A KASP marker for identifying the gelatinization properties of wheat starch under multiple environments and its application

Abstract

The invention discloses a KASP marker for identifying the gelatinization properties of wheat starch under multiple environments and an application thereof, relating to the field of biotechnology. The invention discloses a method for screening or auxiliary screening of wheat with different peak viscosities, comprising the following steps: detecting whether the genotype of the wheat to be tested is the genotype qRVA-Peak7B.1a or the genotype qRVA-Peak7B.1b, wherein the peak viscosity of the wheat with the genotype qRVA-Peak7B.1b is greater than the peak viscosity of the wheat with the genotype qRVA-Peak7B.1a; the wheat with the genotype qRVA-Peak7B.1b is a wheat with a genotype of TT homozygous based on a C101T SNP site; the wheat with the genotype qRVA-Peak7B.1a is a wheat with a genotype of CC homozygous based on the C101T SNP site; and the C101T 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 the use of molecular markers to assist in the selection of wheat germplasm or breeding offspring materials with higher peak viscosity (an important indicator of starch gelatinization properties).

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a KASP marker for identifying the gelatinization properties of wheat starch under multiple environments and its application. Background Art

[0002] Wheat (Triticum aestivum L.) is the most widely cultivated and consumed crop in the world. It is also one of my country's three major grain crops and is of great strategic significance to ensuring national food security. China ranks first in the world in both wheat production and consumption. With the development of social economy and the improvement of people's living standards, wheat quality has received great attention from breeders and consumers, and quality improvement has become one of the main tasks of wheat breeding in my country. Starch, as the main component of wheat grains (accounting for about 75% of the grains), has a significant impact on wheat quality. The gelatinization properties of wheat starch are one of the most important indicators for evaluating the quality of wheat starch and are closely related to the quality of foods such as noodles. Therefore, studying the gelatinization properties of wheat starch and its genetic basis is of great significance to wheat quality improvement.

[0003] Evaluation metrics for wheat starch gelatinization properties include peak viscosity, trough viscosity, thinning value, final viscosity, rebound value, peak time, and gelatinization temperature. Peak viscosity is the most important indicator for measuring starch gelatinization properties. Generally, wheat flour with high peak viscosity produces noodles of better quality. Furthermore, studies have shown a significant positive correlation between peak viscosity and noodle elasticity, smoothness, and softness. Peak year is a quantitative trait, controlled by multiple genes primarily mapped on chromosomes 1A, 1B, 3A, 3B, 4A, 5A, 5B, 6A, 6B, 7A, and 7B. However, due to factors such as the mapping population, genetic background, and mapping method, the results can only reflect the genetic information contained in a specific wheat variety. Furthermore, most QTLs have a low contribution to the phenotype and poor reproducibility across different environments, thus failing to meet the requirements of molecular marker-assisted selection (MAS).

[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 identifying the gelatinization characteristics of wheat starch 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 marker for identifying the gelatinization properties of wheat starch 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 single-nucleotide polymorphism (SNP) site associated with wheat peak viscosity, the SNP site being located on wheat chromosome 7B. The physical position of the SNP site in the wheat genome version IWGSC RefSeq v1.0 is 135576110 bp. The SNP site corresponds to the 101st base from the 5' end of SEQ ID NO: 4. When the site is homozygous for TT, the corresponding genotype is qRVA-Peak7B.1b; when the site is homozygous for CC, the corresponding genotype is qRVA-Peak7B.1a. The peak viscosity is such that the peak viscosity of wheat with the genotype qRVA-Peak7B.1b is greater than, or potentially greater than, the peak viscosity of wheat with the genotype qRVA-Peak7B.1a.

[0008] A reagent or kit for identifying or assisting in identifying the peak viscosity trait of wheat, wherein the reagent or kit is used to detect the above-mentioned SNP site, and comprises a PCR amplification specific primer combination corresponding to the SNP site, as well as template DNA, buffer, dNTPs and other necessary components for gene detection.

[0009] Further preferably, the PCR amplification specific 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.

