CN112430606B - Wheat stripe rust resistant gene yrZ-1949 and molecular marker and application thereof - Google Patents
Wheat stripe rust resistant gene yrZ-1949 and molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a wheat stripe rust resistant gene yrZ-1949 and a molecular marker and application thereof. The wheat stripe rust resistance gene is yrZ-1949 located on the short arm of wheat 7D chromosome, and the physical position of the RefSeqv1.0 genome version is 43.66Mb-46.51Mb. The SNP locus of the molecular marker is located in the yrZ-1949 interval, the polymorphism is A/G, and the molecular marker can accurately track the wheat stripe rust resistant gene yrZ-1949. The invention also discloses a primer group for identifying the wheat stripe rust resistance gene yrZ-1949, and by utilizing the primer group provided by the invention, a wheat variety or strain with the wheat stripe rust resistance gene yrZ-1949 can be quickly screened out for breeding, so that the breeding process of the wheat disease resistance 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 yrZ-1949, and a molecular marker and application thereof.
Background
Wheat is an important food crop, and about 1/3 of the world's population is cultivated. 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 company (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 (Yr 1-Yr 83) are formally named in wheat and related species. Among the genes designated in the official form, 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, yrU, and the like. Due to the continuous emergence of new races, most of the disease-resistant genes gradually or already lose the stripe rust resistance except that Yr5, yr15, yr18, yr36, YRAS2388, yrU and the like have better resistance to the main current popular stripe rust races in China at present. Digging and utilizing a novel anti-source is urgent. 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 one grain wheat (Triticum monococcum ssp. Monococcum,2n =2x =14,a m A m ) Belongs to the cultivation type of wheat (Einkorn) of a first line, is the first wheat cultivated and domesticated by human beings, and is planted and eaten approximately 10000 years before the metric country. The cultivated one-grain wheat is closely related to the Ular pattern wheat of the donor species of the A genome of the common wheat (T.urartu, 2n =2x =14, A. The A genome of the common wheat u A u ) Is an important gene source of common wheat. A thereof m The 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 yrZ-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 yrZ-1949, predict whether a wheat variety contains the characteristics of the stripe rust resistance gene yrZ-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 'SY95-71' is used as a male parent for hybridization, and a hybrid F is obtained 1 ，F 1 Selfing generation individual plant to obtain F 2 ，F 2 Selfing to obtain F 2:3 The family is 124 lines in size, and therefore the genetic mapping population is formed. To F is aligned with 2 ，F 2:3 Identifying the phenotype of stripe rust of the family group, and extracting parents 'Z15-1949', 'SY95-71' and F 2 60 plant DNAs of a population, 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-1949.
Specifically, a wheat stripe rust resistance gene yrZ-1949 comes from the female parent 'Z15-1949', and the gene is located on the short arm of wheat chromosome 7D, and the physical position of the genome version of RefSeqv1.0 is 43.66Mb-46.51Mb (figure 1).
The wheat stripe rust resistance gene yrZ-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 F 2 、F 2:3 The phenotype of the stripe rust of a family group is identified, a genetic map phenotype linkage construction method is combined with a wheat 55K SNP chip to detect a stripe rust resistant gene yrZ-1949, yrZ15-1949 is positioned between SNP sites AX-111656163 and AX-89378255 on a short arm of a 7D chromosome, the region marker is physically positioned, molecular markers are developed for obtained polymorphic sites, primers are designed, and finally the obtained marker KASP-Z15-1949-1 is closely linked with the stripe rust resistant gene yrZ-1949.
A molecular marker KAS P-Z15-1949-1 closely linked with a wheat stripe rust resistant gene yrZ-1949, is an SNP marker which is co-localized on the short arm of wheat 7D chromosome with the wheat stripe rust resistant gene yrZ-1949, and the SNP site thereof is positioned in the interval of yrZ-1949 gene (43.66 Mb-46.51 Mb), and the polymorphism of the molecular marker is C/T.
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.
Furthermore, the primers with the nucleotide sequences shown in SEQ ID NO. 1-2 are added with different fluorescent modifying groups at the 5 'ends respectively, or are added with different fluorescent modifying groups at the 3' ends respectively.
Preferably, the fluorescence modifying group includes, but is not limited to, FIFC, FAM, TET, HEX, JOE, TAMRA or BHQ.
On the other hand, the invention also provides a primer group for detecting the wheat stripe rust resistant gene yrZ-1949, wherein the primer group comprises 3 primers, and the nucleotide sequences of the primers are respectively shown in 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, transgenic wheat cultivation or improvement of wheat disease-resistant resources 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-1949.
