CN117535442B - KASP (KASP-based sequence analysis) mark for identifying wheat precipitation value and application - Google Patents
KASP (KASP-based sequence analysis) mark for identifying wheat precipitation value and application Download PDFInfo
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Abstract
The invention discloses a KASP marker for identifying a wheat precipitation value and application thereof. The primer set for identifying the KASP markers of wheat precipitation values consists of SEQ ID NO:1, the upstream primer F1, SEQ ID NO:2 and the upstream primer F2 shown in SEQ ID NO:3, and a downstream primer R indicated in the formula (3). Experiments prove that the primer group provided by the invention can detect the genotype of wheat to be detected based on a T100G SNP locus, wherein the T100G SNP locus is SEQ ID NO in a wheat genome: 4 from the 5' end, thereby rapidly and efficiently identifying the property of the precipitation value of the wheat. The invention has important application value for accelerating the cultivation of new varieties of high-quality wheat.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a KASP (KASP) marker for identifying a wheat precipitation value and application thereof.
Background
Wheat (Triticum aestivum L.) is one of the most important grain crops in China, and has important strategic significance for guaranteeing grain safety. With the development of social economy and the improvement of living standard, the quality of wheat is greatly concerned by breeders and consumers, and the quality improvement has become one of main targets of wheat breeding in China. According to the current wheat variety quality classification standard (GB/T17320-2013) in China, the wheat quality indexes mainly comprise hardness, crude protein content (grains), wet gluten content, precipitation value, water absorption, stability time, maximum stretching resistance, energy (wheat flour) and the like.
The sedimentation value is fast in measurement speed, simple in method, capable of reflecting the content and quality of the gluten protein, and is an extremely important evaluation index for the quality of the wheat. In addition, the precipitation value has high genetic power, and can be used as an index for early generation screening of wheat quality breeding. The sediment value is inherited in quantitative characters, the inheritance of the sediment value is controlled by multiple genes, and the gene effect is mainly based on the additive effect of the multiple genes. At present, QTLs related to wheat precipitation values are located, but the results only reflect the gene information contained in specific wheat varieties due to the influence of factors such as mapping population, genetic background, mapping method and the like, and most QTLs have small contribution rate to the types and poor repeatability among different environments, so that the requirement of molecular marker assisted Selection (MARKER ASSISTED Selection, MAS) cannot be met. Therefore, the development of a KASP marker for screening wheat sedimentation values is of great importance for improving wheat quality.
Competitive allele-specific PCR (Kompetitive Allele-SPECIFIC PCR, KASP) is a fluorescence-based genotyping technique. The primer is designed according to specific SNP or InDels contained in the target allele, different fluorophores are added at the tail end of the primer, and the target sequence is accurately double-allele typed based on the reading of the fluorescent signal of the PCR terminal, so that the method has the advantages of accuracy, high efficiency and low cost, and has wide application prospect in agricultural breeding.
Disclosure of Invention
The purpose of the invention is how to screen or assist in screening wheat with different sedimentation values.
The invention firstly protects a method for screening or assisting in screening wheat with different precipitation values, which comprises the following steps: detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b, wherein the sedimentation value of the wheat of genotype qSV1D.1b is greater than the sedimentation value of the wheat of genotype qSV1D.1a;
The wheat with the genotype qSV1D.1b is wheat with the genotype TT homozygote based on a T100G SNP locus;
The wheat with the genotype qSV1D.1a is wheat with the genotype GG homozygosity based on the T100G SNP locus;
The T100G SNP locus is SEQ ID NO in wheat genome: 4 from the 5' end.
In the above method, the step of detecting whether the genotype of the wheat to be tested is genotype qsv1d.1a or genotype qsv1d.1b may be as follows:
(a1) Taking genomic DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting the primer group 1 to obtain a PCR amplification product;
The primer group 1 consists of SEQ ID NO:1, the upstream primer F1, SEQ ID NO:2 and the upstream primer F2 shown in SEQ ID NO:3, a downstream primer R shown in the specification;
(a2) After the step (a 1) is completed, detecting a fluorescent signal of the PCR amplification product by adopting an instrument, and obtaining the genotype of the wheat to be detected according to the color of the fluorescent signal.
