CN109468330B - Barley yellow mosaic disease resistance gene eIF4EHOR3298And identification method and application thereof - Google Patents

Abstract

The invention discloses a barley yellow mosaic disease resistance gene eIF4E_HOR3298And identification method and application thereof, namely eIF4E_HOR3298Comprises the nucleotide sequence shown as SED ID NO. 1. dCAPS detection eIF4E_HOR3298A genetic engineering method and PCR primer molecules comprising: an upstream primer having a nucleotide sequence shown by SED ID NO.2, and a downstream primer having a nucleotide sequence shown by SED ID NO. 3. The resistance gene eIF4E of the invention_HOR3298Has resistance to barley yellow mosaic virus and barley temperate mosaic virus toxic strains, and can be obtained by antagonizing resistance gene eIF4E_HOR3298Detecting and identifying to obtain resistant plants, and can be used for barley yellow mosaic disease resistance breeding. dCAPS molecular marker detection eIF4E of the invention_HOR3298Gene technology method capable of effectively detecting the resistance gene eIF4E_HOR3298And the molecular marker-assisted breeding is realized.

Description

Barley yellow mosaic disease resistance gene eIF4EHOR3298And identification method and application thereof

Technical Field

The invention relates to a barley yellow mosaic disease resistance gene, in particular to a barley yellow mosaic disease resistance gene eIF4E_HOR3298And an identification method and application thereof.

Background

Barley yellow mosaic disease is a soil-borne virus disease, is caused by barley yellow mosaic virus (BaYMV) or barley temperate mosaic virus (BaMMV) or a coexisting complex of the barley yellow mosaic virus and the barley temperate mosaic virus, belongs to potyviridae, is one of important diseases of winter barley production areas such as Europe, east Asia and the like, is reduced by 10-50 percent on average in a disease-sensitive field according to the difference of the disease degree and the disease time, and seriously threatens the production of winter barley grains in the Europe area.

BaYMV and BaMMV are parasitized in dormant spores of polymyxa graminis in soil, and due to soil cultivation, wind blowing of soil particles and short-distance transportation of water containing zoospores, viruses are completely transmitted in different plants and different areas along with the transmission of fungal spores. Under natural conditions, BaYMV and BaMMV usually infect the root of a seedling in autumn, and a typical mosaic disease appears in the new leaf of the plant in early spring in the next year, and the leaf yellowing, the short plant, the few tillers, the reduction of the yield of grains, or early withering or no heading and fruit setting are caused along with the increasing of disease spots. The dormant spores of the polymyxa graminis can survive in soil for more than 10 years, and the dormant spores carrying the viruses germinate to infect host plants again under the condition of proper external environment, so that the disease becomes a long-term and lasting disease stress.

The yellow mosaic disease of barley is the most main disease of barley production areas in the middle and lower reaches of Yangtze river in China, and the barley yield in the areas is seriously influenced. The existing chemical and biological control measures have limited effect of reducing the content of polymyxa graminis in soil, can not prevent diseases, and can pollute farmlands and environment due to the large amount of pesticide. Under natural conditions, part of barley materials have genetic disease resistance to barley yellow mosaic disease, and disease resistance genes are introduced into cultivated barley varieties to effectively prevent diseases, so that the barley material is a prevention and control means widely adopted in the current agricultural production. Since genetic variation of viruses frequently occurs under natural selection conditions, the emergence of new virulent strains may cause the current disease resistance genes to fail, and the identification of new resistance genes (or new types of resistance variation of known genes) is of great breeding value.

At present, 18 barley yellow mosaic disease resistance sites are known, and only 2 genes PDIL5-1 and eIF4E are map-cloned. Resistance genetic sites rym4(rym, Resistance to yellow mosaic disease), rym5 and rym6 are alleles, resulting from mutations in the eukaryotic translation initiation factor (eukakyotic translation initiation factor4E) eIF4E sequence, the coding sequence of the eIF4E gene in the disease-resistant material being different from that of the disease-sensitive material, thereby resulting in an alteration in the coding amino acid sequence; resistance genetic loci rym1 and rym11 are alleles (rym is a recessive gene) resulting from loss of function of the Protein Disulfide Isomerase gene (Protein Disulfide isomerease Like) PDIL 5-1. rym4/rym5, rym1/11 are resistance resources mainly used in barley production.

