Efficient breeding method for synergistically improving fruit rot resistance and high oleic acid character of peanuts

Abstract

The invention discloses an efficient breeding method for synergistically improving fruit rot resistance and high oleic acid characters of peanuts, and belongs to the field of plant breeding. The peanut breeding method comprises the following steps: hybridizing a peanut inbred line with high fruit rot resistance with another peanut inbred line with oleic acid content of more than or equal to 75% and excellent comprehensive characters, and performing field natural disease nursery identification and indoor artificial inoculation fruit rot pathogenic bacterium identification on generations in a crossing manner, single plants which are free of rotten fruits and high in oleic acid are selected for continuous selfing in each generation, and the fruit rot-resistant and high-oleic-acid peanut stable strain is bred. According to the method, an efficient breeding way is provided for finally obtaining peanut varieties with high peanut rot resistance and high oleic acid content, compared with a traditional screening method for counting disease levels, the efficiency is greatly improved, a large amount of manpower and material resources are saved, high-resistance materials can be accurately screened out, and the method has great application value in peanut breeding.

Description

Technical Field

[0001] This application belongs to the field of plant breeding, specifically relating to an efficient breeding method for synergistically improving peanut resistance to rot and high oleic acid traits. Background Technology

[0002] Peanut pod rot (also known as peanut fruit rot) is a plant disease caused by soil-borne pathogens such as Fusarium. It is prevalent in fields where peanuts have been continuously cropped for many years, and is exacerbated in rainy years during the pod-setting period. This disease primarily affects peanut pods, causing browning and poor kernel development in mild cases, and rotting and necrosis of the entire pod in severe cases. In recent years, it has caused serious damage in many major peanut-producing areas, often resulting in yield reductions of over 15%, or even total crop failure. Peanut pod rot is a pressing problem in current peanut production that needs to be solved. Although some progress has been made in research, such as identifying key pathogens like Fusarium neospora and establishing a preliminary resistance evaluation system, there is still a significant lack of high-quality, highly resistant or immune peanut varieties in production.

[0003] Oleic acid is a representative of monounsaturated fatty acids. Studies have found that it may help regulate blood lipid balance by lowering low-density lipoprotein cholesterol (LDL) and raising high-density lipoprotein cholesterol (HDL), thereby reducing the risk of arteriosclerosis. The main advantage of high-oleic peanuts lies in their high oleic acid content (over 70%). This type of fatty acid belongs to monounsaturated fatty acids and has characteristics such as high stability, high temperature resistance, and antioxidant properties. Long-term moderate consumption may have certain benefits for cardiovascular health and metabolic regulation.

[0004] Synergistic improvement of peanut resistance to pod rot and oleic acid content, and breeding of high-oleic-acid peanut varieties resistant to pod rot, play an important role in promoting increased yield and improved quality in the peanut industry. Summary of the Invention

[0005] To address the above problems, this invention innovatively provides a highly efficient breeding method for synergistically improving peanut resistance to rot and high oleic acid traits. The method includes the following steps: crossing a peanut inbred line with high resistance to rot (HR) with another peanut inbred line with oleic acid content ≥75% and excellent overall traits; then cross-pollinating each generation to identify the disease in a field nursery where the disease naturally occurs and to identify the pathogen of rot through artificial inoculation in the laboratory; and selecting single plants without rot and with high oleic acid in each generation for continuous self-pollination to breed stable peanut lines that are resistant to rot and have high oleic acid.

[0006] In the above method, the pathogen used for artificial inoculation of fruit rot disease can be Fusarium solani (Fusarium solani). Fusarium nightshade ) and / or Fusarium oxysporum ( Fusarium oxysporum ).

[0007] In one embodiment of the present invention, the pathogen is Fusarium solani (…). Fusarium solani ) strain HSGF09.

