A SNP molecular marker tightly linked to the barley tillering angle QTL Qtac.cdny.3H, a KASP primer set, and their applications
By developing the SNP molecular marker and KASP primer set of closely linked barley tiller angle QTL Qtac.cdny.3H, the accuracy of tiller angle selection in traditional breeding was solved, efficient molecular marker assisted selection was achieved, and barley breeding efficiency and yield were improved.
Patent Information
- Application Number
- CN202510333697.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-20
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2045-03-20
AI Technical Summary
In traditional barley breeding, relying on breeders' experience, the accuracy of selecting tiller angles is poor, the breeding efficiency is low, it is difficult to meet the high yield needs, and there is a lack of tightly linked molecular markers for molecular marker-assisted selection.
The SNP molecular marker and KASP primer set closely linked to the barley tiller angle QTL Qtac.cdny.3H were developed, and accurate genetic testing was performed through KASP technology, and PCR amplification was used for KASP primer set and genotyping was performed according to the amplification results, and plants with ideal tiller angles were screened out.
Accurate tracking and efficient screening of the tiller angle of barley is achieved, the accuracy and speed of breeding are improved, the barley population structure is optimized, and yield and quality are improved.
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Figure CN120119023B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of barley molecular breeding, and in particular to a SNP molecular marker tightly linked to a barley tillering angle QTL Qtac.cdny.3H, a KASP primer set and applications thereof. Background Art
[0002] Barley (Hordeum vulgare L.) is a major cereal crop, playing a crucial role in numerous sectors, including feed production, brewing, and food processing, and possessing irreplaceable economic value. With rapid economic development, rising living standards, and the rapid growth of the brewing and animal husbandry industries, my country's demand for barley production is becoming increasingly urgent. However, due to limited arable land in my country, cultivating high-yield barley varieties has become a top priority for achieving sustainable development in agriculture, animal husbandry, and brewing.
[0003] A reasonable plant structure contributes to high barley yields, and tiller angle is a key factor influencing barley plant structure, yield, and quality. The tiller angle is the angle between the main stem and the tiller. A reasonable tiller angle helps optimize the barley colony structure, enabling the plant to more effectively utilize light, maintain good ventilation, and distribute nutrients appropriately, thereby improving overall yield potential and quality.
[0004] Traditional barley breeding relies primarily on breeders' empirical selection. Tillering angle is a complex quantitative trait (QTL) controlled by the interactions of multiple gene loci and environmental factors. When phenotypes alone are used to screen for genotypes with a reasonable tillering angle, the accuracy is poor, resulting in low breeding efficiency and a long breeding cycle, making it difficult to meet the urgent demand for barley varieties in agricultural production. With the rapid development of modern molecular biology techniques, marker-assisted selection (MAS) technology has become one of the important methods in crop breeding today, greatly improving breeding efficiency and accuracy. Competitive allele-specific PCR (KASP) can accurately detect biallelic mutations in SNPs and indels at specific sites in a wide range of genomic DNA samples. This detection method has the advantages of simple operation, good specificity, high throughput, rapidity, low detection cost, and accurate results. It has also achieved true closed-tube operation and has attracted widespread attention.
[0005] Previously, some scholars conducted QTL positioning analysis on the tiller angle of barley and found that the related QTLs are distributed on the seven chromosomes of barley. For example, Zhou Hong used the Middle Eastern wild barley material "AWCS276" and the barley cultivars "Morex" and "Baudin" to construct two recombinant inbred lines (RIL) populations, and located six QTLs that control the tiller angle. However, there is currently a lack of tightly linked molecular markers that are related to the barley tiller angle trait and can be used for actual molecular breeding. Therefore, it is crucial to develop molecular markers that are tightly linked to the barley tiller angle QTL, and further use molecular marker-assisted breeding technology to select plants with ideal tiller angles, optimize the barley population structure, improve the light and ventilation conditions between plants, and ultimately achieve the goal of breeding new barley varieties with increased yields, which is of great significance in barley breeding. Summary of the Invention
[0006] The purpose of the present invention is to provide a SNP molecular marker tightly linked to the barley tillering angle QTL Qtac.cdny.3H, a KASP primer set and applications thereof, so as to solve the problems existing in the above-mentioned prior art.