In another aspect of the invention, the invention also provides a method for identifying the wheat stripe rust resistant gene yrZ-1949, which comprises the steps of taking the DNA of a material to be identified as a template, carrying out PCR amplification by using specific primer pairs with the sequences respectively shown in SEQ ID NO. 1-3, and reading a fluorescence value, wherein if the fluorescence marked by the primer shown in SEQ ID NO.2, the wheat containing the stripe rust resistant gene yrZ-1949 can be judged.
Specifically, the method comprises the following steps:
1) Extracting 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) Detecting the fluorescence of the PCR amplification product, wherein if HEX fluorescence can be read, the plant to be detected is wheat with the stripe rust resistant gene yrZ-1949.
Further, the PCR amplification system is: 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 primer is prepared by adding 120 mu L, 120 mu L and 300 mu L of primers SEQ ID No.1, 2 and 3 according to the concentration of 10 ng/mu L respectively, and adding ddH 2 O460. Mu.L was mixed.
Further, the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s, renaturation/elongation at 55 ℃ for 60s, and 26 cycles; after completion, fluorescence readings were taken.
The beneficial effects of the invention are as follows:
the invention discloses a molecular marker KASP-Z15-1949-1 located on a wheat 7D chromosome and linked with a wheat stripe rust resistance gene yrZ-1949, wherein the molecular marker is a flanking marker of the wheat stripe rust resistance gene yrZ-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 is closely linked with a wheat stripe rust resistance gene yrZ-1949 on a 7D chromosome, and can be used for positioning the character of wheat stripe rust resistance, so that a novel stripe rust resistance source is increased in a breeding process, 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 yrZ-1949 from a wheat cultivation introgression line 'Z15-1949' for the first time, which is positioned on a wheat 7D chromosome short arm and can obviously increase the wheat stripe rust resistance. 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 remarkably related to the stripe rust resistant gene yrZ-1949, presents the characteristic 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 yrZ-1949 on the 7D chromosome from the cultivated wheat grain introgression line 'Z15-1949' of the present invention;
FIG. 2 is a schematic diagram showing the data scanning of 55K chips for the area of the stripe rust resistance genes yrZ-1949;
FIG. 3 shows the result of the cultivation of the introgression line Z15-1949 'for wheat variety Avocet S' of one-grain wheat in example 2 of the present invention 2 Verifying the fluorescence reading result detected by the plant molecular marker KASP-Z15-1949-1 of the population; 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 stripe rust resistance Gene yrZ-1949 and its molecular marker KASP-Z1949-1
The invention utilizes a cultivated wheat introgression line Z15-1949 with exogenous disease-resistant gene source as a female parent and a wheat variety SY95-71 as a male parent for hybridization to obtain a hybrid F 1 ，F 1 Selfing generation individual plant to obtain F 2 ，F 2 Selfing to obtain F 2:3 The 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 2018 2 、F 2:3 Phenotypic identification of stripe rust of family groupThe data is combined with a wheat 55K SNP chip to construct a genetic map to locate the stripe rust resistant gene yrZ-1949. yrZ15-1949 is positioned between the SNP sites AX-111656163 and AX-89378255 on the short arm of the 7D chromosome (FIG. 2), the markers of the region are physically positioned, the polymorphic sites are obtained and the molecular markers are developed, a total of 12 pairs of KASP primers are designed, and finally the markers KASP-Z15-1949-1 are tightly linked with the stripe rust resistant gene yrZ-1949 (FIG. 1).
The specific process is as follows:
1) Mixing parent material Z15-1949, SY95-71 and F 1 、F 2 、F 2:3 The 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 SY95-71 is fully developed (severity)>50% and prevalence>80%) were subjected to resistance identification. Resistance response survey reference Wellings&Grading criteria 1-9 published by Bariana (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', 'SY95-71' and F by CTAB method 2 ，F 2:3 DNA of plant of 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 the projects of the Beijing Boao crystal and Atlantic Biotechnology company Limited (http:// www.capitalbiotech.co m) and Gu Jizeng.
c) Constructing a linkage map and obtaining molecular markers: according to the 55K SNP chip data, a genetic map is constructed by utilizing 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 2018 2 ，F 2:3 The phenotypic identification data of the stripe rust of the family group is combined with wheat 55K SNP chips to construct a genetic map so as to position the stripe rust resistant gene yrZ-1949. yrZ15-1949 is positioned between SNP loci AX-111656163 and AX-89378255 on the short arm of the 7D chromosome (FIG. 2), the region marker is physically positioned, the polymorphic loci are obtained and molecular markers are developed, a total of 12 pairs of KASP primers (Table 2) are designed, and finally the marker KASP-Z15-1949-1 is tightly linked with the stripe rust resistant gene yrZ-1949.
