CN116121444A - Functional KASP molecular marker of wheat stripe rust resistance gene YrNP63 and application thereof - Google Patents
Functional KASP molecular marker of wheat stripe rust resistance gene YrNP63 and application thereof Download PDFInfo
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Abstract
The invention relates to a functional KASP molecular marker of wheat stripe rust resistance gene YrNP63 and application thereof, wherein the functional KASP molecular marker is KASP2683, and the functional KASP molecular marker is a nucleotide sequence obtained by amplifying a DNA template by a primer of KASP2683 designed according to a specific SNP locus G2683A; the SNP locus G2683A is positioned at 2683 th site of YrNP63 coding gene CDS sequence, and the polymorphism of the SNP locus G2683A is A/G respectively. The functional KASP molecular marker KASP2683 developed by the invention can be simply, conveniently, rapidly and high-flux applied to auxiliary breeding of wheat molecules and germplasm resource screening of YrNP63 genes.
Description
Technical Field
The invention belongs to the technical field of wheat gene detection, and particularly relates to a functional KASP molecular marker of a wheat stripe rust resistance gene YrNP63 and application thereof.
Background
Wheat stripe rust is one of the most important wheat diseases, occurs in almost all wheat production areas worldwide, seriously jeopardizes the safe production of wheat, and causes millions of tons of yield loss each year. As wheat stripe rust belongs to air-borne diseases, the wheat stripe rust has the characteristics of wide epidemic range, high transmission speed and the like, is difficult to prevent and control by spraying bactericides, not only consumes money and labor, but also causes environmental pollution. Compared with the method, the method has the advantages of convenience, economy, effectiveness, environmental friendliness and the like by cultivating and planting disease-resistant varieties for prevention and control.
Although 84 stripe rust resistance genes and more than 380 QTLs are positioned and reported at present, the genes/QTLs are not deeply researched, and most of the genes/QTLs are in an initial positioning stage, so that the problems of large positioning interval, easy recombination of linked molecular markers, unclear genetic effect, linkage encumbrance and the like exist, and the genes/QTLs are not applied successfully in production. In addition, rapid mutation of the rust and the appearance of new physiological race often lead to the loss of resistance of disease-resistant genes or disease-resistant varieties, and particularly since the 21 st century, the occurrence of strong pathogenic race such as CYR32, CYR33 and CYR34 is accompanied, so that disease resistance of disease-resistant genes Yr9, yr26, yrZH84 and the like which are most widely used in production in China is lost successively, and therefore, the introduction of new disease-resistant genes into wheat in China is urgently required.
The YrNP63 gene is a full-growth-period anti-stripe gene found and cloned from a durable anti-stripe wheat variety Napo63 of the International corn wheat improvement Center (CIMMYT) by a map cloning method, keeps good resistance to the epidemic wheat species of the wheat stripe rust in China at present, is positioned at the position of 157696282 (-strand) bp of 2BS chromosome 157688966 version 1.0 of a Chinese spring reference genome, and is an important contribution to the breeding of the wheat anti-stripe rust variety in China in the next years or even decades, however, the research on functional molecular markers for identification of the gene is not available at present, so that the functional KASP molecular markers of the wheat anti-stripe rust gene YrNP63 are developed, the application of the YrNP63 gene in molecular breeding is promoted, and the gene has important significance for accelerating the auxiliary breeding of wheat anti-stripe rust molecules.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a functional KASP molecular marker of a wheat stripe rust resistance gene YrNP63 and application thereof, wherein primers are designed by utilizing co-separated specific SNP loci to identify the stripe rust resistance gene YrNP63, so that the problems of easiness in recombination, unclear genetic effect, linkage encumbrance and the like of a linkage molecular marker technology are solved, and the application of the YrNP63 gene in molecular breeding is promoted.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a functional KASP molecular marker of a wheat stripe rust resistance gene YrNP63, wherein the functional KASP molecular marker is KASP2683, and the functional KASP molecular marker is a nucleotide sequence obtained by amplifying a DNA template according to a primer of KASP2683 designed for a specific SNP site G2683A; the SNP locus G2683A is positioned at 2683 th site of YrNP63 coding gene CDS sequence, and the polymorphism of the SNP locus G2683A is A/G respectively.
