Molecular marker-assisted breeding method for high-yield stress-resistant character of cyperus esculentus

Abstract

The invention discloses a molecular marker-assisted breeding method for high-yield stress-resistant characters of cyperus esculentus, which comprises the following steps: collecting cyperus esculentus germplasm resources, and establishing a germplasm resource library: collecting the cyperus esculentus germplasm resources with various genetic backgrounds from different geographic areas, including wild species, local species, bred species and the like, so as to ensure abundant genetic diversity, and improve the breeding efficiency. The collected germplasm resources are subjected to detailed morphological identification, agronomic character investigation and basic biological characteristic analysis, indexes such as the growth cycle, the plant height, the stem diameter, the leaf characteristics, the tuber yield, the oil content and the protein content of the germplasm resources are recorded, systematic arrangement and archiving are conducted on the germplasm resources, and a germplasm resource database is constructed. The invention relates to the technical field of cyperus esculentus breeding. By screening the molecular marker closely linked with the high-yield stress resistance character of the cyperus esculentus, an efficient breeding process is established, and rapid cultivation of good varieties of the cyperus esculentus is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oil sedge breeding, and particularly relates to a molecular marker assisted breeding method for high-yield and stress-resistant traits of oil sedge. BACKGROUND

[0002] Oil sedge (Cyperus esculentus L), also known as tiger nuts, is a perennial herbaceous plant belonging to the Cyperaceae family. The tubers of oil sedge are rich in starch, oil, protein and various nutrients, and have important economic value and development potential. In recent years, with the growing demand for healthy diets and new agricultural products, the planting area of oil sedge has gradually expanded. However, in the actual planting process, oil sedge often faces many problems, such as being susceptible to pests and diseases, poor tolerance to drought and salinity, and unstable yield, which seriously restricts the sustainable development of the oil sedge industry.

[0003] Traditional breeding methods for oil sedge mainly rely on phenotypic selection and hybridization, which have long breeding cycles, low efficiency, and are easily affected by environmental factors when screening for excellent varieties with high yield and stress-resistant traits, making it difficult to accurately identify and efficiently aggregate these excellent traits. The emergence of molecular marker assisted breeding technology provides a new way to solve this problem. Molecular markers can directly detect DNA fragments closely linked to target traits and are not affected by the environment, allowing for the early screening of individuals with target traits, greatly shortening the breeding cycle and improving breeding efficiency and accuracy.

[0004] Currently, although there are some reports on molecular markers for oil sedge, a complete molecular marker assisted breeding method system for high yield and stress-resistant traits of oil sedge has not yet been established. Therefore, there is an urgent need for a practical molecular marker assisted breeding method for high yield and stress-resistant traits of oil sedge to cultivate new varieties of oil sedge that are more suitable for different environmental conditions, have higher yield and stronger stress resistance, and promote the healthy development of the oil sedge industry.

[0005] Therefore, the present application provides a molecular marker assisted breeding method for high yield and stress-resistant traits of oil sedge to solve the above problems. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides a molecular marker assisted breeding method for high yield and stress-resistant traits of oil sedge, which screens molecular markers closely linked to high yield and stress-resistant traits of oil sedge, establishes an efficient breeding process, and realizes the rapid cultivation of excellent varieties of oil sedge, thereby solving the above problems.

[0007] To achieve the above purpose, the present application realizes the following technical scheme: a molecular marker assisted breeding method for high yield and stress-resistant traits of oil sedge, comprising the following steps:

[0008] Collection of Cyperus esculentus germplasm resources and establishment of germplasm bank: Collect Cyperus esculentus germplasm resources with diverse genetic backgrounds from different geographical regions, including wild species, local varieties, and bred varieties, to ensure rich genetic diversity. Perform detailed morphological identification, agronomic trait investigation, and basic biological property analysis on the collected germplasm resources, record their growth cycle, plant height, stem diameter, leaf characteristics, tuber yield, oil content, protein content, and other indicators, and systematically organize and archive them to construct a germplasm resource database.

[0009] Trait evaluation and screening: Conduct multi-year, multi-point field planting tests on Cyperus esculentus materials in the germplasm bank under different ecological environments to evaluate their performance in high-yield traits (such as tuber yield, oil content, etc.) and stress resistance traits (including drought resistance, salt tolerance, disease and pest resistance, etc.) under various environmental conditions. Analyze the trait data using statistical methods to screen out excellent germplasm resources with significant high-yield and stress resistance traits as breeding parent materials. At the same time, determine the phenotypic selection criteria for each target trait to accurately identify and screen individuals in subsequent breeding processes.

