CN105695605B - Wheat stripe rust resisting ospc geneYr69And its chain SSR molecular marker and application method - Google Patents

Abstract

The present invention relates to Plant genetics and field of wheat breeding, the in particular to molecular labeling of wheat stripe rust resisting disease shape, specially wheat stripe rust resisting ospc geneYr69And its chain SSR molecular marker and application method, solve existing molecular labeling and Stripe Rust Resistance GeneYr69Genetic distance it is remote, using Wheat DNA as template mark not accurate enough problem, application method: 1), 2SA33 is primer amplification；2), program are as follows: initial denaturation；Denaturation is annealed, and is extended, circulation；Extend；It saves backup；3) point sample after, mixing product and sample-loading buffer after electrophoresis, electrophoresis poststaining are taken pictures；4) specific band, amplified illustrates existYr69.Advantage: 1,2AS33 is obtained；2, dominant marker, genetic distance 1.9cM, quick, precise IdentificationYr69Gene simultaneously predicts powder mildew resistance, acceleratesYr69Utilization.

Description

Wheat stripe rust resisting ospc geneYr69And its chain SSR molecular marker and use Method

Technical field

The present invention relates to Plant genetics and field of wheat breeding, in particular to the molecule mark of wheat stripe rust resisting characteristic of disease shape Note, specially wheat stripe rust resisting ospc geneYr69And its chain SSR molecular marker and application method.

Background technique

By wheat stripe rust (Puccinia striiformis west f.sp. tritici(Pst)) caused by wheat Stripe rust is most important worldwide fungal disease.It has the characteristics that epidemic rate is fast, onset area is wide, the extent of injury is high, When disease is serious, 40% or more wheat yield, or even total crop failure can be made.It is rationally the prevention and treatment most economical ring of the disease using disease-resistant variety Guarantor, safely and effectively method.But the new Pathogenic Types CYR31 of variability and wheat stripe rust due to puccinia striiformis, The appearance of CYR32 and CYR33 etc. and popular and disease-resistant variety large area single cropping, cause most of varietal resistances cannot Persistently, it is easy to be overcome by the new microspecies of strip rust bacteria.Therefore, it constantly looks for, identify new Resistance resource, the new resistance of breeding is small Wheat variety, and polymerize with other gene resistant to stripe rust, polymerization kind, multiline variety and the how anti-kind of Durable resistance are cultivated, it is right Delay the Toxicity Variation of strip rust bacteria pathological race, widens wheat breed hereditary basis, increases the service life of disease-resistant variety very Necessity, this is also the essential measure for realizing the sustainable control of China's stripe rust of wheat.

The Spherical scanning plant of wheat is richly stored with Stripe Rust Resistance Gene resource.With wheat distance edge hybrid and dyeing Body engineering is carried out in a deep going way, in the wheat that many Stripe Rust Resistance Genes that wheat relative belongs to are transferred to.Currently, in the world Many of the gene resistant to stripe rust of definite designation and QTL site derive from the Spherical scanning plant of wheat.E. elongata is (also known as Peng mentions card couchgrass,Th. ponticum, 2n=70, JJJJ^sJ^s/StStE^eE^bE^x), it is the relative genus plant of wheat, has Spend more the good characteristics such as how real, drought-resistant, saline-alkali tolerant, a variety of wheat diseases of high-quality, immune or highly resistance.E. elongata and wheat There is height crossability, and its excellent genes is easy to express in Wheat Background, is the transfer of wheat foreign gene and distant hybridization Rare favorable genes donor.CH7086, which is that the octoploid of wheat-wheatgrass derived from E. elongata and common wheat is small, to lay down 7430 small wheat introgression line of laying down, it is immune to wheat powdery mildew and stripe rust.Stripe rust Disease Resistance Identification and genetic analysis show CH7086 carries a dominant disease-resistant gene, and international genome naming committee is named asYr69。