As a further technical scheme, the DNA single-stranded sequences of 90nt before and after the SNP locus G2683A are shown as SEQ ID NO. 1;
as a further technical scheme, the SNP locus G2683A is positioned at the 91 st base in the sequence shown in SEQ ID NO. 1, and the base mutation is A/G.
A functional KASP molecular marker primer of wheat stripe rust resistance gene YrNP63, wherein the functional KASP molecular marker is KASP2683, and the functional KASP molecular marker primer comprises a disease-sensitive site forward primer KASP2683-FAM, a disease-resistant site forward primer KASP2683-HEX and a Common reverse primer KASP2683-Common;
the nucleotide sequence of the forward primer KASP2683-FAM of the infection site is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward primer KASP2683-HEX of the disease resistance site is shown as SEQ ID NO. 3;
the nucleotide sequence of the Common reverse primer KASP2683-Common is shown in SEQ ID NO. 4.
The functional KASP molecular marker of the wheat stripe rust resistance gene YrNP63 is applied to the identification of whether the wheat material contains the stripe rust resistance gene YrNP63, the germplasm resource screening of the wheat stripe rust resistance gene YrNP63 or the auxiliary selection breeding of wheat molecules.
The functional KASP molecular marker primer of the wheat stripe rust resistance gene YrNP63 is applied to the identification of whether the wheat material contains the stripe rust resistance gene YrNP63, the germplasm resource screening of the wheat stripe rust resistance gene YrNP63 or the auxiliary selective breeding of wheat molecules.
A method for identifying whether a wheat material contains a stripe rust resistance gene YrNP63 comprising the steps of:
step 1, extracting genome DNA of a wheat material to be detected;
step 2, taking genomic DNA of a wheat material to be detected as a template, and carrying out PCR (polymerase chain reaction) amplification on genomic DNA of a sample to be detected by using a primer of the functional KASP molecular marker KASP2683 to obtain an amplification product;
the primer of the functional KASP molecular marker KASP2683 comprises a disease-causing site forward primer KASP2683-FAM, a disease-causing site forward primer KASP2683-HEX and a Common reverse primer KASP2683-Common;
the nucleotide sequence of the forward primer KASP2683-FAM of the infection site is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward primer KASP2683-HEX of the disease resistance site is shown as SEQ ID NO. 3;
the nucleotide sequence of the Common reverse primer KASP2683-Common is shown in SEQ ID NO. 4.
And 3, transferring the amplified product to an enzyme-labeled instrument to read fluorescence data, and then introducing the fluorescence data into KlumterCaller software to analyze to obtain genotype data, so as to obtain the genotype.
Wherein, the homozygous allele with HEX fluorescence is "Y:Y", the homozygous allele with FAM fluorescence is "X:X", the heterozygote genotype is "X:Y" or "Y:X", the deletion value genotype is "? ". The genotype is Y, Y or X, Y or Y, X, the wheat material to be tested contains the stripe rust resistance gene YrNP63, and the genotype is X, the wheat material to be tested does not contain the stripe rust resistance gene YrNP63.
As a further technical scheme, the reaction procedure of the PCR amplification is: pre-denaturation at 94℃for 15min; denaturation at 94 ℃ for 20s, annealing at 65 ℃ for 60s,10 cycles, and annealing temperature reduction of 0.6 ℃ per cycle; denaturation at 94℃for 20s, annealing at 55℃for 60s,32 cycles.