At present, pathogenic toxic strains aiming at rym4, rym5, rym6 and rym1/11 (such as European toxic isolates BaYMV-II, BaMMV-SIL and BaMMV-Teil, Japanese toxic isolate BaYMV-III, and Chinese toxic isolates BaYMV-C and BaMMV-C) appear in countries such as Germany, France, Japan and the like and parts of middle and lower reaches of Yangtze river in China, which means that the existing resistance resources can not meet the breeding use, and new resistance genes and matched molecular marker selection methods must be developed.

Disclosure of Invention

The invention aims to provide a barley yellow mosaic disease resistance gene eIF4E_HOR3298And an identification method and application thereof, solves the problem that pathogenic toxic strains appear when carrying barley yellow mosaic disease resistant sites rym4/rym5 and rym1/11, and carries a resistance gene eIF4E_HOR3298The barley material of (1) shows complete resistance to barley yellow mosaic virus and barley temperate mosaic virus in a plurality of disease nursery fields (pathogenic virulent strains carried by the disease nursery can infect rym4/rym5 and rym1/11) respectively, and can be used for barley yellow mosaic virus resistance breeding.

In order to achieve the aim, the invention provides a barley yellow mosaic disease resistance gene eIF4E_HOR3298The eIF4E_HOR3298Comprises the nucleotide sequence shown as SED ID NO. 1.

The invention also provides barley carrying a barley yellow mosaic disease resistance gene eIF4E_HOR3298The eIF4E_HOR3298Comprises the nucleotide sequence shown as SED ID NO. 1.

The invention also provides a barley yellow mosaic disease resistance gene eIF4E_HOR3298The method of (3), comprising:

(1) will carry eIF4E_HOR3298Crossing the gene barley with the barley carrying the resistance variation gene rym1/11 of PDIL5-1 gene as parent material to obtain F₁Generation of genetic material, F₁Selfing the genetic material to obtain F₂Segregating genetic populations;

(2) f is to be₂Randomly sowing generation and parent materials in diseased soil containing BaYMV/BaMMV viruses for cultivation, carrying out phenotype investigation after plants grow up, and judging disease resistance or susceptibility;

(3) extracting total RNA of the plant, carrying out reverse transcription, detecting a gene resistance variation gene rym1/11 of PDIL5-1 in DNA of the plant by adopting PCR (polymerase chain reaction), and carrying out genetic analysis and sequence analysis on the plant not carrying rym1/11 so as to obtain eIF4E_HOR3298Gene, detection of eIF4E by dCAPS molecular marker technique_HOR3298A gene.

Wherein the eIF4E_HOR3298Comprises the nucleotide sequence shown as SED ID NO. 1.

Wherein, the primer molecule of the dCAPS molecular marking technology comprises: nucleotide sequences shown as SED ID NO.2 and SED ID NO. 3.

Preferably, in step (2), the incubation conditions are: light at 12 ℃ and dark at 8 ℃ were alternated.

Preferably, in step (3), the total RNA is extracted by RNAzol reagent.

Preferably, in step (3), the reverse transcription is performed using RT KitWidth gDNAeraser.

Preferably, in step (3), the rym1/11 genetic variation is detected by PCR using the reaction system: the volume ratio is 10: 10: 2: 2: 2: 1: 73 cDNAsemplate, 10x containing Mg²⁺Taq buffer, dNTP mix, upstream primer PDI _45_743_ f, downstream primer PDI _45_743_ r, 5U/. mu.L Taq DNApolymerase, ddH₂O; wherein the upstream primer PDI _45_743_ f comprises: the nucleotide sequence shown in SED ID NO.4, wherein the downstream primer PDI _45_743_ r comprises: nucleotide as shown in SED ID NO.5And (4) sequencing.

