CN114931092B

CN114931092B - Wheat scab resistant molecular breeding method capable of polymerizing multiple traits and facilitating allelic variation

Info

Publication number: CN114931092B
Application number: CN202210554102.2A
Authority: CN
Inventors: 胡文静; 陆成彬; 高德荣; 吴宏亚; 张勇; 张晓祥; 朱冬梅; 吕国锋; 张春梅; 廖森; 赵蝶
Original assignee: JIANGSU LIXIAHE REGION AGRICULTURAL RESEARCH INSTITUTE
Current assignee: JIANGSU LIXIAHE REGION AGRICULTURAL RESEARCH INSTITUTE
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2023-02-28
Anticipated expiration: 2042-05-19
Also published as: CN114931092A

Abstract

The invention discloses a wheat scab resistance molecular breeding method for polymerizing multiple traits to facilitate allelic variation, which comprises the steps of selecting a parent strain carrying resistance genotype of a scab resistance gene Fhb1 and resistance allelic variation of a scab resistance locus Qfhb.3B, carrying out genotype complementation of synergistic allelic variation of a small ear locus QSNS.2B and a thousand grain weight locus QTKW.2B, hybridizing or double crossing the selected parent strain, and harvesting the hybridized or double crossed seeds. Then combining the molecular marker selection favorable for allelic variation and comprehensive agronomic character investigation to carry out multi-generation identification screening to obtain the wheat variety (line) with gibberellic disease and high yield synergistic improvement. The wheat variety (line) with resistance to gibberellic disease bred by the method has polymerization resistance to gibberellic disease and related yield, favorable genotype or allelic variation, and synergistically improved resistance to gibberellic disease and yield level, can be used as a new generation of green and safe wheat new variety (line), and solves the problem of neck sticking in the contemporary breeding industry.

Description

Wheat scab resistant molecular breeding method capable of polymerizing multiple traits and facilitating allelic variation

Technical Field

The invention belongs to the technical field of wheat molecular breeding methods, and relates to a wheat scab resistant molecular breeding method capable of polymerizing multiple traits and facilitating allelic variation.

Background

China is the first large production country and consumption country of wheat, and the production condition of wheat has extremely important significance for national food safety, social and economic development and improvement of the living standard of people. At present, wheat production still faces threats of various diseases, insect pests, abiotic stress and the like. Wheat scab (FHB) is one of the most harmful fungal diseases, and is mainly caused by Fusarium graminearum (Fusarium graminearum) and the like. Pathogenic bacteria infect small flowers at the ear part in the wheat flowering period, continuously expand along the cob in the grain filling and maturing process, and toxins such as Deoxynivalenol (DON), nivalenol (NIV) and Zearalenone (ZEN) are generated and accumulated, so that the cob is blackened, the whole cob is dead, the grains are shriveled, the yield and the quality are reduced, and the generated DON toxin is easy to cause vomiting, poisoning and abortion of people and animals and cause huge damage to the health of the people and the animals. The wheat scab hazard is on a gradually increasing trend worldwide. The Huang-Huai-Mai area and the middle-lower wheat area of Yangtze river are the main production areas of the first and second major wheat in China, and account for about 70 percent of the total annual planting area of the wheat in China. The wheat areas in the middle and lower reaches of the Yangtze river are the common area and the retransmission area of wheat scab. In recent years, wheat scab has become a common disease in Huang-Huai-Mai areas due to the influence of factors such as climate warming, northern moving of rainfall zones, returning of rice and corn straws and the like. In the last decade, 5 major outbreaks of head blight occur in China, and the annual average disease area accounts for about 1/4 of the total planting area. The scab is very popular in China in 2012, the occurrence area is up to 990 ten thousand hectares, and the occurrence areas in 2016 and 2018 are 680 ten thousand hectares and 570 ten thousand hectares respectively. In "approval Standard for crop variety of Jiangsu province", approval Standard for wheat variety resistant to gibberellic disease: the resistance of the Huainan wheat area reaches resistance, and the resistance of the Huaibei wheat area reaches resistance more than that of the Huaibei wheat area. However, at present, few wheat varieties which are examined in Huainan, huaibei and even nationwide can reach the resistance. The cultivation of wheat variety with scab resistance is still the most economic, safe and effective way to solve the harm of scab of wheat, but the contradiction between scab resistance and high yield still restricts the neck of wheat breeding in China.

The current choice for wheat scab and resistance is predominantly phenotypic. The gibberellic disease resistance of wheat is a typical quantitative character, is not single-gene controlled and is easily influenced by multiple factors including environment, the identification of the gibberellic disease resistance of multiple generations in breeding is very complicated and tedious, a large amount of manpower, material resources and financial resources are required to be invested, and in addition, in recent years, the climatic conditions are abnormal and extreme climates frequently occur, so that great challenges are brought to the phenotypic identification of the gibberellic disease. At present, the fusion development of the biological technology, the information technology and the intelligent technology is advancing, and the rapid development of the new generation biological breeding technology is being promoted. "China will accelerate the implementation of the variety industry revivification action, develop key seed source technology and develop and popularize the cultivation of new species. In recent years, the basic theory of science and technology in the breeding industry is breaking through continuously, and biological key technologies such as molecular design breeding and gene editing are applied to breeding by many units. Generally, an important agronomic trait is controlled by a plurality of quantitative loci, the magnitude of the effect exerted by the quantitative trait loci is possibly related to genetic background, and a certain degree of correlation exists among loci of different agronomic traits, and modular regulation often occurs. The complexity becomes the bottleneck of traditional breeding, for example, how to overcome the relationship of the development of the mutual restriction among the spike number, the spike grain number and the thousand grain weight, and how to overcome the difficult problem of the coordination of disease resistance and yield. The key footholds of the biological breeding era are the reasonable design of parent combination, the planning of hybridization schemes, selection indexes and the like, and the breeding efficiency is improved. To achieve this goal, it is extremely important to optimize the regulatory genetic basis for disease resistance and agronomic traits.

Wheat scab resistance and yield are complex quantitative traits of crops, a large number of related QTLs are positioned at home and abroad, and partial genes are cloned, but for breeding, disease-resistant sites/genes and yield-related sites/genes which are most suitable for being utilized in the wheat region are selected to develop molecular markers for biological breeding.

Therefore, the research on the inheritance and breeding of wheat scab resistance is enhanced, and the wheat variety with synergistically improved resistance, yield and the like is very urgent to cultivate.

Disclosure of Invention

In order to overcome the technical problems, the invention provides a wheat scab resistant molecular breeding method for polymerizing multiple traits and favorable allelic variation, which respectively adopts parents with complementary genotypes containing the favorable allelic variation of a scab resistant gene Fhb1, a scab resistant site Qfhb.3B, a spikelet site QSNS.2B and a thousand kernel weight site QTKW.2B to carry out hybridization or double crossing, and hybrid seeds after hybridization or double crossing are harvested; and the accurate identification of disease resistance, agronomic characters and yield of multiple generations is combined, so that the breeding target of wheat scab and high yield synergistic improvement is achieved.

