CN116949200A - KASP primer closely linked with wheat solid stem inhibition gene and application thereof - Google Patents
KASP primer closely linked with wheat solid stem inhibition gene and application thereof Download PDFInfo
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Abstract
The invention discloses a KASP primer closely linked with a wheat solid stem inhibition gene and application thereof, and relates to the technical field of genetic breeding, wherein the KASP primer comprises 3 primers with nucleotide sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively. The KASP molecular marker KASP-669 obtained by amplification of the KASP primer is co-dominant marker. The KASP primer provided by the invention can accurately track the wheat solid stem inhibition gene, identify the property of wheat stems, can improve the accuracy of solid stem wheat creation, can be used for lodging-resistant wheat breeding, and greatly accelerates the breeding process of solid stem wheat varieties.
Description
Technical Field
The invention relates to the technical field of genetic breeding, in particular to a KASP primer closely linked with a wheat solid stem suppression gene and application thereof.
Background
The common wheat can be improved by introducing an exogenous beneficial gene through distant hybridization, but the exogenous beneficial gene is sometimes not expressed or not fully expressed in the common wheat. Therefore, novel suppressor genes are discovered and utilized, and the action mechanism of the suppressor genes is analyzed, so that the effective utilization of exogenous beneficial genes can be promoted.
A single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refers to a polymorphism in a DNA sequence caused by a change such as a transition, a transversion, an insertion, a deletion, etc., at a specific nucleotide position of a DNA in a genome. The technology is that known sequence information is utilized to compare and search SNP loci, then a specific primer is designed by utilizing the discovered variation loci to carry out PCR amplification on genome DNA or cDNA, a specific polymorphism product based on the SNP loci is obtained, and finally the polymorphism of the product is analyzed by utilizing an electrophoresis technology. The SNP markers have the advantages of large quantity and wide distribution; uneven distribution in individual genes and throughout the genome; SNP allele frequencies are easily estimated.
KASP is a novel genotyping technology with low cost and high throughput by competitive allele-specific PCR technology (Kompetitive Allele Specific PCR, KASP) developed by LGC company (Laboratory of the Government Chemist, http:// www.lgcgenomics.com), and is widely applied to molecular marker-assisted selection of crops such as rice, wheat, soybean and the like by accurately performing double allele typing on SNP and InDel loci through specific matching of primer terminal bases.
Scientists have the phenomenon that the expression of exogenous genes is weakened or even lost for many times when exogenous beneficial genes are introduced into common wheat. The reported suppressor genes are mostly derived from a plurality of chromosomes of the D genome, and comprise powdery mildew-resistant suppressor gene Su-Pm21, leaf rust-resistant suppressor gene Su-Lr23, stem rust-resistant suppressor gene SuSr-D1 and the like. Arthrodesis (aegilopseusci, 2n=2x=14, dd) is a donor of the D genome of common wheat and is an important gene source for genetic improvement of common wheat. However, with respect to the solid stem suppression gene of tetraploid wheat, the presence has been reported so far only on the 3D chromosome of common wheat. However, studies on the localization and mechanism of the suppressor gene remain very limited. To date, only one stem rust inhibitor gene, su sr-D1, has been located on 7DL of common wheat cantcatch, which mediates stem rust resistance through the subunits encoding the mediator complex. However, there are still few studies, and therefore, it is necessary to dig more suppressor genes, and it is important to analyze the mechanism of action and the functional pathways of more suppressor phenomena.
Disclosure of Invention
The invention aims to provide a KASP primer closely linked with a wheat solid stem suppression gene and application thereof, wherein the KASP primer comprises 3 primers, can accurately track the wheat solid stem suppression gene, identify the character of wheat stems, improve the accuracy of solid stem wheat creation, create lodging-resistant wheat breeding with solid stems and accelerate the breeding process of solid stem wheat varieties.
In order to achieve the above purpose, the invention provides a KASP primer closely linked with wheat solid stem inhibition genes, which comprises 3 primers with nucleotide sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively;
wherein, the KASP primer is developed from SNP locus located on short arm of wheat 7D chromosome in the wheat genome version of RefSeqv1.0; the SNP locus is positioned at a physical position of 6.70Mbp, the polymorphism is C/T, and the KASP primer can be used for identifying the characters of wheat stems.
The invention also provides a KASP molecular marker KASP-669 obtained by amplifying the KASP primer.
The invention also provides an identification method of the wheat stalk character, which comprises the following steps:
(1): extracting genome DNA of a wheat sample to be detected;
(2): performing PCR amplification by using the extracted DNA as a template and the KASP primer;
(3): detecting fluorescence of the PCR amplification product, wherein when the fluorescence of the amplification product is blue fluorescence, the stalk character representing the wheat is solid stalk; when the fluorescence of the amplified product is yellow fluorescence, the stalk character representing the wheat is hollow stalk (homozygous); when the fluorescence of the amplified product is green fluorescence, the stalk character representing the wheat is hollow stalk (heterozygous).