Preferably, in step (3), the eIF4E_HOR3298The reaction adopted by the molecular marker detection of the gene dCAPS is divided into two steps:

the first step of reaction is to perform PCR amplification on the eIF4E gene by adopting a reaction system as follows: the volume ratio is 10: 10: 2: 2: 2: 1: DNAsemplate, 10x of 73 contains Mg²⁺TaqBuffer, dNTP mix, and upstream primer dCAPS _ HOR3298_ f₂Downstream primers dCAPS _ HOR3298_ r, 5U/. mu.L Taq DNApolymerase, ddH₂O; wherein the upstream primer dCAPS _ HOR3298_ f₂Comprises the following steps: the nucleotide sequence shown as SED ID NO.2, wherein the downstream primer dCAPS _ HOR3298_ r comprises: a nucleotide sequence shown as SED ID NO. 3;

the second reaction is to eIF4E_HOR3298Carrying out gene enzyme digestion, wherein the adopted reaction system is as follows: the volume ratio is 1000: 200: 5: 795 PCR product, 10 XFlyCut Buffer, 20U/. mu.L restriction enzymes EcoRI, ddH₂O。

The invention also provides a dCAPS molecular marker detection eIF4E_HOR3298A kit of genes, the kit comprising: the primer molecule comprises an upstream primer with a nucleotide sequence shown as SED ID NO.2 and a downstream primer with a nucleotide sequence shown as SED ID NO. 3.

The barley yellow mosaic disease resistance gene eIF4E_HOR3298The identification method and the application thereof have the following advantages:

the resistance gene eIF4E of the invention_HOR3298The strain has resistance to pathogenic toxic strains of barley yellow mosaic virus and barley temperate mosaic virus, and can be used for barley yellow mosaic virus resistance breeding. The invention provides a dCAPS molecular marker detection method, which can effectively detect resistance gene eIF4E_HOR3298The method is used for selecting disease-resistant plants in the barley breeding process.

Drawings

FIG. 1 is an electrophoretogram for quality detection of total RNA of barley according to example 1 of the present invention.

FIG. 2 shows example 3 of the present invention at F₂And (3) detecting the detection result electrophoretogram of the PDIL5-1 genotype in the population.

FIG. 3 shows eIF4E of example 3 of the present invention_HOR3298The cleavage result of the gene was electrophoretogram (Maker in the figure is 50bp DNAsader).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 barley yellow mosaic resistance Gene eIF4E_HOR3298Genetic mapping of

The barley genetic material HOR3298 is a local variety of barley from Iran, and W757/612 is a breeding material of barley from Germany (carrying rym1/11 resistance gene whose resistance is derived from loss of function of PDIL5-1 gene).

(1) Hybridization of HOR3298 with W757/612 to obtain F₁Generation of genetic material, construction of F by bagging and selfing₂Segregating genetic populations;

(2) f is to be₂Randomly sowing generation and parent materials in disease soil containing BaYMV/BaMMV viruses, culturing in a light incubator (12 ℃,10h day/8 ℃,14h night), after seeds germinate, beginning to investigate the phenotype of the plants and sampling for later use after the plants grow for 4 months, wherein the phenotype investigation is carried out for three times, and the disease resistance or infection is judged at one week interval;

(3) the method comprises the steps of extracting total RNA from cut plant leaves to be detected by an RNAzol reagent (Beijing Kinbot Biotechnology Co., Ltd., product No. R011-100), measuring the concentration of the extracted RNA by a Nanodrop fluorescence spectrophotometer, and carrying out electrophoresis gel running by using 1% (w/v) agarose gel to detect the extraction quality, wherein the experimental result is shown in figure 1 and is a quality detection electrophoresis chart of the barley total RNA in the embodiment 1 of the invention. Reverse RNA using RT KitWidth gDNAeraser kit (Beijing Kinbot Biotechnology Co., Ltd.; P514-100) which is a tubular reverse transcription premixed Mix comprising: 4x gDNA Erasermix, 5x RT Mastermix, RNAase Free H₂And O, diluting the successfully inverted cDNA by 5 times, and storing the cDNA to the temperature of minus 20 ℃ for later use.