Molecular labeling and the linkage map foundation of disease-resistant gene are to carry out deliberate genetic positioning and figure position gram to target gene The basis of grand disease-resistant gene, while the powerful of even more molecular mark.Simple repeated sequence (simple Sequence repeats, SSR) it is that one kind is widely present in string on genome, being made of several nucleotide repeating units Join repetitive sequence.Since quantity in the genome is abundant, polymorphism height, codominant inheritance, easy to operate, result is reliable and stable etc. Advantage and be widely used with Germplasm Identification, gene Molecular mapping and molecular mark in.Therefore, it utilizes SSR molecular marker positioningYr69The molecular labeling chain with it is for cloning and utilizing gene resistant to stripe rust with exploitationYr69With weight Want meaning.

Summary of the invention

The present invention solves existing molecular labeling and Stripe Rust Resistance GeneYr69Genetic distance it is remote, mark not accurate enough Problem provides a kind of wheat stripe rust resisting ospc geneYr69And its chain SSR molecular marker and application method.

The present invention is achieved by the following technical solutions: wheat stripe rust resisting ospc geneYr69Chain SSR molecular marker, The SSR molecular marker is primer 2 AS33, the nucleotide sequence of the primer are as follows: upstream sequence 5'- AGAAGGCACACTGCTGGAAC-3', downstream sequence 5'-ACATGAGTGAGTTGTGAGTC-3'.

Wheat stripe rust resisting ospc geneYr69Chain SSR molecular marker marks Stripe Rust Resistance Gene in assisted SelectionYr69In Application.

Wheat stripe rust resisting ospc geneYr69Chain SSR molecular marker, with wheat stripe rust resisting ospc geneYr69Heredity away from From for 1.9cM.

Wheat stripe rust resisting ospc geneYr69Chain SSR molecular marker is for identifying in wheat whether contain stripe rust resisting base CauseYr69Application method, including following operating procedure:

1), using Wheat volatiles DNA as PCR amplification template, using SSR molecular marker 2AS33 as primer, PCR amplification is carried out, Reaction system is 20 μ L, and the reaction system includes: 50-100 ng/ μ L Wheat volatiles DNA, 10 × PCR buffer, tool Body are as follows: 10mM Tris-HCl, pH value 8.3,50mM KCl, 1.5mM MgCl₂；0.2mM dNTP, 1U Taq enzyme, 0.25 μM is drawn Object is eventually adding a drop mineral oil；

2), PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 1min, 55 DEG C of annealing 45s, 72 DEG C extend 1min, 35 circulations；72 DEG C of extension 10min；4 DEG C save backup；

3), electrophoresis is 8% native polyacrylamide gel electrophoresis, after 5 μ lPCR products and 5 μ L sample-loading buffers are mixed Point sample, electrophoresis 2h under 180V voltage, cma staining are taken pictures；

4) it, analyzes and identifies, the specific band of 269bp can be amplified, then illustrate that there are Stripe Rust Resistance Genes in wheat germplasmYr69, otherwise, Stripe Rust Resistance Gene is not present in wheat germplasm to be measuredYr69。

Wheat stripe rust resisting ospc geneYr69From E. elongata, the wheat stripe rust resisting ospc geneYr69Positioned at wheat Hybridize top of the wheat breed 2AS chromosome far from centromere obtained with E. elongata.

Compared with the prior art, the present invention has the following advantages: 1, present invention acquisition and Stripe Rust Resistance GeneYr69Chain Molecular labeling 2AS33；2, the label is dominant marker, withYr69Genetic distance be 1.9cM, can quickly, precise Identification it is small WheatYr69The presence or absence of gene and existence simultaneously predict stripe rust resistance, to accelerate to wheat stripe rust resisting ospc geneYr69Utilization；3, the diversification of wheat stripe rust resisting disease antigen is realized.

