KASP molecular marker related to sorghum spike tightness, primer and application of KASP molecular marker

Abstract

The invention relates to the technical field of molecular marker development and molecular marker assisted breeding, and particularly discloses a KASP molecular marker related to sorghum panicle tightness, a primer and application thereof, the KASP molecular marker is at least one of PT1.1 as shown in SEQ ID NO.1 and PT1.2 as shown in SEQ ID NO.2, and basic groups at 25bp positions of molecular marker sequences of PT1.1 and PT1.2 have polymorphism. The molecular marker provided by the invention has relatively high correlation with the sorghum panicle tightness character, rapid, accurate and high-throughput detection of the sorghum panicle tightness can be realized by utilizing the molecular marker on the genotype level, and the marker has an earlier time advantage, higher accuracy and higher speed when being applied to assisted breeding.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular marker development and molecular marker-assisted breeding, and particularly relates to a KASP molecular marker related to the tightness of sorghum panicles, primers thereof, and applications thereof. Background Art

[0002] Sorghum (Sorghum bicolor L.), as a typical gramineous C4 crop, exhibits extensive ecological adaptability globally due to its excellent environmental plasticity. From the ecological gradient with an annual precipitation of 200 mm to 1500 mm to the wide distribution area with a soil pH of 5.0 to 8.5, sorghum can maintain stable yields, and one of the core mechanisms of this environmental adaptability is the highly differentiated panicle types. Under the combined action of long-term natural selection and artificial domestication, sorghum has formed significantly different panicle structures in different ecological regions. For example, in the humid and hot ecological regions in the south, sorghum has evolved into a loose and drooping panicle shape to expand the panicle space, thereby enhancing the transpiration and heat dissipation efficiency and effectively reducing the risk of grain rot in high-temperature and high-humidity environments; while in the arid regions in the north, sorghum shows an upright and compact spindle-shaped panicle, and its optimized population canopy structure not only improves the light energy capture efficiency but also enhances the yield per plant and the resistance to strong winds. This ecological adaptation strategy of "one region, one panicle type" is not only a key factor for sorghum to successfully cross the north-south climate barrier but also provides important trait improvement targets for modern molecular design breeding.

[0003] The introduction of the KASP (Kompetitive Allele Specific PCR) molecular marker technology provides an efficient tool for solving this problem. As the third-generation molecular marker technology, KASP shows significant advantages in terms of accuracy, throughput, and application convenience compared with the first-generation markers (such as RFLP, AFLP) and the second-generation markers (such as SSR, RAPD). Compared with the first-generation markers, KASP does not rely on enzyme digestion and electrophoresis, avoids the use of radioactive isotope probes, and does not require silver staining. The experimental process is safe and non-toxic, making the operation more convenient and the results easier to interpret. Compared with the second-generation markers, KASP uses allele-specific fluorescence probes, can accurately identify single-base variations (SNP), and has a higher throughput, enabling rapid genotyping in large-scale populations.

[0004] Currently, the research on the genetic regulation mechanism of sorghum panicle types is still relatively limited, and there is a lack of efficient molecular marker tools to assist in precise breeding. It is necessary to develop a KASP molecular marker related to the tightness of sorghum panicles. Summary of the Invention

[0005] To develop a KASP molecular marker related to the tightness of sorghum panicles, the present invention provides a KASP molecular marker related to the tightness of sorghum panicles, primers thereof and applications. The molecular marker provided by the present invention has a high correlation with the trait of the tightness of sorghum panicles. At the genotype level, the use of this molecular marker can achieve rapid, accurate and high-throughput detection of the tightness of sorghum panicles. Applying this marker to assisted breeding has earlier time advantages, higher accuracy and faster speed.

