CN107058529B - Method for breeding durable resistance material of wheat stripe rust - Google Patents
Method for breeding durable resistance material of wheat stripe rust Download PDFInfo
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Abstract
The invention belongs to the field of wheat disease-resistant breeding, and particularly discloses a breeding method for efficiently breeding a stripe rust lasting-resistant breeding material by combining a seedling-stage disease-resistant gene with an adult-stage disease-resistant gene such as Yr18 and the like by adopting a mixed small-seed high-pressure identification and multi-generation backcross technology. The method can provide excellent intermediate material for rust-resistant breeding.
Description
Technical Field
The invention belongs to the field of wheat disease resistance breeding, and particularly relates to a method for breeding a durable wheat stripe rust resistant material.
Background
Wheat stripe rust is a fungal airborne disease caused by Puccinia striiformis f.sp.tritici, which can cause regional yield reduction of 0.1-5% and in severe cases, yield reduction of 5-25% in wheat. China is the biggest wheat stripe rust disease circulation area in the world, stripe rust disease is pandemic after multiple outbreaks, and according to statistics, the average generation area of stripe rust disease in China in 2002-2Per year, the method poses a great threat to wheat production. The wheat stripe rust fungus summer spore can be spread in a long distance along with the air flow, so that the wheat stripe rust fungus summer spore can quickly cause the circulating prevalence of the disease in the area with the defect, and belongs to the epidemic disease in the large area.
The selection of disease-resistant varieties by using the stripe rust resistance gene is the most economic and environment-friendly way for controlling stripe rust. Based on resistance responses to wheat stripe rust, wheat stripe rust resistance genes fall into two categories: resistance at the whole growth stage (seedling stage resistance) and resistance at the adult stage. The resistance in the whole growth period (seedling stage resistance) generally lasts for the whole growth period, and the resistance gene has microspecies specialization, high resistance level, easy observation and quality trait inheritance. The disease-resistant genes in the adult plant stage generally have certain resistance to a wide range of pathogenic bacteria, have low resistance level, are not easy to observe and belong to quantitative trait inheritance. In the breeding of wheat stripe rust, breeders tend to use a resistance gene with high resistance level and easy observation in the whole growth period, and great selective pressure is applied to wheat stripe rust, so that the dominant physiological race of the stripe rust is changed quickly, and new physiological races are easy to appear, which is a main reason for quick loss of the resistance of the wheat stripe rust. The development and utilization of the adult plant stage resistance gene with durable resistance and no microspecies specialization is a long-term effective measure for controlling stripe rust. But face the problem that the resistance provided by single or few adult stage resistance genes or QTLs does not meet the level of resistance required for wheat varieties in production.
Disclosure of Invention
The objects of the present invention include:
provides a breeding method for solving the problem of the limitation of single utilization of resistance genes in the seedling stage and the adult stage;
provides a method for breeding a stable breeding material with durable wheat stripe rust resistance.
Specifically, the invention provides a method for breeding a durable resistant material for wheat stripe rust, which comprises the following steps:
1) collecting wheat stripe rust strain, inoculating, identifying and screening out resistance material P in whole growth period1；
2) With P1Using Yr 18-containing mature-stage resistant material P as female parent2Preparing hybridization for male parent to obtain hybrid F1；
3) With F1As the female parent, with P1Carrying out multi-generation backcross for recurrent parents; from BC1F1To BC5F1Before backcross of each generation, disease-resistant plants in the full-growth period in backcross offspring are selected, single plant leaves are picked before heading to extract DNA, and a Yr18 plant-forming period and resistance type codominant marker is adopted for detection, so that the selected female parent is ensured to contain a Yr18 plant-forming period and resistance type gene, and the material with stable resistance is obtained.
Preferably, the wheat stripe rust strains in step 1) are CYR29, CYR30, CYR31, CYR32, CYR33, V26 and/or water source 14, and the identification is performed by room-temperature mixed microspecies high-pressure identification;
the inoculated inoculating material in the step 1) is 10P4-8, 13P2-7 and/or 14P 532.
Preferably, the bir 18-containing paternal material in the adult stage is 13KG458 and/or 13KG662 in the step 2).
