A breeding method for creating new germplasm by using wheat landraces to improve stripe rust resistance and yield

Abstract

The application relates to the field of breeding, in particular to a breeding method for creating new germplasm by using wheat folk varieties to improve stripe rust resistance and yield. The method is characterized in that a two-stage breeding improvement method is established, hybridization, first-stage backcrossing and second-stage selfing are adopted to gradually aggregate excellent allelic sites and eliminate adverse genes, resistance and yield are improved, the stripe rust resistance gene of a wheat folk variety is transplanted into the background of a bred wheat variety which has lost stripe rust resistance, and a series of intermediate materials with good comprehensive traits are created for wheat stripe rust resistance breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of breeding, in particular to a breeding method for creating new germplasm by using wheat folk varieties to improve stripe rust resistance and yield. BACKGROUND

[0002] Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important airborne and epidemic diseases in global wheat production, which seriously affects the yield and quality of wheat.

[0003] Wheat folk varieties are germplasm resources accumulated by ancestors through long-term production practice, with a long history of cultivation, and are precious germplasm resources preserved by natural selection and human intervention, and are important gene sources for breeding improvement of stripe rust resistance. At present, Yr18, Yr81, YrBai and YrYL, etc. Yr genes have been mined from wheat folk varieties, but only Yr18 has been cloned and widely used, and few other Yr genes from wheat folk varieties have been used in breeding. The reason is that compared with modern varieties, folk varieties are late-maturing, prone to lodging, have low thousand-grain weight, and the comprehensive agronomic traits cannot meet the expected selection of resistance parents, so they are often discarded. How to find the lost stripe rust resistance genes from the old wheat folk varieties and effectively transfer and apply them to the current wheat stripe rust resistance breeding has become the focus of attention of resource scientists and breeders.

[0004] A large number of studies have shown that due to the control of wheat folk varieties on growth period, plant height, yield-related traits, which are often controlled by micro-effect polygenes, it is difficult to eliminate undesirable traits and effectively transfer excellent traits by existing breeding methods. Therefore, it is necessary to develop a breeding method for creating new germplasm by using wheat folk varieties to improve stripe rust resistance and yield, effectively eliminating genetic burdens such as late-maturing, high-stalk, and low-thousand-grain weight in folk varieties, and creating breeding "good" intermediate materials with coordinated resistance, yield, and comprehensive agronomic traits, in order to realize effective breeding application of stripe rust resistance genes from folk varieties. SUMMARY

[0005] The key problem to be solved by the present application is how to effectively use folk varieties to improve wheat stripe rust resistance and yield-related traits.

[0006] The purpose of the present application is to provide a breeding method for creating new germplasm by using wheat folk varieties to improve stripe rust resistance and yield.

[0007] The breeding method for creating new germplasm by using wheat folk varieties to improve stripe rust resistance and yield according to the present application comprises the following steps:

[0008] The F1 hybrids were obtained by crossing the early-maturing, short-stemmed, large-grained, and highly susceptible wheat variety Chuannong 16 as the female parent and recipient, and the late-maturing, tall-stemmed, low-1000-grain-weight Chinese wheat variety Benqizimai as the male parent and donor. The wheat variety carries the stripe rust resistance genes QYr.BYZ-5BS and Yr18 and is resistant to stripe rust in its adult stage.

[0009] The lower generations were subjected to continuous backcrossing using Sichuan Agricultural University 16 as the recurrent parent. Specific markers were detected for each individual plant in the backcross segregating generation, and only the plant height and stripe rust resistance genotype and phenotype at the adult stage were identified. Finally, a segregating population BC3F1 was obtained, consisting of mixed-harvest plants with stripe rust resistance and carrying the QYr.BYZ-5BS and Yr18 genes, and plant height meeting the generation-by-generation improvement selection criteria.

[0010] The higher generations were continuously self-pollinated to achieve homozygosity, and molecular identification was performed on each separated higher generation. The growth period and yield-related traits were investigated to obtain BC3F4, and new wheat germplasm with high resistance to stripe rust in the adult stage and excellent comprehensive traits was obtained through plant selection.

[0011] According to the breeding method of the present application for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farm varieties, molecular marker-assisted selection is performed using KASP markers KP5B_78.49, KP5B_83.68 and the Yr18 functional marker Lr34-KASP-E11, which are tightly linked to the stripe rust resistance gene QYr.BYZ-5BS in the adult stage, and real-time fluorescence quantitative PCR is used to detect and analyze the transformed plants of each generation.