[0006] The present invention provides a KASP molecular marker related to the tightness of sorghum panicles, and the KASP molecular marker is at least one of PT1.1 and PT1.2;

[0007] The nucleotide sequence of the molecular marker PT1.1 is shown in SEQ ID NO.1, and the base at the 25th bp of the molecular marker PT1.1 has T / C polymorphism;

[0008] The nucleotide sequence of the molecular marker PT1.2 is shown in SEQ ID NO.2, and the base at the 25th bp of the molecular marker PT1.2 has A / G polymorphism.

[0009] The molecular marker provided by the present invention has a high correlation with the trait of the tightness of sorghum panicles. By genotyping the molecular marker PT1.1 and / or the molecular marker PT1.2 and determining the tightness of the sorghum panicles to be tested according to the genotyping results, a loose-panicle variety can be determined. At the genotype level, the use of this molecular marker can achieve rapid, accurate and high-throughput detection of the tightness of sorghum panicles. Applying this marker to assisted breeding has earlier time advantages, higher accuracy and faster speed.

[0010] The present invention also provides a primer set for amplifying the KASP molecular marker related to the tightness of sorghum panicles. The primer set for amplifying the molecular marker PT1.1 includes two specific forward primers shown in SEQ ID NO.3 to SEQ ID NO.4 and a universal reverse primer shown in SEQ ID NO.5;

[0011] The primer set for amplifying the molecular marker PT1.2 includes two specific forward primers shown in SEQ ID NO.6 to SEQ ID NO.7 and a universal reverse primer shown in SEQ ID NO.8.

[0012] The present invention also provides a kit, and the kit contains any one or a combination of several of the primer set for amplifying the molecular marker PT1.1 and the primer set for amplifying the molecular marker PT1.2.

[0013] The present invention also provides a chip, which contains any one or several combinations of the primer sets for amplifying the molecular marker PT1.1 and the primer sets for amplifying the molecular marker PT1.2.

[0014] Further, the primer set is in powder solid state or liquid state.

[0015] The present invention also provides a method for identifying the tightness of sorghum panicles, comprising the following steps:

[0016] Extract the DNA of the sorghum to be tested;

[0017] Perform PCR amplification on the extracted DNA of the sorghum to be tested by using the primer set or the kit as described above to obtain a PCR product;

[0018] Perform genotyping on the PCR product by using a fluorescence quantitative PCR instrument, and determine the tightness of the sorghum panicles to be tested according to the genotyping result, and determine the loose-panicle variety;

[0019] The standard for determining the tightness of the sorghum panicles to be tested according to the genotyping result is as follows: When using the primer set for amplifying the molecular marker PT1.1, when the fluorescence signal of the PCR product is close to the Y-axis and the genotype is C / C, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the middle and the genotype is C / T, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the X-axis and the genotype is T / T, it is determined that the sorghum sample to be tested is a tight-panicle variety.

[0020] When using the primer set for amplifying the molecular marker PT1.2, when the fluorescence signal of the PCR product is close to the Y-axis and the genotype is G / G, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the middle and the genotype is G / A, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the X-axis and the genotype is A / A, it is determined that the sorghum sample to be tested is a tight-panicle variety.

[0021] The present invention also provides an application of the KASP molecular marker, the primer set or the kit as described above in identifying loose-panicle sorghum and tight-panicle sorghum.

[0022] Further, the determination criteria for loose-panicle sorghum and tight-panicle sorghum are as follows:

[0023] When using the molecular marker PT1.1 for identification, when the fluorescence signal of the PCR product is close to the Y-axis and the genotype is C / C, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the middle and the genotype is C / T, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the X-axis and the genotype is T / T, it is determined that the sorghum sample to be tested is a tight-panicle variety.

[0024] When identified using the molecular marker PT1.2, when the fluorescence signal of the PCR product is close to the Y-axis and the genotype is G / G, the tested sorghum sample is determined to be a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the middle and the genotype is G / A, the tested sorghum sample is determined to be a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the X-axis and the genotype is A / A, the tested sorghum sample is determined to be a compact-panicle sorghum variety.

