KASP molecular marker for identifying wheat precipitation value in multiple environments and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker for identifying wheat precipitation values in multiple environments and application, and relates to the technical field of biology, in particular to a method for screening or assisting in screening wheat with different precipitation values, which comprises the following steps: detecting whether the genotype of wheat to be detected is genotype qSV6A.1a or genotype qSV6A.1b, and detecting the precipitation value gt of the wheat with the genotype qSV6A.1b; the precipitation value of wheat of the genotype qSV6A.1a; the wheat of which the genotype is qSV6A.1b is wheat of which the genotype is GG homozygous based on a C101G SNP (Single Nucleotide Polymorphism) site; the wheat of which the genotype is qSV6A.1a is the wheat of which the genotype is CC homozygous based on a C101G SNP (Single Nucleotide Polymorphism) site; the C101G SNP site is the 101 nucleotide from the 5'terminal of SEQ ID NO.4 in a wheat genome. The molecular marker has important theoretical significance and economic value for molecular marker-assisted selection of wheat germplasm or breeding progeny materials with high precipitation value.

Description

Technical Field

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

[0002] Wheat (Triticum aestivum L.) is the most widely cultivated and consumed crop in the world, and it is also one of the three major food crops in China, which has important strategic significance for 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 current wheat breeding in China. According to the current "Classification Standard for Wheat Variety Quality (GB / T 17320-2013)" in China, wheat quality indicators mainly include hardness, crude protein content (grains), wet gluten content, sedimentation value, water absorption, stability time, maximum tensile resistance, energy (wheat flour), etc.

[0003] The sedimentation value determination is fast, the method is simple, and it can reflect the content and quality of gluten proteins. It is an extremely important evaluation index for wheat quality. In addition, the sedimentation value has high heritability and can be used as an index for early generation screening in wheat quality breeding. The sedimentation value shows quantitative trait inheritance, and its heredity is controlled by multiple genes, and the gene action is mainly based on the additive effect of multiple genes. At present, some QTLs related to wheat sedimentation value have been mapped. However, due to the influence of factors such as mapping population, genetic background, and mapping methods, the results can only reflect the gene information contained in specific wheat varieties, and most QTLs have a small contribution rate to the phenotype and poor repeatability among different environments. Therefore, it still cannot meet the needs of marker-assisted selection.

[0004] Marker-assisted selection is a modern breeding method that uses DNA molecular markers tightly linked to target traits to perform genotype selection on target traits, and has the advantage of being unaffected 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 target alleles, and different fluorescent groups are added to the primer ends. Based on the reading of terminal fluorescence signals of PCR, the target sequence is typed, which has the advantages of high efficiency, accuracy, and low cost, and has broad application prospects in crop breeding. Therefore, developing KASP markers for detecting wheat sedimentation value under multiple environments can provide an effective detection means for the breeding of new high-quality wheat varieties, which is of great significance for improving the level of high-quality wheat breeding in China. 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 sedimentation value of wheat in multiple environments and its application.

[0006] 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, and the sedimentation value of wheat with genotype qSV6A.1b > the sedimentation value of wheat with genotype qSV6A.1a;

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

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

[0010] The C101G SNP locus 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, performing PCR amplification with a primer combination to obtain a PCR amplification product;

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

[0014] (a2) After completing step (a1), using an instrument to detect the fluorescence signal of the PCR amplification product, and obtaining the genotype of the wheat to be tested according to the color of the fluorescence 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, performing PCR amplification with a primer combination to obtain a PCR amplification product;

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

[0018] (b2) Take the PCR amplification product obtained in step (b1) and sequence it.

[0019] (b3) According to the sequencing result obtained in step (b2), obtain the genotype of the wheat to be tested.

[0020] A kit for identifying or assisting in the identification of the sedimentation value of wheat, comprising substances 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 a GG homozygous genotype based on the C101G SNP locus;

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

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

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

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

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

[0027] Use of the above kit or the above molecular marker in identifying or assisting in the identification of the sedimentation value of wheat.

[0028] Use of the above kit or the above molecular marker in screening or assisting in the screening of wheat with different sedimentation values.

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

[0030] Use of the primer combination in the directional cultivation or assisted directional cultivation of wheat lines with high sedimentation values, wherein the primer combination consists of the upstream primer F1 shown in SEQ ID NO: 1, the upstream primer F2 shown in SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3.