In the above method, the reaction procedure for PCR amplification using the primer set 1 may specifically be: pre-denaturation at 94℃for 15min; denaturation at 94℃for 20s, renaturation/extension for 20s (first renaturation/extension at 61℃with a decrease of 0.6℃per cycle) for 10 cycles; denaturation at 94℃for 20s, renaturation/extension at 55℃for 1min for 26 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
In the above method, the reaction system for PCR amplification using the primer set 1 may specifically be 1.6. Mu.L, consisting of 0.4. Mu.L of 2X KASP MASTER mix (LGC, lotNo.13426773), 0.022. Mu.L of primer working solution, 0.8. Mu.L of template and ultrapure water. The primer working solution was prepared by mixing 12 parts by volume of an aqueous solution of the upstream primer F1 (concentration: 100. Mu.M), 12 parts by volume of an aqueous solution of the upstream primer F2 (concentration: 100. Mu.M), 30 parts by volume of an aqueous solution of the downstream primer R (concentration: 100. Mu.M) and 46 parts by volume of 10mM Tris-HCl (pH 8.0).
In the above method, the step of detecting whether the genotype of the wheat to be tested is genotype qsv1d.1a or genotype qsv1d.1b may be as follows:
(b1) Taking genomic DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer group 2 to obtain a PCR amplification product;
The primer group 2 consists of SEQ ID NO:1 from the 5' end at positions 22 to 46, the upstream primer F1, SEQ ID NO:2 from the 5' end at positions 22 to 49 and the upstream primer F2 and SEQ ID NO:3, a downstream primer R shown in the specification;
(b2) Taking the PCR amplification product obtained in the step (b 1), and sequencing;
(b3) And (3) obtaining the genotype of the wheat to be tested according to the sequencing result obtained in the step (b 2).
The invention also provides a kit for identifying or assisting in identifying the precipitation value of wheat. The kit can comprise a substance for detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b;
the genotype qSV1D.1b is a genotype based on a T100G SNP locus and is TT homozygosity;
The genotype qSV1D.1a is homozygous GG based on the genotype of the SNP locus of T100G;
The T100G SNP locus is SEQ ID NO in wheat genome: 4 from the 5' end.
The kit can specifically comprise a substance for detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b.
Any of the above substances for detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b may specifically be primer set 1; the primer group 1 consists of SEQ ID NO:1, the upstream primer F1, SEQ ID NO:2 and the upstream primer F2 shown in SEQ ID NO:3, and a downstream primer R indicated in the formula (3).
Any of the above substances for detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b may specifically be primer set 2; the primer group 2 consists of SEQ ID NO:1 from the 5' end at positions 22 to 46, the upstream primer F1, SEQ ID NO:2 from the 5' end at positions 22 to 49 and the upstream primer F2 and SEQ ID NO:3, and a downstream primer R indicated in the formula (3).
The preparation method of the kit also belongs to the protection scope of the invention. The preparation method of the kit comprises the step of packaging each primer in any one of the primer groups 1 or 2 separately.
The invention also protects SEQ ID NO:4 or SEQ ID NO:4 from the 5' end to the 76 th to 133 th positions.
The invention also provides the application of any one of the kit or any one of the molecular markers in the identification or auxiliary identification of the wheat sediment value.
The invention also protects the application of any one of the above kits or any one of the above molecular markers in screening or assisting in screening wheat with different precipitation values.
The invention also protects the application of any one of the above kits or any one of the above molecular markers in wheat breeding.
In the above, when the genotype of the T100G SNP site in the molecular marker is TT homozygous, wheat of genotype qSV1D.1b is judged. When the genotype of the T100G SNP site in the molecular marker is GG homozygous, wheat of genotype qSV1D.1a is judged. The T100G SNP locus is SEQ ID NO in wheat genome: 4 from the 5' end.
Precipitation value of wheat of genotype qsv1d.1b > precipitation value of wheat of genotype qsv1d.1a.
Above, the > may be in particular statistical >.
Above, SEQ ID NO: the nucleotide sequences shown in positions 1 to 21 from the 5' -end of 2 are fluorescent tag sequences (i.e., FAM fluorescent tag sequences), and the fluorescent signal is particularly green. SEQ ID NO: the nucleotide sequences shown at positions 1 to 21 from the 5' end of 1 are also fluorescent tag sequences (i.e., HEX fluorescent tag sequences), and the fluorescent signal is in particular pink.
Experiments prove that the method provided by the invention can be used for detecting the genotype of the wheat to be detected based on the T100G SNP locus, and screening or assisting in screening the property of the precipitation value of the wheat, and simultaneously has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resources saving and the like. The invention can accelerate the cultivation process of the new variety of high-quality wheat and has important application value.
Drawings
FIG. 1 shows the T100G SNP site and 3 primers in SEQ ID NO:4, and a position on the DNA molecule shown in FIG. 4.