[0025] When jointly identified using the molecular markers PT1.1 and PT1.2, when the genotype identified by PT1.1 is C / C and the genotype identified by PT1.2 is G / G, the tested sorghum sample is a loose-panicle sorghum variety; when the genotype identified by PT1.1 is T / T and the genotype identified by PT1.2 is A / A, the tested sorghum sample is a compact-panicle sorghum variety.

[0026] Furthermore, the sorghum is sorghum 2381 or a derivative line of sorghum 2381.

[0027] Furthermore, the PCR amplification system includes: 2.5 μL of 50 ng / μL DNA template, 5 μL of 2×KASP Master mix, 0.1 μL of each of the two 10 μM upstream primers, 0.3 μL of the 10 μM downstream primer, and the volume is made up to 10 μL with water.

[0028] Furthermore, the reaction program for PCR amplification includes: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing and extension at 62 °C for 60 s, for 9 cycles; denaturation at 95 °C for 15 s, annealing and extension at 57 °C for 60 s, for 31 cycles.

[0029] The present invention also provides an application of the KASP molecular marker, the primer set or the kit in the detection of the purity of sorghum hybrids.

[0030] The molecular marker, primer set and identification method provided by the present invention can be used for the following applications:

[0031] For the identification of the loose or compact panicle trait of sorghum;

[0032] For sorghum molecular marker-assisted breeding.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] The molecular markers provided by the present invention have a high correlation with the trait of the tightness of sorghum panicles. By genotyping the molecular marker PT1.1 and / or the molecular marker PT1.2, the tightness of the sorghum panicles to be tested is determined according to the genotyping results to identify loose-panicle varieties: when using the molecular marker PT1.1 for identification, when the fluorescence signal of the PCR product is close to the Y-axis and the genotype is C / C, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the middle and the genotype is C / T, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the X-axis and the genotype is T / T, it is determined that the sorghum sample to be tested is a tight-panicle variety.

[0035] When using the molecular marker PT1.2 for identification, when the fluorescence signal of the PCR product is close to the Y-axis and the genotype is G / G, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the middle and the genotype is G / A, it is determined that the sorghum sample to be tested is a loose-panicle sorghum variety; when the fluorescence signal of the PCR product is close to the X-axis and the genotype is A / A, it is determined that the sorghum sample to be tested is a tight-panicle variety.

[0036] When using the molecular markers PT1.1 and PT1.2 for combined identification, when the genotype identified by PT1.1 is C / C and the genotype identified by PT1.2 is G / G, the sorghum sample to be tested is a loose-panicle sorghum variety; when the genotype identified by PT1.1 is T / T and the genotype identified by PT1.2 is A / A, the sorghum sample to be tested is a tight-panicle variety.

[0037] The present invention uses marker primers to quickly and efficiently identify the panicle type phenotype of sorghum at various stages. In contrast, traditional breeding requires waiting until the heading stage to determine the panicle type. Compared with this, the application of this marker in assistant breeding has the advantages of early time, cost saving, accuracy and rapidity.

[0038] The method of KASP molecular markers provided by the present invention for identifying the trait of the tightness of sorghum panicles only needs to perform PCR for detection compared with other marker type identification methods such as SSR and InDel, and does not require operations such as sequencing, silver staining and band reading. The operation is simple, rapid and low-cost. Brief Description of the Drawings

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0040] Figure 1 It is the QTL mapping diagram of the RIL of 2381 / LNR-6.

[0041] Figure 2 Genotyping map of KASP molecular marker PT1.1 in 42 sorghum individuals.

[0042] Figure 3 Genotyping map of KASP molecular marker PT1.2 in 42 sorghum individuals. Detailed implementation manners

[0043] The following is a detailed description of the specific implementation manners of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0044] Example 1: A KASP molecular marker, primer related to the tightness of sorghum panicles and their applications.

[0045] I. QTL mapping and determination of molecular markers

[0046] 1. Construction of the population

[0047] Using the tight panicle variety LNR-6 as the male parent and the loose panicle variety 2381 as the female parent. The hybrid combination was configured in 2021, and Table 1 shows the process of obtaining the families.

