Application of KASP molecular marker combination in identification of barley varieties and its development method

Abstract

The invention provides an application of a KASP molecular marker combination in barley variety identification and a development method thereof. The KASP molecular marker combination is helpful to promote barley breeding and germplasm resource management. The development method comprises: extracting the same number of markers on chromosomes chr1H, chr2H, chr3H, chr4H, chr5H, chr6H and chr7H; no other markers can exist within 200 bp upstream and downstream of each marker; and removing sequences with high N base content in the sequence. The barley KASP molecular marker combination for distinguishing barley variety resources of the invention can effectively distinguish barley varieties from different sources, has the characteristics of high stability, accuracy and low cost, improves the efficiency and accuracy of variety identification, is not affected by the environment, is suitable for large-scale promotion and application, and supplements a molecular marker library for barley variety identification. The marker development method is simple, ingeniously designed, and easy and quick to operate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an application of a KASP molecular marker combination in barley variety identification and a development method thereof. Background Art

[0002] Barley (Hordeum vulgare L.) belongs to the Poaceae family and is one of the oldest crops in the world. It is the fourth largest food crop after rice, wheat and corn. It is also one of the model plants widely used in genetic research. It is mainly used as food, feed, beer raw material, medicine and health food. In my country, barley is the main food crop in Tibetan areas, and China is also the world's largest importer of beer raw materials. Therefore, strengthening barley genetic breeding research and new variety breeding is an important way to develop my country's barley industry and ensure food security in Tibetan areas.

[0003] At present, the methods for identifying barley varieties and purity are mainly field crop phenotypic identification (morphological markers), seed isozyme and storage protein detection (biochemical markers) and DNA molecular marker detection. [1-3] Due to the increase in barley varieties and the close relationship between some varieties, it is difficult to quickly identify the authenticity and purity of varieties using morphological and biochemical markers. Therefore, DNA molecular markers have been widely used in the authenticity identification of crop varieties. Commonly used DNA molecular markers mainly include RFLP [4-5] , RAPD [6-9] , AFLP [10-11] 、SSR [12-16] 、SNP [14,17-18] and InDel

[19] . In barley variety identification, RFLP, RAPD, and AFLP molecular markers are difficult to be widely used in variety identification due to their cumbersome operation, poor stability, and long cycle. The commonly used molecular markers for barley identification are SSR and SNP. SSR molecular markers have the advantages of stability and co-dominance, and the marker cost is low and the application technology is simple. However, the disadvantage is that SSR markers may have "amplification loss", messy banding, fuzzy banding and other phenomena in the experiment, which increases the probability of genotype misidentification and cannot detect large quantities of samples at one time. The development of new primers is costly and difficult. At the same time, as the number of barley samples increases, the number of markers used for SSR marker detection is a key factor affecting identification efficiency and cost. InDel markers have the advantages of high stability, high polymorphism and co-dominance. Genotyping can be performed through simple PCR and gel electrophoresis. The disadvantage is that the reagents required for gel electrophoresis are harmful to the human body.

[0004] SNP markers have the advantages of wide distribution, good genetic stability and easy automation, and have rapidly replaced traditional molecular markers. The main detection methods for SNP markers are: CAPS (enzyme amplification polymorphism sequence), AS-PCR (allele-specific PCR), KASP (competitive allele-specific PCR), sequencing, mass spectrometry, HRM (high-resolution melting curve). With the rapid development of high-throughput sequencing technology, the barley genome sequencing has been completed. [20-21] Based on this, the SNP chips of barley 9K and 50K were developed.

[22] These SNP loci can be developed into KASP markers for the identification of barley variety resources.

[0005] Therefore, the successful development of KASP markers can effectively, accurately, quickly and safely identify barley varieties. On the one hand, it can scientifically and standardizedly manage barley germplasm resources, and on the other hand, it can enrich the molecular markers for barley variety identification. It has certain theoretical significance and application value in barley variety identification, barley resource kinship analysis and population division.

