A primer set, kit, detection method and application of a single base insertion variation KASP molecular marker closely linked to a rice FZP gene

Abstract

The application provides a primer set of a single base insertion variation KASP molecular marker closely linked with a rice FZP gene, a kit and a detection method and application thereof, and belongs to the technical field of plant molecular breeding and molecular marker. The application provides a primer set of a single base insertion variation KASP molecular marker closely linked with a rice FZP gene, and the single base insertion variation KASP molecular marker is FZP-1bp-KASP, the variation site is located at the physical position 28,297,569 bp of the 7th chromosome of rice, and is a single base A insertion / non-insertion variation. The marker is not located in the FZP gene, but is close to the FZP gene in physical distance and keeps close linkage, can be used as a substitute determination marker of FZP allele source and genotype in a verified genetic background, and is used for equivalent identification of FZP alleles.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding and molecular marker technology, and in particular to a primer set, kit, detection method and application of a single-base insertion variant KASP molecular marker closely linked to the rice FZP gene. Background Technology

[0002] Rice yield is determined by factors such as the number of panicles per unit area, the number of grains per panicle, and grain weight. The branching and spikelet formation processes directly affect the number of spikelets per panicle and the final yield potential. FZP is a crucial gene regulating the transition from branch meristem to spikelet meristem in rice, and its allelic variations and genetic differences in surrounding regulatory regions are closely related to panicle development. Therefore, establishing tightly linked markers that are close to FZP, have good co-dominance, and are suitable for high-throughput detection has significant application value in FZP gene mapping, allelic tracing, and molecular breeding.

[0003] Previous studies observed panicle phenotypic segregation, such as panicle top degeneration, in segregating materials with the N22-transferred Zhenshan 97 (ZS97) genetic background, and pinpointed the target region to the vicinity of FZP on chromosome 7 through genetic mapping. In gene mapping and breeding selection, if a polymorphic locus is sufficiently close to the target gene and maintains stable linkage within the target genetic background, the marker genotype can be used to trace the parental origin of the chromosome segment containing the target gene. When no recombination occurs between the marker and the target gene, the target genotype can be indirectly inferred based on linkage relationships. Existing marker schemes mainly use conventional PCR and polyacrylamide gel electrophoresis for typing small insertion / deletion sites. While this can achieve co-dominance detection, it involves post-amplification electrophoresis, staining, and manual banding, which is not conducive to large-sample, automated, and standardized typing.

[0004] KASP (Kompetitive Allele-Specific PCR) utilizes two allele-specific primers and one universal primer for competitive amplification, and achieves genotype clustering through different fluorescent reporter systems. It is suitable for detecting SNPs and small insertion / deletion variants. For variants differing by only one base, KASP can complete closed-tube genotyping without relying on gel length resolution, making it particularly suitable for large-scale detection of breeding populations and germplasm resources. Summary of the Invention

[0005] The purpose of this invention is to provide a KASP molecular marker for a single-base insertion variant tightly linked to the rice FZP gene, along with its primers, kits, detection methods, and applications. This addresses the lack of tightly linked markers that are close to FZP, have good co-dominance, and are suitable for high-throughput detection, as well as the difficulty in achieving stable, high-throughput genotyping of 1 bp insertion / deletion variants using conventional gel electrophoresis. Furthermore, by utilizing the tight linkage between this variant site and FZP, it enables the tracing of FZP allele origins, indirect determination of FZP genotypes, gene localization, and marker-assisted selection within a validated genetic background.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a primer set for a single-base insertion variant KASP molecular marker tightly linked to the rice FZP gene. The single-base insertion variant KASP molecular marker is FZP-1bp-KASP (this invention uses this site as a neighboring tightly linked marker of the FZP gene, rather than limiting it to a functional variation of FZP itself; in populations or breeding backgrounds where the marker has been verified to be linked to FZP and no recombination has occurred between them, the FZP genotype or parental allele origin can be inferred from the genotype of this marker). The mutation site of FZP-1bp-KASP is located at physical position 28,297,569 bp on the 7th chromosome of rice, and is a single base A insertion / non-insertion mutation; The primer set includes a first allele-specific core forward primer F1 (SEQ ID No. 4: 5′-CATGATCCAAAGAAAACTCGA-3′), a second allele-specific core forward primer F2 (SEQ ID No. 5: 5′-CATGATCCAAAGAAAACTCGT-3′), and a universal reverse primer R (SEQ ID No. 3: 5′-TGAACTAAATAGTAGTTTAC-3′).