[0012] The KASP specific marker primers of the KASP marker KP5B_78.49 closely linked to the adult stage stripe rust resistance gene QYr.BYZ-5BS include:

[0013] Forward Primer 1: 5'-GAAGGTGACCAAGTTCATGCTGGTTGCAGGAGGGTGTAAAAG-3',

[0014] Forward primer 2:

[0015] 5'-GAAGGTCGGAGTCAACGGATTGGTTGCAGGAGGGTGTAAAAC-3',

[0016] Reverse universal primer: 5′-TTCTTGTACGACGGCCTTTGA-3′;

[0017] The KASP specific marker primers of the molecule KP5B_83.68 include:

[0018] Forward primer 1:

[0019] 5'-GAAGGTGACCAAGTTCATGCTTCCAAATCATCGTTCCTTTGATCC-3',

[0020] Forward primer 2:

[0021] 5'-GAAGGTCGGAGTCAACGGATTTCCAAATCATCGTTCCTTTGATCA-3',

[0022] Reverse universal primer: 5′-GTCGCCTCAGTGTCTCGAAA-3′;

[0023] The KASP specific marker primers of the Yr18 functional marker Lr34-KASP-E11 include:

[0024] Forward primer 1:

[0025] 5'-GAAGGTGACCAAGTTCATGCTAATGTATCGTGAGAGATTTGCAG-3',

[0026] Forward primer 2:

[0027] 5'-GAAGGTCGGAGTCAACGGATTAATGTATCGTGAGAGATTTGCAT-3',

[0028] Reverse universal primer: 5'-AGGTGAATAAATATGAGCATCAGT-3'.

[0029] According to the breeding method of the present application for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farm varieties, the identification of resistance phenotypes by artificially induced inoculation of stripe rust at the adult stage in the field includes:

[0030] When wheat reaches the third leaf and one heart stage, stripe rust is inoculated using the smear method: a mixture of stripe rust fungus and talcum powder at a ratio of 1:50 is applied to the second-to-last leaf of the induced and susceptible control materials. Once the severity of stripe rust on the flag leaf of the susceptible control material reaches 100%, wheat stripe rust resistance assessment is conducted according to the national technical specifications for wheat stripe rust monitoring and reporting.

[0031] According to the breeding method of the present application for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farm varieties, the wheat stripe rust resistance evaluation reaction type (IT) grading standard at the adult stage includes:

[0032] Reactive type 0: no obvious signs or symptoms, resistance evaluation is immune;

[0033] Reaction type 0; Grade: Necrosis or chlorotic spots, no spore formation, resistance evaluation is near immunity

[0034] The reaction type is level 1: necrosis or chlorotic spots or streaks, mild sporulation, and the resistance evaluation is high resistance;

[0035] The reaction type is level 2: necrotic or chlorotic spots or streaks, moderate spore formation, and the resistance evaluation is moderate resistance;

[0036] The reaction type is level 3: necrosis or chlorotic spots or streaks, massive spore formation, and the resistance evaluation is moderate;

[0037] The reaction type is level 4: no necrosis or yellowing, abundant spores, and the resistance evaluation is highly susceptible.

[0038] According to the breeding method of the present application for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farmer varieties, the yield-related trait survey and excellent plant screening include:

[0039] The survey of yield-related traits and the screening of excellent individual plants were conducted in the experimental field and indoors. The high-generation groups in the second improvement section were sown in mid-to-late October, with a row length of 2m, a row spacing of 0.3m, and a plant spacing of 0.1m. Single seeds were sown, with 20 seeds planted per row. One row of susceptible varieties was planted every 20 rows as a control for stripe rust phenotypic identification, and susceptible varieties were planted around the four rows for stripe rust induction. In mid-January of the following year, the middle of the penultimate leaf of the susceptible control and induced varieties was artificially inoculated with a mixed strain of stripe rust fungi currently prevalent in my country's wheat production using the smear method; the experimental plot had uniform fertility, and except for not spraying fungicides during the entire growing period, other field management measures were carried out according to routine management; the growing period was recorded in the field; when the stripe rust disease was fully developed (the severity of stripe rust on the flag leaf of the susceptible control reached 100%), the stripe rust resistance phenotype of all individual plants of each generation was identified at the adult stage; indoor inspections of yield and agronomic traits included six traits: plant height, number of effective tillers, ear length, number of spikelets, number of grains per ear, and 1000-grain weight.

[0040] Compared with the prior art, the advantages and positive effects of the present invention are as follows: (1) In view of the genetic burden of wheat farmer varieties, the present invention establishes a segmented breeding target trait selection, focusing on plant height and resistance in the lower generation and yield-related traits in the higher generation, thereby greatly improving the field selection efficiency; (2) Since the growth period, plant height, yield and agronomic traits of wheat farmer varieties are controlled by micro-effect polygenes, the present invention establishes a "two-stage" breeding improvement method, adopts hybridization, backcrossing (first stage) and self-pollination (second stage) to gradually aggregate its excellent alleles and eliminate its unfavorable genes, synergistically improve resistance and yield, and transfer the stripe rust resistance gene from the wheat farmer varieties in the adult stage into the background of the bred wheat variety that has lost stripe rust resistance, thereby creating a series of intermediate materials that can be used for wheat stripe rust resistance breeding and have good comprehensive traits. The above scheme not only provides a technical strategy for effectively breeding target genes for stripe rust resistance in wheat farmer varieties, but also provides a model for quickly and effectively eliminating genetic linkage drags in wheat farmer varieties and creating new germplasm for disease-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 、 Figure 2 Shows the results of molecular detection of individual plants of different segregating generations using SNP-specific tracking markers of stripe rust resistance genes in the adult stage;

[0042] Figure 3 Shown are wheat farm varieties and their improved individual plants with stripe rust resistance genes at the adult stage (from left to right: Benqizi wheat and improved lines H1, H2, and H3). DETAILED DESCRIPTION

[0043] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in literature, technical specifications, and standards in the field, or according to product specifications. The materials and reagents involved in the following examples, unless otherwise specified, were all commercially available.

