CN114931092A - Wheat scab resistant molecular breeding method for polymerizing multiple characters and facilitating allelic variation - Google Patents

Abstract

The invention discloses a wheat scab resistance molecular breeding method for polymerizing multiple traits and facilitating allelic variation, which comprises the steps of selecting a parent 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 spikelet locus QSNS.2B and a thousand seed weight locus QTKW.2B, hybridizing or double crossing the selected parent, and harvesting 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 gibberellic disease resistance and yield related favorable genotype or allelic variation are polymerized by the gibberellic disease resistance and yield related favorable wheat variety (line) bred by the method, the resistance and the yield level of the gibberellic disease are synergistically improved, and the fusarium head blight resistance and yield level can be used as a new generation of green and safe wheat variety (line) to solve the problem of neck sticking in the contemporary breeding industry.

Description

Wheat scab resistant molecular breeding method for polymerizing multiple characters 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 major producing country and consuming 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. Currently, 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 global risk of wheat scab is on a gradually increasing trend. 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 Yangtze river are the wheat scab frequently-growing area and the wheat scab repeatedly-growing area. 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 gibberellic disease 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. Standard examination of wheat variety for resistance to gibberellic disease in "Standard examination of crop variety in Jiangsu province: the resistance of Huainan wheat area reaches resistance, and the resistance of Huaibei wheat area reaches resistance more than resistance. 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 resistance to gibberellic disease is still the most economic, safe and effective way to solve the harm of gibberellic disease of wheat, but cannot be cultivated into a variety with high yield and resistance to gibberellic disease which is widely popularized so far, and the contradiction between resistance to gibberellic disease and high yield is still the 'Kanji' problem which restricts 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 controlled by a single gene, is easily influenced by multiple factors including environment, the identification of the gibberellic disease resistance of multiple generations of breeding is very complex and tedious, a large amount of manpower, material resources and financial resources are required to be invested, and in addition, in recent years, the climate conditions are abnormal, extreme climates frequently occur, and huge 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 loci may be related to genetic background, and a certain degree of correlation exists between 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 wheat variety with synergistic improvement of wheat scab resistance heredity and breeding research, resistance cultivation, yield and the like is very urgent.

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 favorable allelic variation genotype complementation, including 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 perform hybridization or double crossing, and hybrid seeds after hybridization or double crossing are harvested; and the method combines the accurate identification of disease resistance, agronomic characters and yield of multiple generations to achieve the breeding target of wheat scab and high yield synergistic improvement.

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 a base carrying disease-resistant genotype of a scab-resistant gene Fhb1, resistance allelic variation of a scab-resistant site Q fhb.3B and synergistic allelic variation of a spikelet-resistant site QSNS.2B and a thousand kernel weight site QTKW.2BThe parents with complementary genotype are hybridized or backcrossed, and the hybridized or backcrossed seeds F are harvested ₁ ；

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

Step S3, planting F in 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 ；

Step S4, planting F in greenhouse ₃ In generation, each plant is taken to extract DNA, favorable allelic variation screening of Fhb1, Qfhb.3B, QSNS.2B and QTKW.2B is carried out, the plant hanging plate which contains heterozygous positive favorable allelic variation is selected for genotype detection, simultaneously screening of powdery mildew resistance, yellow mosaic disease resistance, lodging resistance and tillering resistance is carried out in the whole growing period of wheat, the single plant which is easy to lodging, has the plant height of more than 80cm and is late-maturing is eliminated, and F is selected according to the seed collection of the single plant ₄ ；

Step S5, planting F in field ₄ Firstly, screening and retaining plant rows which contain heterozygous positive favorable allelic variation and are simultaneously provided with Fhb1, Qfhb.3B, QSNS.2B and QTKW.2B by utilizing molecular markers in the seedling stage, tagging the plant rows, comprehensively inspecting the comprehensive agronomic characters, the growth period and other disease resistances 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 ；

Step S6, planting F in the field ₅ The molecular marker is used to screen and reserve the strain line which is simultaneously provided with favorable allelic variation of Fhb1, Qfhb.3B, QSNS.2B and QTKW.2B which are homozygosis positive at the seedling stage, the tag marker is used to drop-inoculate the strain line with the gibberellic disease pathogenic bacteria reservation and identification result of 'anti' at the flowering stage, then the comprehensive agronomic characters, growth period and other disease resistances of the selected strain line are comprehensively inspected according to the breeding target, the excellent strain line is selected, the selected strain line is mixed and harvested, the yield identification is carried out after harvesting, and the high yield level is selectedIn a control strain;

step S7, planting F in field ₆ According to the breeding goal, comprehensively examining the comprehensive agronomic traits and other disease resistance of the cell lines, carrying out yield identification after harvesting, selecting the cells with the yield level 5% higher than that of a control, and entering 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 specific detection primer sequences of the thousand-grain-weight locus QTKW.2B are shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

Further, planting F 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 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 compared with Yangmai 25.

