CN112430606A - Wheat stripe rust resistant gene yrZ15-1949 and molecular marker and application thereof - Google Patents
Wheat stripe rust resistant gene yrZ15-1949 and molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a wheat stripe rust resistance gene yrZ15-1949, and a molecular marker and application thereof. The wheat stripe rust resistance gene is yrZ15-1949 located on the short arm of wheat 7D chromosome, and the physical position of the genome version of RefSeqv1.0 is 43.66Mb-46.51 Mb. The SNP locus of the molecular marker is located in an interval of yrZ15-1949, the polymorphism is A/G, and the molecular marker can accurately track wheat stripe rust resistant gene yrZ 15-1949. The invention also discloses a primer group for identifying the wheat stripe rust resistant gene yrZ15-1949, and the primer group provided by the invention can be used for quickly screening a wheat variety or line with the wheat stripe rust resistant gene yrZ15-1949 for breeding, so that the breeding process of the wheat disease resistant variety can be greatly accelerated.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a wheat stripe rust resistant gene yrZ15-1949, and a molecular marker and application thereof.
Background
Wheat is an important food crop, and lives approximately 1/3 people worldwide. Wheat stripe rust is a worldwide important disease caused by wheat stripe rust. The stripe rust disease is pandemic for many times in history in China, and the wheat yield can be reduced by more than 40% in severe years, even the wheat is completely harvested. Due to frequent variation of the physiological race of the stripe rust fungus and appearance of a new toxic physiological race, the resistance of wheat varieties is rapidly lost, the stripe rust disease pandemic is caused, and the safety production of wheat is threatened. The continuous and effective prevention and control of the wheat stripe rust is a long-term international problem. Therefore, the development and utilization of the new stripe rust resistance gene, the increase of the diversity of the stripe rust resistance gene and the cultivation and popularization of the stripe rust resistance wheat variety are the most economic, safe and environment-friendly measures for effectively controlling the stripe rust resistance.
Single Nucleotide Polymorphism (SNP) refers to a DNA sequence Polymorphism caused by a change such as a transition, a transversion, an insertion, or a deletion at a specific Nucleotide position in DNA in a genome. The technology is that known sequence information is utilized to compare and search SNP sites, and then specific primers are designed by utilizing the discovered variation sites to carry out PCR amplification on genome DNA or cDNA, so as to obtain specific polymorphic products based on the SNP sites, and finally, the polymorphism of the products is analyzed by utilizing the electrophoresis technology. The SNP markers have the advantages of large quantity and wide distribution; uneven distribution among individual genes and the entire genome; SNP allele frequencies are easily estimated.
KASP is a novel genotyping technology with low cost and high throughput characteristics by competitive Allele Specific PCR (KASP) developed by LGC (Laboratory of the Goverment Chemist) (http:// www.lgcgenomics.com), carries out accurate double-Allele genotyping on SNP and InDel sites by Specific matching of terminal bases of primers, and is widely applied to molecular marker-assisted selection of crops such as rice, wheat, soybean and the like.
At present, 83 stripe rust resistance genes (Yr1-Yr83) are formally named in wheat and related species thereof. Among the genes designated in full name, only a few genes such as Yr2, Yr6, Yr7, Yr19, Yr23 and Yr51 are recessive genes, and the majority are dominant genes. Only a few stripe rust resistant genes have been cloned so far, such as the Yr5/Yr7/YRSP gene cluster, Yr15, Yr18, Yr36, Yr46, YRAS2388, YrU1, and the like. Due to the continuous emergence of new races, most of the disease-resistant genes are gradually or already losing the stripe rust resistance, except that Yr5, Yr15, Yr18, Yr36, YRAS2388 and YrU1 and the like have better resistance to the main stripe rust races currently prevalent in China. The excavation utilizes a novel anti-source and is imminent. Therefore, the research obtains a new stripe rust resistance gene of the wheat, and a new stripe rust resistance source is increased by utilizing a molecular biology technology, so that the stripe rust resistance of the wheat is increased, the aim of breeding a new stripe rust resistance wheat variety is finally achieved, and the method has significance in wheat breeding work.
