Application of SNP loci combination and KASP primers in identification of rice landraces

Abstract

The application belongs to the technical field of biology, and particularly relates to SNP site combination and application of primers thereof in identification of rice local varieties. The application is based on whole genome resequencing data of 114 rice local varieties in Taihu Lake basin, and more than one million SNP sites are obtained by mining, 27 core SNP markers and matching KASP primers are identified and screened from the SNP sites, and the identification efficiency of 12 KASP markers among the 114 rice local germplasm resources can reach 100%. The application provides a set of reliable and convenient SNP markers for later use for biological research of the rice local varieties in the Taihu Lake basin. The set of SNP markers and detection primers can be used for development of a rice SNP detection kit, construction of a DNA fingerprint map of the rice local varieties, genotyping of the rice local germplasm resources and genetic populations, detection of a background recovery rate of a rice directional improvement variety, detection of purity / true degree of the rice local varieties, rice molecular marker assisted selection breeding and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a SNP site combination of rice local varieties based on whole genome resequencing and KASP technology and application of primers thereof in identification of rice local varieties. BACKGROUND

[0002] China has a long history of rice cultivation and domestication, and has formed multiple secondary centers of origin across the country, which play an important role in rice genetic diversity and germplasm conservation. The middle and lower reaches of the Yangtze River, especially the Taihu Basin (covering Jiangsu, Zhejiang, Anhui and parts of Shanghai), as one of the secondary centers of origin of rice cultivation, is particularly important. The Taihu Basin is not only a key area of rice diversity, but also contains rich rice germplasm resources, which have immeasurable value for modern rice variety improvement and future breakthroughs in rice production.

[0003] However, with the rapid development of modern agricultural technology and the widespread popularity of high-yield varieties, many traditional rice local varieties are at risk of disappearing or being mixed. This trend not only leads to the loss of genetic diversity, but also makes it difficult to accurately distinguish these valuable local varieties through traditional phenotype analysis. Therefore, protecting and accurately identifying rice local varieties in the Taihu Basin has become a key challenge for current rice germplasm conservation and sustainable use.

[0004] In the field of genetics and genomics, single nucleotide polymorphism (SNP) is a popular and reliable molecular marker due to its co-dominant inheritance, high abundance, wide distribution, and suitability for high-throughput automated genotyping. With the continuous progress of sequencing technology, the identification ability of SNP has been significantly improved, making it widely used in many fields such as agriculture, medicine and evolutionary biology. Compared with traditional molecular markers, SNP has higher resolution and accuracy, and has become an indispensable tool in modern genomic research and breeding projects.

[0005] Among various SNP genotyping methods, the competitive allele-specific PCR (KASP) assay technique has become a benchmark technique worldwide due to its high reliability, cost-effectiveness, and simplicity, scalability and flexibility. The KASP technique uses the principle of fluorescence resonance energy transfer (FRET) to detect predetermined co-dominant alleles at specific genomic loci, and realizes accurate detection of SNPs by using quantitative PCR and allele-specific primers. By focusing on a carefully selected subset of SNPs, the KASP technique not only maintains high reliability in large-scale genotyping, but is also particularly suitable for distinguishing closely related or phenotypically similar rice landraces. Therefore, the KASP technique has broad prospects and important value in applications such as germplasm characterization and marker-assisted selection (MAS). However, the current SNP detection of rice has the problems of low discrimination degree of KASP technique and complicated identification method. SUMMARY

[0006] The purpose of the present application is to provide a SNP site combination and its KASP primer in the identification of rice landraces.

[0007] The technical solutions provided by the present application are as follows:

[0008] The application of the SNP site combination in the identification of rice landraces, the SNP site combination comprises Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25, Os27;

[0009] Os03 is located at position 1232061 on chromosome 4, and its deoxynucleotide is G or A;

[0010] Os04 is located at position 23145738 on chromosome 4, and its deoxynucleotide is T or A;

[0011] Os11 is located at position 14510534 on chromosome 8, and its deoxynucleotide is C or G;

[0012] Os14 is located at position 12401152 on chromosome 10, and its deoxynucleotide is G or A;

[0013] Os17 is located at position 12927938 on chromosome 7, and its deoxynucleotide is G or A;

[0014] Os18 is located at position 9336153 on chromosome 10, and its deoxynucleotide is T or C;

[0015] Os19 is located at position 12872110 on chromosome 1, and its deoxynucleotide is C or T;

[0016] Os21 is located at 6162500 on chromosome 6, and its deoxynucleotide is G or T;

[0017] Os22 is located at 28946285 on chromosome 7, and its deoxynucleotide is G or T;

[0018] Os24 is located at 9771123 on chromosome 81, and its deoxynucleotide is C or T;

[0019] Os25 is located at 4264851 on chromosome 10, and its deoxynucleotide is G or A;

[0020] Os27 is located at 22989532 on chromosome 1, and its deoxynucleotide is T or C.

[0021] Further, the single nucleotide polymorphisms of the 12 SNP sites are shown in Table 2.

