CN109811077B - KASP marker closely linked with wheat dwarf gene and application thereof - Google Patents
KASP marker closely linked with wheat dwarf gene and application thereof Download PDFInfo
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Abstract
The invention discloses a KASP marker closely linked with a wheat dwarf gene and application thereof. The KASP primer pair closely linked with the wheat dwarf gene provided by the invention comprises ph3 and ph 4; wherein, ph3 is composed of a primer 1 or a derivative thereof shown in a sequence 1 in a sequence table, a primer 2 or a derivative thereof shown in a sequence 2 in the sequence table and a primer 3 shown in a sequence 3 in the sequence table; the ph4 is composed of a primer 4 or a derivative thereof shown in a sequence 4 in a sequence table, a primer 5 or a derivative thereof shown in a sequence 5 in the sequence table and a primer 6 shown in a sequence 6 in the sequence table. And the ph3 and the ph4 are molecular markers closely linked with the dwarf gene, and can be used for molecular marker-assisted selection and screening of new wheat dwarf materials. The KASP marker tightly linked with the dwarf gene can realize high-throughput and rapid auxiliary selection in the seedling stage and accelerate the dwarf breeding process.
Description
Technical Field
The invention relates to the technical field of molecular biology and crop breeding, in particular to a KASP marker closely linked with a wheat dwarf gene and application thereof.
Background
Wheat is one of three main grain crops in China, and the wheat yield is guaranteed to be closely related to the grain safety in China. The plant height directly affects the lodging resistance of wheat, and the cultivation of short-stalk and semi-short-stalk varieties is particularly important for stable and high yield of wheat. At present, nearly 30 wheat dwarf genes have been discovered and named. The green revolution dwarf genes Rht-B1B and Rht-D1B are respectively positioned at 4BS and 4DS, and because DELLA protein coding is terminated early, GA signal conduction pathways are influenced, and plants are dwarfed. As the wheat genome is huge and the location is complex, most dwarf genes are still in the initial location stage except that Rht-B1B, Rht-D1B and homologous genes thereof are cloned. Research shows that Rht4, Rht5, Rht8, Rht9, Rht12, Rht13, Rht22, Rht23 and Rht24 are respectively positioned at 2BL, 3BS, 2DS, 7BS, 5AL, 7BL, 7AS, 5DL and 6 AL; rht7, Rht21, and Rht-NM9 are located at 2 AS; rht14, Rht16, Rht18, Rht25 are located at6 AS. Among them, Rht8, Rht12, Rht24 and Rht25 have been relatively deeply studied. Studies have shown that hyperdwarfing of Rht8 strain is not due to loss of gibberellin synthesis or signaling pathways, but may be due to reduced sensitivity to brassinolide. Rht8 was located between the 1.29cM distance of the 2DS marker DG279 and DG371 by constructing a genetic map. The Rht12 gene obtained by gamma ray mutagenesis is tightly linked with the marker Xwmc410, the Rht12 gene is further finely located and located to the end of a 5AL chromosome at about 11.21Mb interval, and the gene is tightly linked with the SSR marker Xw5ac207, and the analysis of a transcriptome expression profile shows that the Rht12 reduces the plant height by activating the expression of a TaGA2ox8 gene. The widely used gibberellin sensitive dwarf gene Rht24 has stalk reducing effect of about 6.0-7.9cM, can obviously improve thousand grain weight, and is positioned in a section of 1.85cM between 6AL markers TaAP2 and TaFAR.
Although a large number of wheat dwarf genes are found, only a few wheat dwarf genes are utilized in breeding and production in China. Mainly comprises Rht-B1B, Rht-D1B from agriculture and forestry No. 10, Rht8 or Rht9 of red wheat and Rht 24. The molecular marker identification of 220 parts of wheat resources in China shows that the distribution frequency of Rht-D1B is 45.5%, the distribution frequency of Rht-B1B is 24.5%, and the distribution frequency of Rht8 is 46.8%. Recent research shows that 76% of 242 wheat varieties in China contain Rht24 genes.
The utilization of relatively single dwarf gene leads to deficient genetic variation of wheat breeding parents and limited yield-increasing potential, so that the excavation of excellent new dwarf gene and the development of closely linked molecular markers are urgently needed, and a foundation is laid for the cultivation of new varieties of dwarf and semi-dwarf wheat.