[0008] In the above method, the identification of diseased plants in the field and the identification of pathogens of fruit rot by artificial inoculation in the room are carried out in the cross-generational process. Specifically, the first generation of hybrids (F1) are identified in the field by natural disease, the second generation of hybrids (F2) are identified in the room by artificial inoculation of pathogens of fruit rot, the third generation of hybrids (F3) are identified in the field by natural disease, the fourth generation of hybrids (F4) are identified in the room by artificial inoculation of pathogens of fruit rot, and the fifth generation of hybrids (F5) are identified in the field by natural disease.

[0009] In the above method, the selection criteria for high oleic acid are as follows: in the first generation of hybrids (F1), single plants with seed oleic acid content ≤40% are eliminated, and in each subsequent generation, single plants with seed oleic acid content ≥75% are selected.

[0010] The efficient breeding method of the present invention for synergistically improving peanut resistance to rot and high oleic acid traits also includes the steps of yield identification of stable peanut lines that are resistant to rot and high in oleic acid, field natural disease nursery identification of selected lines with a yield of ≥300 kg / mu, and selection of highly resistant lines as candidate peanut varieties.

[0011] In the above method, the identification of natural disease nurseries in the field refers to the identification of natural disease nurseries of peanut rot in peanut fields where peanuts are continuously cropped year after year and where the disease is severe.

[0012] In the above method, the identification of the pathogen of fruit rot by indoor artificial inoculation is carried out by inoculating the pathogen strain of fruit rot into the pod layer of potted plants after culturing oat grains.

[0013] In the above methods, the calculation method for the disease index in both the naturally occurring disease nursery and the artificial inoculation identification in the field is as follows: During the peanut maturity period, the disease incidence of plants planted in the naturally occurring disease nursery in the field is investigated and classified into the following disease levels:

[0014] Grade 0: Pods without disease spots; Grade 1: Lesions covering less than 10% of the pods; Grade 3: Lesions covering 10%-25% of the pods; Grade 5: Lesions cover 25%-50% of the pods; Grade 7: Pod lesions cover 50%-75% of the surface area; Grade 9: Pod lesion area >75%; Then calculate the diseased finger according to Formula 1: Disease Index (DI) = ∑(Number of pods at each grade × Representative value of each grade) / (Total number of pods × Representative value of the highest grade) × 100 Formula 1.

[0015] In the above method, the high resistance standard for identification of naturally occurring disease nurseries in the field is: High resistance (HR): 0 ≤ disease index ≤ 5.0.

[0016] In the above method, the peanut inbred line with high resistance to rot disease is selected based on the results of identification in the naturally occurring disease nursery in the field and the results of identification based on the pathogen of rot disease after artificial inoculation. In one embodiment of the present invention, the peanut inbred line with high resistance to rot disease (HR) is Yuhanghua 1.

[0017] In one embodiment of the present invention, the peanut inbred line with an oleic acid content ≥75% and excellent overall traits is Jihua 11.

[0018] The beneficial effects of this invention are: This invention rigorously selects peanut lines that exhibit high resistance to rot disease pathogens identified through both indoor artificial inoculation and field natural disease nursery testing as the source of resistance genes. It then improves the resistance traits of peanuts with high oleic acid content and excellent overall characteristics. Cross-pollination testing is conducted across generations, using both field natural disease nursery testing and indoor artificial inoculation for rot disease pathogen identification. Each generation selects single plants without rot and with high oleic acid content for continuous self-pollination. Through multiple generations, the rot resistance gene and high oleic acid gene are purified, enhancing the stability and consistency of rot resistance traits and oleic acid content. This invention provides an efficient breeding pathway for obtaining peanut varieties highly resistant to rot disease and with high oleic acid content. It significantly improves efficiency compared to traditional statistical disease-level screening methods, saving considerable manpower and resources, and accurately screening for highly resistant materials. It has significant application value in peanut breeding. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] The pathogen causing peanut rot in the following examples is Fusarium solani (…). Fusarium solani The strain HSGF09 has been published in the literature “Fan Wanwan, Li Shaojian, Sang Suling, Zhang Haiyan, Gao Meng, Wang Zhenyu. Isolation and identification of pathogens of peanut fruit rot in Henan Province. Chinese Journal of Oil Crops. 2024, 46(2): 377-384. doi: 10.19802 / j.issn.1007-9084.2022289”, and the public can obtain it from the applicant.