[0048] Table 1 Process of obtaining the families

[0049] Time Location Generation Harvest May 2021 Shenyang Experimental Base Configure Hybrid Combinations <![CDATA[Obtain 12 F 1 seeds]]> October 2021 Ledong Experimental Base, Hainan <![CDATA[F 1 plant]]> <![CDATA[Harvest 1 F 1 plant and obtain 1004 F 2 seeds]]> May 2022 Shenyang Experimental Base <![CDATA[F 2 plant]]> <![CDATA[Single-plant harvesting yields F 3 seeds]]> October 2022 Ledong Experimental Base, Hainan <![CDATA[F 3 plant]]> <![CDATA[Single-plant harvesting to obtain F 4 seeds]]> May 2023 Shenyang Experimental Base <![CDATA[F 4 plant]]> <![CDATA[Single plant harvesting to obtain F 5 seeds]]> October 2023 Ledong Experimental Base, Hainan <![CDATA[F 5 plant]]> <![CDATA[Single-plant harvesting yields F 6 seeds]]> May 2024 Shenyang Experimental Base <![CDATA[305 strains of F 6 single strain]]> <![CDATA[Single plant harvesting to obtain F 7 seeds]]>

[0050] 2. Field experiment

[0051] The RIL population was planted in the experimental base of the Sorghum Research Institute of Liaoning Academy of Agricultural Sciences in Shenyang in 2024. The experiment adopted a randomized block design. Each line was planted in 1 row, with 3 replicates, 20 plants in each row, the row length was 3 m, and the row spacing was 0.6 m. Field fertilization and management referred to local field cultivation production, and diseases, insects and weeds were controlled in a timely manner. Each plant was harvested separately at maturity.

[0052] 3. Phenotypic statistics

[0053] A three-level standard was formulated for the panicle type phenotype, namely loose panicle, medium loose panicle, and medium tight panicle. Using this standard, the phenotypes of individuals in the F 6 population were investigated.

[0054] 4. QTL mapping

[0055] Thirty randomly selected individual plants of loose-panicle sorghum and thirty individual plants of medium-compact-panicle sorghum were used to construct a loose-panicle mixed pool and a medium-compact-panicle mixed pool respectively, and then re-sequenced in the second generation together with the two parents. The obtained Raw data was quality-controlled to obtain Clean data, and then the Clean data was aligned to the sorghum reference genome Btx623-T2T Genome v1.0 using the BWA software. The average depth of the two parents exceeded 10×, the coverage of the mixed pool exceeded 30×, and the 1× coverage of all samples exceeded 90% (as shown in Table 2). Subsequently, GATK was used to detect variations in all samples, and the G value was calculated by comparing the deviation between the actual allele counts at each locus in the two groups and the expected counts under the assumption of no QTL. A higher G value indicates a significant difference in allele frequencies at this locus between the two groups. Finally, the interval controlling the panicle type was mapped to 74.29 - 79.83 MB on chromosome 3, named the PT1 (Panicletype 1) locus( Figure 1 ).

[0056] Table 2 Statistics of sequencing depth and coverage

[0057] Sample Alignment Rate Average Depth 1× Coverage 5× Coverage 2381 46689908(96.81％) 10.21 92.81 78.9 LNR-6 52444061(98.62％) 11.51 93.06 82.9 Medium-compact Panicle Mixed Pool 159922605(98.12％) 33.44 97.82 94.78 Loose Panicle Mixed Pool 181750458(98.33％) 37.82 97.86 94.72

[0058] 5. Development of markers

[0059] According to the position information of the PT1 locus, the variant information of the two parents was extracted on both sides of it. Finally, SNP variations between the two parents were found at Chr03:74,375,416 and Chr03:79,822,410 respectively, which could be developed into molecular markers and named PT1.1 and PT1.2 respectively.

[0060] The nucleotide sequence of the molecular marker PT1.1 is shown in SEQ ID NO.1, and the base at the 25bp of this sequence has T / C polymorphism.