[0022] The application of the SNP site combination in identifying rice local varieties, the SNP site combination comprises 27 SNP sites, respectively Os01-Os27;

[0023] Os01 is located at 24407095 on chromosome 1, and its deoxynucleotide is T or G;

[0024] Os02 is located at 5928030 on chromosome 3, and its deoxynucleotide is G or A;

[0025] Os03 is located at 1232061 on chromosome 4, and its deoxynucleotide is G or A;

[0026] Os04 is located at 23145738 on chromosome 4, and its deoxynucleotide is T or A;

[0027] Os05 is located at 32014814 on chromosome 4, and its deoxynucleotide is C or T;

[0028] Os06 is located at 34415769 on chromosome 4, and its deoxynucleotide is G or A;

[0029] Os07 is located at 29706892 on chromosome 6, and its deoxynucleotide is C or T;

[0030] Os08 is located at 3154160 on chromosome 7, and its deoxynucleotide is C or T;

[0031] Os09 is located at 10370491 on chromosome 7, and its deoxynucleotide is A or C;

[0032] Os10 is located at 28631021 on chromosome 7, and its deoxynucleotide is T or G;

[0033] Os11 is located at 14510534 on chromosome 8, and its deoxynucleotide is C or G;

[0034] Os12 is located at 19429341 on chromosome 8, and its deoxynucleotide is A or G;

[0035] Os13 is located at 7814769 on chromosome 9, and its deoxynucleotide is A or G;

[0036] Os14 is located at 12401152 on chromosome 10, and its deoxynucleotide is G or A;

[0037] Os15 is located at 13941647 on chromosome 3, and its deoxynucleotide is T or A;

[0038] Os16 is located at 6718908 on chromosome 1, and its deoxynucleotide is A or G;

[0039] Os17 is located at 12927938 on chromosome 7, and its deoxynucleotide is G or A;

[0040] Os18 is located at 9336153 on chromosome 10, and its deoxynucleotide is T or C;

[0041] Os19 is located at 12872110 on chromosome 1, and its deoxynucleotide is C or T;

[0042] Os20 is located at 3646002 on chromosome 5, and its deoxynucleotide is G or C;

[0043] Os21 is located at 6162500 on chromosome 6, and its deoxynucleotide is G or T;

[0044] Os22 is located at 28946285 on chromosome 7, and its deoxynucleotide is G or T;

[0045] Os23 is located at 8470327 on chromosome 7, and its deoxynucleotide is G or C;

[0046] Os24 is located at 9771123 on chromosome 81, and its deoxynucleotide is C or T;

[0047] Os25 is located at 4264851 on chromosome 10, and its deoxynucleotide is G or A;

[0048] Os26 is located at 3866539 on chromosome 10, and its deoxynucleotide is C or G;

[0049] Os27 is located at 22989532 on chromosome 1, and its deoxynucleotide is T or C;

[0050] The application also provides a primer combination for detecting all or part of the above-mentioned 27 SNP sites.

[0051] Further, the primer combination is preferably a primer for detecting the following 12 SNP sites: Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25, and Os27.

[0052] Each KASP primer corresponding to each SNP site to be detected comprises two forward primers and one reverse primer; the two forward primers are respectively denoted as forward primer 1 and forward primer 2; the 5' end of the forward primer 1 is connected to a fluorescent label sequence, and the 5' end of the forward primer 2 is connected to another fluorescent label sequence.

[0053] The nucleotide sequence of the forward primer 1 for detecting Os01 is shown in SEQ ID No. 1, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 28;

[0054] The nucleotide sequence of the forward primer 1 for detecting Os02 is shown in SEQ ID No. 2, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 29, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 56;

[0055] The nucleotide sequence of the forward primer 1 for detecting Os03 is shown in SEQ ID No. 3, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 30, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 57;

[0056] The nucleotide sequence of the forward primer 1 for detecting Os04 is shown in SEQ ID No. 4, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 31, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 58;

[0057] The nucleotide sequence of the forward primer 1 for detecting Os05 is shown in SEQ ID No. 5, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 32, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 59;

[0058] The nucleotide sequence of the forward primer 1 for detecting Os06 is shown as SEQ ID No. 6, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 33, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 60;

[0059] The nucleotide sequence of the forward primer 1 for detecting Os07 is shown as SEQ ID No. 7, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 34, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 61;

[0060] The nucleotide sequence of the forward primer 1 for detecting Os08 is shown as SEQ ID No. 8, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 35, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 62;

[0061] The nucleotide sequence of the forward primer 1 for detecting Os09 is shown as SEQ ID No. 9, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 36, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 63;

[0062] The nucleotide sequence of the forward primer 1 for detecting Os10 is shown as SEQ ID No. 10, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 37, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 64;

[0063] The nucleotide sequence of the forward primer 1 for detecting Os10 is shown as SEQ ID No. 10, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 37, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 64;

[0064] The nucleotide sequence of the forward primer 1 for detecting Os11 is shown as SEQ ID No. 11, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 38, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 65;

[0065] The nucleotide sequence of the forward primer 1 for detecting Os12 is shown as SEQ ID No. 12, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No. 39, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 66;

[0066] The nucleotide sequence of forward primer 1 for detecting Os13 is shown as SEQ ID No. 13, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 40, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 67;

[0067] The nucleotide sequence of forward primer 1 for detecting Os14 is shown as SEQ ID No. 14, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 41, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 68;

[0068] The nucleotide sequence of forward primer 1 for detecting Os15 is shown as SEQ ID No. 15, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 42, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 69;

[0069] The nucleotide sequence of forward primer 1 for detecting Os16 is shown as SEQ ID No. 16, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 43, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 70;

[0070] The nucleotide sequence of forward primer 1 for detecting Os17 is shown as SEQ ID No. 17, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 44, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 71;

[0071] The nucleotide sequence of forward primer 1 for detecting Os18 is shown as SEQ ID No. 18, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 45, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 72;

[0072] The nucleotide sequence of forward primer 1 for detecting Os19 is shown as SEQ ID No. 19, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 46, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 73;

[0073] The nucleotide sequence of forward primer 1 for detecting Os20 is shown as SEQ ID No. 20, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 47, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 74;

[0074] The nucleotide sequence of forward primer 1 for detecting Os21 is shown as SEQ ID No. 21, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 48, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 75;

[0075] The nucleotide sequence of forward primer 1 for detecting Os22 is shown as SEQ ID No. 22, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 49, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 76;

[0076] The nucleotide sequence of forward primer 1 for detecting Os23 is shown as SEQ ID No. 23, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 50, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 77;

[0077] The nucleotide sequence of forward primer 1 for detecting Os24 is shown as SEQ ID No. 24, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 51, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 78;

[0078] The nucleotide sequence of forward primer 1 for detecting Os25 is shown as SEQ ID No. 25, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 52, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 79;

[0079] The nucleotide sequence of forward primer 1 for detecting Os26 is shown as SEQ ID No. 26, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 53, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 80;

[0080] The nucleotide sequence of forward primer 1 for detecting Os27 is shown as SEQ ID No. 27, the nucleotide sequence of forward primer 2 is shown as SEQ ID No. 54, and the nucleotide sequence of reverse primer is shown as SEQ ID No. 81.