[0010] Molecular marker development and screening: Extract genomic DNA from Cyperus esculentus parent materials and hybrid offspring, and use appropriate genome sequencing strategies (such as reduced genome sequencing, transcriptome sequencing, etc.) for high-throughput sequencing of Cyperus esculentus to obtain a large amount of DNA sequence data. Based on the sequencing data, use bioinformatics tools to develop diverse molecular markers, including simple sequence repeats (SSR), single nucleotide polymorphisms (SNP), etc. Preliminarily screen the developed molecular markers, remove low polymorphic or poor quality markers, and retain molecular markers with high polymorphic information content (PIC) and significant polymorphism between parents for subsequent linkage analysis and gene mapping studies.

[0011] Molecular marker and target trait linkage analysis and gene mapping: Construct hybrid combinations with selected excellent high-yield and stress resistance parents, obtain F1 generation through sexual hybridization, and produce segregation populations (such as F2 generation, BC1F1 generation, etc.) through selfing or backcrossing. Perform phenotypic identification of individuals in the segregation population for target traits, and simultaneously use developed molecular markers for genotypic analysis to construct genetic linkage maps. Use quantitative genetics analysis software to perform linkage analysis of molecular markers and target traits, determine molecular markers tightly linked to high-yield and stress resistance traits, and then preliminarily map genes controlling these traits. Estimate the genetic distance between markers and genes by analyzing the recombination rate between linked markers and target genes, and screen out molecular markers closely linked to target traits and with short genetic distance as markers for assisted selection.

[0012] Molecular marker assisted selection breeding: according to the linkage analysis results, determine the key molecular markers for assisted selection. In the process of Cyperus esculentus hybrid breeding, genotype detection is carried out on early generations (such as seedling stage) of hybrid offspring, and these molecular markers are used to quickly screen individuals carrying high yield and stress resistance related alleles. Phenotypic verification is carried out on the selected excellent individuals, which are planted in the field, and their growth and development, yield and stress resistance are comprehensively evaluated under different environmental conditions. Combined with the results of molecular marker assisted selection and phenotype screening, the breeding process is gradually promoted, and multiple generations of selection and purification are carried out, and finally a stable genetic high yield and stress resistance Cyperus esculentus new variety is bred.

[0013] Beneficial effects

[0014] The application provides a molecular marker assisted breeding method for high yield and stress resistance traits of Cyperus esculentus. Compared with the prior art, the following beneficial effects are achieved:

[0015] (1) Improve breeding efficiency: through molecular marker assisted selection, genotype detection can be carried out at an early stage of the plant and in a non-growing season, individuals with target traits can be screened in advance, the breeding cycle is shortened, and the breeding process of excellent varieties is accelerated.

[0016] (2) Precise selection: direct selection of gene loci related to high yield and stress resistance traits avoids the problem of inaccurate phenotype selection caused by environmental factors in traditional breeding, improving the accuracy and reliability of selection.

[0017] (3) Multi-trait comprehensive improvement: simultaneous improvement of multiple high yield and stress resistance traits of Cyperus esculentus is achieved, which can breed new varieties of Cyperus esculentus with excellent comprehensive traits, meet production needs, and improve the yield and planting range of Cyperus esculentus. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the molecular marker assisted breeding flowchart of high yield and stress resistance traits of Cyperus esculentus of the application. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0020] Example 1: Cyperus esculentus germplasm resource collection and evaluation

[0021] As shown in Figure 1 , the application provides a molecular marker assisted breeding method for high yield and stress resistance traits of Cyperus esculentus;

[0022] In this embodiment, 300 Cyperus esculentus germplasm resources, including 80 wild species, 150 local varieties and 70 improved varieties, were introduced from different regions of China, such as northeast China, north China, east China, central China and southwest China, and some foreign countries. These germplasm resources were planted in the test field according to unified cultivation and management measures.

[0023] Morphological identification was performed, and morphological characteristics of the plants, such as plant height, stem diameter, tiller number, leaf shape and color, were recorded. After the growth cycle ended, the tubers were harvested, and indexes such as tuber yield, oil content and protein content were measured. Meanwhile, some germplasm resources were subjected to drought resistance, salt and alkali tolerance and disease and pest resistance identification tests. Through a combination of field natural disease investigation and artificial inoculation identification, the resistance levels of the germplasm resources to main diseases and pests (such as aphids and root rot) were evaluated; pot drought simulation tests and saline-alkali land planting tests were performed to measure the growth indexes and yield performance of the germplasm resources under drought and saline-alkali stress.

