Application of SNP (Single Nucleotide Polymorphism) site combination and KASP primer thereof in identifying local rice variety

Abstract

The invention belongs to the technical field of biology, and particularly relates to an application of an SNP (Single Nucleotide Polymorphism) site combination and a primer thereof in identifying local varieties of rice. On the basis of whole genome re-sequencing data of 114 parts of rice local varieties in the Taihu Lake basin, more than millions of SNP loci are mined, 27 core SNP markers and matched KASP primers are identified and screened from the SNP loci, and the identification efficiency of the 12 KASP markers on the 114 parts of rice local germplasm resources can reach 100%. The invention provides a set of credible SNP markers convenient for later use for biological research of local rice varieties in the Taihu Lake basin, and the set of SNP markers and detection primers can be used for development of rice SNP detection kits, construction of DNA fingerprints of local rice varieties and genetic typing of local rice germplasm resources and genetic populations, and can be applied to development of rice SNP detection primers. Detecting the background recovery rate of the directionally improved rice variety, detecting the purity / authenticity of the local rice variety, and carrying out molecular marker-assisted selective breeding on the rice.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the development of SNP site combinations of rice local varieties based on whole genome resequencing and KASP technology and the application of primers thereof in identifying rice local varieties. Background Art

[0002] China has a long history of rice cultivation and domestication, and has formed several secondary origin centers across the country, which play an important role in the preservation of rice genetic diversity and germplasm resources. The middle and lower reaches of the Yangtze River, especially the Taihu Lake Basin (covering Jiangsu, Zhejiang, Anhui provinces and parts of Shanghai), is particularly important as one of the secondary origin centers of rice cultivation. The Taihu Lake Basin is not only a key area for rice diversity, but also contains rich rice germplasm resources, which are of immeasurable value for the improvement of modern rice varieties 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 landraces are facing the risk of disappearance or admixture. This trend not only leads to the loss of genetic diversity, but also makes it difficult to accurately distinguish these precious landraces through traditional phenotypic analysis. Therefore, protecting and accurately identifying rice landraces in the Taihu Lake Basin has become a key challenge for the current conservation and sustainable use of rice germplasm resources.

[0004] In the field of genetics and genomics, single nucleotide polymorphism (SNP) is a universal and reliable molecular marker that is favored due to its co-dominant inheritance, high abundance, wide distribution, and suitability for high-throughput automated genotyping. With the continuous advancement of sequencing technology, the recognition 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 genomics research and breeding projects.

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

[0006] The purpose of the present invention is to provide an application of a SNP site combination and a KASP primer thereof in identifying rice local varieties.

[0007] The technical solution provided by the present invention is as follows:

[0008] Application of a SNP locus combination in identifying rice local varieties, wherein the SNP locus combination includes Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25, and 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 deoxynucleotides are T or A;

[0011] Os11 is located at position 14510534 on chromosome 8, and its deoxynucleotides are 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 deoxynucleotides are T or C;

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

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

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

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

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

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

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

[0022] Application of SNP loci combination in identifying rice local varieties, the SNP loci combination includes 27 SNP loci, namely Os01-Os27;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0049] Os27 is located at position 22,989,532 on chromosome 1, and its deoxynucleotides are T or C;

[0050] The present invention also provides a primer combination, wherein the primer combination is used to detect all or part of the KASP primers in the above 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, Os27;

[0052] Among them, the KASP primers corresponding to each SNP site to be tested include two forward primers and one reverse primer respectively; the two forward primers are respectively recorded 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 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;

[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 used to detect 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 in SEQ ID No. 6, the nucleotide sequence of the 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;

[0059] The nucleotide sequence of the forward primer 1 used to detect Os07 is shown in SEQ ID No. 7, the nucleotide sequence of the 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;

[0060] The nucleotide sequence of the forward primer 1 for detecting Os08 is shown in SEQ ID No. 8, the nucleotide sequence of the 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;

[0061] The nucleotide sequence of the forward primer 1 used to detect Os09 is shown in SEQ ID No. 9, the nucleotide sequence of the 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;

[0062] The nucleotide sequence of the forward primer 1 used to detect Os10 is shown in SEQ ID No. 10, the nucleotide sequence of the 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;

[0063] The nucleotide sequence of the forward primer 1 used to detect Os10 is shown in SEQ ID No. 10, the nucleotide sequence of the 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;