[0081] Further, the fluorescent tag sequence connected to the 5' end of the forward primer 1 is a FAM fluorescent tag sequence; and the fluorescent tag sequence connected to the 5' end of the forward primer 2 is a HEX fluorescent tag sequence.

[0082] Still further, the FAM fluorescent tag sequence is shown as SEQ ID No. 82; and the HEX fluorescent tag sequence is shown as SEQ ID No. 83.

[0083] In the present application, the above primer combination is referred to as OsSNP KASP Primermix.

[0084] The present application also provides a kit of the above primer combination OsSNP KASP Primermix.

[0085] Further, the reagent or kit can also contain KASP 2xMastermix, MgCl2 and ddH2O.

[0086] The KASP 2xMastermix in the reagent or kit contains fluorescent probe A, fluorescent probe B, quencher probe A and quencher probe B.

[0087] The nucleotide sequence of the fluorescent probe A is identical to the nucleotide sequence of the fluorescent tag sequence connected to the 5' end of the forward primer 1, and a fluorescent group A is connected to the 5' end; the nucleotide sequence of the quencher probe A is reverse complementary to the nucleotide sequence of the tag sequence A, and a quencher group is connected to the 3' end.

[0088] The nucleotide sequence of the fluorescent probe B is identical to the nucleotide sequence of the fluorescent tag sequence connected to the 5' end of the forward primer 2, and a fluorescent group B is connected to the 5' end; the nucleotide sequence of the quencher probe B is reverse complementary to the nucleotide sequence of the tag sequence B, and a quencher group is connected to the 3' end.

[0089] In the specific embodiment of the present application, the fluorescent group A is FAM; the fluorescent group B is HXE; and the quencher group is BHQ.

[0090] The present application also provides the use of the above SNP site combination or the above primer combination or the above kit in any one of the following;

[0091] (1) developing a SNP site detection kit for rice; the SNP site referred to herein is all or part of the following 27 SNP sites:

[0092] (2) genotyping rice germplasm resources and / or genetic populations;

[0093] (3) constructing a DNA fingerprint map of rice germplasm resources / variety in Taihu Lake Basin;

[0094] (4) detecting the purity and / or authenticity of rice varieties.

[0095] The present application also provides a method for genotyping rice germplasm resources and / or genetic populations, comprising the following steps:

[0096] (a1) extracting genomic DNA from all rice samples in the test rice germplasm resources and / or genetic populations, respectively;

[0097] (a2) PCR reaction system was prepared for each SNP site in the DNA of the test rice sample according to the 27 SNP sites described above, and PCR amplification was performed;

[0098] (a3) The FAM and HEX fluorescence values of the PCR amplification products of the DNA of the test rice sample at the 27 SNP sites described above were detected by using a KASP fluorescence detector, and the genotyping results of the test rice sample at the 27 SNP sites were obtained (i.e. the specific nucleotides at the SNP sites, the same below). The data of a single SNP site is recorded as XX, YY or XY, wherein X and Y are the single nucleotide polymorphism at the site, specifically A, T, G or C, arranged in this order. The missing or invalid data is recorded as --. If the sum of the proportions of XY and -- genotypes of a sample is higher than 10%, the sample is not used.

[0099] The application also provides a method for constructing a DNA fingerprint of a rice local germplasm resource / variety in the Taihu Lake Basin, comprising the following step (b1):

[0100] (b1) Extracting the genomic DNA of all rice germplasm resources or varieties used for constructing the DNA fingerprint, detecting the genotypes of the 27 SNP sites described in claim 1, and arranging the genotypes of each rice germplasm resource or variety at the 27 SNP sites in order of site number to form a specific sequence, which is the DNA fingerprint of the rice germplasm resource or variety;

[0101] (b2) According to the following, the genotype combination of the 27 core characteristic SNP sites in step (b1) is converted into a computer string label:

[0102] The genotyping results of the 27 core characteristic SNP sites consist of XX, YY, XY or --, wherein X and Y are the single nucleotide at the site, which is A, T, G or C, and -- represents missing or invalid data. Each SNP site genotype combination has 11 possible genotype combinations, including AA, TT, GG, CC, AT, AG, AC, TG, TC, GC and NN;

[0103] According to the above rules, the genotype combination of the 27 core characteristic SNP sites in the DNA fingerprint of the rice germplasm resource or variety is converted into a string of 54 characters, which is the core code of the DNA fingerprint of the rice germplasm resource or variety;

[0104] (b3) The core code of the DNA fingerprint of the rice germplasm resource or variety constructed in step (b2) is converted into a two-dimensional code or a bar code that can be recognized by a device terminal, so as to intelligently identify and manage the rice germplasm resource or variety;

[0105] (b4) based on the core code of the DNA fingerprint of the (b2) rice germplasm or variety, the identification efficiency of the rice material is evaluated, wherein only 12 markers including Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25 and Os27 are required to identify 114 rice materials tested at 100%.