[0024] After comprehensive analysis and evaluation, 30 Cyperus esculentus germplasm resources with high yield potential (tuber yield increased by more than 15% compared with the control variety, and oil content reached more than 25%) and strong stress resistance (yield reduction under drought conditions was not more than 10%, salt and alkali tolerance concentration was more than 3‰, and resistance to main diseases and pests was moderate or above) were selected as parent materials for subsequent breeding. The specific performance of the traits is shown in Table 1.

[0025] Table 1: Performance of the selected excellent Cyperus esculentus parent materials

[0026]

[0027] Example 2: Development and screening of molecular markers

[0028] As shown in Figure 1 Example 1, on the basis of Example 1;

[0029] In this embodiment, genomic DNA of the above-mentioned 30 excellent parent materials was extracted, and the genomic DNA was sequenced using the simplified genomic sequencing technology (Restriction site-associated DNA sequencing, RAD-seq). A genomic DNA library was constructed, and the DNA was digested with a suitable restriction enzyme. After end repair, linker ligation and fragment selection, DNA fragments suitable for sequencing were obtained. Deep sequencing was performed using a high-throughput sequencing platform, and a large amount of sequencing data was obtained.

[0030] The sequencing data is subjected to quality control and filtering to remove low-quality reads, adapter sequence contamination, and repetitive sequences, etc., to obtain high-quality clean data. Bioinformatics software is used to analyze the clean data, and SSR and SNP molecular markers are mined based on the reference genome (if no reference genome, then de novo assembly). A total of 500 SSR molecular markers and 2000 SNP molecular markers are developed.

[0031] The developed molecular markers are subjected to preliminary screening, and 150 SSR markers and 500 SNP markers with obvious polymorphism between parents are selected. Further, these polymorphic markers are used to verify part of the known genotypes of Cyperus esculentus materials, to evaluate their amplification stability and accuracy, and finally 100 high-quality SSR markers and 300 high-quality SNP markers are selected for subsequent linkage analysis and gene mapping research, part of the screened molecular marker information is shown in Table 2.

[0032] Table 2: Part of the screened molecular marker information

[0033]

[0034] Example 3: Genetic linkage map construction and gene mapping

[0035] The selected excellent high-yield stress-resistant parent material A (with high yield, drought resistance, salt and alkali tolerance, etc.) and parent material B (with high yield, disease and pest resistance, etc.) are crossed to obtain F1 generation. The F1 generation is selfed to produce F2 segregation population, and a total of 500 F2 generation plants are planted.

[0036] The plants of the F2 segregation population are subjected to phenotypic identification of target traits, including determination of tuber yield, oil content, drought resistance, salt and alkali tolerance, disease and pest resistance, etc. At the same time, the F2 segregation population is subjected to genotype analysis using the molecular markers screened in Example 2.

[0037] Genetic mapping software Mapmaker / Exp3.0 and JoinMap4.0 are used to perform linkage analysis of molecular markers and target traits with a threshold value of LOD value 3.0, to construct a genetic linkage map. Through analysis, 8 molecular markers closely linked to high yield traits, 6 molecular markers closely linked to drought resistance traits, 5 molecular markers closely linked to salt and alkali tolerance traits, and 7 molecular markers closely linked to disease and pest resistance traits are located in the Cyperus esculentus genome. The genetic distance of these markers to the target genes is less than 10 cM, which can be used as auxiliary selection molecular markers for subsequent breeding process, part of the linkage analysis results are shown in Table 3.

[0038] Table 3: Part of the linkage analysis results of molecular markers and target traits

[0039] Target traits Molecular marker name Genetic distance (cm) LOD value High yield trait SSR-1 2.1 5.8 High yield trait SSR-3 4.3 6.2 ... ... ... ... Drought resistance trait SNP-5 3.7 5.1 Drought resistance trait SNP-8 6.5 5.3 ... ... ... ... Salt-alkali tolerance trait SSR-5 2.8 4.9 Salt-alkali tolerance trait SNP-11 5.2 5.0 ... ... ... ... Disease and pest resistance trait SNP-15 3.3 5.5 Disease and pest resistance trait SSR-7 4.1 5.7

[0040] Example 4: Marker-assisted selection breeding

[0041] Based on the hybrid combination constructed by parent material A and parent material B, the F1 generation thereof is backcrossed with parent material A to generate a BC1F1 generation segregation population, and 300 BC1F1 generation plants are planted.

[0042] At the seedling stage of the BC1F1 generation plants, the leaf genomic DNA is extracted, and the genotype is detected by using the molecular markers determined in Example 3 to be closely linked to the high-yield stress resistance traits. According to the detection results of the molecular markers, 60 BC1F1 generation plants carrying the alleles related to high yield, drought resistance, salt and alkali tolerance, and disease and pest resistance are screened out.

