SNP markers, kasp primer combinations, kits and applications related to rice yield and lodging resistance traits

Abstract

This invention discloses a SNP marker, KASP primer combination, kit, and application related to rice yield and lodging resistance traits. The SNP marker is located at position 19,576,953 bp on chromosome 11 of the rice reference genome MSU7.0, with an allelic variation of G / A, enabling simultaneous and precise screening for high yield and lodging resistance traits in rice. The KASP primer combination developed based on this SNP marker exhibits stable biological associations with the traits, demonstrating high reliability in different genetic backgrounds. It solves the long-standing problem of genetic trade-offs between traits, achieving synergistic selection of superior traits and overcoming breeding bottlenecks. Furthermore, the genotyping method based on these KASP primers features high throughput, speed, low cost, and high automation, meeting the practical needs of modern breeding for early and rapid screening of large-scale populations, and providing a practical molecular tool for high-yield and stress-resistant rice breeding.

Description

Technical Field

[0001] This invention relates to molecular marker-assisted breeding, and more particularly to an SNP marker, KASP primer combination, kit, and application related to rice yield and lodging resistance traits. Background Technology

[0002] Rice is one of the world's most important food crops, and its yield is directly related to food security and social stability. Breeding practices over the past thirty years have shown that increasing the number of grains per panicle is a key way to overcome the bottleneck in rice yield. However, increasing the number of grains per panicle often leads to an increase in panicle weight. If stem strength is not improved simultaneously, lodging is highly likely, causing severe yield losses of 10%–30% annually and quality deterioration. Therefore, developing synergistically improved varieties that combine high grain number per panicle with high lodging resistance has become an important strategic direction in current rice breeding.

[0003] Traditional breeding relies on phenotypic selection, but grain number per ear and stem strength are complex quantitative traits controlled by multiple genes, making them susceptible to environmental interference. Phenotypic selection is not only inefficient, but also often requires more than ten years to develop a new variety, a lengthy process. In recent years, the rise of molecular marker-assisted selection technology has provided an efficient means to simultaneously improve multiple complex traits. Existing research has successfully identified several key genes regulating grain number per ear or stem strength, such as those controlling grain number per ear. Gn1a and FZP and regulating stem strength STRONG1 and OsTB1 However, existing technologies have significant limitations: First, most reported gene or molecular markers only target a single trait, such as ear number or stem strength, making them difficult to use directly for synergistic improvement; second, even if genes that simultaneously affect multiple traits exist, it remains a gap to determine whether there are specific superior allelic variations in their natural variations that can synergistically optimize multiple target traits, and how to accurately identify and utilize such variations. Summary of the Invention

[0004] Objectives of the invention: The first objective is to provide an SNP marker that is simultaneously associated with rice yield and lodging resistance; the second objective is to provide a KASP primer combination and kit for detecting the SNP marker; and the third objective is to provide the application of the above products in marker-assisted selection breeding of rice.

[0005] Technical solution: The SNP markers related to rice yield and lodging resistance described in this invention are located at position 19576953 bp on chromosome 11 of the rice reference genome MSU7.0 (MSU Rice Genome Annotation Project Release 7), and their allelic variation bases are G / A.

[0006] The KASP primer combination described in this invention is used to detect the aforementioned SNP markers related to rice yield and lodging resistance.

[0007] Preferably, the primer GC content in the KASP primer combination is between 51% and 56%, the melting temperature is 60-64℃, and the length is 18-41 bases.

[0008] Preferably, the KASP primer combination consists of an allele 1-specific forward primer as shown in SEQ ID NO: 1, an allele 2-specific forward primer as shown in SEQ ID NO: 2, and a universal reverse primer as shown in SEQ ID NO: 3.

[0009] Preferably, the molar ratio of the allele 1-specific forward primer, the allele 2-specific forward primer, and the universal reverse primer is 1:1:(2-3).

[0010] The kit described in this invention contains the aforementioned KASP primer combination.

[0011] The application of the SNP markers, KASP primer combinations, or kits described in this invention in marker-assisted selection breeding of rice.

[0012] Preferably, the application is in molecular marker-assisted selection breeding of high-yield and highly lodging-resistant rice.

