Development and application of excellent genotypes and kasp markers of a functional gene applicable to early growth and rapid development of rice
By developing SNP markers and KASP primers for identifying early-growing and fast-emerging traits in rice, the problem of weed competition inhibition in direct-seeding rice technology was solved, and the growth rate of rice seedlings was improved and an environmentally friendly breeding method was achieved, reducing costs and pollution.
Patent Information
- Application Number
- CN202310428618.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-20
- Publication Date
- 2025-10-21
- Estimated Expiration
- 2043-04-20
AI Technical Summary
One of the main limiting factors for the widespread application of direct seeding technology in my country is that weed competition inhibits the growth of rice seedlings. Existing breeding technology cannot effectively control the growth rate of rice seedlings, resulting in increased costs and environmental pollution.
SNP markers and KASP primers were developed for identifying the early-maturing and fast-growing traits of rice. Through PCR detection and fluorescence analysis, genotypes were identified to select early-maturing and fast-growing rice varieties. The superior genotype Hap2LOC_Os01g68500 was used to increase seedling height and reduce weed competition.
It has achieved precision breeding, increased the growth rate of rice seedlings, reduced the use of chemical pesticides, lowered costs and reduced environmental pollution.
Smart Images
Figure CN116656856B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to the development and application of an excellent genotype and KASP marker of a functional gene applicable to early growth and rapid development of rice. Background Art
[0002] In recent years, rice production has faced labor shortages, and the time-saving and labor-saving direct seeding method has become increasingly popular. However, weeds are a significant limiting factor in the widespread adoption of direct seeding in my country. Direct seeding requires the subsequent use of large amounts of chemical pesticides to suppress weed growth. The use of pesticides not only increases the cost of rice cultivation but also seriously pollutes the environment on which we depend.
[0003] After direct seeding of rice seeds, rice seedlings and weeds engage in a competitive inhibition relationship for nutrients, light, and other factors. After sowing, if weeds grow quickly, rice seedlings will grow more slowly, or even die from nutrient and light deficiency. Conversely, if rice seeds germinate and grow faster than weeds, they can absorb nutrients and capture light first, thus suppressing weed growth. Rice seedling growth rate is a complex trait controlled by multiple genes, making it difficult to achieve effective and precise breeding using traditional breeding techniques. Only by understanding the molecular genetic mechanisms at the genetic level and implementing molecular breeding can breakthroughs in molecular breeding for rapid rice seedling growth be achieved. Summary of the Invention
[0004] A first object of the present invention is to provide a SNP marker for identifying a genotype having an early-growing and fast-emerging trait in rice. The SNP marker is located at base 727 of the nucleotide sequence shown in SEQ ID NO. 1, and the base is G or T. If the base at the SNP site is G, the genotype is short, and if the base at the SNP site is T, the genotype is tall.
[0005] The second object of the present invention is to provide KASP primers for amplifying the SNP markers. The KASP primer sequences are shown in SEQ ID NOs. 5-7, specifically:
[0006] F1: GAAGGTGACCAAGTTCATGCT CGTCCCCTTCATTGCGACGC;
[0007] F2: GAAGGTCGGAGTCAACGGATT CGTCCCCTTCATTGCGACGA;
[0008] R:GTGTGTGAGGACGGATCGGT.
[0009] The underlined portion of the F1 primer is the FAM fluorescent specific tag sequence, and the underlined portion of the F2 primer is the HEX fluorescent specific tag sequence.
[0010] The third object of the present invention is to provide a kit containing the primers.
[0011] The fourth object of the present invention is to provide the use of the SNP marker, primer or kit in identifying genotypes with early-growing and fast-developing traits in rice.
[0012] The fifth object of the present invention is to provide the use of the SNP marker, primer or kit in rice breeding.
[0013] Preferably, the purpose of the breeding is to breed rice with early-growing and fast-developing traits.
[0014] A sixth object of the present invention is to provide a method for identifying a genotype having the early-growing and fast-developing trait of rice, comprising the following steps:
[0015] (1) Extracting DNA from the rice genome to be tested;
[0016] (2) Using rice genomic DNA as a template, primers
[0017] F1: GAAGGTGACCAAGTTCATGCT CGTCCCCTTCATTGCGACGC;
[0018] F2: GAAGGTCGGAGTCAACGGATT CGTCCCCTTCATTGCGACGA;
[0019] R: GTGTGTGAGGACGGATCGGT for PCR detection;
[0020] (3) Use a fluorescence detector to analyze the genotype of the PCR amplification product.