Preferably, the backcross in the step 3) is performed by adopting a summer propagation generation-adding technology;
step 3) the YR18 adult stage concurrent resistant co-dominant STS marker is csLv 34;
the detection method in the step 3) is molecular marker detection, wherein the PCR amplification system accounts for 20 mu L, and comprises 2 mu L of 10 xbuffer, 1UTaq DNA polymerase, 0.4 mu L of dNTPs, 0.5 mu L of primers and 50ng of template DNA, wherein the concentration of 4 dNTPs is 10mmol L–1The concentration of Taq DNA polymerase was 2.5U. mu.L–1The concentration of the forward primer and the reverse primer is 4 mu mol L-1; the PCR amplification procedure was: denaturation at 94 deg.C for 1min, annealing at 57 deg.C for 1min, extension at 72 deg.C for 2min, and 5 cycles; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; finally, denaturation at 94 ℃ for 1min, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 5 min;
the sequence of the primer is as follows:
a forward primer: 5'-GTTGGTTAAGACTGGTGATGG-3', respectively;
reverse primer: 5'-TGCTTGCTATTGCTGAATAGT-3' are provided.
Further, electrophoresis detection is carried out on the PCR amplification product in the step 3), and a 150bp band appears in the amplification product, so that the material contains the YR18 adult-stage and anti-type gene.
The invention also discloses application of the material bred by any one of the methods as a wheat stripe rust resistant strain to participate in strain comparison experiments or as a breeding intermediate material.
The nucleotide sequence of the YR18 strain-stage concurrent resistance gene can be found in Krattiger S G, Lagudah E S, Spielmeyer W, Singh R P, Huerta-Espeno J, McFadden H, Bossolini E, Filter L L, Keller B.A reactive ABC transporter reduced resistance to multiple gene expression in great science 2009,323: 1360-
The Yr18 adult stage and anti-type codominant STS marker csLv34 can be found in the literature: lagudah E S, McFadden H, Singh R P, Huerta-Espino J, Bariana H S, Spielmeyer W. molecular genetic characterization of the Lr34/Yr18slow running restriction gene region in the where Appl Genet,2006,114: 21-30. Advantageous effects
The method adopts mixed small seed high-pressure identification and multi-generation backcross technology to combine the disease-resistant genes at the seedling stage with the disease-resistant genes at the adult plant stage such as Yr18 and the like, can efficiently culture and obtain the stripe rust lasting resistance breeding material, and provides an excellent intermediate material for the rust resistance breeding.
Drawings
FIG. 1 is a flow chart of a wheat stripe rust lasting resistance efficient selection technology
FIG. 2 is a graph showing the results of polyacrylamide gel electrophoresis
Wherein, the symbols indicate that the individual plant contains the target gene and can be selected as a backcross female parent.
Detailed Description
Example one high pressure identification of Mixed seeds at Room temperature for selection of Whole growth stage resistant Material P1And (4) testing.
Mixing collected strains such as CYR29, CYR30, CYR31, CYR32, CYR33, V26 and a water source 14 for later use, planting materials to be screened in a pot culture mode in a greenhouse, sowing 5-8 seeds on each material, inoculating the mixed strains by a smearing method when the materials grow to 1 heart and 1 leaf, humidifying the inoculated strains for 24 hours by a humidifier after inoculation, keeping the dark condition, controlling the room temperature to be 10-16 ℃, then adjusting the room temperature to be about 20 ℃, lighting the time for 12 hours, identifying the seedling disease resistance of the materials to be tested after the materials are fully infected by a susceptible control Sichuan breeding 12, and screening excellent materials with good seedling disease resistance such as 10P4-8, 13P2-7 and 14P 532.
Example II, P1×P2Hybridization gave rise to the F1 generation trial.
The excellent materials (10P4-8, 13P2-7, 14P532, etc.) selected in example 1 were used as the parent P1Using known material (such as 13KG458, 13KG662, etc.) containing Yr18 with resistance in adult stage as male parent P2Then, hybridization and assembly were carried out to obtain F1 material.
