue is marked with FAM fluorescence, and the position of the blue scatter plot represents the same as the parent connected to the FAM linker (for example, TT), and red represents heterozygosity (for example, AT).

[0030] Figure 3 Distribution diagram of the 94 developed KASP markers on 10 maize chromosomes.

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

[0032] Figure 5 It is the genotype result of identifying the ploidy of plants showing haploid and diploid phenotypes in the field using the KASP markers of KA01 - KA47.

[0033] Figure 6 It is the genotype result of identifying the ploidy of plants showing haploid and diploid phenotypes in the field using the KASP markers of KA48 - KA94.

[0034] Figure 5 and Figure 6Among them, A to N are in turn the B73 inbred line, the haploid in the F1 of B73×CAU5, the Mo17 inbred line, the haploid in the F1 of Mo17×CAU5, the Z58 inbred line, the haploid in the F1 of Z58×CAU5, the HZ4 inbred line, the haploid in the F1 of HZ4×CAU5, the W22 inbred line, the haploid in the F1 of W22×CAU5, the Qi319 inbred line, the haploid in the F1 of Qi319×CAU5, the TX inbred line, and the haploid in the F1 of TX×CAU5. Detailed implementation mode

[0035] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0036] Example 1

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

[0038] Eight maize inbred lines (Mo17, B73, Qi319, HZ4, Z58, W64A, W22, TX) were respectively crossed with the common inducer line CAU5. For the seeds on the F1 plants obtained, after first obtaining pseudo-haploid seeds through R1-nj color screening, these pseudo-haploid seeds were germinated. After extracting the DNA of each pseudo-haploid, 12 KASP markers with polymorphisms between the known parents on maize chromosome 7 were used for haploid screening (Table 1). The individuals that all showed the maternal genotype among all 12 markers were retained as true haploids for the next Maize 60K chip screening experiment. 10 haploid seeds were selected as replicates for each inbred line combination, and their DNA was extracted respectively and then entered the next round of screening ( Figure 1 ).

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

[0040] 1) Transfer 8.7 μl of the haploid DNA of each combination above to a 96-well PCR plate in turn, centrifuge briefly at 250 xg and 20 °C for standby.

[0041] 2) Prepare the denaturing solution (DMM) according to Table 2 and vortex to mix evenly, then centrifuge briefly at 250 xg at room temperature. Add 8.7 μl of the denaturing solution to each well, centrifuge briefly at 1000 xg, then vortex and mix evenly for 10 min. Add 56.6 μl of the neutralizing solution to each well to terminate denaturation, centrifuge briefly at 250 xg, then vortex and mix evenly, and let stand.

[0042] 3) Prepare the amplification solution according to Table 3. Invert it up and down and then continue to vortex and mix evenly. After adding 100.1 μl of the amplification solution to each well, centrifuge briefly at 250 x g, vortex and mix evenly, and then centrifuge at 1000 rpm for 1 min at room temperature. Subsequently, place the AMP Plate in a 37°C constant temperature hybridization oven for 22 - 24 hours.

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

[0044] 3. Identification method for the complete elimination sites of paternal chromosomes

[0045] After genotyping the data downloaded from the 60K chip, they are divided into four categories in total. "--" indicates that no genotype is detected at this site, "AA / TT / GG / CC" indicates that this site is a homozygous genotype, and "AT / AC / AG / TC / TG / GC" indicates that this site is heterozygous. Taking the hybridization combination of Mo17 and CAU5 as an example, first screen the sites that are homozygous and polymorphic in both parents among 61,224 markers (polymorphism means that the genotypes of the two parents are different at this site, such as AA and TT); secondly, evenly divide each chromosome into 10 parts, and record 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%, 90 - 100% from top to bottom respectively. Count the ratio (Table 5) of the number of sites where the paternal chromosomes are completely eliminated (complete elimination means that at the same site, the genotype of the haploid individual is the same as that of the maternal genotype, and the specific genotype information is shown in Table 4) in each interval to the total number of sites in that interval. Subsequently, count the number of completely eliminated sites on each chromosome of 8 combinations respectively (Table 6), and after taking the intersection, finally screen out 145 sites that show 100% stable elimination of paternal chromosomes in each of the 10 haploids of the F1 generation of the above 8 hybridization combinations (Table 4). Subsequently, according to the values of CR (>96%) and FLD (>3.6) within the group where these markers are located, evaluate the sites that can be used to develop KASP markers ( Figure 2 ).