The invention also provides a kit for identifying the wheat stalk traits, which comprises the KASP primer.
The KASP primer provided by the invention can be used for wheat breeding, and comprises the steps of screening wheat containing solid stem characters or creating wheat lines with solid stem characters.
The KASP primer closely linked with the wheat solid stem suppression gene can identify whether a wheat plant contains the solid stem suppression gene or not, can predict the stalk character of wheat, solves the problem of difficult detection of the stalk character of wheat, improves the accuracy of solid stem wheat creation, and has the following advantages:
the invention discloses a wheat solid stem suppression gene Su-TdDof from Artemisia interna AS92 for the first time, which is positioned on a short arm of an artificially synthesized wheat 7D chromosome, the gene significantly suppresses the expression of the solid gene, and the gene has important utilization value in the creation and lodging-resistant breeding of the solid stem wheat.
The invention discloses a method for accurately detecting a molecular marker KASP-669 from Artemisia princeps Pampanini AS92 based on a fluorescence quantitative PCR platform, which is a co-dominant marker, and has the advantages of accurate and efficient detection and convenient and stable amplification.
The molecular marker KASP-669 disclosed by the invention is extremely obviously related to the solid stem inhibition gene Su-TdDof, presents the characteristic of closely linked markers, and has high accuracy and high success rate when used for auxiliary selection of molecular markers and primers thereof.
Drawings
FIG. 1 is a map of the localization of the KASP molecular marker and solid stem suppression gene Su-TdDof on the wheat 7D chromosome in the present invention.
FIG. 2 shows the KASP typing results of 139 strains by the 3 KASP primer pairs of KASP-669 in the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental example 1 solid Stem suppressor Su-TdDof, KASP molecular marker and obtaining of KASP primer
1) Population family creation
The invention utilizes durum wheat Ma to be hybridized with two festival wheat AS92 and AS96 respectively, chromosome doubling is carried out under natural conditions, artificially synthesized wheat is created and respectively named AS wheat Syn-SAU-117 and Syn-SAU-119, ma is semi-dry durum wheat provided by GeorgeFedak of the Woltai research and development center of Canada, and the festival wheat is stored in the wheat research institute of Sichuan agricultural university by taking AS AS code. Hybridization with wheat Syn-SAU-117 as female parent and wheat Syn-SAU-119 as male parent to obtain hybrid F 1 Instead of F 1 Obtaining F by single plant selfing 2 Instead of F 2 Obtaining F by substitution selfing 2:3 Family, F 2 The population size contained 139 lines, named Syn-7D-1, syn-7D-2, respectively.....about.Syn-7D-139, thereby constituting a population of genetic maps.
2) Identification of stalk phenotype
Parental materials Syn-SAU-117, syn-SAU-119 and F 1 、F 2 、F 2:3 The families are planted in Wenjianghui and a base according to plant spacing of 10cm, line length of 2m and line spacing of 30cm, and the field stalk solidity is identified. In the wheat jointing stage Zadoksstage34, the main tillering of the material is obtained, the middle part of the second internode of the stalk base is cut, and the solidity identification is carried out on the cross section of the wheat. The stem thickness and pulp cavity diameter of the stem were measured, and the stem solidity was reflected in the pulp cavity ratio (the ratio of the pulp cavity diameter to the stem thickness). And grading according to the filling condition of the medullary cavity marrow tissue: 1 is completely hollow and 5 is completely solid.
3) Transcriptome data analysis
(a) Sampling a sample and extracting RNA: parent Syn-SAU-117, syn-SAU-119 and F pairs at the jointing stage Zadoksstage34 2 The 20 stem base second internodes of the group are completely solid homozygous individual plants and the 20 stem base second internodes are completely hollow homozygous individual plants for sampling, and the first four nodes appear on the stems. About 0.5cm of stalks are measured from the bottom of the lowermost section to the uppermost section for the second internode of the base. The sample was immediately placed in a 1.5mL microcentrifuge tube and placed in dry ice for storage and sample delivery. The RNA was extracted and mixed by https:// www.tcuni.com /) which was sent to the Chengdu future technology Co.
(b) Transcriptome data analysis: the samples taken in step (a) were sent to all day future technologies Inc. (https:// www.tcuni.com /) for sequencing and completion of transcriptome analysis.