[0007] To achieve the above object, the present invention provides the following solutions:
[0008] The present invention provides a single-nucleotide polymorphism (SNP) molecular marker (molecular marker KASP3H5) tightly linked to the barley tiller angle QTL Qtac.cdny.3H. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 21. A G or A mutation exists at base 44 of the sequence. This polymorphism is associated with barley tiller angle. Molecular marker KASP3H5 is located within the confidence interval of the barley tiller angle QTL Qtac.cdny.3H.
[0009] The present invention provides a KASP primer set for detecting the above-mentioned SNP molecular marker, wherein the KASP primer set comprises an upstream primer F1 having a nucleotide sequence as shown in SEQ ID NO.15, an upstream primer F2 having a nucleotide sequence as shown in SEQ ID NO.16, and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.17.
[0010] The present invention provides the use of the above-mentioned KASP primer set in preparing a detection product for identifying the size of barley tillering angle.
[0011] Further preferably, the detection product includes a detection chip, a detection kit or a detection reagent.
[0012] The present invention provides a detection product for identifying the size of barley tillering angles, wherein the detection product comprises the above-mentioned KASP primer set.
[0013] Further preferably, the detection product includes a detection chip, a detection kit or a detection reagent.
[0014] The present invention provides the use of the above-mentioned SNP molecular marker, the above-mentioned KASP primer set or the above-mentioned detection product in identifying the size of the tillering angle of barley.
[0015] The present invention provides a method for identifying the angle size of barley tillers, comprising using the genome of a barley sample to be tested as a template, performing PCR amplification on the template using the above-mentioned KASP primer set, and performing genotyping according to the amplification result.
[0016] Preferably, if the genotyping result is AA, the barley sample to be tested is determined to be a barley variety with a large tillering angle; if the genotyping result is GG, the barley sample to be tested is determined to be a barley variety with a small tillering angle.
[0017] That is, in the present invention, the barley samples tested that carry the barley tiller angle QTL Qtac.cdny.3H all show the same fluorescent signal as the fluorescent probe labeled with the upstream primer F2 shown in SEQ ID NO. 16, while the barley materials that do not carry the barley tiller angle QTL Qtac.cdny.3H all show the same fluorescent signal as the fluorescent probe labeled with the upstream primer F1 shown in SEQ ID NO. 15.
[0018] The present invention provides the use of the above-mentioned SNP molecular marker, the above-mentioned KASP primer set or the above-mentioned detection product in screening or predicting barley varieties with large tillering angles.
[0019] The present invention provides the use of the above-mentioned SNP molecular marker, the above-mentioned KASP primer set or the above-mentioned detection product in improving barley germplasm resources.
[0020] The present invention provides the use of the above-mentioned SNP molecular marker, the above-mentioned KASP primer set or the above-mentioned detection product in barley molecular marker-assisted breeding.
[0021] As an additional solution, the present invention provides the use of the above-mentioned SNP molecular marker or the above-mentioned KASP primer set in the preparation of a product for identifying the barley tillering angle QTL Qtac.cdny.3H.
[0022] As an additional solution, the present invention provides the use of the above-mentioned SNP molecular marker, the above-mentioned KASP primer set or the above-mentioned product in identifying the barley tillering angle QTL Qtac.cdny.3H.