FIG. 2 shows the results of genotyping 359 wheat germplasm in a natural population (CK is a negative control; TT is TT homozygous, i.e., genotype qSV1D.1b; GG is GG homozygous, i.e., genotype qSV1D.1a).
FIG. 3 is a correlation analysis of the average of the wheat germplasm and the sedimentation value of genotype qSV1D.1b and genotype qSV1D.1a in natural populations under different environments; the method comprises the steps that 18-19 paddy fields_g are genotypes qSV1D.1a of 2018-2019 paddy fields, 18-19 paddy fields_T are genotypes qSV1D.1b of 2018-2019 paddy fields, 19-20 paddy fields_g are genotypes qSV1d.1a of 2019-2020 paddy fields, 19-20 paddy fields_T are genotypes qSV1b of 2019-2020 paddy fields, 20-21 paddy fields_g are genotypes qSV1D.1a of 2020-2021 paddy fields, 20-21 paddy fields_T are genotypes qSV1D.1b of 2020-2021 water fields, 18-19 dry fields_G are genotypes qSV1d.1a of 2018-2019 dry fields, 18-19 dry fields_T are genotypes qSV1d.1b of 2019 dry fields, 19-20 dry fields_G are genotypes qSV1d.1b of 2019-2020 dry fields_20-202, and the differences of the genotypes qSV1d.1b of the 20-20 dry fields are significant levels of the genotypes of the 2020-20-2021 dry fields, and 20-21 dry fields_g are the genotypes of the 2020-2021 dry fields.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The wheat material used in the examples described below (specific wheat germ plasm names are described in ：Jie Zhao,Lijing Sun,et al.Genome-wide association study ofyield-related traits in common wheat(Triticum aestivum L.)under normal and drought treatment conditions.Frontiers in Plant Science.DOI:10.3389/fpls.2022.1098560) in the following documents, which are all from the institute of grain and oil crops, the academy of agricultural and forestry sciences in Hebei, and which are available to the public from the applicant, and which are used only for repeated experiments of the present invention and not for other uses.
Example 1 discovery of T100G SNP site in wheat genome and establishment of genotyping method of wheat based on T100G SNP site
1. Discovery of T100G SNP locus in wheat genome
Through a great deal of experiments, the inventor of the invention discovers a SNP locus in the wheat genome, and the SNP locus is named as a T100G SNP locus. The T100G SNP site is located in SEQ ID NO:4 from the 5' end, genotypes are TT homozygote and GG homozygote. In the Chinese spring wheat genome IWGSC REFSEQ V1.0.0, the physical position of the T100G SNP locus is 411733093 of the 1D chromosome; SEQ ID NO:4 is the 411732994-411733193 th position of the 1D chromosome.
2. Synthesis of primer set for amplifying T100G SNP locus
Based on the KASP technical principle, the inventors of the present invention have devised and synthesized a primer set for amplifying a T100G SNP site through a large number of experiments. The primer set consists of an upstream primer F1, an upstream primer F2 and a downstream primer R. The nucleotide sequences of the respective primers are shown in Table 1.
TABLE 1
Note that: the FAM fluorescent tag sequence is underlined singly, and the HEX fluorescent tag sequence is underlined doubly.
The T100G SNP site and 3 primers (parts) are set forth in SEQ ID NO:4 (red background represents T100G SNP site, box labeled primer).
3. Establishment of genotyping method of wheat to be tested based on T100G SNP locus
Through a large number of experiments, the inventor establishes a genotyping method of the wheat to be tested based on the T100G SNP locus. The method comprises the following specific steps:
1. Acquisition of genomic DNA of wheat to be tested
Extracting genome DNA of young leaves of the wheat to be detected by adopting a CTAB method to obtain the genome DNA of the wheat to be detected.
The quality and the concentration of the genome DNA of the wheat to be detected all meet the PCR requirement, and the standard is as follows: 1% agarose electrophoresis shows that the DNA band is clear, no obvious impurity and no degradation; ultraviolet spectrophotometer Nanodrop2000 (Thermo) detects that A260/A280 ratio is about 1.8, and A260/A230 ratio is greater than 1.8.
The genome DNA of the wheat to be tested is diluted with deionized water to a concentration of 28.3 ng/. Mu.L, and a template for PCR amplification in the next step is obtained.
2. And (3) taking genomic DNA of wheat to be detected or water (serving as negative control) as a template, and adopting the primer group synthesized in the step two to carry out PCR amplification to obtain a PCR amplification product.
