A rice core SNP marker combination based on liquid phase microarray and KASP technology and its application

Abstract

The present invention discloses a set of rice core SNP marker combinations developed based on liquid phase chip and KASP technology and their applications. The present invention provides the following applications of 60 SNP sites in the rice genome: preparing a rice SNP site detection kit; performing genotyping on rice germplasm resources or genetic populations; constructing a rice germplasm resource or variety DNA molecular fingerprint database; and performing genetic diversity analysis on rice germplasm resources. The 60 SNP site combinations provided by the present invention can be applied to the construction of a rice molecular fingerprint database, variety identification, and genetic diversity analysis of germplasm resources; and the application of these 60 core site combinations can greatly improve detection accuracy, shorten detection time, and improve detection levels compared to the currently widely used biochemical detection methods and SSR methods. This will provide more effective and reliable technical means for molecular detection; and will provide important technical support for rice variety identification.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a set of rice core SNP marker combinations developed based on liquid phase chips and KASP technology and applications thereof. Background Art

[0002] Rice is one of the important food crops in my country. The selection and promotion of new rice varieties are of great significance to rice production. According to statistics, there are thousands of new rice varieties approved nationwide every year, and the homogeneity problem between varieties is becoming more and more serious. The continuous improvement of breeding technology has made the differences between varieties more subtle, and the traditional variety identification methods are stretched. Molecular markers can essentially reflect the differences between biological individuals, have high individual specificity and stable reliability, and can accurately and quickly identify new and existing varieties. With the rapid development of molecular biology and biotechnology, various DNA fingerprinting technologies have been introduced one after another. These technologies have their own characteristics, making molecular markers play an important role in variety identification.

[0003] There are many types of molecular markers used to distinguish varieties, such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), single nucleotide polymorphisms (SNP), etc. Different types of molecular markers can be used to distinguish polymorphisms between varieties. RFLP and AFLP markers are not widely used due to the complexity of marker development, detection stability, inconvenient detection methods and data integration. Currently, the most widely used is SSR markers. However, SSR markers also have certain limitations: (1) Designing SSR primers requires knowing the target sequence information (2) There are species restrictions, and only a very small number of SSR primers can be used in different species with close kinship. (3) The operation is cumbersome and inefficient, and the color development process is prone to background interference; data reading is not easy to automate. Due to the limitations of SSR itself, this technology can no longer meet the current development needs of the seed industry.

[0004] SNP refers to a genetic marker formed by the variation of a single nucleotide in the genome. It is numerous and rich in polymorphism, and is one of the most polymorphic genetic variations in the genome. SNP markers are widely used in the development and application of molecular markers due to their high density distribution in the genome, high throughput detection methods, and easy automated analysis. With the rapid development of high-throughput sequencing technology, the genome resequencing of more and more crops has made SNP markers increasingly abundant. Based on these rich SNP data, the genotyping platform based on SNP chip arrays and resequencing SNP typing technology has developed rapidly. On June 20, 2021, the National Agricultural Technology Center organized an agricultural industry standard review meeting in Beijing, and three standards, "SNP Marker Method for Authenticity Identification of Rice Varieties", "SNP Marker Method for Authenticity Identification of Corn Varieties", and "SNP Marker Method for Authenticity Identification of Wheat Varieties", were approved. This indicates that the SNP marker method has broad development prospects in the field of biological detection, and provides strong technical support and basis for maintaining crop variety innovation and market circulation order.

[0005] Competitive allele-specific PCR (KASP) technology is a high-throughput genotyping technology mainly based on SNPs that has been developed in recent years. Compared with traditional molecular markers, it has unique advantages. It is a simple gel-free fluorescent polymerase chain reaction that can meet the requirements of low-, medium- and high-throughput genotyping based on ordinary laboratory operations. It has certain flexibility and is suitable for experimental designs with large changes in target sites and sample quantities. It has important application value in future crop variety identification. Summary of the invention

[0006] The purpose of the present invention is to provide a set of SNP site combinations for rice germplasm identification.

[0007] In the first aspect, the present invention claims to protect the use of 60 SNP sites in the rice genome in any of the following:

[0008] (1) Preparing a rice SNP site detection kit;

[0009] (2) Genotyping of rice germplasm resources or genetic populations;

[0010] (3) Construct a DNA molecular fingerprint database of rice germplasm resources or varieties;

[0011] (4) Conduct genetic diversity analysis on rice germplasm resources.

[0012] The physical positions of the 60 SNP sites are determined based on the whole genome sequence comparison of the rice variety Nipponbare, the version number of the whole genome sequence of the rice variety Nipponbare is MSU7.0; the 60 SNP sites are OsK01-OsK60;

[0013] Osk1 is located at position 1228512 on chromosome 1, and its deoxynucleotides are G or A;

[0014] Osk2 is located at position 4645481 on chromosome 1, and its deoxynucleotides are G or A;

[0015] Osk3 is located at position 6644387 on chromosome 1, and its deoxynucleotides are C or A;

[0016] Osk4 is located at position 9305876 on chromosome 1, and its deoxynucleotides are G or A;

[0017] Osk5 is located at position 12854426 on chromosome 1, and its deoxynucleotides are C or G;

[0018] Osk6 is located at position 24749480 on chromosome 1, and its deoxynucleotides are A or G;

[0019] Osk7 is located at position 30545662 on chromosome 1, and its deoxynucleotides are A or G;

[0020] Osk8 is located at position 5785532 on chromosome 2, and its deoxynucleotides are G or A;

[0021] Osk9 is located at position 21772969 on chromosome 2, and its deoxynucleotides are G or C;

[0022] Osk10 is located at position 34474061 on chromosome 2, and its deoxynucleotides are C or A;

[0023] Osk11 is located at position 35882696 on chromosome 2, and its deoxynucleotides are A or G;

[0024] Osk12 is located at position 7956017 on chromosome 3, and its deoxynucleotides are G or A;

[0025] Osk13 is located at position 13775121 on chromosome 3, and its deoxynucleotides are G or A;

[0026] Osk14 is located at position 16579520 on chromosome 3, and its deoxynucleotides are C or T;

[0027] Osk15 is located at position 25181277 on chromosome 3, and its deoxynucleotides are A or G;

[0028] Osk16 is located at position 15846017 on chromosome 4, and its deoxynucleotides are T or C;

[0029] Osk17 is located at position 19209543 on chromosome 4, and its deoxynucleotides are T or C;

[0030] Osk18 is located at position 21,469,290 on chromosome 4, and its deoxynucleotides are A or G;

[0031] Osk19 is located at position 22842238 on chromosome 4, and its deoxynucleotides are A or G;

[0032] Osk20 is located at position 31997187 on chromosome 4, and its deoxynucleotides are T or C;

[0033] Osk21 is located at position 7384864 on chromosome 5, and its deoxynucleotides are T or C;

[0034] Osk22 is located at position 17035145 on chromosome 5, and its deoxynucleotides are T or C;

[0035] Osk23 is located at position 23305515 on chromosome 5, and its deoxynucleotides are G or C;

[0036] Osk24 is located at position 1529649 on chromosome 6, and its deoxynucleotides are G or T;

[0037] Osk25 is located at position 3166267 on chromosome 6, and its deoxynucleotides are T or C;

[0038] Osk26 is located at position 8454606 on chromosome 6, and its deoxynucleotides are A or G;

[0039] Osk27 is located at position 10345230 on chromosome 6, and its deoxynucleotides are G or A;

[0040] Osk28 is located at position 25220061 on chromosome 6, and its deoxynucleotides are T or C;

[0041] Osk29 is located at position 2865195 on chromosome 7, and its deoxynucleotides are A or G;

[0042] Osk30 is located at position 4660773 on chromosome 7, and its deoxynucleotides are T or A;

[0043] Osk31 is located at position 8809828 on chromosome 7, and its deoxynucleotides are A or G;

[0044] Osk32 is located at position 10756612 on chromosome 7, and its deoxynucleotides are A or T;

