A KASP molecular marker for identifying wheat sedimentation value under multiple environments and its application

Abstract

The present invention discloses a KASP molecular marker for identifying the sedimentation value of wheat under multiple environments and its application, relating to the field of biotechnology. The method includes screening or assisting screening of wheat with different sedimentation values, comprising the following steps: detecting whether the genotype of the wheat to be tested is the genotype qSV6A.1a or the genotype qSV6A.1b, wherein the sedimentation value of the wheat with the genotype qSV6A.1b is greater than the sedimentation value of the wheat with the genotype qSV6A.1a; the wheat with the genotype qSV6A.1b is a wheat with a GG homozygous genotype based on the C101G SNP site; the wheat with the genotype qSV6A.1a is a wheat with a CC homozygous genotype based on the C101G SNP site; the C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome. The present invention has important theoretical significance and economic value for using molecular markers to assist in the selection of wheat germplasm or breeding offspring materials with higher sedimentation values.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a KASP molecular marker for identifying wheat precipitation values ​​under multiple environments and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is the world's most widely cultivated and consumed crop, and one of my country's three major grain crops, holding strategic importance for ensuring national food security. China leads the world in both wheat production and consumption. With socioeconomic development and improvements in people's living standards, wheat quality has garnered significant attention from both breeders and consumers, and quality improvement has become a major priority in wheat breeding in my country. According to my country's current "Wheat Variety Quality Classification Standard (GB / T 17320-2013)," wheat quality indicators primarily include hardness, crude protein content (kernels), wet gluten content, sedimentation value, water absorption, stability time, maximum tensile resistance, and energy (wheat flour).

[0003] Sedimentation value is a fast and simple method to determine, and it reflects the content and quality of gluten, making it a crucial indicator for evaluating wheat quality. Furthermore, sedimentation value has high heritability, making it a useful indicator for early-generation screening of wheat quality breeding. Sedimentation value is a quantitative trait, controlled by multiple genes, with the effects of these genes primarily acting as additive polygenic factors. Several QTLs associated with wheat sedimentation value have been identified, but due to factors such as the mapping population, genetic background, and mapping method, the results only reflect the genetic information contained in a specific wheat variety. Furthermore, most QTLs have a low contribution to the phenotype and exhibit poor reproducibility across different environments, thus failing to meet the needs of molecular marker-assisted selection.

[0004] Molecular marker-assisted selection is a modern breeding method that uses DNA molecular markers that are closely linked to the target traits to select the genotype of the target traits. It has the advantage of not being affected by external environmental factors. KASP (Kompetitive Allele-Specific PCR) is a fluorescence-based homogeneous genotyping technology developed in recent years. Primers are designed according to specific SNPs or InDels in the target alleles, and different fluorescent groups are added to the ends of the primers. The target sequence is typed based on the reading of the PCR terminal fluorescence signal. It has the advantages of high efficiency, accuracy and low cost, and has broad application prospects in crop breeding. Therefore, the development of KASP markers for detecting wheat sedimentation values ​​under multiple environments can provide an effective detection method for the selection and breeding of new high-quality wheat varieties, which is of great significance to improving the level of high-quality wheat breeding in my country. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a KASP molecular marker for identifying the precipitation value of wheat under multiple environments and its application.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] A method for screening or assisting in screening wheat with different sedimentation values, comprising the following steps: detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b, wherein the sedimentation value of the wheat with genotype qSV6A.1b is greater than the sedimentation value of the wheat with genotype qSV6A.1a;

[0008] The wheat of the genotype qSV6A.1b is a wheat of homozygous GG genotype based on the C101G SNP site;

[0009] The wheat of the genotype qSV6A.1a is a wheat of CC homozygous genotype based on the C101G SNP site;

[0010] The C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0011] Further preferably, the step of detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b is as follows:

[0012] (a1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification to obtain a PCR amplification product;

[0013] The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3;

[0014] (a2) After completing step (a1), the fluorescent signal of the PCR amplification product is detected by an instrument, and the genotype of the wheat to be tested is obtained based on the color of the fluorescent signal.