[0007] Preferably, the 5′ ends of F1 and F2 are respectively connected to KASP universal connector sequences that can be recognized by different fluorescence detection systems.

[0008] Preferably, the sequences of the adapter primers F1 and F2 are as shown in SEQ ID NO: 1~2 (the universal reverse primer R remains unchanged, as shown in SEQ ID No. 3).

[0009] SEQ ID No. 1: 5′-GAAGGTGACCAAGTTCATGCTCATGATCCAAAGAAAACTCGA-3′; SEQ ID No. 2: 5′-GAAGGTCGGAGTCAACGGATTCATGATCCAAAGAAAACTCGT-3′.

[0010] The present invention also provides a kit for detecting the FZP-1bp-KASP site in rice, comprising the aforementioned primer set.

[0011] Preferably, the kit further includes a KASP 2×PCR Mix, which may contain DNA polymerase, a buffer system, dNTPs, a fluorescent reporter system, and other components required for the KASP reaction.

[0012] This invention also provides a method for detecting the FZP-1bp-KASP locus genotype in rice using the FZP-1bp-KASP molecular marker, the primer set described above, or the kit described above, comprising the following steps: (1) Extract genomic DNA from the rice to be tested; (2) Using the genomic DNA as a template, allele-specific PCR amplification is performed using primers designed based on the FZP-1bp-KASP molecular marker, or the primer set described in any one of claims 1 to 3, or the kit described in claim 4; (3) Two types of fluorescence signals were collected on a real-time fluorescence PCR instrument; (4) Determine whether the material to be tested is A-insertion homozygous, non-insertion homozygous, or heterozygous based on the relative intensity and clustering position of the two types of fluorescence signals.

[0013] Preferably, the first fluorescent signal corresponds to the F1 amplification product and indicates the insertion of the A allele; the second fluorescent signal corresponds to the F2 amplification product and indicates the non-inserted allele; samples with significant fluorescence signals of both types are identified as heterozygous genotypes.

[0014] Preferably, the CFX96 real-time fluorescence PCR system is used for amplification and plate reading.

[0015] The present invention also provides an application of the FZP-1bp-KASP molecular marker, or the primer set or the kit described herein, in detecting single-base insertion variants closely linked to the rice FZP gene.

[0016] Preferably, the FZP genotype or parental allele origin of the segregating population is inferred from the results of single-base insertion variation, and used for molecular breeding.

[0017] Preferably, the results of single-base insertion variation are used for FZP gene localization or fine localization, recombinant screening, germplasm resource typing, and molecular marker-assisted selection of FZP-related spike type, number of grains per spike, or spike degeneration traits.

[0018] Based on a re-verified sequence of the FZP gene and its neighboring regions, this invention identifies a single-base A insertion / non-insertion variant at 28,297,569 bp on chromosome 7. N22 exhibits the A insertion variant, while ZS97 exhibits the non-insertion variant. The FZP gene is located between 28,299,602 and 28,301,089 bp in the same coordinate system, with the nearest boundary between these variants approximately 2,033 bp. This close physical distance is suitable as a close-linked marker for allele tracking of FZP. To transform this variant into a high-throughput marker directly usable for gene mapping and molecular breeding, this invention designs KASP allele-specific primers for this site and establishes a detection system based on the CFX96 platform. Specifically: This invention provides a single-base insertion variant KASP molecular marker FZP-1bp-KASP that is tightly linked to the rice FZP (FRIZZY PANICLE) gene, as well as an allele-specific primer set for detecting this site, a kit containing the primer set, and a detection method based on KASP amplification and dual-channel fluorescence clustering. In particular, it relates to the application of this tightly linked marker in gene localization or molecular breeding to track the origin of FZP alleles and indirectly infer the FZP genotype.

[0019] The beneficial effects of this invention are: 1) The marker site is about 2,033 bp away from the FZP gene boundary, and is physically close to it, so it can be used as a tightly linked marker for the FZP gene; the marker genotype can be used to trace the origin of FZP alleles and indirectly infer the FZP genotype.