[0023] The present invention discloses the following technical effects:
[0024] This invention discloses for the first time a single-nucleotide polymorphism (SNP) locus (Qtac.cdny.3H) located on chromosome 3H of barley and significantly associated with the tiller angle trait. Based on this SNP, a molecular marker tightly linked to the barley tiller angle QTL (Qtac.cdny.3H) and a KASP primer set for detecting this marker were obtained. This molecular marker and KASP primer set offer the advantages of accurate and efficient detection, as well as convenient and stable amplification. Detection and analysis have demonstrated that the molecular marker provided by this invention can accurately track the barley tiller angle QTL (Qtac.cdny.3H), demonstrates high accuracy for marker-assisted selection, and can accelerate the breeding of barley varieties with different tiller angles adapted to different environments. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0026] Figure 1 is the physical interval of the barley tillering angle QTL Qtac.cdny.3H on chromosome 3H identified by QTL-seq analysis in Example 1 of the present invention;
[0027] Figure 2 The results of genotyping the DNA of individual strains in the mapping population using fluorescent quantitative PCR primers in Example 1 of the present invention are as follows;
[0028] Figure 3 This is the genetic linkage map of the barley tillering angle QTL Qtac.cdny.3H in Example 1 of the present invention;
[0029] Figure 4 The results of genotyping of the F2 verification population constructed from the mutant strains tac-1 and Morex in Example 3 of the present invention using fluorescent quantitative PCR primers;
[0030] Figure 5 This is the tillering angle distribution of the F2 verification population constructed from the mutant lines tac-1 and Morex in Example 3 of the present invention. DETAILED DESCRIPTION
[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.
[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0036] Materials preparation:
[0037] The large tillering angle mutant strain tac-1 was created by the applicant in the early stage using the barley material BOWMAN through EMS mutagenesis; the material is preserved in the Cereals Institute of Chengdu Agricultural Science and Technology Vocational College; the barley varieties Fleet and Morex with small tillering angles are conventional varieties in this field and can be purchased by technicians in this field.
[0038] Example 1 Obtaining SNP sites associated with barley tillering angle and KASP3H5 primer pairs
[0039] (1) The large tillering angle mutant line tac-1 was used as the female parent and the small tillering angle barley variety Fleet was used as the male parent to obtain the hybrid F1. The F1 generation individual plants were self-pollinated to obtain the F2 segregating population.
[0040] (2) DNA extraction: At the five-leaf stage of barley, DNA of the parents and each individual plant was extracted using the CTAB method.
[0041] (3) Phenotypic identification of tiller angle of F2 segregating population: At the maturity stage of barley, the tiller angle of each individual plant in the F2 population was measured.
[0042] (4) Construction of extreme pools: Sort by tillering angle, and take DNA from the first 30 plants and the last 30 plants to construct extreme pools B-Pool and S-Pool, respectively.
[0043] (5) Sequencing and data processing: DNA from extreme pools B-Pool and S-Pool was sent to the company for resequencing. Paired-end sequencing was performed using the Illumina high-throughput sequencing platform, with a read length of 150bp. SNP Calling and QTL-Seq analysis were based on the method of Takagi et al. (Takagi H, Abe A, Yoshida K, et al. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations [J]. The Plant Journal, 2013, 74 (1): 174-183). Briefly, Fastp was used to filter the raw data, BWA was used to align the clean data to the Morex v3 reference genome, BCFtools was used to detect and extract SNP information, and the ΔSNP-index method was used to determine that the candidate interval of the barley tiller angle QTL was within 572.13-591.31Mb on the barley chromosome 3H ( Figure 1 ).
[0044] (6) Combined with the information from SNP Calling, high-confidence SNP sites near the candidate interval were screened to develop fluorescent quantitative PCR primers for subsequent detection. Using the PolyMarker website, 6 pairs of available fluorescent quantitative PCR primers were successfully designed (Table 1). Fluorescent quantitative PCR primer design standards: amplification primer length 18-25bp, amplification product length 45-60bp, annealing temperature 57-62℃, GC content between 40%-60%. The synthetic primer sequence composition is:
[0045] Forward primer 1: FAM probe + amplification primer sequence;
[0046] Forward primer 2: HEX probe + amplification primer sequence;
[0047] Reverse primer: Amplification primer sequence.