Cultivation of wheat (Triticum monococcum ssp. monococcum,2n ═ 2x ═ 14, amAm) Belongs to the cultivation type of wheat (Einkorn) of a single line, is the first wheat cultivated and domesticated by human beings, and begins to be cultivated and eaten approximately 10000 years before the metric yuan. The cultivated one-grain wheat is closely related to the common wheat A genome donor species Uraltu wheat (T. urartu, 2 n-2 x-14, AuAu) Is an important gene source of common wheat. A thereofmThe chromosome set has higher homology with the chromosome set A of the common wheat, and the beneficial genes of the chromosome set can be transferred to the common wheat through sexual hybridization. The cultivated wheat has rich genetic diversity and a large number of transformable disease-resistant genes.
Disclosure of Invention
The invention aims to provide a wheat stripe rust resistance gene yrZ15-1949, a molecular marker closely linked with the gene and application thereof, wherein the molecular marker can accurately track the wheat stripe rust resistance gene yrZ15-1949, predict whether a wheat variety contains the characteristics of the stripe rust resistance gene yrZ15-1949, and further facilitate molecular design breeding.
In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:
based on the purposes, a cultivated wheat introgression line Z15-1949 with exogenous disease-resistant gene source is used as a female parent, a wheat variety 'SY 95-71' is used as a male parent for hybridization, and a hybrid F is obtained1，F1Selfing the single plant to obtain F2，F2Selfing to obtain F2:3The family is 124 lines in size, and therefore the genetic mapping population is formed. To F2，F2:3Identifying the phenotype of stripe rust of the family group, and extracting parents 'Z15-1949', 'SY 95-71' and F 260 plant DNAs of a group, and the invention uses a method for constructing phenotype linkage of a genetic map by using a wheat 55K SNP chip to position a wheat stripe rust resistant gene yrZ 15-1949.
Specifically, a wheat stripe rust resistant gene yrZ15-1949 comes from a female parent 'Z15-1949', and the gene is positioned on the 7D short arm of wheat chromosome, and the physical position of the genome version of RefSeqv1.0 is 43.66Mb-46.51Mb (figure 1).
The wheat stripe rust resistance gene yrZ15-1949 can obviously increase wheat stripe rust resistance.
In another aspect of the present invention, a genetic map was constructed using JoinMap4.0 based on the 55K SNP chip data. Combined with the phenotypic data of the population for stripe rust resistance, 2017 and 2018 for F2、F2:3Identifying the phenotype of stripe rust of a family group, detecting a stripe rust resistant gene yrZ15-1949 by combining a wheat 55K SNP chip construction genetic map phenotype linkage method, positioning yrZ15-1949 between SNP loci AX-111656163 and AX89378255 on a short arm of a 7D chromosome, physically positioning the region markers, developing molecular markers of obtained polymorphic loci, designing primers, and finally obtaining the markers KASP-Z15-1949-1 and KASP-Z15-1949-1The stripe rust resistance gene yrZ15-1949 is closely linked.
A molecular marker KAS P-Z15-1949-1 closely linked with a wheat stripe rust resistant gene yrZ15-1949 is an SNP marker which is co-localized on the short arm of a wheat 7D chromosome with the wheat stripe rust resistant gene yrZ15-1949, the SNP locus of the molecular marker is located in the interval of yrZ15-1949 genes (43.66Mb-46.51Mb), and the polymorphism of the molecular marker is A/G.
The molecular marker KASP-Z15-1949-1 can be obtained by amplifying a primer group with a nucleotide sequence shown in SEQ ID NO. 1-3.
Further, different fluorescent modifying groups are respectively added to the 5 'end or the 3' end of the primer with the nucleotide sequence shown as SEQ ID No. 1-2.
Preferably, the fluorescence modifying group includes, but is not limited to, FIFC, FAM, TET, HEX, JOE, TAMRA or BHQ.
In another aspect of the invention, the invention also provides a primer group for detecting the wheat stripe rust resistance gene yrZ15-1949, wherein the primer group comprises 3 primers, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 1-3.
Meanwhile, a kit containing the primer group is also within the protection scope of the invention.