[0044] The farm variety wheat carrying the wheat stripe rust resistance genes QYr.BYZ-5BS and Yr18 in the following examples is derived from the germplasm bank of the Wheat Research Institute of Sichuan Agricultural University; the recipient variety Chuannong 16 used for Yr gene transfer is a commercial variety bred by Sichuan Agricultural University in 2003 and approved by the state, with the approval number: Guoshenmai 2003023, and is currently collected and stored in the germplasm bank of the Wheat Research Institute of Sichuan Agricultural University.

[0045] Example 1: Using the "two-stage" breeding method to improve wheat stripe rust resistance and yield-related traits in farmer varieties

[0046] The early-maturing, short-stem, large-grain, and highly susceptible to stripe rust wheat variety Sichuan Agricultural University 16 was used as the female parent, and the late-maturing, tall-stem, low 1000-grain weight, Chinese wheat farmer variety Benqiezimai, which carries the stripe rust resistance genes QYr.BYZ-5BS and Yr18 and has stripe rust resistance in the adult stage, was used as the male parent. F1 hybrid seeds were obtained by hybridization; the F1 was planted in the wheat production season of the following year and backcrossed with the recipient material Sichuan Agricultural University 16 as the recurrent parent to obtain BC1F1 seeds.

[0047] In the third wheat production season, BC1F1 segregating generation populations were planted in the disease identification nursery, and a standard for individual plant selection with progressively improved plant height (plant height ≤ 100 cm) was set. Phenotypic identification of mixed physiological races of stripe rust was carried out in the field by artificial inoculation, and specific marker tracking molecular detection was performed through single plant DNA extraction and resistance target gene KASP gene analysis technology. Individual plants carrying the adult stripe rust resistance genes QYr.BYZ-5BS and Yr18, with a plant height ≤ 100 cm and showing high resistance to stripe rust in the adult stage were retained. They were backcrossed with the recipient recurrent parent Sichuan Agricultural University 16 during the flowering period, and the hybrid seeds were mixed and harvested after maturity to obtain BC2F1 seeds.

[0048] In the fourth wheat production season, BC2F1 segregating generation populations were planted in the disease identification nursery, and a standard for individual plant selection with progressively improved plant height (plant height ≤ 95 cm) was set. Phenotypic identification of mixed physiological races of stripe rust was continued by artificial inoculation in the field, and specific marker tracking molecular detection was performed through individual plant DNA extraction and resistance target gene KASP gene analysis technology. Individual plants carrying the adult stripe rust resistance genes QYr.BYZ-5BS and Yr18, with plant height ≤ 95 cm and showing high resistance to stripe rust in the adult stage were retained. They were backcrossed with the recipient recurrent parent Sichuan Agricultural University 16 during the flowering period, and the hybrid seeds were mixed and harvested after maturity to obtain BC3F1 seeds.

[0049] In the fifth production season, BC3F1 segregating generation populations were planted in the disease identification nursery, and the selection criteria for individual plants with progressive plant height improvement were set, including growth period (growth period ≤ 190 days), plant height (plant height ≤ 90 cm), and yield-related trait selection criteria (number of effective tillers ≥ 5, spike length ≥ 9 cm, number of spikelets ≥ 11, number of grains per spike ≥ 55, and 1000-grain weight ≥ 45 g). Phenotypic identification of mixed physiological races of stripe rust was continued in the field, and specific marker tracking molecular detection was performed through single plant DNA extraction and resistance target gene KASP gene analysis technology. Individual plants carrying the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18, with plant height ≤ 90 cm, field phenotypes meeting the yield-related trait selection criteria, and showing high resistance to stripe rust in the adult stage were retained. After maturity, self-pollinated seeds were mixed and harvested to obtain BC3F2 seeds.

[0050] In the sixth production season, BC3F2 segregating generation populations were planted in the disease identification nursery. The selection criteria for individual plant height were set, including growth period (growth period ≤ 190 days), plant height (plant height ≤ 90 cm), and yield-related traits (number of effective tillers ≥ 5, spike length ≥ 9 cm, number of spikelets ≥ 11, number of grains per spike ≥ 55, and 1000-grain weight ≥ 45 g). Phenotypic identification of mixed physiological races of stripe rust was continued by artificial field inoculation, and specific marker tracking molecular detection was performed through single plant DNA extraction and resistance target gene KASP gene analysis technology. Individual plants carrying the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18 homozygous, with plant height ≤ 90 cm, field phenotypes meeting the yield-related traits selection criteria, and showing high resistance to stripe rust in the adult stage were retained. After maturity, self-pollinated seeds were harvested from individual plants to obtain BC3F3 seeds, and each individual plant seed became a stable strain.