[0045] Osk33 is located at position 12924029 on chromosome 7, and its deoxynucleotides are G or A;

[0046] Osk34 is located at position 21324970 on chromosome 7, and its deoxynucleotides are T or C;

[0047] Osk35 is located at position 24622118 on chromosome 7, and its deoxynucleotides are T or C;

[0048] Osk36 is located at position 1530355 on chromosome 8, and its deoxynucleotides are C or T;

[0049] Osk37 is located at position 2275008 on chromosome 8, and its deoxynucleotides are T or C;

[0050] Osk38 is located at position 7890733 on chromosome 8, and its deoxynucleotides are A or G;

[0051] Osk39 is located at position 19466177 on chromosome 8, and its deoxynucleotides are G or A;

[0052] Osk40 is located at position 24070735 on chromosome 8, and its deoxynucleotides are G or A;

[0053] Osk41 is located at position 39319 on chromosome 9, and its deoxynucleotides are A or T;

[0054] Osk42 is located at position 10515653 on chromosome 9, and its deoxynucleotides are T or C;

[0055] Osk43 is located at position 12725653 on chromosome 9, and its deoxynucleotides are T or C;

[0056] Osk44 is located at position 13665180 on chromosome 9, and its deoxynucleotides are T or G;

[0057] Osk45 is located at position 17753206 on chromosome 9, and its deoxynucleotides are T or C;

[0058] Osk46 is located at position 369133 on chromosome 10, and its deoxynucleotides are T or C;

[0059] Osk47 is located at position 2335637 on chromosome 10, and its deoxynucleotides are C or T;

[0060] Osk48 is located at position 3787248 on chromosome 10, and its deoxynucleotides are T or C;

[0061] Osk49 is located at position 10724291 on chromosome 10, and its deoxynucleotides are T or G;

[0062] Osk50 is located at position 19029893 on chromosome 10, and its deoxynucleotides are C or T;

[0063] Osk51 is located at position 4374875 on chromosome 11, and its deoxynucleotides are C or T;

[0064] Osk52 is located at position 18935157 on chromosome 11, and its deoxynucleotides are G or A;

[0065] Osk53 is located at position 20525980 on chromosome 11, and its deoxynucleotides are T or C;

[0066] Osk54 is located at position 26085857 on chromosome 11, and its deoxynucleotides are G or A;

[0067] Osk55 is located at position 28010037 on chromosome 11, and its deoxynucleotides are T or G;

[0068] Osk56 is located at position 215397 on chromosome 12, and its deoxynucleotides are C or T;

[0069] Osk57 is located at position 1794727 on chromosome 12, and its deoxynucleotides are T or G;

[0070] Osk58 is located at position 2738115 on chromosome 12, and its deoxynucleotides are A or T;

[0071] Osk59 is located at position 13784750 on chromosome 12, and its deoxynucleotides are A or G;

[0072] Osk60 is located at position 24090217 on chromosome 12, and its deoxynucleotides are T or C.

[0073] In a second aspect, the present invention claims a set of KASP primers.

[0074] The set of KASP primers claimed in the present invention is KASP primers for detecting the 60 SNP sites described in the first aspect above.

[0075] 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 forward primer 1 is obtained by connecting a fluorescent label sequence to the 5' end of specific primer 1, and the forward primer 2 is obtained by connecting another fluorescent label sequence to the 5' end of specific primer 2; the reverse primer is a universal primer.

[0076] The specific primer 1 for detecting Osk01 is composed of nucleotides 22 and thereafter of SEQ ID No.1, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.61, and the reverse primer is composed of nucleotides shown in SEQ ID No.121;

[0077] The specific primer 1 for detecting Osk02 is composed of nucleotides 22 and later of SEQ ID No.2, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.62, and the reverse primer is composed of nucleotides shown in SEQ ID No.122;

[0078] The specific primer 1 for detecting Osk03 is composed of nucleotides 22 and later of SEQ ID No.3, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.63, and the reverse primer is composed of nucleotides shown in SEQ ID No.123;

[0079] The specific primer 1 for detecting Osk04 is composed of nucleotides 22 and later of SEQ ID No.4, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.64, and the reverse primer is composed of nucleotides shown in SEQ ID No.124;

[0080] The specific primer 1 for detecting Osk05 consists of nucleotides 22 and later of SEQ ID No.5, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.65, and the reverse primer consists of nucleotides shown in SEQ ID No.125;

[0081] The specific primer 1 for detecting Osk06 consists of nucleotides 22 and later of SEQ ID No.6, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.66, and the reverse primer consists of nucleotides shown in SEQ ID No.126;

[0082] The specific primer 1 for detecting Osk07 is composed of nucleotides 22 and later of SEQ ID No.7, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.67, and the reverse primer is composed of nucleotides shown in SEQ ID No.127;

[0083] The specific primer 1 for detecting Osk08 is composed of nucleotides 22 and later of SEQ ID No.8, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.68, and the reverse primer is composed of nucleotides shown in SEQ ID No.128;

[0084] The specific primer 1 for detecting Osk09 consists of nucleotides 22 and later of SEQ ID No.9, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.69, and the reverse primer consists of nucleotides shown in SEQ ID No.129;

[0085] The specific primer 1 for detecting Osk10 is composed of nucleotides 22 and later of SEQ ID No.10, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.70, and the reverse primer is composed of nucleotides shown in SEQ ID No.130;

[0086] The specific primer 1 for detecting Osk11 is composed of nucleotides 22 and later of SEQ ID No.11, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.71, and the reverse primer is composed of nucleotides shown in SEQ ID No.131;

[0087] The specific primer 1 for detecting Osk12 is composed of nucleotides 22 and later of SEQ ID No.12, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.72, and the reverse primer is composed of nucleotides shown in SEQ ID No.132;

[0088] The specific primer 1 for detecting Osk13 is composed of nucleotides 22 and later of SEQ ID No.13, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.73, and the reverse primer is composed of nucleotides shown in SEQ ID No.133;

[0089] The specific primer 1 for detecting Osk14 is composed of nucleotides 22 and later of SEQ ID No.14, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.74, and the reverse primer is composed of nucleotides shown in SEQ ID No.134;

[0090] The specific primer 1 for detecting Osk15 consists of nucleotides 22 and later of SEQ ID No.15, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.75, and the reverse primer consists of nucleotides shown in SEQ ID No.135;

[0091] The specific primer 1 for detecting Osk16 consists of the nucleotides 22 and thereafter of SEQ ID No.16, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.76, and the reverse primer consists of the nucleotides shown in SEQ ID No.136;

[0092] The specific primer 1 for detecting Osk17 is composed of nucleotides 22 and later of SEQ ID No.17, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.77, and the reverse primer is composed of nucleotides shown in SEQ ID No.137;

[0093] The specific primer 1 for detecting Osk18 is composed of nucleotides 22 and later of SEQ ID No.18, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.78, and the reverse primer is composed of nucleotides shown in SEQ ID No.138;

[0094] The specific primer 1 for detecting Osk19 consists of nucleotides 22 and later of SEQ ID No.19, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.79, and the reverse primer consists of nucleotides shown in SEQ ID No.139;

[0095] The specific primer 1 for detecting Osk20 consists of nucleotides 22 and later of SEQ ID No.20, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.80, and the reverse primer consists of nucleotides shown in SEQ ID No.140;

[0096] The specific primer 1 for detecting Osk21 consists of nucleotides 22 and later of SEQ ID No.21, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.81, and the reverse primer consists of nucleotides shown in SEQ ID No.141;

[0097] The specific primer 1 for detecting Osk22 consists of nucleotides 22 and later of SEQ ID No.22, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.82, and the reverse primer consists of nucleotides shown in SEQ ID No.142;

[0098] The specific primer 1 for detecting Osk23 consists of nucleotides 22 and later of SEQ ID No.23, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.83, and the reverse primer consists of nucleotides shown in SEQ ID No.143;