In another aspect of the invention, the application of the molecular marker KASP-Z15-1949-1 or the primer group in wheat molecular breeding and breeding of transgenic wheat or improvement of disease-resistant resources of wheat is also provided.
Specifically, the molecular marker KASP-Z15-1949-1 or the primer group is applied to the cultivation of stripe rust resistant wheat or the identification of wheat varieties with stripe rust resistant genes yrZ 15-1949.
In another aspect of the invention, the invention also provides a method for identifying the wheat stripe rust resistant gene yrZ15-1949, using DNA of a material to be identified as a template, carrying out PCR amplification by using specific primer pairs with sequences respectively shown as SEQ ID NO. 1-3, and reading a fluorescence value, wherein if the fluorescence of the primer label shown as SEQ ID NO.2 is judged to be the wheat containing the stripe rust resistant gene yr Z15-1949.
Specifically, the method comprises the following steps:
1) extracting the genome DNA of a plant to be detected;
2) taking the genome DNA of a plant to be detected as a template, carrying out PCR amplification reaction by using the primer group and reading a fluorescence value;
3) and detecting the fluorescence of the PCR amplification product, and if HEX fluorescence can be read, determining that the plant to be detected is wheat with the stripe rust resistant gene yrZ 15-1949.
Further, the PCR amplification system is as follows: mu.L Master Mix, Mix primer 1.4. mu.L, template DNA 5ng, double distilled water to a total of 10. mu.L, at least 3 independent blanks with double distilled water instead of DNA template.
The mixed primers are obtained by adding 120 mu L, 120 mu L and 300 mu L of primers SEQ ID No.1, 2 and 3 respectively according to the concentration of 10 ng/mu L, and adding ddH2O460. mu.L was mixed.
Further, the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s and renaturation/elongation at 55 ℃ for 60s for 26 cycles; after completion, fluorescence readings were taken.
The invention has the beneficial effects that:
the invention discloses a molecular marker KASP-Z15-1949-1 which is located on a wheat 7D chromosome and linked with a wheat stripe rust resistance gene yrZ15-1949, wherein the molecular marker is a flanking marker of the wheat stripe rust resistance gene yrZ15-1949 on a short arm of the wheat 7D chromosome, and the linkage degree is high. The marker can be used for detecting the stripe rust resistance gene on the 7D chromosome of wheat, and rapidly screening plants with the locus, thereby facilitating the molecular assisted breeding of high stripe rust resistance wheat. The molecular marker KASP-Z15-1949-1 provided by the invention is closely linked with a wheat stripe rust resistance gene yrZ15-1949 on a 7D chromosome, and can be used for positioning the character of stripe rust resistance of wheat, so that a novel stripe rust resistance source is increased in the breeding process, the stripe rust resistance is further increased, and the purpose of breeding a new stripe rust resistance wheat variety is finally achieved.
1) The invention discloses a stripe rust resistance gene yrZ15-1949 from a cultivated wheat one-grain introgression line Z15-1949' for the first time, which is positioned on a short arm of a wheat 7D chromosome and can obviously increase the stripe rust resistance of wheat. The gene has higher utilization value in increasing a novel stripe rust resistant source in wheat stripe rust resistant breeding.
2) The invention discloses a method for accurately detecting a molecular marker KASP-Z15-1949-1 from a cultivated wheat-one-grain introgression line 'Z15-1949' based on a fluorescent quantitative PCR platform for the first time, wherein the molecular marker KASP-Z15-1949-1 is a codominant marker, the detection is accurate and efficient, and the amplification is convenient and stable.
3) The molecular marker KASP-Z15-1949-1 disclosed by the invention is significantly related to the stripe rust resistant gene yrZ15-1949, presents the characteristics of a tightly linked marker, and has high accuracy and high success rate when used for molecular marker-assisted selection.