[0051] During the seventh production season, individual BC3F3 seeds obtained were sown in plots in the disease identification plots according to the following field design: 6-row plots. Susceptible material was sown in the first row as stripe rust induction material, and individual BC3F3 seeds were sown in rows 2-6. Row length was 2 m, row spacing was 0.3 m, and plant spacing was 0.1 m. Single seeds were sown at random, with 20 seeds planted per row. A control plot, using the recurrent parent, Sichuan Agricultural University 16, was set up every 10 plots. The field design and sowing method were the same as above. Susceptible varieties were planted around the identification plots for stripe rust induction. Molecular identification of the stripe rust resistance genes QYr.BYZ-5BS and Yr18 was performed on each strain in the adult stage; the growth period was recorded during the growth period, including the seedling stage, tillering stage, jointing stage, booting stage, flowering stage, filling stage, and maturity stage; after maturity, 10 individual plants were selected for indoor investigation and analysis of six traits including plant height, number of effective tillers, spike length, number of spikelets, number of grains per spike, and 1000-grain weight.

[0052] The 10 μL fluorescence quantitative PCR system for tracking the QYr.BYZ-5BS and Yr18-specific KASP markers for adult-stage stripe rust resistance genes consisted of 2 μL DNA (50 ng / μL), 2 μL primer mix (12% Primer F, 12% Primer H, 30% Primer R, 46% ddH2O), 4.5 μL HiGeno2×Probe Mix B, and 1.5 μL ddH2O. The real-time fluorescence quantitative PCR amplification protocol was as follows: 95°C pre-denaturation for 15 min; 10 cycles of 95°C denaturation for 20 s, 61°C annealing for 40 s, with a 0.6°C decrease between cycles; and 38 cycles of 95°C denaturation for 20 s, 55°C annealing for 40 s. Data were read using a BIO-RAD CFX96, and genotyping analysis was performed using BIO-RAD CFX96 Manager v3.1.

[0053] Phenotypic evaluation of wheat stripe rust resistance at the adult stage under an artificial inoculation-induced environment was performed as follows: A stripe rust identification plot was established at the Chongzhou base of Sichuan Agricultural University. Plants were sown in mid-to-late October, with rows 2 m long, 0.3 m apart, and 0.1 m apart. Single seeds were sown, with 20 seeds per row. Susceptible varieties were planted around the perimeter for stripe rust induction. In mid-January of the following year, the penultimate leaves of susceptible control and induced varieties were inoculated with a mixture of stripe rust strains currently prevalent in Chinese wheat production. The experimental plots were uniformly fertile, and conventional field management practices were followed throughout the growing season, with the exception of no fungicide spraying. When the severity of flag leaf stripe rust of the susceptible control material reaches 100%, the investigation and disease grade classification are carried out in accordance with the agricultural industry standard of the People's Republic of China "Technical Specifications for Evaluation of Wheat Disease and Insect Resistance Part 1: Technical Specifications for Evaluation of Wheat Stripe Rust (NY / T 1443.1-2007)": Grade 0: No obvious signs or symptoms, the resistance evaluation is immune; Grade 0: Necrosis or chlorotic spots, no spore formation, the resistance evaluation is near immunity; Grade 1: Necrosis or chlorotic spots or stripes, mild spore formation, the resistance evaluation is high resistance; Grade 2: Necrosis or chlorotic spots or stripes, moderate spore formation, the resistance evaluation is moderate resistance; Grade 3: Necrosis or chlorotic spots or stripes, a large number of spores are formed, the resistance evaluation is moderately susceptible; Grade 4: No necrosis or yellowing, abundant spores, the resistance evaluation is highly susceptible.

[0054] The specific investigation of growth period and yield-related traits is as follows: relevant traits are investigated with reference to the "Wheat Germplasm Resource Description Specifications and Data Standards". During the wheat growth period, the investigation is conducted, and when the material matures, the growth period is investigated, including the emergence stage, tillering stage, jointing stage, booting stage, flowering stage, grain filling stage, and maturity stage; after maturity, the target candidate individual plants or representative individual plants of the plant line (select 5-10 representative plants in each plot) are investigated for yield-related traits, including six traits: plant height, number of effective tillers, ear length, number of spikelets, number of grains per ear, and 1000-grain weight.

[0055] result:

[0056] 1. Detection of stripe rust resistance and Yr target gene in wheat farmers' varieties of Benqiezi wheat and the improved recipient material Sichuan Agricultural University 16 at the adult stage, as well as phenotypic characteristics of yield-related traits

[0057] During the 2016-2017 wheat production season, phenotypic identification of stripe rust resistance in adult wheat varieties of Benhezimai and Chuannong 16 was conducted. The results showed that Benhezimai was highly susceptible to stripe rust in adult wheat, with Chuannong 16 being the recipient material. Detection of QYr.BYZ-5BS and Yr18 KASP-specific molecular tracking markers revealed that Benhezimai carried both QYr.BYZ-5BS and Yr18, while Chuannong 16 did not carry these two Yr genes (Table 1). Furthermore, growth-stage and yield-related traits of Benhezimai and Chuannong 16 were investigated (Table 2).