As a further technical scheme, the reaction system of the PCR amplification is: DNA 0.2. Mu.g, 2 XKASP V4.0Mastermix 2. Mu.L, primer mix 0.044. Mu.L, ddH 2 O 1.956mL。
As a further technical scheme, in the primer mixture, the concentration of the forward primer KASP2683-FAM of the disease-sensing site is 12mM/uL, the concentration of the forward primer KASP2683-HEX of the disease-sensing site is 12mM/uL, and the concentration of the Common reverse primer KASP2683-Common is 30mM/uL.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, a specific SNP locus G2683A which is coseparated with the wheat stripe rust resistance gene YrNP63 is found through research, and a functional KASP molecular marker KASP2683 of the YrNP63 is developed based on the specific SNP locus G2683A, which belongs to a functional marker.
The functional KASP molecular marker KASP2683 can be simply, conveniently, rapidly and high-flux applied to auxiliary breeding of wheat molecules and germplasm resource screening of YrNP63 genes.
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FIG. 1 is a KASP2683 marker pair F in example 3 2 Genotyping results for the genetically isolated population;
FIG. 2 is a genotyping result of KASP2683 marker on genetically diverse populations in example 4;
Detailed Description
The technical solutions of the present invention will be clearly and fully described below with reference to specific embodiments, and it is apparent that the described embodiments are 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.
Example 1: development of functional KASP molecular markers
The development of functional KASP molecular marker of wheat stripe rust resistance gene YrNP63 includes the following steps:
the nucleotide sequence of YrNP63 was obtained by aligning the CDS sequence of the cloned YrNP63 gene (see SEQ ID NO. 5) with CDS sequences of the other 41 infectious alleles from the gene in different wheat varieties (sequence numbers in NCBI: MW788048.1, MW788047.1, MW788060.1, MW788051.1, MW788050.1, MW788063.1, MW788059.1, MW788062.1, MW756037.1, MW788061.1, MW788053.1, MW788052.1, MW788055.1, MW788054.1, MW788064.1, MW788056.1, MW756040.1, MW656173.1, MW788058.1, MW756039.1, MW756036.1, LR890103.1, MW788046.1, MW788045.1, MW788044.1, MW788043.1, MW788042.1, MW788041.1, 788040.1, MW788039.1, MW788038.1, MW788037.1, MW788036.1, MW788035.1, MW788034.1, MW788033.1, MW788032.1, MW788031.1, MW756041.1, 788057.1, MW 788049.1), obtaining a specific KASP position of YrG 63, and designing a specific marker of KASP position of KASP 83 to the final marker of PCR by designing the specific marker of KASP position of the final marker;
the specific SNP locus G2683A is positioned at 2683 th site of CDS sequence of YrNP63 coding gene, and the molecular marker polymorphism is A/G respectively.
The DNA sequence of each DNA single-stranded sequence of 90nt before and after the SNP locus G2683A is shown as SEQ ID 1;
the primers of the functional KASP molecular marker KASP2683 comprise a disease-causing site forward primer KASP2683-FAM, a disease-causing site forward primer KASP2683-HEX and a Common reverse primer KASP2683-Common;
the nucleotide sequence of the forward primer KASP2683-FAM of the infection site is shown as SEQ ID NO. 2; the 5' end ' GAAGGTGACCAAGTTCATGCT ' of the SEQ ID NO. 1 is a fluorescent tag sequence;
the nucleotide sequence of the forward primer KASP2683-HEX of the disease resistance site is shown as SEQ ID NO. 3; the 5' end ' GAAGGTCGGAGTCAACGGATT ' of the SEQ ID NO. 1 is a fluorescent tag sequence;
the nucleotide sequence of the Common reverse primer KASP2683-Common is shown in SEQ ID NO. 4.