[0106] Method C: a method for detecting the purity and / or authenticity of a rice variety, comprising the following steps:

[0107] (c1) preparing 50 or more seeds of the rice variety to be tested and the standard control rice variety, germinating and sowing, and when the seedlings grow to one leaf one heart, taking equal amounts of tissue from single seedlings, and then mixing and sampling according to the rice variety to be tested and the standard control rice variety, and extracting genomic DNA;

[0108] (c2) detecting the genotypes of the 27 SNP sites described above, thereby obtaining the genotype data of the 27 SNP sites of the rice variety to be tested and the standard control rice variety. When the genotype of the standard control rice variety sample at a SNP site is XX or YY, the site is an effective site, and when the genotype of the site is XY or -- type, the site is an ineffective site. If the proportion of ineffective sites of the standard control rice variety sample is higher than 10%, it is not used, and the test is invalid.

[0109] (c3) comparing the genotypes of the rice variety to be tested and the standard control rice variety at the effective sites of the 27 SNP sites described above for the standard control rice variety sample, and the results are two: the same or different;

[0110] (c4) the purity (P) of the variety is expressed in % and calculated according to formula (1):

[0111]

[0112] In the formula:

[0113] P - variety purity;

[0114] S1 - the number of effective sites of the standard control rice variety;

[0115] S2 - the number of sites with different genotypes between the rice variety to be tested and the standard control rice variety among the effective sites of the standard control rice variety;

[0116] (c5) when the purity of the to-be-tested rice variety is ≤ 95%, it is determined that the to-be-tested rice variety and the standard control variety are different varieties, when the purity of the to-be-tested rice variety is > 95% and < 100%, it is determined that the to-be-tested rice variety is an approximate variety or a suspected same variety of the standard control variety, and when the purity of the to-be-tested rice variety is 100%, it is determined that the to-be-tested rice variety and the standard control variety are the same variety;

[0117] In the above-mentioned methods, the rice varieties are all conventional local varieties, and are of japonica rice or indica rice type.

[0118] In the specific embodiments of the present application, the rice is specifically selected from the 114 rice samples shown in Table 1.

[0119] Further, in the above-mentioned methods, the genotype detection of the 27 SNP sites can be completed by using the above-mentioned primer combination or kit.

[0120] Beneficial effects

[0121] Based on the more than one million SNP variation site data obtained from the whole genome resequencing work of rice local varieties in the Taihu Basin carried out by Suzhou Seed Management Station, after strict quality control and sequencing verification of the variation site data, 27 SNP markers and matching KASP primers are identified and screened by using the KASP technology platform. Further, it is found that only 12 markers including Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25 and Os27 are needed to identify the tested rice materials at 100%. The present application provides a set of reliable and convenient SNP markers for later use for rice biological research. The SNP markers and detection primers can be used for the development of rice SNP detection kits, the construction of rice variety DNA fingerprint, the genotyping of rice germplasm resources and genetic population, the construction of rice germplasm resources / variety DNA fingerprint, the detection of rice variety purity / authenticity, etc.

[0122] The present application aims to use modern genomics technologies such as KASP to efficiently and accurately identify and protect rice local varieties in the Taihu Basin, so as to play an important role in maintaining rice genetic diversity, promoting rice variety improvement and ensuring food security, etc. BRIEF DESCRIPTION OF DRAWINGS

[0123] Figure 1 Data record for the screening process of the rice SNP markers of the present application.

[0124] Figure 2 Efficiency evaluation for the identification of 114 rice germplasm resources by using 27 KASP markers.

[0125] Figure 3 An example of KASP experimental detection results is shown in Figure 1. DETAILED DESCRIPTION

[0126] The application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0127] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0128] Example 1, Screening and development of SNP markers and KASP primers for rice local varieties in Taihu Basin

[0129] Based on the SNP variation data of whole genome resequencing of 114 rice germplasm resources / variety materials (Table 1) completed by Suzhou Seed Management Station (provided by Suzhou Seed Management Station, file name all.raw.snp.vcf, number of markers 6186437), combined with mathematical statistical analysis and strict filtering, as well as first-generation sequencing verification and KASP test verification, 27 high-confidence SNP markers and matching KASP primers were screened out. The specific steps for screening and developing SNP markers and KASP primers are as follows:

[0130] (1) Use GATK (v4.1.4) software to set hard filtering for original SNP sites to exclude false positive variations caused by sequencing quality problems, execute the following statement:

[0131] gatk --java-options "-Xmx16g" # Set memory usage limit

[0132] -D java.io, tmpdir =. / tmp" # Set temporary file directory

[0133] VariantFiltration # Variant filtration

[0134] -R GCF_001433935.1_IRGSP-1.0_genomic.fna # Reference genome

[0135] # Set input file, set filter criteria

[0136] # Output population SNP file with hard filter criteria

[0137] (2) Remove sites that do not meet hard filter criteria, execute the following statement:

[0138] # Set memory usage limit

[0139] # Set temporary file directory

[0140] # Extract variants

[0141] # Reference genome

[0142] # Remove variants that do not meet criteria

[0143] # Output hard filtered SNP file

[0144] (3) Use vcftools (v0.1.16) software to filter the extracted SNPs, select sites with only two alleles, maximum missing rate less than 5%, and minimum allele frequency MAF higher than 5%, execute the following statement:

[0145] # Recode --recode-INFO-all --stdout | bgzip -c -f > all.snp.filtered.vcf.gz

[0146] The number of markers reserved after data quality control in this step is 1078168.

[0147] (4) The sequence of 50bp before and after each SNP site on the chromosome is extracted, and its conservation is checked. Further quality control is performed to reserve sites with an average sequencing depth of more than 5x and a quality value greater than 30, and 139325 SNP sites are filtered.

[0148] (5) The sequence of 100bp upstream and downstream of each site is intercepted, and the sequence is aligned to the reference genome using blast (v2.10.1+) software to check its specificity, and SNP sites aligned to multiple positions are excluded. The polymorphic information content PIC of the remaining sites is calculated, and sites with PIC>0.35 are reserved, and primer design software primer3 (v2.4.0) is used to design primers for SNP markers and convert them to KASP markers.