Further, the step S6 of instilling gibberellic disease pathogenic bacteria to the single flowers of the hanging plant row at the flowering stage to retain the plant row whose identification result is "resistant" is specifically: preparation of a suspension of gibberellic spores 4X 10 ⁵ ～5× 10 ⁵ spore/mL, in a field, in the flowering period of wheat, adopting a single flower drip method for inoculation, randomly selecting 20 spikes of each plant row or strain, inoculating small flowers in the middle of each spike, marking, and after inoculation, 7: 00-18:00, spraying water to the seed ears every 2 hours, uniformly and fully spraying the water to the wheat ear parts, wherein the water spraying is stopped immediately after the wheat blooms for 20 days every time for 10 minutes; after 21 days of inoculation, the disease occurrence of inoculated spikes is investigated, the number of small spikes and the total number of small spikes are detected per spike, the severity of gibberellic disease PSS is equal to the number of small spikes detected/the total number of small spikes multiplied by 100%, plant rows or plant lines with PSS less than 25% and 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 25 is used as an anti-resistant control, and Yangma 13 is used as a sensitive control.

Further, 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.

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

Further, the comprehensive agronomic traits of the selected plant rows, lines and strains in the comprehensive examination 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 of the agronomic trait and the growth period in the steps S5, S6 and S7 is yangma 25.

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

the invention uses the ordinary wheat variety Yangmai 16 which is different from the known resistance source such as Sumai No.3, Wangshiba and the like as the source of the gibberellic disease resistant site for the first time, does not carry Fhb1, has excellent agronomic characteristics 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 that the Yangmai 16 carries a new gibberellic disease resistant site Qfhb.3B for the first time and has no negative effect on the yield characteristics. 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, thus not only obviously improving the resistance of gibberellic disease, but also improving the yield. 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 breakthrough in gibberellic disease resistance and yield, greatly shortens breeding period, reduces field workload and pesticide and fertilizer application, 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 closely linked molecular markers at 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 invention pertains.

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

step S1, parent selection: selecting main cultivars in a wheat natural population and derivatives thereof as objects, extracting seeds or seedling stage leaf DNA, selecting varieties (lines) carrying disease-resistant genotypes of a scab resistant gene Fhb1 and scab resistant loci Qfhb.3B, wherein the varieties (lines) carry synergistic allelic variation complementation of the positions QSNS.2B of every panicle and the QTKW.2B of thousand grain weight loci as parents, selecting a strain Yangma 28 carrying the resistance allelic variation of the scab resistant gene Fhb1, a Yangma 30 carrying the resistance allelic variation of the scab resistant loci Qfhb.3B, wherein the Yangma 28 carries the synergistic genotype of the thousand grain weight loci QTKW.2B, the Yangma 30 carries the synergistic genotype of the positions QSNS.2B of the panicles, and the genotypes/allelic variation of the disease-resistant loci and the yield-related loci of the two parents realize complementation.

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

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

3) Step 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: zhenmi 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) step S4, planting F in greenhouse in 2018 month 1 ₃ 1 line per 1 single plant harvested in the first generation, 2m line length, 0.25m line spacing, 40 grains per line, taking leaf from each single plant, screening favorable allelic variation of Fhb1, Qfhb.3B, QSNS.2B and QTKW.2B, selecting single plant brands containing all favorable allelic variation (including heterozygous positives) for genotype detection, and simultaneously, obtaining the DNA sequence of the single plant varietyScreening powdery mildew and yellow mosaic disease resistance, lodging resistance and tillering resistance in the whole growth period of the wheat, and eliminating single plants which are easy to lodging, have plant heights of more than 80cm and are later than Yangmai 25 in the mature period. Harvesting single plants in the final harvest in 6 months;