Disclosure of Invention
In order to make up for the defects in the field, the invention provides a KASP marker closely linked with a wheat dwarf gene and application thereof.
The technical scheme of the invention is as follows:
the invention provides KASP primer pairs closely linked with wheat dwarf genes, which comprise ph3 and ph 4; wherein,
the ph3 is composed of a primer 1 shown in a sequence 1 in a sequence table or a derivative thereof, a primer 2 shown in a sequence 2 in the sequence table or a derivative thereof and a primer 3 shown in a sequence 3 in the sequence table;
the ph4 is composed of a primer 4 or a derivative thereof shown in a sequence 4 in a sequence table, a primer 5 or a derivative thereof shown in a sequence 5 in the sequence table and a primer 6 shown in a sequence 6 in the sequence table.
The derivative of the primer 1 shown in the sequence 1 in the sequence table is a fluorescent sequence connected with the 5' end of a single-stranded DNA molecule shown in the sequence 1;
the derivative of the primer 2 shown in the sequence 2 in the sequence table is that the 5' end of a single-stranded DNA molecule shown in the sequence 2 is connected with another fluorescent sequence.
The derivative of the primer 4 shown in the sequence 4 in the sequence table is a fluorescent sequence connected with the 5' end of a single-stranded DNA molecule shown in the sequence 4;
the derivative of the primer 5 shown in the sequence 5 in the sequence table is that the 5' end of the single-stranded DNA molecule shown in the sequence 5 is connected with another fluorescent sequence.
The derivative of the primer 1 shown in the sequence 1 in the sequence table is a fluorescent sequence FAM connected with the 5' end of a single-stranded DNA molecule shown in the sequence 1;
the derivative of the primer 2 shown in the sequence 2 in the sequence table is a fluorescent sequence HEX connected with the 5' end of the single-stranded DNA molecule shown in the sequence 2.
The derivative of the primer 1 shown in the sequence 4 in the sequence table is a fluorescent sequence FAM connected with the 5' end of the single-stranded DNA molecule shown in the sequence 1;
the derivative of the primer 5 shown in the sequence 5 in the sequence table is a fluorescent sequence HEX connected with the 5' end of the single-stranded DNA molecule shown in the sequence 5.
The nucleotide sequences of the two allele forward primers with different terminal bases of ph3 are respectively as follows: 5 'GAAGGTGACCAAGTTCATGCTgaACaagAtggatctccgctaA 3' (SEQ ID NO: 7 in the sequence Listing), 5 'GAAGGTCGGAGTCAACGGATTgaACaagAtggatctccgctaG 3' (SEQ ID NO: 8 in the sequence Listing),
the nucleotide sequence of the ph3 universal reverse primer is as follows: 5 'actgcaggagtatctaGcactG 3' (SEQ ID NO: 3 in the sequence Listing);
the nucleotide sequences of the two allele forward primers with different terminal bases of ph4 are respectively as follows: 5 'GAAGGTGACCAAGTTCATGCTgtttcgcatccggcttgtT 3' (SEQ ID NO: 9 in the sequence Listing), 5 'GAAGGTCGGAGTCAACGGATTgtttcgcatccggcttgtC 3' (SEQ ID NO: 10 in the sequence Listing),
the nucleotide sequence of the ph4 universal reverse primer is as follows: 5 'ccgttccttgtccttctgga 3' (SEQ ID NO: 6 in the sequence Listing).
The invention also provides a PCR reagent containing the KASP primer pair.
The invention also provides a kit containing the KASP primer pair or the PCR reagent.
The application of the KASP primer pair or the PCR reagent or the kit in at least one of the following 1) to 5) also belongs to the protection scope of the invention:
1) identifying or assisting in identifying the plant height and/or stalk strength of the wheat;
2) preparing a product for identifying or assisting in identifying the plant height and/or stalk strength of the wheat;
3) detecting or screening or breeding dwarf or semi-dwarf wheat;
4) preparing, detecting, screening or breeding dwarf or semi-dwarf wheat products;
5) and identifying or assisting in identifying the wheat dwarf genotype.
