SNP molecular marker closely linked with barley tillering angle QTL Qtac.cdny.3H, KASP primer group and application thereof

Abstract

The invention relates to the technical field of barley molecular breeding, in particular to an SNP molecular marker closely linked with a barley tillering angle QTL Qtac.cdny.3H, a KASP primer group and application of the SNP molecular marker and the KASP primer group. The invention discloses the SNP locus-Qtac.cdny.3H which is located in the barley 3H chromosome and is remarkably related to the barley tillering angle character for the first time, the molecular marker closely linked with the barley tillering angle QTL Qtac.cdny.3H and the KASP primer group used for detecting the molecular marker are obtained based on the SNP locus, and the molecular marker and the KASP primer group have the advantages of being accurate and efficient in detection and convenient and stable in amplification. The result of the embodiment shows that the molecular marker can accurately track the barley tillering angle QTL Qtac.cdny.3H, the accuracy of molecular marker-assisted selection is high, and the breeding process of barley varieties with different tillering angles adapting to different environments can be accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of barley molecular breeding, and particularly to an SNP molecular marker, a KASP primer set and their applications that are closely linked to the barley tiller angle QTL Qtac.cdny.3H. Background Art

[0002] Barley (Hordeum vulgare L.) is an important cereal crop and occupies an extremely important position in many fields such as feed production, brewing industry and food processing, with irreplaceable economic value. With the rapid development of the economy, the living standards of the people have been continuously improved, and the rapid development of the beer brewing industry and animal husbandry, the demand for barley yield in China has become increasingly urgent. However, due to the limited arable land area in China, cultivating high-yield barley varieties has become an urgent task for the sustainable development of agriculture, animal husbandry and beer brewing industry in China.

[0003] A reasonable plant type structure is conducive to high yield of barley, and the tiller angle is one of the key factors affecting the plant type structure, yield and quality of barley. The barley tiller angle is the angle between the main stem and the tiller. A reasonable tiller angle helps to optimize the population structure of barley, enabling the plant to more effectively utilize light, have good ventilation and reasonably allocate nutrients, thereby enhancing the overall yield potential and quality.

[0004] Traditional barley breeding mainly relies on the experience selection of breeders. The tiller angle is controlled by complex quantitative traits (Quantitative trait locus, QTL) and is affected by the interaction of multiple gene loci and environmental factors. When screening genotypes with reasonable tiller angles based on phenotypes alone, the accuracy is poor, resulting in low breeding efficiency and long breeding cycles, making it difficult to meet the urgent needs of agricultural production for barley varieties. With the rapid development of modern molecular biology techniques, marker-assisted-selection (MAS) technology has become one of the important means in the field of crop breeding today, greatly improving the breeding efficiency and accuracy. Kompetitive Allele Specific PCR (KASP) can accurately perform dual-allele detection of SNPs and Indels at specific loci in a wide range of genomic DNA samples. This detection method has the advantages of simple operation, good specificity, high throughput, fast speed, low detection cost, accurate results, etc., and has received widespread attention due to its realization of true closed-tube operation.

[0005] Previously, some scholars conducted QTL mapping analysis on barley tiller angle and found that the QTLs related to it were distributed on 7 chromosomes of barley. For example, Zhou Hong used the wild barley material "AWCS276" from the Middle East and the barley cultivars "Morex" and "Baudin" to construct two recombinant inbred line (RIL) populations respectively, and mapped 6 QTLs controlling tiller angle. However, there is a severe lack of closely linked molecular markers related to the barley tiller angle trait that can be used in actual molecular breeding. Therefore, it is crucial to develop molecular markers closely linked to the barley tiller angle QTL. Further use of molecular marker-assisted breeding technology to select plants with ideal tiller angles, optimize the barley population structure, improve the light and ventilation conditions among plants, and ultimately achieve the goal of breeding new high-yield barley varieties is of great significance in barley breeding work. Summary of the Invention

[0006] The object of the present invention is to provide an SNP molecular marker, a KASP primer set and their applications that are closely linked to the barley tiller angle QTL Qtac.cdny.3H 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 an SNP molecular marker (molecular marker KASP3H5) that is closely 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, and there is a mutation of G or A at the 44th base of this sequence. This polymorphism is related to the barley tiller angle. The 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 SNP molecular marker. The KASP primer set includes an upstream primer F1 with a nucleotide sequence shown in SEQ ID NO.15, an upstream primer F2 with a nucleotide sequence shown in SEQ ID NO.16, and a downstream primer R with a nucleotide sequence shown in SEQ ID NO.17.