5) step 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 simultaneously carrying with scab resistant gene Fhb1, scab resistant site Qfhb.3B resistant allelic variation, NS.2B per ear spikelet site and thousand kernel weight site QTKW.2B favorable allelic variation (containing heterozygosis positive), listing mark, and comprehensively inspecting comprehensive agronomic characters and other disease resistance of the selected plant rows according to breeding targets: identification of yellow mosaic, powdery mildew and rust (leaf rust) adopts natural onset identification, and Ningmai 13 is used as a yellow mosaic disease resistance (R) control; zhenmai No.9 was used as a control for powdery mildew resistance (R). Selecting the rows with the disease identification results of yellow mosaic disease, powdery mildew and rust disease all showing disease resistance (R) level, inspecting the agronomic traits, taking Yangmai 25 as a basic control, selecting the row listing mark with the plant height less than 90cm, good lodging resistance, the number of single plant spikes greater than or equal to 8 and the number of grains per spike greater than or equal to 45. And (5) 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 control variety Yangmai 25.

6) Step S6, planting F in field in 2019 and 10 months ₅ Planting adult plant line, 6-line region, line length 2m, line spacing 0.25m and 50 grains in each line, 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 with homozygosis resistance allelic variation of gibberellic disease resistance gene Fhb1 and Qfh b.3B locus and beneficial allelic variation (homozygosis) of spikelet locus QSNS.2B and thousand grain weight locus Q TKW.2B by molecular marker, and making into gibberellic spore suspension 4X 10 ⁵ ～5×10 ⁵ spore/mL, in the field of 4-5 months in 2020, in the wheat flowering period, adopting single flower instillation method to inoculate, randomly selecting 20 ears of each strain, inoculating in the small flower opened in the middle of each ear,and marking, spraying water to the seed ears 7: 00-18: 00 every day after inoculation every 2 hours, uniformly spraying the water to the wheat ears for 10 minutes every time, and stopping spraying water immediately after the wheat blooms for 20 days. After 21 days of inoculation, the disease incidence of the inoculated spikes is investigated, the number of the small spikes and the total small spikes of each spike are detected, the scab severity PSS is equal to the number of the small spikes of the disease/the total small spikes multiplied by 100%, strains with the resistance level close to that of Sumai No.3 are reserved, the PSS is less than 25%, Sumai No.3 and Annong 8455 are respectively used as disease-resistant and disease-susceptible controls, Yangma 25 is used as an anti-resistant control, Yangma 13 is used as a 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; zhenmai No.9 is a control for powdery mildew resistance (R). Selecting a strain listing mark with disease resistance identification results of powdery mildew and yellow mosaic disease both showing R-grade plant row agronomic traits, taking Yangmai 25 as a basic control, selecting a strain listing mark with plant height lower than 90cm, good lodging resistance, single plant spike number more than or equal to 8 and per spike number more than or equal to 45. 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 the Yangmai 25. The gibberellic disease severity of these two 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 mean severity is based on the mean condition of the disease. The disease resistance investigation and identification standard is according to agricultural industry standard NY/T2954-2016 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 10 months in 2020 ₆ Each selected strain is planted into 10 rows of regions with the row length of 3.2 meters and the row spacing of 0.23 meter, and according to the breeding target, the comprehensive investigation is carried outThe comprehensive agronomic traits and other disease resistance of the cell strains are identified, the identification of powdery mildew and yellow mosaic disease adopts natural morbidity identification, and Zhenmai No.9 is a powdery mildew resistance (R) control; ningmai 13 is a control for yellow mosaic disease resistance (R). And selecting strains with disease resistance identification results of powdery mildew and yellow mosaic disease both in R grade to investigate agronomic traits, selecting strain branding marks with good lodging resistance, the number of single plant ears being more than or equal to 8 and the number of grains per ear being more than or equal to 45 by taking Yangmai 25 as a basic control. The yield identification is carried out after 6 months of harvest in 2021, the yield level is completely converted into the 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 571.3 kg/mu and 578.5 kg/mu, the yield is increased by 5.48% and 6.81% respectively compared with that of the control Yangmai 25, and the yield identification enters the next year of multi-point yield identification.

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 30n g/. mu.L, and detecting the linkage His label of the wheat scab resistant gene Fhb 1.