[0020] 2) This marker can be directly used for FZP gene localization and fine mapping: it can quickly obtain codominant genotype data in segregating populations and combine it with phenotype or other region markers to screen recombinant individuals, thereby improving the efficiency of genotype determination in target regions.

[0021] 3) In the polymorphism screening, six, six and four standard materials of N22 homozygous, ZS97 homozygous and N22 / ZS97 heterozygous formed three clearly separated fluorescent clusters, indicating that the marker has stable polymorphism and codominant typing ability between the N22 and ZS97 genetic backgrounds.

[0022] 4) In the F2 segregating population detection, the vast majority of valid samples can be stably classified into the three expected genotypes: N22 homozygous, ZS97 homozygous, or heterozygous. A small number of samples that fall outside the standard cluster or have abnormal fluorescence signals can be treated as invalid / retestable samples and not as new allele types, thus facilitating the establishment of a standardized genotyping quality control process.

[0023] 5) KASP dual-fluorescence closed-tube detection eliminates the need for post-amplification electrophoresis, staining, and manual banding, reducing the risk of amplified product contamination and improving throughput and standardization. The 10 μL reaction system is suitable for large-scale detection in breeding populations and germplasm resources. It should be noted that this marker is a tightly linked FZP marker, not a proven causal variant. When used across genetic backgrounds or for final material identification, it can be verified by combining FZP direct sequencing, neighboring markers, or the target phenotypic pattern. Attached Figure Description

[0024] Figure 1 This is a schematic diagram showing the relative positions of the single base variation site at 28,297,569 bp on chromosome 7 and the FZP gene (28,299,602–28,301,089 bp). The closest boundary between the two is approximately 2,033 bp apart.

[0025] Figure 2 A schematic diagram of the FZP-1bp-KASP primer design.

[0026] Figure 3 The flowchart shows the detection process for the FZP-1bp-KASP molecular marker.

[0027] Figure 4 The image shows the polymorphism screening diagram of the FZP-1bp-KASP standard material. Six, six, and four samples of N22 homozygous, ZS97 homozygous, and N22 / ZS97 heterozygous materials, respectively, form three clearly separated fluorescent clusters. In the image, red and blue dots represent the N22 and ZS97 genotypes, respectively, while green dots represent heterozygous genotypes.

[0028] Figure 5 This is a typical allele discrimination cluster diagram of FZP-1bp-KASP in the F2 segregating population; the main samples formed three clusters: N22 homozygous (red dots), heterozygous (green dots), and ZS97 homozygous (blue dots), and a few abnormal scattered points (pink dots) were treated as invalid / retestable samples. Detailed Implementation

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1: Determination of FZP-1bp-KASP site and primer design

[0031] Based on previous research on FZP candidate regions, the FZP gene and its neighboring sequences were re-verified. According to the reference genome coordinate system used in this study, the FZP gene is located on chromosome 7, 28,299,602–28,301,089 bp; a single-base A insertion / non-insertion variation (e.g., ...) was detected at 28,297,569 bp. Figure 1 (As shown). Rice N22 is an A-insertion type, while rice ZS97 is a non-insertion type. This locus is approximately 2,033 bp from the nearest boundary of the FZP gene, located in the vicinity of FZP, and is physically close. Combining the previous results of locating the target region near FZP, this invention uses this locus as a tightly linked genotyping target for FZP, named FZP-1bp-KASP. The role of this marker is to track adjacent chromosomal segments that are co-inherited with FZP; in individuals that have been genetically verified and where no recombination has occurred between the marker and FZP, the marker genotype can serve as a substitute indicator for determining the FZP genotype or parental allele origin.

[0032] Based on the KASP allele-specific PCR design principle, two forward primers with allele recognition differences at their 3′ ends were designed on one side of the mutation site, and a universal reverse primer was designed on the other side. Different universal KASP fluorescent detection adapters were added to the 5′ ends of the two forward primers, forming F1′ and F2′ primers that can competitively amplify in a dual-channel fluorescence system. In this example, F1′ corresponds to the N22 type A inserted allele, and F2′ corresponds to the non-inserted allele. A schematic diagram of the primer design is shown below. Figure 2 As shown, primer information is shown in Table 1.

[0033] Table 1 Primer Information

[0034] Note: The 5′ ends of F1′ and F2′ carry different KASP universal detection adapters FAM and VIC, respectively; the R primers do not contain adapters. If an equivalent KASP fluorescence system is used, universal adapters matching the selected system can be used while maintaining the core allele-specific sequence.