[0061] The nucleotide sequence of the molecular marker PT1.2 is shown in SEQ ID NO.2, and the base at the 25bp of this sequence has A / G polymorphism.

[0062] SEQ ID NO.1:

[0063] GGGGGTTGTATGTATGGTCAGAAGNGGAGGCAGCGAAGAGAGAATC GGCT, where N represents "T" or "C".

[0064] SEQ ID NO.2:

[0065] TCTATGCAGCGCCCTACAGATACTNATTGACGAGGACGACTATGCCTC TG, where N represents "A" or "G".

[0066] II. Design of Molecular Marker Primer Sets

[0067] Specific primer sets were designed for the nucleotide sequences of PT1.1 and PT1.2 respectively.

[0068] For the PT1.1 locus, the primer set includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3, another upstream primer with a nucleotide sequence as shown in SEQ ID NO.4, and a common downstream primer with a nucleotide sequence as shown in SEQ ID NO.5. At the same time, the 5'-end of the upstream primer shown in SEQ ID NO.3 is linked with a fluorescent tag sequence FAM, and the 5'-end of the other upstream primer shown in SEQ ID NO.4 is linked with a fluorescent tag sequence HEX. The 3'-end of the upstream primer is the allelic variant base T / C at this locus, and the downstream primer ensures the specificity of Chr03 chromosome for PCR amplification. The primer sequences are shown in Table 3.

[0069] For the PT1.2 locus, the primer set includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.6, another upstream primer with a nucleotide sequence as shown in SEQ ID NO.7, and a common downstream primer with a nucleotide sequence as shown in SEQ ID NO.8. At the same time, the 5'-end of the upstream primer shown in SEQ ID NO.6 is linked with a fluorescent tag sequence FAM, and the 5'-end of the other upstream primer shown in SEQ ID NO.7 is linked with a fluorescent tag sequence HEX. The 3'-end of the upstream primer is the allelic variant base T / C at this locus, and the downstream primer ensures the specificity of Chr03 chromosome for PCR amplification. The primer sequences are shown in Table 3.

[0070] Table 3 Specific Primer Combinations for PT1.1 and PT1.2 Loci

[0071]

[0072] Note: The lowercase parts are the linker sequences FAM and HEX.

[0073] III. Verification of Molecular Markers

[0074] Select 42 Fs in Example 1 6Family plants, using the improved CTAB method to extract DNA from the sorghum samples to be tested. The extracted DNA was subjected to PCR amplification using primer sets for amplifying PT1.1 and PT1.2 respectively to obtain PCR amplification products. The PCR products were detected in a real-time fluorescence quantitative PCR instrument, the fluorescence intensity signal values were read, and cluster analysis was performed to obtain the gene typing at polymorphic sites. According to the gene typing results, the panicle morphology of the sorghum samples to be tested was determined. Among them, in the molecular marker PT1.1 detection experiment, 17 samples were close to the Y-axis and were determined to be allele 2 with a genotype of C / C; 2 samples were close to the middle and were determined to be heterozygous with a genotype of C / T. A total of 19 samples close to the Y-axis and close to the middle should have a loose panicle phenotype; 23 samples were close to the X-axis and were determined to be allele 1 with a genotype of T / T. There should be 23 samples with a compact panicle phenotype( Figure 2 ). Combining the phenotypic data, it was found that 20 out of 42 F 6 family plants had a loose panicle phenotype and 22 had a compact panicle phenotype. Therefore, the detection accuracy rate of the molecular marker PT1.1 was 95%.