The invention also provides a method for identifying or assisting in identifying the plant height and/or stalk strength of wheat, which comprises the following steps: carrying out KASP reaction on the wheat to be detected by using the primer pair, and detecting a reaction product, wherein the height of the wheat to be detected, which is generated by the reaction product and has the color of connecting the 5 'end of the DNA molecule shown in the sequence 2 or the sequence 5 with the fluorescent sequence, is less than the height of the wheat to be detected, which is generated by the reaction product and has the color of connecting the 5' end of the DNA molecule shown in the sequence 1 or the sequence 4 with the fluorescent sequence; and/or
The stalk intensity of the wheat to be detected with the color of the DNA molecule with the 5 'end connected with the fluorescence sequence shown in the sequence 2 or the sequence 5 generated by the reaction product is greater than the stalk intensity of the wheat to be detected with the color of the DNA molecule with the 5' end connected with the fluorescence sequence shown in the sequence 1 or the sequence 4 generated by the reaction product.
The invention also provides a method for identifying or assisting in identifying the wheat dwarf genotype, which comprises the following steps: and (3) carrying out KASP reaction on the wheat to be detected by using the primer pair, and detecting a reaction product, wherein the reaction product generates a DNA molecule shown in a sequence 2 or a sequence 5, and the wheat to be detected with the color of connecting the 5' end of the DNA molecule with a fluorescent sequence is dwarf genotype wheat.
The dwarf gene is positioned on the 2D chromosome through the initial positioning analysis of the 660k SNP chip; the dwarf gene is positioned in the interval of about 0.85cM of 2DL markers ph3 and ph4 by utilizing KASP molecular marker development and genetic linkage map construction. And the ph3 and the ph4 are molecular markers closely linked with the dwarf gene, and can be used for molecular marker-assisted selection and screening of new wheat dwarf materials.
The KASP marker tightly linked with the dwarf gene can realize high-throughput and rapid auxiliary selection in the seedling stage and accelerate the dwarf breeding process.
Drawings
FIG. 1 shows the comparison of plant height and stalk strength of wild type and dwarf mutant JE 0124.
FIG. 2 is the primary mapping analysis of dwarf gene; wherein, A, nong Da 5181/JE 0124F2A population; B. JE 0124/Nongda 5181F2And (4) a group.
FIG. 3 genetic mapping of dwarf genes on 2DL chromosomes; wherein, A, nong Da 5181/JE 0124F2A population; B. JE 0124/Nongda 5181F2And (4) a group.
FIG. 4 ph3 and ph4 KASP marker detection results; blue dots represent samples of the nong 5181 genotype, red dots represent samples of the JE0124 genotype, green dots represent heterozygous samples, and black dots are negative controls; wherein A, ph3 KASP marks the detection result; B. ph4 KASP marks the detection result.
Detailed Description
The source of the biological material is as follows:
wild type wheat: jing 411, a known variety (from seed company, Beijing) that has been approved by variety approval.
Wheat dwarf mutant JE 0124: is a stable dwarf mutant material which is obtained by mutating Jing 411 through EMS, and can be obtained by the public from the institute of crop science of Chinese academy of agricultural sciences. The mutagenesis method is as follows: after soaking the Beijing 411 dry seeds in water for 10 hours, treating the seeds for 4 hours by using 1.0% EMS (enhanced message service) and slightly shaking the seeds at 50rpm under a dark condition, flushing the treated seeds for 4 hours under running water and then planting the seeds in the ground. After 6 generations of continuous planting, a stable dwarf mutant JE0124 is screened out.
The agricultural land is 5181: the Chinese agricultural university grows in 2014, and the known variety is approved by the variety.
Example 1 comparison of plant height and stalk Strength of wild type and dwarf mutant JE0124
1. Experimental materials and methods
Wild-type Jing 411 and a dwarf mutant JE0124 are planted in a nursery test field of the institute of crop science of the Chinese academy of agricultural sciences, 15 plants are planted in each row, the row length is 2 meters, and the plant height of the plants is measured when the wheat is mature and harvested in 6 months. The plant height is determined by continuously planting for three years in 2016-2018, and at least 5 times of the planting are repeated. The stalk strength comparison is determined by the stalk breaking resistance determination index, and the specific determination method comprises the following steps: the high stable main stem of the plant was cut 20cm at its base and the resistance to breakage of the stem was measured perpendicular to the stem in its central area (10cm position) using a digital display type push-pull force meter, with about 9-10 replicates per material.