[0006] Morphological marking, or sensory identification, is the most commonly used method for identifying germplasm resources. The external morphological characteristics of seeds (such as grain shape, color, wheat awn, color of barley aleurone layer, base axis, base mark, etc.) are used to identify varieties. For example, my country's national standard "Crop Seed Inspection Procedures for Authenticity and Variety Purity Identification" (GB / T 3543.5-1995) stipulates the morphological identification marks of barley seed varieties. [1] The barley variety standards issued by the Australian Grain Trade Association every year provide specific standard sample images and morphological identification characteristics of the varieties. For example, the sensory identification atlas for the identification of Australian beer barley varieties describes eight characteristics: grain color, grain shape, lemma base shape, spikelet axis, spikelet axis hairs, lateral dorsal vein teeth, lemma and palea wrinkles, and ventral groove expansion, which are further used to identify Australian imported barley.

[23] .

[0007] In actual applications, due to the wide variety of barley varieties, very limited seed traits, and unclear morphological differences between different varieties, the test results are subject to subjective errors and low accuracy. For a wide variety of barley varieties, this technology is inefficient and time-consuming.

[0008] Biochemical markers are mainly based on the electrophoresis technology of seed isozymes and storage proteins such as alcohol-soluble proteins. According to the entry-exit inspection and quarantine industry standard of the People's Republic of China "SN / T 1048-2002 Protein Electrophoresis Analysis Method for Identification of Imported and Exported Grain and Feed Barley Varieties", alcohol-soluble proteins in barley grains are extracted, separated by vertical plate electrophoresis, and the electrophoresis pattern is obtained after silver staining. The electrophoresis pattern of alcohol-soluble proteins in barley is an inherent characteristic of the variety, and comparative analysis can identify barley varieties to a certain extent. [2] .

[0009] This technology has problems such as incomplete barley biochemical marker database, poor staining effect during detection, and difficult to identify band images; it is difficult to distinguish complex protein samples and closely related varieties, and its application range is narrow; the protein extraction process is time-consuming and inefficient, and the reagents are toxic to a certain extent, which affects physical health.

[0010] Identification of barley varieties using DNA molecular markers (a genetic marker developed based on the polymorphism of biological macromolecules) [3] . A technical solution for identifying barley varieties using simple sequence repeats (SSR) molecular markers. According to the agricultural industry standard of the People's Republic of China NY / T 2466-2013 "Technical Procedure for Identification of Barley Varieties - SSR Molecular Marker Method", the DNA sequence between primers is amplified using SSR markers using PCE amplification technology. The amplified products are electrophoresed and separated according to the size differences of the amplified fragments, and distinguished by silver nitrate staining or fluorescent dye labeling. This method uses denaturing polyacrylamide gel electrophoresis with silver staining detection and capillary electrophoresis with fluorescence detection.

[0011] For example, Wang Yanping et al. used 28 pairs of polymorphic SSR markers to analyze genetic diversity and construct fingerprint maps of 29 barley DUS test standard varieties.

[14] A total of 125 alleles were amplified, and the PIC value of each primer pair in 29 varieties was 0.25-0.81. Cluster analysis showed that the 29 barley DUS test standard varieties could be clustered into 4 categories with distant genetic relationships. These materials were first clustered according to the spike ridge type, indicating that there were large genetic differences between varieties with different spike ridge types. Summary of the invention

[0012] In view of the deficiencies in the prior art, the first object of the present invention is to provide an application of the KASP molecular marker combination in barley variety identification.

[0013] The second object of the present invention is to provide a method for developing the above-mentioned KASP molecular marker combination.

[0014] To achieve the above first purpose, the solution of the present invention is:

[0015] Application of KASP molecular marker combination in identification of barley varieties.

[0016] Furthermore, the KASP molecular marker combination facilitates barley breeding and germplasm resource management.