[0064] The nucleotide sequence of the forward primer 1 for detecting Os11 is shown in SEQ ID No. 11, the nucleotide sequence of the 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;

[0065] The nucleotide sequence of the forward primer 1 for detecting Os12 is shown in SEQ ID No. 12, the nucleotide sequence of the 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;

[0066] The nucleotide sequence of the forward primer 1 for detecting Os13 is shown in SEQ ID No. 13, the nucleotide sequence of the 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;

[0067] The nucleotide sequence of the forward primer 1 for detecting Os14 is shown in SEQ ID No. 14, the nucleotide sequence of the 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;

[0068] The nucleotide sequence of the forward primer 1 for detecting Os15 is shown in SEQ ID No. 15, the nucleotide sequence of the 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;

[0069] The nucleotide sequence of the forward primer 1 for detecting Os16 is shown in SEQ ID No. 16, the nucleotide sequence of the 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;

[0070] The nucleotide sequence of the forward primer 1 for detecting Os17 is shown in SEQ ID No. 17, the nucleotide sequence of the 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;

[0071] The nucleotide sequence of the forward primer 1 for detecting Os18 is shown in SEQ ID No. 18, the nucleotide sequence of the 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;

[0072] The nucleotide sequence of the forward primer 1 for detecting Os19 is shown in SEQ ID No. 19, the nucleotide sequence of the 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;

[0073] The nucleotide sequence of the forward primer 1 for detecting Os20 is shown in SEQ ID No. 20, the nucleotide sequence of the 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;

[0074] The nucleotide sequence of the forward primer 1 for detecting Os21 is shown in SEQ ID No. 21, the nucleotide sequence of the 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;

[0075] The nucleotide sequence of the forward primer 1 for detecting Os22 is shown in SEQ ID No. 22, the nucleotide sequence of the 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;

[0076] The nucleotide sequence of the forward primer 1 for detecting Os23 is shown in SEQ ID No. 23, the nucleotide sequence of the 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;

[0077] The nucleotide sequence of the forward primer 1 for detecting Os24 is shown in SEQ ID No. 24, the nucleotide sequence of the 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;

[0078] The nucleotide sequence of the forward primer 1 for detecting Os25 is shown in SEQ ID No. 25, the nucleotide sequence of the 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;

[0079] The nucleotide sequence of the forward primer 1 for detecting Os26 is shown in SEQ ID No. 26, the nucleotide sequence of the 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;

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

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

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

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

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

[0085] Furthermore, the reagent or kit may also contain KASP 2×Mastermix, MgCl2 and ddH2O.

[0086] The KASP 2×Mastermix in the reagent or kit contains fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B.

[0087] The nucleotide sequence of the fluorescent probe A is consistent with the nucleotide sequence of the fluorescent label sequence connected to the 5' end of the forward primer 1, and the 5' end is connected to the fluorescent group A; the nucleotide sequence of the quenching probe A is reverse complementary to the nucleotide sequence of the label sequence A, and the 3' end is connected to the quenching group.

[0088] The nucleotide sequence of the fluorescent probe B is consistent with the nucleotide sequence of the fluorescent label sequence connected to the 5' end of the forward primer 2, and the 5' end is connected to the fluorescent group B; the nucleotide sequence of the quenching probe B is reverse complementary to the nucleotide sequence of the label sequence B, and the 3' end is connected to the quenching group.

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

[0090] The present invention also provides the use of the above-mentioned SNP site combination or the above-mentioned primer combination or the above-mentioned kit in any of the following items;

[0091] (1) Developing a rice SNP site detection kit; the SNP site described herein is all or part of the following 27 SNP sites;

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

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

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

[0095] The present invention 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 tested rice germplasm resources and / or genetic populations;

[0097] (a2) For the rice sample DNA, PCR reaction systems were prepared in sequence according to the 27 SNP sites described above, and PCR amplification was performed;

[0098] (a3) Using a KASP fluorescence detector, the FAM and HEX fluorescence values ​​of the PCR amplification product of the test rice sample DNA at the 27 SNP sites mentioned above are detected, and the genotyping results of the test rice sample at the 27 SNP sites are calculated (i.e., the specific nucleotides at the SNP sites are detected, the same below). The data of a single SNP site is recorded as XX, YY or XY, where X and Y are the single nucleotide polymorphisms of the site, specifically A, T, G or C, arranged in this order. Missing or invalid data is recorded as -. If the sum of the proportions of XY and - genotypes of a sample is higher than 10%, it will not be adopted.