[0013] Preferably, the application steps include: (1) Extract genomic DNA from the rice samples to be tested; (2) Using the genomic DNA obtained in step 1 as a template, competitive allele-specific PCR amplification is performed using the KASP primer combination described in any one of claims 2 to 5, or the kit described in claim 6; (3) After the amplification is completed, the endpoint fluorescence signal is read, the genotype is identified and screened for breeding.

[0014] Preferably, the reaction conditions for competitive allele-specific PCR amplification in step 2 are: pre-denaturation at 93-95℃ for 15 minutes, annealing at 61-55℃ for 60 seconds, decreasing by 0.6℃ per cycle, for 10 cycles; denaturation at 93-95℃ for 20 seconds, annealing at 50-60℃ for 60 seconds, for 35-45 cycles.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention provides an SNP marker that can simultaneously and accurately screen for high yield and lodging resistance traits in rice. The KASP primer combination developed based on this SNP marker has a stable biological association with the trait, and has strong reliability in different genetic backgrounds. It solves the long-standing problem of genetic trade-offs between traits, realizes the synergistic selection of superior traits, and breaks through the breeding bottleneck; 2. The genotyping method based on this KASP primer has the characteristics of high throughput, fast speed, low cost, and high degree of automation. It can meet the actual needs of modern breeding for early and rapid screening of large-scale populations, and provide a practical molecular tool for high-yield and stress-resistant rice breeding. Attached Figure Description

[0016] Figure 1 for GNP11 Manhattan plot of association analysis between gene regions and rice panicle grain number and stem strength; Figure 2 This is a comparative analysis diagram of ear grain number and stem strength among different allele materials classified based on key SNP sites. Figure 3 For representative materials carrying different alleles GNP11 Gene relative expression level analysis graph; Figure 4 Scatter plot of fluorescence signals for genotyping of the test population using the GNP11-KASP primer combination; Figure 5 Phenotypic comparison analysis of different genotypes identified in the test population using the GNP11-KASP primer combination. Detailed Implementation

[0017] The technical solution of the present invention will be further described below.

[0018] Example 1: Screening of SNP markers co-associated with rice panicle grain number and lodging resistance trait The rice reference genome MSU7.0 from 300 core rice germplasm resources (provided by the College of Agriculture, Yangzhou University) was analyzed at chromosome 11, positions 19572400-19578539 bp. GNP11Gene region sequence analysis identified 42 single nucleotide polymorphisms (SNPs) and 3 insertion / deletion (InDel) variants. Based on field phenotypic data, candidate gene association analyses were conducted for grain number per panicle and stem strength. Grain number per panicle was determined by randomly selecting three panicles from each material at maturity, averaging the total number of grains per panicle. Stem strength was measured at maturity using a stem strength meter on the second internode at the base of the main stem, averaging the strength of three main stems from each material. Population structure analysis (PCA) was performed using TASSEL v5.0 software, and association analysis was conducted using a general linear model. P <10 -5 As a threshold for significant association.

[0019] The results of the candidate gene association analysis for grain number per ear are as follows: Figure 1 As shown in Figure A, the results of the candidate gene association analysis for stem strength are as follows: Figure 1 As shown in B, the results indicate that... GNP11 Two SNP sites (SNP11:19576953 and SNP11:19576997) were identified in the gene and its promoter region that were significantly associated with both grain number per ear and lodging resistance.

[0020] According to the results described in Example 4 of the specific implementation of the prior patent (CN202310026378.8), it can be seen that when the base at SNP11:19576953 mutates from G to A, GNP11 Gene promoter activity was significantly increased. Therefore, this site was chosen as the optimal target for developing molecular markers.

[0021] Based on the SNP11:19576953 site, the tested germplasm was divided into two categories: materials carrying allele 1, with the sequence "G / G" at this site, totaling 248 accessions; and materials carrying allele 2, with the sequence "A / A" at this site, totaling 52 accessions. Further phenotypic statistical analysis was then performed.