(4) Molecular marker detection of the F2 population PDIL5-1 genotype, reference example 3, at F₂Plants not carrying rym1/11 are selected from the population, RNA of 16 susceptible samples and RNA of 16 disease-resistant samples are respectively mixed into pools according to the phenotype investigation result, each sample is equally mixed according to the concentration of 2 mu g/mu L, and mixed pool transcriptome sequencing (Bulked segegant RNA-Seq, BSR-Seq) and bioinformatics analysis are carried out, and the result proves that only one resistance locus is contained in the barley variety HOR3298 and is positioned at the tail end of the long arm of the chromosome 3H.

(5) F obtained by crossing barley variety HOR3298 with barley variety Franka (carrying rym4 resistance gene)₁An alternative to allelic testing. Hybridizing to obtain F₁Seeding the generation group and the parent material in a seedling tray, putting the seedling tray into an illumination incubator for sprouting (24 ℃,14h day/18 ℃,10h night), mechanically rubbing and inoculating the barley yellow mosaic virus until two leaves and one heart period are reached, and finding F₁All the plants of the generation show the resistance to the barley mosaic disease. The resistance gene carried by the barley variety HOR3298 is shown to have allelic relation with rym4, and the resistance of HOR3298 is proved to be provided by eIF4E gene.

Example 2 resequencing of the eIF4E gene in barley cultivar HOR3298

The nucleotide sequence variation of the eIF4E gene is identified by PCR amplification and first-generation sequencing of a nucleic acid fragment of 648bp (SED ID NO.1) in the full-length coding region of the eIF4E gene in the barley variety HOR3298, which is shown in the following table 1, which is a reaction system for PCR amplification of the barley variety HOR3298 in example 2 of the present invention.

Table 1 shows the reaction system for PCR amplification of the barley variety HOR3298 of example 2 of the present invention

The upstream primer eIF4E-54s comprises: the nucleotide sequence (GCCCGTCCGTCSTAGAAAAG) as shown in SED ID No.6, wherein the downstream primer eIF4E-849as comprises: the nucleotide sequence shown in SED ID NO.7 (GAAACAGCATCCACCCGCTA).

Table 2 is a program chart of PCR reaction of barley variety HOR3298 of example 2 of the present invention

The sequencing result is subjected to sequence analysis by using Sequencher5.4.6 software, and sequence differences between other published variation types of the hordeum vulgare HOR3298 gene and the eIF4E gene are compared to find eIF4E_HOR3298Different from the published sequences, as shown in the following table 3, which is a table of sequence differences between the eIF4E gene of the barley variety HOR3298 of the present invention and eIF4E genes of other published variation types, it is indicated that the resistance gene of HOR3298 is another novel variation haplotype of eIF 4E.

Table 3 shows the sequence differences between the eIF4E gene of the barley variety HOR3298 of the present invention and the eIF4E genes of other variants

Note: 1. reference is made to Ping Yangtal, the Theoretical & applied genetics (2017),130(2): 331-344; 2. 3: reference is made to Nils Stein et al, the plant journal (2005),42(6): 912-); 4. 5: reference is made to Dragon periodic et al, the scientific & Applied Genetics (2014),127(5) 1061-1071.

Example 3 molecular marker assay F₂Population PDIL5-1 genotype

Selection of F that does not contain the loss of function variation of the PDIL5-1 gene, i.e., the resistance gene rym1/11, using published molecular markers (Yang et al 2014, therapeutic and Applied Genetics, 127: 1625-1634)₂The plants, as shown in Table 4 below, are a PCR reaction system for detecting the resistance gene rym1/11 in example 2 of the present invention.

Table 4 is a PCR reaction system table for detecting the resistance gene rym1/11 in example 2 of the present invention

Experimental materials: EasyTaq DNApolymerase (cat # AP111) and 10x EasyTaq Buffer (+ Mg)²⁺) (product No. GK101-01), all purchased from Beijing Quanjin Biotechnology, Inc.; dNTP mix solution (cat. No. B500056-0500) was purchased from Biotechnology engineering (Shanghai) Ltd.

The upstream primer PDI _45_743_ f includes: the nucleotide sequence shown in SED ID NO.4, wherein the downstream primer PDI _45_743_ r comprises: the nucleotide sequence shown as SED ID No. 5.