The invention provides a wheat scab resistant molecular breeding method for aggregating multiple traits and favorable allelic variation, which comprises the following steps,

s1, selecting parents carrying the disease-resistant genotype of a scab-resistant gene Fhb1 and the resistance allelic variation of a scab-resistant site Q fhb.3B and the genotype complementation allelic variation of a spikelet-resistant site QSNS.2B and a thousand kernel weight site QTKW.2B, hybridizing or double crossing the selected parents, and harvesting the hybridized or double crossed seeds F ₁ ；

S2, planting F ₁ Generation of hybrid seed, harvesting self-bred seed F ₂ ；

S3, planting F in a greenhouse ₂ Screening powdery mildew and yellow mosaic disease resistance, lodging resistance and tillering resistance, eliminating single plants which are susceptible to diseases, easy to lodging, have plant heights of more than 80cm and late maturity under natural conditions, and harvesting selected single plant seeds F ₃ ；

S4, planting F in the greenhouse ₃ The generation, taking leaf to extract DNA from each plant, screening Fhb1, qfhb.3B, QSNS.2B and QTKW.2B for favorable allelic variation, selecting the plant tags containing positive favorable allelic variation for genotype detection, screening powdery mildew and yellow mosaic disease resistance, lodging resistance and tillering resistance in the whole growth period of wheat, eliminating the plants which are easy to lodging and have plant height of more than 80cm and late maturity, and selecting F seeds according to the harvested plants ₄ ；

S5, planting F in the field ₄ The method comprises the steps of firstly utilizing molecular markers to screen and reserve plant rows which contain heterozygous positive favorable allelic variation and simultaneously carry Fhb1, qfhb.3B, QSNS.2B and QTKW.2B in the seedling stage, utilizing tag markers to select and reserve the plant rows, comprehensively examining the comprehensive agronomic characters, the growth period and other disease resistance of the selected plant rows according to breeding targets, selecting excellent plant rows, harvesting 5-6 single plants with consistent characters, carrying out yield identification after harvesting, and selecting plant row seeds F with the yield level higher than that of a contrast ₅ ；

S6, planting F in the field ₅ The method comprises the following steps of firstly screening and retaining strains which are homozygous and positive and are beneficial to allelic variation by utilizing molecular markers in a seedling stage and also carry Fhb1, qfhb.3B, QSNS.2B and QTKW.2B, listing markers, instilling single flowers of the listing strains with gibberellic disease pathogens in a flowering stage to retain strains with identification results of resistance, then comprehensively inspecting comprehensive agronomic characters, growth periods, other disease resistances and the like of selected strains according to breeding targets, selecting excellent strains, mixing selected strains, performing yield identification after harvesting, and selecting strains with yield levels higher than those of controls;

s7, planting F in the field ₆ According to breeding targets, comprehensively inspecting comprehensive agronomic characters and other disease resistances of cell lines, performing yield identification after harvesting, selecting cells with yield levels 5% higher than those of controls, and performing next-generation multipoint yield identification.

Furthermore, the sequences of the specificity detection primers of the gibberellic disease resistant gene Fhb1 are shown in SEQ ID NO.1 and SEQ ID NO. 2.

Furthermore, the sequences of the specificity detection primers of the gibberellic disease resistance locus Qfhb.3B are shown as SE Q ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

Furthermore, the specific detection primer sequences of the granule weight site QSNS.2B are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

Further, the sequence of the specificity detection primer of thousand-grain-weight locus QTKW.2B is shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

Further, F is planted in the steps S3 and S4 ₂ Generation and F ₃ Screening powdery mildew resistance, yellow mosaic disease resistance, lodging resistance and tillering resistance in generations, and specifically eliminating single plants which are susceptible to diseases, easy to lodging, have plant heights of more than 80cm and are late-maturing: uniformly planting Sumai No.3 (high-susceptibility powdery mildew) in advance for 4-6 days at intervals of single plants in a greenhouse by taking Zhemai No.9 as a control for resisting powdery mildew; taking Ningmai 13 as a control for resisting yellow mosaic disease, and screening single plants for resisting yellow mosaic disease in comparison in the whole growth period; judging tillering performance by taking Yangmai 25 as a reference, and eliminating single plants with the spike number less than that of the Yangmai 25 per single plant; the selection of lodging and maturity is compared with Yangmai 25.

Further, the step S6 of single flower instillation inoculation of the scab pathogenic bacteria to the listing plant row in the flowering phase and the plant row with the reserved identification result of 'resistant' are specifically as follows: preparation of a suspension of gibberellin spores 4X 10 ⁵ ～5× 10 ⁵ spore/mL, in a field, in the flowering period of wheat, inoculating by adopting a single flower drip method, randomly selecting 20 spikes for each plant row or strain, inoculating at the small flower opened in the middle of each spike, marking, spraying water to the seed spikes every 2 hours, uniformly and fully spraying the water to the wheat spikes for 10 minutes every time, and immediately stopping spraying the water after the wheat flowers for 20 days, wherein the ratio of each plant row or strain is 7-00; after 21 days of inoculation, the disease condition of the inoculated spikes is investigated, the number of the small spikes and the total small spikes of each spike are detected, the severity of gibberellic disease PSS = the number of the small spikes detected/the number of the total small spikes multiplied by 100%, the plant rows or the plant lines of which the PSS is less than 25% and is close to the 'resistant' level of Sumai No.3 are reserved, sumai No.3 and Annong 8455 are respectively used as disease-resistant and susceptible controls, yangma No. 25 is used as an anti-resistant control, and Yangma No.13 is used as a neutral control.

Further, the identification of other disease resistance in steps S5, S6 and S7 is specifically: identifying yellow mosaic disease and powdery mildew by adopting natural morbidity identification; selecting the plant-row listing mark with disease-resistant (R) grade disease-resistant identification results of yellow mosaic disease and powdery mildew.

Further, the identification of powdery mildew and yellow mosaic disease in the steps S5, S6 and S7 also comprises that the Zhenmai No.9 is a powdery mildew resistance (R) control; ningmai 13 is a control for yellow mosaic disease resistance (R).

Further, the comprehensive agronomic traits of the selected plant rows, lines and strains in the comprehensive investigation in the steps S5, S6 and S7 are as follows: selecting the plant rows with the plant height of less than 90cm, good lodging resistance, the number of single plant ears of more than or equal to 8, the number of grains per ear of more than or equal to 45 and the maturity period equivalent to Yangmai 25.

Further, the control variety for the agronomic characters and the growth period in the steps S5, S6 and S7 refers to Yangmai 25.

Compared with the prior art, the invention has the following technical effects:

the invention uses the common wheat variety Yangmai 16 which is different from the known resistance sources such as Sumai No.3, wangshuibai and the like as a source of the scab resistant site for the first time, does not carry Fhb1, has excellent agronomic characters and once is one of the varieties with the largest popularization area in the middle and lower reaches of Yangtze river, obtains the first-class prize of Chinese agriculture science and technology of the rural part of agriculture, and the applicant discovers for the first time that the Yangmai 16 carries a new scab resistant site Qfhb.3B and has no negative effect on yield characters. Newly discovering a spikelet number locus QSNS.2B and a thousand kernel weight locus QTKW.2B in the elytrigia repens No.1 and Yangmai No.12 by the applicant, wherein the synergistic allelic variation of the yield loci has no negative effect on the gibberellic disease, and the adverse effect of the disease-resistant genes on the yield traits can be overcome through the favorable allelic variation polymerization of the resistance loci and the yield loci. Therefore, favorable allelic variation carrying Fhb1+ Qfhb.3B + QSNS.2B + QTKW.2B is selected in breeding, so that the resistance to gibberellic disease can be obviously improved, and the yield can be improved at the same time. The wheat scab resistant molecular breeding method which has polymerized multiple characters and is beneficial to allelic variation can become a model of biological breeding to be popularized and applied. The wheat variety bred by the method of the invention has break-through of gibberellic disease resistance and yield, greatly shortens the breeding period, reduces the field workload and the application of pesticides and fertilizers, and is beneficial to ensuring the safe and sustainable development of wheat raw grain production in China.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 is a molecular breeding method of wheat scab resistance with aggregated multiple traits and favorable allelic variation.

FIG. 2 is a schematic diagram showing the amplification results of the molecular markers closely linked to the Fhb1 locus of the gibberellic disease resistant gene in the parent and the progeny in example 2, and the arrow indicates a positive band of the gibberellic disease resistant gene.

FIG. 3 is a diagram showing the results of the amplification assay of the site Qfhb.3B for gibberellic disease resistance in the natural population in example 3.

FIG. 4 is a diagram showing the results of the amplification detection of the small ear number site QSNS.2B (a) and the thousand kernel weight site QTKW.2B (b) in the natural population in example 6.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the present invention belongs.