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

[0100] (b1) extracting genomic DNA from all rice germplasm resources or varieties used to construct a 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 the order of site numbers to form a specific sequence, which is the DNA fingerprint of the rice germplasm resource or variety;

[0101] (b2) For the 27 core characteristic SNP sites in step (b1), the genotype combinations are converted into computer string labels as follows:

[0102] The genotyping results of the 27 core characteristic SNP sites are composed of XX, YY, XY or --, where X and Y are single nucleotides of the site, which are A, T, G or C, and -- indicates 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 resources or varieties is converted into a string consisting of 54 characters, which is the core code of the DNA fingerprint of the rice germplasm resources or varieties;

[0104] (b3) converting the rice germplasm resources or varieties DNA fingerprint core code constructed in step (b2) into a two-dimensional code or bar code that can be recognized by the device terminal, so as to perform intelligent identification and management of the rice germplasm resources or varieties;

[0105] (b4) Based on the core code of the DNA fingerprint of the rice germplasm resources or varieties described in (b2), the identification efficiency of the rice materials was evaluated, wherein only 12 markers including Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25, and Os27 were required, and 100% of the 114 rice materials tested could be identified.

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

[0107] (c1) preparing more than 50 seeds of the rice variety to be tested and the standard control rice variety, germinating and sowing, and taking equal amounts of tissue from each seedling when the seedlings grow to one leaf and one heart, and then dividing the seedlings into two groups according to the rice variety to be tested and the standard control rice variety for mixed sampling, and extracting genomic DNA;

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

[0109] (c3) comparing the genotype differences between the rice variety to be tested and the standard reference rice variety at the effective sites of the 27 SNP sites mentioned above in the standard reference rice variety sample, and there are two results: the genotypes are the same or different;

[0110] (c4) The value of the purity (P) of the variety is expressed in %, and is 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 in the effective sites of the standard control rice variety where the genotypes of the rice variety to be tested are different from those of the standard control rice variety;

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

[0117] In the above methods, the rice varieties are all conventional local varieties, japonica rice or indica rice types.

[0118] In a specific embodiment of the present invention, the rice is specifically selected from the 114 rices shown in Table 1.

[0119] Furthermore, in the above methods, the above primer combination or kit can be used to complete the genotype detection of the 27 SNP sites.

[0120] Beneficial Effects

[0121] The present invention is based on more than one million SNP variant site data obtained from the whole genome resequencing of local rice varieties in the Taihu Basin carried out by the Suzhou Seed Management Station. These variant site data have been strictly quality controlled and sequenced for verification, and 27 SNP markers and matching KASP primers have been identified and screened using the KASP technology platform. It is further found that only 12 markers including Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25, and Os27 are needed to identify 100% of the rice materials tested. The present invention provides a set of reliable and easy-to-use SNP markers for rice biology research. This set of SNP markers and detection primers can be used for the development of rice SNP detection kits, the construction of rice variety DNA fingerprints, the genotyping of rice germplasm resources and genetic populations, the construction of rice germplasm resources / variety DNA fingerprints, and the detection of rice variety purity / authenticity.

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

[0123] Figure 1 This is the data record of the rice SNP marker screening process of the present invention.

[0124] Figure 2 To evaluate the identification efficiency of 114 rice germplasm resources using 27 KASP markers.

[0125] Figure 3 This is an example of the KASP experimental detection results. DETAILED DESCRIPTION

[0126] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0127] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0128] Example 1: Screening and development of SNP markers and KASP primers for rice landraces in the Taihu Lake Basin

[0129] Based on the SNP variation data of the whole genome resequencing of 114 rice germplasm resources / variety materials (Table 1) completed by Suzhou Seed Management Station (provided by Suzhou Seed Management Station, the file name is all.raw.snp.vcf, the number of markers is 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. 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 the original SNP sites to exclude some false positive variants caused by sequencing quality issues. Execute the statement:

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

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

[0133] VariantFiltration\#Variation filtering

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

[0135] -V all.raw.snp.vcf --filter-expression "QD<2.0||MQ<40.0||FS>60.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0" --filter-name'SNP_filter'\#Set input file and filter criteria

[0136] -O all.snp.hardfilter.vcf.gz # Output population SNP file with hard filter criteria