[0010] A primer combination is provided for detecting a single nucleotide polymorphism at the following SNP site in a wheat genome, wherein the SNP site is located on wheat chromosome 7B, the physical position of the SNP site in the wheat genome version number IWGSC RefSeq v1.0 is 135576110 bp, the SNP site corresponds to the 101st base from the 5' end of SEQ ID NO:4, when the site is homozygous for TT, the corresponding genotype is qRVA-Peak7B.1b; when the site is homozygous for CC, the corresponding genotype is qRVA-Peak7B.1a; the peak viscosity is such that the peak viscosity of wheat with the genotype qRVA-Peak7B.1b is greater than, or is candidate greater than, the peak viscosity of wheat with the genotype qRVA-Peak7B.1a; the PCR amplification-specific 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; and this primer combination is used to detect the aforementioned SNP site.

[0011] Application of the above SNP sites in wheat molecular marker-assisted selection breeding.

[0012] The above-mentioned SNP sites are used for primary screening, and / or rescreening, and / or identification, and / or classification, and / or auxiliary identification of wheat peak viscosity in the early stage of wheat molecular marker-assisted selection breeding.

[0013] The primer combination is used in the directional breeding or auxiliary directional breeding of wheat lines with high peak viscosity.

[0014] The method for identifying or assisting in identifying the peak viscosity of wheat in the early stages of breeding comprises the following steps:

[0015] (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using a primer combination to obtain a PCR amplification product;

[0016] 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;

[0017] (2) After completing step (1), an instrument is used to detect the fluorescent signal of the PCR amplification product, and the genotype of the wheat to be tested is obtained according to the color of the fluorescent signal;

[0018] (3) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than that of wheat with genotype qRVA-Peak7B.1a.

[0019] The method for identifying or assisting in identifying the peak viscosity of wheat in the early stages of breeding comprises the following steps:

[0020] (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using a primer combination to obtain a PCR amplification product;

[0021] 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;

[0022] (2) taking the PCR amplification product obtained in step (1) and sequencing it;

[0023] (3) obtaining the genotype of the wheat to be tested based on the sequencing results obtained in step (2);

[0024] (4) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than that of wheat with genotype qRVA-Peak7B.1a.

[0025] Based on the above SNP sites, KASP molecular markers related to wheat peak viscosity were developed.

[0026] The beneficial effects of the above technical solution are as follows: the present invention provides a KASP marker Kasp_qRVA-Peak7B.1 for identifying allelic variations of qRVA-Peak7B.1a and qRVA-Peak7B.1b, and its correlation with peak viscosity, an important indicator of wheat starch gelatinization properties. The SNP molecular markers of the present invention are applied to molecular marker-assisted selection of wheat starch gelatinization properties to quickly and efficiently screen out wheat varieties (germplasm) with higher peak viscosity, thereby accelerating the breeding process of high-quality new wheat varieties. The present invention has important theoretical significance and economic value for the use of molecular markers to assist in the selection of wheat germplasm or breeding offspring materials with higher peak viscosity (an important indicator of starch gelatinization properties). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the KASP primer locations on chromosome 7B for different allele types of the QTL qRVA-Peak7B.1 related to starch gelatinization properties of common wheat; the sequence in the figure is the sequence at physical position 135576010bp-135576209bp on wheat chromosome 7B, the upstream and downstream primer positions of the KASP marker are marked with boxes, and the red background represents the SNP at physical position 135576110bp on chromosome 7B.

[0028] Figure 2Schematic diagram of the genotyping test results of 474 wheat germplasms in a natural population; CC is the CC homozygous type, that is, the genotype qRVA-Peak7B.1a; TT is the TT homozygous type, that is, the genotype qRVA-Peak7B.1b; CK is the negative control.