Detailed description of the invention

Fig. 1 is primer 2 AS33 in CH7086, director 29 and 29 × CH7086 of director F₂For the amplification knot of part single plant Fruit.M:Marker in figure; P_R: disease-resistant parent (CH7086); P_S: Susceptible parent (director 29);BR: disease-resistant gene Pond;Bs: susceptible gene pool;R: homozygous disease-resistant strain;H: separation strains;S: pure and mild susceptible strain；Arrow show primer 2AS33 is in disease-resistant parent and single plant with disease-resistant geneYr69Chain band；

Fig. 2 is gene resistant to stripe rustYr69Genetic linkage map and China spring 2A Deletion line genetic map comparison diagram.In figure (a) it isYr69Genetic linkage map；It (b) is China spring 2A Deletion line genetic map.

Specific embodiment

Disease-resistant parent CH7086 for resistant analysis hybridizes with director 29, is returned the segregating population for generating offspring F₁、F₂、BC₁And F_2:3Family constitutes genetic mapping group；

One, strain-forming period resistance is identified

Material to be tested is planted by following type of seeding in crop field: disease-resistant parent CH7086, Susceptible parent director 29 and silk floss Sun 11 and CH7086 × director 29 and CH7086 × Mianyang 11 F₁For 1 row of each plantation, F₂10 rows of generation plantation, BC₁7 rows of generation plantation, Every the program request of row simple grain 18；F_2:3Family and parent respectively plant 1 row, every row simple grain program request 15-20；Long 1.6 m of row, line-spacing 0.25 m；To ensure that expert evidence is sufficiently fallen ill, 1 row inscription virtuous 169 is planted every 10 rows and does susceptible control, and in experimental material four The high sense stripe rust kind Mianyang 11 of week plantation is as inducing materials；Experimental material is inoculated with after jointing, by ready fresh spore Sub- suspension be sprayed on leaf gone on the wheat seeding of wax (in sprayer be added CYR32 Fresh spores, it is first wet with a small amount of clear water Paste is stirred into after profit, adds enough water to forsythia)；Investigation, which is recorded, after the onset of to susceptible control inscription virtuous 169 sufficiently invades Dye type, by the response type of each wheat lines of strain investigation records, response type is recorded by 6 grade standards；According to anti-× sense hybrid F₁It is anti- Answer type and F₂、F_2:3And BC₁Resist in group, feel single plant segregation ratio, analysis new strain of wheat CH7086 is disease-resistant to strip rust bacteria CRY32 Number of genes and aobvious recessiveness；According toχ ²Test determines the matching degree of its observed value and theoretical value, is finally inferred to Rust resistance product It is number, the aobvious recessive and interaction mode of resistant gene entrained by CH7086.

Two, F₂(this step only proves side of the present invention with a kind of specific embodiment to the molecular marker analysis of group Method is set up, and not representing only following wheat breeds can be identified with the method for the inventionYr69The presence or absence of gene)

1, Genome DNA extraction uses SDS method；

2, the foundation in disease-resistant pond and susceptible pond: 29 cross combination F of CH7086 × director is chosen₂205 single plants of segregating population For ssr analysis and disease-resistant gene chromosome mapping；Segregating population bulked segregant analysis according to propositions such as Michelmore (bulked segregation analysis, BSA) chooses 10 plants of immune single plants (disease resistance response grade is 0 grade) and 10 respectively The DNA mixed in equal amounts of plant height sense single plant (susceptible level of reaction is 4 grades) constructs disease-resistant pond (BR) and susceptible pond (BS)；