Example 1 a molecular breeding method for wheat scab resistance by aggregating multiple traits and favorable allelic variation according to the scheme shown in fig. 1, comprises the following steps,

step S1, parent selection: selecting a main cultivated variety and a derivative thereof in a wheat natural population as objects, extracting seeds or seedling stage leaf DNA, selecting a variety (line) carrying complementation of disease-resistant genotype of a fusarium head blight-resistant gene Fhb1 and disease-resistant genotype of a fusarium head blight-resistant site Qfhb.3B, selecting a variety (line) carrying complementation of synergistic allelic variation of each ear of spikelet site QSNS.2B and a thousand kernel weight site QTKW.2B as parents, selecting a strain Yangma 28 carrying resistance allelic variation of the fusarium head blight-resistant gene Fhb1, a Yangma 30 carrying resistance allelic variation of the fusarium head blight-resistant site Qfhb.3B, simultaneously carrying the synergistic genotype of the thousand kernel weight site QTKW.2B on the Yangma 28 and carrying the synergistic genotype of the spike number site QSNS.2B on the Yangma 30, and realizing complementation of the disease-resistant/allelic variation of the disease-resistant sites and yield-related sites of the two parents.

Planting selected parents in a greenhouse in 2016, performing hybridization and grouping in the last 10 th ten days, and harvesting F in 2016 years and 12 months ₁ Generation hybrid seeds;

2) Step S2, planting F in greenhouse in 1 month in 2017 ₁ Generation hybrid, 6 months harvest inbred seed F ₂ ；

3) S3, planting F in 8 months in 2017 greenhouse ₂ Planting F in greenhouse ₂ Screening powdery mildew and yellow mosaic disease resistance, lodging resistance, tillering resistance and the like in generations, and eliminating single plants which are susceptible to diseases, easy to lodging, have plant heights of more than 80cm and are late-maturing: zhenmai No.9 is used as a control for resisting powdery mildew; taking Ningmai 13 as a control for resisting yellow mosaic disease, and screening single plants for resisting yellow mosaic disease in comparison in the whole growth period; tillering, lodging and mature period are judged by taking Yangmai 25 as a reference. Selecting single seeds in the harvest in 12 months;

4) S4, planting F in the greenhouse in 2018 in 1 month ₃ In the generation, 1 row of every 1 single plant harvested in the last generation, 2m in row length, 0.25m in row spacing and 40 grains in each row are taken, DNA is extracted from leaves of each single plant, favorable allelic variation screening of Fhb1, qfhb.3B, QSNS.2B and QTKW.2B is carried out, single plant brands containing all favorable allelic variation (including heterozygous positivity) are selected for genotype detection, screening of powdery mildew resistance, yellow mosaic disease resistance, lodging resistance and tillering resistance is carried out in the whole growth period of wheat, and single plants which are easy to lodging, have plant heights of more than 80cm and are later than Yangmai 25 in the mature period are eliminated. Harvesting the single plant in 6 months;

5) S5, planting F in field in 2018 and 10 months ₄ Generation, plant adult plant row, each single plant harvested in the previous generation 2 rows, 2 row length, 0.25m row spacing, 40 grains in each row, firstly randomly selecting leaves of 10 single plants in each row in seedling stage, mixing, extracting DNA, identifying and reserving the rows of the plants simultaneously carrying with scab resistant gene Fhb1, scab resistant site Qfhb.3B resistant allelic variation, QSNS.2B of spikelet site and thousand kernel weight site QTKW.2B favorable allelic variation (containing heterozygosis positive), listing marks, and comprehensively inspecting comprehensive agronomic characters, other disease resistances and the like of the selected plant rows according to breeding targets: identifying yellow mosaic disease, powdery mildew and rust disease (leaf rust) by adopting natural onset identification, and taking Ningmai 13 as a yellow mosaic disease resistance (R) control; zhenmi No.9 served as a control for powdery mildew resistance (R). Selecting plants with disease identification results of yellow mosaic disease, powdery mildew and rust disease all showing disease resistance (R) level to investigate agronomic performanceAnd (3) selecting a plant row tag mark with the plant height of less than 90cm, good lodging resistance, the number of single plant ears of more than or equal to 8 and the number of grains per ear of more than or equal to 45 by taking Yangmai 25 as a basic control. And (4) carrying out mixed harvest on the selected excellent plant rows for 5-6 individual plants with consistent personality, and carrying out yield identification after 6-month harvest. And selecting the plant rows with the yield level higher than that of the Yangmai 25 of the control variety.

6) S6, planting F in field in 2019 and 10 months ₅ Planting adult plant line, 6-row region, 2m row length, 0.25m row spacing and 50 grains in each row, firstly randomly selecting 10 leaves of single plant in each plant line at seedling stage, mixing them, extracting DNA, identifying and retaining the plant lines carrying homozygous resistant allelic variation of sites of gibberellic disease resistant genes Fhb1 and Qfh b.3B and beneficial allelic variation (homozygous) of sites of panicle small ear QSNS.2B and thousand grain weight site Q TKW.2B by molecular marker, making listing mark, preparing gibberellic spore suspension 4X 10 ⁵ ～5×10 ⁵ spore/mL, in a field of 4-5 months in 2020, in the flowering period of wheat, adopting a single-flower instillation method for inoculation, randomly selecting 20 spikes of each strain, inoculating small flowers in the middle of each spike, marking, spraying water to the inoculation spikes every 2 hours, uniformly spraying the water to the wheat spikes for 10 minutes every time, and immediately stopping spraying water after the wheat flowers for 20 days. After 21 days of inoculation, the disease incidence of inoculated spikes is investigated, the number of small spikes and the total small spikes are detected per spike, the severity of gibberellic disease PSS = the number of small spikes/the total small spikes multiplied by 100%, strains with PSS less than 25% and resistance level close to that of Sumai No.3 are reserved, sumai No.3 and Annong 8455 are respectively used as disease-resistant and susceptible controls, yangma 25 is used as an anti-resistant control, yangma 13 is used as an anti-sensitive control, and the average severity of 6 strains is less than 25%. Then according to breeding targets, comprehensively investigating the comprehensive agronomic characters, other disease resistance and the like of the selected strains: identification of powdery mildew and yellow mosaic disease adopts natural morbidity identification, and Ningmai 13 is a yellow mosaic disease resistance (R) control; zhenmi No.9 is a control for powdery mildew resistance (R). Selecting plants with disease resistance identification results of powdery mildew and yellow mosaic disease both in R grade to investigate agronomic traits, selecting plant height of less than 90cm by taking Yangmai 25 as basic control, good lodging resistance, single plant spike number of more than or equal to 8, and grain number per spike of more than or equal to 845 strain listing and marking. For the selected excellent strains, about 15 single plants with consistent characters are harvested, yield and grain weight identification is carried out after 6 months of harvest, the yield level is completely converted into the numerical value of the yield per mu, the average yield of Yangmai 25 in the current year is 530.6 kg/mu, the selected strains with the yield remarkably higher than that of the control Yangmai 25 are 2 selected strains, the yield is respectively 20-16 and 20-88, the yield is respectively 559.9 kg/mu and 568.3 kg/mu, and the yield is increased by 5.52 percent and 7.11 percent compared with that of Yangmai 25. The gibberellic disease severity of both lines was 8.1% and 9.4%, respectively, and was significantly below 25%, close to the severity of soymi No.3 in the year (PSS = 6.4%). The conversion to average severity is based on the average incidence of the disease. Disease resistance investigation and identification standards are according to agricultural industry standard NY/T2954-201 of the people's republic of China: in the technical specification for identifying scab resistance of wheat regional test varieties, the wheat regional test varieties with the small ear rate of less than 25 percent, namely the wheat regional test varieties with the severity of less than 2 grade, are regarded as R grade.