Drawings
FIG. 1 shows the location of the stripe rust resistance gene yrZ15-1949 on the 7D chromosome from the cultivated wheat monocot introgression line 'Z15-1949' according to the invention;
FIG. 2 is a schematic diagram showing the data scanning of the 55K chip for the regions where the yellow rust resistant genes yrZ15-1949 are located;
FIG. 3 shows F of the single grain wheat introgression line 'Z15-1949' Xwheat variety 'Avocet S' cultivated in example 2 of the present invention2Verifying the fluorescence reading result detected by a population plant molecular marker KASP-Z15-1949-1; wherein FAM (blue square, 'Avocet S') fluorescence is a stripe rust susceptible plant, and HAX (yellow circle, 'Z15-1949') fluorescence is a stripe rust resistant plant; green triangle fluorescence is a heterozygous strain; black diamond fluorescence is blank.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 acquisition of the stripe rust resistance Gene yrZ15-1949 and its molecular marker KASP-Z1949-1
The invention utilizes the cultivation one with exogenous disease-resistant gene sourceThe wheat grain introgression line Z15-1949 is used as a female parent and the wheat variety 'SY 95-71' is used as a male parent for hybridization to obtain a hybrid F1，F1Selfing the single plant to obtain F2，F2Selfing to obtain F2:3The family is 124 lines in size, and therefore the genetic mapping population is formed.
According to 55K SNP chip data, a genetic map is constructed by using JoinMap4.0. Combining with stripe rust resistance phenotype data of a population, setting a threshold LOD (LOD) to be more than or equal to 2.5 by using an inclusion complex interstitial Mapping-ADD (ICIM-ADD) in QTL IciMapping 4.0 under the condition of setting the threshold LOD to be more than or equal to 2.5, and carrying out two-year F pair on 2017 and 20182、F2:3The phenotypic identification data of the stripe rust of the family group is combined with a wheat 55K SNP chip to construct a genetic map to position the stripe rust resistant gene yrZ 15-1949. yrZ15-1949 was located between SNP sites AX-111656163 and AX89378255 on the short arm of the 7D chromosome (FIG. 2), the region markers were physically located and the polymorphic sites were obtained and molecular markers were developed, a total of 12 pairs of KASP primers were designed, and finally the obtained marker KASP-Z15-1949-1 was closely linked to the rust resistance gene yrZ15-1949 (FIG. 1).
The specific process is as follows:
1) mixing parent material Z15-1949, SY95-71 and F1、F2、F2:3The family is planted in Wenjianghui and Hei base according to the plant spacing of 10cm, the row length of 2m and the row spacing of 30cm for field stripe rust resistance identification. Before wheat jointing, using puccinia striiformis mixed with physiological race (32 in bar, 33 in bar, 34 in bar, 11-4 water source and 11-5 water source) and talcum powder according to the ratio of 1: 250 parts of the mixture was mixed and inoculated by a smear method in an induction system. When the material SY95-71 to be induced has enough morbidity (severity)>50% and prevalence>80%) were identified for resistance. Resistance response survey reference Wellings&Bariana published grading criteria for fractions 1-9 (Table 1). The resistance survey was conducted three times in total, once every ten days, based on the most severe disease of flag leaves of the susceptible parent. In the grading standard, 1-4 are disease-resistant, 5 is intermediate, and 6-9 are susceptible.
TABLE 1 wheat stripe rust seedling stage reactive grading and identification criteria
2)55K SNP chip analysis
a) DNA extraction: extraction of parent 'Z15-1949', 'SY 95-71' and F by CTAB method2，F2:3DNA of the plant of the family group.
b) The extracted DNA is subjected to quality detection by using an ultramicro spectrophotometer, and is sent to a company for genotype analysis after being qualified, and the genotype analysis of the double affinity mapping group is completed by a 55K SNP chip developed by combining Beijing Boo Ato crystal technology limited (http:// www.capitalbiotech.com) and Jia Suo Jian project.
c) Constructing a linkage map and obtaining molecular markers: according to 55K SNP chip data, a genetic map is constructed by using JoinMap4.0. Combining with stripe rust resistance phenotype data of a population, setting a threshold LOD (LOD) to be more than or equal to 2.5 by using an inclusion complex interstitial Mapping-ADD (ICIM-ADD) in QTL IciMapping 4.0 under the condition of setting the threshold LOD to be more than or equal to 2.5, and carrying out two-year F pair on 2017 and 20182，F2:3The phenotypic identification data of the stripe rust of the family group is combined with a wheat 55K SNP chip to construct a genetic map to position the stripe rust resistant gene yrZ 15-1949. yrZ15-1949 were located between SNP sites AX-111656163 and AX89378255 on the short arm of the 7D chromosome (FIG. 2), the region markers were physically located and a total of 12 pairs of KASP primers (Table 2) were designed for obtaining polymorphic sites and developing molecular markers, and finally the marker KASP-Z15-1949-1 was closely linked to the yellow rust resistant gene yrZ 15-1949.