[0099] The specific primer 1 for detecting Osk24 consists of nucleotides 22 and later of SEQ ID No.24, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.84, and the reverse primer consists of nucleotides shown in SEQ ID No.144;

[0100] The specific primer 1 for detecting Osk25 consists of nucleotides 22 and later of SEQ ID No.25, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.85, and the reverse primer consists of nucleotides shown in SEQ ID No.145;

[0101] The specific primer 1 for detecting Osk26 consists of the nucleotides 22 and thereafter of SEQ ID No.26, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.86, and the reverse primer consists of the nucleotides shown in SEQ ID No.146;

[0102] The specific primer 1 for detecting Osk27 consists of nucleotides 22 and later of SEQ ID No.27, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.87, and the reverse primer consists of nucleotides shown in SEQ ID No.147;

[0103] The specific primer 1 for detecting Osk28 is composed of nucleotides 22 and later of SEQ ID No.28, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.88, and the reverse primer is composed of nucleotides shown in SEQ ID No.148;

[0104] The specific primer 1 for detecting Osk29 consists of nucleotides 22 and later of SEQ ID No.29, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.89, and the reverse primer consists of nucleotides shown in SEQ ID No.149;

[0105] The specific primer 1 for detecting Osk30 is composed of nucleotides 22 and later of SEQ ID No.30, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.90, and the reverse primer is composed of nucleotides shown in SEQ ID No.150;

[0106] The specific primer 1 for detecting Osk31 is composed of nucleotides 22 and later of SEQ ID No.31, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.91, and the reverse primer is composed of nucleotides shown in SEQ ID No.151;

[0107] The specific primer 1 for detecting Osk32 consists of nucleotides 22 and later of SEQ ID No.32, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.92, and the reverse primer consists of nucleotides shown in SEQ ID No.152;

[0108] The specific primer 1 for detecting Osk33 consists of nucleotides 22 and later of SEQ ID No.33, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.93, and the reverse primer consists of nucleotides shown in SEQ ID No.153;

[0109] The specific primer 1 for detecting Osk34 consists of nucleotides 22 and later of SEQ ID No.34, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.94, and the reverse primer consists of nucleotides shown in SEQ ID No.154;

[0110] The specific primer 1 for detecting Osk35 consists of nucleotides 22 and later of SEQ ID No.35, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.95, and the reverse primer consists of nucleotides shown in SEQ ID No.155;

[0111] The specific primer 1 for detecting Osk36 consists of nucleotides 22 and later of SEQ ID No.36, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.96, and the reverse primer consists of nucleotides shown in SEQ ID No.156;

[0112] The specific primer 1 for detecting Osk37 is composed of nucleotides 22 and later of SEQ ID No.37, the specific primer 2 is composed of nucleotides 22 and later of SEQ ID No.97, and the reverse primer is composed of nucleotides shown in SEQ ID No.157;

[0113] The specific primer 1 for detecting Osk38 is composed of nucleotides 22 and thereafter of SEQ ID No.38, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.98, and the reverse primer is composed of nucleotides shown in SEQ ID No.158;

[0114] The specific primer 1 for detecting Osk39 consists of nucleotides 22 and later of SEQ ID No.39, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.99, and the reverse primer consists of nucleotides shown in SEQ ID No.159;

[0115] The specific primer 1 for detecting Osk40 consists of nucleotides 22 and later of SEQ ID No.40, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.100, and the reverse primer consists of nucleotides shown in SEQ ID No.160;

[0116] The specific primer 1 for detecting Osk41 consists of nucleotides 22 and thereafter of SEQ ID No.41, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.101, and the reverse primer consists of nucleotides shown in SEQ ID No.161;

[0117] The specific primer 1 for detecting Osk42 consists of nucleotides 22 and later of SEQ ID No.42, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.102, and the reverse primer consists of nucleotides shown in SEQ ID No.162;

[0118] The specific primer 1 for detecting Osk43 consists of nucleotides 22 and later of SEQ ID No.43, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.103, and the reverse primer consists of nucleotides shown in SEQ ID No.163;

[0119] The specific primer 1 for detecting Osk44 consists of nucleotides 22 and thereafter of SEQ ID No.44, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.104, and the reverse primer consists of nucleotides shown in SEQ ID No.164;

[0120] The specific primer 1 for detecting Osk45 consists of nucleotides 22 and thereafter of SEQ ID No.45, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.105, and the reverse primer consists of nucleotides shown in SEQ ID No.165;

[0121] The specific primer 1 for detecting Osk46 consists of nucleotides 22 and later of SEQ ID No.46, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.106, and the reverse primer consists of nucleotides shown in SEQ ID No.166;

[0122] The specific primer 1 for detecting Osk47 consists of nucleotides 22 and thereafter of SEQ ID No.47, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.107, and the reverse primer consists of nucleotides shown in SEQ ID No.167;

[0123] The specific primer 1 for detecting Osk48 is composed of nucleotides 22 and thereafter of SEQ ID No.48, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.108, and the reverse primer is composed of nucleotides shown in SEQ ID No.168;

[0124] The specific primer 1 for detecting Osk49 consists of nucleotides 22 and later of SEQ ID No.49, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.109, and the reverse primer consists of nucleotides shown in SEQ ID No.169;

[0125] The specific primer 1 for detecting Osk50 consists of nucleotides 22 and later of SEQ ID No.50, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.110, and the reverse primer consists of nucleotides shown in SEQ ID No.170;

[0126] The specific primer 1 for detecting Osk51 consists of nucleotides 22 and later of SEQ ID No.51, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.111, and the reverse primer consists of nucleotides shown in SEQ ID No.171;

[0127] The specific primer 1 for detecting Osk52 consists of nucleotides 22 and thereafter of SEQ ID No.52, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.112, and the reverse primer consists of nucleotides shown in SEQ ID No.172;

[0128] The specific primer 1 for detecting Osk53 consists of nucleotides 22 and thereafter of SEQ ID No.53, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.113, and the reverse primer consists of nucleotides shown in SEQ ID No.173;

[0129] The specific primer 1 for detecting Osk54 consists of nucleotides 22 and later of SEQ ID No.54, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.114, and the reverse primer consists of nucleotides shown in SEQ ID No.174;

[0130] The specific primer 1 for detecting Osk55 consists of nucleotides 22 and thereafter of SEQ ID No.55, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.115, and the reverse primer consists of nucleotides shown in SEQ ID No.175;

[0131] The specific primer 1 for detecting Osk56 consists of nucleotides 22 and later of SEQ ID No.56, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.116, and the reverse primer consists of nucleotides shown in SEQ ID No.176;

[0132] The specific primer 1 for detecting Osk57 consists of nucleotides 22 and later of SEQ ID No.57, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.117, and the reverse primer consists of nucleotides shown in SEQ ID No.177;

[0133] The specific primer 1 for detecting Osk58 is composed of nucleotides 22 and thereafter of SEQ ID No.58, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.118, and the reverse primer is composed of nucleotides shown in SEQ ID No.178;

[0134] The specific primer 1 for detecting Osk59 consists of nucleotides 22 and later of SEQ ID No.59, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.119, and the reverse primer consists of nucleotides shown in SEQ ID No.179;

[0135] The specific primer 1 for detecting Osk60 consists of the 22nd and subsequent nucleotides of SEQ ID No.60, the specific primer 2 consists of the 22nd and subsequent nucleotides of SEQ ID No.120, and the reverse primer consists of the nucleotides shown in SEQ ID No.180.

[0136] In a specific embodiment of the present invention, in the forward primer 1, the fluorescent label sequence connected to the 5' end may be a FAM fluorescent label sequence; in the forward primer 2, the fluorescent label sequence connected to the 5' end is a HEX fluorescent label sequence.

[0137] Specifically, the FAM fluorescent label sequence is shown as positions 1 to 21 of SEQ ID No.1; the HEX fluorescent label sequence is shown as positions 1 to 21 of SEQ ID No.2.