[0029] Figure 3 Schematic diagram of the association analysis results between wheat germplasm of genotype qRVA-Peak7B.1a and genotype qRVA-Peak7B.1b and the mean value of wheat peak viscosity (an important indicator of starch gelatinization characteristics) in natural populations under different environments; among them, 19-20Normal_C is the genotype qRVA-Peak7B.1a of normal water and fertilizer in 2019-2020, and 19-20Normal_T is the genotype of normal water and fertilizer in 2019-2020. genotype qRVA-Peak7B.1b, 20-21normal_C is the genotype qRVA-Peak7B.1a of normal water and fertilizer in 2020-2021, 20-21normal_T is the genotype qRVA-Peak7B.1b of normal water and fertilizer in 2020-2021, 19-20drought_C is the genotype qRVA-Peak7B.1a of drought treatment in 2019-2020, 19-20drought_T is 2 The genotype qRVA-Peak7B.1b was treated with drought in 2019-2020, the genotype qRVA-Peak7B.1a was treated with drought in 2020-2021, the genotype qRVA-Peak7B.1b was treated with drought in 2020-2021, the genotype qRVA-Peak7B.1b was treated with drought in 2020-2021, and the genotype qRVA-Peak7B.1b was treated with low nitrogen in 2019-2020. B.1a, 19-20 Low Nitrogen_T is the genotype qRVA-Peak7B.1b treated with low nitrogen in 2019-2020, 20-21 Low Nitrogen_C is the genotype qRVA-Peak7B.1a treated with low nitrogen in 2020-2021, and 20-21 Low Nitrogen_T is the genotype qRVA-Peak7B.1b treated with low nitrogen in 2020-2021; ** indicates P < 0.01, that is, the difference reaches an extremely significant level. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Example 1: Detection of different allele types of wheat starch gelatinization property-related QTL qRVA-Peak7B.1 using KASP marker Kasp_qRVA-Peak7B.1

[0037] The KASP marker Kasp_qRVA-Peak7B.1 was used to detect different allele types of the wheat starch gelatinization QTL qRVA-Peak7B.1 at physical position 135576110 bp on chromosome 7B (referring to the Chinese spring wheat genome IWGSC RefSeq v1.0). 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.

[0038] (1) PCR amplification system and procedure

[0039] 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.

[0040] Preparation of KASP marker primer working solution: Two KASP upstream primers were designed based on the SNP at the physical location 135576110 bp in the wheat genome (IWGSC RefSeq v1.0) for the wheat starch gelatinization QTL qRVA-Peak7B.1. The polymorphism at this SNP site is a C / T 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 consisted 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.

[0041] Table 1 Sequences of KASP marker primers used to identify allelic variation of QTLqRVA-Peak7B.1 in common wheat

[0042]

[0043] 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.

[0044] 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.

[0045] (2) Genotyping

[0046] After the PCR reaction was completed, the fluorescence signal was converted into an analyzable numerical value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya). The fluorescence scanning results were displayed graphically using GraphPad Prism. qRVA-Peak7B.1a (CC homozygous) showed FAM fluorescence, distributed near the x-axis; qRVA-Peak7B.1b (TT 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 ).

[0047] Example 2: Detection of starch gelatinization properties of common wheat germplasm using KASP marker Kasp_qRVA-Peak7B.1

[0048] 474 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 (2019-2020 and 2020-2021). 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.

[0049] After harvest, the peak viscosity, an important indicator of wheat starch gelatinization characteristics, was measured using a Perten rapid viscometer (RVA). KASP marker detection showed that among 474 Chinese wheat germplasm materials, 20 germplasms were of the qRVA-Peak7B.1a allele type and 454 germplasms were of the qRVA-Peak7B.1b allele type. The allele types of wheat germplasm materials and the peak viscosity (an important indicator of starch gelatinization characteristics) of wheat in different years and environments are shown in Table 2. The statistical results showed that the mean peak viscosity of wheat germplasm materials carrying the allele qRVA-Peak7B.1b in different years and different environments was higher than the mean peak viscosity of wheat germplasm materials carrying the allele qRVA-Peak7B.1a, and the two were extremely significantly different (P<0.01). Table 2 Wheat germplasm Kasp_qRVA-Peak7B.1 marker detection results and peak viscosity, an important indicator of wheat starch gelatinization characteristics in different years and different environments

[0050]

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[0059]

[0060]

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[0063]

[0064]

[0065]

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[0068]

[0069] Note: NA indicates that the average peak viscosity data is missing.

[0070] The statistical results showed that the mean peak viscosity of wheat germplasm materials carrying the allele qRVA-Peak7B.1b in different years and under different environments was higher than that of wheat germplasm materials carrying the allele qRVA-Peak7B.1a, and the difference between the two was extremely significant (P<0.01) (Table 3, Figure 3 ).