2. Results of the experiment
The three-year plant height determination result shows that the wild-type Jing 411 plant height is 90-95cm, the mutant JE0124 plant height is 60-65cm, and the JE0124 plant height is obviously reduced compared with the wild-type plant height and is reduced by about 30cm (A in figure 1). The stalk breaking resistance test result shows that the JE0124 stalk breaking resistance is obviously higher than that of the wild type, and the short stalk mutant JE0124 has higher stalk strength (B in figure 1) compared with the wild type. By integrating the measurement results of plant height and stalk breaking resistance, JE0124 is presumed to have better lodging resistance.
Example 2 Primary mapping of dwarf Gene based on 660k SNP chip
1. Genetic population construction and experiment method
Carrying out positive and negative cross on the short stalk mutant JE0124 and the relatively high stalk backbone parent Pidao 5181 to obtain 2F2The population was subjected to subsequent gene mapping analysis. F obtained by hybridization with Nongda 5181 as female parent and JE0124 as male parent (Nongda 5181/JE0124)2The number of the individual plants in the population is 312; f obtained by hybridization with JE0124 as female parent and Panaxanthus 5181 as male parent (JE 0124/Panaxanthus 5181)2The number of individuals in the population was 282. Take two F2And (4) extracting DNA from the leaf samples of the individual plants of the population for subsequent positioning analysis. After the plant height phenotype in the field is stable, the plant height of each individual plant is measured.
Selecting two F according to plant height phenotype data220-30 high-stalk and short-stalk extreme individuals in the population are subjected to equal amount mixing of DNA to construct an extreme high-stalk pool and a short-stalk pool, and SNP chip scanning is performed on the mixed pool DNA and the amphiphilic DNA by using Affymetrix Wheat660k chip. According to the genotype data scanned by the SNP chip, two parents and two mixed pools are screened to have difference, simultaneously, the loci of the dwarf pool and the dwarf parent are consistent, the SNP locus number of each chromosome after screening is counted, and the chromosome with more enrichment of the screened SNP is the dwarf gene locus.
2. Results of the experiment
In the agricultural level 5181/JE 0124F2In the population, the screened SNPs were mainly enriched in the 2D chromosome, with a relatively small number of other chromosomal SNPs (a in fig. 2); at the same time, in JE 0124/Nongda 5181F2The number of SNPs enriched for the 2D chromosome was also highest in the population (B in FIG. 2). The synthesis shows that the dwarf gene is positioned on the 2D chromosome.
Example 3 genetic linkage mapping analysis and verification of dwarf genes
1. Experimental methods
According to polymorphic sites on 2D chromosomes of two parent agro-megas 5181 and JE0124 of an SNP chip, based on known flanking sequences, two allele forward primers and a reverse primer with different terminal bases are designed, the 5' ends of the two forward primers are respectively connected with sequences with different fluorescent probes, and the genotype of a sample can be determined according to a fluorescent signal detected after PCR amplification by using KASP Master mix (LGC). According to SNP loci of the pesticide 5181 and the JE0124 on a 2D chromosome, 48 pairs of KASP marker primers are designed in total, and 8 pairs of KASP markers with good genotyping are obtained by screening through detecting genotypes in parents and single strains of partial groups and are used for constructing a genetic linkage map.
For agricultural crops 5181/JE 0124F2312 individuals in the population were analyzed, the genotype of each individual was determined by KASP marker detection, and the plant height of each individual was investigated. And combining genotype and plant height phenotype data, and performing genetic linkage positioning analysis on the dwarf gene by using QTL IciMapping4.0 software. According to the positioning result, the marker with the LOD value more than or equal to 3 and relatively higher is the marker linked with the dwarf gene, the KASP marker comprising the linked marker is selected to JE 0124/Nongda 5181F2282 individuals in the population are subjected to genotype and phenotype identification, and genetic linkage positioning analysis is carried out by using QTL IiMapping 4.0 software, and if a QTL with a higher LOD value is detected between the same two markers, the accuracy of the original positioning segment is verified.