[0010] The present invention provides the application of the above KASP primer set in the preparation of a detection product for identifying the size of barley tiller 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 tillering angle size of barley, and the detection product contains 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 application of the above-mentioned SNP molecular marker, the above-mentioned KASP primer set or the above-mentioned detection product in identifying the tillering angle size of barley.

[0015] The present invention provides a method for identifying the tillering angle size of barley, which includes using the genome of a barley sample to be tested as a template, performing PCR amplification on the template with the above-mentioned KASP primer set, and performing genotype typing according to the amplification result.

[0016] Preferably, if the gene typing result is AA, it is determined that the barley sample to be tested is a barley variety with a large tillering angle; if the gene typing result is GG, it is determined that the barley sample to be tested is a barley variety with a small tillering angle.

[0017] That is, in the present invention, the barley samples to be tested carrying the barley tillering angle QTL Qtac.cdny.3H all show the same fluorescence signal as the fluorescence probe marked by the upstream primer F2 shown in SEQ ID NO.16, while the barley materials without the barley tillering angle QTL Qtac.cdny.3H all show the same fluorescence signal as the fluorescence probe marked by the upstream primer F1 shown in SEQ ID NO.15.

[0018] The present invention provides the application 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 application 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 application 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 aspect, the present invention provides the application of the above-mentioned SNP molecular marker or the above-mentioned KASP primer set in preparing a product for identifying the barley tillering angle QTL Qtac.cdny.3H.

[0022] As an additional aspect, the present invention provides the application 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] The present invention firstly discloses an SNP locus - Qtac.cdny.3H located on chromosome 3H of barley and significantly related to the tillering angle trait of barley. Based on this SNP locus, molecular markers tightly linked to the barley tillering angle QTL Qtac.cdny.3H and a KASP primer set for detecting this molecular marker are obtained. This molecular marker and KASP primer set have the advantages of accurate and efficient detection, convenient and stable amplification. Detection and analysis show that the molecular marker provided by the present invention can accurately track the barley tillering angle QTL Qtac.cdny.3H, with high accuracy for molecular marker-assisted selection, and can accelerate the breeding process of barley varieties with different tillering angles adapted to different environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 It 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 It is the result of genotyping the single-plant DNA of the mapping population using the fluorescence quantitative PCR primer pair in Example 1 of the present invention;

[0028] Figure 3 It is the genetic linkage map of the barley tillering angle QTL Qtac.cdny.3H in Example 1 of the present invention;

[0029] Figure 4 It is the result of genotyping the F constructed from the mutant line tac-1 and Morex in Example 3 of the present invention 2 using the fluorescence quantitative PCR primer for the validation population;

[0030] Figure 5 It is the F constructed from the mutant line tac-1 and Morex in Example 3 of the present invention 2 tillering angle distribution of the validation population. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0036] Material preparation:

[0037] The large tiller angle mutant line tac-1 is a material created by the applicant through EMS mutagenesis using the barley material BOWMAN in the early stage; this material is stored in the Miscellaneous Grain Institute of Chengdu Agricultural Science and Technology Vocational College; the barley varieties Fleet and Morex with small tiller angles are conventional varieties in the art and can be obtained by the routine purchase of those skilled in the art.

[0038] Example 1 Obtaining SNP loci related to barley tiller angle and KASP3H5 primer pairs

[0039] (1) Using the large tiller angle mutant line tac-1 as the female parent and the barley variety Fleet with a small tiller angle as the male parent for hybridization to obtain the hybrid F 1 F 1Self-cross the first-generation single plant to obtain F 2 segregating population.

[0040] (2) DNA extraction: At the five-leaf stage of barley, use the CTAB method to extract the DNA of the parents and each single plant.

[0041] (3) F 2 Phenotypic identification of tiller angle in the segregating population: At the maturity stage of barley, measure the tiller angle of each single plant in the F 2 population.

[0042] (4) Construct extreme pools: Sort according to the tiller angle size, and take the DNA of the first 30 plants and the last 30 plants respectively to construct the extreme pools B-Pool and S-Pool.