3, polymorphic molecular marker screens: first passing through PCR method to microsatellite mark with anti-sense parent and disease-resistant pond, susceptible pond Polymorphism screening is carried out, specifically: (1) it is 20 μ L that PCR, which react total system, including 10 × PCR buffer, specifically: 10 mM Tris-HCl, pH value 8.3,50 mM KCl, 1.5 mM MgCl₂；0.2 mM dNTP, 1 U Taq enzyme, 0.25 μM is drawn Object and 50-100 ng/ μ L Wheat volatiles DNA, are eventually adding a drop mineral oil；(2) PCR amplification program are as follows: 94 DEG C of initial denaturations 5 min；94 DEG C of 1 min of denaturation, 55 DEG C of 45 s of annealing, 72 DEG C of 1 min of extension, 35 recycle；72 DEG C of 10 min of extension；4 ℃ It saves backup；(3) electrophoresis is 8% native polyacrylamide gel electrophoresis, and 5 μ LPCR products and 5 μ L sample-loading buffers are mixed Point sample afterwards, electrophoresis 2h under 180V voltage, cma staining are taken pictures；(note: this part is being found used in the gene process Method, belong to universal method, the program in " application method of molecular labeling " is to carry out the gene using molecular labeling 2AS33 Used method when the detection of presence or absence.Universal method is not necessarily suitable all molecular labelings, so 2AS33's makes Although identical as universal method with program, individually it is described.)

4, the exploitation of Functional marker: using being positioned at wheat 2AS-0.78-1.00 in GrainGenes database EST on section goes to compare wheat 2AS exploration sequence (alliance 2014 is sequenced in international Wheat volatiles), comparison condition BLASTn's E value is set as 0.00001；The Contig compared is submitted to SSR IT software, finds SSR marker；

5, genetic map construction: the F of the polymorphic molecular marker screened and building in anti-sense pond is utilized₂Group carries out Genotyping calculates the genetic distance (cM) of molecular labeling and target gene with Kosambi function, and uses Joinmap 4.0 Software analyzes the linkage relationship of polymorphism mark and disease-resistant gene, and constructs genetic linkage maps；

Three, result

1、Yr69Genetic analysis: in cross combination CH7086 × 29 F of director₂205 single plants in, 152 plants disease-resistant (to invade Dye type is 0-0;96 plants, 56 plants of 1-2 grade of grade), 53 plants susceptible (infecting type for 10 plants of 3 grades, 43 plants of 4 grades), χ²= 0.08, P=0.78, it is as shown in table 1 below to show that the segregation ratio for resisting, feeling meets 3R:1S()；

Table 1 resists ' strain-forming period resistance separation of the sense cross combination different generations group to strip rust bacteria CYR32

2、Yr69Chromosome mapping:

205 plants of F of cross combination CH7086 × director 29 are had chosen in this test₂Group is used for Microsatellite marker analysis； 608 pairs of SSR primers being evenly distributed on 2l chromosome of wheat are chosen, confrontation, sense parent carry out polymorphism screening, and discovery has 4 pairs of SSR primersXgwm210、Xwmc382、Xbarc124WithXgwm636There are polymorphisms between anti-perceptual shape pond, and through F₂ It is chain with the disease-resistant gene in material that segregating population verifies this 4 pairs of SSR markers；By the website Graingenes (http: // The wheat microsatellites genetic map and R der that wheat.pw.usda.gov/cgi-bin/graingenes) can inquire, The wheat high density microsatellite genetic map that Somers etc. is constructed respectively, which is studied, to be compared, it is found that this 4 pairs of primers are all positioned in 2AS On chromosome, therefore the preliminary disease-resistant gene for inferring CH7086 is located on 2AS；In order to which further antagonism gene is accurately positioned, There is polymorphism to this 4 pairs using China spring and a full set of China spring nulli-tetrasomes material, both-end body material and missing based material Primer is expanded, as a result by the target gene Primary Location chain with resistance marker in the section bin 2AS5-0.78-1.00；