[0017] Furthermore, the KASP molecular marker combination specifically detects 18 SNP loci of barley; the SNP loci information corresponding to the KASP molecular markers are shown in Table 1:

[0018] Table 1

[0019]

[0020] Furthermore, each KASP molecular marker of the amplified KASP molecular marker combination includes two forward primers and one reverse primer. The sequences of the 18 KASP molecular markers are shown in SEQ ID NO.1 to SEQ ID NO.54, and the KASP marker information is shown in Table 2:

[0021] Table 2

[0022]

[0023]

[0024] To achieve the above second purpose, the solution of the present invention is:

[0025] A method for developing a KASP molecular marker combination comprises the following steps:

[0026] (1) Extract the same number of markers on chromosomes chr1H, chr2H, chr3H, chr4H, chr5H, chr6H, and chr7H;

[0027] (2) No other markers can exist within 200 bp upstream and downstream of each marker;

[0028] (3) Remove sequences with high N base content.

[0029] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0030] First, the markers selected by the present invention cover as wide a range of chromosome segments as possible, and use limited markers to reveal the differences between varieties as widely as possible. The barley KASP molecular marker combination for distinguishing barley variety resources of the present invention can effectively distinguish barley varieties from different sources, and has the characteristics of high stability, accuracy and low cost. The KASP marker can accurately judge the SNPs sites of a wide range of DNA samples (including DNA samples of complex genomes); in addition, high-throughput automatic nucleic acid extraction, automated genomic DNA liquid separation and PCR system construction can be performed, and there are 96-well plates, 384-well plates and 1536-well plates. The water bath PCR instrument for high-throughput PCR and DNA sample genotyping can improve the efficiency and accuracy of variety identification to a certain extent, and is not affected by the environment, so it is suitable for large-scale promotion and application, and is further used to supplement the molecular marker library for barley variety identification.

[0031] Second, the labeling development method of the present invention is simple, ingeniously designed, easy and fast to operate, and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a sensory map of Australian beer barley in the prior art (1: Arapiles; 2: Baudin; 3: Buloke; 4: Commander; 5: Dhow; 6: Fitzroy; 7: Flagship; 8: Gairdner; 9: Grimmett; 10: Hamelin; 11: Schooner; 12: Sloop; 13: Stirling; 14: Tallon; 15: Vlamingh).

[0033] Figure 2 The figure is the SDS-PAGE map of Canadian barley varieties in the prior art.

[0034] Figure 3 This is the amplification result of 29 materials using primer EBmac0635 in the prior art.

[0035] Figure 4 This is the SSR molecular clustering diagram of 29 materials in the prior art.

[0036] Figure 5 The figure shows the distribution of the KASP molecular marker in the present invention on the barley genome.

[0037] Figure 6 This is the genotyping diagram of the KASP molecular marker in the present invention.

[0038] Figure 7 This is a cluster diagram of KASP molecular markers of 169 materials in the present invention. DETAILED DESCRIPTION

[0039] The invention provides an application of a KASP molecular marker combination in barley variety identification and a development method thereof.

[0040] 1. Genotype data and data quality control

[0041] The BarleySNP50K chip developed by Illumina was used, which includes 44,040 SNP markers and 37,789 markers on chromosomes. There are 7 chromosomes in total, namely chr1H, chr2H, chr3H, chr4H, chr5H, chr6H and chr7H. Figure 5 As shown in Figure 2, 288 natural barley populations from around the world were scanned for whole genome chips to obtain raw data for SNP gene analysis.

[0042] Data filtering: 1) Filter out samples with unknown genotypes accounting for more than 10% of the total number of samples; 2) Filter out samples with an expected heterozygosity less than 0.3. After filtering under the above two conditions, 24,435 dimorphic SNP loci markers remain. Cut 200 bp upstream and downstream of the above markers, a total of 400 bp sequences, a total of 24,435 sequences, and then compare them to the barley reference genome to select specific sequences. If, in addition to comparing to the original position, it also compares to other positions in the genome for more than 80 bp (the comparison software uses blastn (v2.6.0+), and the evalue is set to 10 -5 ), then filter these specific sequences. Finally, 915 markers were obtained after filtering (most of the markers were co-dominant types, among which H type was less).

[0043] 2. Development and screening of KASP molecular markers

[0044] For these 915 markers, markers are uniformly extracted based on their length on the reference genome. The extraction principle is as follows:

[0045] 1) In principle, the same number of markers are extracted from each chromosome.