The sequences of a specific primer group for detecting the linked His label of the gibberellic disease resistant gene Fhb1 by molecular markers are shown in Table 1:

TABLE 1 primer sequence information of linkage marker Fhb1 for gibberellic disease resistance gene

Method for detecting gibberellic disease resistance by adopting PCR amplificationThe corresponding linkage mark His of the major gene Fhb1, and the PCR amplification method comprises the following steps: the PCR amplification system is 10 mu L, and comprises 1.0 mu L of wheat genome DNA 30 ng/mu L, 1.0 mu L of 10 XPCR buffer, 0.2 mu L of 10Mm dNTP and 10Mm MgCl 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 deg.C for 5 min; 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, 32 cycles; (3) extension at 72 ℃ for 5 min; storing at 4 ℃.

The His primers were used to detect wheat material in 1% agarose electrophoresis solution as in example 1, and the target genotype was the same as Ningmai 13, which was the material of choice. FIG. 2 is a schematic diagram showing the amplification results of the Fhb1 site closely linked molecular marker His in parents and offspring, and an arrow indicates a target band for positive amplification.

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

And (3) field test: 174 parts of DH derived from 'Yangmai 16/Zhongmai 895' is taken as a material, the DH and the parents of the DH are planted in a gibberellic disease identification garden (Yangzhou, Jiangsu) of Wanfu test base of agricultural science institute in Rihe river area in 2016, 2017 and 2018 for 3 continuous growing seasons, the sowing period of Yangzhou wheat in the current year is set to 10 months and 20 days, the test adopts a random block design, 2 rows of areas and 2 times of repetition, each row comprises 30 grains, the row length is 1.5m, and the row spacing is 0.23 m. 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 incidence condition of the inoculated ears after 28 days, counting the number of small ears per ear and the total number of small ears per ear, 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 and BIN functions of IcMapping v4.1 software (http:// www.isbr feeding. net), primarily constructing a genetic linkage MAP by utilizing a MAP function, correcting the genetic MAP by utilizing JoinMap v4.0, and drawing the genetic MAP by utilizing MapChart2.3(https:// www.wur.nl/en/show/Mapct. htm). QTL (quantitative trait locus) significantly related to the severity of gibberellic disease (mean panicle rate, PSS) was detected by using an IcMap ping v4.1 software complete interval mapping (IC IM), and the LOD threshold was set to 3.0. For comparison with the previous results, the linkage 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 molecular markers: considering that the Qfhb.3B initial location region 705.28-742.44 Mb is large and not beneficial to further utilization, the applicant divides the DH families into 2 mixed pools according to the gibberellic disease phenotype identification result, 15 families each of the anti-gibberellic disease mixed pool (PSS < 25%) and the gibberellic disease sensing mixed pool (PSS > 75%), performs exon capture and mixed pool RNA sequencing (BSR) of the wheat leaf genome mixed pool, also performs wheat 660K SNP chip scanning and re-sequencing on the parent Yangmai 16 and the Miao 895 to dig more polymorphic SNP sites, develops KASP markers corresponding to 14 SNPs in the target region by using Primer 5.0 software according to the version 2.1 information of the wheat Chinese spring reference genome, scans and verifies in the original population, develops a polymorphic SNP marker at the 726.43Mb physical chromosome position, and can be linked with the gibberellic disease in the best phenotype, therefore, this marker was named YM16-fhb-3B, and the information on the primers is shown in Table 3.

TABLE 3 YM 16-fhb-3B-related information

Preparation of 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 muL (about 30 ng/muL) of the wheat DNA template to be detected, 0.06 muL of primer working solution and 2.5 muL of KASP Master Mix (LGC company, KBS-1016-002) are supplemented to 5 muL by using sterile ultrapure water; PCR reaction procedure: (1) pre-denaturation at 94 ℃ for 10 min; (2) denaturation at 95 ℃ for 20s, 61-55 ℃ (0.6 ℃ per cycle) for 45s, for 10 cycles; (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-. The FAM excitation wavelength is 485nm, and the emission wavelength is 520 nm; the VIC excitation wavelength is 535nm, the emission wavelength is 556nm, the system reference fluorescence ROX excitation wavelength is 575nm, and the emission wavelength is 610 nm. 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 Zhongmai 895 DH line' 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 parting result fluorescence signal coordinate system, 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, which is the same as that of Yangmai 16; 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 at the SNP site is proved to be T when the genotype is different from the genotype of Yangmai 16. The results of T-test of genotype at Qfhb.3B and severity of gibberellic disease of "DH line of Yangmai 16X Mizhong 895" are shown in Table 4.