[0137] (2) Eliminate sites that do not meet the hard filtering conditions and execute the statement:

[0138] gatk--java-options"-Xmx16g\#Set memory usage limit

[0139] -Djava.io,tmpdir=. / tmp"\#Set temporary file directory

[0140] SelectVariants\#Extract variants

[0141] -R GCF_001433935.1_IRGSP-1.0_genomic.fna\#reference genome

[0142] -V all.snp.hardfilter.vcf.gz--exclude-filtered\#Remove mutations that do not meet the standards

[0143] -O all.snp.hardfiltered.vcf.gz#Output hard-filtered SNP file

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

[0145] vcftools--gzvcf all.snp.hardfiltered.vcf.gz--min-alleles 2--max-alleles 2--max-missing 0.95--maf0.05--recode--recode-INFO-all--stdout|bgzip-cf>all.snp.filtered.vcf.gz

[0146] The number of markers retained after data quality control in this step is 1,078,168.

[0147] (4) The sequences of 50 bp before and after each SNP site on the chromosome were extracted to check its conservation. Further quality control was performed to retain sites with an average sequencing depth of more than 5× and a quality value greater than 30, and 139,325 SNP sites were filtered out.

[0148] (5) Sequences of 100 bp upstream and downstream of each site were intercepted, and the sequences were aligned to the reference genome using blast (v2.10.1+) software to check its specificity and exclude SNP sites that were aligned to multiple positions. The polymorphic information content (PIC) of the remaining sites was calculated, and sites with PIC>0.35 were retained. Primers were designed for SNP markers using primer design software primer3 (v2.4.0) and converted into KASP markers.

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

[0150] (6) Combine steps (3) to (5) to manually select SNP markers, with the criteria being:

[0151] Step (5) includes designing a marker for a KASP primer;

[0152] Preferably, the marker is located at or near the coding region of the gene;

[0153] A maximum of 2 markers per haplotype block;

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

[0155] In this step, a total of 65 SNP markers were selected for subsequent experimental verification.

[0156] (7) Select three rice varieties with large phenotypic differences (TH_10, TH_67, TH_113, see Table 1 for details), and perform first-generation sequencing to verify the genotype of the SNP site selected in step (6). If the sequencing is successful and the result is consistent with the resequencing result, it indicates that the SNP site is real and suitable as a KASP marker.

[0157] (8) Based on the results of (7), the artificial optimization of synthetic primers was repeated again to verify the results. Finally, 27 SNP markers were selected as usable markers.

[0158] The positional genotype information of the 27 SNP markers obtained in the present invention is shown in Table 2. The physical positions of the SNP marker sites are annotated based on the IRGSP-1.0 version of the reference genome of the rice variety Nipponbare.

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

[0160]

[0161]

[0162]

[0163] Table 2 Information about 27 SNP loci

[0164] Serial number chromosome Location 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 above 27 SNP markers, each corresponding KASP primer of the SNP marker includes two forward primers and one reverse primer. The 5' end of the forward primer 1 sequence is connected to the HEX fluorescent label sequence, 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID No. 82), and the 5' end of the forward primer 2 sequence is connected to the HEX fluorescent label sequence, 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID No. 83). 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 corresponding detection kit for 27 SNP markers of rice (see Table 2). The kit contains the 27 sets of primers (see Table 3), which are synthesized by Sangon Biotech (Shanghai) Co., Ltd., wherein the forward primer 1 and the reverse primer 2 have been added with FAM fluorescent sequence and HEX fluorescent sequence respectively, and the product is recorded as OsSNP KASP Primermix.

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

[0171] The kit is equipped with MgCl2 and ddH2O, wherein the MgCl2 is a product of Beijing Wokai Biotechnology Co., Ltd., with a CAS number of 7786-30-3, and the ddH2O is high-pressure sterilized double distilled water.