TABLE 25 pairs of KASP primer sequences
Wherein, the underlined part is FAM tag sequence, and the wavy line part is HEX tag sequence.
d) Comparison of the stripe rust resistance gene loci: the prior reports reported more resistance to stripe rust, but relatively less resistance to stripe rust was detected on the short arm of the 7D chromosome. To date, stripe rust resistance genes YrY212, YrM8003, YrWV, YrYL, YrHY, and adult plant resistance gene Yr18, all of which are dominant disease resistance genes, have been reported on wheat chromosome 7 DS. Wherein YrY212 is from Equisetum arvense, YrM8003 is from Austria rye, YRWV is from Haynaldia villosa, and YRYL and YRHY are from local varieties of wheat in China. Yr18 is derived from common wheat, has been formally named and cloned, and exhibits partial resistance during the plant stage. However, yrZ15-1949 is a recessive gene, and shows resistance to the rust stripe at 34 seedling stage and resistance to the rust stripe mixed physiological race (34 in stripe, 33 in stripe, 32 in stripe, 11-4 water source and 11-5 water source) in the field, and shows resistance at the whole growth stage. The resistance genes yrZ15-1949 of the introgression lines Z15-1949 appeared different from the other resistance genes (YR18, YrY212, YrM8003, YRWV, YRYL and YRHY) on the 7DS chromosome according to pedigree analysis, anti-pedigree analysis and chromosomal location, indicating yrZ15-1949 is a novel gene.
2 molecular markers are finally obtained from 4 pairs of KASP primers, wherein KASP-Z15-1949-1 is closely linked with the stripe rust resistant gene yr Z15-1949 of the cultivated single-grain wheat introgression line 'Z15-1949'.
Example 2 molecular marker KASP-Z15-1949-1 in validation of population 'Z15-1949' x wheat variety 'Avocet S' F2In (1)
1) The population plants were verified using F2 cultivated in the single grain wheat introgression line 'Z15-1949' x wheat variety 'Avocet S', and 80 individuals were randomly selected in the progeny lines.
2) The KASP-Z15-19491 marker detection is carried out on the obtained 80 individuals, and the specific method comprises the following steps: extracting the DNA of 80 strains; taking the DNA as a template, carrying out PCR amplification by taking a specific primer pair of a molecular marker KASP-Z15-1949-1 as a primer, and carrying out fluorescence reading, wherein the primer is as follows:
primer on FAM tag: (FAM tag sequence is underlined) 5-GAAGGTGACCAAGTTCATGCTTGGATGGTCGCTTTCCATTC-3’；
Primers on HEX tag: (wave line part HEX tag sequence)
A universal downstream primer: 5'-AAGGAAAAGGAAAGACCAGA-3' are provided.
The amplification system of the PCR amplification is as follows: mu.L Master Mix, three primers SEQ ID Nos. 1, 2 and 3 at a concentration of 10 ng/. mu.L, 120. mu.L, and 300. mu.L, respectively, and ddH was added2O460. mu.L was mixed and used as a mixed primer, 1.4. mu.L of the mixed primer, 5ng of template DNA, and double distilled water were added to a total amount of 10. mu.L, and at least 3 independent blanks were added in which the DNA template was replaced by the double distilled water.
The procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s and renaturation/elongation at 55 ℃ for 60s for 26 cycles; after completion, fluorescence readings were taken.