Example 2: genotyping method
A method for identifying whether a wheat material contains a stripe rust resistance gene YrNP63 comprising the steps of:
step 1, extracting genome DNA as an amplification template;
respectively extracting genome DNA of wheat material to be detected as amplification template
Step 2, taking genomic DNA of a wheat material to be detected as a template, and carrying out PCR amplification on genomic DNA of a sample to be detected by using the primer of the functional KASP molecular marker KASP2683 of the embodiment 1 to obtain an amplified product;
the PCR amplification reaction system comprises: DNA 0.2. Mu.g, 2 XKASP V4.0Mastermix 2. Mu.L, primer mix 0.044. Mu.L, ddH 2 O 1.956mL。
In the primer mixture, the concentration of the forward primer KASP2683-FAM of the disease-resistant site is 12mM/uL, the concentration of the forward primer KASP2683-HEX of the disease-resistant site is 12mM/uL, and the concentration of the Common reverse primer KASP2683-Common is 30mM/uL.
The PCR amplification reaction program is as follows: pre-denaturation at 94℃for 15min; denaturation at 94 ℃ for 20s, annealing at 65 ℃ for 60s,10 cycles, and annealing temperature reduction of 0.6 ℃ per cycle; denaturation at 94℃for 20s, annealing at 55℃for 60s,32 cycles.
Step 3, transferring the amplified product to a lu star omega multifunctional enzyme-labeled instrument to read fluorescence data, and then introducing the fluorescence data into Klu ter Caller software to analyze to obtain genotype data, so as to obtain genotype; wherein, the homozygous allele with HEX fluorescence is "Y:Y", the homozygous allele with FAM fluorescence is "X:X", the heterozygote genotype is "X:Y" or "Y:X", the deletion value genotype is "? ". The genotype is Y, Y or X, Y or Y, X, the wheat material to be tested contains the stripe rust resistance gene YrNP63, and the genotype is X, the wheat material to be tested does not contain the stripe rust resistance gene YrNP63. The greenhouse and field stripe rust resistance phenotype identification results of the wheat to be tested in the embodiment prove that the wheat material containing YrNP63 is disease-resistant to stripe rust.
Example 3: application in parental genetic population
Cross breeding is the most important breeding mode of wheat, wheat Naop63 is a stripe rust resistant wheat variety, contains YrNP63 gene, avocet S is a wheat variety which shows complete disease to stripe rust, does not contain YrNP63 gene, and uses the functional KASP molecular marker KASP2683 marker described in example 1 and the method described in example 2 to F of Napo63/Avs group 2 Genotyping of offspring showed that F can be isolated 2 Offspring are clearly classified into three types and co-segregate with disease resistance (fig. 1). The result shows that KASP2683 can replace phenotype selection to select YrNP63, can be applied to molecular auxiliary selection breeding of YrNP63, and accelerates the breeding process.
Example 4: use in genetically diverse populations
The stripe rust resistance genes contained in the wheat disease resistance germplasm resources are clear, and the application of the germplasm in disease resistance breeding is of guiding significance.
A molecular test was performed on 318 parts of wheat stripe rust disease resistant germplasm resources consisting of domestic and foreign farms, varieties (lines), partially known gene vector varieties (lines), 28 parts of International wheat stripe rust identified hosts and 19 parts of Chinese wheat stripe rust identified hosts using the functional KASP molecular marker KASP2683 marker described in example 1 and the method described in example 2, and the results showed that 6 parts of wheat germplasm in total were consistent with the results of the Napo63 grouping, namely Yaco "S", arduini, P9964, HD2204, attila and Selkirk, respectively, and that these wheat exhibited disease resistance to stripe rust (FIG. 2). Arduini, P9964, HD2204 and Attila are all CIMMYT germplasm and Attila was previously reported with a stripe rust resistant QTL site, so these wheat varieties (lines) contain the YrNP63 gene, these results indicate that KASP2683 is able to distinguish YrNP63 from other alleles. The results show that KASP2683 can be used as a functional diagnostic marker for screening germplasm resources of wheat stripe rust resistance gene YrNP63.