[0046] 2) No other markers can exist within 200bp upstream and downstream of each marker (excluded based on the original data).

[0047] 3) Remove sequences with high N base content.

[0048] Finally, a total of 124 markers were obtained.

[0049] 3. The distribution of the number of marker types is as follows Figure 5 shown.

[0050] 4. Genotyping steps

[0051] The genomic DNA of the sample to be tested was extracted according to the operating steps of the Shanghai Pudi Plant Genome Extraction Kit. The total KASP reaction system is 10μL, including 2×KASPMaster Mix 5μL, KASPAssayMix (primer mixed working solution) 0.125μL, and 4.875μL of template DNA with a concentration of 20ng / μL. The KASP reaction procedure is as follows: step one: 95℃, 10min; step two: 95℃, 20s, 61-55℃, 1min, each cycle is reduced by 0.6℃, for a total of 10 cycles; step three: 95℃, 20s, 55℃, 1min, for a total of 35 cycles, and the PCR reaction is carried out in a Hydrocycler purchased from LGC. 16 The PCR products were scanned and analyzed by KASP fluorescence analyzer (LGC model: FLUOstar Omega fluorescent plate reader).

[0052] 5. Selection and evaluation of barley KASP molecular markers

[0053] In order to further screen out representative markers, this study used 50 genomic DNAs of barley materials with distant genetic relationships as templates to filter 124 KASP molecular markers, mainly based on the ability to accurately type the HEX and FAM genotypes and PIC>0.30. Finally, 51 KASP molecular markers were screened out, with PIC ranging from 0.308 to 0.500, with an average of 0.449, and MAF ranging from 0.190 to 0.467, with an average of 0.368. Figure 6 shown.

[0054] 6. Application of KASP molecular markers in variety identification

[0055] In order to test the role of the developed KASP molecular markers in the identification of barley varieties, 18 KASP markers with high specificity and stability were selected with the standards of MAF>0.4 and PIC>0.45. Cluster analysis was performed on 169 barley resources from Jiangsu Province. The clustering results showed that these barley varieties were clustered according to geographical location, which could well reflect the genetic relationship of the tested materials. Figure 7 shown.

[0056] Based on the genotyping data of 18 KASP markers, a molecular ID card of 169 materials was established (Table 3), which can effectively distinguish most of the barley variety resources in Jiangsu Province, such as some barley sister lines 2000Jian25, 2000Jian26, and 2000Jian27 can be distinguished. However, a small number of barley lines cannot be distinguished, such as the barley lines from Yancheng, 96AC19-8, 96AC19-28, and 96AC19-14, which require more KASP markers for identification. For sister lines or near-isogenic lines that cannot be distinguished, specific marker sites can be further added to distinguish them. The technical method developed by the present invention is scalable.

[0057] Table 3 Molecular ID information of some barley materials

[0058]

[0059] References (such as patents / papers / standards)

[0060] [1] National Standard “Inspection Procedure for Crop Seeds - Authenticity and Purity of Varieties” (GB / T 3543.5-1995)

[0061] [2] Entry-Exit Inspection and Quarantine Industry Standard of the People’s Republic of China: “SN / T 1048-2002 Protein Electrophoresis Analysis Method for Identification of Barley Varieties for Import and Export Grain and Feed”

[0062] [3] Agricultural Industry Standard of the People’s Republic of China NY / T 2466-2013 “Technical Procedures for Identification of Barley Varieties—SSR Molecular Marker Method”.

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[15] Yang Zhenhua. Establishment and optimization of PCR reaction system for SSR molecular markers in beer barley. Heilongjiang Agricultural Science, 2015, (5): 26-29.