Fluorescence readings (see FIG. 3) where FAM (blue square, ` Avocet ` S), HAX (yellow circle, ` Z15-1949 `); green triangle fluorescence is a heterozygous strain; black diamond fluorescence is blank. The genotype of the plant which detects FAM (blue) fluorescence consistent with 'Avocet S' is recorded as B, the plant is the stripe rust-sensitive plant, the genotype of the plant which shows HAX (yellow) fluorescence like 'Z15-1949' is recorded as A, the plant is the stripe rust-resistant plant, the green triangle fluorescence is the heterozygous strain and is recorded as H, and the plant is the stripe rust-sensitive plant. The genotype and field phenotype values for each individual plant are shown in table 3. The actual result is basically consistent with the expected result, which shows that the stripe rust resistance gene yrZ15-1949 of the invention has the effect of obviously increasing the stripe rust resistance of wheat; meanwhile, the molecular marker KASP-Z15-1949-1 can be used for identifying the anti-stripe rust gene yrZ15-1949 by tracking.
TABLE 3 'Z15-1949' × 'Avocet S' recombinant inbred line KASP-Z15-1949-1 genotype corresponds to phenotype results
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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Claims (10)
1. The wheat stripe rust resistance gene yrZ15-1949 is characterized in that the wheat stripe rust resistance gene yrZ15-1949 is located on the short arm of wheat 7D chromosome, and the physical location of the genome version of RefSeqv1.0 is 43.66Mb-46.51 Mb.
2. A molecular marker closely linked with a wheat stripe rust resistance gene yrZ15-1949 is characterized in that the molecular marker and the wheat stripe rust resistance gene yrZ15-1949 are co-located on the short arm of a wheat 7D chromosome, the SNP locus of the molecular marker is located in the interval of yrZ15-1949, and the polymorphism is A/G.
3. The molecular marker closely linked with the wheat stripe rust resistance gene yrZ15-1949 of claim 2, wherein the molecular marker is obtained by amplification of a primer set having a nucleotide sequence shown in SEQ ID No. 1-3.
4. The molecular marker tightly linked with the wheat stripe rust resistance gene yrZ15-1949 of claim 3, wherein the primers with the nucleotide sequences shown in SEQ ID No. 1-2 are added with different fluorescent modifying groups at the 5 'end or the 3' end.
5. A primer group for detecting a wheat stripe rust resistant gene yrZ15-1949 is characterized by comprising 3 primers, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 1-3.
6. The molecular marker of claim 2 or the primer set of claim 5, wherein the molecular marker or the primer set is used for wheat molecular breeding, transgenic wheat cultivation or improvement of wheat disease-resistant resources.
7. Use of the molecular marker of claim 2 or the primer set of claim 5 for breeding stripe rust resistant wheat or identifying wheat varieties having stripe rust resistant gene yrZ 15-1949.
8. A method for identifying a wheat stripe rust resistance gene yrZ15-1949, which is characterized by comprising the following steps:
1) extracting the genome DNA of a plant to be detected;
2) taking the genome DNA of a plant to be detected as a template, performing PC R amplification reaction by using the primer group, and reading a fluorescence value;
3) and detecting the fluorescence of the PCR amplification product, and if HEX fluorescence can be read, determining that the plant to be detected is wheat with the stripe rust resistant gene yrZ 15-1949.
9. The method for identifying the wheat stripe rust resistance gene yrZ15-1949 of claim 8, wherein the PCR amplification system is: 5 μ L MasterMix, claim 5 primer set mix primer 1.4 μ L, template DNA 5ng, double distilled water to a total of 10 μ L, at least 3 independent blanks with double distilled water instead of DNA template.