[0138] Furthermore, the KASP primers used to detect each SNP site are as follows:

[0139] The nucleotide sequence of the forward primer 1 for detecting Osk01 is shown in SEQ ID No. 1, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 61, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 121;

[0140] The nucleotide sequence of the forward primer 1 for detecting Osk02 is shown in SEQ ID No. 2, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 62, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 122;

[0141] The nucleotide sequence of the forward primer 1 for detecting Osk03 is shown in SEQ ID No.3, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.63, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.123;

[0142] The nucleotide sequence of the forward primer 1 for detecting Osk04 is shown in SEQ ID No.4, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.64, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.124;

[0143] The nucleotide sequence of the forward primer 1 for detecting Osk05 is shown in SEQ ID No.5, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.65, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.125;

[0144] The nucleotide sequence of the forward primer 1 for detecting Osk06 is shown in SEQ ID No.6, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.66, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.126;

[0145] The nucleotide sequence of the forward primer 1 for detecting Osk07 is shown in SEQ ID No.7, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.67, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.127;

[0146] The nucleotide sequence of the forward primer 1 for detecting Osk08 is shown in SEQ ID No.8, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.68, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.128;

[0147] The nucleotide sequence of the forward primer 1 for detecting Osk09 is shown in SEQ ID No.9, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.69, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.129;

[0148] The nucleotide sequence of the forward primer 1 for detecting Osk10 is shown in SEQ ID No.10, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.70, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.130;

[0149] The nucleotide sequence of the forward primer 1 for detecting Osk11 is shown in SEQ ID No.11, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.71, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.131;

[0150] The nucleotide sequence of the forward primer 1 for detecting Osk12 is shown in SEQ ID No.12, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.72, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.132;

[0151] The nucleotide sequence of the forward primer 1 for detecting Osk13 is shown in SEQ ID No.13, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.73, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.133;

[0152] The nucleotide sequence of the forward primer 1 for detecting Osk14 is shown in SEQ ID No. 14, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 74, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 134;

[0153] The nucleotide sequence of the forward primer 1 for detecting Osk15 is shown in SEQ ID No.15, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.75, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.135;

[0154] The nucleotide sequence of the forward primer 1 for detecting Osk16 is shown in SEQ ID No.16, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.76, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.136;

[0155] The nucleotide sequence of the forward primer 1 for detecting Osk17 is shown in SEQ ID No. 17, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 77, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 137;

[0156] The nucleotide sequence of the forward primer 1 for detecting Osk18 is shown in SEQ ID No.18, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.78, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.138;

[0157] The nucleotide sequence of the forward primer 1 for detecting Osk19 is shown in SEQ ID No.19, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.79, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.139;

[0158] The nucleotide sequence of the forward primer 1 for detecting Osk20 is shown in SEQ ID No.20, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.80, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.140;

[0159] The nucleotide sequence of the forward primer 1 for detecting Osk21 is shown in SEQ ID No.21, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.81, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.141;

[0160] The nucleotide sequence of the forward primer 1 for detecting Osk22 is shown in SEQ ID No.22, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.82, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.142;

[0161] The nucleotide sequence of the forward primer 1 for detecting Osk23 is shown in SEQ ID No.23, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.83, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.143;

[0162] The nucleotide sequence of the forward primer 1 for detecting Osk24 is shown in SEQ ID No.24, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.84, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.144;

[0163] The nucleotide sequence of the forward primer 1 for detecting Osk25 is shown in SEQ ID No.25, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.85, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.145;

[0164] The nucleotide sequence of the forward primer 1 for detecting Osk26 is shown in SEQ ID No.26, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.86, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.146;

[0165] The nucleotide sequence of the forward primer 1 for detecting Osk27 is shown in SEQ ID No.27, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.87, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.147;

[0166] The nucleotide sequence of the forward primer 1 for detecting Osk28 is shown in SEQ ID No.28, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.88, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.148;

[0167] The nucleotide sequence of the forward primer 1 for detecting Osk29 is shown in SEQ ID No.29, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.89, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.149;

[0168] The nucleotide sequence of the forward primer 1 for detecting Osk30 is shown in SEQ ID No.30, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.90, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.150;

[0169] The nucleotide sequence of the forward primer 1 for detecting Osk31 is shown in SEQ ID No.31, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.91, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.151;

[0170] The nucleotide sequence of the forward primer 1 for detecting Osk32 is shown in SEQ ID No.32, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.92, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.152;

[0171] The nucleotide sequence of the forward primer 1 for detecting Osk33 is shown in SEQ ID No.33, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.93, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.153;

[0172] The nucleotide sequence of the forward primer 1 for detecting Osk34 is shown in SEQ ID No.34, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.94, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.154;

[0173] The nucleotide sequence of the forward primer 1 for detecting Osk35 is shown in SEQ ID No.35, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.95, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.155;

[0174] The nucleotide sequence of the forward primer 1 for detecting Osk36 is shown in SEQ ID No.36, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.96, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.156;

[0175] The nucleotide sequence of the forward primer 1 for detecting Osk37 is shown in SEQ ID No.37, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.97, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.157;

[0176] The nucleotide sequence of the forward primer 1 for detecting Osk38 is shown in SEQ ID No.38, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.98, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.158;

[0177] The nucleotide sequence of the forward primer 1 for detecting Osk39 is shown in SEQ ID No.39, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.99, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.159;

[0178] The nucleotide sequence of the forward primer 1 for detecting Osk40 is shown in SEQ ID No.40, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.100, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.160;

[0179] The nucleotide sequence of the forward primer 1 for detecting Osk41 is shown in SEQ ID No.41, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.101, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.161;

[0180] The nucleotide sequence of the forward primer 1 for detecting Osk42 is shown in SEQ ID No.42, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.102, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.162;

[0181] The nucleotide sequence of the forward primer 1 for detecting Osk43 is shown in SEQ ID No.43, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.103, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.163;

[0182] The nucleotide sequence of the forward primer 1 for detecting Osk44 is shown in SEQ ID No.44, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.104, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.164;

[0183] The nucleotide sequence of the forward primer 1 for detecting Osk45 is shown in SEQ ID No.45, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.105, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.165;

[0184] The nucleotide sequence of the forward primer 1 for detecting Osk46 is shown in SEQ ID No.46, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.106, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.166;

[0185] The nucleotide sequence of the forward primer 1 for detecting Osk47 is shown in SEQ ID No.47, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.107, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.167;

[0186] The nucleotide sequence of the forward primer 1 for detecting Osk48 is shown in SEQ ID No.48, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.108, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.168;

[0187] The nucleotide sequence of the forward primer 1 for detecting Osk49 is shown in SEQ ID No.49, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.109, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.169;

[0188] The nucleotide sequence of the forward primer 1 for detecting Osk50 is shown in SEQ ID No.50, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.110, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.170;

[0189] The nucleotide sequence of the forward primer 1 for detecting Osk51 is shown in SEQ ID No.51, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.111, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.171;

[0190] The nucleotide sequence of the forward primer 1 for detecting Osk52 is shown in SEQ ID No.52, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.112, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.172;

[0191] The nucleotide sequence of the forward primer 1 for detecting Osk53 is shown in SEQ ID No.53, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.113, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.173;

[0192] The nucleotide sequence of the forward primer 1 for detecting Osk54 is shown in SEQ ID No.54, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.114, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.174;

[0193] The nucleotide sequence of the forward primer 1 for detecting Osk55 is shown in SEQ ID No.55, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.115, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.175;

[0194] The nucleotide sequence of the forward primer 1 for detecting Osk56 is shown in SEQ ID No.56, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.116, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.176;

[0195] The nucleotide sequence of the forward primer 1 for detecting Osk57 is shown in SEQ ID No.57, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.117, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.177;

[0196] The nucleotide sequence of the forward primer 1 for detecting Osk58 is shown in SEQ ID No.58, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.118, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.178;

[0197] The nucleotide sequence of the forward primer 1 for detecting Osk59 is shown in SEQ ID No.59, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.119, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.179;

[0198] The nucleotide sequence of the forward primer 1 for detecting Osk60 is shown as SEQ ID No.60, the nucleotide sequence of the forward primer 2 is shown as SEQ ID No.120, and the nucleotide sequence of the reverse primer is shown as SEQ ID No.180.