EXAMPLE III backcross test and field identification
Using the F1 material obtained in example two as the parent material, the same Yr18 strain-stage-concurrently-resistant material (e.g., 13KG458, 13K) as described in example two was usedG662, etc.) P2Hybridizing to obtain BC as male parent1F1Germplasm; the next generation is further BC1F1As the female parent, with P2Hybridizing to obtain BC as male parent2F1(ii) a In this manner, up to BC6F1And obtaining the material YrP with stable disease-resistant target character and agronomic character. The implementation process is carried out under the condition that the field dominance is induced by various seeds, and the resistance phenotype of the selected backcross female parent in the single plant seedling stage and after heading is high resistance. The inducing material and the backcross test material are planted alternately, mixed dominant seeds are inoculated by a smearing method respectively at the 1 st-heart-1-leaf stage, the 3-leaf stage and before heading, the seedling stage identification is carried out 20-30d after the first inoculation, and the adult stage identification is carried out after heading. The test farm is developed in the new growth of the current generation, and the propagation of the Spirospermum in summer is carried out in the Spirospermum of West Chang.
Example four, test for identifying disease-resistant Yr18 strain-stage concurrent resistance gene in the whole growth stage of backcross progeny.
Before hybridization of the female parent material selected in the third embodiment, DNA was extracted, and screening of Yr18 gene of concurrent resistance at the adult stage was performed by using codominant STS marker csLv34, wherein the total PCR reaction system was 20. mu.L, and the PCR reaction system included 2. mu.L of 10 XBuffer, 1UTaq DNA polymerase (2.5 uL-1), 0.4. mu.L of dNTPs (the concentration of 4 dNTPs is 10mmol L-1), 0.5. mu.L of primer (4. mu. mol L-1), and 50ng of template DNA. The PCR program is 94 ℃ denaturation for 1min,57 ℃ annealing for 1min,72 ℃ extension for 2min,5 cycles; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; finally, denaturation at 94 ℃ for 1min, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 5 min. The PCR amplification product was detected by 6% denaturing polyacrylamide gel electrophoresis, and the expected amplification products were 150bp (containing the target gene) and 229bp (not containing the target gene). A single strain containing 150bp of amplification product is selected as a backcross female parent in the third embodiment.
6% denaturing polyacrylamide gel electrophoresis detection test:
1. preparing a glass plate: and fully washing one flat plate and the other groove plate with clear water, scrubbing with absolute ethyl alcohol once, and then ventilating and drying.
2. Coating a plate: after the alcohol is dried, uniformly coating stripping silane on the groove plate, and drying; affinity silane (8 ul of affinity silane stock solution, 8ul of glacial ethanol and 2ml of absolute ethanol) is uniformly coated on the plate and dried.
3. Plate installation: the glass plate is arranged at the lower part, the electrophoresis tank plate is arranged at the upper part, the edge strips are arranged at two sides between the 2 plates, after alignment, the wave plate and the electrophoresis tank plate are clamped by a flange pair clamp and are horizontally arranged.
4. Pouring glue; 50mL of 6% polypropylene tyramine gel solution is measured, 350ul of APS (namely 10% of pastel persulfate and 28ul of TEMED) is added, the mixture is poured into a glue filling device and mixed evenly, glue is slowly and evenly filled from a glue filling opening, then the straight surface of a comb is inserted into the glue opening, no bubbles are generated, and the comb is placed for about 1 hour.
5. Pre-electrophoresis: taking down the clamp, pulling out the comb, putting the rubber plate into the electrophoresis tank, clamping, and filling buffer solution, namely IxTBE. And 80W balanced power electrophoresis for half an hour.
6. Sample application: and pulling out the comb, blowing the waste glue at the glue filling opening clean by using a liquid transfer gun, inserting the comb into the glue filling opening in the forward direction, blowing each comb hole clean by using the liquid transfer gun, and starting sample application.
7. Electrophoresis: electrophoresis at 80W for about 1 hr.
8. Silver staining: and taking the rubber plate down from the electrophoresis tank, separating the two glass plates, putting the glass plate with the rubber surface facing upwards into a silver dyeing tank for 15min, and keeping the silver dyeing tank away from light.
9. Washing with water: the glass plate was taken out of the silver staining solution and washed with distilled water rapidly for 15 s.
10. And (3) developing: and (4) putting the washed glass plate into a developing solution until the strips are clear.
11. And (3) washing with water again: washing with tap water for a while, washing off NaOH on the glue surface, and standing for air drying.
12. And (4) statistical observation: the juice strip data can be directly collected by placing the glass plate on a film viewer;
the gel detection result of partial backcross progeny is shown in the attached figure 2 of the specification.