TABLE 4T test results of the isogenic variation of gibberellic disease severity (PSS) and Qfhb.3B loci in DH lines

As can be seen from table 4, at the site qfhb.3b, the average value of the severity of wheat scab with allele T is significantly higher than that of wheat with allele C overall, and the genotype and phenotype of a part of DH families were tested using the double sample T of Excel 2019 (genotype was not detected in 7 families), and the results indicated that: the genotype of 92 families is C as same as that of Yangmai 16, the genotype of 75 families is T as same as that of Zhongmai 895, the average value of the severity of the gibberellic disease of the families with the genotype of C is reduced by 20.57 percent compared with that of the families 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 locus and the genotype detection system can be applied to wheat scab resistant molecular marker assisted breeding.

Example 4 application of KASP primer set breeding of site Qfhb.3B of gibberellic disease resistance and analysis of polymerization effect of the primer set breeding and Fh b1

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 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 repeated for 2 times, each row comprises 40 grains, the row length is 1.5m, and the row spacing is 0.23 m. The field fertilization and management refer to local field cultivation production, and the cordyceps sinensis is prevented and treated in timeAnd (4) harming. In 2015, a gibberellic spore suspension was prepared at 4X 10 ⁵ ～5×10 ⁵ spore/mL, 4 in the middle of the 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 of the inoculated ears after 21 days, counting the number of the small ears and the total small ears which are attacked in each ear, and finally taking the average value of 2 times of repetition as the average severity PSS of the family.

And (3) genotype detection: the 77 varieties (lines) were genotyped using the primer set for the Qfhb.3B site KASP obtained in example 3 and the primer set for Fhb1 of example 2. The fluorescence signal data of the amplification products of the primer group of KASP marker YM16-fhb-3B at the 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 variety at the 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 site is proved to be T, and the attached figure 3 shows the result of amplifying the fluorescence signal of the natural population by 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, where R indicates resistance allelic variation carrying Fhb1 and S indicates resistance allelic variation not carrying the gene.

TABLE 577 average values of gibberellic disease severity and Fhb1, Qfhb.3B locus genotype measurements

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 disease-resistant genotype), 26 families carried no resistant allelic variation at the qfhb.3b site, had the susceptible allelic variation TT, and 32 families carried the qfhb.3b site resistant allelic variation CC, with the results of T-test shown in table 6.

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

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, with a double sample T test using Exc el 2019, with the removal of the effect of Fhb1, and the results indicated that: 32 varieties (lines) and Yangmai 16 have the same genotype of C, 26 varieties (lines) and Zhongmai 895 have the same genotype of T, the average value of the scab severity of the varieties (lines) with the genotypes of C is reduced by 22.48 percent compared with that of the varieties (lines) with the genotypes of T, and the obvious difference exists on the level of p <0.01, which shows that the primer group of KASP marker YM16-fhb-3B at the 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 allelic variation of gibberellic disease severity (PSS) and Qfhb.3B locus resistance 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 of p <0.01, which indicates that the primer group of KASP marker YM16-fhb-3B at the 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 on polymerization with the Qfhb.3B site, with S representing the Fhb1 disease genotype and R representing the Fhb1 disease resistance genotype, and the results are shown in Table 8.

TABLE 8 analysis of the combined Effect of resistant allelic variation of Fhb1 and Qfhb.3B loci

Note: different lower case letters after PSS (%) indicate that the numbers 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% and 43.22% 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 resistance effect than the single gene/locus resistance genotypes. Meanwhile, the primer group His of the Fhb1 and the primer group YM16-fh B-3B of the Qfhb.3B site 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-grain weight site QTKW.2B and KASP thereof