The above described embodiments are only preferred examples of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Claims (9)
1. A functional KASP molecular marker of a wheat stripe rust resistance gene YrNP63, characterized in that the functional KASP molecular marker is KASP2683, and the functional KASP molecular marker is a nucleotide sequence obtained by amplifying a DNA template with a primer of KASP2683 designed according to a specific SNP site G2683A; the SNP locus G2683A is positioned at 2683 th site of YrNP63 coding gene CDS sequence, and the polymorphism of the SNP locus G2683A is A/G respectively.
2. A functional KASP molecular marker of wheat stripe rust resistance gene YrNP63 is characterized in that the DNA single-chain sequence of 90nt before and after SNP locus G2683A is shown as SEQ ID NO. 1.
3. A functional KASP molecular marker primer of a wheat stripe rust resistance gene YrNP63, which is characterized in that the functional KASP molecular marker is KASP2683, and the functional KASP molecular marker primer comprises a disease-sensitive site forward primer KASP2683-FAM, a disease-resistant site forward primer KASP2683-HEX and a Common reverse primer KASP2683-Common;
the nucleotide sequence of the forward primer KASP2683-FAM of the infection site is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward primer KASP2683-HEX of the disease resistance site is shown as SEQ ID NO. 3;
the nucleotide sequence of the Common reverse primer KASP2683-Common is shown in SEQ ID NO. 4.
4. Use of a functional KASP molecular marker of the wheat anti-stripe rust gene YrNP63 according to any one of claims 1-2 for identifying whether a wheat material contains the anti-stripe rust gene YrNP63, germplasm resource screening of the wheat anti-stripe rust gene YrNP63 or wheat molecular assisted selection breeding.
5. Use of a primer of a functional KASP molecular marker of the wheat stripe rust resistance gene YrNP63 according to claim 3 for identifying whether a wheat material contains stripe rust resistance gene YrNP63, germplasm resource screening of the wheat stripe rust resistance gene YrNP63 or wheat molecular assisted selection breeding.
6. A method for identifying whether a wheat material contains a stripe rust resistance gene YrNP63, comprising the steps of:
step 1, extracting genome DNA of a wheat material to be detected;
step 2, taking genomic DNA of a wheat material to be detected as a template, and carrying out PCR (polymerase chain reaction) amplification on genomic DNA of a sample to be detected by using a primer of the functional KASP molecular marker KASP2683 to obtain an amplification product;
step 3, transferring the amplified product to an enzyme-labeled instrument to read fluorescence data, and then introducing the fluorescence data into KlumterCaller software to analyze to obtain genotype data, so as to obtain genotype;
wherein, the homozygous allele with HEX fluorescence is "Y:Y", the homozygous allele with FAM fluorescence is "X:X", the heterozygote genotype is "X:Y" or "Y:X", the deletion value genotype is "? "; when the obtained genotype is Y, X, Y or Y, the wheat material to be tested contains the stripe rust resistance gene YrNP63, and when the obtained genotype is X, the wheat material to be tested does not contain the stripe rust resistance gene YrNP63.
7. The method for identifying whether wheat material contains the yellow rust resistance gene YrNP63 according to claim 1, wherein the PCR amplification reaction procedure is: pre-denaturation at 94℃for 15min; denaturation at 94 ℃ for 20s, annealing at 65 ℃ for 60s,10 cycles, and annealing temperature reduction of 0.6 ℃ per cycle; denaturation at 94℃for 20s, annealing at 55℃for 60s,32 cycles.
8. The method for identifying whether wheat material contains the yellow rust resistance gene YrNP63 according to claim 1, wherein the PCR amplification reaction system is: DNA 0.2. Mu.g, 2 XSKASPV4.0Mastermix 2. Mu.L, primer mix 0.044. Mu.L, ddH 2 O1.956mL。
9. The method for determining whether or not a wheat material contains the yellow rust resistance gene YrNP63 according to claim 8, wherein the concentration of the forward primer for a disease site KASP2683-FAM is 12mM/uL, the concentration of the forward primer for a disease site KASP2683-HEX is 12mM/uL, and the concentration of the Common reverse primer KASP2683-Common is 30mM/uL.
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