TABLE 2 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 former report that there are more genes resistant to stripe rust, but relatively few genes were detected on the short arm of the 7D chromosome. Hitherto, stripe rust resistance genes YrY, 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 YrY is from Olympic wheat, yrM8003 is from Austria rye, YRWV is from Haynaldia villosa, and YRYL and YRHY are from local variety of Chinese wheat. Yr18 is derived from common wheat, has been formally named and cloned, and shows partial resistance in the strain stage. However, yrZ-1949 is a recessive gene which shows 34 seedling stage resistance to the rust stripe fungus physiological race and field resistance to the rust stripe fungus mixed physiological race (34 in stripe, 33 in stripe, 32 in stripe, 11-4 water source and 11-5 water source), showing full-growth stage resistance. The resistance genes yrZ-1949 of the introgression lines Z15-1949 differed from the other resistance genes on the 7DS chromosome (Yr 18, yrY, yrM8003, yrWV, yrYL and YrHY) by pedigree analysis, resistance profiling analysis and location on chromosome, indicating yrZ-1949 as a new 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 yrZ-1949 of the cultivated wheat one-grain introgression line 'Z15-1949'.
Example 2 molecular marker KASP-Z15-1949-1 in validation of population 'Z15-1949' x wheat variety 'Avocet S' F 2 In (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 labeling detection is carried out on the obtained 80 single plants, 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'.
The amplification system of the PCR amplification is as follows: mu.L Master Mix, three primers SE Q ID No.1, 2 and 3 at a concentration of 10 ng/. Mu.L, 120. Mu.L and 300. Mu.L, respectively, and ddH was added 2 O460. 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 double distilled water were added to replace the DNA templateBlank of the plate.
The procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 15min; 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 in which FAM (blue) fluorescence consistent with 'Avocet S' is detected is recorded as B, the plant is a stripe rust-sensitive plant, the genotype of the plant which shows HAX (yellow) fluorescence like 'Z15-1949' is recorded as A, the plant is a stripe rust-resistant plant, the green triangle fluorescence is a heterozygous strain and is recorded as H, and the plant is a 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 yrZ-1949 of the invention has the function of obviously increasing the stripe rust resistance of wheat; meanwhile, the molecular marker KASP-Z15-1949-1 can be used for identifying the stripe rust resistant gene yrZ-1949 by tracking.
Table 3'Z15-1949' × 'Avocet S' recombinant inbred line KASP-Z15-1949-1 genotype corresponds to phenotype
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 (5)
1. A molecular marker closely linked with a wheat stripe rust resistance gene yrZ-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 yrZ-1949, and the polymorphism is C/T;
the molecular marker is obtained by amplifying a primer group with a nucleotide sequence shown in SEQ ID NO. 1-3.
2. The molecular marker tightly linked with the wheat stripe rust resistance gene yrZ-1949 of claim 1, wherein the primer with the nucleotide sequence shown in SEQ ID No. 1-2 has different fluorescent modifying groups added to the 5 'end or the 3' end.
3. A method for identifying a wheat stripe rust resistance gene yrZ-1949, which is characterized by comprising the following steps:
1) Extracting 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 a primer group shown as SEQ ID NO. 1-3, and reading a fluorescence value;
3) And detecting the fluorescence of the PCR amplification product, wherein if HEX fluorescence can be read, the plant to be detected is wheat with the stripe rust resistant gene yrZ-1949.
4. The method for identifying the wheat stripe rust resistance gene yrZ-1949 of claim 3, wherein the PCR amplification system is: 5 uL MasterMix, primer set mixed primer 1.4 uL shown in SEQ ID NO. 1-3, template DNA 5ng, double distilled water added to total amount of 10 uL, at least 3 independent blanks with double distilled water replacing DNA template.
5. The method for identifying the wheat stripe rust resistance gene yrZ-1949 of claim 3, wherein the PCR amplification procedure is: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s, renaturation/elongation at 55 ℃ for 60s, and 26 cycles; after completion, fluorescence readings were taken.
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| CN113897457B (en) * | 2021-11-29 | 2023-07-18 | 四川农业大学 | KASP molecular marker linked with wheat stripe rust resistance QTL and application |
| 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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| CN116254277B (en) * | 2023-05-12 | 2023-07-11 | 西北农林科技大学深圳研究院 | Wheat WRKY transcription factor gene TaWRKY40 and application thereof |
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