KASP primers tightly linked to wheat solid stem suppression gene and their application

Abstract

The invention discloses a KASP primer closely linked to a wheat solid stem inhibition gene and an application thereof, and relates to the field of genetic breeding technology. The KASP primer comprises three primers whose nucleotide sequences are respectively shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6. The KASP molecular marker KASP-669 obtained by amplifying the KASP primer is a co-dominant marker. The KASP primer provided by the invention can accurately track the wheat solid stem inhibition gene, identify the properties of wheat stalks, improve the accuracy of solid stem wheat creation, can be used for wheat lodging resistance breeding, and greatly accelerate the breeding process of solid stem wheat varieties.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding, and in particular to a KASP primer tightly linked to a wheat solid stem inhibition gene and an application thereof. Background Art

[0002] my country is a major wheat producer and consumer. Due to the increase in population, decrease in land, and environmental degradation, the pressure of food demand continues to increase, so we must work hard to improve wheat yield and quality. Common wheat can be improved by introducing exogenous beneficial genes through distant hybridization, but exogenous beneficial genes sometimes cannot be expressed or cannot be fully expressed in common wheat. Therefore, discovering and utilizing new inhibitory genes and analyzing the mechanism of action of inhibitory genes can promote the effective use of exogenous beneficial genes.

[0003] Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by changes such as conversion, transversion, insertion, and deletion at a specific nucleotide position in the genome. The technology uses known sequence information to compare and find SNP sites, and then uses the discovered variant sites to design specific primers to perform PCR amplification on genomic DNA or cDNA to obtain specific polymorphic products based on SNP sites, and finally uses electrophoresis technology to analyze the polymorphism of the products. The advantages of SNP markers are large number and wide distribution; uneven distribution in a single gene and the entire genome; and easy estimation of SNP allele frequency.

[0004] KASP is a competitive allele-specific PCR technology (Kompetitive Allele Specific PCR, KASP) developed by LGC (Laboratory of the Government Chemist, http: / / www.lgcgenomics.com). It is a new genotyping technology with low cost and high throughput. It can accurately perform double allele typing of SNP and In Del sites through specific matching of primer terminal bases. It has been widely used in molecular marker-assisted selection of crops such as rice, wheat, and soybean.

[0005] When scientists introduce exogenous beneficial genes into common wheat, the phenomenon of weakened or even lost expression of exogenous genes has occurred many times. So far, most of the reported suppressor genes come from multiple chromosomes of the D genome, including the suppressor gene Su-Pm21 for powdery mildew resistance, the suppressor gene Su-Lr23 for leaf rust resistance, and the suppressor gene SuSr-D1 for stem rust resistance. Aegilops tauschii (2n=2x=14, DD) is the donor of the D genome of common wheat and an important gene source for genetic improvement of common wheat. However, so far, the solid stem suppressor gene of tetraploid wheat has only been reported on chromosome 3D of common wheat. However, the location and mechanism of suppressor genes are still very limited. So far, only one stem rust suppressor gene SuSr-D1 has been located on 7DL of common wheat Canthatch, which mediates stem rust resistance by encoding a subunit of the mediator complex. However, related research is still relatively small, so it is necessary to explore more suppressor genes and analyze the mechanism of action and functional pathways of more suppressor phenomena. Summary of the invention

[0006] The purpose of the present invention is to provide a KASP primer tightly linked to a wheat solid stem inhibition gene and an application thereof. The KASP primer comprises three primers, which can accurately track the wheat solid stem inhibition gene, identify the properties of wheat stalks, improve the accuracy of solid stem wheat creation, create lodging-resistant wheat breeding with solid stems, and accelerate the selection process of solid stem wheat varieties.

[0007] In order to achieve the above object, the present invention provides a KASP primer tightly linked to a wheat solid stem inhibition gene, the KASP primer comprising three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively;

[0008] Among them, the KASP primer was developed from the SNP site on the short arm of wheat chromosome 7D located in the wheat genome version RefSeqv1.0; the SNP site is located at a physical position of 6.70Mbp, and its polymorphism is C / T. The KASP primer can be used to identify the traits of wheat stems.

[0009] The present invention also provides a KASP molecular marker KASP-669 obtained by amplification of the above KASP primers.

[0010] The present invention also provides a method for identifying wheat stem traits, comprising the following steps:

[0011] (1): Extracting genomic DNA from the wheat sample to be tested;

[0012] (2): Using the extracted DNA as a template, PCR amplification was performed using the above-mentioned KASP primers;

[0013] (3): Detect the fluorescence of the PCR amplification product. When the fluorescence of the amplification product is blue fluorescence, it means that the stem trait of the wheat is a solid stem; when the fluorescence of the amplification product is yellow fluorescence, it means that the stem trait of the wheat is a hollow stem (homozygous); when the fluorescence of the amplification product is green fluorescence, it means that the stem trait of the wheat is a hollow stem (heterozygous).