7) Step S7, planting F in field in 10 months in 2020 ₆ Each selected strain is planted into a 10-row area, the row length is 3.2 meters, the row spacing is 0.23 meter, comprehensive agronomic traits, other disease resistance and the like of the strains in the cell are comprehensively considered according to breeding targets, identification of powdery mildew and yellow mosaic disease adopts natural morbidity identification, and Zhenmai No.9 is a control of powdery mildew resistance (R); ningmai 13 is used as a control for yellow mosaic disease resistance (R). And selecting a strain with the disease resistance identification results of powdery mildew and yellow mosaic disease both in R grade to investigate the agronomic traits, selecting a strain branding mark with good lodging resistance, the number of grains per plant being more than or equal to 8 and the number of grains per plant being more than or equal to 4 by taking Yangmai 25 as a basic control. And (3) performing yield identification after 6-month harvest in 2021, wherein the yield level is completely converted into a numerical value of the yield per mu, the yield levels of 20-16 and 20-88 are still 5% higher than that of the control Yangmai 25, the yield level of the Yangmai 25 in the current year is 541.6 kg/mu, the yields of 20-16 and 20-88 respectively reach 5.3kg/mu and 578.5 kg/mu, the yields are respectively increased by 5.48% and 6.81% compared with that of the control Yangmai 25, and the yield identification is performed at multiple points in the next year.

FIG. 1 is a molecular breeding method of wheat scab resistance with aggregated multiple traits and favorable allelic variation, and it can be seen that the method of the present invention can aggregate favorable genotypes or allelic variation of disease-resistant and yield-related trait genes/loci through reasonable parent selection and design, thereby greatly improving breeding efficiency and trait improvement accuracy.

Example 2 establishment of molecular marker assisted selection method of gibberellic disease resistant gene Fhb1

Extraction of candidate parent Material, F, related to example 1 by CTAB method ₃ Individual leaf, F ₄ Plant row leaves and F ₅ And diluting the genome DNA of the mixed leaves of the strain to obtain a template solution with the DNA concentration of about 30 ng/mu L, and detecting the linkage His label of the gibberellic disease resistant gene Fhb1 of wheat.

The sequences of the specific primer group for detecting the linkage His tag of the gibberellic disease resistant gene Fhb1 by using the molecular marker are shown in Table 1:

TABLE 1 primer sequence information of Fhb 1-linked marker of gibberellic disease resistance gene

Detecting a corresponding linkage marker His of a major gene Fhb1 resisting the fusarium head blight by adopting a PCR amplification method, wherein the PCR amplification method comprises the following steps: the PCR amplification system is 10 μ L, and comprises wheat genome DNA 30ng/μ L1.0 μ L, PCR buffer 10 × 1.0 μ L, dNTP 10Mm 0.2 μ L, mgCl 10Mm ₂ 1.0 mul, 5U Taq polymerase 0.2 mul, 5 mul upstream primer 0.4 mul, 5 mul downstream primer 0.4 mul and sterile deionized water 5.8 mul; the PCR amplification procedure is as follows: (1) pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 20s, and extension at 68 ℃ for 2-3.5 min; (2) Denaturation at 94 ℃ for 20s, annealing at 57 ℃ for 20s, extension at 72 ℃ for 1min, and 32 cycles; (3) extension at 72 ℃ for 5 minutes; storing at 4 ℃.

Detection of example 1 in 1% agarose electrophoresis using His primers for wheat material, the target genotype was the same as ningmai 13, which was the material of choice. FIG. 2 is a schematic diagram of the amplification results of Fhb1 locus closely linked molecular marker His in parent and offspring, and an arrow indicates the target band with positive amplification.

Example 3 excavation of stable wheat scab resistant site Qfhb.3B and development of effective KASP primer set

And (3) field test: with 174 DH lines from Yangmai 16/Zhongmai 895 are materials, the DH lines and parents of the DH lines are planted in a gibberellic disease identification garden (Yangsu Yangzhou) of a Wanfu test base of agricultural science institute in the Rituyuwa area in 2016, 2017 and 2018 for 3 continuous growing seasons, the sowing period of Yangzhou wheat in the test is set to be 10 months and 20 days, the test adopts a random block design, 2 rows of regions and 2 times of repetition, each row comprises 30 grains, the row length is 1.5m, and the row spacing is 0.23m. The field fertilization and management refer to local field cultivation and production, the pests and weeds are prevented and controlled in time, and the gibberellic spore suspension is prepared by 4 multiplied by 10 ⁵ ～5×10 ⁵ spore/mL, adopting a single flower instillation method to inoculate gibberellic disease in the flowering period of wheat, inoculating 10 mu l of spore solution in 1 floret in the middle of each ear, randomly marking 20 ears in each line, investigating the morbidity of the inoculated ears 28 days later, counting the number of diseased spikelets per ear and the total spikelet number, and finally taking the average value of 2 repetitions as the average severity PSS of the family.

Genotype detection and QTL mapping: the method comprises the steps of extracting genome DNA by adopting a CTAB method, obtaining a genotype by utilizing a Wheat Wheat 660K chip, filtering and removing redundant genotype data by utilizing SNP (single nucleotide polymorphism) and BIN (binary polymorphism) functions of IcMapping v4.1 software (http:// www.isbr feeding. Net), primarily constructing a genetic linkage MAP by utilizing an MAP function, correcting the genetic MAP by utilizing JoinMap v4.0, and drawing the genetic MAP by utilizing MapChart2.3 (https:// www.www.wur.nl/en/show/Mapchart.htm). QTL significantly correlated with the severity of head blight (mean panicle rate, PSS) was detected using an Integrated composite interval mapping (IC IM) method with IcMap ping v4.1 software, and the LOD threshold was set to 3.0. For comparison with the previous results, the linked markers or gene sequences were aligned with the EnsemblPlants database (http:// 202.194.139.32/blast. Ht ml) of the Chinese spring 2.1 reference genomic sequence. The site Qfhb.3B with obvious synergism on wheat scab resistance is obtained by experiments, and the resistance genotype is from Yangmai 16, as shown in Table 2. The applicant discovers that the resistant allelic variation (favorable allelic variation) of the site has no negative effect on the agronomic traits and the yield by carrying out agronomic traits and yield investigation on the DH line of Yangmai 16/Migmai 895 and carrying out T test on the related SNP genotyping of the site and the phenotypes of the agronomic traits and the yield, and therefore, the resistant allelic variation of the site is considered to be a scab resistant genotype which is very suitable for production.

TABLE 2 Qfhb.3B site cases

Developing a molecular marker: considering that the Qfhb.3B primary localization interval 705.28-742.44 Mb is large and not beneficial to further utilization, the applicant divides DH families into 2 pools, 15 pools with gibberellic disease resistance (PSS < 25%) and gibberellic disease susceptibility (PSS > 75%), performs exon capture and pool RNA sequencing (BSR) of mixed wheat leaf genome pools, also performs wheat 660K SNP chip scanning and resequencing on parent Yangmai 16 and Yangmai 895 to find more polymorphic SNP sites, develops KASP markers corresponding to 14 SNPs in the target interval according to the 2.1 version information of Chinese spring reference genome of wheat, performs scanning and verification in the original population, develops a polymorphic KASP marker developed at 726.43Mb chromosome physical position 726.43Mb according to the Primer of Chinese spring reference genome version 2.1 of wheat, and can be best linked with gibberellic disease phenotype, therefore, the marker is named as KASP marker 16-YM-3 Mb related Primer, such as related Primer of gibberellic disease, such as related Primer 16-3B-3 Mb.

TABLE 3 YM16-fhb-3B-related information

Preparing KASP labeled primer working solution: mu.L (100. Mu.M) of each of the forward primers and 12. Mu.L (100. Mu.M) of each of the downstream primers were taken, and supplemented with sterile ultrapure water to 100. Mu.L, and the mixture was thoroughly mixed to prepare a KASP-labeled primer working solution for use. PCR amplification reaction System: 2.2. Mu.L (about 30 ng/. Mu.L) of wheat DNA template to be tested, 0.06. Mu.L of primer working solution and 2.5. Mu.L of KASP Master Mix (LGC company, KBS-1016-002) are supplemented to 5. Mu.L with sterile ultrapure water; PCR reaction procedure: (1) pre-denaturation at 94 ℃ for 10min; (2) Denaturation at 95 ℃ for 20s, 61-55 ℃ (0.6 ℃ per cycle) for 45s, 10 cycles total; (3) denaturation at 94 ℃ for 20s, renaturation at 56 ℃ for 45s, 31 cycles; storing at 4 ℃. The experiment was carried out while setting up blank controls (NTC) without template DNA added to the reaction system, 1 or more blank controls per plate.

