KASP primers for identifying wheat grain hardness Pinu-D1e and Pinb-D1i genes and application of KASP primers for identifying wheat grain hardness Pinu-D1e and Pinb-D1i genes

Abstract

The invention discloses KASP primers for identifying wheat grain hardness Pinu-D1e and Pinb-D1i genes and application of the KASP primers, the KASP primers comprise two groups, namely six primers, and the nucleotide sequences of the primers are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively. The KASP molecular marker obtained by amplification of the KASP primer is a co-dominant marker. The KASP primer provided by the invention can accurately track the wheat grain hardness Pinu-D1e gene and Pinb-D1i gene, can improve the accuracy of soft wheat creation, can be used for wheat quality breeding, and greatly accelerates the breeding process of high-quality wheat varieties.

Description

Technical Field

[0001] The invention relates to the technical field of molecular genetic breeding, and in particular to a KASP primer for identifying wheat grain hardness Pina-D1e and Pinb-D1i genes and an application thereof in wheat germplasm identification and wheat breeding. Background Art

[0002] Wheat is the third largest food crop, accounting for about 40% of the world's population's staple food, and provides more than 20% of energy and protein. Grain hardness is an important parameter for evaluating wheat quality, and is also an important evaluation indicator for wheat classification, grading, pricing and processing, and determines the final use of wheat. It not only affects the processing quality and nutritional quality of wheat, but also has an important impact on its deep processing. With the improvement of people's living standards, the demand for high-quality wheat continues to grow, resulting in a structural imbalance in the supply and demand relationship. Therefore, it is necessary to introduce exogenous favorable genes through distant hybridization, strengthen the exploration of excellent resources of grain hardness genes, improve wheat quality, and meet people's demand for high-quality life.

[0003] SNP (Single Nucleotide Polymorphism) is a small variation at a specific position in a DNA sequence, which includes the replacement, inversion, increase or decrease of nucleotides. These SNP sites can be located by comparing known DNA sequences. Specific primers are designed based on these variation sites, and then PCR amplification is performed on DNA or its complementary sequence to produce genetic polymorphism products based on SNP. The genetic variation pattern of these products can be revealed through electrophoresis analysis. As a genetic marker, SNP has significant application value for genetic research due to its wide distribution in the genome, large number and easy-to-estimate allele frequency.

[0004] KASP (Kompetitive Allele Specific PCR) is a cost-effective, easy-to-use, and highly accurate genotyping technology suitable for high-throughput and automated gene detection. It has been widely used in germplasm resource identification and kinship research, molecular marker-assisted breeding, genetic map construction and gene localization, and seed purity identification of crops such as wheat, rice, and soybean.

[0005] Wheat grain hardness is a quantitative trait with multiple quantitative trait loci (QTLs). The main QTL locus is controlled by the Ha (hardness) locus located in the subterminal region on the short arm of chromosome 5D. The Ha locus is composed of Pina and Pinb. When both Pina and Pinb are wild type, the grain is soft. When one of them is missing or mutated, the grain is hard, and softness is dominant over hardness. Aegilops tauschii (2n=2x=14, DD) is an annual plant of the genus Aegilops and the donor of the D genome of common wheat. It has many Pina and Pinb allele variants such as Pina-D1c, Pina-D1d, Pina-D1e, Pina-D1f, Pina-D1g, Pina-D1h, Pinb-D1h, Pinb-D1i, Pinb-D1k, Pinb-D1m, Pinb-D1n, Pinb-D1o. These variants are multi-point mutations and show soft texture. In addition, the allele Pina-D1e / Pinb-D1i (coding protein PINAc / PINBh) from Aegilops tauschii can be expressed in artificial wheat, and the grains are softer than those containing Pina-D1a / Pinb-D1a (coding protein PINAa / PINBa). Studies have shown that the PIN gene may regulate grain size while affecting grain hardness. Therefore, the grain hardness genes Pina and Pinb of Aegilops tauschii can be used to improve the grain hardness trait of common wheat. Using synthetic wheat as a bridge and combining molecular marker-assisted breeding, they can be transferred to common wheat to breed wheat varieties with softer grains. Summary of the invention

[0006] The purpose of the present invention is to provide a KASP primer for identifying wheat grain hardness Pina-D1e and Pinb-D1i genes and their application, which can quickly identify wheat plants containing Pina-D1e and Pinb-D1i genes in a population.

[0007] In order to achieve the above object, the present invention provides a KASP primer linked to the wheat grain hardness Pina-D1e gene and Pinb-D1i gene, which can realize the identification of wheat grain hardness, assist and accelerate wheat germplasm identification and wheat breeding efficiency.