[0015] Further preferably, the step of detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b is as follows:

[0016] (b1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification to obtain a PCR amplification product;

[0017] The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3;

[0018] (b2) taking the PCR amplification product obtained in step (b1) and sequencing it;

[0019] (b3) Obtaining the genotype of the wheat to be tested based on the sequencing results obtained in step (b2).

[0020] A kit for identifying or assisting in identifying the sedimentation value of wheat, comprising a substance for detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b;

[0021] The genotype qSV6A.1b is based on the C101G SNP site, which is a homozygous GG genotype;

[0022] The genotype qSV6A.1a is a CC homozygous genotype based on the C101G SNP site;

[0023] The C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0024] Further preferably, the substance for detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b is a primer combination;

[0025] The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3.

[0026] The molecular marker shown in SEQ ID NO:4.

[0027] Application of the above kit or the above molecular marker in identifying or assisting in identifying wheat precipitation value.

[0028] Application of the above kit or the above molecular marker in screening or auxiliary screening of wheat with different precipitation values.

[0029] Application of the above kit or the above molecular marker in wheat breeding.

[0030] The primer combination is used in the directional breeding or auxiliary directional breeding of wheat lines with high sedimentation values. The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3.

[0031] The beneficial effects of the above technical solution are as follows: the present invention provides a KASP marker, Kasp_qSV6A.1, for identifying allelic variations of qSV6A.1a and qSV6A.1b, and its correlation with wheat sedimentation value. The KASP marker of the present invention is applied to molecular marker-assisted selection of wheat sedimentation values, enabling rapid and efficient screening of wheat varieties (germplasm) with high sedimentation values, thereby accelerating the breeding of new high-quality wheat varieties. This invention has important theoretical significance and economic value for the use of molecular marker-assisted selection of wheat germplasm or breeding material with high sedimentation values. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the KASP primer locations for different allele types of the common wheat sedimentation value QTL qSV6A.1 on chromosome 6A; the sequence in the figure is the physical position 63057734bp-63057933bp of wheat chromosome 6A. The positions of the upstream and downstream primers of the KASP marker are marked with boxes, and the red background represents the SNP at the physical position 63057834bp of chromosome 6A.

[0033] Figure 2 Schematic diagram of the genotyping results of 329 wheat germplasms in a natural population. In the figure, CC is the CC homozygous type, i.e., genotype qSV6A.1a; GG is the GG homozygous type, i.e., genotype qSV6A.1b; CK is the negative control;

[0034] Figure 3Schematic diagram of the association analysis results of wheat germplasm with genotype qSV6A.1a and genotype qSV6A.1b and the mean sedimentation value in natural populations under different environments; among them, 20-21normal_C is the genotype qSV6A.1a with normal water and fertilizer in 2020-2021, 20-21normal_G is the genotype qSV6A.1b with normal water and fertilizer in 2020-2021, 21-22normal_C is the genotype qSV6A.1a with normal water and fertilizer in 2021-2022, 21-22normal_G is the genotype qSV6A.1b with normal water and fertilizer in 2021-2022, 20-21drought_C is the genotype qSV6A.1a with drought treatment in 2020-2021, and 20-21drought_G is the genotype qSV6A.1b with normal water and fertilizer in 2020-202 The genotype under 1-year drought treatment is qSV6A.1b, 21-22 drought_C is the genotype under 2021-2022 drought treatment, 21-22 drought_G is the genotype under 2021-2022 drought treatment, 20-21 low nitrogen_C is the genotype under 2020-2021 low nitrogen treatment, 20-21 low nitrogen_G is the genotype under 2020-2021 low nitrogen treatment, 21-22 low nitrogen_C is the genotype under 2021-2022 low nitrogen treatment, qSV6A.1a, 21-22 low nitrogen_G is the genotype under 2021-2022 low nitrogen treatment; ** indicates P < 0.01, that is, the difference reaches the extremely significant level. DETAILED DESCRIPTION

[0035] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained through commercial purchase. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0036] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or collections thereof.

[0037] It will also be understood that the term "and / or" used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0038] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more but not all embodiments," unless otherwise specifically emphasized. The terms "including," "comprising," "having," and variations thereof all mean "including but not limited to," unless otherwise specifically emphasized.