[0172] Example 2: Rice germplasm resources / variety genotyping

[0173] The present invention refers to the technical documents of the British LGC company and combines the actual operation to provide a method system for genotyping rice germplasm resources / variety (specifically 114 rice materials in Table 1) using the 27 SNP markers (Table 2 and Table 3) in Example 1, including the following steps:

[0174] (1) Extraction of DNA from rice samples: 200 mg to 300 mg of seedlings or leaves of the rice variety to be tested were placed in a 1.5 ml centrifuge tube, and fully ground with liquid nitrogen. 700 μL of preheated 65°C solution was added to each tube. CTAB extraction solution; place the centrifuge tube in a 65℃ water bath for 30min~1h, gently shake the centrifuge tube every 10min or so; cool for 2min after the water bath, add 0.5mL of chloroform-isoamyl alcohol mixture, shake vigorously to mix, and centrifuge at 12000rmp for 5~10min; take the supernatant in the centrifuge tube to a new centrifuge tube, add 2 times the volume of pre-cooled isopropanol, mix by inversion, place at -20℃ to precipitate for more than 30min, centrifuge at 12000rmp for 10min, and discard the supernatant; wash twice with 75% ethanol, centrifuge at 12000rmp for 3min, discard the supernatant, and dissolve the precipitate in 50μL deionized water or TE after natural drying, detect the concentration, dilute the concentration to 100ng / μl, use immediately or store at -20℃.

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

[0176] (2) For the 27 SNP markers (Table 2 and Table 3), KASP Mastermix and OsSNP KASP Primermix were added in sequence to prepare the PCR reaction system for PCR amplification. OsSNP KASP Primermix consisted of forward primer 1 with a fluorescent label FAM connected to the 5' end, forward primer 2 with a fluorescent label HEX connected to the 5' end, and reverse primer, which were mixed with ddH2O at 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 PCR reaction system construction

[0179] Components 96-well plate 2×KASP mastermix 2.5ul OsSNP KASP Primermix 1.25ul Template DNA 1.25ul <![CDATA[ddH2O]]> 0ul Total 5ul 1Reaction

[0180] Table 5 PCR reaction conditions

[0181]

[0182] PCR reaction: 1) Dilute the DNA into 200 μL of a 96-well plate, use an 8-well pipette (manufacturer: Eppendorf-10 μL) to draw 1.25 μL into a 96-well PCR plate, centrifuge to the bottom for use (manufacturer: AXYGEN); 2) Mix KASP Mastermix, OsSNP KASP Primermix, ddH2O, etc. in proportion to form PCR-mix; 3) Dispense 3.75 μL of PCR-mix into each reaction well, centrifuge to the bottom, and seal with a silicone cap or sealing film; 4) Follow the above reaction procedure in a 96-well PCR instrument (model: CFX Connet TM The reaction was completed using a flow cytometry system (Real-Time System, Bio-Read, USA).

[0183] (3) Fluorescence detection and genotyping: The 96-well plate after PCR was completed was removed, and after cooling, the silicone cover or sealing film was removed and the plate was placed on a SNP typing detector (model FLUOstar Omega SNP) to detect the FAM and HEX fluorescence values. The genotyping results were analyzed using KlusterCaller software to obtain the genotype.

[0184] The genotyping results of a single SNP marker site are recorded as "XX" or "XY" or "YY". "XX" and "YY" represent homozygous and "XY" represents heterozygous, where X and Y are single nucleotides at the site, such as A, T, G, and C. If a single nucleotide A, T, G, or C cannot be determined for a certain SNP marker, it is invalid data and is recorded as "--".

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

[0186] If the proportion of invalid data of a mark is higher than 10%, the mark is an invalid mark; if the proportion of invalid data of a sample is higher than 10%, the sample is an invalid sample and will not be adopted.

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

[0188] Example 3: Construction of the DNA fingerprint core code of 114 rice local germplasm resources in the Taihu Lake Basin

[0189] The present invention uses the proposed rice germplasm resource / variety fingerprint method to construct a fingerprint map for 114 rice germplasm resources (Table 1) stored in Suzhou Seed Management Station and draws its core code, including the following steps:

[0190] (1) According to the steps of Example 2, genotyping of 114 rice germplasm resources / variety (Table 1) at the 27 SNP markers (Table 2) was completed. 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 representations thereof.

[0191] (2) For the 27 core characteristic SNP sites in step (1), the genotype combinations are converted into computer string labels as follows:

[0192] First, the genotyping results of the 27 core characteristic SNP sites are composed of XX, YY, XY or --, where X and Y are single nucleotides of the site, which are A, T, G or C, and -- indicates 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;

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

[0194] (3) The core codes of the DNA fingerprints of the 114 rice germplasm resources / varieties were obtained in sequence. The core codes can be converted into two-dimensional codes (Table 6) as the characteristics of the corresponding rice germplasm resources / varieties, and used as the basic standard for authenticity verification in the process of germplasm resource / variety exchange and sales.