[0058] Table 1 Information on the phenotypic and genotypic stripe rust resistance of Benqiezi wheat and Sichuan Agricultural University 16

[0059] adult plant resistance (IT) resistance evaluation QYr.BYZ-5BS Yr18 Lagurus ovatus 0； near-immune + + Chuan Nong 16 4 highly susceptible - -

[0060] Note: “+” indicates that the gene locus is carried; “-” indicates that the gene locus is not carried

[0061] 2. Improvement of stripe rust resistance by backcrossing using specific marker-assisted selection of stripe rust resistance genes in adult plants

[0062] Using Sichuan Agricultural University 16 as the recipient parent and wheat variety Benqiezimai carrying the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18 as the donor material, the following steps were performed:

[0063] (1) 2016-2017 wheat production season: Wheat was sown in mid-to-late October 2016. In early April 2017, a hybrid was conducted in the stripe rust identification nursery of the Chongzhou base of Sichuan Agricultural University using Sichuan Agricultural 16 as the female parent and a farmer's variety of Benqiezi wheat carrying the adult stripe rust resistance genes QYr.BYZ-5BS and Yr18 as the male parent. A total of 35 Sichuan Agricultural 16 × Benqiezi wheat F1 seeds were obtained.

[0064] (2) 2017-2018 wheat production season: In mid-to-late October 2017, single seeds of F1 and the recurrent parent Sichuan Agricultural University 16 were sown in the stripe rust identification nursery of the Chongzhou base of Sichuan Agricultural University. Backcrossing was carried out with the hybrid F1 as the female parent and Sichuan Agricultural University 16 as the male parent during the flowering period. After maturity, the hybrid ears were mixed and harvested, and a total of 218 BC1F1 seeds were obtained.

[0065] (3) 2018-2019 wheat production season: In mid-to-late October 2018, the BC1F1 segregating generation and the recurrent parent Sichuan Agricultural University 16 were sown as single seeds in the stripe rust identification nursery of the Chongzhou base of Sichuan Agricultural University, and a total of 215 BC1F1 plants were obtained; in December 2018, molecular detection was performed using QYr.BYZ-5BS and Yr18 specific tracking markers; in early January 2019, artificial inoculation of stripe rust was carried out in the field; and stripe rust identification was carried out at the adult stage from late March to early April. Based on a progressively improved plant height selection standard (plant height ≤ 100 cm) and specific marker tracking molecular testing, individual plants carrying both the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18 and exhibiting high stripe rust resistance in adult stages were retained. Based on target gene detection and field identification of adult-stage stripe rust resistance phenotypes, a total of three target plants with plant height ≤ 100 cm, carrying both QYr.BYZ-5BS and Yr18, and exhibiting a stripe rust resistance phenotype were identified. Backcrosses were conducted at flowering using these three target BC1F1 plants as female parents and Sichuan Agricultural University 16 as the male parent. Hybrid ears were mixed and harvested at maturity, yielding a total of 422 BC2F1 seeds.

[0066] (4) 2019-2020 wheat production season: In mid-to-late October 2019, the BC2F1 segregating generation and the recurrent parent Sichuan Agricultural University 16 were sown as a single seed in the stripe rust identification nursery of the Chongzhou base of Sichuan Agricultural University, and a total of 417 BC2F1 plants were obtained; in December 2019, molecular detection was performed using QYr.BYZ-5BS and Yr18 specific tracking markers; in early January 2020, artificial inoculation of stripe rust was carried out in the field; and stripe rust identification was carried out at the adult plant stage from late March to early April. Based on a progressively improved plant height selection standard (plant height ≤ 100 cm) and specific marker tracking molecular testing, individual plants carrying both the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18 and exhibiting high stripe rust resistance were retained. Through target gene detection and field identification of adult-stage stripe rust resistance phenotypes, a total of four target plants with plant height ≤ 95 cm, carrying both QYr.BYZ-5BS and Yr18, and exhibiting a stripe rust resistance phenotype were identified. Backcrosses were conducted at flowering using these four target BC2F1 plants as female parents and Sichuan Agricultural University 16 as the male parent. Hybrid ears were harvested at maturity, yielding a total of 511 BC3F1 seeds.

[0067] (5) 2020-2021 wheat production season: In mid-to-late October 2020, the BC3F1 segregation generation was sown as a single seed in the stripe rust identification nursery of the Chongzhou base of Sichuan Agricultural University, and a total of 510 BC3F1 plants were obtained; in December 2020, molecular detection was performed using QYr.BYZ-5BS and Yr18 specific tracking markers; in early January 2021, artificial inoculation of stripe rust was carried out in the field; and stripe rust identification was carried out at the adult plant stage from late March to early April. Based on the selection criteria for progressive plant height improvement (growing period ≤ 190 days), plant height ≤ 90 cm, and yield-related traits (number of effective tillers ≥ 5, spike length ≥ 9 cm, number of spikelets ≥ 11, number of grains per spike ≥ 55, and 1000-grain weight ≥ 45 g), target gene detection, and field stripe rust resistance phenotypic identification at the mature plant stage, six target plants were identified that simultaneously carried QYr.BYZ-5BS and Yr18, had plant height ≤ 90 cm, carried both QYr.BYZ-5BS and Yr18, and exhibited stripe rust resistance and yield-related traits that met the selection criteria. Each ear of these six plants was bagged and self-pollinated at heading, and each plant was harvested at maturity to produce six BC3F2 seed pools.