[0149] The number of markers successfully designed in this step is 14552.

[0150] (6) Combined with steps (3)-(5), the SNP markers are manually optimized, and the standard is:

[0151] The markers in step (5) have designed KASP primers;

[0152] The markers located in the coding region of the gene or near the coding region are preferred;

[0153] There are at most 2 markers on each haplotype block;

[0154] The markers are relatively evenly distributed on the chromosome.

[0155] A total of 65 SNP markers are selected for subsequent experimental verification in this step.

[0156] (7) Three rice varieties with large phenotypic differences (TH_10, TH_67, TH_113, see Table 1) are selected, and the genotypes of the SNP sites selected in step (6) are verified by one-generation sequencing. If the sequencing is successful and the result is consistent with the resequencing result, it indicates that the SNP site really exists and is suitable as a KASP marker.

[0157] (8) According to the results of (7), the manual optimization and synthesis primer experimental verification operations are repeated again. Finally, 27 SNP markers are selected as available markers.

[0158] The position genotype information of the 27 SNP markers obtained in the application is shown in Table 2. Among them, the physical position of the SNP marker site is annotated based on the rice variety Nipponbare reference genome IRGSP-1.0 version.

[0159] Table 1. 114 local rice germplasm resources / varieties in the Taihu Lake Basin

[0160]

[0161]

[0162]

[0163] Table 2 Information about 27 SNP loci

[0164] SEQ ID NO Chromosome Position Allelic variation Os01 Chr01 24407095 T / G Os02 Chr03 5928030 G / A Os03 Chr04 1232061 G / A Os04 Chr04 23145738 T / A Os05 Chr04 32014814 C / T Os06 Chr04 34415769 G / A Os07 Chr06 29706892 C / T Os08 Chr07 3154160 C / T Os09 Chr07 10370491 A / C Os10 Chr07 28631021 T / G Os11 Chr08 14510534 C / G Os12 Chr08 19429341 A / G Os13 Chr09 7814769 A / G Os14 Chr10 12401152 G / A Os15 Chr03 13941647 T / A Os16 Chr01 6718908 A / G Os17 Chr07 12927938 G / A Os18 Chr10 9336153 T / C Os19 Chr01 12872110 C / T Os20 Chr05 3646002 G / C Os21 Chr06 6162500 G / T Os22 Chr07 28946285 G / T Os23 Chr07 8470327 G / C Os24 Chr08 9771123 C / T Os25 Chr10 4264851 G / A Os26 Chr10 3866539 C / G Os27 Chr11 22989532 T / C

[0165] The data records of rice SNP marker screening process are as follows Figure 1 As shown, the KASP primers developed by the present invention for detecting the aforementioned 27 SNP markers include two forward primers and one reverse primer for each SNP marker. Forward primer 1 has a HEX fluorescent tag sequence, 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID No. 82), attached to its 5' end. Forward primer 2 has a HEX fluorescent tag sequence, 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID No. 83), attached to its 5' end. The primers corresponding to each marker of the present invention are shown in Table 3.

[0166] Table 3. Primers corresponding to each marker of the present invention

[0167]

[0168]

[0169] The present invention provides a detection kit for 27 rice SNP markers (see Table 2). The kit contains the 27 primer sets (see Table 3), synthesized by Sangon Biotech (Shanghai) Co., Ltd. Forward Primer 1 and Reverse Primer 2 have been pre-coated with FAM and HEX fluorescent sequences, respectively. The product is labeled OsSNP KASP Primermix.

[0170] The kit also contains KASP 2×Mastermix (product catalog number KBS-1016-002) from LGC, UK. 2×KASP Mix consists of fluorescent probe A, fluorescent probe B, quencher probe A and quencher probe B, as well as high-fidelity Taq enzyme, dNTP, MgCl2, and 1% dNTP. 2+The nucleotide sequence of the fluorescent probe A is 5'-gaaggtgaccaagttcatgct-3' (SEQ ID No. 82), and a FAM fluorescent group is connected to the 5' end; the nucleotide sequence of the fluorescent probe B is 5'-gaaggtcggagtcaacggatt-3' (SEQ ID No. 83), and a HEX fluorescent group is connected to the 5' end; the nucleotide sequence of the quenched probe A is 5'-AGCATGAACTTGGTCACCTTC-3' (SEQ ID No. 84), and a quenched group BHQ is connected to the 3' end; and the nucleotide sequence of the quenched probe B is 5'-AATCCGTTGACTCCGACCTTC-3' (SEQ ID No. 85), and a quenched group BHQ is connected to the 3' end.

[0171] The kit is also provided with MgCl2 and ddH2O, wherein the MgCl2 is a product of Beijing Woke Biological Technology Co., Ltd., the product CAS number is 7786-30-3, and the ddH2O is autoclaved double distilled water.

[0172] Example 2, rice germplasm resource / variety genotyping

[0173] Referring to the technical document of the British LGC company and combining with the actual operation, the application provides a method system for genotyping rice germplasm resources / variety (specifically, 114 rice materials in Table 1) by using 27 SNP markers (Tables 2 and 3) in Example 1, which comprises the following steps:

[0174] (1) Extraction of DNA of the rice sample to be tested: 200-300 mg of seedlings or leaves of the rice variety to be tested is placed in a 1.5 ml centrifuge tube, and liquid nitrogen is added for grinding; 700 μL of 65 ℃ preheated CTAB extraction solution is added to each tube; the centrifuge tube is placed in a 65 ℃ water bath for 30-60 min, and the centrifuge tube is gently shaken every 10 min or so during the period; after the water bath is completed, 0.5 mL of chloroform-isopentanol mixed solution is added, and the mixture is mixed by vigorous shaking, and centrifuged at 12000 rmp for 5-10 min; the supernatant in the centrifuge tube is taken into a new centrifuge tube, 2 times the volume of pre-cooled isopropyl alcohol is added, and the mixture is mixed by inversion, and then placed in a-20 ℃ refrigerator for precipitation for 30 min or more, and centrifuged at 12000 rmp for 10 min, and the supernatant is discarded; the precipitate is washed twice with 75% ethanol, and centrifuged at 12000 rmp for 3 min, and the supernatant is discarded; the precipitate is naturally dried, dissolved in 50 μL of deionized water or TE, the concentration is detected, the concentration is diluted to 100 ng / μl, and then used immediately or stored in a-20 ℃ refrigerator.