[0022] The peanut variety Yuhanghua No. 1 in the following examples is an early-maturing processing peanut variety bred by Puyang Academy of Agricultural and Forestry Sciences, with the approval number Jin Shenhua (Ren) 2015002. It has been disclosed in the literature "Chen Cuixia. Breeding of the new peanut variety Yuhanghua No. 1". The public can obtain it from the applicant to repeat the experiment of this application.

[0023] The peanut variety Jihua 11 in the following examples is a variety bred by the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences. Its national registration number in 2018 is GPD Peanut (2018) 130072; its variety right number is CNA20120744.3, and the authorization announcement date is January 1, 2016, with authorization announcement number CNA006809G. The public can obtain the experiments to replicate this application from the applicant.

[0024] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0025] Example 1 1. Construction of a field-based natural disease nursery screening method Different peanut germplasm were randomly divided into blocks and replicated three times. They were planted in a natural disease nursery for peanut rot in Licaopo Village, Er'an Township, Neihuang County (peanuts are continuously cropped year after year, and the disease is severe). The disease was identified for three consecutive years. The disease incidence was investigated at the peanut maturity period. Referring to the method of Li Shuchen, Li Buyang, et al. (Li Shuchen, Jia Haimin, Zhao Juying, et al. Identification and pathogenicity of peanut rot in Hebei Province[J]. Hebei Agricultural Sciences, 2011, 15 (05): 37-39. [2] Li Buyang. Establishment of resistance evaluation standards for peanut rot and screening of resistant germplasm resources[D]. Hebei University of Science and Technology, 2023.), the disease severity of peanut rot was divided into 9 levels, namely: Grade 0: Pods without disease spots; Grade 1: Lesions covering less than 10% of the pods; Grade 3: Lesions covering 10%-25% of the pods; Grade 5: Lesions cover 25%-50% of the pods; Grade 7: Pod lesions cover 50%-75% of the surface area; Grade 9: Diseased area of ​​pods >75%.

[0026] Based on the severity classification of peanut rot disease and in combination with actual production feasibility, pods with lesions covering more than 50% of their surface area are classified as rotten.

[0027] Disease Index (DI) = ∑(Number of pods at each grade × Representative value of each grade) / (Total number of pods × Representative value of the highest grade) × 100 (Formula 1) Rotten fruit rate = (Number of peanuts with diseased area greater than 50% / Total number) × 100% (Formula 2) Calculate the rotten fruit rate (Formula 2) and the disease index (Formula 1). Analyze the correlation between the disease index and the rotten fruit rate, and a significant positive correlation is found. Cluster analysis is performed on the experimental materials based on the disease index. Different peanut germplasms are clustered into six categories: HR, MR, R, S, and HS, according to the following criteria: High resistance (HR): 0 ≤ disease index ≤ 5.0; Disease resistance (R): 5.0 < disease index ≤ 10.0; Metabolic MRI (MR): 10.0 < disease index ≤ 20.0; Sickness (S): 20.0 < Sickness Index ≤ 30.0; High susceptibility (HS): 30.0 < disease index ≤ 100.0.

[0028] Based on the clustering results, the resistance of varieties is distinguished. If the experimental materials show changes in adjacent resistance types over three years, it is considered that the experimental materials have relatively consistent performance across years.

[0029] Yuhanghua 1 showed high resistance (HR), while Jihua 11 showed high sensitivity (HS).

[0030] 2. Construction of a method for identifying pathogens induced by artificial inoculation in the laboratory for peanut rot disease. 2.1 Selection of pathogenic strains for peanut rot disease Peanut pods with different degrees of disease and different symptoms were collected during the peanut ripening period in a natural disease nursery.

[0031] Tissue isolation was used: small tissue pieces, 3-5 mm on each side, were cut from the lesion, immersed in 70% alcohol for 15 seconds, then transferred to 0.1% mercuric chloride aqueous solution for 3-5 minutes, and rinsed three times with sterile water. The tissues were then transferred to culture media in petri dishes for incubation. The cultured pathogens were then subjected to routine morphological identification and molecular sequencing verification.