[0039] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0040] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] Example 1: Detection of Different Allele Types of Wheat Sedimentation Value QTL qSV6A.1 Using KASP Marker Kasp_qSV6A.1

[0042] The KASP marker Kasp_qSV6A.1 was used to detect different allele types of the wheat sedimentation value QTL qSV6A.1 at physical position 63057834 bp on chromosome 6A (referring to the Chinese spring wheat genome IWGSC RefSeq v1.0). The following two steps were used: PCR amplification and genotyping. The experimental methods in the examples are all conventional methods unless otherwise specified. Common wheat germplasm materials were all stored at the Wheat Research Center of the Institute of Cereals and Oils Crops, Hebei Academy of Agricultural and Forestry Sciences.

[0043] (1) PCR amplification system and procedure

[0044] Extract common wheat genomic DNA using the CTAB method and dissolve in 300 μL of ultrapure water. Check DNA quality by electrophoresis on a 1% agarose gel, ensuring clear bands, no obvious impurities, and no degradation. Measure the concentration and dilute the DNA to 28.3 ng / μL. PCR amplification was performed using the diluted genomic DNA as a template.

[0045] Preparation of KASP marker primer working solution: Two KASP upstream primers were designed based on the SNP at the physical location 63057834 bp in the wheat genome (IWGSC RefSeq v1.0) for the wheat sedimentation value QTL qSV6A.1. The polymorphism at this SNP site is a C / G base difference. FAM and HEX fluorescent linker sequences were added to the 5' ends of the primers, respectively. A universal KASP downstream primer was also designed. The primer sequences are shown in Table 1. The KASP marker primer working solution consists of: 12 μL of each of the two upstream primers (100 μM), 30 μL of the downstream primer (100 μM), and 46 μL of ultrapure water. Mix thoroughly and store at -20°C until use.

[0046] Table 1 Sequences of KASP marker primers used to identify allelic variation of QTLqSV6A.1 in common wheat

[0047]

[0048]

[0049] PCR amplification system: 1.5 μL template DNA, 0.0417 μL primer working solution, 0.75 μL 2×KASP Master Mix (LGC Company, Lot No. 13426773), and the reaction system was supplemented to 3 μL with sterile ultrapure water.

[0050] PCR reaction program: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension for 20 s (the first annealing / extension temperature was 61°C, decreased by 0.6°C for each cycle), 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 1 min, 26 cycles; extension at 72°C for 3 min; storage at 4°C.

[0051] (2) Genotyping

[0052] After the PCR reaction was completed, the fluorescence signal was converted into an analyzable value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya). The fluorescence scanning results were displayed graphically using GraphPad Prism. qSV6A.1a (CC homozygous) showed FAM fluorescence, distributed near the x-axis; qSV6A.1b (GG homozygous) showed HEX fluorescence, distributed near the y-axis; the negative control (CK) had no detectable signal and was distributed near the origin (e.g., Figure 2 ).

[0053] Example 2: Detection of Sedimentation Values ​​of Common Wheat Germplasm Using KASP Marker Kasp_qSV6A.1

[0054] 329 Chinese wheat germplasm materials were planted at the Dishang Experimental Station of the Institute of Cereals and Oils Crops, Hebei Academy of Agricultural and Forestry Sciences for two consecutive years (2020-2021 and 2021-2022). They were sown every year under normal water and fertilizer conditions (one watering each at the jointing stage and the filling stage, with an irrigation volume of 50m3 / s). 3 / mu, nitrogen fertilizer 12Kg / mu), drought treatment (no watering during the whole growth period, nitrogen fertilizer 12Kg / mu) and low nitrogen treatment (watering once at the jointing stage and once at the filling stage, irrigation volume 50m 3 / mu, nitrogen fertilizer 6Kg / mu) under three environments, 3m row length, randomized block design, and three replications.

[0055] After harvest, wheat grains were ground into flour using a Brabender mill. Sedimentation values ​​were determined according to the agricultural industry standard, "Zeleny Method for Determination of Sedimentation Value of Wheat (NY / T 1095-2006)." KASP marker testing revealed that among 329 Chinese wheat germplasm accessions, 184 had the qSV6A.1a allele and 145 had the qSV6A.1b allele. Table 2 shows the allele types and sedimentation values ​​of wheat germplasm accessions in different years and environments.