[0043] (5) Sequencing and data processing: Send the DNA of the extreme pools B-Pool and S-Pool to the company for resequencing. Use the Illumina high-throughput sequencing platform for paired-end sequencing, and the read length is 150 bp. SNP Calling and QTL-Seq analysis are 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, use Fastp to filter the raw data, use BWA to align the clean data to the Morex v3 reference genome, use BCFtools to detect and extract SNP information, and use the ΔSNP-index method to determine that the candidate interval of the barley tiller angle QTL is within 572.13-591.31 Mb on chromosome 3H of barley ( Figure 1 ).

[0044] (6) Combine the information of SNP Calling, screen high-confidence SNP sites near the candidate interval to develop fluorescence quantitative PCR primers for subsequent detection. Using the PolyMarker website, 6 pairs of available fluorescence quantitative PCR primers were successfully designed (Table 1). Fluorescence quantitative PCR primer design criteria: The length of the amplification primer is 18-25 bp, the length of the amplification product is 45-60 bp, the annealing temperature is 57-62 °C, and the GC content is between 40% and 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 sequences of FAM probe and HEX probe are as follows:

[0049] FAM probe: 5’-GAAGGTGACCAAGTTCATGCT-3’, SEQ ID NO.1 (can bind to FAM fluorophore);

[0050] HEX probe: 5’-GAAGGTCGGAGTCAACGGATT-3’, SEQ ID NO.2 (can bind to HEX fluorophore).

[0051] (7) Genetic map construction and QTL mapping analysis: Select 6 pairs of primers designed above, using the genomic DNA of the F population constructed by crossing the large tiller angle mutant line tac-1 and the small tiller angle barley variety Fleet as a template, perform PCR amplification and genotype analysis. The genotyping results of the F population using the KASP3H5 primer pair are shown in 2 Obtain molecular marker data, use JoinMap4.0 to construct a genetic map, and use MapQTL6.0 for QTL mapping analysis, successfully verifying the results of QTL-seq analysis. The SNP locus Qtac.cdny.3H is located in the 9.17 Mb interval between KASP3H6 and KASP3H2, and is tightly linked to KASP3H5. The linkage map between the position of the barley tiller angle QTL Qtac.cdny.3H on chromosome 3H and its tightly linked molecular marker KASP3H5 is shown in 2 The genotyping results of the F population using the KASP3H5 primer pair are shown in Figure 2 After obtaining the molecular marker data, use JoinMap4.0 to construct a genetic map and use MapQTL6.0 for QTL mapping analysis, successfully verifying the results of QTL-seq analysis. The SNP locus Qtac.cdny.3H is located in the 9.17 Mb interval between KASP3H6 and KASP3H2, and is tightly linked to KASP3H5. The linkage map between the position of the barley tiller angle QTL Qtac.cdny.3H on chromosome 3H and its tightly linked molecular marker KASP3H5 is shown in Figure 3 as follows.

[0052] Table 18 pairs of KASP primer sequences and amplified fragment lengths

[0053]

[0054]

[0055] Example 2 Development of the molecular marker KASP3H5 related to barley tiller angle

[0056] Based on the amplification results of the barley tiller angle KASP3H5 primer pair designed according to Example 1, the molecular marker KASP3H5 was obtained.

[0057] The sequence of the molecular marker KASP3H5 is shown in SEQ ID NO.21, specifically as follows:

[0058] 5’-GGCCCCTTTATTGGTTGGTGATTGAAACTTTGCTTTCTTAGGC[G]TCGACTCAGACATCAGCAGA-3’, there is a G / A mutation at the 44th base of this sequence.

[0059] Application of the molecular marker KASP3H5 closely linked to the barley tiller angle QTL Qtac.cdny.3H in Example 3

[0060] (1) Experimental materials: Select the barley variety Morex with a small tiller angle as the male parent, and the large tiller angle mutant line tac-1 as the female parent to cross and obtain F 1 F 1 The single plants of the generation are self-crossed to obtain F 2 segregating population.

[0061] (2) DNA extraction: Use the CTAB method to extract the genomic DNA of the single plants in the above F 2 segregating population.

[0062] (3) Phenotypic identification: At the maturity stage of barley, measure the tiller angle of each single plant in the above F 2 population.