[0008] The present invention provides a KASP primer for identifying wheat grain hardness Pina-D1e and Pinb-D1i genes. The KASP primer comprises 6 primers whose nucleotide sequences are shown as SEQ ID NOs.1 to 3 and SEQ ID NOs.4 to 6, wherein the polymorphism of the KASP primer for identifying the Pina-D1e gene is T / C, and the polymorphism of the KASP primer for identifying the Pinb-D1i gene is G / A.

[0009] The present invention also provides a kit for identifying wheat grain hardness genes, which comprises six primers with nucleotide sequences as shown in SEQ ID NOs. 1 to 3 and SEQ ID NOs. 4 to 6. The kit can be used for identifying wheat grain hardness genes, wherein the grain hardness genes are Pina-D1e and Pinb-D1i genes.

[0010] The KASP primers or kit provided by the invention can be used in wheat breeding, including the creation of wheat lines with different grain hardness.

[0011] The present invention also provides a method for identifying wheat grain hardness, comprising the following steps:

[0012] (1) Extracting genomic DNA of the wheat sample to be tested using the CTAB method;

[0013] (2) Using the extracted DNA as a template, PCR amplification was performed using the above 6 KASP primers; the reaction system was 10 μL: 5 μL KASP Mastermix, 1.4 μL primer mixture, 100 ng genomic DNA, and 2.6 μL ddH2O. Amplification program: 94°C for 15 min, 10 cycles of 20 s at 94°C, 60 s at 65-57°C, 32 cycles of 20 s at 94°C, and 60 s at 57°C;

[0014] (3) Detect the fluorescence of the PCR amplification product. When the fluorescence of the amplification product is yellow, it means that the grain hardness gene of the wheat is Pina-D1e / Pinb-D1i and is homozygous; when the fluorescence of the amplification product is green, it means that the wheat has the Pina-D1e / Pinb-D1i grain hardness gene and is heterozygous; when the fluorescence of the amplification product is blue, it means that the wheat does not have the Pina-D1e / Pinb-D1i grain hardness gene.

[0015] The present invention has the following advantages:

[0016] The present invention discloses for the first time molecular marker primers for accurately detecting the grain hardness genes Pina-D1e and Pinb-D1i from Aegilops tauschii AS60 based on a fluorescent quantitative PCR platform, and the nucleotide sequences thereof are shown in SEQ ID NO.1 to 6, respectively. Among them, the KASP primers for the Pina-D1e gene can distinguish Pina-D1a, Pina-D1c, and Pina-D1d; the KASP primers for the Pinb-D1i gene can distinguish Pinb-D1a and Pinb-D1h. And it is a co-dominant marker, with accurate and efficient detection, convenient and stable amplification.

[0017] The KASP molecular marker primer disclosed in the present invention is extremely significantly correlated with the grain hardness genes Pina-D1e and Pinb-D1i, presents linkage marker characteristics, and has high accuracy and success rate for molecular marker and primer-assisted selection. It can be used for the identification of wheat grain hardness, assist and accelerate wheat germplasm identification and wheat breeding efficiency, and has significant practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the KASP molecular marker typing result of the Pina-D1e gene in the present invention.

[0019] Figure 2 This is the KASP molecular marker typing result of the Pinb-D1i gene in the present invention. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0022] The wheat seed resources used in the embodiments of the present invention are all from the germplasm resource bank of Professor Zhang Lianquan of Sichuan Agricultural University.

[0023] Wheat grain hardness is a quantitative trait with multiple quantitative trait loci (QTLs). The main QTL locus is controlled by the Ha (hardness) locus located in the subterminal region on the short arm of chromosome 5D. The Ha locus is composed of Pina and Pinb. When both Pina and Pinb are wild type, the grain is soft. When one of them is missing or mutated, the grain is hard, and softness is dominant over hardness. Aegilops tauschii (2n=2x=14, DD) is an annual plant of the genus Aegilops and the donor of the D genome of common wheat. It has many Pina and Pinb allele variants such as Pina-D1c, Pina-D1d, Pina-D1e, Pina-D1f, Pina-D1g, Pina-D1h, Pinb-D1h, Pinb-D1i, Pinb-D1k, Pinb-D1m, Pinb-D1n, Pinb-D1o. These variants are multi-point mutations and show soft quality. In addition, the Aegilops tauschii allele type Pina-D1e / Pinb-D1i (coding protein PINAc / PINBh) can be expressed in artificial synthetic wheat, and the grains are softer than Pina-D1a / Pinb-D1a (coding protein PINAa / PINBa), and are superior to other allele types in grain length and grain width. Therefore, the grain hardness genes Pina-D1e and Pinb-D1i of Aegilops tauschii can be used to identify and improve the grain hardness trait of common wheat. With synthetic wheat as a bridge, they can be transferred to common wheat in combination with molecular marker-assisted breeding to cultivate wheat varieties with softer grains. To date, the Pina-D1e and Pinb-D1i genes only exist in Aegilops tauschii and synthetic wheat and have not been used in wheat production. This study was the first to discover the presence of the Pina-D1e and Pinb-D1i genes in the new wheat variety Shumai 830 bred by improving synthetic wheat. Therefore, the present invention provides a KASP primer linked to the wheat grain hardness Pina-D1e gene and Pinb-D1i gene, which can realize the identification of wheat grain hardness, assist and accelerate wheat germplasm identification and wheat breeding efficiency.