[0068] (6) 2021-2022 wheat production season: Six BC3F2 segregating generations were sown in the stripe rust identification field at the Chongzhou base of Sichuan Agricultural University in mid-to-late October 2021, yielding 152 to 207 plants. Molecular detection was performed in December 2021 using QYr.BYZ-5BS and Yr18-specific tracking markers. Artificial inoculation of stripe rust was performed in the field in early January 2022. Stripe rust identification was performed at the adult stage in late March to early April. Based on the selection criteria for progressive plant height improvement, including growth period (growth period ≤ 190 days), plant height (plant height ≤ 90 cm), and yield-related traits (effective tiller number ≥ 5, spike length ≥ 9 cm, spikelet number ≥ 11, grain number per spike ≥ 55, and 1000-grain weight ≥ 45 g), target gene detection, and stripe rust resistance phenotype identification at the adult plant stage in the field, a total of 7 strains carrying both QYr.BYZ-5BS and Yr18 (plant height ≤ 90 cm, and strains carrying both QYr.BYZ-5BS and Yr18 ( Figure 1 、 Figure 2 ), and target individual plants with stripe rust resistance and yield-related traits that met the selection criteria. Each ear of the seven individual plants was bagged and self-pollinated at heading. Upon maturity, the individual plants were harvested to form lines. A total of seven BC3F3 line seeds were obtained, with each line yielding 211 to 236 seeds.

[0069] (6) 2022-2023 wheat production season: 200 seeds of 7 BC3F3 strains were taken and divided into two equal parts. They were planted in Chongzhou and Wenjiang experimental bases of Sichuan Agricultural University in mid-to-late October 2022. The plot was designed as a 5-row area with a row length of 2m, a row spacing of 0.3m, and a plant spacing of 0.1m. Artificial inoculation of stripe rust fungi was carried out in the field in early January 2022; stripe rust disease identification was carried out in the adult stage from late March to early April. During the growth period, 10 individual plants of each strain were selected after maturity for investigation and analysis of plant height, number of effective tillers, spike length, number of spikelets, number of grains per spike, and 1,000-grain weight. Four excellent strains were selected based on the target genotype, stripe rust resistance phenotype in the adult stage, and agronomic traits. The excellent strains were the disease-resistant strains obtained by screening and cultivation (Table 2, Figure 3 ).

[0070] Table 2 Identification of synergistically improved strains of Sichuan Agricultural University 16, Benqiezi wheat, and their stripe rust resistance and yield-related traits

[0071]

[0072]

[0073] Note: “+” indicates that the locus is carried; “-” indicates that the locus is not carried; plant height, number of effective tillers, spike length, number of spikelets, number of grains per spike, and 1000-grain weight refer to the weighted average of single plant tested at the Wenjiang and Chongzhou test sites.

[0074] Example 2: Using a single-cross pedigree breeding method different from the "two-stage" breeding improvement method of this application to improve wheat stripe rust resistance and yield-related traits in farmer varieties

[0075] The early-maturing, short-stem, large-grain, and highly susceptible stripe rust wheat variety Sichuan Agricultural University 16 was used as the female parent, and the late-maturing, tall-stem, low 1000-grain weight, Chinese wheat farmer variety Benqiezimai, which carries the stripe rust resistance genes QYr.BYZ-5BS and Yr18 and has stripe rust resistance in the adult stage, was used as the male parent. F1 hybrid seeds were obtained by hybridization; the F1 was planted in the wheat production season of the following year, and all individual plants were harvested after maturity to obtain F2 seeds.

[0076] In the third wheat production season, F2 segregating generation populations were planted in the disease identification nursery. According to the breeding goals of the wheat area, improved individual plant selection criteria were set for plant height (plant height ≤ 90 cm), growing period (growing period ≤ 190 days) and yield-related traits (number of effective tillers ≥ 5, spike length ≥ 9 cm, number of spikelets ≥ 11, number of grains per spike ≥ 55, and 1000-grain weight ≥ 45 g). Phenotypic identification of mixed physiological races of stripe rust was carried out by artificial field inoculation, and specific marker tracking molecular detection was performed through single plant DNA extraction and resistance target gene KASP gene analysis technology. Individual plants carrying the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18, showing high resistance to stripe rust in the adult stage, and whose growing period and yield-related traits met the selection criteria were retained. After maturity, self-pollinated seeds were harvested from each individual plant to obtain F2 seeds.

[0077] The detection of the stripe rust resistance gene QYr.BYZ-5BS and Yr18-specific KASP tracking molecular markers in the adult stage was carried out according to Example 1.