Table 5 is a PCR reaction schedule for detecting the resistance gene rym1/11 in example 2 of the present invention

Electrophoresis was performed on a 1% (w/V) agarose gel, 2. mu.L of loading dye (6X) was added to 10. mu.L of the amplified product, and a fragment size indicator of '5000 + DNA ladder' (Beijing Bomaide Gene technology Co., Ltd.) was used for electrophoresis at 180V for 30min, and the results of the experiment are shown in FIG. 2, which is F of example 2 of the present invention₂The detection result chart of detecting the PDIL5-1 genotype in the population shows that the band is deleted in FIG. 2, which indicates that the gene function is lost due to deletion of a certain segment sequence of the PDIL5-1 gene of the plant, and the plant is disease-resistant at the PDIL5-1 locus, otherwise, the plant is disease-sensitive.

Example 4 detection of eIF4E Using the molecular marker dCAPS _ HOR3298_HOR3298Gene

Based on eIF4E_HOR3298The specific mononucleotide polymorphism at 565bp is used in developing dCAPS molecular marker with upstream primer dCAPS _ HOR3298_ f₂And a downstream primer dCAPS _ HOR3298_ r and an upstream primer dCAPS _ HOR3298_ f₂Comprises the following steps: the nucleotide sequence shown in SED ID NO.2, and the downstream primer dCAPS _ HOR3298_ r comprises: as defined in SED ID NO.3The nucleotide sequence shown, was specifically amplified to obtain a 224bp fragment spanning intron 3 and exon 4 of the eIF4E gene. Cutting eIF4E by using FlycutEcoRI through restriction enzyme digestion reaction_HOR3298The 33bp fragment of (1).

Table 6 shows the detection of eIF4E in example 4 of the present invention_HOR3298PCR reaction System Table

Table 7 shows the detection of eIF4E in example 4 of the present invention_HOR3298PCR reaction schedule of

And (3) carrying out enzyme digestion on the PCR product, incubating for 1.5h at 37 ℃ in a PCR instrument, and carrying out full enzyme digestion to obtain an enzyme digestion product.

Table 8 shows the results of example 4 on eIF4E_HOR3298Reaction system table of PCR product enzyme digestion

Experimental materials: FlyCutEcoRI (cat # JE201-01) and 10x FlyCut Buffer were both purchased from Beijing Quanjin Biotechnology Ltd.

3% (w/V) agarose gel (note: the fragment size difference of the enzyme digestion product is only 30bp, high concentration gel is needed to effectively distinguish the fragment difference), 20 uL enzyme digestion product is added into 3 uL loading dye for loading, 50bp DNAladder' (Beijing Bomaide Gene technology Co., Ltd.) is used as the fragment size indication, 180V voltage electrophoresis is carried out for 30-35min, as shown in FIG. 3, the eIF4E of the embodiment 3 of the invention_HOR3298Enzyme cutting electrophoresis knot of geneAccording to the result, the amplified band obtained by PCR, namely the PCR product with the fragment size of 224bp is subjected to enzyme digestion reaction by using a restriction enzyme FlycutEcoRI and contains eIF4E_HOR3298The PCR product of the gene is cut out to obtain a 34bp fragment, namely 190bp, and a band less than 224bp appears (sample HOR 3298); does not contain eIF4E_HOR3298The fragment of the gene is not cut by enzyme, namely 224bp (sample W757/612, sample HTX from local Chinese variety); the two fragments of 224bp and 190bp (sample HOR3298+ W757/612 and sample HOR3298+ HTX) appeared simultaneously in the heterozygous state, and the result proves that the molecular marker dCAPS _ HOR3298 can be used for effectively identifying eIF4E in the homozygous and heterozygous states_HOR3298A gene.