[0046] 4. Development process of KASP markers

[0047] 1) Primer and probe design

[0048] First, design two forward PCR primers targeting specific SNPs. Each primer corresponds to an allele of an SNP by adjusting its 3'-end. Secondly, add a tag sequence to the 5'-end of each forward primer. In addition, design a fluorescent probe corresponding to the tag sequence. The consensus sequence of specific primer 1 is 5′-GAAGGTGACCAAGTTCATGCT-3′, as shown in SEQ ID NO.1, and a specific sequence capable of binding to FAM fluorescence is added to the tail. The consensus sequence of specific primer 2 is 5′-GAAGGTCGGAGTCAACGGATT-3′, as shown in SEQ ID NO.2, and a specific sequence capable of binding to HEX fluorescence is added to the tail. The two primers share the reverse primer sequence. The primer sequences are shown in Table 7.

[0049] 2) Conventional PCR amplification

[0050] In the first round of PCR amplification, the allele-specific primer can recognize the specific allele template to complete allele recognition. Starting from the second round of PCR amplification, templates carrying the universal tag sequence appear in the products, and this step completes the introduction of the universal tag sequence into the PCR products corresponding to SNPs. Subsequently, during the PCR amplification process, the fluorescent probe is added to the PCR products by binding to the DNA strand complementary to the universal sequence. After multiple rounds of PCR amplification, more fluorescent probes anneal to the newly synthesized complementary strands without quenching groups, gradually enhancing the fluorescence intensity of the PCR products.

[0051] 5. Usage method of the newly developed KASP marker

[0052] According to the above method, 145 pairs of designed KASP markers were verified by ordinary PCR amplification. Finally, 94 pairs of KASP markers could amplify correct bands, which were recorded as successfully developed KASP markers. Subsequently, the fluorescence quantitative PCR method could be used for accurate identification of haploids (the reaction procedure is shown in Table 8). Each 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 were as follows: in the first round, 94 °C for 15 min; 94 °C for 20 s, 61 - 55 °C for 60 s, with a decrease of 0.6 °C for each cycle, and a total of 10 cycles were carried out; in the second round, 94 °C for 20 s, 55 °C for 60 s, and a total of 26 cycles were carried out. PCR amplification was completed on a qPCR instrument 7900 (Applied Biosystems, USA). Fluorescence signals were collected by the end-point method, and then the detection data were read using the SNP viewer 2.0 software developed by LGC Company. This method is suitable for individuals or small-scale laboratories that need to identify haploids, can operate with a relatively small sample size conveniently, and the results can be obtained in as fast as 3 hours. It can also perform batch and accurate biallelic determination using the DOUGLUS platform with high throughput. It has the advantages of short cycle, high verification rate, low cost, and strong flexibility, can efficiently, conveniently and accurately complete the screening work of haploids, and can combine with the field phenotypic identification work to confirm that the detection results of KASP markers are consistent with the field phenotypes of mono / diploids.

[0053] Figure 4 For the ploidy identification of a newly developed KASP marker KA77 randomly selected, it was found that the plants identified as haploids (with the same genotype as the female parent) had characteristics such as narrow leaves, larger leaf angles, shorter plants, pollen sterility, etc. compared with diploid plants (genotypes are heterozygotes of the male and female parent genotypes), which were in line with the field phenotypes of haploid plants.

[0054] Figure 5 and Figure 6 The genotype results of using KASP markers KA01 - KA94 to identify the ploidy of plants that showed haploid and diploid phenotypes in the field.