[0199] In a third aspect, the present invention claims a kit comprising the set of KASP primers described in the second aspect above.

[0200] Furthermore, the reagent or kit may also contain KASP 2×Mastermix, MgCl2 and ddH2O.

[0201] The reagent or kit KASP 2×Mastermix (LGC, UK, KBS-1016002) contains fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B, as well as high-fidelity Taq enzyme, dNTP, Mg 2+ And so on.

[0202] The nucleotide sequence of the fluorescent probe A: 5'-gaaggtgaccaagttcatgct-3' 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.

[0203] The nucleotide sequence of the fluorescent probe B: 5'-gaaggtcggagtcaacggatt-3' 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.

[0204] In a specific embodiment of the present invention, the fluorescent group A is FAM; the fluorescent group B is HEX; and the quenching group is BHQ.

[0205] In a fourth aspect, the present invention claims the use of the set of KASP primers described in the second aspect above or the kit described in the third aspect above in any of the following:

[0206] (1) Preparing a rice SNP site detection kit;

[0207] (2) Genotyping of rice germplasm resources or genetic populations;

[0208] (3) Construct a DNA molecular fingerprint database of rice germplasm resources or varieties;

[0209] (4) Conduct genetic diversity analysis on rice germplasm resources.

[0210] In a fifth aspect, the present invention claims a method for genotyping rice germplasm resources or genetic populations.

[0211] The method for genotyping rice germplasm resources or genetic populations claimed in the present invention may include the following steps:

[0212] (a1) extracting genomic DNA from all rice samples in the tested rice germplasm resources or genetic populations;

[0213] (a2) for the rice sample DNA obtained in (a1), PCR reaction systems are prepared in sequence according to the 60 SNP sites described in the first aspect above, and PCR amplification is performed respectively using the corresponding primers in the set of KASP primers described in the second aspect above;

[0214] (a3) performing fluorescence scanning on the amplified product, reading and analyzing the fluorescence signal, thereby obtaining the genotyping results of the test rice sample DNA at the 60 SNP sites.

[0215] In a sixth aspect, the present invention claims protection for a method for constructing a DNA molecular fingerprint database of rice germplasm resources or varieties.

[0216] The method for constructing a DNA molecular fingerprint database of rice germplasm resources or varieties claimed in the present invention may include the following steps: extracting genomic DNA of rice germplasm resources or varieties used to construct the rice DNA molecular fingerprint database, detecting the genotypes of the 60 SNP sites mentioned in the first aspect above, and the genotype data of the detected rice germplasm resources or varieties at the 60 SNP sites obtained constitute the DNA molecular fingerprint database of rice germplasm resources or varieties.

[0217] In a specific embodiment of the present invention, after the genotypes of the 60 SNP sites are detected, the following steps are also included: the genotype data of each rice germplasm resource or variety at each SNP site are sorted into the format of 0, 1, 2, and arranged in combination according to the numbering order of the SNP sites, wherein the genotype consistent with the reference genome is recorded as 0, the genotype inconsistent with the reference genome is recorded as 1, the heterozygous genotype is recorded as 2, and the deletion is recorded as N. The reference genome is the whole genome sequence of the rice variety Nipponbare version number MSU7.0, and each rice germplasm resource or variety obtains a digital string consisting of 0, 1, 2, and the digital string can also be a rice germplasm resource or variety DNA fingerprint database.

[0218] In a seventh aspect, the present invention claims protection for a method for genetic diversity analysis of rice germplasm resources.

[0219] The method for genetic diversity analysis of rice germplasm resources claimed in the present invention may include the following steps: extracting genomic DNA of rice germplasm resources used for genetic diversity analysis, detecting the genotypes of the 60 SNP sites mentioned in the first aspect above, and then constructing a phylogenetic tree of rice resources using the neighbor-joining method based on the genetic distance between each rice germplasm resource.

[0220] Furthermore, in the sixth and seventh aspects, detecting the genotypes of the 60 SNP sites can be achieved by the method described in the fifth aspect above.

[0221] In a specific embodiment of the present invention, the rice is specifically selected from the rice shown in Table 5 and Table 6 in the examples.

[0222] The KASP molecular marker combination for distinguishing rice varieties provided by the present invention can efficiently, stably and accurately identify and distinguish rice varieties and germplasm resources, can achieve high-throughput, high-discrimination and low-cost large-scale identification of rice varieties, and is suitable for large-scale promotion and application.

[0223] The 60 SNP loci combination provided by the present invention can be applied to the construction of rice molecular fingerprint database, variety identification and analysis of genetic diversity of germplasm resources; and the application of this 60 core loci combination can greatly improve the detection accuracy, shorten the detection time and improve the detection level compared with the currently widely used biochemical detection method and SSR method. This will provide a more effective and reliable technical means for molecular detection and provide important technical support for rice variety identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0224] Figure 1 The following are the cluster diagrams of the genotyping of the four major KASP markers. (a) is the genotyping diagram of KASP marker K4; (b) is the typing analysis of KASP marker K82; (c) is the genotyping of KASP marker K49; (d) is the genotyping of KASP marker K138.

[0225] Figure 2 Polymorphism analysis of 60 KASP markers. (A) The physical location of the 60 SNP sites on the chromosome. The numbers on the right are the numbers of the 60 KASP markers, and the numbers on the left are the physical locations of the 60 KASP markers on the chromosome, in Mb; (B) The minimum allele frequency distribution (MAF) and polymorphism information content distribution (PIC) of the 60 SNP sites.

[0226] Figure 3 It is a DNA molecular fingerprint database of 80 rice samples.

[0227] Figure 4 This is a cluster analysis of 122 rice resources. DETAILED DESCRIPTION

[0228] 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.

[0229] 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.

[0230] Example 1: Acquisition of 60 core SNP loci combinations in rice germplasm resources

[0231] 1. Genotype data filtering analysis

[0232] The GenoBaits Rice 10K Panel developed by Boridi was used to perform a whole-genome scan of 314 rice germplasm resources in the Jiangsu Crop Germplasm Resource Bank to obtain the original data of whole-genome SNP genotyping of the 314 rice resources.

[0233] The raw sequencing data were filtered using fastp (version 0.20.0, parameters: -n 10-q 20-u 40) to obtain Clean Reads. Based on the alignment results of Clean Reads in the reference genome, the UnifiedGenotyper module of the GATK (version v3.5-0-g36282e4) software was used to detect variations. The mutation results were extracted from the genotyping information of the SNP sites using a self-written Perl script. When the coverage depth of a sample at a certain SNP site was <5X, it indicated that the sequencing depth of the sample at that site was insufficient. To ensure the accuracy of the genotyping results, the site was treated as missing and represented by NA in the table. When the sample had a mutation at a certain SNP site, it was necessary to judge based on the mutation frequency of the site. If the mutation frequency was ≥0.8 or ≤0.2, the site was homozygous. If the mutation frequency was between 0.2-0.8, each allele in the heterozygous genotype had at least 4 reads support, then the SNP site was a heterozygous mutation, otherwise the site was treated as missing and represented by NA. Finally, the genotyping results of the sample at all SNP sites were summarized, and a total of 22,428 dimorphic SNP site markers were obtained. The obtained SNP data were further filtered. Plink was used to convert the vcf file into a ped / map file, and then plink was used to filter the data. The screening criteria were based on sequencing depth greater than 10, minimum allele frequency greater than 0.05, genotype missing rate less than 0.1, and Hardy-Weinberg equilibrium significance threshold P<0.001. After filtering, 20,260 high-quality SNP sites were obtained.