The purpose chain with label is encrypted and further determined that in order to which disease-resistant gene designation of chromosome section is marked The accuracy and reliability of gene, this research, which is chosen, comes from the website Graingenes (http://wheat.pw.usda.gov/ Cgi-bin/graingenes) wheat microsatellites genetic map and more people's buildings such as R der, Somers, Eriksen and Marone Wheat high density microsatellite genetic map on 50 be positioned on chromosome 2AS pair SSR primer, 9 pairs of STS primers and fixed 154 pairs of EST primers, confrontation sense parent and anti-sense are designed positioned at the wheat est sequence of 2AS5-0.78-1.00 chromosome interval It is screened in character pond；As a result, it has been found that except aforementionedXgwm210、Xwmc382、Xbarc124WithXgwm636Outside, other 2 couples of SSR PrimerXgpw7101WithXwmc25, 1 pair of STS primerXmag3807And 1 pair of EST primer2AS33(Fig. 1) in anti-sense parent and resists Also the polymorphic differences of stable and consistent are presented between perceptual shape pond and in group's progeny material；It is analyzed by JoinMap4.0 software, This 6 pairs of SSR markers, 1 pair of STS label and 1 pair of EST label and disease-resistant geneYrCH86In the same linkage group, position is suitable Sequence are as follows:Xgwm636-Xgwm210-Xwmc382-Xwmc25-X2AS33-YrCH86-Xmag3807-Xgpw7101-Xbarc124, Genetic distance is respectively 4.5 cM, 2.3 cM, 2.5 cM, 2.7 cM, 1.9 cM, 3.1 cM, 3.4 cM, 4.8cM(Fig. 2)；Mesh Preceding world wheat cdna naming committee is formally by resistant geneYrCH86Name forYr69。

<110>Institute of Crop Science, Shanxi Academy of Agricultural Sciences

<120>wheat stripe rust resisting ospc gene Yr69 and its chain SSR molecular marker and application method

<160>2

<210>1

<211>total

<212>DNA

<213>artificial sequence

<221>primer sequence

<222>(1) ... (20)

<400>1

5'-AGAAGGCACACTGCTGGAAC-3'

20

<210>2

<211>total

<212>DNA

<213>artificial sequence

<221>primer sequence

<222>(1) ... (20)

<400>2

5'-ACATGAGTGAGTTGTGAGTC-3'

20

Claims (2)

1. a kind of wheat stripe rust resisting ospc geneYr69Chain SSR molecular marker is for identifying in wheat whether contain stripe rust resisting GeneYr69Application method, the SSR molecular marker be primer 2 AS33, the nucleotide sequence of the primer are as follows: upstream sequence For 5'-AGAAGGCACACTGCTGGAAC-3', downstream sequence 5'-ACATGAGTGAGTTGTGAGTC-3'；It is characterized by: Including following operating procedure:

1), using Wheat volatiles DNA as PCR amplification template, using SSR molecular marker 2AS33 as primer, PCR amplification, reaction are carried out System is 20 μ L, and the reaction system includes: 50-100 ng/ μ L Wheat volatiles DNA, 10 × PCR buffer, specifically: 10mM Tris-HCl, pH value 8.3,50mM KCl, 1.5mM MgCl₂；0.2mM dNTP, 1U Taq enzyme, 0.25 μM of primer, most A drop mineral oil is added afterwards；

2), PCR amplification program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 1min, 55 DEG C of annealing 45s, 72 DEG C of extension 1min, 35 A circulation；72 DEG C of extension 10min；4 DEG C save backup；

3), electrophoresis is 8% native polyacrylamide gel electrophoresis, point after 5 μ LPCR products and 5 μ L sample-loading buffers are mixed Sample, electrophoresis 2h under 180V voltage, cma staining are taken pictures；

4) it, analyzes and identifies, the specific band of 269bp can be amplified, then illustrate that there are Stripe Rust Resistance Genes in wheat germplasmYr69, Otherwise, Stripe Rust Resistance Gene is not present in wheat germplasm to be measuredYr69。

2. application of the wheat stripe rust resisting ospc gene Yr69 chain SSR molecular marker in Stripe Rust Resistance Gene Yr69 identification, institute Stating SSR molecular marker is primer 2 AS33, the nucleotide sequence of the primer are as follows: upstream sequence 5'- AGAAGGCACACTGCTGGAAC-3', downstream sequence 5'-ACATGAGTGAGTTGTGAGTC-3'.

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