[0048] The FAM probe and HEX probe sequences are as follows:
[0049] FAM probe: 5'-GAAGGTGACCAAGTTCATGCT-3', SEQ ID NO. 1 (can bind to FAM fluorescent group);
[0050] HEX probe: 5'-GAAGGTCGGAGTCAACGGATT-3', SEQ ID NO. 2 (can be combined with a HEX fluorescent group).
[0051] (7) Genetic map construction and QTL location analysis: The six primer pairs designed above were selected and the F2 population DNA constructed by hybridization of the large tillering angle mutant line tac-1 and the small tillering angle barley variety Fleet was used as a template for PCR amplification and genotyping analysis. The results of genotyping the F2 population using the KASP3H5 primer pair are shown in Figure 2 After obtaining the molecular marker data, JoinMap4.0 was used to construct a genetic map, and MapQTL6.0 was used to perform QTL positioning analysis, which successfully verified the results of the QTL-seq analysis. The SNP site Qtac.cdny.3H is located in the 9.17Mb interval between KASP3H6 and KASP3H2, and is tightly linked to KASP3H5. The linkage map between the position of the barley tillering angle QTL Qtac.cdny.3H on chromosome 3H and the molecular marker KASP3H5 that is tightly linked to it is shown in the figure. Figure 3 shown.
[0052] Table 18 Pairs of KASP primer sequences and amplified fragment lengths
[0053]
[0054]
[0055] Example 2 Development of molecular marker KASP3H5 associated with barley tillering angle
[0056] According to the amplification results of the barley tillering angle KASP3H5 primer pair designed in Example 1, the molecular marker KASP3H5 was obtained.
[0057] The sequence of the molecular marker KASP3H5 is shown in SEQ ID NO. 21, and is as follows:
[0058] 5'-GGCCCCTTTATTGGTTGGTGATTGAAACTTTGCTTTCTTAGGC[G]TCGACTCAGACATCAGCAGA-3', there is a G / A mutation at the 44th base of the sequence.
[0059] Example 3 Application of molecular marker KASP3H5 tightly linked to barley tillering angle QTL Qtac.cdny.3H
[0060] (1) Experimental materials: The barley variety Morex with a small tillering angle was selected as the male parent and the mutant strain tac-1 with a large tillering angle was selected as the female parent to obtain F1. The F1 generation of individual plants was self-pollinated to obtain the F2 segregating population.
[0061] (2) DNA extraction: The CTAB method was used to extract the DNA of individual strains of the F2 segregating population.
[0062] (3) Phenotypic identification: During the barley maturity period, the tillering angle of each individual plant in the above F2 population was measured.
[0063] (4) Genotype detection: Sort by tillering angle, select 40 DNA samples of individual plants with extremely large tillering angles and 40 DNA samples of individual plants with extremely small tillering angles as templates, perform fluorescence quantitative PCR amplification with the primer pair of KASP3H5, and perform genotyping on the DNA to be tested based on the PCR amplification results.
[0064] The reaction system for fluorescent quantitative PCR amplification was as follows: 5 μL of 2×KASP Mastermix, 0.14 μL of KASPAssay Mix, 50 ng of template DNA, and DNase / RNase-free deionized water to a total volume of 10 μL; the nucleotide sequences of the primers contained in the KASPAssay Mix were shown as SEQ ID NOs. 15-17, and the volume ratio of the three primers was 2:2:5.
[0065] In the embodiment of the present invention, the fluorescence quantitative PCR program is as follows: activation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 59°C for 60 s, 36 cycles; and fluorescence signal acquisition at 37°C for 60 s.