Taking wheat seedlings, and extracting the genomic DNA of the wheat to be detected by adopting a CTAB method. And (3) performing PCR amplification by using the wheat genome DNA to be detected as a template and adopting the KASP primer group and the PCR reagent to obtain a PCR amplification product. The PCR reaction was performed on an ABI Veriti 384PCR instrument (Thermo Fisher), and the fluorescence value was read by scanning the PCR amplification product with an Omega F SNP typing detector (LGC Genomics Ltd, KBS-0024-0 02). The FAM excitation wavelength is 485nm, and the emission wavelength is 520nm; the excitation wavelength of VIC is 535nm, the emission wavelength is 556nm, the excitation wavelength of system reference fluorescence ROX is 575nm, and the emission wavelength is 610nm. Genotyping was performed using Kluster Calle rTM (KBioscience) and the genotype of the Qfhb.3B site was determined from the results of the analysis. Amplifying part of 'Yangmai 16 multiplied by the DH line of Zhongmai 895' and two parents according to the method, analyzing the fluorescence signal data of the amplified products by Kluster Caller software to gather at the position (blue) close to the X axis in the fluorescence signal coordinate system of the typing results, and confirming that the genotypes of the 36 th bases (SNP sites) of the wheat at KASP marker flanking nucleotide sequences (such as SEQ ID NO.3 and 4) are respectively C; and the fluorescence signal data of the amplified products are analyzed by Kluster C aller software and gathered at the position (red) close to the Y axis in the coordinate system, and the genotype of the coordinate system is different from the genotype of Yangmai 16, so that the genotypes of the coordinate systems at the SNP site are respectively T. The results of T-test of genotype at Qfhb.3B and severity of gibberellic disease of "Yangmai 16X Migmai 895 DH line" are shown in Table 4.

TABLE 4 partial T test results of infection allelic variation of DH family gibberellic disease severity (PSS) and Qfhb.3B locus

As can be seen from table 4, at site qfhb.3b, average severity of wheat scab with allele T was significantly higher overall than wheat with allele C, and the genotype and phenotype of part of DH lines were examined using double-sample T of Excel 2019 (genotype was not detected in 7 lines), indicating that: the genotype of 92 families is the same as that of Yangmai 16 and C, the genotype of 75 families is the same as that of Zhongmai 895 and T, the average value of the scab severity of the families with the genotype of C is reduced by 20.57 percent compared with the average value of the scab severity of the families with the genotype of T, and the obvious difference exists on the level of p <0.01, which indicates that the primer group of KASP marker YM16-fhb-3B of Qfhb.3B site and the genotype detection system can be applied to wheat scab resistance molecular marker assisted breeding.

Example 4 application of KASP primer set for breeding of gibberellic disease resistant site Qfhb.3B and analysis of polymerization effect of the KASP primer set and Fhb1

And (3) field test: in the embodiment, 77 parts of wheat varieties (lines) planted in an identification garden of a gulf head experiment base in 2019 are used as materials, the sowing period of the wheat in Yangzhou in the current year is set to 10 months and 20 days, the experiment adopts a random block design, 3 rows of areas are repeated for 2 times, 40 grains are planted in each row, the row length is 1.5m, and the row spacing is 0.23m. The field fertilization and management refers to local field cultivation and production, and timely prevents and controls insect pests and weeds. In 2015, a suspension of gibberellic spores was prepared at 4X 10 ⁵ ～5×10 ⁵ spore/mL, 4 mid-month, adopting a single flower instillation method to inoculate gibberellic disease in the flowering period of wheat, inoculating 10 mu l of spore solution at 1 floret in the middle of each ear, randomly marking 10 ears in each row, investigating 30 ears in total for each family, investigating the incidence condition of the inoculated ears after 21 days, counting the number of the small ears per ear and the total small ears per ear, and finally taking the average value of 2 repetitions as the average severity PSS of the family.

And (3) genotype detection: the 77 varieties (lines) were genotyped using the primer set for KASP at the Qfhb.3B site obtained in example 3 and the primer set for Fhb1 of example 2. The fluorescence signal data of the primer group amplification product of KASP marker YM16-fhb-3B of Qfhb.3B locus is analyzed by Kluster Caller software and gathered in the typing result, and the fluorescence signal data is analyzed by Kluster Caller and gathered to be the same as Yangmai 16, namely, the genotype of the wheat line at Qfhb.3B locus is proved to be C; if the fluorescence signal data of the amplified products of the wheat strains are analyzed and gathered by Kluster Caller software and are different from the typing of Yangmai 16, the genotype of the wheat strains at the SNP locus is proved to be T, and the attached figure 3 shows the result of the fluorescence signal of the amplified natural population of the primer group of YM 16-fhb-3B. The results of genotyping at both Fhb1 and Qfhb.3B for 77 varieties (lines) are shown in Table 5, R indicates that they carry a resistant allelic variation of Fhb1, and S indicates that they do not carry a resistant allelic variation of the gene.

TABLE 5 average values of gibberellic disease severity and the results of genotype detection at Fhb1 and Qfhb.3B sites for 77 varieties (lines)

Note: fhb1: S represents a disease-resistant genotype not carrying the gene; r represents the disease-resistant genotype carrying the gene

Under the effect of removing Fhb1 (i.e. picking up material carrying Fhb1 resistance genotype), 26 families carried no resistant allelic variation at the site of qfhb.3b, were susceptible allelic variation TT, and 32 carried resistant allelic variation CC at the site of qfhb.3b, with the results of T-test as shown in table 6.

TABLE 6T test results for removing the Userai variation of gibberellic disease severity (PSS) and Qfhb.3B locus resistance under the influence of Fhb1 Effect

As can be seen from table 6, the mean severity of wheat scab with the qfhb.3b locus allele T was significantly higher overall than wheat with allele C under the influence of Fhb1 removal, and the two-sample T test with Exc el 2019 for the phenotypes of both alleles, the results indicated: 32 varieties (lines) and Yangmai 16 have the same genotype of C,26 varieties (lines) and Migmai 895 have the same genotype of T, the average scab severity of the varieties (lines) with the genotype of C is reduced by 22.48 percent compared with the average scab severity of the varieties (lines) with the genotype of T, and the obvious difference exists on the level that p is less than 0.01, which indicates that the primer group of KASP marker YM16-fhb-3B of Qfhb.3B site and the genotype detection system can be applied to wheat scab resistance molecular marker assisted breeding.

Table 7T test results considering the susceptibility allelic variation of gibberellic disease severity (PSS) and Qfhb.3B loci under the influence of the effect of Fhb1

As can be seen from table 7, without removing the effect of Fhb1, the mean severity of wheat scab with the qfhb.3b locus allele T was overall significantly higher than wheat with allele C, and the phenotypes of both alleles were tested using double sample T of E xcel 2019, which indicates that: the genotype of 40 varieties (lines) is the same as that of Yangmai 16 and C, the genotype of 37 varieties (lines) is the same as that of Zhongmai 895 and T, the average scab severity of the varieties (lines) with the genotypes C is reduced by 19.10 percent compared with that of the varieties (lines) with the genotypes T, and the obvious difference exists on the level that p is less than 0.01, which indicates that the primer group of KASP marker YM16-fhb-3B of Qfhb.3B site and the genotype detection system can be applied to wheat scab resistance molecular marker assisted breeding. We also analyzed the effect of Fhb1 polymerization with Qfhb.3B, S for Fhb1 disease genotype and R for Fhb1 disease resistance genotype, with the results shown in Table 8.

TABLE 8 analysis of the Combined Effect of resistant allelic variants at Fhb1 and Qfhb.3B sites

Note: after PSS (%), the numbers indicated by different lower case letters are significantly different at the p <0.01 level

As can be seen from table 8, the analysis of variance results show that: the average value of the scab severity of the variety (line) carrying both Fhb1 and Qfhb.3B resistant alleles (R + CC) is reduced by 24.13 percent and 43.22 percent compared with the average value of the scab severity of the variety (line) carrying only Fhb1 resistant alleles (R + TT) or carrying only Qfhb.3B resistant alleles (CC), and the obvious difference exists on the level of p <0.01, which indicates that the aggregation of the Fhb1 and Qfhb.3B disease resistant genotypes has more obvious scab resistant effect than the single gene/locus resistant genotypes. Meanwhile, the primer group of Fhb1 His and the primer group of Qfhb.3B YM16-fh B-3B and the genotype detection system can be applied to wheat scab-resistant molecular marker-assisted breeding.