[0032] Molecular identification and sequencing methods: Mycelia of purified cultured strains were used to extract genomic DNA from each strain using a fungal genomic DNA extraction kit. PCR amplification of the genomic DNA of each strain was performed using primers for the ribosomal DNA intratranscribed spacer region (ITS) and ITS4, and primers for translation elongation factors (EF1 and EF2). The PCR reaction volume was 25 μL, and the reaction procedure followed the methods of White and Rahjoo. After PCR product detection by 1% agarose gel electrophoresis, the samples were sent to the company for sequencing. The obtained sequences were then analyzed for BLAST homology comparison in GenBank.

[0033] Morphological and molecular biological identification of the pathogens using tissue isolation and molecular identification sequencing methods revealed that the pathogens in the natural disease nursery of peanut rot were all Fusarium, with Fusarium solani and Fusarium oxysporum being the dominant strains.

[0034] Fusarium solani, a pathogen of peanut fruit rot in Henan Province, was selected from the literature “Fan Wanwan, Li Shaojian, Sang Suling, Zhang Haiyan, Gao Meng, Wang Zhenyu. Isolation and identification of pathogens of peanut fruit rot in Henan Province. Chinese Journal of Oil Crops. 2024, 46(2): 377-384. doi: 10.19802 / j.issn.1007-9084.2022289”. Fusarium solani The strain HSGF09 was used for subsequent screening for resistance to fruit rot disease.

[0035] 2.2 Methods for identifying the pathogen induced by artificial inoculation in the field of peanut rot disease Peanut germplasm with consistent performance across years in step 1 of this embodiment was selected, including those with different resistances, and identified through artificial inoculation of disease nurseries.

[0036] The specific steps for field artificial inoculation identification are as follows: First, Fusarium solani was cultured using oat grains (… Fusarium solani Apply strain HSGF09 evenly to the surface after rotary tillage, till and irrigate, and sow after the soil has been loosened. Manage the seedlings normally, and maintain sufficient soil moisture during the flowering and pegging stages and the fruit filling stage, keeping the soil moisture >90%.

[0037] The experiment was conducted with three replicates, each containing 10 plants. Disease incidence and disease index were assessed after harvest. The grading standards, disease index calculation formula, and disease resistance evaluation grading standards were the same as those described above for screening in natural disease nurseries in the field.

[0038] The results showed that materials such as Yuhanghua No. 1 belonged to high sensitivity (HR), while materials such as Jihua No. 11 belonged to high sensitivity (HS).

[0039] 3. Comprehensive evaluation Based on the screening criteria and calculation methods of natural disease nurseries in the field, peanut materials with consistent resistance performance across years were identified through artificial inoculation with pathogens. Those with resistance results consistent with those in the natural disease nurseries were considered the final resistance results. Based on these final resistance results, the different resistances of different materials were determined. The comprehensive evaluation criteria are as follows: High resistance (HR'): HR (0 ≤ disease index ≤ 5.0) was screened in natural field disease nurseries, and HR (0 ≤ disease index ≤ 5.0) was induced by artificial inoculation with pathogens; Disease resistance (R'): Screening R' in natural disease nurseries in the field (5.0 < disease index ≤ 10.0), and R'' induced by artificial inoculation with pathogens (5.0 < disease index ≤ 10.0); Mid-resistance (MR'): MR was screened in natural disease nurseries in the field (10.0 < disease index ≤ 20.0), and MR was induced by artificial inoculation with pathogens (10.0 < disease index ≤ 20.0); Disease susceptibility (S'): Screening for S' in natural disease nurseries in the field (20.0 < disease index ≤ 30.0), and S' induced by artificial inoculation with pathogens (20.0 < disease index ≤ 30.0); High susceptibility (HS'): HS was screened in natural field disease nurseries (30.0 < disease index ≤ 100.0) and HS was induced by artificial inoculation with pathogens (30.0 < disease index ≤ 100.0).