[0066] (5) The specific method for analyzing PCR products is as follows: the samples containing the barley tiller angle QTL Qtac.cdny.3H all showed the same type of fluorescence signal as the large tiller angle mutant line tac-1, which is marked as A; while the samples not containing the barley tiller angle QTL Qtac.cdny.3H all showed the same type of fluorescence signal as the small tiller angle material Morex, which is marked as B. The results are as follows: Figure 4Among the 80 accessions, 39 amplified the same type of fragment as the mutant line tac-1, indicating that they carry the barley tiller angle QTL Qtac.cdny.3H and are predicted to have larger tiller angles. 33 amplified the same B-type fragment as Morex and are not carriers of the barley tiller angle QTL Qtac.cdny.3H, indicating that they have smaller tiller angles.
[0067] (6) The typing results are as follows Figure 4 As shown, the typing results were then combined with the actual tiller angle data of these individual plants (Table 2). The average tiller angle of plants with the same type as the mutant line tac-1 was 28.35, which was significantly higher than the average tiller angle of plants with the same type as Morex (22.30). Figure 5 ), that is, the tillering angle of barley plants with genotype AA is greater than that of barley plants with genotype GG. The actual results (Table 2) are different from the expected results ( Figure 4 ), indicating that the locus provided by the present invention - barley tiller angle QTL Qtac.cdny.3H and the molecular marker KASP3H5 developed based on the locus do have the effect of significantly increasing the barley tiller angle, and the KASP3H5 primer pair can be used to track and identify the barley tiller angle QTL Qtac.cdny.3H.
[0068] Table 2 Correspondence between KASP3H5 genotype and phenotype in the F2 population of “tac-1”דMorex”
[0069]
[0070]
[0071] Note: A represents genotype AA; B represents genotype GG.
[0072] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. Use of the KASP primer set in the preparation of a detection product for identifying the angle of barley tillers, characterized in that: The KASP primer set includes an upstream primer F1 having a nucleotide sequence as shown in SEQ ID NO.15, an upstream primer F2 having a nucleotide sequence as shown in SEQ ID NO.16, and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.
17.
2. Use of a KASP primer set or a detection product containing the KASP primer set in identifying the tillering angle of barley, characterized in that: The KASP primer set includes an upstream primer F1 having a nucleotide sequence as shown in SEQ ID NO.15, an upstream primer F2 having a nucleotide sequence as shown in SEQ ID NO.16, and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.
17.
3. A method for identifying the angle of barley tillers, characterized in that: The method comprises using the genome of a barley sample to be tested as a template, performing PCR amplification on the template using a KASP primer set, and performing genotyping according to the amplification result, wherein the KASP primer set comprises an upstream primer F1 having a nucleotide sequence as shown in SEQ ID NO.15, an upstream primer F2 having a nucleotide sequence as shown in SEQ ID NO.16, and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.17; If the genotyping result is AA, the barley sample to be tested is determined to be a barley variety with a large tillering angle; if the genotyping result is GG, the barley sample to be tested is determined to be a barley variety with a small tillering angle.
4. Use of a KASP primer set or a detection product containing the KASP primer set in screening or predicting barley varieties with large tillering angles, characterized in that: The KASP primer set includes an upstream primer F1 having a nucleotide sequence as shown in SEQ ID NO.15, an upstream primer F2 having a nucleotide sequence as shown in SEQ ID NO.16, and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.
17.
5. Use of a KASP primer set or a detection product containing a KASP primer set in improving barley germplasm resources, characterized in that: The KASP primer set includes an upstream primer F1 having a nucleotide sequence as shown in SEQ ID NO.15, an upstream primer F2 having a nucleotide sequence as shown in SEQ ID NO.16, and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.
17.
6. Use of a KASP primer set or a detection product containing the KASP primer set in molecular marker-assisted breeding of barley tillering angle, characterized in that: The KASP primer set includes an upstream primer F1 having a nucleotide sequence as shown in SEQ ID NO.15, an upstream primer F2 having a nucleotide sequence as shown in SEQ ID NO.16, and a downstream primer R having a nucleotide sequence as shown in SEQ ID NO.17.