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

[0196] Table 6 Core codes of DNA fingerprints of 114 local rice germplasm resources / varieties in the Taihu Lake Basin

[0197]

[0198]

[0199]

[0200]

[0201] Based on 114 rice whole genome resequencing data, the present invention uses the data analysis method explored in the early stage to mine and obtain more than one million SNP sites. These variable site data are strictly quality controlled and sequenced for verification, and 27 SNP markers and matching KASP primers are identified and screened using the KASP technology platform. The present invention provides a set of reliable and easy-to-use SNP markers for rice biology research. In addition to being used for the development of rice SNP detection kits, the construction of rice variety DNA fingerprints, and the genotyping of rice germplasm resources and genetic populations, this set of SNP markers and detection primers can also be used for rice variety purity / authenticity detection, etc.

[0202] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of SNP locus combination in identifying rice varieties, characterized in that: The SNP site combination includes Os03, Os04, Os11, Os14, Os17, Os18, Os19, Os21, Os22, Os24, Os25, and Os27; Os03 is located at position 1232061 on chromosome 4, and its deoxynucleotide is 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 deoxynucleotide is G or A; Os17 is located at position 12927938 on chromosome 7, and its deoxynucleotide is 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 deoxynucleotide is G or T; Os24 is located at position 9771123 on chromosome 81, and its deoxynucleotides are C or T; Os25 is located at position 4264851 on chromosome 10, and its deoxynucleotide is G or A; Os27 is located at position 22989532 on chromosome 1, and its deoxynucleotide is T or C.

2. Application of SNP locus combination in identifying rice varieties, characterized in that: The SNP locus combination includes 27 SNP loci, namely Os01-Os27; Os01 is located at position 24407095 on chromosome 1, and its deoxynucleotide is T or G; Os02 is located at position 5928030 on chromosome 3, and its deoxynucleotide is G or A; Os03 is located at position 1232061 on chromosome 4, and its deoxynucleotide is 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 deoxynucleotide is 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 deoxynucleotide is 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 deoxynucleotide is 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 deoxynucleotide is 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 81, and its deoxynucleotides are C or T; Os25 is located at position 4264851 on chromosome 10, and its deoxynucleotide is 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 1, and its deoxynucleotide is T or C.

3. A primer combination, characterized in that: The primer combination is used to detect the SNP site combination for detecting 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 respectively; the two forward primers are respectively recorded 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; 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. 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 the forward primer 1 used to detect 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; The nucleotide sequence of the forward primer 1 used to detect 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; The nucleotide sequence of the forward primer 1 for detecting Os06 is shown in SEQ ID No. 6, the nucleotide sequence of the 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 the forward primer 1 used to detect Os07 is shown in SEQ ID No. 7, the nucleotide sequence of the 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 the forward primer 1 for detecting Os08 is shown in SEQ ID No. 8, the nucleotide sequence of the 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 the forward primer 1 used to detect Os09 is shown in SEQ ID No. 9, the nucleotide sequence of the 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 the forward primer 1 used to detect Os10 is shown in SEQ ID No. 10, the nucleotide sequence of the 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 the forward primer 1 used to detect Os10 is shown in SEQ ID No. 10, the nucleotide sequence of the 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 the forward primer 1 for detecting Os11 is shown in SEQ ID No. 11, the nucleotide sequence of the 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 the forward primer 1 for detecting Os12 is shown in SEQ ID No. 12, the nucleotide sequence of the 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 the forward primer 1 for detecting Os13 is shown in SEQ ID No. 13, the nucleotide sequence of the 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 the forward primer 1 for detecting Os14 is shown in SEQ ID No. 14, the nucleotide sequence of the 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 the forward primer 1 for detecting Os15 is shown in SEQ ID No. 15, the nucleotide sequence of the 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 the forward primer 1 for detecting Os16 is shown in SEQ ID No. 16, the nucleotide sequence of the 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 the forward primer 1 for detecting Os17 is shown in SEQ ID No. 17, the nucleotide sequence of the 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 the forward primer 1 for detecting Os18 is shown in SEQ ID No. 18, the nucleotide sequence of the 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 the forward primer 1 for detecting Os19 is shown in SEQ ID No. 19, the nucleotide sequence of the 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 the forward primer 1 for detecting Os20 is shown in SEQ ID No. 20, the nucleotide sequence of the 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 the forward primer 1 for detecting Os21 is shown in SEQ ID No. 21, the nucleotide sequence of the 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 the forward primer 1 for detecting Os22 is shown in SEQ ID No. 22, the nucleotide sequence of the 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 the forward primer 1 for detecting Os23 is shown in SEQ ID No. 23, the nucleotide sequence of the 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 the forward primer 1 for detecting Os24 is shown in SEQ ID No. 24, the nucleotide sequence of the 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 the forward primer 1 for detecting Os25 is shown in SEQ ID No. 25, the nucleotide sequence of the 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 the forward primer 1 for detecting Os26 is shown in SEQ ID No. 26, the nucleotide sequence of the 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 as SEQ ID No.27, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No.54, and the nucleotide sequence of the reverse primer is shown as 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 a FAM fluorescent label sequence; The 5' end of the forward primer 2 is connected to a HEX fluorescent tag sequence.