[0022] The statistical results of the number of grains per ear are as follows: Figure 2 As shown in Figure A, the statistical results of stem strength are as follows: Figure 2 As shown in Figure B, the number of grains per panicle and stem strength of the material carrying allele 2 were significantly higher than those of the material carrying allele 1, indicating that this SNP variation is closely related to the synergistic regulation of grain number per panicle and stem strength in rice.

[0023] Example 2: Detection of GNP11 expression levels in different alleles From the 300 rice core germplasm resources in Example 1, 20 homozygous germplasm materials carrying allele 1 and 20 carrying allele 2 were selected respectively. Total RNA was extracted from the stems of each material using a plant total RNA extraction kit (Vazyme, R711-01); then, 1 µg of the total RNA obtained from each germplasm material was used to synthesize first-strand cDNA using a reverse transcription kit (CWBIO, CW2020M) as a template for subsequent quantitative PCR.

[0024] Use primers as shown below: GNP11 -F upstream primer: 5'-agctgaagttccagatgattga-3'; GNP11 -R downstream primer: 5'-actttctcaaatgactcgcaac-3'; Actin -F upstream primer: 5'-gatgacccagatcatgtttg-3'; Actin -R downstream primer: 5'-gggcgatgtaggaaagc-3'; Quantitative PCR was performed using a SYBR Green I kit (CWBIO, CW3360M), with three technical replicates per sample. Fluorescence values ​​were collected at the extension phase of each cycle. Actin As an intrinsic parameter, the Ct value is calculated via 2 -ΔΔCt Method calculation GNP11 The relative expression level of mRNA.

[0025] The results are as follows Figure 3 As shown, allele 2 material GNP11 Gene expression levels were significantly higher than in allele 1 material, indicating that genotypic variation at the key SNP locus (Chr11: 19576953) is related to... GNP11 The gene expression levels were significantly correlated, providing a direct molecular biological basis for developing this SNP into a functional molecular marker.

[0026] Example 3: Genotyping and trait association analysis of rice varieties using GNP11-KASP molecular markers 1. Preparation of GNP11-KASP primer combination Based on the allele at SNP11:19576953, we designed a forward primer specific to allele 1 (as shown in SEQ ID NO: 1): 5'-gaaggtgaccaagttcatgcttatcggctattggctaggcg-3', a forward primer specific to allele 2 (as shown in SEQ ID NO: 2): 5'-gaaggtcggagtcaacggatttatcggctattggctaggca-3', and a universal reverse primer (as shown in SEQ ID NO: 3): 5'-tgccttaaacccctccacg-3'.

[0027] The above primers and probes were synthesized by Qingke Biotechnology Co., Ltd. The GNP11-KASP primer combination was prepared by mixing allele 1-specific forward primers, allele 2-specific forward primers, and universal reverse primers at a volume ratio of 1:1:2.5, all at a concentration of 100 μM.

[0028] 2. Verification of the accuracy and stability of the GNP11-KASP primer combination From the 300 core germplasm resources in Example 1, 60 germplasm materials were randomly selected as verification samples, including 42 materials with allele 1 and 18 materials with allele 2.

[0029] Genomic DNA was extracted from various materials using the cetyltrimethylammonium bromide (CTAB) method as templates. KASP was detected using the KASP-TF Master Mix kit (LGC, KBS-1050-103) based on KASP primer combinations.

[0030] The reaction conditions for KASP detection were as follows: pre-denaturation at 94℃ for 15 min; followed by 10 cycles of landing PCR: denaturation at 94℃ for 20 s, annealing / extension at 61-55℃ for 60 s (the annealing / extension temperature was reduced by 0.6℃ per cycle); then 40 cycles of amplification: denaturation at 94℃ for 20 s, annealing / extension at 55℃ for 60 s.

[0031] After the reaction, the endpoint fluorescence signal was read using a fluorescence detection instrument. Clustering and typing were performed based on the signal intensity of the FAM and HEX channels to determine the genotype as G / G homozygous, A / A homozygous, or G / A heterozygous. The typing results were compared with the genotype data obtained from previous sequencing. The results showed that the KASP typing results and sequencing results had a 100% consistency rate, indicating that the developed GNP11-KASP primer combination has high accuracy and stability and can be used for subsequent genotype identification of unknown materials.