[0024] Experimental Example 1 Development of KASP primers for wheat grain hardness Pina-D1e gene and Pinb-D1i gene

[0025] 1. Use NCBI (https: / / www.ncbi.nlm.nih.gov / ) to download different alleles of wheat Pina gene and Pinb gene, including Pina-D1a (DQ363911), Pina-D1c (AY649746), Pina-D1e (AY252034), Pina-D1g (AY252029), Pina-D1i (AY573899), Pina-D1m (EF620907), Pina-D1n (EF620908), Pina-D1o (KP136427), Pina-D1p (AY599893), Pina-D1q (AB181238), Pina-D1t (JN680739), Pina-D1u (KJ446781), Pina-D1v (KJ44678 0), Pina-D1w (KJ446779); Pinb-D1a (KM189132), Pinb-D1aa (EF620912), Pinb-D1ab (AB3028 94), Pinb-D1ad(KT885199), Pinb-D1b(DQ363914), Pinb-D1i(AY649747), Pinb-D1p(AY58188 9), Pinb-D1q(WF620909), Pinb-D1r(AJ619022), Pinb-D1s(AJ619021), Pinb-D1t(EF620910 ), Pinb-D1u(EF620911), Pinb-D1v(AY598029), Pinb-D1w(AY640304), Pinb-D1x(AM909618).

[0026] DNAMAN software was used for comparison, and KASP markers were developed based on the SNP differences between different allele types, and compared with the Chinese Spring reference genome (IWGSC Reference Sequence v1.2). Among them, the 66th base of Pina-D1e is T, and the base of this site in other Pina allele types is C; the 48th base of Pinb-D1i is G, and the base of this site in other Pinb allele types is A. Specific KASP primers were designed according to the differences in SNP sites, and the sequences are shown in Table 1 below.

[0027] Table 1 KASP primer sequences

[0028]

[0029] 2. The designed primers were used to verify the results in different materials, including: (1) 2 wheat varieties: Chinese Spring and Shumai 830; (2) 14 Aegilops tauschii materials (materials from the germplasm resource bank of Professor Zhang Lianquan of the State Key Laboratory of Sichuan Agricultural University): AS87, AS90, AS92, AS94, AS95, AS96, AS2386, AS2387, AS2388, AS2389, AS2392, AS60, AS63, AS69; (3) 18 breeding isolates (material sources: Shumai 2278 / Mianmai 902 / / Shumai 114F2, among which Shumai 2278 contains Pina -D1e gene and Pinb-D1i gene, Mianmai 902 and Shumai 114 contain Pina-D1a gene and Pinb-D1a gene): WJZ583-1, WJZ583-2, WJZ583-3, WJZ583-4, WJZ583-5, WJZ583-6, WJZ583-7, WJZ583-8, WJZ583-9, WJZ583-10, WJZ583-11, WJZ583-12, WJZ583-13, WJZ583-14, WJZ583-15, WJZ583-16, WJZ583-17, WJZ583-18.

[0030] Extract the DNA of the above materials as a template, the specific method is as follows:

[0031] (1) Take 0.5 g of the leaves of the material to be tested and put them into a 2 mL centrifuge tube and add steel balls.

[0032] (2) Soak the centrifuge tube in liquid nitrogen for about 1 min, and then grind it in a tissue grinder (30 hours, 30 seconds).

[0033] (3) After the sample is ground into powder, add 600 μL of preheated (65°C) CTAB buffer, shake well, and place in a 65°C water bath for 25 min, shaking every 5 min.

[0034] (4) After incubation in a water bath, add 600 μL of chloroform to the centrifuge tube, shake well, and let stand at room temperature for 10 min.

[0035] (5) Centrifuge the sample at 12000 rpm, 4°C for 10 min.

[0036] (6) The supernatant solution was transferred to a new 1.5 mL centrifuge tube and 800 μL of pre-chilled (-20°C) isopropanol was added.

[0037] (7) Centrifuge the sample at 12,000 rpm, 4°C for 10 min.

[0038] (8) Discard the supernatant and wash the precipitate twice with 800 μL of anhydrous ethanol.

[0039] (9) After the precipitate was dried in a fume hood, 100 μL of ddH2O was added.