Sequence listing
<110> institute of agricultural sciences of Sichuan province
<120> method for breeding durable resistant material of wheat stripe rust
<130> 2017
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
gttggttaag actggtgatg g 21
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<400> 2
tgcttgctat tgctgaatag t 21
Claims (1)
1. A method for breeding a wheat stripe rust durable resistance material comprises the following steps:
1) collecting wheat stripe rust strains, inoculating, identifying and screening a resistance material P1 in the whole growth period;
2) p1 as female parent and Yr 18-containing plant-growing period concurrently resistant material P2Preparing hybridization for male parent to obtain hybrid F1；
3) With F1As the female parent, with P1Backcrossing for many generations for recurrent parents by adopting a summer propagation generation-adding technology; from BC1F1To BC5F1Before backcrossing of each generation, firstly selecting disease-resistant plants in the full-growth period in backcrossed offspring, picking individual plant leaves before heading, extracting DNA, and detecting by adopting a YR18 adult-period and anti-type codominant marker csLv34 to ensure that the selected female parent contains a YR18 adult-period and anti-type gene until a material with stable resistance is obtained;
the wheat stripe rust strains in the step 1) are CYR29, CYR30, CYR31, CYR32, CYR33, V26 and a water source 14, the identification is carried out by the room-temperature mixed microspecies high-pressure identification, and the inoculated materials in the step 1) are 10P4-8, 13P2-7 and/or 14P 532;
step 2) the Yr 18-containing concurrently resistant male parent material in the adult stage is 13KG458 and/or 13KG 662;
the detection method in the step 3) is molecular marker detection, wherein the PCR amplification system accounts for 20 muL, and comprises 2 muL of 10 xbuffer, 1UTaq DNA polymerase, 0.4 muL of dNTPs, 0.5 muL of primers and 50ng of template DNA, wherein the concentration of 4 kinds of dNTPs is 10mmol L-1The concentration of Taq DNA polymerase was 2.5U. mu.L-1The concentration of the forward and reverse primers was 4. mu. molL-1(ii) a The PCR amplification procedure was: denaturation at 94 deg.C for 1min, annealing at 57 deg.C for 1min, extension at 72 deg.C for 2min, and 5 cycles; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for lmin for 30 cycles; finally, denaturation at 94 ℃ for 1min, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 5 min;
the sequence of the primer is as follows:
a forward primer: 5'-GTTGGTTAAGACTGGTGATGG-3', respectively;
reverse primer: 5'-TGCTTGCTATTGCTGAATAGT-3', respectively;
and 3) performing electrophoresis detection on the PCR amplification product, wherein a band of 150bp appears in the amplification product, and the material contains the YR18 adult-stage and anti-type gene.
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| CN107586868B (en) * | 2017-09-11 | 2021-01-15 | 四川农业大学 | Molecular marker linked with stripe rust resistance locus from introgression line of wheatgrass P genome in adult stage and application thereof |
| CN110663542A (en) * | 2019-11-19 | 2020-01-10 | 中国科学院西北高原生物研究所 | Breeding method for long-acting resistance of wheat stem rust Ug99 |
| CN112704005A (en) * | 2020-12-29 | 2021-04-27 | 石河子大学 | Rapid directional improvement technology for wheat target character |
| CN114600767A (en) * | 2022-04-07 | 2022-06-10 | 四川省农业科学院作物研究所 | Method for rapidly breeding wheat durable stripe rust resistant variety |
| CN114731947B (en) * | 2022-05-20 | 2023-07-18 | 四川省农业科学院作物研究所 | Breeding method of novel micro-effect polygenic durable disease-resistant wheat variety |
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| CN101558735A (en) * | 2009-06-03 | 2009-10-21 | 西北农林科技大学 | Novel method for cultivating wheat stripe rust resistant polygene polymerized flower |
| CN103931484A (en) * | 2014-04-04 | 2014-07-23 | 福建农林大学 | Breeding method of widely-resistive wheat variety |
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| CN101558735A (en) * | 2009-06-03 | 2009-10-21 | 西北农林科技大学 | Novel method for cultivating wheat stripe rust resistant polygene polymerized flower |
| CN103931484A (en) * | 2014-04-04 | 2014-07-23 | 福建农林大学 | Breeding method of widely-resistive wheat variety |
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