[0075]

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[0082]

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[0083] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Sequence Listing <110> Jiangsu Academy of Agricultural Sciences <120> Application of KASP molecular marker combination in identification of barley varieties and its development method <160> 54 <170> SIPOSequenceListing 1.0 <210> 1 <211> 44 <212> DNA <213> Artificial Sequence <400> 1 gaaggtgacc aagttcatgc ttgccttggt ggcctttgct tgct 44 <210> 2 <211> 43 <212> DNA <213> Artificial Sequence <400> 2 gaaggtcgga gtcaacggat tgccttggtg gcctttgctt gcc 43 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <400> 3 tagtgcctga catacctgct gcattgt 27 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <400> 4 gaaggtgacc aagttcatgc ttataaagca accatcaccg aaca 44 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <400> 5 gaaggtcgga gtcaacggat tataaagcaa ccatcaccga acc 43 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <400> 6 gctgcgcgtc tgtgtgtgtt tatatcg 27 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <400> 7 gaaggtgacc aagttcatgc ttacagatca accattgtcg agcgat 46 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <400> 8 gaaggtcgga gtcaacggat tacagatcaa ccattgtcga gcgac 45 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <400> 9 taagacactt gagggtaaaa ttggggatg 28 <210> 10 <211> 47 <212> DNA <213> Artificial Sequence <400> 10 gaaggtgacc aagttcatgc ttttcaggag aaatatctta ccgtaat 47 <210> 11 <211> 46 <212> DNA <213> Artificial Sequence <400> 11 gaaggtcgga gtcaacggat tttcaggaga aatatcttac cgtaac 46 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <400> 12 atgcaccctg gttggaagaa cgacaag 27 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <400> 13 gaaggtgacc aagttcatgc tgagtacatg cgagctgaat cgtc 44 <210> 14 <211> 44 <212> DNA <213> Artificial Sequence <400> 14 gaaggtcgga gtcaacggat tgagtacatg cgagctgaat cgtg 44 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <400> 15 tctgaagcaa ccaaacacgt cagacgtc 28 <210> 16 <211> 47 <212> DNA <213> Artificial Sequence <400> 16 gaaggtgacc aagttcatgc ttgattatct gttcctttac tgtctct 47 <210> 17 <211> 46 <212> DNA <213> Artificial Sequence <400> 17 gaaggtcgga gtcaacggat tgattatctg ttcctttact gtctcc 46 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <400> 18 caacaaggac tggtcaayaa atcaaatg 28 <210> 19 <211> 47 <212> DNA <213> Artificial Sequence <400> 19 gaaggtgacc aagttcatgc tataatactc ctaaggtcag tgcacct 47 <210> 20 <211> 46 <212> DNA <213> Artificial Sequence <400> 20 gaaggtcgga gtcaacggat ttaatactcc taaggtcagt gcaccc 46 <210> twenty one <211> 29 <212> DNA <213> Artificial Sequence <400> twenty one caaccacaga aagttaccaa atggaactc 29 <210> twenty two <211> 46 <212> DNA <213> Artificial Sequence <400> twenty two gaaggtgacc aagttcatgc ttgtaatttt ctcatgaata gtcttc 46 <210> twenty three <211> 46 <212> DNA <213> Artificial Sequence <400> twenty three gaaggtcgga gtcaacggat ttgtaatttt ctcatgaata gtcttg 46 <210> twenty four <211> 29 <212> DNA <213> Artificial Sequence <400> twenty four cacaaaccgt tcgtgttttc tctaggctg 29 <210> 25 <211> 45 <212> DNA <213> Artificial Sequence <400> 25 gaaggtgacc aagttcatgc tcatccttgt magagtaact ctaga 45 <210> 26 <211> 45 <212> DNA <213> Artificial Sequence <400> 26 gaaggtcgga gtcaacggat tcatccttgt magagtaact ctagc 45 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <400> 27 gatttctggg atggacgttt atcggatc 28 <210> 28 <211> 45 <212> DNA <213> Artificial