[0234] 2. SNP site screening

[0235] Further screening was performed based on the conditions of polymorphic information content PIC>0.4 and dimorphism to obtain 1192 candidate SNP sites. From the candidate SNP sites, the candidate SNP sites with no other variable sites within 100 bp before and after were screened out. Then, the target SNPs that were conserved among the different rice varieties were selected from the candidate SNP sites. Finally, 170 SNP sites were screened out for the development of KASP markers.

[0236] 3. Development of KASP molecular markers

[0237] KASP primer combinations for PCR amplification were designed from the website http: / / www.snpway.com / based on 170 target SNP sites and flanking sequences to develop KASP molecular markers. Each KASP primer combination is designed with two SNP-specific primers (F1 and F2) and one universal primer (R). A specific sequence that can bind to FAM fluorescence is added to the tail of F1, and a specific sequence that can bind to HEX fluorescence is added to the tail of F2.

[0238] IV. Sample DNA Extraction and Quality Inspection

[0239] 46 7-day-old rice seedlings were sampled and plant genomic DNA was extracted using the CTAB method. The DNA concentration and quality of the samples were measured using the BioDrop uLite nucleic acid micro-analyzer, and all samples were uniformly diluted to an appropriate concentration for the instrument (about 10-20 ng / μl) based on the test results.

[0240] 5. PCR Amplification Process

[0241] 1. Aliquot the DNA sample into a 96-well PCR plate

[0242] Transfer the diluted DNA to a 96-well PCR sample plate and add 2 ddH2O (NTC) to each plate as a control.

[0243] 2. Preparation of KASP genotyping mixture

[0244] Referring to Table 1, the mixed solutions required for the reaction were prepared respectively.

[0245] Table 1. KASP genotyping mixture

[0246] 96-well array tape Wet DNA 0.8μl / well 2×KASP Master mix+Assay 0.8μl / well Total reaction volume 1.6μl / well

[0247] Note: All reagents should be vortexed briefly before use, and the lost volume should be added during preparation to ensure sufficient genotyping mix.

[0248] Add the KASP genotyping mixture to the array tape containing the DNA template. Use the IntelliQube SNP gene detection platform to compile a program, and place the 96 array tape, DNA sample plate, and KASP genotyping mixture into the machine in sequence. Operate the machine to execute the program, and automatically perform the process of dispensing the DNA sample dilution solution and the KASP genotyping mixture into the 96-well array tape and sealing the membrane.

[0249] 3. PCR Cycling Reaction

[0250] PCR reactions can be performed in IntelliQube machine in SNP genotyping inline mode (for single membrane) or in Hydrocycler in SNP genotyping outline mode (for multiple membranes) for waterbath PCR.

[0251] The program settings are shown in Table 2.

[0252] Table 2. PCR cycle reaction program

[0253]

[0254] 6. SNP genotyping

[0255] The PCR amplification products are read using a fluorescence quantitative PCR instrument or an enzyme reader that can detect FAM and HEX wavelength fluorescence, and SNP typing is performed based on the fluorescence ratio.

[0256] The genotyping results were determined according to the following criteria: the sample genotypes in red that were clustered close to the X-axis in the genotyping diagram were alleles combined with the FAM fluorescent label sequence, the sample genotypes in blue that were clustered close to the Y-axis were alleles connected to the HEX fluorescent label sequence, the sample genotypes in purple in the middle were heterozygous for the two alleles, the black samples were negative controls, and the gray samples were not clearly clustered and were treated as missing.

[0257] Data is recorded as XX or XY, where X and Y are single nucleotides at the site, such as A, T, C, G, arranged in this order. Invalid data is recorded as NA. If the invalid data ratio of a sample is higher than 10%, it will not be used. After testing, 122 pairs of high-quality KASP molecular marker primers were finally obtained. These marker sites can accurately and clearly cluster varieties with the same genotype (clustering forms such as Figure 1 ), while the negative control NTC (No Template Control) always aggregated together and did not produce any signal, with a success rate of 72% ( Figure 1 (a) and (b)). The main reasons why other primers were eliminated are: (1) they could not cluster different genotypes, and even clusters of the same genotype were relatively scattered ( Figure 1 (c)), (2) the primer amplification efficiency in the population is not high ( Figure 1 (d)).

[0258] VII. Screening of core KASP molecular markers

[0259] In order to further screen out representative markers, the 122 identified markers were filtered. According to the genotyping results and the distribution of the KASP primer combination on rice chromosomes (mainly based on the ability to accurately type the HEX type and FAM type genotypes and MAF>0.40, PIC>0.3, and uniform distribution of positions on chromosomes, etc.), 60 sites were finally screened out (the genomic location information and variant bases of the 60 SNP sites are shown in Table 3, and the corresponding primer sequences and their numbers are shown in Table 4) to form a KASP molecular marker combination for distinguishing rice varieties and germplasm resources. These sites cover 12 chromosomes of the rice genome, with an average interval of 6.2Mb ( Figure 2 (A)). The genetic diversity analysis software PowerMarker3.25 was used to conduct an overall evaluation of the allele frequencies of these sites. The PIC of the 60 SNP sites ranged from 0.34 to 0.50, with an average of 0.46, and the MAF ranged from 0.50 to 0.78, with an average of 0.604 ( Figure 2 Middle (B)), indicating that the 60 SNP sites have high specificity and high stability.

[0260] Table 3. Genomic location information and variant bases of 60 SNP sites

[0261]

[0262]

[0263] Note: The SNP site location information in the table is calculated based on the whole genome sequence of the rice variety Nipponbare version MSU7.0 as the reference genome.

[0264] Table 4. Primer sequence information of 60 KASP markers (5'-3')

[0265]

[0266]

[0267]

[0268]

[0269] Note: The 5' end of all "forward primers 1" is the FAM fluorescent label sequence 5'-GAAGGTGACCAAGTTCATGCT-3' (such as the underlined part in SEQ ID No.1); the 5' end of all "forward primers 2" is the HEX fluorescent label sequence 5'-GAAGGTCGGAGTCAACGGATT-3' (such as the underlined part in SEQ ID No.61).

[0270] Example 2: Application of 60 core SNP loci combination in constructing rice DNA molecular fingerprint database

[0271] 80 bred varieties that were widely promoted in Jiangsu Province between 1970 and 2020 were selected as experimental materials. The names of the varieties are shown in Table 5.

[0272] Table 5. 80 varieties that were widely promoted in Jiangsu Province between 1970 and 2020

[0273] Nanjing 23 Nanjing 52 Wu Xiangjing 19 Wulianjing No.1 Su Xiangjing 100 Ningjing No.1 Yanjing No.5 Lianjing No.6 Nanjing 34 Nanjing0212 Wuyunjing No.8 Wulingjing No.1 Sujing No.2 Ningjing No.3 Yanjing No.6 Lianjing No.7 Nanjing 35 Nanjing9108 Wuyunjing No.7 Wujing No. 4 Yangjing 186 Ningjing No.4 Yanjing No.9 Lianjing No.9 Nanjing 39 Nanjing 5055 Wuyunjing No.11 Wujing No. 13 Yangfujing No.8 Ningjing No.6 Yanjing No. 10 Lianjing No.11 Nanjing 41 Wuxiangjing No.1 Wuyunjing No. 23 Wujing No. 15 Yangyujing 2 Ningjing No.7 Yanjing No. 16 Lianjiajing No.1 Nanjing 44 Wuyujing No.2 Wuyunjing No. 24 Changnongjing No.4 Yangjing 4038 Ningjing No.8 Salt Rice No. 10 Xudao No.3 Nanjing 45 Wuyujing No.3 Wuyunjing No. 21 Changnongjing No.5 Huaidao No.5 Zhendao No.4 Salt Rice No. 8 Xudao No.4 Nanjing 46 Wuyujing No.5 Wuyunjing 27 Changnongjing No.8 Huaidao No.6 Zhendao No.11 Salt Rice No.9 Xudao No.5 Nanjing 49 Wuxiangjing No.9 Wuyunjing No. 31 Suxiangjing No.1 Huaidao No.11 Zhendao No.12 Lianjing No.3 Xudao No.9 Nanjing 51 Wuxiangjing No.14 Wu Yujing 20 Suxiangjing No.2 Huaidao No.13 Zhendao 88 Lianjing No.4 Xudao No.10