[0075] In the molecular marker PT1.2 detection experiment, 16 samples were close to the Y-axis and were determined to be allele 2 with a genotype of G / G; 2 samples were close to the middle and were determined to be heterozygous with a genotype of G / A. A total of 18 samples close to the Y-axis and close to the middle should have a loose panicle phenotype. 24 samples were close to the X-axis and were determined to be allele 1 with a genotype of A / A. There should be 24 samples with a compact panicle phenotype( Figure 3 ). Combining the phenotypic data, it was found that 20 of them had a loose panicle phenotype and 22 had a compact panicle phenotype. It can be seen that the detection accuracy rate of the molecular marker PT1.2 was 95%. The accuracy rate was as high as 100% when the two markers were jointly identified (Table 4).

[0076] Table 4 PT1.1, PT1.2 and joint identification

[0077]

[0078]

[0079] The PCR amplification system of the KASP marker primers includes:

[0080] 2.5 μL of 50 ng / μL DNA template, 5 μL of 2×KASP Master mix, 0.1 μL of each of the two upstream primers at 10 μM, 0.3 μL of the downstream primer at 10 μM, and the volume was made up to 10 μL with water.

[0081] The PCR reaction program of the KASP marker primers is as follows:

[0082] Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing and extension at 62°C for 60 s, with 9 cycles; denaturation at 95°C for 15 s, annealing and extension at 57°C for 60 s, with 31 cycles. A blank control (NTC) without adding template DNA was set in the reaction system during the experiment, and 3 blank controls were set for each plate.

[0083] IV. Marker-assisted selection breeding

[0084] When using 2381 to cross with a tight-panicle variety by emasculation, and artificially selecting loose-panicle offspring based on phenotype in the F 2 generation, it is often difficult to fix the phenotype in the offspring due to the existence of heterozygous individuals. Now, the above molecular markers PT1.1 and PT1.2 can be used for detection, and marker-assisted selection can be carried out in the F 2 generation, so as to accelerate the breeding process. The specific implementation is as follows:

[0085] (1) Extract the DNA of the sorghum seedlings to be tested by the improved CTAB method.

[0086] (2) Using the DNA extracted in step (1) as a template, perform PCR amplification with the primer sets of the molecular markers PT1.1 and PT1.2 shown; the PCR amplification system and program are the same as above.

[0087] (3) Put the PCR products amplified in step (2) into a fluorescence quantitative PCR instrument, read the FAM and HEX fluorescence intensities, perform cluster analysis, obtain the genotyping at the polymorphic loci, select the homozygous individuals with allele 2, genotype C / C identified by PT1.1 and genotype G / G identified by PT1.2, and eliminate other individuals.

[0088] (4) In the next year, plant the homozygous individuals with genotype C / C identified by PT1.1 and genotype G / G identified by PT1.2 in the field. At this time, the phenotype of the tightness of the panicle does not segregate and is quickly fixed.

[0089] The SNP variations provided by the present invention, as well as the PCR detection primers and corresponding kits developed based on this SNP variation, can be used for the identification or breeding of the tightness of the sorghum panicle, and can realize the conversion of phenotype judgment into genotype identification, and quickly identify or screen sorghum loose-panicle and tight-panicle materials from the genotype level.

[0090] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept.

[0091] Obviously, those skilled in the art can make various modifications and variations 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 also intends to include these modifications and variations.

Claims

1. A KASP molecular marker related to the tightness of sorghum ear, characterized in that: The KASP molecule marker is at least one of PT1.1 and PT1.2; The nucleotide sequence of the molecular marker PT1.1 is shown in SEQ ID NO.1, and the base at 25 bp of the molecular marker PT1.1 has a T / C polymorphism; The nucleotide sequence of the molecular marker PT1.2 is shown in SEQ ID NO.

2. The base at 25 bp of the molecular marker PT1.2 has an A / G polymorphism.

2. A primer set for amplifying the KASP molecular marker associated with sorghum ear tightness according to claim 1, characterized in that: The primer set for amplifying the molecular marker PT1.1 includes two specific forward primers shown in SEQ ID NO.3 to SEQ ID NO.4 and a universal reverse primer shown in SEQ ID NO.5; The primer set for amplifying the molecular marker PT1.2 includes two specific forward primers shown in SEQ ID NO.6 to SEQ ID NO.7 and a universal reverse primer shown in SEQ ID NO.