Example 5 development of primers sets for mining spikelet number site QSNS.2B, thousand kernel weight site QTKW.2B and KASP thereof

And (3) field test: in the embodiment, 205 families of Yangmai No. 12/Thiampyrum 1 RIL group planted in yield identification nursery of Bay experiment base in 2017-2019 are used as materials, the sowing period of Yangmai wheat in the current year is set to 10 months and 20 days, the test adopts a random block design, 3 rows of areas are repeated for 2 times, 50 grains in each row are used, the row length is 2m, and the row spacing is 0.3m. The field fertilization and management refers to local field cultivation production, and diseases, pests and weeds can be prevented and controlled in time. Heading date was recorded for the RIL population and the parents (approximately 50% of wheat heading in row 3). 2018. And (3) surveying the number of spikelets per ear in the late filling stage-mature stage of wheat in 2019 and 2020, randomly marking 10 single plants in each family, surveying the number of spikelets per ear of 10 spikes per single plant, and finally taking the average value of 10 single plants as the number of spikelets per ear (SNS) of the family. Harvesting the 10 single plants when the plants are mature, threshing, removing diseased grains, reserving large grains and small grains, observing the weight of 3 300 grains, taking an average value, finally converting the average value into the weight of 1000 grains, namely the thousand-grain weight, and finally taking the average value of 2 times of repetition as the average thousand-grain weight (TGW) of the family.

Genotype detection and QTL mapping: the method comprises the steps of extracting genome DNA by adopting a CTAB method, obtaining a genotype by utilizing a Wheat Wheat 55K chip, filtering and removing redundant genotype data by utilizing SNP (single nucleotide polymorphism) and BIN (binary polymorphism) functions of IcMapping v4.1 software (http:// www. Aspect edition. Net), primarily constructing a genetic linkage MAP by utilizing an MAP function, and drawing the genetic MAP by utilizing MapChart2.3 (https:// www. Wur.wur.nl/en/show/Ma hart.htm). An ICIM (I nclusive composite interval mapping) method of Icimapping v4.1 software is used for detecting QTL (quantitative trait locus) obviously related to the number of spikelets and thousand kernel weight of each spike, and an LOD threshold value is set to be 3.0. For comparison with previous results, the linked markers or gene sequences were aligned with the EnsemblPlants database (http:// 202.194.139.32/blast. Html) of the Chinese spring 2.1 reference genomic sequence. The site QSN S.2B of the number of spikelets of each spike and the thousand kernel weight synergistic site QTKW.2B of each spike of the wheat are obtained through experiments and are shown in the table 9. The applicant simultaneously identifies the scab resistance of Yangmai No. 12/Thiampyrum 1R IL population, and performs T test on the SNP genotypes of the located spikelet locus QSNS.2B and thousand kernel weight synergistic locus QTKW.2B regions and the scab phenotype of the population, and finds that the synergistic genotypes (allelic variation) of the SNP target regions of the two loci have no negative effect on the scab resistance, so that the synergistic allelic variation of the two loci is considered to be the genotype suitable for being utilized in production.

TABLE 9 situation of spikelet number site QSNS.2B and thousand kernel weight synergistic site QTKW.2B per ear

Developing a molecular marker: according to SNP information of QSNS.2B and QTKW.2B target intervals and phenotype identification data of characters, SNPs most significantly related to the number of spikelets and thousand grain weight of each spike are mined from 5-7 SNPs significantly related to the target intervals and are AX109358385 and AX109907473, corresponding KASP Primer groups YZ1-SNS-2B and YM12-TKW-2B are successfully developed by utilizing SNP flanking sequences, reference genome version 2.1 information and Primer 5.0 software, and specific information is shown in Table 10

TABLE 10 YZ1-SNS-2B and YM12-TKW-2B primer set information

Preparation of KASP-labeled YZ1-SNS-2B primer working solution: mu.L (100. Mu.M) of each forward primer was taken, 30. Mu.L (100. Mu.M) of the downstream primer was taken, and the amount of each forward primer was made 100. Mu.L with sterile ultrapure water, and the mixture was thoroughly mixed to prepare a KASP-labeled primer working solution for use. Preparation of KASP marker YM12-TKW-2B primer working solution: mu.L (100. Mu.M) of the forward primer and 12. Mu.L (100. Mu.M) of each of the downstream primers were taken, and supplemented to 100. Mu.L with sterile ultrapure water, and the mixture was thoroughly mixed to prepare a KASP-labeled primer working solution for use.

KASP marker YZ1-SNS-2B and KASP marker YM 12-TKW-2B: 2.2. Mu.L (about 30 ng/. Mu.L) of the wheat DNA template to be tested, 0.06. Mu.L of the primer working solution, and 2.5. Mu.L of KASP Master Mix (LGC Co., ltd., KBS-1016-002) were supplemented with sterile ultrapure water to 5. Mu.L; PCR reaction procedure: (1) pre-denaturation at 96 ℃ for 10min; (2) Denaturation at 94 ℃ for 20s, 61-55 ℃ (0.6 ℃ per cycle) for 40s, for 10 cycles; (3) denaturation at 94 ℃ for 20s, renaturation at 57 ℃ for 45s, and 32 cycles; storing at 4 ℃. The experiment was carried out while setting a blank control (NTC) without adding template DNA to the reaction system, and 1 or more blank controls were set for each plate.

Taking wheat seedlings, and extracting the genomic DNA of the wheat to be detected by adopting a CTAB method. And (3) performing PCR amplification by using the wheat genome DNA to be detected as a template and adopting the KASP primer group and the PCR reagent to obtain a PCR amplification product. The PCR reaction was performed on an ABI Veriti 384PCR instrument (Thermo Fisher), and the fluorescence value was read by scanning the PCR amplification product with an Omega F SNP typing detector (LGC Genomics Ltd, KBS-0024-0 02). The FAM excitation wavelength is 485nm, and the emission wavelength is 520nm; the excitation wavelength of VIC is 535nm, the emission wavelength is 556nm, the excitation wavelength of system reference fluorescence ROX is 575nm, and the emission wavelength is 610nm. Genotyping is carried out by Kluster Calle rTM (Kbioscience), and the genotypes of the QSNS.2B and QTKW.2B loci are determined according to the analysis result. Amplifying a part of a Yangmai 12/Elytrigia 1 RIL line and two parents according to the method, wherein for a QSNS.2B site, the genotype T with the same Elytrigia 1 is excellent allelic variation (favorable allelic variation), and the fluorescence signal data of an amplification product is analyzed by Kluster Caller to gather at a position (blue) close to an X axis in a parting result fluorescence signal coordinate system, is the same as Yangmai 12 and different from Elytrigia 1, namely the genotypes of the wheat at KASP marker flanking nucleotide sequences (such as 36 th base (SNP site) of SEQ ID NO. 7) are respectively C and are allelic variation for reducing the number of spikelets per ear; and the fluorescence signal data of the amplification products are analyzed by Kluster Caller software to gather at the position (red) close to the Y axis in the coordinate system, are different from the typing of Yangmai 12 and are the same as No.1 of elytrigia repens, so that the genotypes of the SNP sites of the families are respectively T and are the allelic variation (favorable allelic variation) for increasing the number of spikelets per spike, for the QTKW.2B site, the genotype C which is the same as that of the Yangmai 12 is the synergistic allelic variation (excellent allelic variation), the fluorescence signal data of the amplification products are analyzed by Kluster Caller software to gather at the position (blue) close to the X axis in the typing result fluorescence signal coordinate system, are the same as that of the Yangmai 12 and are different from that of the elytrigia repens 1, namely, the genotypes of the flanking nucleotide sequences of the K ASP markers (such as the 36 th base (SNP site) of SEQ ID NO. 7) of the wheat are respectively C and are the allelic variation for increasing the thousand weight, and the fluorescence signal data of the amplification products are analyzed by Kluster Caller software to gather at the position (red) close to the Y axis in the coordinate system, different from Yangmai 12 typing, the same as elytrigia repens No.1, proves that the genotypes of the families at the SNP sites are respectively T, and the allelic variation is reduced in thousand grain weight.