(c) Constructing a linkage map and obtaining a molecular marker: based on the resulting transcriptome data, combining the parental and pooled phenotypes, using R-studio to construct a SNP density map, represented by F 2 And (5) sequencing a pool constructed by group materials to obtain the polymorphic site density. As is clear, a relatively high-density SNP site was scanned in the region of 4.26Mbp to 12.46Mbp on the short arm of the 7D chromosome, and the region was physically mapped, and the obtained polymorphic site was developed with KASP molecular markers, using F 2 The population is subjected to a screening process,a total of 4 KASP molecular markers, respectively designated as KASP-533, KASP-669, KASP-1055 and KASP-1166, were developed, and each KASP molecular marker contained three primers, namely 12 KASP molecular marker primers were obtained, the specific sequences of which are shown in Table 1 below, wherein the localization of the solid stem inhibitor gene (designated Su-TdDof) on the wheat 7D chromosome is shown in FIG. 1. Genotyping the 139 strains obtained above using 4 sets of KASP primers, and combining the phenotype of the strains (shown in Table 2 below) to obtain a molecular marker KASP-669 closely linked to the solid stem suppression gene Su-TdDof, wherein the molecular marker is developed from a SNP site co-located on the short arm of the wheat 7D chromosome with the solid stem suppression gene Su-TdDof of wheat, and the SNP site is located at the physical position 6.70Mbp of the wheat refSeqv1.0 genome version, and the polymorphism is C/T; wherein, the KASP typing results of 139 strains of 3 primer pairs of the molecular marker KASP-669 are shown in FIG. 2. The linkage map constructed using JoinMap4.0 is shown in FIG. 1, in combination with the results of analysis of the colony stalk solidity phenotype data and KASP molecular tagged primers. At F 2 In the generation group, the genotype of the plant with FAM (blue) fluorescence consistent with the parent Syn-SAU-119 is marked as B, the plant is marked as solid stem plant, and the phenotype is marked as S; the genotype of the plant which shows HEX (yellow) fluorescence like the parent Syn-SAU-117 is marked as A, the plant is a hollow stem plant, and the phenotype is marked as H; the green triangle fluorescence is a heterozygous strain, the genotype is marked as C, the genotype is marked as a hollow stem plant, and the phenotype is marked as R. F obtained above 2:3 The field phenotype values of 139 individual plant genotypes and stalk solidity of the family are shown in the following table 2, and the actual detection result is basically consistent with the expected result, so that the solid stalk suppression gene Su-TdDof provided by the invention does have the effect of obviously suppressing the stalk solidity of wheat, and meanwhile, the molecular marker KASP-669 and the KASP primer thereof provided by the invention can be used for tracking and identifying the solid stalk suppression gene Su-TdDof and can also be used for predicting the characters of the stalk of the wheat.
TABLE 14 KASP primer sequences for KASP molecular markers
TABLE 2F 2 Genotypic and phenotypic consequences of populations identified by the molecular marker KASP-669
d) Comparison of the repressor loci: there are many reported inhibitors, the stem rust inhibitor gene mainly located on the 7D chromosome and one solid stem inhibitor gene located on the 3D chromosome, and Su-TdDof is also from Artemisia annua (Aegilops tauschii), but it is located on the 7DS chromosome. Based on pedigree analysis and chromosomal location, the solid stem repressor gene Su-TdDof of wheat Syn-SAU-117 appears to be different from other repressors, indicating that Su-TdDof is a novel gene.
In conclusion, the wheat solid stem suppression gene Su-TdDof is identified for the first time, and the KASP molecular marker and the KASP primer thereof provided by the invention can be used for rapidly screening the festival wheat with solid stem characters, so that the accuracy of solid stem wheat creation can be improved, the created wheat with solid stem can be used for wheat lodging-resistant breeding, and the breeding process of solid stem wheat varieties can be greatly accelerated.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (6)
1. The KASP primer closely linked with the wheat solid stem inhibition gene is characterized by comprising 3 primers with nucleotide sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively;
wherein the KASP primer is developed from SNP loci located on short arms of wheat 7D chromosomes in a wheat genome version of RefSeqv1.0; the SNP locus is positioned at a physical position of 6.70Mbp, the polymorphism of the SNP locus is C/T, and the KASP primer can be used for identifying the characters of wheat stems.
2. A KASP molecular marker amplified from the KASP primer of claim 1, wherein the molecular marker is KASP-669.
3. The identification method of the wheat stalk character is characterized by comprising the following steps:
(1): extracting genome DNA of a wheat sample to be detected;
(2): PCR amplification using the KASP primer of claim 1 using the extracted DNA as a template;
(3): detecting fluorescence of the PCR amplification product, wherein when the fluorescence of the amplification product is blue fluorescence, the stalk character representing the wheat is solid stalk; when the fluorescence of the amplified product is yellow fluorescence, the stalk character representing the wheat is hollow stalk; when the fluorescence of the amplified product is green fluorescence, the stalk character representing the wheat is hollow stalk.
4. A kit for identifying a wheat stalk trait, comprising a KASP primer of claim 1.
5. Use of a KASP primer according to claim 1 in wheat breeding.
6. The use according to claim 5, comprising screening wheat containing solid stalk traits or creating wheat lines with solid stalk traits.
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