10. The method for identifying the wheat stripe rust resistance gene yrZ15-1949 of claim 9, wherein the PCR amplification procedure is: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s and renaturation/elongation at 55 ℃ for 60s for 26 cycles; after completion, fluorescence readings were taken.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113637790A (en) * | 2021-08-13 | 2021-11-12 | 四川农业大学 | KASP molecular marker of stripe rust resistant gene YRAS2388R, primer, kit and application |
| CN113897457A (en) * | 2021-11-29 | 2022-01-07 | 四川农业大学 | KASP molecular marker linked with wheat stripe rust resistant QTL and application thereof |
| CN114410652A (en) * | 2022-01-21 | 2022-04-29 | 西南科技大学 | Stripe rust resistant gene QYRsv and swust-1BL tightly linked molecular marker in adult plant stage and application thereof |
| CN115896339A (en) * | 2023-02-14 | 2023-04-04 | 四川农业大学 | Specific SNP molecular marker related to wheat stripe rust resistance gene Yr81 and application thereof |
| CN116254277A (en) * | 2023-05-12 | 2023-06-13 | 西北农林科技大学深圳研究院 | Wheat WRKY transcription factor gene TaWRKY40 and application thereof |
| CN116855504A (en) * | 2023-05-11 | 2023-10-10 | 四川农业大学 | Wheat red glume red stalk gene RgM G52 and KASP molecular marker and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140359898A1 (en) * | 2013-06-03 | 2014-12-04 | The Board Of Regents For Oklahoma State University | Transgenic cereal plants with increased resistance to rust diseases |
| WO2019082175A1 (en) * | 2017-10-23 | 2019-05-02 | Carmel Haifa University Economic Corporation Ltd. | STRIPE RUST RESISTANCE GENE WTK1 (Yr15) AND USE THEREOF |
| CN109706263A (en) * | 2019-02-22 | 2019-05-03 | 四川农业大学 | Chain SNP marker and application with wheat stripe rust resisting ospc gene QYr.sicau-1B-1 |
| CN109913577A (en) * | 2019-04-24 | 2019-06-21 | 中国农业科学院棉花研究所 | Molecular labeling and its application with wheat stripe rust resisting ospc gene Yr1152 close linkage |
| CN112481275A (en) * | 2020-12-11 | 2021-03-12 | 四川农业大学 | Wheat stripe rust resistant gene yrZ15-1370 and molecular marker and application thereof |
-
2020
- 2020-12-11 CN CN202011460142.8A patent/CN112430606B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140359898A1 (en) * | 2013-06-03 | 2014-12-04 | The Board Of Regents For Oklahoma State University | Transgenic cereal plants with increased resistance to rust diseases |
| WO2019082175A1 (en) * | 2017-10-23 | 2019-05-02 | Carmel Haifa University Economic Corporation Ltd. | STRIPE RUST RESISTANCE GENE WTK1 (Yr15) AND USE THEREOF |
| CN109706263A (en) * | 2019-02-22 | 2019-05-03 | 四川农业大学 | Chain SNP marker and application with wheat stripe rust resisting ospc gene QYr.sicau-1B-1 |
| CN109913577A (en) * | 2019-04-24 | 2019-06-21 | 中国农业科学院棉花研究所 | Molecular labeling and its application with wheat stripe rust resisting ospc gene Yr1152 close linkage |
| CN112481275A (en) * | 2020-12-11 | 2021-03-12 | 四川农业大学 | Wheat stripe rust resistant gene yrZ15-1370 and molecular marker and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| CHAONAN SHI等: "Identification of herbicide resistance loci using a genome-wide association study and linkage mapping in Chinese common wheat", 《THE CROP JOURNAL》 * |
| 张明虎 等: "栽培一粒小麦渐渗系抗条锈病基因的分子标记", 《第十届小麦基因组学及分子育种大会摘要集》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113637790A (en) * | 2021-08-13 | 2021-11-12 | 四川农业大学 | KASP molecular marker of stripe rust resistant gene YRAS2388R, primer, kit and application |
| CN113897457A (en) * | 2021-11-29 | 2022-01-07 | 四川农业大学 | KASP molecular marker linked with wheat stripe rust resistant QTL and application thereof |
| CN114410652A (en) * | 2022-01-21 | 2022-04-29 | 西南科技大学 | Stripe rust resistant gene QYRsv and swust-1BL tightly linked molecular marker in adult plant stage and application thereof |
| CN114410652B (en) * | 2022-01-21 | 2023-07-21 | 西南科技大学 | Molecular marker closely linked with adult-stage stripe rust resistance genes QYrsv and swust-1BL and application thereof |
| CN115896339A (en) * | 2023-02-14 | 2023-04-04 | 四川农业大学 | Specific SNP molecular marker related to wheat stripe rust resistance gene Yr81 and application thereof |
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