Example 5 detection kit for molecular marker dCAPS _ HOR3298

Detection of resistance gene eIF4E by molecular marker dCAPS _ HOR3298_HOR3298A kit (50 times) comprising the components (as in table 1): two dCAPS primers (upstream and downstream), and a positive control (eIF 4E)_HOR3298The plasmid DNA of (1), Taq DNApolymerase, Taqbuffer, dNTPmix, FlyCutEcoRI, 10x FlyCutBuffer, and ddH₂O。

TABLE 9 Components Table of the detection kit for molecular marker dCAPS _ HOR3298 of the present invention

The test procedure was as described in example 4, and the results are shown in FIG. 3.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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Claims (7)

1. Barley yellow mosaic disease resistance gene eIF4E_HOR3298The method of (2), comprising:

(1) will carry eIF4E_HOR3298Crossing the gene barley with the barley carrying the resistance variation gene rym1/11 of PDIL5-1 gene as parent material to obtain F₁Generation of genetic material, F₁Selfing the genetic material to obtain F₂Segregating genetic populations;

(2) f is to be₂Randomly sowing generation and parent materials in diseased soil containing BaYMV/BaMMV viruses for cultivation, carrying out phenotype investigation after plants grow up, and judging disease resistance or susceptibility;

(3) extracting total RNA of the plant, carrying out reverse transcription, detecting a gene resistance variation gene rym1/11 of PDIL5-1 in DNA of the plant by adopting PCR (polymerase chain reaction), and carrying out genetic analysis and sequence analysis on the plant not carrying rym1/11 so as to obtain eIF4E_HOR3298Gene, detection of eIF4E by dCAPS molecular marker technique_HOR3298A gene;

wherein the eIF4E_HOR3298The sequence of the gene is a nucleotide sequence shown as SED ID NO. 1;

wherein, the primer molecule of the dCAPS molecular marking technology is a nucleotide sequence shown as SED ID NO.2 and SED ID NO. 3.

2. The barley yellow mosaic disease resistance gene eIF4E according to claim 1_HOR3298The method of (2), wherein the incubation conditions are: light at 12 ℃ and dark at 8 ℃ were alternated.

3. The barley yellow mosaic disease resistance gene eIF4E according to claim 1_HOR3298The method of (3), wherein the total RNA is extracted using an RNAzol reagent.

4. The barley yellow mosaic disease resistance gene eIF4E according to claim 1_HOR3298The method for identifying (1), wherein in the step (3), RT KitWidth gDNAeraser is used for the reverse transcription.

5. The barley yellow mosaic disease resistance gene eIF4E according to claim 1_HOR3298The method for identifying (2), wherein in step (3), the genetic variation of rym1/11 is detected by PCR using the reaction system: the volume ratio is 10: 10: 2: 2: 2: 1: 73 cDNAsemplate, 10x containing Mg²⁺Taq Buffer, dNTP mix, upstream primer PDI _45_743_ f, downstream primer PDI _45_743_ r, 5U/. mu.LTaq DNApolymerase, ddH₂O; wherein the upstream primer PDI _45_743_ f is the nucleotide sequence shown in SED ID NO.4, and the downstream primer PDI _45_743_ r is the nucleotide sequence shown in SED ID NO. 5.

6. The barley yellow mosaic disease resistance gene eIF4E according to claim 1_HOR3298In step (3), the method of identifying eIF4E, wherein the step (3) comprises_HOR3298The reaction adopted by the molecular marker detection of the gene dCAPS is divided into two steps:

the first step of reaction is to perform PCR amplification on the eIF4E gene by adopting a reaction system as follows: the volume ratio is 10: 10: 2: 2: 2: 1: DNAsemplate, 10x of 73 contains Mg²⁺Taq Buffer, dNTP mix, and upstream primer dCAPS _ HOR3298_ f₂Downstream primers dCAPS _ HOR3298_ r, 5U/. mu.L Taq DNA polymerase, ddH₂O; wherein the upstream primer dCAPS _ HOR3298_ f₂The downstream primer dCAPS _ HOR3298_ r is a nucleotide sequence shown as SED ID NO. 3;

the second reaction is to eIF4E_HOR3298Carrying out gene enzyme digestion, wherein the adopted reaction system is as follows: the volume ratio is 1000: 200: 5: 795 PCR product, 10 XFlyCut Buffer, 20U/. mu.L restriction enzymes EcoRI, ddH₂O。

7. dCAPS molecular marker detection eIF4E_HOR3298A kit of genes, comprising: the primer molecules are an upstream primer with a nucleotide sequence shown as SED ID NO.2 and a downstream primer with a nucleotide sequence shown as SED ID NO. 3; eIF4E_HOR3298The sequence of the gene is the nucleotide sequence shown in SED ID NO. 1.

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