[0040] The Pina gene and Pinb gene in the materials were amplified and sequenced, and the amplification primers were shown in Table 2 below; it was found that the allele types of the Pina gene and Pinb gene contained in 35 materials were shown in Table 3 below, and were verified using the KASP molecular marker developed by the present invention.

[0041] Table 2 Primers for amplification of Pina gene and Pinb gene

[0042]

[0043] Table 3 Allele types of Pina gene and Pinb gene in 35 materials

[0044]

[0045]

[0046] The typing results of the two sets of KASP primers are as follows: Figure 1 and Figure 2 As shown, Figure 1 This is the result of KASP molecular marker typing of the Pina-D1e gene. The fluorescence of the amplified product in the figure is yellow fluorescence, which means that the 66th base of the Pina gene contained in the plant is T, and its grain hardness gene is Pina-D1e, and it is homozygous; the fluorescence of the amplified product is green fluorescence, which means that the 66th base of the Pina gene contained in one 5D chromosome of the plant is T, and the 66th base of the Pina gene contained in the other 5D chromosome is C, and the Pina-D1e grain hardness gene exists, and it is heterozygous; when the fluorescence of the amplified product is blue fluorescence, it means that the 66th base of the Pina gene contained in the plant is C, and the Pina-D1e grain hardness gene does not exist.

[0047] Figure 2 This is the result of KASP molecular marker typing of Pinb-D1i gene. The fluorescence of the amplified product in the figure is yellow fluorescence, which means that the 48th base of Pinb gene contained in the plant is G, and its grain hardness gene is Pinb-D1i, and it is homozygous; the fluorescence of the amplified product is green fluorescence, which means that the 48th base of Pinb gene contained in one 5D chromosome of the plant is G, and the 48th base of Pinb gene contained in the other 5D chromosome is A, and Pinb-D1i grain hardness gene exists, and it is heterozygous; the fluorescence of the amplified product is blue fluorescence, which means that the 48th base of Pinb gene contained in the plant is A, and Pinb-D1i grain hardness gene does not exist.

[0048] In summary, the present invention identified wheat grain hardness Pina-D1e and Pinb-D1i for the first time, and the KASP primers provided by the present invention can quickly identify Pina-D1e and Pinb-D1i genes, and can quickly screen out individual plants with Pina-D1e and Pinb-D1i, which can improve the accuracy of soft wheat identification. That is, when Pina-D1e and Pinb-D1i genes exist in wheat plants at the same time, the grains appear soft, otherwise they are all hard. It can be seen that by identifying wheat germplasm through the KASP primers provided by the present invention, the hardness of wheat grains can be quickly identified, which can be applied to the creation of wheat lines with different grain hardness, and can also be used in wheat breeding, greatly accelerating the breeding process of high-quality wheat varieties.

[0049] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A KASP primer for identifying wheat grain hardness Pina-D1e and Pinb-D1i genes, characterized in that: The KASP primers include 6 primers with nucleotide sequences as shown in SEQ ID NOs. 1 to 3 and SEQ ID NOs. 4 to 6, wherein the polymorphism of the KASP primers for identifying the Pina-D1e gene is T / C, and the polymorphism of the KASP primers for identifying the Pinb-D1i gene is G / A.

2. A kit for identifying wheat grain hardness genes, characterized in that: The kit comprises the KASP primers as claimed in claim 1, wherein the grain hardness genes are Pina-D1e and Pinb-D1i genes.

3. Use of the KASP primers according to claim 1 or the kit according to claim 2 in identifying wheat grain hardness genes.

4. Use of the KASP primer according to claim 1 or the kit according to claim 2 in wheat breeding.

5. The use according to claim 4, characterized in that: The application includes the creation of wheat lines with different kernel hardness.

6. A method for identifying wheat grain hardness, characterized in that: The following steps are included: (1) Extracting genomic DNA of the wheat sample to be tested using the CTAB method; (2) Using the extracted DNA as a template, PCR amplification was performed using the KASP primers described in claim 1; the reaction system was 10 μL: 5 μL KASP Master mix, 1.4 μL primer mixture, 100 ng genomic DNA, and 2.6 μL ddH2O. Amplification program: 94°C for 15 min, 10 cycles of 20 s at 94°C, 60 s at 65-57°C, 32 cycles of 20 s at 94°C, and 60 s at 57°C; (3) Detect the fluorescence of the PCR amplification product. When the fluorescence of the amplification product is yellow, it means that the grain hardness gene of the wheat is Pina-D1e / Pinb-D1i and is homozygous; when the fluorescence of the amplification product is green, it means that the wheat has the Pina-D1e / Pinb-D1i grain hardness gene and is heterozygous; when the fluorescence of the amplification product is blue, it means that the wheat does not have the Pina-D1e / Pinb-D1i grain hardness gene.

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