[0014] The invention also provides a kit for identifying wheat stalk traits, and the kit comprises the above-mentioned KASP primers.

[0015] The KASP primers provided by the present invention can be used in wheat breeding, including screening wheat with solid stem traits or creating wheat lines with solid stem traits.

[0016] The KASP primers of the present invention, which are closely linked to the wheat solid stem inhibition gene, can identify whether wheat plants contain the solid stem inhibition gene, can predict the stalk traits of wheat, solve the problem of difficult detection of wheat stalk traits, improve the accuracy of solid stem wheat creation, and have the following advantages:

[0017] The present invention discloses for the first time a wheat solid stem inhibition gene Su-TdDof from Aegilops tauschii AS92, which is located on the short arm of artificial wheat 7D chromosome. The gene significantly inhibits the expression of solid stem genes and has important utilization value in the creation of solid stem wheat and lodging resistance breeding.

[0018] The present invention discloses for the first time a molecular marker KASP-669 for accurately detecting Aegilops tauschii AS92 based on a fluorescent quantitative PCR platform, which is a co-dominant marker with accurate and efficient detection and convenient and stable amplification.

[0019] The molecular marker KASP-669 disclosed in the present invention is extremely significantly correlated with the solid stem inhibition gene Su-TdDof, presents a tightly linked marker feature, and is used for molecular markers and primer-assisted selection with high accuracy and success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the location map of the KASP molecular marker and the solid stem inhibition gene Su-TdDof in the present invention on wheat chromosome 7D.

[0021] Figure 2 The KASP typing results of 139 strains were obtained by using three KASP primers of KASP-669 in the present invention. DETAILED DESCRIPTION

[0022] 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.

[0023] Experimental Example 1: Obtaining the solid stem suppression gene Su-TdDof, KASP molecular markers and KASP primers

[0024] 1) Group family creation

[0025] The present invention uses durum wheat Ma to hybridize with two Aegilops tauschii AS92 and AS96, respectively, and performs chromosome doubling under natural conditions to create artificially synthesized wheat and name them wheat Syn-SAU-117 and Syn-SAU-119, respectively. Ma is a semi-dry durum wheat provided by George Fedak of the Ottawa Research and Development Center in Canada, and Aegilops tauschii is stored in the Wheat Research Institute of Sichuan Agricultural University with the code AS. Wheat Syn-SAU-117 is used as the female parent and wheat Syn-SAU-119 is used as the male parent to hybridize to obtain the hybrid F1 generation, the F1 generation individual plant is self-pollinated to obtain the F2 generation, and the F2 generation is self-pollinated to obtain the F 2:3 The family,F2 population size contained 139 strains, named Syn-7D-1, Syn-7D-2~......~Syn-7D-139, to constitute the genetic mapping population.

[0026] 2) Stem phenotype identification

[0027] The parent materials Syn-SAU-117, Syn-SAU-119 and F1, F2, F 2:3 The families were planted at the Wenjiang Huihe base with a plant spacing of 10 cm, a row length of 2 m, and a row spacing of 30 cm to identify the solidity of the stems in the field. At the wheat jointing stage Zadoks Stage 34, the main tillers of the material were taken, the middle of the second internode at the base of the stem was cut, and the solidity of the cross section was identified. The stem diameter and medullary cavity diameter of the stem were measured, and the medullary cavity ratio (the ratio of medullary cavity diameter to stem diameter) was used to reflect the solidity of the stem. At the same time, the medullary cavity was graded according to the fullness of the medullary tissue: 1 was completely hollow and 5 was completely solid.

[0028] 3) Transcriptome data analysis

[0029] (a) Sample collection and RNA extraction: 20 homozygous plants with completely solid second internodes at the base of the stems of the parents Syn-SAU-117, Syn-SAU-119 and F2 populations and 20 homozygous plants with completely hollow second internodes at the base of the stems were sampled at the jointing stage Zadoks Stage 34, when the first four nodes appeared on the stems. The second internode at the base was measured from the bottom of the lowest node to the uppermost node for about 0.5 cm of the stem. The samples were immediately placed in 1.5 mL microcentrifuge tubes and stored in dry ice for sample delivery. They were sent to Chengdu Tiancheng Future Technology Co., Ltd. (https: / / www.tcuni.com / ) for RNA extraction and sample mixing.

[0030] (b) Transcriptome data analysis: The samples taken in step (a) were sent to Chengdu Tiancheng Future Technology Co., Ltd. (https: / / www.tcuni.com / ) for sequencing and transcriptome analysis.