[0063] (4) Genotype detection: According to the tiller angle size ranking, select the genomic DNA of 40 single plants with extremely large tiller angles and 40 single plants with extremely small tiller angles as templates, and perform fluorescence quantitative PCR amplification with the primer pair of KASP3H5. Genotype the DNA to be tested according to the PCR amplification results.

[0064] Reaction system for fluorescence quantitative PCR amplification: 2×KASP Mastermix 5 μL, KASPAssay Mix 0.14 μL, template DNA 50 ng, Dnase / RNase-free deionized water is added to a total volume of 10 μL; among them, the nucleotide sequences of the primers in KASPAssay Mix are shown in SEQ ID NO.15-17, and the volume ratio of the three primers is 2:2:5.

[0065] In the examples of the present invention, the fluorescence quantitative PCR program: activate at 95 °C for 10 min; denature at 95 °C for 20 s, anneal and extend at 65 °C for 60 s, cycle 10 times, and the annealing and extension temperature is reduced by 1 °C each time; denature at 94 °C for 20 s, anneal and extend at 59 °C for 60 s, cycle 36 times; 60 s at 37 °C, collect fluorescence signals.

[0066] (5) The specific method for analyzing the PCR products is as follows: 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, denoted as A; while samples without the barley tiller angle QTL Qtac.cdny.3H all showed the same type of fluorescence signal as the small tiller angle material Morex, denoted as type B. The results are as Figure 4 shown. Among the 80 plants, 39 plants could amplify fragments of the same type as the mutant line tac-1, which were plants carrying the barley tiller angle QTL Qtac.cdny.3H, and it was predicted that these plants had larger tiller angles; 33 plants could amplify the type B fragments of the same type as Morex, which were plants not carrying the barley tiller angle QTL Qtac.cdny.3H, and it was predicted that these plants had smaller tiller angles.

[0067] (6) The genotyping results are as Figure 4 shown. Then, the genotyping results were combined with the actual tiller angle data of these individual plants (Table 2). The average tiller angle of the plants of the same type as the mutant line tac-1 was 28.35, which was significantly higher than the average tiller angle of the plants of the same type as Morex, which was 22.30 ( Figure 5 ). That is, the tiller angle of barley plants with the genotype AA was greater than that of barley plants with the genotype GG. The actual results (Table 2) were consistent with the expected results ( Figure 4 ), indicating that the locus - barley tiller angle QTL Qtac.cdny.3H provided by the present invention and the molecular marker KASP3H5 developed based on this locus indeed had the effect of significantly increasing the barley tiller angle, and the KASP3H5 primer pair could be used to track and identify the barley tiller angle QTL Qtac.cdny.3H.

[0068] Table 2 Corresponding results of KASP3H5 genotypes and phenotypes in the F 2 population of "tac-1" × "Morex"

[0069]

[0070]

[0071] Note: A represents the genotype AA; B represents the genotype GG.

[0072] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A SNP molecular marker tightly linked to the barley tillering angle QTL Qtac.cdny.3H, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.21, and there is a G or A mutation at the 44th base of the sequence.

2. A KASP primer set for detecting the SNP molecular marker according to claim 1, 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. Use of the KASP primer set according to claim 2 in preparing a detection product for identifying the angle of barley tillering.

4. A detection product for identifying the angle of barley tillering, characterized in that: The detection product comprises the KASP primer set according to claim 2.

5. Use of the SNP molecular marker according to claim 1, the KASP primer set according to claim 2 or the detection product according to claim 4 in identifying the tillering angle of barley.

6. A method for identifying the angle of barley tillering, characterized in that: The method comprises taking the genome of a barley sample to be tested as a template, performing PCR amplification on the template using the KASP primer set described in claim 2, and performing genotyping according to the amplification result.

7. The method according to claim 6, characterized in that 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.

8. Use of the SNP molecular marker according to claim 1, the KASP primer set according to claim 2, or the detection product according to claim 4 in screening or predicting barley varieties with large tillering angles.

9. Use of the SNP molecular marker according to claim 1, the KASP primer set according to claim 2 or the detection product according to claim 4 in improving barley germplasm resources.

10. Use of the SNP molecular marker according to claim 1, the KASP primer set according to claim 2, or the detection product according to claim 4 in barley molecular marker-assisted breeding.

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