[0055] The KASP markers of the present invention have the following advantages:

[0056] 1) High marker reliability

[0057] Using the data of Maize 60K Chip of the hybrid progeny F1 haploids of 8 representative inbred lines and the inducer line CAU5 is the most comprehensive detection method for chip sites under the existing experimental conditions. In the induced F1, for each site, there will be three different situations: a. complete elimination of the paternal parent (the F1 genotype is the same as that of the maternal parent); b. partial elimination of the paternal parent (the F1 genotype is a heterozygous type of the genotypes of the paternal and maternal parents); c. elimination of the maternal parent (the F1 genotype is the same as that of the paternal parent).

[0058] When we screened the sites, we preferentially screened the sites where complete paternal chromosome elimination occurred (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 marker use.

[0059] 2) Wide marker coverage and uniform distribution

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

[0061] 3) Simple identification method

[0062] The identification method of KASP markers mentioned in the present invention can utilize the Douglas 7900 and microplate reader platforms to directly perform accurate biallelic determination. This method has a short cycle, high verification rate, low cost, and strong flexibility. It can efficiently, conveniently, and accurately complete the screening work of haploids, and can combine with the field phenotypic identification work to confirm that the detection results of KASP markers are consistent with the field phenotypes of haploids / diploids (narrow and small leaves, large leaf angles, short plants, pollen sterility, etc.). Compared with the traditional gel electrophoresis detection method, which is limited to processing a maximum of 96-well samples per test cycle and has low efficiency. The Douglas 7900 platform mentioned in the present invention can process 720 384-well plates each time, which not only has high efficiency but also eliminates the error of manual detection in the traditional method.

[0063] Table 1 KASP marker numbers and physical positions 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 Reaction system of DMM denaturing solution

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

[0067] Table 3 Reaction system of Amp MM amplification solution

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

[0069] Table 4 Genotypes and corresponding gene numbers of 145 loci with complete elimination of paternal chromosomes in the 60K chip data in the inducer line CAU5 and 8 inbred lines

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Note: Spaces represent no corresponding gene numbers in the current version.

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

[0077] Proportion / % 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 sites where paternal chromosomes are completely eliminated in each inbred 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 of 94 KASP markers developed from 145 sites where paternal chromosomes are completely eliminated

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] Table 8 KASP marker PCR amplification reaction system

[0092] Ingredient Volume KASP Master mix 1.5 μL KASP Primer mix 0.042 μL Template DNA 1.5 μL (50 ng / μL) Total 3.042 μl

[0093] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A KASP marker for identifying maize haploids, characterized in that: The KASP marker is any one of KA01 to KA94: The KASP marker is determined based on the whole genome sequence alignment of the corn variety B73, and the version number of the whole genome sequence of the corn variety B73 is B73 RefGen V3.

2. A primer set for amplifying the KASP marker according to claim 1.

3. The primer set according to claim 2, characterized in that The primer set consists of ninety-four primer pairs, which correspond to KASP markers numbered KA01 to KA94 in sequence. Each primer pair consists 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 is shown in SEQ ID NO.1, and the common sequence of all specific primers 2 is shown in SEQ ID NO.

2.

4. The primer set according to claim 3, characterized in that: A specific sequence capable of binding to FAM fluorescence is added to the tail of the specific primer 1, and the FAM fluorescent group is labeled. A specific sequence capable of binding to HEX fluorescence is added to the tail of the specific primer 2, and the HEX fluorescent group is labeled.

5. A kit comprising the primer set according to claim 2.

6. Use of the KASP marker according to claim 1, the primer set according to claim 2 or the kit according to claim 5 in identifying maize haploids.

7. A method for identifying maize ploidy, characterized in that: The following steps are involved: 1) Using the genomic DNA of the corn to be tested and its parents as templates, obtaining genotype data of at least one SNP site in KA01 to KA94 of the KASP marker of claim 1 for the corn to be tested and its parents; 2) According to the genotype data, if the genotype of the corn to be tested is the same as that of the maternal plant, the corn to be tested is a haploid plant; if the genotype of the corn to be tested is a heterozygous genotype of the paternal and maternal plants, the corn to be tested is a diploid plant.

8. The method for identifying maize ploidy according to claim 7, characterized in that: Methods for obtaining genotype data include fluorescent quantitative PCR detection method and high-throughput SNP detection and analysis method.

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