[0021] Preferably, the genotype of the PCR amplification product is analyzed using a fluorescence detector, specifically: if only FAM fluorescence is detected, the SNP site is a G / G homozygous genotype, which is a genotype for short seedlings; if only HEX is detected, the SNP site is a T / T homozygous genotype, which is a genotype for tall seedlings; if both FAM and HEX fluorescence are detected, the SNP site is a G / T heterozygous genotype.
[0022] The seventh object of the present invention is to provide the application of the above method in breeding rice varieties with early-growing and fast-developing traits.
[0023] The present invention provides a SNP marker for identifying a genotype having the early-growing and fast-developing trait of rice. The marker can predict rice varieties having the early-growing and fast-developing trait of rice, thereby providing a basis for breeding rice varieties having the early-growing and fast-developing trait of rice. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 : KASP test results of different genotype materials. DETAILED DESCRIPTION
[0025] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.
[0026] Example 1
[0027] Through genome-wide association analysis of 391 rice accessions, we found a gene that controls plant height at the seedling stage. This gene has two different genotypes in natural populations: Hap1 LOC_Os01g68500 and Hap2 LOC_Os01g68500 The sequences of the two genotypes are shown in SEQ ID NO.1 and SEQ ID NO.2. The comparison results of the two genotypes are as follows: the 727th base of SEQ ID NO.1 is G, and the 727th base of SEQ ID NO.2 is T, resulting in the amino acid sequences of the encoded proteins of LOC_Os01g68500 as shown in SEQ ID NO.3 and SEQ ID NO.4. The difference between them is that the 169th amino acid of SEQ ID NO.3 is Ala (A), and the 169th amino acid of SEQ ID NO.4 is Ser (S). Among them, Hap2 LOC_Os01g68500 It is an excellent genotype for controlling rice seedling height. It can increase seedling height, make rice grow early and quickly, avoid weed competition, and is suitable for molecular breeding of direct-seeded rice.
[0028] SEQ ID NO.1(Hap1 LOC_Os01g68500 ):
[0029]
[0030] SEQ ID NO.2(Hap2 LOC_Os01g68500 ):
[0031]
[0032] SEQ ID NO.3(Hap1 LOC_Os01g68500 ):
[0033] MASSSSWWVVMLLMVVAAAGWGGVAAATAAEAAHEVLRAHGLPRGLLPAGIADFRHDEGSGRFEAALGESCTAQFEVGLRYNATVAGVISYGRIASLSGVSAQDLFLWFPVRGIRVDVPSSGVIYFDVGVVFKHFPLAVFEAPPPCTPDPLLLLTQVCEDGSVAGGGAASQ*.
[0034] SEQ ID NO.4(Hap2 LOC_Os01g68500 ):
[0035] MASSSSWWVVMLLMVVAAAGWGGVAAATAAEAAHEVLRAHGLPRGLLPAGIADFRHDEGSGRFEAALGESCTAQFEVGLRYNATVAGVISYGRIASLSGVSAQDLFLWFPVRGIRVDVPSSGVIYFDVGVVFKHFPLAVFEAPPPCTPDPLLLLTQVCEDGSVAGGGASSQ*.
[0036] We conducted direct seeding of 391 rice varieties under three direct seeding modes (see Table 1 below). We detected their genotypes and measured their seedling height 14 days after sowing. The following results were obtained:
[0037] Method 1 (GST): seeds were pre-germinated and sown in plastic trays (35.0 cm × 23.0 cm × 6.0 cm);
[0038] Method 2 (GSF): seeds are pre-germinated and sown in rice fields;
[0039] Method 3 (DST): Seeds are sown directly in plastic trays without pre-germination.
[0040] Table 1
[0041]
[0042]
[0043] ** indicates P < 0.01, and the numbers in brackets indicate the number of plants measured.
[0044] As can be seen from the table above, Hap2 LOC_Os01g68500 It is an excellent genotype for controlling rice seedling height.