[0056] Table 2 Kasp_qSV6A.1 marker detection results of wheat germplasm and precipitation values ​​in different years and environments

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Note: NA indicates missing data for mean sedimentation value.

[0076] The statistical results showed that the mean precipitation values ​​of wheat germplasm materials carrying the allele qSV6A.1b in different years and under different environments were higher than those of wheat germplasm materials carrying the allele qSV6A.1a, and the difference between the two reached a very significant level (P<0.01) (Table 3, Figure 3 ).

[0077] Table 3 Statistical analysis of the relationship between allelic variation types and sedimentation values ​​of QTLqSV6A.1 in common wheat

[0078]

[0079] Note: P<0.01 indicates that the difference reached an extremely significant level.

[0080] After extensive experiments, the inventors discovered a single nucleotide polymorphism (SNP) site in the wheat genome, designated the C101G SNP site. The C101G SNP site is located at position 101 from the 5' end of SEQ ID NO: 4, and the genotypes are CC homozygous and GG homozygous. In the Chinese spring wheat genome (IWGSC RefSeq v1.0), the physical location of the C101G SNP is at position 63,057,834 on chromosome 6A. Based on the SNP (C / G) differential site, a KASP marker, Kasp_qSV6A.1, was developed for identifying the wheat quality indicator, sedimentation value. The allele qSV6A.1a (CC homozygous), which carries FAM fluorescence and is distributed near the x-axis, indicates a reduced sedimentation value. The allele qSV6A.1b (GG homozygous), which carries HEX fluorescence and is distributed near the y-axis, indicates a higher sedimentation value. The marker was validated using 329 Chinese wheat germplasm samples under three conditions: normal water and fertilizer, drought, and low nitrogen. The results showed that the marker can effectively and accurately genotype both qSV6A.1a and qSV6A.1b alleles. This method provides an effective detection method for the accurate and rapid screening of wheat germplasm with high sedimentation values ​​under multiple environments.

[0081] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0082] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0083] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. A method for assisting in screening wheat with different sedimentation values, characterized in that: The method comprises the following steps: detecting whether the genotype of the wheat to be tested is the genotype qSV6A.1a or the genotype qSV6A.1b, and the precipitation value of the wheat of the genotype qSV6A.1b is greater than the precipitation value of the wheat of the genotype qSV6A.1a; The wheat of the genotype qSV6A.1b is a wheat of homozygous GG genotype based on the C101G SNP site; The wheat of the genotype qSV6A.1a is a wheat of CC homozygous genotype based on the C101G SNP site; The C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

2. The method according to claim 1, characterized in that The steps of detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b are as follows: (a1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification to obtain a PCR amplification product; The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3; (a2) After completing step (a1), the fluorescent signal of the PCR amplification product is detected by an instrument, and the genotype of the wheat to be tested is obtained based on the color of the fluorescent signal.

3. The method according to claim 1, characterized in that The steps of detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b are as follows: (b1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification to obtain a PCR amplification product; The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3; (b2) taking the PCR amplification product obtained in step (b1) and sequencing it; (b3) Obtaining the genotype of the wheat to be tested based on the sequencing results obtained in step (b2).

4. The purpose of the kit, characterized in that: The kit includes a substance for detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b; The genotype qSV6A.1b is based on the C101G SNP site, which is a homozygous GG genotype; The genotype qSV6A.1a is a CC homozygous genotype based on the C101G SNP site; The C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome; The use is to assist in identifying the sedimentation value of wheat, and the sedimentation value of wheat with genotype qSV6A.1b is greater than the sedimentation value of wheat with genotype qSV6A.1a.

5. The use of the kit according to claim 4, characterized in that: The substance for detecting whether the genotype of the wheat to be tested is genotype qSV6A.1a or genotype qSV6A.1b is a primer combination; The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO:

3.

6. Use of the kit according to any one of claims 4 to 5 in assisting the screening of wheat with different precipitation values.

7. Use of a primer combination in assisting the directional breeding of wheat lines with high sedimentation values, the primer combination consisting of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3.

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