Method for directionally breeding rapid wheat breeding material by combining Vrn-Ala and Ppd-D1b

Abstract

The invention discloses a rapid wheat breeding method combining Vrn-A1a and Ppd-D1b genes, which comprises the following steps: taking wheat germplasm SN0594 with characteristics of no vernalization, short growth cycle and safe overwintering as a parent, obtaining an F2 group through hybridization and artificial climate chamber selfing generation adding, and finally breeding grains capable of safe overwintering by combining genotype identification and agronomic character screening. According to the method, the freezing resistance of the winter wheat and the short growth period characteristic of the spring wheat are integrated, the created novel germplasm adapts to the rapid generation adding stability of the phytotron, field performance can be verified through autumn sowing, and the problems that the winter wheat is long in character stability period, large in loss and the like are effectively solved. The method comprises the steps of (1) selecting SN0594 to be hybridized with a target parent, (2) cultivating an F2 population in an artificial climate chamber, (3) screening genotypes and agronomic traits, and (4) sowing and validing.The method is easy and convenient to operate and capable of being popularized on a large scale, and an efficient scheme is provided for rapid stabilization of wheat traits and field verification.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and specifically relates to a method for combining Vrn-A1a with Ppd-D1b for directional cultivation of wheat rapid breeding materials and application thereof. Background Art

[0002] The long breeding cycle of wheat limits the efficiency of breeding new varieties. The wheat materials used in production must be strains with stable genetic backgrounds. After hybridization, they need to undergo 7-8 generations of self-pollination and selection to stabilize the target traits. Selecting suitable breeding materials is crucial to shortening the breeding cycle. Winter wheat has strong frost resistance and can safely survive the winter when sown in the field in autumn, but it requires 4-8 weeks of vernalization to transition to the reproductive growth stage, and can only achieve 2-3 generations a year in an artificial climate chamber environment; spring wheat does not require vernalization and can continuously multiply generations in an artificial climate chamber, achieving 4-5 generations a year, but has poor frost resistance, and the cultivated strains cannot verify the traits and functions in the field in autumn sowing. There is an urgent need to create a wheat germplasm that is suitable for rapid stabilization of traits in artificial climate chambers and can be used to verify strain functions in autumn sowing in the field. The wheat germplasm SN0594 created in our laboratory in the early stage does not require vernalization, has a heading period of 30 days in the artificial climate chamber, and can achieve rapid and stable traits under artificial climate chamber conditions; compared with the sister line material carrying the Vrn-A1a+Ppd-D1a gene, SN0594 carries the Vrn-A1a+Ppd-D1b gene, and field trials in 2022-2023 and 2023-2024 have proven that it can safely overwinter, but the plant height is higher and the agronomic traits are poor. Summary of the invention

[0003] The purpose of the present invention is to overcome the above-mentioned problems and provide a method and application thereof for directing the cultivation of wheat rapid breeding materials by combining Vrn-A1a and Ppd-D1b. The method can achieve the accelerated generation of stable strains in an artificial climate chamber, and then verify the trait function of the strains in a field autumn sowing environment. The generation stabilization time is shortened, the efficiency of creating stable strains is improved, and the function of the target traits can be verified in a field sowing environment.

[0004] The specific technical solutions are as follows:

[0005] The present invention provides a method for combining Vrn-A1a with Ppd-D1b for directional cultivation of wheat rapid breeding materials and its application, comprising the following steps ( Figure 1 ):

[0006] Step 1. Select parent material A with excellent agronomic traits and hybridize it with wheat germplasm SN0594

[0007] In October, the parent material A and wheat germplasm SN0594 with better agronomic traits are planted in the field. According to the investigation results of the flowering period of the parents, the sowing date of parent A and SN0594 is arranged in time to ensure that the flowering periods of the parents coincide when hybridization is performed; during the flowering period of wheat, parent A and SN0594 are hybridized, and the hybrid ears are harvested, threshed, and grains are harvested in June of the first year when the wheat is mature;

[0008] Step 2. Produce F by self-crossing in artificial climate chamber 2

[0009] The F obtained in step 1 1 The seeds were germinated at room temperature, and the germinated seeds were sown in a seedling pot with a diameter of 30 cm and a height of 20 cm containing a substrate, 6 seeds were sown in each pot, and placed in an artificial climate room at 25°C, with a light duration of 22 hours per day, a light intensity of 600 Lux or more, and a humidity of 60-70%. During the growth period, watering was paid attention to; about 20-25 days after flowering, the ears were harvested, and the harvested ears were placed in a drying room at 55°C for 10-12 hours, and the progeny F was obtained by manual threshing. 2 ; The entire step 2 takes about 2 months;