[0035] Example 2 DNA extraction and KASP reaction system

[0036] Fresh rice leaves were collected for testing, and genomic DNA was extracted using the CTAB method. The DNA was appropriately diluted and used as a template for the KASP reaction. KASP 2×PCR Mix from Guangzhou Good Biotechnology Co., Ltd. was preferred, and the reaction was performed in a 10 μL volume.

[0037] Table 2 Reaction System

[0038] Note: In the above system, the final concentrations of F1′ and F2′ are both 100 nM, and the final concentration of R is 300 nM. Alternatively, the three primers can be premixed in a 1:1:3 molar ratio or an equal concentration-volume ratio, but the final concentrations of the two allele-specific primers should be maintained within an appropriate range during the reaction. A positive control with a known genotype and a template-free control should be included in each batch of tests.

[0039] Example 3: CFX96 Amplification Procedure and Fluorescence Interpretation

[0040] Amplification and endpoint fluorescence reading were performed using the CFX96 real-time fluorescence PCR system. When using KASP 2×PCR Mix V1 or V2, the following procedure can be followed: Table 3 Reaction Procedure

[0041] Note: If clustering is still insufficient after 28 amplification cycles, the amplification step can be performed for another 5 to 7 cycles without changing other conditions, and the fluorescence can be read again; if there is no increase in background without template control and the genotyping is sufficient, the number of amplification cycles can be appropriately reduced.

[0042] After amplification, allele discrimination or endpoint fluorescence clustering analysis was performed in CFX96 software. The FAM channel and the HEX / VIC channel, compatible with the second KASP reporter system, were preferred for acquisition. Samples with dominant F1′ amplification were identified as homozygous N22 type A insertion; samples with dominant F2′ amplification were identified as non-insertion homozygous; samples with both types of signals clearly visible and located between two homozygous clusters were identified as heterozygous; template-free controls should be located in the low-fluorescence region.

[0043] Example 4: Polymorphism Screening and F2 Segregation Population Genotyping Validation

[0044] To evaluate the polymorphism and co-dominant genotyping ability of the FZP-1bp-KASP marker in the N22 / ZS97 genetic background, six homozygous N22 materials, six homozygous ZS97 materials, and four heterozygous N22 / ZS97 materials of known types were selected. KASP amplification and endpoint fluorescence clustering were performed according to the systems described in Examples 2 and 3 (detection flowchart shown in Figure 3). Figure 3 (As shown). The results are as follows. Figure 4 As shown, the three types of standard materials form three mutually separated clusters: N22 homozygous materials cluster into A-insertion homozygous clusters, ZS97 homozygous materials cluster into non-insertion homozygous clusters, and heterozygous materials are located between the two homozygous clusters and form independent heterozygous clusters. This result indicates that FZP-1bp-KASP exhibits clear polymorphism between N22 and ZS97, can stably distinguish between the two homozygous and heterozygous genotypes, and can serve as a co-dominant linkage marker for FZP neighboring regions.

[0045] Further, FZP-1bp-KASP was applied to the F2 segregating population from N22 / ZS97 for detection, and the results were as follows: Figure 5 As shown, the vast majority of samples with valid fluorescence signals can be classified into the three expected clusters: N22 homozygous, ZS97 homozygous, or heterozygous. A small number of samples are outside these three standard clusters, close to low-fluorescence regions, or exhibit anomalous scattering. These samples are classified as "invalid genotyping / awaiting retesting" in this invention, and are not interpreted as a fourth genotype, nor included in the valid genotype statistics. Such anomalous signals may be related to technical factors such as DNA degradation, excessively low or high template concentrations, residual inhibitors during extraction, sample loading errors, or occasional amplification failures. For these samples, DNA can be re-extracted, template concentration adjusted, and KASP detection repeated. The presence of a small number of invalid samples does not affect the formation of stable co-dominant clusters among the remaining valid samples.