[0078] The method for evaluating the stripe rust resistance phenotype of wheat at the adult stage under the artificial inoculation induction environment is carried out according to Example 1.

[0079] The growth period and yield-related traits were investigated according to Example 1.

[0080] result:

[0081] 1. The stripe rust resistance and Yr target gene detection and yield-related phenotypic characteristics of the wheat farmer variety Benqiezimai and the improved recipient material Sichuan Agricultural University 16 at the adult stage are detailed in Example 1.

[0082] 2. Marker-assisted selection of F2 isolates of stripe rust resistance genes at the adult stage

[0083] Using Chuannong 16 as the recipient parent and the wheat variety Benqiezimai carrying the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18 as the donor material, the following steps were performed:

[0084] (1) 2016-2017 wheat production season: Wheat was sown in mid-to-late October 2016. In early April 2017, a hybrid was carried out in the stripe rust identification nursery of the Chongzhou base of Sichuan Agricultural University using Sichuan Agricultural 16 as the female parent and a farmer's variety of Benqiezi wheat carrying the adult stripe rust resistance genes QYr.BYZ-5BS and Yr18 as the male parent. A total of 47 Sichuan Agricultural 16 × Benqiezi wheat F1 seeds were obtained.

[0085] (2) 2017-2018 wheat production season: In mid-to-late October 2017, F1 seeds were sown individually in the stripe rust identification field of the Chongzhou base of Sichuan Agricultural University, and the seeds were self-pollinated in bags. After maturity, the hybrid ears were mixed and harvested, and a total of 329 F2 seeds were obtained.

[0086] (3) 2018-2019 wheat production season: In mid-to-late October 2018, the F2 segregation generation was sown in the stripe rust identification nursery of the Chongzhou base of Sichuan Agricultural University, and a total of 322 F2 plants were obtained; in December 2018, molecular detection was performed using QYr.BYZ-5BS and Yr18 specific tracking markers; in early January 2019, artificial inoculation of stripe rust was carried out in the field; and stripe rust identification was carried out at the adult plant stage from late March to early April. Based on a progressive plant height improvement selection standard (plant height ≤ 100 cm) and specific marker tracking molecular testing, individual plants carrying both the adult-stage stripe rust resistance genes QYr.BYZ-5BS and Yr18 and exhibiting high stripe rust resistance in adult stages were retained. F2 segregating plants were selected based on target gene detection, field identification of adult-stage stripe rust resistance phenotypes, and improved individual plant selection criteria (growing period ≤ 190 days, plant height ≤ 90 cm, number of effective tillers ≥ 5, spike length ≥ 9 cm, number of spikelets ≥ 11, number of kernels per spike ≥ 55, and 1000-kernel weight ≥ 45 g). A total of 12 individual plants carrying both QYr.BYZ-5BS and Yr18 and exhibiting stripe rust resistance were identified. However, all of these plants were > 100 cm tall, which did not meet the breeding requirements of the local wheat region, so further breeding and improvement experiments were discontinued.

[0087] The method of the present application can create a series of intermediate materials that can be used for the synergistic improvement of stripe rust resistance and yield-related traits in wheat farmer varieties. The present application not only provides a technical strategy for the effective transfer of target genes for stripe rust resistance in wheat farmer varieties, but also provides an example for quickly and effectively eliminating genetic linkage drag in wheat farmer varieties and creating useful new germplasms for synergistic improvement of disease resistance and yield in breeding.

[0088] The application is further described in detail in conjunction with the specific embodiments, and the listed examples are only for illustrating the application and do not constitute limitation of the application.