[0010] Step 3. F 2 The F 3 seed

[0011] In August of the first year, the F obtained in step 2 will be 2 The seeds were planted in an artificial climate chamber; they were germinated at room temperature, and the germinated seeds were directly sown into a 96-well culture plate containing a substrate without vernalization treatment, with one seed sown in each well. The culture conditions were the same as in step 2; the vernalization photoperiod molecular markers developed by Yan et al. were used to screen individual plants carrying the Vrn-A1a+Ppd-D1b genotype, and F plants with better agronomic traits were selected. 2 Individual plants that meet the requirements are marked with a card, and the marked ears are harvested about 20-25 days after flowering; the progeny F with the Vrn-A1a+Ppd-D1b genotype combination and good agronomic traits are obtained. 3 ; The entire step 3 takes about 2 months;

[0012] Step 4. Sowing and harvesting F that can safely survive the winter 4 Grains

[0013] In October of the first year, the F obtained in step 3 will be 3The seeds were sown in the field, with a 3m×3m plot, 30cm row spacing and 15cm plant spacing. No pesticides were applied to the seeds. Winter hardiness under field conditions was evaluated in February of the following year, using a visual rating system of 1-9, where 1 = 100% plant death and 9 = all plants survived. The ears of individual plants that survived the winter safely were harvested in June of the following year at the wheat maturity stage, and the F 4 The individual ears are dried and threshed manually and packed into different seed bags; 4 The generation meets the conditions of carrying the Vrn-A1a+Ppd-D1b genotype homozygous, short growth cycle, good agronomic traits, and safe wintering. The material can not only continuously and quickly increase the generation under the conditions of the artificial climate chamber, but also safely survive the winter after autumn sowing, and identify phenotypic or resistance and other trait indicators in the field environment.

[0014] Preferably, in step 1, each hybrid ear is ensured to have at least 10 spikelets.

[0015] Preferably, in step 1, F is produced 2 The population is used for genotype identification and phenotypic screening, and the number of hybrid ears is not less than 3.

[0016] Compared with the prior art, the present invention has significant beneficial effects:

[0017] The new wheat germplasm created by the present invention combines the dual advantages of excellent frost resistance of winter wheat and no need for vernalization of spring wheat, and has excellent agronomic traits. It can realize breeding in the dual environment of artificial climate chamber and field, accelerate the generation in the artificial climate chamber, shorten the stable years of wheat strains, and accurately identify the phenotype and resistance of materials in the autumn sowing in the field. Using this method, we hybridized SN0594 with winter wheat SN34 and Yannong 15 with excellent agronomic traits, and obtained wheat materials that do not require vernalization, have a short growth cycle, and can safely overwinter. This proves the feasibility of this method. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of hybridizing winter wheat SN34 with excellent agronomic traits with wheat germplasm SN0594 to obtain a stable strain.

[0019] Figure 2 is the calculation formula for the group. Among them:

[0020] N:F 1 Number of seeds; n: number of generations; 20: seeds per ear; 0.9: survival rate.

[0021] First 1 / 4: F 2 The probability of Vrn-A1a / Vrn-A1a in the generation; the second 1 / 4: F 2The probability of Ppd-D1b / Ppd-D1b in the generation; 1 / 2: F 2 The probability of better agronomic traits in the next generation. DETAILED DESCRIPTION

[0022] Example 1: Wheat SN34 is a parent example.

[0023] The Vrn gene is a vernalization gene. When the genotype is Vrn / Vrn or Vrn / vrn, heading can be produced without vernalization. When the genotype is vrn / vrn, vernalization is required for heading to occur.

[0024] Step 1: Hybridize SN34 with wheat germplasm SN0594 to obtain F 1

[0025] SN34 is a winter wheat material with good agronomic traits, and its genotype at the vernalization photoperiod locus was identified as vrn-1+Ppd-D1a.

[0026] The results of the early flowering period survey showed that the flowering period of the two parents was quite different, and SN34 bloomed about 10 days earlier. In October, the parents SN34 and SN0594 were planted in the field. SN34 was sown in 4 batches, with an interval of about 1 week between each batch.

[0027] During the flowering period of wheat, hybridization is performed using SN34 as the female parent and SN0594 as the male parent, or using SN34 as the male parent and SN0594 as the female parent.

[0028] Subsequent work is to construct an F-type for vernalization photoperiod genotype identification and screening of excellent agronomic traits. 2 The population size needs to be around 500, with a total of 3 hybrid ears. Each emasculated ear must contain 10 spikelets, and about 30 hybrid grains can be obtained.

[0029] In June of the first year, hybrid ears are harvested when the wheat matures and threshed manually.

[0030] Step 2: Self-crossing in an artificial climate chamber to produce hybrid F 2

[0031] The hybrid seeds (F 1), germinate at room temperature (25°C), sow the germinated seeds (seed root length is about 1 cm) into a 96-well culture plate containing a substrate, and place it in an artificial climate chamber at 25°C, 22 hours of light per day, light intensity greater than or equal to 600Lux, and humidity of 60-70%. Pay attention to watering during the growth period, and harvest the ears about 20-25 days after flowering, when the grains are green to a certain extent. The harvested ears are placed in a drying room at 55°C for 10-12 hours, and threshed manually, at which time the progeny F is obtained. 2 This step takes a total of 2 months, during which time pay attention to pests and diseases such as aphids and powdery mildew.