Example 6 verification of KASP primer set for spikelet-based quantitative trait loci (QSNS.2B) and thousand-grain weight locus (QTKW.2B)

And (3) field test: in the embodiment, 173 natural wheat populations planted in a yield identification garden of a gulf-head experiment base in 2019 are used as materials, the seeding time of Yangzhou wheat in the current year is set to be 10 months and 20 days, the experiment adopts a random block design, 3 rows of areas are arranged, 2 times of repetition are carried out, each row is 50 grains, the row length is 2m, and the row spacing is 0.3m. The field fertilization and management refers to local field cultivation production, and diseases, pests and weeds can be prevented and controlled in time. Heading date was recorded for the RI L population and the parents (approximately 50% of wheat heading in the 3-row zone). And (3) surveying the number of spikelets per ear in the late filling stage-mature stage of wheat in 2020, randomly marking 10 single plants per family, surveying the number of spikelets per ear of 10 main stem spikes per single plant, and finally taking the average value of the 10 single plants as the number of spikelets per ear (SNS) of the family. Harvesting the 10 single plants when the plants are mature, threshing, removing diseased grains, reserving large grains and small grains, observing the weight of 3 300 grains, taking an average value, finally converting the average value into the weight of 1000 grains, namely the thousand-grain weight, and finally taking the average value of 2 times of repetition as the average thousand-grain weight (TGW) of the family.

And (3) genotype detection: the 173 varieties (lines) were genotyped using the KASP primer sets YZ1-SNS-2B and YM12-Q-2B of QSNS.2B and QTKW.2B obtained in example 5. For QSNS.2B sites, the same genotype T of the Elytrigia repens No.1 is excellent allelic variation, the fluorescence signal data of the amplification product is analyzed by Kluster Caller software and gathered at the position (blue) close to the X axis in the fluorescence signal coordinate system of the typing result, the position is the same as Yangmai 12 and is different from the No.1 Elytrigia repens, namely, the genotypes of the wheat at KASP marker flanking nucleotide sequences (such as 36 th base (SN P site) of SEQ ID NO. 7) are respectively C which is allelic variation for reducing the number of small spikes of each spike; the fluorescence signal data of the amplification products are analyzed by Kluster Caller software to gather at the position (red) close to the Y axis in a coordinate system, are different from Yangmai 12 in typing and are the same as No.1 Elytrigia repens, and the genotype of the amplification products at the S NP site is respectively T, and is allelic variation for improving the number of spikelets per spike; and the fluorescence signal data of the amplified product is analyzed by K lustrer Caller software and gathered at the position (red) close to the Y axis in the coordinate system, the position is different from Yangmai 12 in typing and is the same as No.1 of elytrigia, so that the genotypes of the SNP sites of the families are respectively T, and are allelic variation for reducing thousand seed weight, and the primer groups of YZ1-SNS-2B and YM12-TKW-2B in the attached figures 4a and 4B are respectively used for amplifying the fluorescence of natural groups And (4) light signal results. The results of genotype and phenotype identification and T test of 173 varieties (lines) at two loci of QSNS.2B and QTKW.2B are shown in tables 11 and 12.

TABLE 11 number of spikelets per ear (SNS) and Thousand Kernel Weight (TKW) of 173 varieties (lines) and genotype test results of QSNS.2B and QTKW.2B loci

TABLE 12T test results of the number of spikelets per ear and the thousand kernel weight average value of varieties (lines) carrying QSNS.2B and QTKW.2B with different genotypes at two sites

As can be seen from tables 11 and 12, the QSNS.2B locus carrying the genotype T identical to that of elytrigia repens No.1 is a dominant allelic variation, and can significantly increase the number of spikelets per spike. 93 varieties (lines) carry the same genotype TT as elytrigia repens No.1, 80 varieties (lines) carry the same genotype CC as Yangmai 12, the genotype of TT can be obviously improved by 2.75 percent compared with the genotype of CC, and the difference is obvious on the level that p is less than 0.01, which shows that the primer group of KASP marker YZ1-SNS-2B of QSNS.2B locus and the genotype detection system can be applied to the molecular marker assisted breeding of the number of wheat per spike. For QTKW.2B locus, the genotype C carrying the same gene as Yangmai 12 is dominant allelic variation and has obvious synergistic effect on thousand grain weight. 125 varieties (lines) carry CC with the same genotype as Yangmai 12, 48 varieties (lines) carry TT with the same genotype as elytrigia repens No.1, the CC genotype can be obviously improved by 3.73 percent of thousand kernel weight compared with the TT genotype, and the obvious difference exists on the level that p is less than 0.01, which indicates that the primer group of KASP marker YM12-TKW-2B of QTKW.2B locus and the genotype detection system can be applied to the molecular marker assisted breeding of the small spike number of each spike of wheat.

Example 7 establishment of methods for molecular marker-assisted selection of Qfhb.3B disease-resistant genotypes, QSNS.2B and QTKW.2B synergistic allelic variation

The parent, F, involved in example 1 was examined with a primer set labeled YM16-fhb-3B, YZ1-SNS-2B and YM12-TKW-2B ₃ Individual leaf, F ₄ Plant row mixing blade, F ₅ Whether the mixed leaf material of the strain carries the dominant allelic variation of the character.

1. The extracted parental genome DNA is used as a template, and PCR amplification is carried out by adopting the primer group of the linked KASP marker YM16-fhb-3B for detecting the scab resistant locus of the wheat in the embodiment 3 and the primer groups of the linked KASP markers YZ1-SNS-2B and YM12-TKW-2B for detecting the spikelet number and thousand kernel weight locus of the wheat in the embodiment 5 to obtain an amplification product, and the target material with the Qfhb.3B locus same as the Yangmai 16 genotype, the QSNS.2B locus same as the thinopyrum 1 genotype and the QTKW.2B locus same as the Yangmai 12 gene is determined.

2. Extracting parent and F by CTAB method ₃ Individual plant, F ₄ Plant-row mixing blade, F ₅ The genomic DNA of the leaf was mixed with the strain and diluted to give a template solution having a DNA concentration of about 30 ng/. Mu.L.

The materials in steps 1), 4), 5) and 6) of example 1 were amplified together with the two parents as above. The fluorescence signal data of the Qfhb.3B locus amplification product is analyzed and gathered at the position (blue) close to the X axis in the parting result fluorescence signal coordinate system by Kluster Call software, and is the same as Yangmai 16, namely the genotype of 36 th base (SNP locus) of the wheat in a molecular marker YM16-fhb-3B flanking nucleotide sequence (such as SEQ ID NO. 3) is proved to be C, and the wheat is a selected material; and the fluorescence signal data of the amplified products are analyzed by Kluster Caller software and gathered at a position (red) close to a Y axis in a coordinate system, and the genotype of the wheat is proved to be T at the SN P site and the material is rejected if the genotype of the wheat is different from the genotype of Yangmai 16. The fluorescence signal data of the QSNS.2B locus amplification product is analyzed by Kluster Caller software and gathered at the position (red) close to the Y axis in the parting result fluorescence signal coordinate system, and is the same as the elytrigia repens No.1, so that the genotype of 36 th base (SNP site) of the wheat in the molecular marker YZ1-SNS-2B flanking nucleotide sequence (such as SEQ ID NO. 7) is proved to be T (favorable allelic variation), and the wheat is a selected material; and the fluorescence signal data of the amplified product is analyzed by Kluster Caller software and gathered at the position (blue) close to the X axis in the coordinate system, and the genotype of the wheat at the SNP site is proved to be C if the fluorescence signal data is different from the No.1 elytrigia typing, so that the material is rejected. The fluorescence signal data of the QTKW.2B locus amplification product is analyzed and gathered at the position (blue) close to an X axis in a parting result fluorescence signal coordinate system by Kluster Caller software, and the fluorescence signal data is the same as Yangmai 12, so that the genotype of 36 th base (SNP locus) of the wheat in a molecular marker YM12-TKW-2B flanking nucleotide sequence (such as SEQ ID NO. 11) is proved to be C (favorable allelic variation), and the wheat is a selected material; and the fluorescence signal data of the amplified product is analyzed by Kluster Caller software and gathered at the position (red) close to the Y axis in the coordinate system, and the genotype of the wheat at the SNP site is proved to be T when the fluorescence signal data is different from the genotype of Yangmai 12, so that the material is rejected.

Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not as a limitation, and thus other examples of example embodiments may have different values.

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<211> 41

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

gaaggtgacc aagttcatgc tcgaaactga aagatgtgcg c 41

<210> 9

<211> 41

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

gaaggtcgga gtcaacggat tcgaaactga aagatgtgcg t 41

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

cgcgtggaat caagtaaata cat 23

<210> 11

<211> 71

<212> DNA

<213> Triticum aestivum

<220>

<221> misc_feature

<222> (36)..(36)

<223> r is [ C/T ]

<400> 11

cccacacctt ctcaattagc aacatgatga atggargata gataggtcac ttacgctccg 60

agatggctga g 71

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

gaaggtgacc aagttcatgc tggagcgtaa gtgacctatc tatcg 45

<210> 13

<211> 45

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

gaaggtcgga gtcaacggat tggagcgtaa gtgacctatc tatca 45

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 14

taggataaac acgcgcccac 20

Claims

1. A molecular breeding method for wheat scab resistance with polymerization of multiple traits and favorable allelic variation, which is characterized in that the method comprises the following steps,

step S1, selecting and carrying gibberellic disease resistant geneFhb1Disease resistance genotype of、Qfhb.3BResistance allelic variation of locus, the locus of spikelet per earQSNS.2BAnd thousand kernel weight sitesQTKW.2BThe selected parents are crossed or backcrossed, and seeds F after crossing or backcrossing are harvested ₁ ；

S4, planting F in a greenhouse ₃ Generation, taking leaf from each individual plant to extract DNA, proceedingFhb1、Qfhb.3B、QSNS.2BAndQTKW.2Bselecting the single plant listing containing heterozygous positive favorable allelic variation for genotype detection, simultaneously screening the powdery mildew and yellow mosaic disease resistance, lodging resistance and tillering resistance in the whole growth period of wheat, eliminating the single plants which are easy to lodging, have the plant height of more than 80cm and are late-maturing, and selecting seeds F according to the single plants ₄ ；

S5, planting F in the field ₄ The seeds are planted into plants and firstly are screened and reserved by utilizing molecular markers in the seedling stage and simultaneously carry with the molecular markersFhb1、Qfhb.3B、QSNS.2BAndQTKW.2Bthe heterozygous and positive plant rows which are beneficial to allelic variation are marked, comprehensive agronomic characters, growth periods and other disease resistances of the selected plant rows are comprehensively considered according to breeding targets, excellent plant rows are selected, wherein 5-6 individual plants with consistent characters are harvested, yield identification is carried out after harvesting, and plant row seeds F with yield level higher than that of a control are selected ₅ ；

S6, planting F in the field ₅ The adult plant line is first screened and retained with molecular marker in seedling stageFhb1、Qfhb.3B、QSNS.2BAndQTKW.2Bthe method comprises the following steps of (1) carrying out homozygous positive strains favorable for allelic variation, carrying out listing marking, carrying out instillation on single flowers of listing strains to keep strains with gibberellic disease pathogenic bacteria and identifying results as resistant strains, comprehensively investigating comprehensive agronomic characters, growth periods, other disease resistances and the like of selected strains according to breeding targets, selecting excellent strains, carrying out mixed harvest on selected strains, carrying out yield identification after harvesting, and selecting strains with yield levels higher than those of controls;

s7, planting F in the field ₆ Comprehensively investigating comprehensive agronomic traits and other disease resistance of the cell lines according to a breeding target, performing yield identification after harvesting, selecting cells with the yield level 5% higher than that of a control, and performing next-generation multipoint yield identification;

the gene for resisting gibberellic diseaseFhb1The sequence of the specificity detection primer is shown as SEQ ID NO.1 and SEQ ID NO. 2;

the gibberellic disease resistance locusQfhb.3BThe sequence of the specificity detection primer of (3) is shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;

the number of bits of each earQSNS.2BThe sequence of the specificity detection primer of (3) is shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10;

the thousand kernel weight locusQTKW.2BThe specific detection primer sequences of (1) are shown as SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

2. The molecular breeding method for wheat scab resistance according to claim 1, wherein F is planted in the steps S3 and S4 ₂ Generation and F ₃ Screening powdery mildew resistance, yellow mosaic disease resistance, lodging resistance and tillering resistance, and specifically, eliminating single plants which are susceptible to diseases, easy to lodging, have plant heights of more than 80cm and late-maturing: taking Zhenmai No.9 as a control for resisting powdery mildew, and normally sowing Sunmai No.3 with high powdery mildew at intervals of single plants in a greenhouse for 4-6 days in advance to uniformly plant the Sunmai No.3 with high powdery mildew to induce powdery mildew; taking Ningmai 13 as a control for resisting yellow mosaic disease, and screening individual plants for resisting yellow mosaic disease in comparison in the whole growth period; judging tillering performance by taking Yangmai 25 as a reference, and eliminating the single plants with the ear number less than that of Yangmai 25; the selection of lodging and maturity is controlled by Yangmai 25.

3. The molecular breeding method for wheat scab resistance according to claim 1, wherein the plant rows that are inoculated with scab pathogenic bacteria in single flower instillation to the listing plant rows at the flowering stage in step S6 and have the retention identification result of "resistant" are specifically: preparation of a suspension of gibberellic spores 4X 10 ⁵ ～5×10 ⁵ spore/mL, in field, at wheat flowering stage, useInoculating by a single flower drip method, randomly selecting 20 spikes from each plant row or strain, inoculating the small flowers in the middle of each spike, marking, spraying water to the inoculated spikes every 2 hours, uniformly and fully spraying the water to the wheat spikes for 10 minutes every time, and stopping spraying water immediately after the wheat blooms for 20 days, wherein the weight ratio of the water sprayed to the inoculated spikes is 7-00; after 21 days of inoculation, the disease condition of the inoculated spikes is investigated, the number of the small spikes and the total small spikes of each spike are detected, the severity of gibberellic disease PSS = the number of the small spikes detected/the number of the total small spikes multiplied by 100%, the plant rows or the plant lines of which the PSS is less than 25% and is close to the 'resistant' level of Sumai No.3 are reserved, sumai No.3 and Annong 8455 are respectively used as disease-resistant and susceptible controls, yangma No. 25 is used as an anti-resistant control, and Yangma No.13 is used as a neutral control.

4. The molecular breeding method for wheat scab resistance according to claim 1, wherein the identification of other disease resistance in steps S5, S6 and S7 is specifically: identifying yellow mosaic and powdery mildew by adopting natural morbidity identification; selecting the plant-row listing mark with disease-resistant (R) grade disease-resistant identification results of yellow mosaic disease and powdery mildew.

5. The molecular breeding method for wheat scab resistance according to claim 4, wherein the identification of powdery mildew and yellow mosaic disease in steps S5, S6 and S7 further comprises planting Zhenmai No.9 as a powdery mildew resistance (R) control; ningmai 13 is used as a control for yellow mosaic disease resistance (R).

6. The molecular breeding method for wheat scab resistance according to claim 1, wherein the comprehensive agronomic traits of selected plant rows, lines and strains in the comprehensive examination in steps S5, S6 and S7 are specifically: selecting the plant rows with the plant height of less than 90cm, good lodging resistance, the number of single plant ears of more than or equal to 8, the number of grains per ear of more than or equal to 45 and the maturity period equivalent to Yangmai 25; and the reference variety of the agronomic characters and the growth period in the steps S5, S6 and S7 is Yangmai 25.