And (3) field test: in the embodiment, 205 families of Yangmai No. 12/Thiampyrum exhibition No.1 RIL group planted in yield identification garden of Bay experiment base in 2017 plus 2019 are used as materials, the sowing period of Yangmai 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 arranged, the row length is 2m, and the row distance is 0.3 m. 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 the 3 row region). 2018. And (3) surveying the number of spikelets per ear in the late filling stage-mature period of wheat in 2019 and 2020, randomly marking 10 individual plants in each family, surveying the number of spikelets per ear of 10 spikes per individual plant, and finally taking the average value of the 10 individual 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, keeping large grains and small grains, observing the weight of 3 300 grains, taking an average value, finally converting the weight into the weight of 1000 grains to obtain the thousand grain weight, and finally taking the average value of 2 times of repetition as the mean 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.isbre eating. net), primarily constructing a genetic linkage MAP by utilizing MAP functions, and drawing the genetic MAP by utilizing MapChart2.3(https:// www.wur.nl/en/show/Ma pchart. 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 the previous results, the linkage 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 per spike and the QTKW.2B of the thousand kernel weight synergistic site of the wheat obtained by the experiment 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 molecular markers: according to SNP information of QSNS.2B and QTKW.2B target intervals and phenotype identification data of characters, SNPs which are most significantly related to the number of spikelets and thousand kernel weight of each ear are excavated from 5-7 SNPs which are significantly related in 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 2.1 version 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 of the forward primers and 30. mu.L (100. mu.M) of the downstream primer were collected, supplemented to 100. mu.L with sterile ultrapure water, and mixed thoroughly to prepare a KASP-labeled primer working solution for future 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 muL (about 30 ng/muL) of the wheat DNA template to be detected, 0.06 muL of primer working solution and 2.5 muL of KASP Master Mix (LGC company, KBS-1016-002) are supplemented to 5 muL by using sterile ultrapure water; PCR reaction procedure: (1) pre-denaturation at 96 ℃ for 10 min; (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, 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-. The FAM excitation wavelength is 485nm, and the emission wavelength is 520 nm; the VIC excitation wavelength is 535nm, the emission wavelength is 556nm, the system reference fluorescence ROX excitation wavelength is 575nm, and the emission wavelength is 610 nm. 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. Part of the "Yangmai 12/Elytrigia 1 RIL line" and two parents are amplified according to the method, for QSNS.2B locus, the genotype T with the same Elytrigia 1 is excellent allelic variation (favorable allelic variation), the fluorescence signal data of the amplification product is analyzed by Kluster Caller to gather at the position (blue) close to X axis in the fluorescence signal coordinate system of the typing result, and 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 locus) 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 product is analyzed by Kluster Caller to gather at the position (red) close to Y axis in the coordinate system, is different from Elymia 12 and is the same as Elytrigia 1, so that the genotypes of the SNP locus are respectively T, is an allelic variation (favorable allelic variation) which increases the number of spikelets per ear. For QTKW.2B locus, the genotype C of Yangmai 12 which is the same as that of Yangmai 12 is synergistic allelic variation (excellent allelic variation), the fluorescence signal data of the amplification product is analyzed by Kluster Caller software to gather at the position (blue) close to the X axis in the fluorescence signal coordinate system of the typing result, the fluorescence signal data are the same as those of Yangmai 12 and are different from those of Elytrigia 1, namely the fluorescence signal data of the wheat are analyzed by Kluster Caller software to gather at the position (red) close to the Y axis in the coordinate system, are different from those of Yangmai 12 and are the same as those of Elytrigia 1, and the fluorescence signal data of the amplification product are respectively T at the position (red) close to the Y axis in the coordinate system and are the allelic variation for reducing the thousand grain weight.

Example 6 validation of KASP primer set for spikelet number site QSNS.2B and thousand kernel weight site QTKW.2B per ear