The reaction system was 1.6. Mu.L, and consisted of 0.4. Mu.L of 2X KASPMASTER MIX (LGC, lotNo.13426773), 0.022. Mu.L of primer working solution, 0.8. Mu.L of template and ultrapure water. The primer working solution was prepared by mixing 12. Mu.L of an aqueous solution of the upstream primer F1 (concentration: 100. Mu.M), 12. Mu.L of an aqueous solution of the upstream primer F2 (concentration: 100. Mu.M), 30. Mu.L of an aqueous solution of the downstream primer R (concentration: 100. Mu.M) and 46. Mu.L of 10mM Tris-HCl (pH 8.0).
The reaction procedure is: pre-denaturation at 94℃for 15min; denaturation at 94℃for 20s, renaturation/extension for 20s (first renaturation/extension at 61℃with a decrease of 0.6℃per cycle) for 10 cycles; denaturation at 94℃for 20s, renaturation/extension at 55℃for 1min for 26 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
3. After the step 2 is completed, a fluorescence detection system (Araya) is used for reading fluorescence data of the PCR amplification product, and the result is subjected to graphical display by GRAPHPAD PRISM, namely, the genotype of the wheat to be detected is judged according to the color of the fluorescence signal, wherein the specific principle is as follows: if the wheat to be detected shows a green fluorescent signal, the genotype of the wheat to be detected based on the T100G SNP locus is TT homozygote, and the genotype of the wheat to be detected is named as genotype qSV1D.1b at the moment; if the wheat to be tested shows pink fluorescent signals, the genotype of the wheat to be tested based on the T100G SNP locus is GG homozygote, and the genotype of the wheat to be tested is named as genotype qSV1D.1a. The negative control did not have any detectable signal.
The graphical display result is as follows: genotype qSV1D.1a showing pink fluorescent signal is distributed near the y-axis; genotype qsv1d.1b showing green fluorescent signal is distributed near x-axis; the negative control with no detectable signal was distributed near the origin.
Example 2 correlation analysis of two genotypes (genotype qSV1D.1b and genotype qSV1D.1a) of wheat with the precipitation value
1. Genotyping of individual wheat in natural populations
Genotyping was performed on each wheat variety in the natural population using the method of step three of example 1. The natural population consisted of 359 wheat germplasm (hexaploid in each case) representing the Huang Huaimai region of the main cultivar and the elite line. The wheat germplasm names are detailed in table 2 and column 2 of table 3.
The detection results are shown in FIG. 2.
The genotypes of 359 wheat germplasm are detailed in columns 3-4 of Table 2 and columns 3-4 of Table 3. The genotype of 288 wheat germplasm is genotype qSV1D.1a, and the genotype of 71 wheat germplasm is genotype qSV1D.1b.
TABLE 2
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Note that: NA represents the average precipitate value data missing.
TABLE 3 Table 3
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Note that: NA represents the average precipitate value data missing.
2. Detection of wheat precipitate value traits
359 Wheat germplasm in a natural population is planted on a test station on a dike of a institute of grain and oil crops of the academy of agricultural and forestry sciences in Hebei province for three years (2018-2019, 2019-2020 and 2020-2021 respectively), and simultaneously sowed on water lands (soil moisture sowing, watering once in a jointing period and a grouting period respectively, and the water filling amount is 50m 3/mu) and dry lands (soil moisture sowing, no watering in a whole growth period), 3m rows are long, a random granule design is carried out, and three times of repetition are carried out. Wherein, the water treatment is normal irrigation, and the dry land treatment is drought stress. After harvesting, 30 strains of seeds were randomly selected from each wheat germplasm, the wheat seeds were milled into flour using an experimental mill (Brabender), the sedimentation value of the flour was determined according to the agricultural industry standard wheat sedimentation value determination Zeleny method (NY/T1095-2006) and averaged, and recorded as the average sedimentation value.
Statistics of average sedimentation values of 359 wheat germplasm under 6 environments (i.e. 2018-2019 water land, 2019-2020 water land, 2020-2021 water land, 2018-2019 dry land, 2019-2020 dry land and 2020-2021 dry land) are shown in columns 5-7 (different years water land) in table 2 and columns 5-7 (different years dry land) in table 3, and only part of wheat germplasm does not obtain average sedimentation value data due to planting reasons and the like, and is expressed by NA.
3. Correlation analysis
1. Average sedimentation value of wheat germplasm of the same genotype under the same environment is averaged and is recorded as sedimentation value average value.