[0071] Table 3 Statistical analysis of the relationship between allelic variation types of QTLqRVA-Peak7B.1 in common wheat and peak viscosity, an important indicator of wheat starch gelatinization properties

[0072]

[0073] Note: P<0.01 indicates that the difference reached an extremely significant level.

[0074] After extensive experiments, the inventors discovered a single nucleotide polymorphism (SNP) site in the wheat genome, designated C101T. The C101T SNP site is located at position 101 from the 5' end of SEQ ID NO: 4, and the genotypes are CC homozygous and TT homozygous. In the Chinese spring wheat genome (IWGSC RefSeq v1.0), the physical location of the C101T SNP is at position 135576110 on chromosome 7B. Based on the SNP (C / T) differential site, a KASP marker, Kasp_qRVA-Peak7B.1, was developed for identifying wheat starch gelatinization properties. The alleles carrying FAM fluorescence, located near the x-axis, are qRVA-Peak7B.1a (CC homozygous), which reduces wheat peak viscosity (an important indicator of starch gelatinization properties). The alleles carrying HEX fluorescence, located near the y-axis, are qRVA-Peak7B.1b (TT homozygous), which increases wheat peak viscosity (an important indicator of starch gelatinization properties). The marker was validated using 474 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 qRVA-Peak7B.1a and qRVA-Peak7B.1b alleles. This method provides an effective detection method for the accurate and rapid screening of wheat germplasm samples with high peak viscosity (a key indicator of starch gelatinization) under multiple environments.

[0075] 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.

[0076] 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.

[0077] 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 SNP molecular marker associated with wheat peak viscosity, characterized by: The SNP molecular marker is shown in SEQ ID NO: 4, wherein when the 101st base site of SEQ ID NO: 4 is homozygous for TT, the corresponding genotype is qRVA-Peak7B.1b; when the 101st base site of SEQ ID NO: 4 is homozygous for CC, the corresponding genotype is qRVA-Peak7B.1a; the peak viscosity is: the peak viscosity of wheat with the genotype qRVA-Peak7B.1b is greater than or is potentially greater than the peak viscosity of wheat with the genotype qRVA-Peak7B.1a.

2. A reagent or kit for identifying or assisting in identifying the peak viscosity trait of wheat, characterized in that: The reagent or kit is used to detect the SNP molecular marker described in claim 1, and the reagent or kit comprises a PCR amplification specific primer combination corresponding to the SNP molecular marker, as well as template DNA, buffer, dNTPs and other gene detection components.

3. The reagent or kit according to claim 2, characterized in that: The PCR amplification specific 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.

4. A primer combination, characterized in that: This primer combination is used to detect the SNP molecular marker described in claim 1, and 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.

5. Use of the SNP molecular marker according to claim 1 in wheat molecular marker-assisted selection of high peak viscosity wheat breeding.

6. Use of the SNP molecular marker according to claim 1 in the initial screening, and / or rescreening, and / or identification, and / or classification, and / or assisted identification of wheat peak viscosity in the early stage of wheat molecular marker-assisted selection breeding.

7. Use of the primer combination according to claim 4 in directed breeding or assisted directed breeding of wheat lines with high peak viscosity.

8. A method for identifying or assisting in identifying wheat peak viscosity in the early stages of breeding, characterized in that: The following steps are involved: (1) Using the genomic DNA of the wheat to be tested as a template, a primer combination is used to perform PCR amplification, and the SNP molecular marker described in claim 1 is detected 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; (2) After completing step (1), the fluorescence signal of the PCR amplification product is detected by an instrument, and the genotype of the wheat to be tested is obtained according to the color of the fluorescence signal; (3) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than that of wheat with genotype qRVA-Peak7B.1a.

9. A method for identifying or assisting in identifying wheat peak viscosity in the early stages of breeding, characterized in that: The following steps are involved: (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using a primer combination 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; (2) Sequencing the PCR amplification product obtained in step (1); (3) Obtaining the genotype of the wheat to be tested based on the sequencing results obtained in step (2); (4) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than that of wheat with genotype qRVA-Peak7B.1a.

10. The SNP molecular marker according to claim 1 is used to develop a KASP molecular marker associated with wheat peak viscosity.

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