And (3) field test: in the embodiment, 173 wheat natural populations planted in a yield identification garden of a Bay-head experiment base in 2019 are used as materials, the seeding period of the 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 repeated for 2 times, 50 grains are arranged in each row, the row length is 2m, and the row spacing is 0.3 m. 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 row 3). 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 locus, the genotype T with the same Elaeagnus trix 1 is excellent allelic variation, the fluorescence signal data of the amplification product is analyzed by Kluster Caller software to gather at the position (blue) close to X axis in the fluorescence signal coordinate system of the typing result, the fluorescence signal data are the same as Yangmai 12 and different from Elaeagnus trix 1, namely the genotype of the flanking nucleotide sequence of the wheat on KASP marker (for example, the 36 th base (SN P locus) of SEQ ID NO.7 is C which is allelic variation for reducing the number of spikelets per ear), and the fluorescence signal data of the amplification product is analyzed by Kluster Caller software to gather at the position (red) close to Y axis in the coordinate system, the genotype of the genotype T12 is different from the genotype of Yangmai 1, and the genotype of the Elaeagnus trix 1 is T which is the same as the genotype C of the S NP locus and is allelic variation for increasing the number of spikelets per ear, the genotype C with the same as QT 12 is excellent allelic variation for QT 2B locus, the fluorescence signal data of the amplified products were analyzed by Kluster Caller software to gather at the X-axis position (blue) in the fluorescence signal coordinate system of the typing results, which is the same as Yangmai 12 and different from Elytrigia repens No.1, thus demonstrating that the genotypes of the flanking nucleotide sequences of these wheat at the KASP marker (e.g., the 36 th base (SNP site) of SEQ ID NO.7 are respectively C, which is an allelic variation for increasing thousand seed weight), while the fluorescence signal data of the amplified products were analyzed by K luter Caller software to gather at the Y-axis position (red) in the coordinate system, which is different from Yangmai 12 typing and the same as Elytrigia repens No.1, which demonstrates that the genotypes of these SNP sites are respectively T, which is an allelic variation for decreasing thousand seed weight, and that the primer sets of YZ1-SNS-2B and YM12-TKW-2B amplify the fluorescence signals of natural populations, 173 varieties (lines) at two QTB and KW 2NS loci and QSB The results of the T test are shown in tables 11 and 12.

Table 11173 varieties (lines) with small ear number (SNS) and Thousand Kernel Weight (TKW) per ear and QSNS.2B and QTKW.2B locus genotype detection results

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 the same genotype CC as Yangmai 12, 48 varieties (lines) carry the same genotype TT as elytrigia repens No.1, the CC genotype can be obviously improved by 3.73 percent of thousand grain 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 number of small ears of each ear of wheat.

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

The parent, F, referred to in example 1 was examined with a primer set of molecular markers 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 wheat in the embodiment 3 and the primer group of the linked KASP marker YZ1-SNS-2B and YM12-TKW-2B for detecting the spikelet number and thousand kernel weight locus of wheat in the embodiment 5 to obtain an amplification product, and the target material of which the Qfhb.3B locus is the same as the Yangmai 16 genotype, the QSNS.2B locus is the same as the Yangmai No.1 genotype and the QTKW.2B locus is the 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 Qfhb.3B locus amplification products are analyzed by Kluster Call er software and gathered at the position (blue) close to the X axis in a parting result fluorescence signal coordinate system, and the position is the same as Yangmai 16, namely the genotype of 36 th base (SNP site) 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 the position (red) close to the Y axis in the coordinate system, and the genotype of the wheat at the SN P site is proved to be T when the wheat is different from Yangmai 16 in typing, so that the material is rejected. 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 nucleotide sequence (such as SEQ ID NO.7) flanking the molecular marker YZ1-SNS-2B 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 amplified product of the QTKW.2B locus is analyzed by Kluster Caller software and gathered at a position (blue) close to an X axis in a parting result fluorescence signal coordinate system, and the position 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 restrictive, and thus other examples of example embodiments may have different values.

SEQUENCE LISTING

<110> institute of agricultural science in the region of Ri river of Jiangsu

<120> molecular breeding method for wheat scab resistance with polymerization multiple characters and favorable allelic variation

<130> 202205

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

attcctacta gccgcctggt 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

actggggcaa gcaaacattg 20

<210> 3

<211> 53

<212> DNA

<213> Triticum aestivum

<220>

<221> misc_feature

<222> (36)..(36)

<223> r is [ T/C ]

<400> 3

tccatgctag aaaagggccg caccaccgcc gtcgcrcacc acccacaagc gcc 53

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

gaaggtgacc aagttcatgc tggcgcttgt gggtggtga 39

<210> 5

<211> 39

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

gaaggtcgga gtcaacggat tggcgcttgt gggtggtgg 39

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

tccatgctag aaaagggccg 20

<210> 7

<211> 71

<212> DNA

<213> Triticum aestivum

<220>

<221> misc_feature

<222> (36)..(36)

<223> r is [ C/T ]

<400> 7

aatacccgca agtatacgaa actgaaagat gtgcgrggtc aagacggctt agaacgctac 60

aaaatattca a 71

<210> 8

<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 (10)

1. A wheat scab resistant molecular breeding method for aggregating multiple traits and favorable allelic variation is characterized in that the method comprises the following steps,

s1, selecting parents carrying the disease-resistant genotype of the scab-resistant gene Fhb1 and the resistant allelic variation of the Qfhb.3B locus, the genotype complementation of the synergistic allelic variation of the QSNS.2B and the thousand kernel weight locus QTKW.2B of each ear, hybridizing or double crossing the selected parents, and harvesting the hybridized or double crossed seeds F ₁ ；