Claims

1. A breeding method for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farm varieties, characterized in that: The method comprises the following steps: The early-maturing, short-stem, large-grain, and highly susceptible wheat variety Sichuan Agricultural University 16 was used as the female parent, and the late-maturing, tall-stem, low-1000-grain-weight wheat variety carrying the stripe rust resistance gene was used as the female parent. QYr.BYZ-5BS and Yr18 The Chinese wheat farmer variety Benqiezimai, which is resistant to stripe rust in the adult stage, was used as the male parent to obtain F1 hybrid seeds; In the following wheat production season, F1 was planted and backcrossed with the recipient material Chuannong 16 as the recurrent parent to obtain BC1F1 seeds; In the third wheat production season, the BC1F1 segregating generation population was planted, and the selection criteria for individual plants with progressive plant height improvement were set to plant height ≤ 100 cm. Field stripe rust resistance identification was performed, and DNA from individual plants was extracted for specific molecular testing. The plants carrying the stripe rust resistance gene in the adult stage were retained. QYr.BYZ-5BS and Yr18 Individual plants with a plant height of ≤100 cm and high resistance to stripe rust in the mature stage were backcrossed with the recipient recurrent parent, Sichuan Agricultural University 16, at the flowering stage. After maturity, the hybrid seeds were mixed and harvested to obtain BC2F1 seeds. In the fourth wheat production season, the BC2F1 segregating generation population was planted, and the selection standard for individual plants with progressive plant height improvement was set to plant height ≤ 95 cm. Field stripe rust resistance identification was performed, and DNA from individual plants was extracted for specific molecular testing. The plants carrying the stripe rust resistance gene in the adult stage were retained. QYr.BYZ-5BS and Yr18 , and the plant height is ≤95cm, and the individual plants showing high resistance to stripe rust in the mature stage are backcrossed with the recipient recurrent parent Sichuan Agricultural University 16 at the flowering stage, and the hybrid seeds are mixed and harvested after maturity to obtain BC3F1 seeds; In the fifth production season, the BC3F1 segregating generation population was planted, and the selection criteria for individual plants with progressive plant height improvement were set to plant height ≤ 90 cm, and the selection criteria for yield-related traits were set to growth period ≤ 190 days, number of effective tillers ≥ 5, spike length ≥ 9 cm, number of spikelets ≥ 11, number of grains per spike ≥ 55, and 1000-grain weight ≥ 45 g. Field stripe rust resistance was identified, and DNA from individual plants was extracted for specific molecular testing. Plants carrying the stripe rust resistance gene in the adult stage were retained. QYr.BYZ-5BS and Yr18 , and the plant height is ≤90cm, the field phenotype meets the selection criteria for yield-related traits and the individual plants show high resistance to stripe rust in the adult stage, after maturity, the self-pollinated seeds are mixed and harvested to obtain BC3F2 seeds; In the sixth production season, the BC3F2 segregating generation population was planted, and the selection criteria for individual plant height was set to 90 cm or less, and the selection criteria for yield-related traits were growth period 190 days or less, number of effective tillers 5 or more, spike length 9 cm or more, number of spikelets 11 or more, number of grains per spike 55 or more, and 1000-grain weight 45 g or more. Field stripe rust resistance was identified, and DNA from individual plants was extracted for specific molecular testing. Plants carrying the stripe rust resistance gene in the adult stage were retained. QYr.BYZ-5BS and Yr18 Individual plants that are homozygous, ≤90 cm tall, meet the selection criteria for yield-related traits in the field, and show high resistance to stripe rust in the adult stage will be harvested from self-pollinated seeds to obtain BC3F3 seeds after maturity. Each individual plant seed will become a stable strain. In the seventh production season, the BC3F3 single-plant seeds were sown in plots according to the field design, and the stripe rust resistance was identified in the field. The stripe rust resistance gene of each strain was detected in the adult stage. QYr.BYZ-5BS and Yr18 Molecular identification, investigation of the growth period, which includes the emergence period, tillering period, jointing period, booting period, flowering period, grain filling period, and maturity period. After maturity, individual plants were selected for investigation and analysis of plant height, number of effective tillers, ear length, number of spikelets, number of grains per ear, and 1000-grain weight, to obtain high resistance to stripe rust in the adult stage and the presence of stripe rust resistance genes in the adult stage. QYr.BYZ-5BS and Yr18 New wheat germplasm with excellent traits related to growth period and yield.

2. The breeding method for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farm varieties according to claim 1, characterized in that: Utilization of adult-stage stripe rust resistance genes QYr.BYZ-5BS Tightly linked KASP markers KP5B_78.49, KP5B_83.68 and Yr18 Feature Flags Lr34-KASP-E11 Real-time fluorescence quantitative PCR molecular detection was performed to analyze the transformed plants of each generation, among which, The KASP marker KP5B_78.49 The KASP-specific labeled primers include: Forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTGGTTGCAGGAGGGTGTAAAAG-3', Forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTGGTTGCAGGAGGGTGTAAAAC-3', Reverse universal primer: 5′-TTCTTGTACGACGGCCTTTGA-3′; The KASP marker KP5B_83.68 The KASP-specific labeled primers include: Forward primer 1: 5'- GAAGGTGACCAAGTTCATGCTTCCAAATCATCGTTCCTTTGATCC-3', Forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTTCCAAATCATCGTTCCTTTGATCA-3', Reverse universal primer: 5′-GTCGCCTCAGTGTCTCGAAA-3′; described Yr18 KASP-specific marker primers for functional labeling of Lr34-KASP-E11 include: Forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTAATGTATCGTGAGAGATTTGCAG-3', Forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTAATGTATCGTGAGAGATTTGCAT-3', Reverse universal primer: 5'-AGGTGAATAAATATGAGCATCAGT-3'.

3. The breeding method for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farm varieties according to claim 1, characterized in that: The following steps are used to identify the resistance to stripe rust in adult plants in the field: When wheat has three leaves and one heart, mix the stripe rust fungus with talcum powder in a ratio of 1:100 and apply it to the second leaf from the induced leaf and the susceptible control material. When the severity of stripe rust on the flag leaf of the susceptible control material reaches 100%, the wheat stripe rust resistance is identified.

4. The breeding method for creating new germplasm by synergistically improving stripe rust resistance and yield using wheat farm varieties according to claim 1, wherein the yield-related traits include growth period, number of effective tillers, spike length, number of spikelets, number of grains per spike, and 1000-grain weight.

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