[0175] This step is only a recommended method, and other methods that can achieve the same effect can also be used.

[0176] (2) For 27 SNP markers (Table 2 and Table 3), KASP Mastermix, OsSNP KASP Primermix, and PCR reaction system were added in turn, and PCR amplification was performed. Among them, OsSNP KASP Primermix was composed of forward primer 1 with 5' end connected with fluorescent label FAM, forward primer 2 with 5' end connected with fluorescent label HEX, and reverse primer, which were mixed with ddH2O in a volume ratio of 12:12:30:46.

[0177] The PCR reaction system is shown in Table 4. The PCR reaction conditions are shown in Table 5.

[0178] Table 4 Construction of PCR reaction system

[0179] Component 96-well plate 2x KASP mastermix 2.5ul OsSNP KASP Primermix 1.25ul Template DNA 1.25ul ddH2O 0ul Total 5ul 1 Reaction

[0180] Table 5 PCR reaction conditions

[0181]

[0182] PCR reaction: 1) Diluted DNA was aliquoted into a 200 μL 96-well plate, and 1.25 μL was taken by an 8-hole gun (manufacturer Eppendorf-10 μL) into a 96-well PCR plate, and centrifuged to the bottom (manufacturer AXYGEN) for use; 2) KASP Mastermix, OsSNP KASP Primermix, and ddH2O were mixed in proportion to form PCR-mix; 3) PCR-mix was aliquoted into each reaction well at 3.75 μL per reaction, then centrifuged to the bottom, and sealed with a silica gel cap or sealing film; 4) The reaction was completed according to the previous reaction program in a 96-well PCR instrument (model CFX Connet TM Real-Time System, Bio-Read, USA).

[0183] (3) Fluorescence detection and genotyping: After the PCR was completed, the 96-well plate was removed, the silica gel cap or sealing film was removed after cooling, and the plate was placed on the SNP genotyping instrument (model FLUOstar Omega SNP) to detect FAM and HEX fluorescence values, KlusterCaller software was used to analyze the genotyping results, and genotypes were obtained.

[0184] The single SNP marker site genotyping results were recorded as "XX" or "XY" "YY", "XX" and "YY" indicating homozygous, and "XY" indicating heterozygous, wherein X and Y were single nucleotides such as A, T, G, and C. If a SNP marker could not determine the single nucleotides A, T, G, and C, it was invalid data, and the invalid data was recorded as "--".

[0185] Figure 3 Figure 1 shows an example of KASP test results. Diamond (NTC); circle (XX); square (YY); triangle (XY).

[0186] If the proportion of invalid data of a marker is higher than 10%, the marker is invalid; if the proportion of invalid data of a sample is higher than 10%, the sample is invalid, and is not used.

[0187] (4) Genotyping of 114 rice germplasm resources / variety (Table 1) at the 27 marker sites, and the results are shown in Table 6.

[0188] Example 3, construction of DNA fingerprint core code of 114 rice local germplasm resources in Taihu Basin

[0189] The fingerprint of 114 rice germplasm resources (Table 1) preserved by Suzhou Seed Management Station is constructed by using the fingerprint method of rice germplasm resources / variety, and the core code is drawn, including the following steps:

[0190] (1) According to the steps of Example 2, the genotyping of 114 rice germplasm resources / variety (Table 1) at the 27 SNP markers (Table 2) sites is completed, and the results are shown in Table 6. The genotyping results of the 27 SNP sites corresponding to each material in Table 6 are one of the fingerprint representation methods of the material.

[0191] (2) According to the following, the genotype combination is converted into a computer string label for the 27 core characteristic SNP sites in step (1):

[0192] First, the genotyping results of the 27 core characteristic SNP sites are composed of XX, YY, XY or --, wherein X and Y are single nucleotides of A, T, G or C, and -- represents missing or invalid data. Each SNP site genotype combination has 11 possible genotypes, including AA, TT, GG, CC, AT, AG, AC, TG, TC, GC and NN.

[0193] According to the above rules, the genotype combination of the 27 core characteristic SNP sites in the DNA fingerprint of the rice germplasm resource or variety is converted into a string composed of 27 groups (54 characters), which is the core code of the DNA fingerprint of the rice germplasm resource or variety;

[0194] (3) The core code of the DNA fingerprint of the 114 rice germplasm resources / variety is obtained in turn, which can be converted into a two-dimensional code (Table 6) as the characteristics of the corresponding rice germplasm resource / variety, and is used as the basis for testing the authenticity of the rice germplasm resource / variety in the process of exchange and sale.

[0195] (4) Based on the core code of the DNA fingerprint of the rice germplasm resource or variety in step (2), the identification efficiency of the rice material is evaluated Figure 2 ), wherein only 12 markers including Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25 and Os27 can 100% identify the 114 rice materials tested.