8.

3. A kit, characterized in that: It comprises any one or a combination of the primer set for amplifying the molecular marker PT1.1 and the primer set for amplifying the molecular marker PT1.2 described in claim 2.

4. A chip, characterized in that: It comprises any one or a combination of the primer set for amplifying the molecular marker PT1.1 and the primer set for amplifying the molecular marker PT1.2 described in claim 2.

5. The kit according to claim 3 or the chip according to claim 4, characterized in that: The primer set is in powder solid or liquid state.

6. A method for identifying the tightness of sorghum ears, characterized in that: The following steps are involved: Extract the sorghum DNA to be tested; Using the primer set in claim 2 or the kit in claim 3 to perform PCR amplification on the extracted sorghum DNA to be tested to obtain a PCR product; The PCR products were genotyped using a fluorescent quantitative PCR instrument, and the tightness of the panicle of the sorghum to be tested was determined according to the genotyping results to determine the loose panicle variety; The standard for determining the tightness of the sorghum ear to be tested according to the typing results is: when the primer set for amplifying the molecular marker PT1.1 is used, when the fluorescence signal of the PCR product is close to the Y axis, the genotype is C / C, and the sorghum sample to be tested is determined to be a loose ear variety of sorghum; when the fluorescence signal of the PCR product is close to the middle, the genotype is C / T, and the sorghum sample to be tested is determined to be a loose ear variety of sorghum; when the fluorescence signal of the PCR product is close to the X axis, the genotype is T / T, and the sorghum sample to be tested is determined to be a tight ear variety of sorghum; When the primer set for amplifying the molecular marker PT1.2 is used, when the fluorescence signal of the PCR product is close to the Y-axis, the genotype is G / G, and the sorghum sample to be tested is determined to be a loose-ear sorghum variety; when the fluorescence signal of the PCR product is close to the middle, the genotype is G / A, and the sorghum sample to be tested is determined to be a loose-ear sorghum variety; when the fluorescence signal of the PCR product is close to the X-axis, the genotype is A / A, and the sorghum sample to be tested is determined to be a tight-ear sorghum variety.

7. Use of the KASP molecular marker according to claim 1, the primer set according to claim 2 or the kit according to claim 3 in identifying loose ears of sorghum and tight ears of sorghum.

8. The use according to claim 7, characterized in that: The criteria for determining whether sorghum is loose-eared or tight-eared are as follows: when using molecular marker PT1.1 for identification, when the fluorescence signal of the PCR product is close to the Y axis, the genotype is C / C, and the sorghum sample to be tested is determined to be a loose-eared sorghum variety; when the fluorescence signal of the PCR product is close to the middle, the genotype is C / T, and the sorghum sample to be tested is determined to be a loose-eared sorghum variety; when the fluorescence signal of the PCR product is close to the X axis, the genotype is T / T, and the sorghum sample to be tested is determined to be a tight-eared sorghum variety. When using molecular marker PT1.2 for identification, when the fluorescence signal of the PCR product is close to the Y axis, the genotype is G / G, and the sorghum sample to be tested is determined to be a sorghum loose ear variety; when the fluorescence signal of the PCR product is close to the middle, the genotype is G / A, and the sorghum sample to be tested is determined to be a sorghum loose ear variety; when the fluorescence signal of the PCR product is close to the X axis, the genotype is A / A, and the sorghum sample to be tested is determined to be a sorghum tight ear variety; When molecular markers PT1.1 and PT1.2 are used for joint identification, when the genotype identified by PT1.1 is C / C and the genotype identified by PT1.2 is G / G, the sorghum sample to be tested is a loose-ear sorghum variety; when the genotype identified by PT1.1 is T / T and the genotype identified by PT1.2 is A / A, the sorghum sample to be tested is a tight-ear sorghum variety.

9. The use according to claim 8, characterized in that: The sorghum is sorghum 2381 or a derivative line of sorghum 2381.

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