[0040] The results showed that the following materials were consistent between the screening in the natural disease nursery in the field and the screening induced by artificial inoculation: Yuhanghua No. 1 and other materials were highly resistant (HR'), while Jihua No. 11 was highly susceptible (HS').

[0041] 3. Hybridization and breeding 3.1 Parental selection and hybridization Germplasm with consistent resistance results from natural disease nurseries and artificial inoculation in the field were selected as alternative parents.

[0042] Peanut quality was determined using a near-infrared spectroscopy instrument, primarily by detecting oleic acid content.

[0043] Among the candidate parents, select a material with high resistance to fruit rot disease (HR') as the first parent, and then select a material with oleic acid content ≥75% and excellent overall traits as the second parent.

[0044] This application uses the peanut germplasm Yuhanghua 1, which is highly resistant to rotten fruit disease (HR') and has ordinary oleic acid (oleic acid content 44%), as the female parent, and the peanut germplasm Jihua 11, which is highly susceptible to rotten fruit disease (HS') and has high oleic acid (oleic acid content ≥75%) and excellent comprehensive traits, as the male parent to construct a hybrid combination, and obtain hybrid F1 generation seeds in the same year.

[0045] 3.2 Self-pollination purification and stabilization In May of the first year, a hybrid combination of "Yuhanghua No. 1 × Jihua No. 11" was prepared. In September, F1 hybrid seeds were harvested. In November, individual seeds from the male and female parents and F1 generation were planted. The leaves of the F1 plants were examined using the KASP method to identify true and false hybrids. Based on the results, false hybrids were removed, and true hybrid plants were retained for self-pollination to obtain F2 generation seeds (seeds harvested from F1 plants, and so on below). The plants were then inoculated indoors with the pathogen of fruit rot, *Fusarium solani* (also known as leaf mold). Fusarium solani Strains HSGF09 were used to screen for single plants without rotten fruit. Selected single plants were harvested for F1. ２ For F2 generation seedlings, near-infrared single-plant seed testing was used to eliminate individual plants with oleic acid content ≤40% for planting.

[0046] In May of the second year, F2 generation plants were planted individually with one seed each in a natural disease nursery for fruit rot in the field. Plants without rotten fruit were selected, and F3 generation seeds were harvested from these selected plants. Seeds from plants with an oleic acid content ≥75% were retained for F3 generation planting. In November, F3 generation plants were planted individually with one seed each and inoculated indoors with the fruit rot pathogen *Fusarium solani* (also known as Fusarium solani). Fusarium solaniStrain strain HSGF09 was used to screen for single plants without rotten fruit, and the selected single plants were used to harvest F4 generation seeds. Near-infrared spectroscopy was used to detect the seeds of single plants, and single plants with an oleic acid content ≥75% were retained for planting F4 generation plants.

[0047] In May of the third year, F4 generation plants were planted individually with one seed each and then sent to a natural disease nursery for fruit rot. Plants without rotten fruit were selected, and F5 generation seeds were harvested from these selected plants. Near-infrared spectroscopy was used to identify individual plants with an oleic acid content ≥75%, and these seeds were used to plant F5 generation plants. In November, F5 generation plants were planted individually with one seed each and inoculated indoors with the fruit rot pathogen *Fusarium solani* (also known as Fusarium solani). Fusarium solani Strains of strain HSGF09 were used to screen for single plants without rotten fruit, and the selected single plants were used to harvest F6 generation seeds.

[0048] F6 generation individual plants are planted into rows, and individual plants with consistent traits within the same row are mixed to form a line. Seeds from the mixed line are tested for quality using a near-infrared spectrometer. Lines with an oleic acid content of 75% or higher are selected as stable peanut lines resistant to rotting disease and high in oleic acid.

[0049] The above process of selecting inbred lines enhances the stability and consistency of fruit rot resistance traits and oleic acid content by purifying the fruit rot resistance gene and the high oleic acid gene.

[0050] Single-plant, single-seed planting refers to planting seeds harvested from a single plant in the previous generation as a plot, and sowing each seed individually within the plot.