[0196] Table 6 Core code of DNA fingerprint of 114 rice local germplasm resources / variety in Taihu Lake Basin

[0197]

[0198]

[0199]

[0200]

[0201] Based on the whole genome resequencing data of 114 rice, the present application uses the data analysis method explored in the early stage to obtain more than one million SNP sites, and after strict quality control and sequencing verification of the variation site data, 27 SNP markers and matching KASP primers are identified and screened by using the KASP technology platform. The present application provides a set of reliable and convenient SNP markers for later use for rice biological research. The SNP markers and detection primers can be used for rice SNP detection kit development, rice variety DNA fingerprint construction, rice germplasm resource and genetic population genotyping, and also can be used for rice variety purity / true degree detection.

[0202] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. Application of SNP locus combination in identifying rice varieties, characterized in that: The rice types are japonica and indica rice varieties from the Taihu Lake Basin; the SNP locus combination consists of Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25, and Os27; the physical location of the SNP loci is based on the rice variety Nipponbare reference genome IRGSP-1.0 version; Os03 is located at position 1232061 on chromosome 4, and its deoxynucleotides are G or A; Os04 is located at position 23145738 on chromosome 4, and its deoxynucleotides are T or A; Os11 is located at position 14510534 on chromosome 8, and its deoxynucleotides are C or G; Os14 is located at position 12401152 on chromosome 10, and its deoxynucleotides are G or A; Os17 is located at position 12927938 on chromosome 7, and its deoxynucleotides are G or A; Os18 is located at position 9336153 on chromosome 10, and its deoxynucleotides are T or C; Os19 is located at position 12872110 on chromosome 1, and its deoxynucleotides are C or T; Os21 is located at position 6162500 on chromosome 6, and its deoxynucleotides are G or T; Os22 is located at position 28946285 on chromosome 7, and its deoxynucleotides are G or T; Os24 is located at position 9771123 on chromosome 8, and its deoxynucleotides are C or T; Os25 is located at position 4264851 on chromosome 10, and its deoxynucleotides are G or A; Os27 is located at position 22989532 on chromosome 11, and its deoxynucleotides are T or C.

2. Application of SNP locus combination in identifying rice varieties, characterized in that: The rice types are japonica and indica rice varieties from the Taihu Lake Basin; the SNP locus combination consists of 27 SNP loci, namely Os01-Os27; the physical positions of the SNP loci are based on the rice variety Nipponbare reference genome IRGSP-1.0 version; Os01 is located at position 24407095 on chromosome 1, and its deoxynucleotides are T or G; Os02 is located at position 5928030 on chromosome 3, and its deoxynucleotides are G or A; Os03 is located at position 1232061 on chromosome 4, and its deoxynucleotides are G or A; Os04 is located at position 23145738 on chromosome 4, and its deoxynucleotides are T or A; Os05 is located at position 32014814 on chromosome 4, and its deoxynucleotides are C or T; Os06 is located at position 34415769 on chromosome 4, and its deoxynucleotides are G or A; Os07 is located at position 29706892 on chromosome 6, and its deoxynucleotides are C or T; Os08 is located at position 3154160 on chromosome 7, and its deoxynucleotides are C or T; Os09 is located at position 10370491 on chromosome 7, and its deoxynucleotides are A or C; Os10 is located at position 28631021 on chromosome 7, and its deoxynucleotides are T or G; Os11 is located at position 14510534 on chromosome 8, and its deoxynucleotides are C or G; Os12 is located at position 19429341 on chromosome 8, and its deoxynucleotides are A or G; Os13 is located at position 7814769 on chromosome 9, and its deoxynucleotides are A or G; Os14 is located at position 12401152 on chromosome 10, and its deoxynucleotides are G or A; Os15 is located at position 13941647 on chromosome 3, and its deoxynucleotides are T or A; Os16 is located at position 6718908 on chromosome 1, and its deoxynucleotides are A or G; Os17 is located at position 12927938 on chromosome 7, and its deoxynucleotides are G or A; Os18 is located at position 9336153 on chromosome 10, and its deoxynucleotides are T or C; Os19 is located at position 12872110 on chromosome 1, and its deoxynucleotides are C or T; Os20 is located at position 3646002 on chromosome 5, and its deoxynucleotides are G or C; Os21 is located at position 6162500 on chromosome 6, and its deoxynucleotides are G or T; Os22 is located at position 28946285 on chromosome 7, and its deoxynucleotides are G or T; Os23 is located at position 8470327 on chromosome 7, and its deoxynucleotides are G or C; Os24 is located at position 9771123 on chromosome 8, and its deoxynucleotides are C or T; Os25 is located at position 4264851 on chromosome 10, and its deoxynucleotides are G or A; Os26 is located at position 3866539 on chromosome 10, and its deoxynucleotides are C or G; Os27 is located at position 22989532 on chromosome 11, and its deoxynucleotides are T or C.