[0045] Example 2
[0046] Based on the sequence information of LOC_Os01g68500 and the base sequence of its reverse complementary chain, KASP primers were synthesized. The primer sequences are shown in SEQ ID NOs. 5-7, specifically:
[0047] F1: GAAGGTGACCAAGTTCATGCT CGTCCCCTTCATTGCGACGC;
[0048] F2: GAAGGTCGGAGTCAACGGATT CGTCCCCTTCATTGCGACGA;
[0049] R:GTGTGTGAGGACGGATCGGT;
[0050] The underlined portion of the F1 primer is the FAM fluorescent specific tag sequence, and the underlined portion of the F2 primer is the HEX fluorescent specific tag sequence.
[0051] When synthesizing the above primers, the ULTRPAGE purification method was selected. The operation steps are as follows:
[0052] Dissolve the primer powder to 36 μM concentrations of F1 and F2 and 90 μM concentration of R, then mix the three primers at a volume ratio of 1:1:1 to form a primer mix. Primer screening: Run at least 2 wells for each primer, each parent, and F1.
[0053] The PCR amplification system is as follows:
[0054]
[0055] The PCR amplification procedure is as follows:
[0056]
[0057]
[0058] After amplification, use fluorescent quantitative PCR to read the band: the reading procedure is as follows:
[0059] 25℃ for 5s+Plate Read. After the band reading is completed, select the fluorescence type FAM, HEX, ROX, and select Allelic Discrimination for analysis. 10 accessions of genotype 1 (Allele 1) (accession numbers are 66, 471, 517, 518, 549, 576, 620, 757, 941, 1332) and 13 accessions of genotype 2 (Allele 2) (accession numbers are 463, 477, 521, 525, 542, 589, 616, 1268, 1279, 1341, 1342, 1365, 1370) were selected for verification; this showed that this KASP primer set had a significant clustering effect in rice ( Figure 1 ), which can be applied to production practice.
[0060] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. Use of a SNP marker for identifying a rice seedling height genotype, a SNP-marked KASP primer, or a kit containing a SNP-marked KASP primer in identifying a rice seedling height genotype, characterized in that: The SNP marker is located at base 727 of the nucleotide sequence shown in SEQ ID NO. 1, and the base is G or T; the KASP primer sequence is: F1: GAAGGTGACCAAGTTCATGCT CGTCCCCTTCATTGCGACGC; F2: GAAGGTCGGAGTCAACGGATT CGTCCCCTTCATTGCGACGA; R:GTGTGTGAGGACGGATCGGT; If the base at the SNP site is G, the genotype is short seedlings, and if the base at the SNP site is T, the genotype is tall seedlings.
2. Use of a SNP marker for identifying a rice seedling height genotype, a SNP-marked KASP primer, or a kit containing a SNP-marked KASP primer in rice breeding for increasing seedling height, characterized in that: The SNP marker is located at base 727 of the nucleotide sequence shown in SEQ ID NO. 1, and the base is G or T; the KASP primer sequence is: F1: GAAGGTGACCAAGTTCATGCT CGTCCCCTTCATTGCGACGC; F2: GAAGGTCGGAGTCAACGGATT CGTCCCCTTCATTGCGACGA; R:GTGTGTGAGGACGGATCGGT; If the base at the SNP site is G, the genotype is short seedlings, and if the base at the SNP site is T, the genotype is tall seedlings.
3. A method for identifying high genotypes of rice seedlings, characterized in that: The following steps are involved: (1) Extracting DNA from the rice genome to be tested; (2) Using rice genomic DNA as a template, PCR detection was performed using KASP primers. The KASP primer sequences are: F1: GAAGGTGACCAAGTTCATGCT CGTCCCCTTCATTGCGACGC; F2: GAAGGTCGGAGTCAACGGATT CGTCCCCTTCATTGCGACGA; R:GTGTGTGAGGACGGATCGGT; (3) Use a fluorescence detector to analyze the genotype of the PCR amplification product; if only FAM fluorescence is detected, the SNP site is a G / G homozygous genotype, which is a genotype for short seedlings; if only HEX is detected, the SNP site is a T / T homozygous genotype, which is a genotype for tall seedlings; if both FAM and HEX fluorescence are detected, the SNP site is a G / T heterozygous genotype.
4. Use of the method according to claim 3 in breeding rice varieties with high seedling growth.