[0046] Application directions of tightly linked markers in FZP gene localization and molecular breeding

[0047] 1) In N22, ZS97 or target breeding parents, the genotype at the 28, 297, 569 bp locus on chromosome 7 was detected using FZP-1bp-KASP to confirm the existence of A insertion / non-insertion polymorphism between parents, and the correspondence between "marker allele - FZP allele" was established in combination with the parent FZP genotype or source information; 2) Construct F2, BC, RIL or other segregating populations, extract DNA from leaves during the seedling stage, and perform batch typing using FZP-1bp-KASP; mark abnormal samples outside the standard cluster as to be retested to avoid misjudging abnormal signals caused by low-quality DNA as recombination or new genotypes; 3) In the process of gene mapping or fine mapping, the FZP-1bp-KASP genotype is analyzed in conjunction with the target phenotype and other region markers to determine the source of the chromosomal segment adjacent to the FZP carried by the individual, and to help identify recombinant individuals near the FZP, narrow down the mapping interval, or verify candidate regions. 4) In a genetic background where FZP-1bp-KASP has been verified to be stably and closely linked to FZP through parental and segregating populations, if no recombination occurs between the target individual and FZP, the parental allele source or heterozygous status corresponding to the FZP locus can be inferred based on the N22 homozygous, ZS97 homozygous, or heterozygous genotype of FZP-1bp-KASP, and early generation material screening can be carried out accordingly. 5) For breeding materials that have entered high generations or are intended to be fixed, agronomic traits such as panicle degeneration rate, number of spikelets per panicle, seed setting rate, panicle length and yield can be investigated. If necessary, direct sequencing of the FZP gene or verification with a closer neighbor marker can be combined to exclude the influence of a very small number of recombination events on linkage inference.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set for a single-base insertion variant KASP molecular marker tightly linked to the rice FZP gene, characterized in that, The single-base insertion variant KASP molecule is labeled as FZP-1bp-KASP; The mutation site of FZP-1bp-KASP is located at physical position 28,297,569 bp on the 7th chromosome of rice, and is a single base A insertion / non-insertion mutation; The primer set includes a first allele-specific core forward primer F1, a second allele-specific core forward primer F2, and a universal reverse primer R, with sequences shown in SEQ ID NO: 3~5.

2. The primer set for the single-base insertion variant KASP molecular marker tightly linked to the rice FZP gene as described in claim 1, characterized in that, The 5′ ends of F1 and F2 are respectively connected to KASP universal connector sequences that can be recognized by different fluorescence detection systems.

3. The primer set for a single-base insertion variant KASP molecular marker tightly linked to the rice FZP gene as described in claim 2, characterized in that, The sequences of the primers F1 and F2 with adapters are shown in SEQ ID NO: 1~2.

4. A kit for detecting the FZP-1bp-KASP site in rice, characterized in that, Includes the primer set as described in any one of claims 1 to 3.

5. A method for detecting the FZP-1bp-KASP locus genotype in rice using the FZP-1bp-KASP molecular marker, the primer set according to any one of claims 1 to 3, or the kit according to claim 4, characterized in that, Includes the following steps: (1) Extract genomic DNA from the rice to be tested; (2) Using the genomic DNA as a template, allele-specific PCR amplification is performed using primers designed based on the FZP-1bp-KASP molecular marker, or the primer set described in any one of claims 1 to 3, or the kit described in claim 4; (3) Two types of fluorescence signals were collected on a real-time fluorescence PCR instrument; (4) Determine whether the material to be tested is A-insertion homozygous, non-insertion homozygous, or heterozygous based on the relative intensity and clustering position of the two types of fluorescence signals.

6. The detection method as described in claim 5, characterized in that, The first fluorescent signal corresponds to the F1 amplification product and indicates the insertion of the A allele; the second fluorescent signal corresponds to the F2 amplification product and indicates the non-inserted allele; samples with significant fluorescence signals in both categories are identified as heterozygous genotypes.

7. The use of an FZP-1bp-KASP molecular marker, or the primer set according to any one of claims 1 to 3, or the kit according to claim 4, in detecting single-base insertion variants closely linked to the rice FZP gene.

8. The application as described in claim 7, characterized in that, By using single-base insertion variation results, the FZP genotype or parental allele origin of the segregating population can be inferred and used for molecular breeding.

9. The application as described in claim 7, characterized in that, The results of single-base insertion variation can be used for FZP gene localization or fine mapping, recombinant screening, germplasm resource typing, and molecular marker-assisted selection of FZP-related spike type, number of grains per spike, or spike degeneration traits.

Images