5. The primer combination according to claim 3, characterized in that: 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 site combination according to claim 1 or 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) Preparing a kit for detecting genotypes of rice SNP loci; (2) Genotyping of rice germplasm resources or genetic populations; (3) Construct DNA fingerprints of rice germplasm resources or varieties; (4) Testing the purity or authenticity of rice varieties; The types of rice are local japonica rice and indica rice varieties in the Taihu Lake Basin.

8. A method for genotyping rice germplasm resources or genetic populations, characterized in that: The steps include: (a1) extracting genomic DNA from all rice samples in the tested rice germplasm resources or genetic populations; (a2) preparing PCR reaction systems for the rice sample DNA obtained in (a1) according to the 27 SNP sites described in claim 2, and performing PCR amplification respectively 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 in claim 2, and calculating the genotyping results of the test rice sample DNA at the 27 SNP sites; The types of rice are local japonica rice and indica rice varieties in 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 a 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 site 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 of the site, which are A, T, G or C, and -- indicates missing or invalid data, and each SNP site genotype combination includes 11 genotype combinations including AA, TT, GG, CC, AT, AG, AC, TG, TC, GC and NN; According to the above rules, the genotype combination of the SNP loci in the DNA fingerprint of the rice germplasm resources or varieties is converted into a string composed of characters, and the string is the core code of the DNA fingerprint of the rice germplasm resources or varieties; (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 resources or varieties DNA fingerprint core code constructed in step (b3) into a two-dimensional code or bar code that can be recognized by the device terminal, so as to perform intelligent identification and management of the rice germplasm resources or varieties; The types of rice are local japonica rice and indica rice varieties in the Taihu Lake Basin.

10. A method for detecting the purity or authenticity of rice varieties, characterized in that: The steps include: (c1) preparing more than 50 seeds of the rice variety to be tested and the standard control rice variety, germinating and sowing, and taking equal amounts of tissue from each seedling when the seedlings grow to one leaf and one heart, and then dividing the seedlings into two groups according to the rice variety to be tested and the standard control rice variety for mixed sampling, and extracting genomic DNA; (c2) detecting the genotypes of the 27 SNP sites described in claim 2, thereby obtaining genotype data of the tested rice variety and the standard control rice variety at the 27 SNP sites; When the genotype of the standard reference rice variety sample at a certain SNP site is XX or YY, the site is a valid site, and when the genotype of the site is XY or -- type, the site is an invalid site; wherein X and Y are A, T, G or C, and -- indicates missing or invalid data; (c3) comparing the genotype differences between the rice variety to be tested and the standard reference rice variety at the effective sites of the 27 SNP sites in claim 2 for the standard reference rice variety sample, and the results are as follows: the genotypes are the same or different; (c4) The value of the purity (P) of the variety is expressed in %, and is calculated according to formula (1): In the formula: P——variety purity; S1——the number of effective sites of the standard control rice variety; S2——the number of sites in the effective sites of the standard control rice variety where the genotypes of the rice variety to be tested are different from those of the standard control rice variety; (c5) when the purity of the rice variety to be tested is ≤95%, the rice variety to be tested and the standard reference variety are determined to be different varieties; when the purity of the rice variety to be tested is >95% and <100%, the rice variety to be tested is determined to be a similar variety or a suspected variety of the standard reference variety; when the purity of the rice variety to be tested is 100%, the rice variety to be tested and the standard reference variety are determined to be the same variety; The types of rice are local japonica rice and indica rice varieties in the Taihu Lake Basin.

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