[0274] Rice DNA extraction and detection, PCR amplification and data collection gene analysis are carried out in the same manner as in Example 1. The PCR amplification product is read using a fluorescence quantitative PCR instrument or an enzyme reader that can detect FAM and HEX wavelength fluorescence, and SNP typing is performed based on the fluorescence ratio. The data is recorded as XX or XY, where X and Y are single nucleotides at the site, such as A, T, C, and G, arranged in this order. Invalid data is recorded as NA. If the proportion of invalid data for a sample is higher than 10%, it will not be adopted. The genotype data is organized into the format of 0, 1, and 2; the genotype (homozygous) that is consistent with the reference genome (the whole genome sequence of the rice variety Nipponbare version number MSU7.0) is 0; the inconsistent (homozygous) is 1, the heterozygous type is 2, and the invalid data is N. Two varieties are different varieties if there is a difference in one SNP site. The DNA molecular fingerprint map data of 80 rice varieties are as follows. Figure 3 As shown. It can be seen that the 60 core SNP marker combinations of the present invention can effectively distinguish the 80 rice varieties tested. Among them, Wuyunjing No. 7 and Wuyunjing No. 8 have only one locus difference. Pedigree analysis found that these two varieties have the same parental origin, indicating that the 60 core SNP marker combinations of the present invention can effectively distinguish the main bred varieties in Jiangsu area.

[0275] Example 3: Application of 60 core SNP loci combination in rice genetic diversity analysis

[0276] In order to further test the application of the molecular marker combination in the genetic diversity of rice germplasm resources, the 60 core KASP marker combinations developed by the present invention were used to perform genotyping on 122 germplasm resources (Table 6) preserved in the Jiangsu Province Crop Germplasm Resources Medium-term Bank, including 94 cultivated varieties and 28 local varieties (see Example 1 for specific operations). Then, based on the genetic distance between each variety, the neighbor-joining method was used to construct a phylogenetic tree of the 122 rice resources. The results showed that rice from different sources could be clustered according to pedigree origin and geographical distribution, reflecting the phylogenetic relationship between different rice varieties. The cluster analysis results of the 122 rice resources are shown in Figure 1. Figure 4 shown.

[0277] Table 6. Names of 122 rice germplasm resources

[0278]