2. And (3) carrying out correlation analysis on the average value of the wheat germplasm and the sedimentation value of the genotype qSV1D.1b and the genotype qSV1D.1a in the natural population under the same environment by adopting a double-tail t-test.
The results are shown in Table 4 (the number of statistical wheat germplasm obtained according to tables 2 and 3, i.e. the number of wheat germplasm removed from which the average sedimentation value data were not obtained) and FIG. 3.
TABLE 4 Table 4
Note that: p <0.01 represents that the difference reached an extremely significant level.
The results showed that, in a natural population consisting of 359 wheat germplasm, the sedimentation value of wheat of genotype qsv1d.1b > the sedimentation value of wheat of genotype qsv1d.1a, said ">" is statistically >. Studies on natural populations indicate that genotype qSV1D.1b is an excellent genotype that increases wheat sedimentation.
The result shows that the wheat sediment value character can be screened or assisted screened by detecting the genotype of the wheat to be detected based on the T100G SNP locus, and the method has important application value in the assisted breeding process of the wheat molecular marker.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. A method for screening or assisting in screening wheat of different sedimentation values, comprising the steps of: detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b, wherein the sedimentation value of the wheat of genotype qSV1D.1b is greater than the sedimentation value of the wheat of genotype qSV1D.1a;
The wheat with the genotype qSV1D.1b is wheat with the genotype TT homozygote based on a T100G SNP locus;
The wheat with the genotype qSV1D.1a is wheat with the genotype GG homozygosity based on the T100G SNP locus;
The T100G SNP locus is SEQ ID NO in wheat genome: 4 from the 5' end.
2. The method according to claim 1, characterized in that: the step of detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b is as follows:
(a1) Taking genomic DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting the primer group 1 to obtain a PCR amplification product;
The primer group 1 consists of SEQ ID NO:1, the upstream primer F1, SEQ ID NO:2 and the upstream primer F2 shown in SEQ ID NO:3, a downstream primer R shown in the specification;
(a2) After the step (a 1) is completed, detecting a fluorescent signal of the PCR amplification product by adopting an instrument, and obtaining the genotype of the wheat to be detected according to the color of the fluorescent signal.
3. The method according to claim 1, characterized in that: the step of detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b is as follows:
(b1) Taking genomic DNA of wheat to be detected as a template, and carrying out PCR amplification by adopting a primer group 2 to obtain a PCR amplification product;
The primer group 2 consists of SEQ ID NO:1 from the 5' end at positions 22 to 46, the upstream primer F1, SEQ ID NO:2 from the 5' end at positions 22 to 49 and the upstream primer F2 and SEQ ID NO:3, a downstream primer R shown in the specification;
(b2) Taking the PCR amplification product obtained in the step (b 1), and sequencing;
(b3) And (3) obtaining the genotype of the wheat to be tested according to the sequencing result obtained in the step (b 2).
4. A kit for identifying or assisting in identifying a wheat precipitation value comprises a reagent for detecting whether the genotype of wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b;
the genotype qSV1D.1b is a genotype based on a T100G SNP locus and is TT homozygosity;
The genotype qSV1D.1a is homozygous GG based on the genotype of the SNP locus of T100G;
The T100G SNP locus is SEQ ID NO in wheat genome: 4 from the 5' end.
5. The kit of claim 4, wherein: the reagent for detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b is a primer group 1;
the primer group 1 consists of SEQ ID NO:1, the upstream primer F1, SEQ ID NO:2 and the upstream primer F2 shown in SEQ ID NO:3, and a downstream primer R indicated in the formula (3).
6. The kit of claim 4, wherein: the reagent for detecting whether the genotype of the wheat to be detected is genotype qSV1D.1a or genotype qSV1D.1b is a primer group 2;
the primer group 2 consists of SEQ ID NO:1 from the 5' end at positions 22 to 46, the upstream primer F1, SEQ ID NO:2 from the 5' end at positions 22 to 49 and the upstream primer F2 and SEQ ID NO:3, and a downstream primer R indicated in the formula (3).
SEQ ID NO:4 or SEQ ID NO:4 from the 5' end to the 76 th to 133 th positions.
8. Use of a kit according to any one of claims 4 to 6 or a molecular marker according to claim 7 for the identification or assisted identification of wheat sedimentation values.
9. Use of a kit according to any one of claims 4 to 6 or a molecular marker according to claim 7 for screening or aiding in screening of wheat of different sedimentation values.
10. Use of a kit according to any one of claims 4 to 6 or a molecular marker according to claim 7 in wheat breeding.
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