3. A primer combination, characterized in that: The primer combination is used to detect the SNP site combination detected in the rice genome as claimed in claim 2; The KASP primers corresponding to each SNP site to be tested include two forward primers and one reverse primer; the two forward primers are respectively denoted as forward primer 1 and forward primer 2; the 5' end of the forward primer 1 is connected to a fluorescent tag sequence, and the 5' end of the forward primer 2 is connected to another fluorescent tag sequence; The nucleotide sequence of forward primer 1 for detecting Os01 is shown in SEQ ID No. 1, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 28, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 55; The nucleotide sequence of the forward primer 1 used to detect Os02 is shown in SEQ ID No. 2, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 29, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 56; The nucleotide sequence of the forward primer 1 used to detect Os03 is shown in SEQ ID No. 3, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 30, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 57; The nucleotide sequence of forward primer 1 for detecting Os04 is shown in SEQ ID No. 4, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 31, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 58; The nucleotide sequence of forward primer 1 for detecting Os05 is shown in SEQ ID No. 5, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 32, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 59; The nucleotide sequence of forward primer 1 for detecting Os06 is shown in SEQ ID No. 6, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 33, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 60; The nucleotide sequence of forward primer 1 for detecting Os07 is shown in SEQ ID No. 7, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 34, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 61; The nucleotide sequence of forward primer 1 for detecting Os08 is shown in SEQ ID No. 8, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 35, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 62; The nucleotide sequence of forward primer 1 for detecting Os09 is shown in SEQ ID No. 9, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 36, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 63; The nucleotide sequence of forward primer 1 for detecting Os10 is shown in SEQ ID No. 10, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 37, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 64; The nucleotide sequence of forward primer 1 for detecting Os10 is shown in SEQ ID No. 10, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 37, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 64; The nucleotide sequence of forward primer 1 for detecting Os11 is shown in SEQ ID No. 11, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 38, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 65; The nucleotide sequence of forward primer 1 for detecting Os12 is shown in SEQ ID No. 12, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 39, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 66; The nucleotide sequence of forward primer 1 for detecting Os13 is shown in SEQ ID No. 13, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 40, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 67; The nucleotide sequence of forward primer 1 for detecting Os14 is shown in SEQ ID No. 14, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 41, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 68; The nucleotide sequence of forward primer 1 for detecting Os15 is shown in SEQ ID No. 15, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 42, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 69; The nucleotide sequence of forward primer 1 for detecting Os16 is shown in SEQ ID No. 16, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 43, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 70; The nucleotide sequence of forward primer 1 for detecting Os17 is shown in SEQ ID No. 17, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 44, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 71; The nucleotide sequence of forward primer 1 for detecting Os18 is shown in SEQ ID No. 18, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 45, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 72; The nucleotide sequence of forward primer 1 for detecting Os19 is shown in SEQ ID No. 19, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 46, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 73; The nucleotide sequence of forward primer 1 for detecting Os20 is shown in SEQ ID No. 20, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 47, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 74; The nucleotide sequence of forward primer 1 for detecting Os21 is shown in SEQ ID No. 21, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 48, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 75; The nucleotide sequence of forward primer 1 for detecting Os22 is shown in SEQ ID No. 22, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 49, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 76; The nucleotide sequence of forward primer 1 for detecting Os23 is shown in SEQ ID No. 23, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 50, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 77; The nucleotide sequence of forward primer 1 for detecting Os24 is shown in SEQ ID No. 24, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 51, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 78; The nucleotide sequence of forward primer 1 for detecting Os25 is shown in SEQ ID No. 25, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 52, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 79; The nucleotide sequence of forward primer 1 for detecting Os26 is shown in SEQ ID No. 26, the nucleotide sequence of forward primer 2 is shown in SEQ ID No. 53, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 80; The nucleotide sequence of the forward primer 1 for detecting Os27 is shown in SEQ ID No. 27, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 54, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.

81.

4. The primer combination according to claim 3, characterized in that: The 5' end of the forward primer 1 is connected to the FAM fluorescent tag sequence; The 5' end of the forward primer 2 is connected to a HEX fluorescent tag sequence.

5. The primer combination according to claim 4, wherein: The FAM fluorescent tag sequence is shown in SEQ ID No.82; The HEX fluorescent tag sequence is shown as SEQ ID No.

83.

6. A kit comprising the primer combination according to any one of claims 3 to 5.

7. Use of the SNP locus combination according to claim 2, the primer combination according to any one of claims 3 to 5, or the kit according to claim 6 in any of the following: (1) Genotyping of rice germplasm resources or genetic populations; (2) Construct DNA fingerprints of rice germplasm resources or varieties; (3) Detecting the purity or authenticity of rice varieties; The types of rice are japonica rice and indica rice varieties local to the Taihu Lake Basin.

8. A method for genotyping rice germplasm resources or genetic populations, characterized in that: The steps include: (a1) Extract genomic DNA from all rice samples in the tested rice germplasm resources or genetic populations; (a2) For the test rice sample DNA obtained in (a1), PCR reaction systems are prepared sequentially according to the 27 SNP sites described in claim 2, and PCR amplification is performed using the primer combination described in any one of claims 3 to 5; (a3) using a KASP fluorescence detector to detect the FAM and HEX fluorescence values ​​of the PCR amplification product of the test rice sample DNA at the 27 SNP sites described in claim 2, and calculating the genotyping results of the test rice sample DNA at the 27 SNP sites; The types of rice are japonica rice and indica rice varieties local to the Taihu Lake Basin.

9. A method for constructing a DNA fingerprint of rice germplasm resources or varieties, characterized in that: The steps include: (b1) extracting genomic DNA from all rice germplasm resources or varieties used to construct the DNA fingerprint, detecting the genotypes of the 27 SNP sites described in claim 2, and arranging the genotypes of each rice germplasm resource or variety at the SNP site in order of site number to form a specific sequence, which is the DNA fingerprint of the rice germplasm resource or variety; (b2) For the SNP sites in step (b1), convert the genotype combination into a computer string label according to the following steps: The 27 SNP sites described in claim 2 are genotyped as XX, YY, XY, or --, wherein X and Y are single nucleotides at the site, which are A, T, G, or C, and -- indicates missing or invalid data. The genotype combinations of each SNP site include AA, TT, GG, CC, AT, AG, AC, TG, TC, GC, and NN, totaling 11 genotype combinations; According to the above rules, the genotype combination of the SNP site in the DNA fingerprint of the rice germplasm resource or variety is converted into a character string composed of characters, and the character string is the core code of the DNA fingerprint of the rice germplasm resource or variety; (b3) evaluating the identification efficiency of rice materials based on the core code of the DNA fingerprint of the rice germplasm resources or varieties; (b4) converting the rice germplasm resource or variety DNA fingerprint core code constructed in step (b3) into a QR code or barcode that can be recognized by the device terminal to perform intelligent identification and management of the rice germplasm resource or variety; The types of rice are japonica rice and indica rice varieties local to the Taihu Lake Basin.

Images