[0051] The identification of the pathogen of peanut rot disease by artificial inoculation in the indoor environment was carried out with reference to Yu Jing's "A Method for Inoculating Peanut Fruit Rot Disease (Application Publication No.: CN112243824A)". The pathogen strain of peanut rot disease was cultured in oat grains and then inoculated and buried in the pod layer of potted plants during the peanut pod-setting period for identification.

[0052] 3.3 Variety Identification and Investigation 3.3.1 Production and Quality Assessment Yield assessments were conducted on the aforementioned stable peanut lines resistant to rot and high in oleic acid. Lines with a yield equivalent to ≥300 kg per mu were selected for promotion to varieties.

[0053] 3.3.2 Re-inspection of resistant disease nurseries The selected peanut lines were subjected to field natural disease identification (refer to the steps in section 1 above). Highly resistant (HR) lines were retained as candidate peanut varieties for regional yield evaluation trials.

[0054] 3.3.3 Comprehensive Assessment Selected strains were planted in plots, with regional trial control varieties as controls; randomized block design, three replicates; after maturity and harvest, the strains were dried and weighed; yield comparison tests and quality assessments were conducted; strains with a yield increase of 6% per mu compared to the control, kernel yield ≥70%, and oleic acid content ≥75% were selected.

[0055] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for producing flowering plants, characterized in that: Includes the following steps: A peanut inbred line highly resistant to rot was crossed with another peanut inbred line with an oleic acid content ≥75% and excellent overall traits. After each generation, the disease was identified in the field by natural disease nursery and in the laboratory by artificial inoculation with rot pathogens. Single plants without rot and with high oleic acid were selected from each generation and continuously self-pollinated to breed stable peanut lines resistant to rot and with high oleic acid.

2. The method according to claim 1, characterized in that: The artificial inoculation with the pathogen causing fruit rot was carried out by Fusarium solani (Fusarium solani). Fusarium solani ) and / or Fusarium oxysporum ( Fusarium oxysporum ).

3. The method according to claim 2, characterized in that: The pathogen is *Fusarium solani* (also known as Fusarium solani). Fusarium solani ) strain HSGF09.

4. The method according to any one of claims 1-4, characterized in that: The identification of disease pathogens in natural field disease nurseries and the identification of disease pathogens in artificial inoculation in the laboratory were carried out in the cross-generational process. Specifically, the first generation of hybrids was identified in natural field disease nurseries, the second generation of hybrids was identified in artificial inoculation in the laboratory, the third generation of hybrids was identified in natural field disease nurseries, the fourth generation of hybrids was identified in artificial inoculation in the laboratory, and the fifth generation of hybrids was identified in natural field disease nurseries.

5. The method according to claim 4, characterized in that: The selection criteria for high oleic acid content are as follows: in the first generation of hybrids, single plants with seed oleic acid content ≤40% are eliminated, and in each subsequent generation, single plants with seed oleic acid content ≥75% are selected.

6. The method according to claim 5, characterized in that: The method also includes the steps of yield identification of the stable peanut lines resistant to rot and high in oleic acid, field natural disease identification of selected lines with a yield of ≥300 kg / mu, and selection of highly resistant lines as candidate peanut varieties.

7. The method according to claim 6, characterized in that: The identification of the natural disease nursery in the field was carried out by identifying the natural disease nursery of peanut rot in peanut fields where peanuts are continuously cropped year after year and the disease is severe. The identification of the pathogen of peanut rot by artificial inoculation in the room was carried out by inoculating the pathogen strain of peanut rot into the pod layer of potted plants after culturing it in oat grains.

8. The method according to claim 7, characterized in that: The peanut inbred lines selected for their high resistance to rotten fruit disease were both identified as highly resistant based on the results of identification in the naturally occurring disease nursery in the field and the results of identification based on the pathogen of rotten fruit disease after artificial inoculation.

9. The method according to claim 8, characterized in that: The peanut inbred line with high resistance to rotten fruit disease is Yuhanghua 1.

10. The method according to claim 7, characterized in that: The peanut inbred line with an oleic acid content ≥75% and excellent overall traits is Jihua 11.