The method obtains a pair of KASP markers ph3 and ph4 closely linked with the wheat dwarf gene. The nucleotide sequences of the two allele forward primers with different terminal bases of ph3 are respectively as follows: 5 'GAAGGTGACCAAGTTCATGCTgaACaagAtggatctccgctaA 3' (SEQ ID NO: 7) and 5 'GAAGGTCGGAGTCAACGGATTgaACaagAtggatctccgctaG 3' (SEQ ID NO: 8) of the sequence listing, wherein the nucleotide sequence of the ph3 universal reverse primer is as follows: 5 'actgcaggagtatctaGcactG 3' (sequence 3 in the sequence table), and the length of the amplified band is 53 bp; the nucleotide sequences of the two allele forward primers with different terminal bases of ph4 are respectively as follows: 5 'GAAGGTGACCAAGTTCATGCTgtttcgcatccggcttgtT 3' (SEQ ID NO: 9) and 5 'GAAGGTCGGAGTCAACGGATTgtttcgcatccggcttgtC 3' (SEQ ID NO: 10) in the sequence listing, wherein the nucleotide sequence of the ph4 universal reverse primer is as follows: 5 'ccgttccttgtccttctgga 3' (SEQ ID NO: 6 in the sequence table), and the length of the amplified band is 72 bp.
The sequences of the fluorescent labels connected to the 5' ends of the ph3 and ph4 are respectively as follows: FAM (5 'GAAGGTGACCAAGTTCATGCT), HEX (5' GAAGGTCGGAGTCAACGGATT).
The reaction systems of ph3 and ph4 KASP are as follows: 2 XKASP Master mix 5. mu.L, primer mix 0.12. mu.L (6. mu.L each for the two forward primers, 15. mu.L for the universal primer, and 23. mu.L of ultrapure water to 50. mu.L, which is the primer mix), 50mM MgCl2mu.L of 0.08. mu.L, 200ng of genomic DNA and ultrapure water to 10. mu.L. The PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing at 65 ℃ for 1min for 9 cycles, each cycle decreasing by 0.6 ℃; denaturation at 94 ℃ for 20s, annealing at 57 ℃ for 1min for 30 cycles.
The PCR product result is obtained by detecting a fluorescence signal by a FLUOstar Omega ELISA reader. Amplification products with FAM sequence tags are labeled blue, and amplification products with HEX sequence tags are labeled red. FAM sequence tag primer is A site (ph3) or T site (ph4), HEX sequence tag primer is G site (ph3) or C site (ph 4).
2. Results of the experiment
Agricultural product 5181/JE 0124F2Population mapping results show that the dwarf gene is mapped to the interval between the 2D chromosome long arm markers ph3 and ph4 (calculated by QTL IcMapping 4.0 software) (A in FIG. 3), the LOD value is 5.342, the phenotype contribution rate is 8.27%, and the additive effect is 3.96(LOD value, phenotype contribution rate, additive effect calculation method are shown in Meng et al 2015, QTL IcMapping: Integrated software for genetic linking map construction and qualitative tracking in biological position. crop joint 3, 269-283.).
JE 0124/Nongda 5181F2The population further verifies the interval, and the 5 KASP markers including the ph3 and ph4 linkage markers are selected for genotype identification, a genetic linkage map is constructed, the dwarf gene is positioned in the interval (B in the picture 3) of about 1.19cM between ph3 and ph4 by combining phenotype data, the LOD value is 5.78, the phenotype contribution rate is 8.52 percent, and the additive effect is 3.84. Verification showed that the dwarf gene was located between the markers ph3 and ph 4.
Through fluorescence signal detection (FLUOstar Omega ELISA reader), the genotype (red spot) of the dwarf mutant JE0124 and the genotype (blue spot) of the Piaoda 5181 can be obviously separated by the markers of ph3 (A in figure 4) and ph4 (B in figure 4), and the genotype information obtained by the two markers can be used for predicting the existence of the dwarf gene.