[0031] The beneficial effects of adopting the above technical solutions are as follows: The present invention provides the KASP marker Kasp_qSV6A.1 for identifying the allelic variations of qSV6A.1a and qSV6A.1b and its correlation with the sedimentation value of wheat. Applying the KASP marker in the present invention to the molecular marker-assisted selection of the sedimentation value of wheat can quickly and efficiently screen out wheat varieties (germplasms) with a high sedimentation value, thereby accelerating the breeding process of new high-quality wheat varieties. The present invention has important theoretical significance and economic value for using molecular marker-assisted selection to select wheat germplasms or breeding offspring materials with a high sedimentation value. Description of the Drawings

[0032] Figure 1 It is a schematic diagram of the KASP primer positions of different allelic types of the common wheat sedimentation value QTL qSV6A.1 on chromosome 6A; the sequence in the figure is the sequence of the physical position 63057734bp - 63057933bp of wheat chromosome 6A, and 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 It is a schematic diagram of the detection results of the genotyping of 329 wheat germplasms in the natural population. In the figure, CC is the CC homozygous type, that is, the genotype qSV6A.1a; GG is the GG homozygous type, that is, the genotype qSV6A.1b; CK is the negative control;

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

[0035] The following examples illustrate the present invention in detail. All kinds of raw materials and various equipment used in the present invention are conventional commercially available products and can be directly obtained through market 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 specification of the present application and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their combinations.

[0037] It should also be understood that the term "and / or" used in the specification of the present application and the appended claims refers to any combination and all possible combinations of one or more of the related listed items, and includes these combinations.

[0038] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that specific features, structures, or characteristics described in connection with that embodiment are included in one or more embodiments of this application. Thus, statements such as "in one embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments", etc. that appear in different places in this specification do not necessarily all refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in another way. The terms "comprising", "including", "having" and their variants all mean "including but not limited to", unless otherwise specifically emphasized in another way.

[0039] In addition, in the description of this application's specification and the appended claims, the terms "first", "second", "third", etc. are only used for distinguishing descriptions and should not be construed as indicating or implying relative importance.

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0041] Example 1: Detecting different allelic types of wheat sedimentation value QTL qSV6A.1 using the KASP marker Kasp_qSV6A.1

[0042] Detecting different allelic types of wheat sedimentation value QTL qSV6A.1 at the physical position of 63057834 bp on chromosome 6A (referring to the Chinese Spring wheat genome IWGSC RefSeq v1.0) using the KASP marker Kasp_qSV6A.1 is divided into the following two steps: PCR amplification and genotyping. The experimental methods in the examples are all conventional methods unless otherwise specified. All common wheat germplasm materials are preserved in the Wheat Research Center of the Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences.

[0043] (1) PCR amplification system and procedure

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

[0045] Preparation of KASP marker primer working solution: Two KASP upstream primers were designed according to the SNP at the physical position of 63057834 bp of the wheat sedimentation value QTL qSV6A.1 in the wheat genome version IWGSC RefSeq v1.0. The polymorphism of this SNP site is the base difference between C and G. FAM and HEX fluorescent linker sequences were added to the 5' end of the primers respectively. At the same time, a common KASP downstream primer was designed. The primer sequences are shown in Table 1. The KASP marker primer working solution includes: 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. After mixing, it was stored at -20 °C for later use.

[0046] Table 1 KASP marker primer sequence list for identifying allelic variations of common wheat QTL qSV6A.1

[0047]

[0048]

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

[0050] PCR reaction procedure: 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 is 61 °C, and it decreases 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, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) were used to convert the fluorescence signal into an analyzable value. The fluorescence scanning results were graphically displayed using GraphPad Prism. qSV6A.1a (CC homozygous type) carried FAM fluorescence and was distributed near the x-axis; qSV6A.1b (GG homozygous type) carried HEX fluorescence and was distributed near the y-axis; the negative control (CK) had no detected signal and was distributed near the origin (as Figure 2 ).

[0053] Example 2 Detection of the sedimentation value of common wheat germplasm materials using the KASP marker Kasp_qSV6A.1

[0054] Three hundred and twenty-nine Chinese wheat germplasm materials were planted at the Dishang Experimental Station of the Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences for two consecutive years (2020 - 2021, 2021 - 2022). Each year, they were sown under three environments: normal water and fertilizer (one irrigation of 50 m 3 / mu at jointing stage and filling stage, 12 Kg / mu of nitrogen fertilizer), drought treatment (no irrigation throughout the growth period, 12 Kg / mu of nitrogen fertilizer), and low-nitrogen treatment (one irrigation of 50 m 3 / mu at jointing stage and filling stage, 6 Kg / mu of nitrogen fertilizer). The plot length was 3 m, and a randomized block design was used with three replicates.