Sequence <400> 28 gaaggtgacc aagttcatgc tacaccatct gttcatcctg ttcat 45 <210> 29 <211> 44 <212> DNA <213> Artificial Sequence <400> 29 gaaggtcgga gtcaacggat tcaccatctg ttcatcctgt tcag 44 <210> 30 <211> 29 <212> DNA <213> Artificial Sequence <400> 30 gcagttnaat ttgaactgcc aaaacatct 29 <210> 31 <211> 47 <212> DNA <213> Artificial Sequence <400> 31 gaaggtgacc aagttcatgc tacaagacga ttgaagatat gcattca 47 <210> 32 <211> 46 <212> DNA <213> Artificial Sequence <400> 32 gaaggtcgga gtcaacggat tcaagacgat tgaagatatg cattcg 46 <210> 33 <211> 28 <212> DNA <213> Artificial Sequence <400> 33 ttaggrgactt gtgccaagat acaagaac 28 <210> 34 <211> 47 <212> DNA <213> Artificial Sequence <400> 34 gaaggtgacc aagttcatgc tacgtacctc ctttctggtt taaaagt 47 <210> 35 <211> 47 <212> DNA <213> Artificial Sequence <400> 35 gaaggtcgga gtcaacggat tacgtacctc ctttctggtt taaaagg 47 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <400> 36 gtcgaattat caatctaggt acacatgtg 29 <210> 37 <211> 47 <212> DNA <213> Artificial Sequence <400> 37 gaaggtgacc aagttcatgc ttcttgtcgc cgacgggtcc gaccttt 47 <210> 38 <211> 46 <212> DNA <213> Artificial Sequence <400> 38 gaaggtcgga gtcaacggat tcttgtcgcc gacgggtccg accttg 46 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <400> 39 cacctgcttc cgcgcccatc tctccgac 28 <210> 40 <211> 48 <212> DNA <213> Artificial Sequence <400> 40 gaaggtgacc aagttcatgc ttgctaagtt tagtttcaat tttaaata 48 <210> 41 <211> 47 <212> DNA <213> Artificial Sequence <400> 41 gaaggtcgga gtcaacggat tgctaagttt agtttcaatt ttaaatc 47 <210> 42 <211> 28 <212> DNA <213> Artificial Sequence <400> 42 cggaatacag gaatccgcat ggcgggac 28 <210> 43 <211> 47 <212> DNA <213> Artificial Sequence <400> 43 gaaggtgacc aagttcatgc tatagctggt tcttttcttc attccat 47 <210> 44 <211> 46 <212> DNA <213> Artificial Sequence <400> 44 gaaggtcgga gtcaacggat ttagctggtt cttttcttca ttccac 46 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <400> 45 tatgtagttc father aaaggccac <210> 46 <211> 48 <212> DNA <213> Artificial Sequence <400> 46 gaaggtgacc aagttcatgc tactacacca ctatggaatc aggagaat <210> 47 <211> 47 <212> DNA <213> Artificial Sequence <400> 47 gaaggtcgga gtcaacggat tctacaccac tatggaatca ggagaac <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <400> 48 tgctatgttt ataactctat tacgcgatc <210> 49 <211> 47 <212> DNA <213> Artificial Sequence <400> 49 gaaggtgacc aagttcatgc tggataatct tatatgttaa gcacagt <210> 50 <211> 46 <212> DNA <213> Artificial Sequence <400> 50 gaaggtcgga gtcaacggat tgataatctt atatgttaag cacagc 46 <210> 51 <211> 29 <212> DNA <213> Artificial Sequence <400> 51 tcgcggagta gataagatgc ttaactagg 29 <210> 52 <211> 46 <212> DNA <213> Artificial Sequence <400> 52 gaaggtgacc aagttcatgc tagctcgtag ctacggaaat actcga 46 <210> 53 <211> 45 <212> DNA <213> Artificial Sequence <400> 53 gaaggtcgga gtcaacggat tgctcgtagc tacggaaata ctcgg 45 <210> 54 <211> 29 <212> DNA <213> Artificial Sequence <400> 54 gataagattg tcataaattg accgatcag 29

Claims

1. Application of KASP molecular marker combination in identification of barley varieties, wherein the KASP molecular marker combination detects 18 SNP sites of barley respectively; the details are as follows: Each KASP molecular marker of the amplified KASP molecular marker combination includes two forward primers and one reverse primer, as follows:

2. The use according to claim 1, characterized in that: The KASP molecular marker combination facilitates barley breeding.

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