[0032] Step 3: F 2 The F 3 seed

[0033] Follow these steps to get F in August of the same year. 2 Grains, F obtained in step 2 2 About 600 seeds, considering that some seeds did not germinate, about 540 plants survived. 2 Indoor planting, DNA extraction, vernalization photoperiod genotyping and agronomic trait screening were performed. 2 , and germinated at room temperature. The germinated seeds were directly sown into a 96-well culture plate containing a matrix without vernalization treatment, with one seed sown in each well. The culture conditions were the same as in step 2. The vernalization photoperiod molecular markers developed by Yan et al. were used to screen single plants carrying the Vrn-A1a+Ppd-D1b genotype homozygous, and F with better agronomic traits were selected. 2 Individual plants that meet the requirements are marked with a card, and the marked ears are harvested about 20-25 days after flowering; according to F 2 The ratio of the vernalization gene loci Vrn-A1a / Vrn-A1a: Vrn-A1a / vrn-1: vrn-A1 / vrn-A1=1:2:1, and the photoperiod gene loci Ppd-D1a / Ppd-D1a: Ppd-D1a / Ppd-D1b: Ppd-D1b / Ppd-D1b=1:2:1 (the probability of Vrn-A1a and Ppd-D1b being inherited independently on different chromosomes is 1 / 4×1 / 4=1 / 16) ( Figure 2 ), about 34 plants carrying the Vrn-A1a+Ppd-D1b genotype homozygous can be obtained, and then the plants with excellent agronomic traits can be selected from them, and about 17 progeny F plants with good agronomic traits that do not require vernalization can be obtained. 3 .

[0034] Step 4: Sowing and harvesting F that can safely survive the winter 4 Single ear

[0035] In mid-October of the first year, approximately 340 F 3 The seeds were sown in the field, and eventually about 300 plants survived, with a row spacing of 30 cm and a plant spacing of 15 cm. The seeds were sown spot-sown, and no pesticides were applied to these seeds. Winter hardiness under field conditions was evaluated in February of the following year, using a visual scoring method of 1-9, where 1 = 100% of the plants died and 9 = all plants survived. The ears of individual plants that survived the winter safely were harvested in June of the following year at the wheat maturity stage, and the F 4 The individual ears are dried and threshed manually and packed into different seed bags; 4 The next generation meets the conditions of carrying the Vrn-A1a+Ppd-D1b homozygous gene, having a short growth cycle, good agronomic traits, and being able to safely overwinter.

[0036] The above is only a preferred implementation of this patent. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of this patent. These improvements and substitutions should also be regarded as the scope of protection of this patent.

Claims

1. A method for directing the cultivation of wheat rapid breeding materials by combining Vrn-A1a and Ppd-D1b, characterized in that: The following steps are involved: Step 1. Parental hybridization to obtain F1 generation: The parent material A with excellent agronomic traits was field-hybridized with the wheat germplasm SN0594 carrying the Vrn-A1a+Ppd-D1b gene, and the sowing period was adjusted to ensure that the flowering period coincided, and the hybrid grains were harvested at maturity; Step 2. Generate F2 population in artificial climate chamber: After germination, the F1 seeds were sown in an artificial climate chamber and cultivated at 25°C, 22 hours of light, ≥600 Lux light intensity, and 60-70% humidity. The F2 seeds were harvested 20-25 days after flowering. Step 3. Genotypic and phenotypic screening of F2 generation to obtain F3 grains: Plant the F2 population, use the Vrn-A1a and Ppd-D1b molecular markers to screen for double-gene homozygous plants, simultaneously select plants with excellent agronomic traits, and harvest the target F3 generation grains; Step 4. Field test of cold resistance of F4 generation: The F3 generation grains were sown in autumn, and the overwintering survival rate was evaluated by visual scoring. The F4 generation materials with Vrn-A1a+Ppd-D1b homozygous genes, short growth period and safe overwintering characteristics were harvested.

2. The method according to claim 1, characterized in that: In step 1, retain at least 10 spikelets for each hybrid ear.

3. The method according to claim 1, characterized in that: The number of hybrid ears used to construct the F2 population in step 1 is no less than 3.

4. Application of the method according to any one of claims 1 to 3 in preparing wheat germplasm resources, characterized in that: The germplasm resource carries the Vrn-A1a+Ppd-D1b homozygous genotype.

5. Application of the method according to any one of claims 1 to 3 in preparing wheat germplasm resources, characterized in that: The growth period of the plants corresponding to the germplasm resources is ≤60 days.

6. Application of the method according to any one of claims 1 to 3 in preparing wheat germplasm resources, characterized in that: Rapid breeding of ≥ 4 generations per year can be achieved in the field and artificial climate chamber environment.

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