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

Step S3, planting F in 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-maturing under natural conditions, harvesting and selecting single plant seeds F ₃ ；

Step S4, planting F in greenhouse ₃ In generation, each plant is taken to extract DNA, favorable allelic variation screening of Fhb1, Qfhb.3B, QSNS.2B and QTKW.2B is carried out, the plant hanging plate which contains heterozygous positive favorable allelic variation is selected for genotype detection, simultaneously screening of powdery mildew resistance, yellow mosaic disease resistance, lodging resistance and tillering resistance is carried out in the whole growing period of wheat, the single plant which is easy to lodging, has the plant height of more than 80cm and is late-maturing is eliminated, and F is selected according to the seed collection of the single plant ₄ ；

Step S5, planting F in field ₄ The seed adult plant is first screened and maintained in seedling stage with molecular marker containing Fhb1, Qfhb.3B, QSNS.2B and QTKW.2BHeterozygous 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, single plants with 5-6 individual 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 ；

Step S6, planting F in field ₅ The molecular marker is utilized to screen and reserve strains which are simultaneously provided with homozygous positive favorable allelic variation of Fhb1, Qfhb.3B, QSNS.2B and QTKW.2B in a seedling stage, the marker is hung, the single flower of the marker strain is instilled with a strain with a gibberellic disease pathogenic bacterium reservation and identification result of resistance in a flowering stage, then comprehensive agronomic characters, growth period, other disease resistance and the like of a selected strain are comprehensively inspected according to a breeding target, excellent strains are selected, selected strains are mixed and harvested, the yield is identified after harvesting, and the strains with the yield level higher than that of the contrast are selected;

step S7, planting F in 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.

2. The molecular breeding method for wheat scab resistance according to claim 1, wherein the sequences of the specific detection primers of the scab resistance gene Fhb1 are shown as SEQ ID No.1 and SEQ ID No. 2.

3. A molecular breeding method for wheat with resistance to fusarium head blight according to claim 1, wherein the sequences of specific detection primers of the fusarium head blight resistance locus Qfhb.3B are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

4. The molecular breeding method for wheat scab resistance according to claim 1, wherein the sequences of the specific detection primers for the panicle number site QSNS.2B are shown as SEQ ID No.8, SE Q ID No.9 and SEQ ID No. 10.

5. The molecular breeding method for wheat scab resistance according to claim 1, wherein the specificity detection primer sequences of thousand kernel weight locus QTKW2B are shown as SEQ ID No.12, SEQ ID No.13 and SEQ ID No. 14.

6. 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 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.

7. The molecular breeding method for wheat scab resistance according to claim 1, wherein the step S6 of single-flower instillation of the scab pathogenic bacteria into the hanging plant row at the flowering stage is 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, adopting a single flower drip method for inoculation, randomly selecting 20 spikes from each plant row or strain, inoculating small flowers in the middle of each spike, marking, spraying water to the seed spikes every 2 hours at a rate of 7: 00-18: 00 every day, uniformly and fully spraying the water to the wheat spikes for 10 minutes every time, and immediately stopping spraying the water after the wheat blooms for 20 days; after 21 days of inoculation, the disease condition of the inoculated spikes is investigated, the number of small spikes and the total number of small spikes are detected per spike, the severity PSS of gibberellic disease is equal to the number of small spikes detected/the total number of small spikes multiplied by 100%, plant rows or lines with PSS less than 25% and with the level close to the resistance level of No.3 Sumai are reserved, and No.3 Sumai and Annong 8455 are respectively used as disease resistance and disease resistanceThe susceptible control, Yangmai 25 as the medium resistant control and Yangmai 13 as the medium sensitive control.

8. 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 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.

9. The molecular breeding method for wheat scab resistance according to claim 8, 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 a control for yellow mosaic disease resistance (R).

10. The wheat scab resistant molecular breeding method of claim 1, wherein the comprehensive agronomic traits of selected plant rows, lines and lines comprehensively examined in the steps S5, S6 and S7 are specifically: selecting a plant row 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; the control variety of the agricultural character and the growth period in the steps S5, S6 and S7 is Yangmai 25.

Images