[0055] After harvesting, the wheat grains were ground into flour using an experimental mill (Brabender), and the sedimentation value of the flour was determined according to the agricultural industry standard "Determination of Wheat Sedimentation Value - Zeleny Method (NY / T 1095 - 2006)". Through KASP marker detection, among the 329 Chinese wheat germplasm materials, 184 germplasms were of the qSV6A.1a allele type, and 145 germplasms were of the qSV6A.1b allele type. The allele types of wheat germplasm materials and the sedimentation values under different years and environments are shown in Table 2.

[0056] Table 2 Detection results of Kasp_qSV6A.1 markers for wheat germplasm and sedimentation values under 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 that the average sedimentation value data is missing.

[0076] The statistical results show that the mean sedimentation value of wheat germplasm materials carrying the allele qSV6A.1b is higher than that of wheat germplasm materials carrying the allele qSV6A.1a in different years and different environments, and the difference between the two reaches an extremely significant level (P<0.01) (as shown in Table 3, Figure 3 ).

[0077] Table 3 Statistical analysis of the relationship between allelic variation types of common wheat QTL qSV6A.1 and sedimentation value

[0078]

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

[0080] Through a large number of experiments, the inventors of the present invention found an SNP locus in the wheat genome, named the C101G SNP locus. The C101G SNP locus is located at the 101st position 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 position of C101G SNP is at the 63057834th position on chromosome 6A. According to the SNP (C / G) difference site, a KASP marker Kasp_qSV6A.1 for identifying the sedimentation value of wheat quality index was developed. The allele type qSV6A.1a (CC homozygous) that carries FAM fluorescence and is distributed near the x-axis is the allele type that reduces the wheat sedimentation value, and the allele type qSV6A.1b (GG homozygous) that carries HEX fluorescence and is distributed near the y-axis is the allele type that increases the wheat sedimentation value. Using 329 Chinese wheat germplasm materials to verify this marker for two consecutive years in three environments of normal water and fertilizer, drought treatment and low nitrogen treatment, the results show that this marker can effectively and accurately genotype the two allele types of qSV6A.1a and qSV6A.1b. The present invention provides an effective detection method for accurately and quickly screening wheat germplasm materials with high sedimentation value in multiple environments.

[0081] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions, and variations can be made to these examples without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0082] In the above embodiments, the descriptions of the various embodiments have their own emphases. For parts not detailed or recorded in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0083] The above-described embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent substitutions for some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention and should all be included within the protection scope of the present invention.

Claims

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

2. The method according to claim 1, wherein The steps for 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, performing PCR amplification with a primer combination to obtain a PCR amplification product; The primer combination consists of the upstream primer F1 shown in SEQ ID NO: 1, the upstream primer F2 shown in SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3; (a2) After completing step (a1), using an instrument to detect the fluorescence signal of the PCR amplification product, and obtaining the genotype of the wheat to be tested according to the color of the fluorescence signal.

3. The method according to claim 1, characterized in that The steps for 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, performing PCR amplification with a primer combination to obtain a PCR amplification product; The primer combination consists of the upstream primer F1 shown in SEQ ID NO: 1, the upstream primer F2 shown in SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3; (b2) Taking the PCR amplification product obtained in step (b1) and sequencing; (b3) According to the sequencing result obtained in step (b2), obtaining the genotype of the wheat to be tested.

4. A kit for identifying or assisting in the identification of the sedimentation value of wheat, characterized in that, 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 a GG homozygous genotype based on the C101G SNP locus; The genotype qSV6A.1a is a CC homozygous genotype based on the C101G SNP locus; The C101G SNP locus is the 101st nucleotide from the 5'-end of SEQ ID NO: 4 in the wheat genome.

5. 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 the upstream primer F1 shown in SEQ ID NO: 1, the upstream primer F2 shown in SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO:

3.

6. The molecular marker shown in SEQ ID NO:

4.

7. Use of the kit according to any one of claims 4 to 5 or the molecular marker according to claim 6 in identifying or assisting in identifying the sedimentation value of wheat.

8. Use of the kit according to any one of claims 4 to 5 or the molecular marker according to claim 6 in screening or assisting in screening wheat with different sedimentation values.

9. Use of the kit according to any one of claims 4 to 5 or the molecular marker according to claim 6 in wheat breeding.

10. Use of the primer combination in the directional cultivation or assisting in the directional cultivation of wheat lines with high sedimentation values, wherein the primer combination consists of the upstream primer F1 shown in SEQ ID NO: 1, the upstream primer F2 shown in SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3.

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