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Claims (6)
- A KASP primer pair comprising ph3 and ph 4; wherein,the ph3 is composed of a primer 1 shown in a sequence 1 in a sequence table or a derivative thereof, a primer 2 shown in a sequence 2 in the sequence table or a derivative thereof and a primer 3 shown in a sequence 3 in the sequence table;the ph4 is composed of a primer 4 or a derivative thereof shown in a sequence 4 in a sequence table, a primer 5 or a derivative thereof shown in a sequence 5 in the sequence table and a primer 6 shown in a sequence 6 in the sequence table;the derivative of the primer 1 is that the 5' end of a single-stranded DNA molecule shown in a sequence 1 is connected with a fluorescent sequence, and the fluorescent sequence is a fluorescent sequence FAM;the derivative of the primer 4 is that the 5' end of a single-stranded DNA molecule shown in a sequence 4 is connected with a fluorescent sequence, and the fluorescent sequence is a fluorescent sequence FAM;the derivative of the primer 2 is that the 5' end of a single-stranded DNA molecule shown in the sequence 2 is connected with another fluorescent sequence, and the other fluorescent sequence is a fluorescent sequence HEX;the derivative of the primer 5 is that the 5' end of a single-stranded DNA molecule shown in a sequence 5 is connected with another fluorescent sequence, and the other fluorescent sequence is a fluorescent sequence HEX.
- 2. PCR reagents comprising KASP primer pairs of claim 1.
- 3. A kit comprising the KASP primer pair of claim 1 or the PCR reagent of claim 2.
- 4. Use of the KASP primer pair of claim 1 or the PCR reagent of claim 2 or the kit of claim 3 in at least one of 1) to 5) below:1) identifying or assisting in identifying the plant height and/or stalk strength of the wheat;2) preparing a product for identifying or assisting in identifying the plant height and/or stalk strength of the wheat;3) detecting or screening or breeding dwarf or semi-dwarf wheat;4) preparing, detecting, screening or breeding dwarf or semi-dwarf wheat products;5) and identifying or assisting in identifying the wheat dwarf genotype.
- 5. A method for identifying or assisting in identifying wheat plant height and/or stalk strength comprises the following steps: carrying out KASP reaction on wheat to be detected by using the primer pair as described in claim 1, and detecting a reaction product, wherein the height of a wheat plant to be detected, which generates the color of the fluorescence sequence connected to the 5 'end of the DNA molecule shown in the sequence 2 or the sequence 5, of the reaction product, is less than the height of a wheat plant to be detected, which generates the color of the fluorescence sequence connected to the 5' end of the DNA molecule shown in the sequence 1 or the sequence 4, of the reaction product; and/orThe stalk intensity of the wheat to be detected with the color of the DNA molecule with the 5 'end connected with the fluorescence sequence shown in the sequence 2 or the sequence 5 generated by the reaction product is greater than the stalk intensity of the wheat to be detected with the color of the DNA molecule with the 5' end connected with the fluorescence sequence shown in the sequence 1 or the sequence 4 generated by the reaction product.
- 6. The method for identifying or assisting in identifying the wheat dwarf genotype comprises the following steps: carrying out KASP reaction on wheat to be detected by using the primer pair as claimed in claim 1, and detecting a reaction product, wherein the wheat to be detected, which generates a color that the 5' end of the DNA molecule shown in the sequence 2 or the sequence 5 is connected with a fluorescent sequence, is dwarf genotype wheat.
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| CN116855504A (en) * | 2023-05-11 | 2023-10-10 | 四川农业大学 | Wheat red glume red stalk gene RgM G52 and KASP molecular marker and application thereof |
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| CN104073488A (en) * | 2014-07-16 | 2014-10-01 | 南京农业大学 | Molecular marker for dwarfing wheat marker gene, and primer and application thereof |
| CN104531882A (en) * | 2015-01-12 | 2015-04-22 | 北京农学院 | Molecular marker primer pair, method and application for authenticating new dwarf main effect QTL of wheat |
| CN108179220A (en) * | 2018-02-27 | 2018-06-19 | 西北农林科技大学 | The KASP labels of wheat dwarf stem gene Rht12 close linkages and its application |
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| CN104073488A (en) * | 2014-07-16 | 2014-10-01 | 南京农业大学 | Molecular marker for dwarfing wheat marker gene, and primer and application thereof |
| CN104531882A (en) * | 2015-01-12 | 2015-04-22 | 北京农学院 | Molecular marker primer pair, method and application for authenticating new dwarf main effect QTL of wheat |
| CN108179220A (en) * | 2018-02-27 | 2018-06-19 | 西北农林科技大学 | The KASP labels of wheat dwarf stem gene Rht12 close linkages and its application |
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