[0043] The 60 screened BC1F1 generation plants are planted in the field, and the phenotype verification and comprehensive evaluation are performed under different environmental conditions (including normal planting conditions, drought stress conditions, salt and alkali conditions, and disease and pest occurrence conditions). After two years of multi-point testing, combined with the results of molecular marker-assisted selection and phenotype screening, three new stable genetic high-yield stress-resistant Cyperus esculentus lines are finally bred, which are named as OS-GA1, OS-GA2 and OS-GA3, respectively. These new lines show high tuber yield (20%-30% higher than the local main cultivar), good oil content (26%-28%), and strong stress resistance (5%-8% reduction in yield under drought conditions, salt and alkali tolerance up to 4‰, and high resistance to main diseases and pests) in multi-point testing. The performance of the new lines is shown in Table 4.

[0044] Table 4 Performance of high-yield stress-resistant Cyperus esculentus new lines

[0045]

[0046] It should be noted that the relational terms such as first and second and the like are used merely to distinguish one entity or action from another, without necessarily requiring or implying that there is any such actual relationship or order between such entities or actions. In addition, the terms "comprises", "comprising", or any other variant thereof are intended to cover non-exclusive inclusions, so that a process, method, article, or apparatus that comprises a list of elements does not only include those elements, but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0047] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera, comprising the following steps: Step 1: Collect cyperus oleifera germplasm resources and establish a germplasm resource bank; Step 2: Evaluate and screen the cyperus oleifera materials in the germplasm resource bank to identify high-yield and stress-resistant superior parents; Step 3: Develop and screen molecular markers for Cyperus oleifera; Step 4: Construct hybrid combinations to obtain segregating populations, conduct linkage analysis and gene mapping between molecular markers and target traits, and identify molecular markers that are closely linked to high-yield and stress-resistant traits; Step 5: Using the molecular markers to assist in the selection of hybrid offspring, combined with phenotypic screening, to cultivate new high-yield and stress-resistant cyperus chinensis varieties.

2. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: The jatropha germplasm resources include wild species, local varieties and cultivated varieties, which are collected from different geographical regions to ensure rich genetic diversity.

3. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: The trait evaluation includes identification and analysis of tuber yield, oil content, protein content, drought resistance, salt and alkali resistance, and disease and pest resistance of jatropha, and determination of excellent germplasm resources as breeding parents through statistical methods.

4. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: The molecular markers include simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers, which are developed through genome sequencing and bioinformatics analysis, and are initially screened to retain markers with high polymorphism information content (PIC) and obvious polymorphism between parents.

5. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: The quantitative genetics analysis software used in the linkage analysis and gene mapping is Mapmaker / Exp3.0 and JoinMap4.0, and it is determined that the genetic distance of the molecular markers closely linked to the target trait is less than 10 cM.

6. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: The molecular marker-assisted selection is to perform genotype detection on the early generations of hybrid offspring, screen out individuals carrying the target allele using molecular markers closely linked to high-yield and stress-resistant traits, and then conduct multi-generation selection and purification in combination with field phenotypic verification.

7. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: During the field planting trials, the selected excellent individuals are subjected to multi-point and multi-year phenotypic identification, including growth indicators, yield performance and stress resistance evaluation under different ecological environments, to ensure the stability and adaptability of the bred varieties.

8. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to any one of claims 1 to 7, characterized in that: The tuber yield of the new high-yield and stress-resistant oil bean variety is more than 15% higher than that of the local main cultivated varieties, and the oil content reaches more than 25%. At the same time, it has strong stress resistance, including a yield reduction of no more than 10% under drought conditions, a salt and alkali concentration tolerance of more than 3‰, and a medium resistance level or above to major diseases and pests.

9. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: In the step of collecting cyperus oleifera germplasm resources, detailed morphological identification, agronomic trait investigation and basic biological characteristic analysis are carried out on the germplasm resources, and indicators such as growth cycle, plant height, stem diameter, leaf characteristics, tuber yield, oil content, and protein content are recorded. A germplasm resource database is constructed to fully understand the characteristics of the germplasm resources and provide an accurate basis for parent selection.

10. The molecular marker-assisted breeding method for high-yield and stress-resistant traits of cyperus oleifera according to claim 1, characterized in that: In the molecular marker-assisted selection breeding step, when performing genotype detection on hybrid offspring, the detection method used has high accuracy and repeatability, can reliably distinguish different alleles, ensure the reliability of the screening results, and thus improve breeding efficiency and accuracy.

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