[0032] 3. GNP11-KASP primer combination used for genotyping and trait association analysis of rice varieties. To further verify the predictive effect of this molecular marker in practical breeding applications, an independent test population consisting of 48 japonica and indica rice varieties (as shown in Table 1) was selected to conduct genotype identification and phenotypic association verification.

[0033] Table 1. Information on Independent Test Groups

[0034] The aforementioned 48 materials were field-grown at the Yangzhou Experimental Station in Jiangsu Province in 2025. Leaves of various rice varieties were collected, and genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method as a template. KASP detection was performed using a kit based on KASP primer combinations. After the reaction, the endpoint fluorescence signal was read using a fluorescence detection instrument, and clustering was performed based on the signal intensity of the FAM and HEX channels to identify the genotype as G / G homozygous, A / A homozygous, or G / A heterozygous.

[0035] The results of the identification are as follows Figure 4 As shown, different genotype samples can be clearly distinguished on the endpoint fluorescence signal scatter plot. Among the 48 test materials, the distribution of A / A type and G / G type is 13 and 35 samples, respectively.

[0036] Subsequently, three representative plants were randomly selected from each material, and the stem strength of the second internode at the base of the main stem was measured using a stem strength tester. The number of grains per ear was also investigated. The average value of the three representative plants from each material was taken as the phenotypic value of that material for statistical analysis.

[0037] The statistical analysis results of the number of grains per ear are as follows: Figure 5 As shown in Figure A, the statistical analysis results of stem strength are as follows: Figure 5 As shown in Figure B, the average number of grains per panicle and stem strength of the A / A genotype group were significantly higher than those of the G / G genotype group. These results indicate that this molecular marker can stably and effectively predict the potential of unknown rice materials in terms of high yield and lodging resistance, and has practical value for direct application in marker-assisted selection breeding.

Claims

1. A SNP marker associated with rice yield and lodging resistance, characterized in that, The SNP marker is located at position 19,576,953 bp on chromosome 11 of the rice reference genome MSU7.0, and its allelic variation is G / A.

2. A KASP primer combination, characterized in that, The primer combination detects the SNP markers associated with rice yield and lodging resistance as described in claim 1.

3. The KASP primer combination according to claim 2, characterized in that, The KASP primer combination has a primer GC content of 51-56%, a melting temperature of 60-64℃, and a length of 18-41 bases.

4. The KASP primer combination according to claim 3, characterized in that, The KASP primer combination consists of an allele 1-specific forward primer as shown in SEQ ID NO: 1, an allele 2-specific forward primer as shown in SEQ ID NO: 2, and a universal reverse primer as shown in SEQ ID NO:

3.

5. The KASP primer combination according to claim 4, characterized in that, The molar ratio of the allele 1-specific forward primer, the allele 2-specific forward primer, and the universal reverse primer is 1:1:(2-3).

6. A reagent kit, characterized in that, The kit contains the KASP primer combination as described in any one of claims 2 to 5.

7. The application of the SNP marker of claim 1, or the KASP primer combination of any one of claims 2 to 5, or the kit of claim 6 in molecular marker-assisted selection breeding of rice.

8. The application according to claim 7, characterized in that, The application is in molecular marker-assisted selection breeding of high-yield and highly lodging-resistant rice.

9. The application according to claim 7, characterized in that, The steps of the application include: (1) Extract genomic DNA from the rice samples to be tested; (2) Using the genomic DNA obtained in step 1 as a template, competitive allele-specific PCR amplification is performed using the KASP primer combination described in any one of claims 2 to 5, or the kit described in claim 6; (3) After the amplification is completed, the endpoint fluorescence signal is read, the genotype is determined and screened for breeding.

10. The application according to claim 9, characterized in that, The reaction conditions for competitive allele-specific PCR amplification in step 2 are as follows: pre-denaturation at 93-95℃ for 15 minutes, annealing at 61-55℃ for 60 seconds, decreasing by 0.6℃ per cycle, for 10 cycles; denaturation at 93-95℃ for 20 seconds, annealing at 50-60℃ for 60 seconds, for 35-45 cycles.

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