[0031] (c) Construction of linkage map and acquisition of molecular markers: Based on the obtained transcriptome data, combined with the phenotypes of the parents and the mixed pool, the SNP density map was constructed using R-studio, and the mixed pool constructed from the F2 population material was sequenced to obtain its polymorphic site density. It can be seen that a high-density SNP site was scanned in the 4.26Mbp to 12.46Mbp interval on the short arm of chromosome 7D. The markers in this area were physically located, and KASP molecular markers were developed for the obtained polymorphic sites. The F2 population was used for screening, and a total of 4 KASP molecular markers were developed, namely KASP-533, KASP-669, KASP-1055, and KASP-1166. Each KASP molecular marker contains three primers, that is, 12 KASP molecular marker primers were obtained. The specific sequences are shown in Table 1 below, among which the solid stem inhibition gene (named Su-TdDof) is located on the wheat chromosome 7D as shown in the figure. Figure 1 As shown. The 139 strains obtained above were genotyped using four sets of KASP primers. Combined with the phenotypes of the strains (shown in Table 2 below), it can be concluded that the molecular marker KASP-669 is closely linked to the solid stem inhibition gene Su-TdDof. The molecular marker was developed from a SNP site co-localized with the wheat solid stem inhibition gene Su-TdDof on the short arm of wheat chromosome 7D. The SNP site is located at the physical position of 6.70Mbp in the wheat RefSeqv1.0 genome version, and its polymorphism is C / T. Among them, the KASP typing results of the three primers of the molecular marker KASP-669 for the 139 strains are shown in Figure 2 Combined with the population stem solidity phenotypic data and the analysis results of the primers of the KASP molecular marker, the linkage map constructed using JoinMap4.0 is shown in Figure 1As shown. In the F2 generation population, the plant genotype detected with HEX (blue) fluorescence consistent with the parent Syn-SAU-119 was recorded as B, which was a solid-stem plant, and the phenotype was recorded as S; the plant genotype that showed FAM (yellow) fluorescence like the parent Syn-SAU-117 was recorded as A, which was a hollow-stem plant, and the phenotype was recorded as H; the green triangle fluorescence was a heterozygous plant, the genotype was recorded as C, which was a hollow-stem plant, and the phenotype was recorded as R. The F2 population obtained above 2:3 The genotypes of a total of 139 individual plants in the family and the field phenotypic values ​​of stem solidity are shown in Table 2 below. It can be seen that the actual test results are basically consistent with the expected results, indicating that the solid stem inhibition gene Su-TdDof provided by the present invention does have a significant effect of inhibiting the solidity of wheat stems. At the same time, the molecular marker KASP-669 and its KASP primers provided by the present invention can be used to track and identify the solid stem inhibition gene Su-TdDof, and can also be used to predict the traits of wheat stems.

[0032] Table 1 KASP primer sequences of 4 KASP molecular markers

[0033]

[0034] Table 2 Genotype and phenotype results of F2 population identified by molecular marker KASP-669

[0035]

[0036]

[0037]

[0038]

[0039] d) Comparison of suppressor gene loci: There are many suppressor genes reported publicly. The suppressor genes from Aegilops tauschii are mainly stem rust resistance suppressor genes located on chromosome 7D and a solid stem suppressor gene located on chromosome 3D. Although Su-TdDof also comes from Aegilops tauschii, it is located on chromosome 7DS. According to pedigree analysis and chromosome location, the solid stem suppressor gene Su-TdDof of wheat Syn-SAU-117 behaves differently from other suppressor genes, indicating that Su-TdDof is a new gene.

[0040] In summary, the present invention identified the wheat solid stem inhibition gene Su-TdDof for the first time, and the KASP molecular marker and KASP primer provided by the present invention can quickly screen out Aegilops tauschii with solid stem traits, which can improve the accuracy of solid stem wheat creation, and the created wheat with solid stems can be used for wheat lodging resistance breeding, which can greatly accelerate the breeding process of solid stem wheat varieties.

[0041] 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 tightly linked to a wheat solid stem inhibition gene, characterized in that: The KASP primers include three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively; The KASP primers are developed from SNP sites located on the short arm of wheat chromosome 7D in the wheat genome version RefSeqv1.0; the SNP site is located at a physical position of 6.70 Mbp, and its polymorphism is C / T. The KASP primers can be used to identify the traits of wheat stems.

2. A method for identifying wheat stem traits, characterized in that: The following steps are included: (1): Extracting genomic DNA from the wheat sample to be tested; (2): Using the extracted DNA as a template, PCR amplification is performed using the KASP primers described in claim 1; (3): Detect the fluorescence of the PCR amplification product. When the fluorescence of the amplification product is blue fluorescence, it means that the stem trait of the wheat is a solid stem; when the fluorescence of the amplification product is yellow fluorescence, it means that the stem trait of the wheat is a hollow stem; when the fluorescence of the amplification product is green fluorescence, it means that the stem trait of the wheat is a hollow stem.

3. A kit for identifying wheat stalk traits, characterized in that: The kit comprises the KASP primer as claimed in claim 1.

4. The use of the KASP primer in wheat breeding according to claim 1, characterized in that: The application is to screen wheat with solid stem traits or to create wheat strains with solid stem traits.

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