[0279] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of a set of KASP primers for detecting 60 SNP sites in the rice genome in any of the following: (1) Preparation of a rice SNP site detection kit; (2) Genotyping of rice germplasm resources or genetic populations; (3) Construct a DNA molecular fingerprint database of rice germplasm resources or varieties; (4) Conduct genetic diversity analysis on rice germplasm resources; The physical positions of the 60 SNP sites are determined based on the whole genome sequence comparison of the rice variety Nipponbare, the version number of the whole genome sequence of the rice variety Nipponbare is MSU7.0; the 60 SNP sites are OsK01-OsK60; Osk1 is located at position 1228512 on chromosome 1, and its deoxynucleotide is G or A; Osk2 is located at position 4645481 on chromosome 1, and its deoxynucleotide is G or A; Osk3 is located at position 6644387 on chromosome 1, and its deoxynucleotides are C or A; Osk4 is located at position 9305876 on chromosome 1, and its deoxynucleotide is G or A; Osk5 is located at position 12854426 on chromosome 1, and its deoxynucleotides are C or G; Osk6 is located at position 24749480 on chromosome 1, and its deoxynucleotides are A or G; Osk7 is located at position 30545662 on chromosome 1, and its deoxynucleotides are A or G; Osk8 is located at position 5785532 on chromosome 2, and its deoxynucleotide is G or A; Osk9 is located at position 21772969 on chromosome 2, and its deoxynucleotides are G or C; Osk10 is located at position 34474061 on chromosome 2, and its deoxynucleotides are C or A; Osk11 is located at position 35882696 on chromosome 2, and its deoxynucleotides are A or G; Osk12 is located at position 7956017 on chromosome 3, and its deoxynucleotide is G or A; Osk13 is located at position 13775121 on chromosome 3, and its deoxynucleotide is G or A; Osk14 is located at position 16579520 on chromosome 3, and its deoxynucleotides are C or T; Osk15 is located at position 25181277 on chromosome 3, and its deoxynucleotides are A or G; Osk16 is located at position 15846017 on chromosome 4, and its deoxynucleotides are T or C; Osk17 is located at position 19209543 on chromosome 4, and its deoxynucleotides are T or C; Osk18 is located at position 21469290 on chromosome 4, and its deoxynucleotides are A or G; Osk19 is located at position 22842238 on chromosome 4, and its deoxynucleotides are A or G; Osk20 is located at position 31997187 on chromosome 4, and its deoxynucleotides are T or C; Osk21 is located at position 7384864 on chromosome 5, and its deoxynucleotides are T or C; Osk22 is located at position 17035145 on chromosome 5, and its deoxynucleotides are T or C; Osk23 is located at position 23305515 on chromosome 5, and its deoxynucleotides are G or C; Osk24 is located at position 1529649 on chromosome 6, and its deoxynucleotide is G or T; Osk25 is located at position 3166267 on chromosome 6, and its deoxynucleotides are T or C; Osk26 is located at position 8454606 on chromosome 6, and its deoxynucleotides are A or G; Osk27 is located at position 10345230 on chromosome 6, and its deoxynucleotide is G or A; Osk28 is located at position 25220061 on chromosome 6, and its deoxynucleotides are T or C; Osk29 is located at position 2865195 on chromosome 7, and its deoxynucleotides are A or G; Osk30 is located at position 4660773 on chromosome 7, and its deoxynucleotides are T or A; Osk31 is located at position 8809828 on chromosome 7, and its deoxynucleotides are A or G; Osk32 is located at position 10756612 on chromosome 7, and its deoxynucleotides are A or T; Osk33 is located at position 12924029 on chromosome 7, and its deoxynucleotide is G or A; Osk34 is located at position 21324970 on chromosome 7, and its deoxynucleotides are T or C; Osk35 is located at position 24622118 on chromosome 7, and its deoxynucleotides are T or C; Osk36 is located at position 1530355 on chromosome 8, and its deoxynucleotides are C or T; Osk37 is located at position 2275008 on chromosome 8, and its deoxynucleotides are T or C; Osk38 is located at position 7890733 on chromosome 8, and its deoxynucleotides are A or G; Osk39 is located at position 19466177 on chromosome 8, and its deoxynucleotide is G or A; Osk40 is located at position 24070735 on chromosome 8, and its deoxynucleotides are G or A; Osk41 is located at position 39319 on chromosome 9, and its deoxynucleotides are either A or T; Osk42 is located at position 10515653 on chromosome 9, and its deoxynucleotides are T or C; Osk43 is located at position 12725653 on chromosome 9, and its deoxynucleotides are T or C; Osk44 is located at position 13665180 on chromosome 9, and its deoxynucleotides are T or G; Osk45 is located at position 17753206 on chromosome 9, and its deoxynucleotides are T or C; Osk46 is located at position 369133 on chromosome 10, and its deoxynucleotides are T or C; Osk47 is located at position 2335637 on chromosome 10, and its deoxynucleotides are C or T; Osk48 is located at position 3787248 on chromosome 10, and its deoxynucleotides are T or C; Osk49 is located at position 10724291 on chromosome 10, and its deoxynucleotides are T or G; Osk50 is located at position 19029893 on chromosome 10, and its deoxynucleotides are C or T; Osk51 is located at position 4374875 on chromosome 11, and its deoxynucleotides are C or T; Osk52 is located at position 18935157 on chromosome 11, and its deoxynucleotide is G or A; Osk53 is located at position 20525980 on chromosome 11, and its deoxynucleotides are T or C; Osk54 is located at position 26085857 on chromosome 11, and its deoxynucleotide is G or A; Osk55 is located at position 28010037 on chromosome 11, and its deoxynucleotides are T or G; Osk56 is located at position 215397 on chromosome 12, and its deoxynucleotides are C or T; Osk57 is located at position 1794727 on chromosome 12, and its deoxynucleotides are T or G; Osk58 is located at position 2738115 on chromosome 12, and its deoxynucleotides are A or T; Osk59 is located at position 13784750 on chromosome 12, and its deoxynucleotides are A or G; Osk60 is located at position 24090217 on chromosome 12, and its deoxynucleotides are T or C; The set of KASP primers is a KASP primer for detecting the 60 SNP sites; 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 forward primer 1 is obtained by connecting a fluorescent label sequence to the 5' end of the specific primer 1, and the forward primer 2 is obtained by connecting another fluorescent label sequence to the 5' end of the specific primer 2; The specific primer 1 for detecting Osk01 consists of nucleotides 22 and thereafter of SEQ ID No.1, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.61, and the reverse primer consists of nucleotides shown in SEQ ID No.121; The specific primer 1 for detecting Osk02 consists of nucleotides 22 and thereafter of SEQ ID No.2, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.62, and the reverse primer consists of nucleotides shown in SEQ ID No.122; The specific primer 1 for detecting Osk03 consists of nucleotides 22 and thereafter of SEQ ID No.3, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.63, and the reverse primer consists of nucleotides shown in SEQ ID No.123; The specific primer 1 for detecting Osk04 consists of nucleotides 22 and thereafter of SEQ ID No.4, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.64, and the reverse primer consists of nucleotides shown in SEQ ID No.124; The specific primer 1 for detecting Osk05 consists of nucleotides 22 and thereafter of SEQ ID No.5, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.65, and the reverse primer consists of nucleotides shown in SEQ ID No.125; The specific primer 1 for detecting Osk06 consists of the nucleotides 22 and thereafter of SEQ ID No.6, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.66, and the reverse primer consists of the nucleotides shown in SEQ ID No.126; The specific primer 1 for detecting Osk07 consists of nucleotides 22 and thereafter of SEQ ID No.7, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.67, and the reverse primer consists of nucleotides shown in SEQ ID No.127; The specific primer 1 for detecting Osk08 is composed of nucleotides 22 and thereafter of SEQ ID No.8, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.68, and the reverse primer is composed of nucleotides shown in SEQ ID No.128; The specific primer 1 for detecting Osk09 consists of nucleotides 22 and thereafter of SEQ ID No.9, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.69, and the reverse primer consists of nucleotides shown in SEQ ID No.129; The specific primer 1 for detecting Osk10 consists of nucleotides 22 and thereafter of SEQ ID No.10, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.70, and the reverse primer consists of nucleotides shown in SEQ ID No.130; The specific primer 1 for detecting Osk11 is composed of nucleotides 22 and thereafter of SEQ ID No.11, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.71, and the reverse primer is composed of nucleotides shown in SEQ ID No.131; The specific primer 1 for detecting Osk12 consists of nucleotides 22 and thereafter of SEQ ID No.12, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.72, and the reverse primer consists of nucleotides shown in SEQ ID No.132; The specific primer 1 for detecting Osk13 consists of nucleotides 22 and thereafter of SEQ ID No.13, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.73, and the reverse primer consists of nucleotides shown in SEQ ID No.133; The specific primer 1 for detecting Osk14 consists of the nucleotides 22 and thereafter of SEQ ID No.14, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.74, and the reverse primer consists of the nucleotides shown in SEQ ID No.134; The specific primer 1 for detecting Osk15 consists of nucleotides 22 and thereafter of SEQ ID No.15, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.75, and the reverse primer consists of nucleotides shown in SEQ ID No.135; The specific primer 1 for detecting Osk16 consists of the nucleotides 22 and thereafter of SEQ ID No.16, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.76, and the reverse primer consists of the nucleotides shown in SEQ ID No.136; The specific primer 1 for detecting Osk17 consists of the nucleotides 22 and thereafter of SEQ ID No.17, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.77, and the reverse primer consists of the nucleotides shown in SEQ ID No.137; The specific primer 1 for detecting Osk18 is composed of nucleotides 22 and thereafter of SEQ ID No.18, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.78, and the reverse primer is composed of nucleotides shown in SEQ ID No.138; The specific primer 1 for detecting Osk19 consists of nucleotides 22 and thereafter of SEQ ID No.19, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.79, and the reverse primer consists of nucleotides shown in SEQ ID No.139; The specific primer 1 for detecting Osk20 consists of nucleotides 22 and thereafter of SEQ ID No.20, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.80, and the reverse primer consists of nucleotides shown in SEQ ID No.140; The specific primer 1 for detecting Osk21 consists of nucleotides 22 and thereafter of SEQ ID No.21, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.81, and the reverse primer consists of nucleotides shown in SEQ ID No.141; The specific primer 1 for detecting Osk22 consists of nucleotides 22 and thereafter of SEQ ID No.22, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.82, and the reverse primer consists of nucleotides shown in SEQ ID No.142; The specific primer 1 for detecting Osk23 consists of nucleotides 22 and thereafter of SEQ ID No.23, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.83, and the reverse primer consists of nucleotides shown in SEQ ID No.143; The specific primer 1 for detecting Osk24 consists of nucleotides 22 and thereafter of SEQ ID No.24, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.84, and the reverse primer consists of nucleotides shown in SEQ ID No.144; The specific primer 1 for detecting Osk25 consists of nucleotides 22 and thereafter of SEQ ID No.25, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.85, and the reverse primer consists of nucleotides shown in SEQ ID No.145; The specific primer 1 for detecting Osk26 consists of nucleotides 22 and thereafter of SEQ ID No.26, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.86, and the reverse primer consists of nucleotides shown in SEQ ID No.146; The specific primer 1 for detecting Osk27 consists of nucleotides 22 and thereafter of SEQ ID No.27, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.87, and the reverse primer consists of nucleotides shown in SEQ ID No.147; The specific primer 1 for detecting Osk28 consists of nucleotides 22 and thereafter of SEQ ID No.28, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.88, and the reverse primer consists of nucleotides shown in SEQ ID No.148; The specific primer 1 for detecting Osk29 consists of nucleotides 22 and thereafter of SEQ ID No.29, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.89, and the reverse primer consists of nucleotides shown in SEQ ID No.149; The specific primer 1 for detecting Osk30 consists of nucleotides 22 and thereafter of SEQ ID No.30, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.90, and the reverse primer consists of nucleotides shown in SEQ ID No.150; The specific primer 1 for detecting Osk31 consists of nucleotides 22 and thereafter of SEQ ID No.31, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.91, and the reverse primer consists of nucleotides shown in SEQ ID No.151; The specific primer 1 for detecting Osk32 consists of nucleotides 22 and thereafter of SEQ ID No.32, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.92, and the reverse primer consists of nucleotides shown in SEQ ID No.152; The specific primer 1 for detecting Osk33 consists of nucleotides 22 and thereafter of SEQ ID No.33, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.93, and the reverse primer consists of nucleotides shown in SEQ ID No.153; The specific primer 1 for detecting Osk34 consists of nucleotides 22 and later of SEQ ID No.34, the specific primer 2 consists of nucleotides 22 and later of SEQ ID No.94, and the reverse primer consists of nucleotides shown in SEQ ID No.154; The specific primer 1 for detecting Osk35 consists of nucleotides 22 and thereafter of SEQ ID No.35, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.95, and the reverse primer consists of nucleotides shown in SEQ ID No.155; The specific primer 1 for detecting Osk36 consists of nucleotides 22 and thereafter of SEQ ID No.36, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.96, and the reverse primer consists of nucleotides shown in SEQ ID No.156; The specific primer 1 for detecting Osk37 consists of nucleotides 22 and thereafter of SEQ ID No.37, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.97, and the reverse primer consists of nucleotides shown in SEQ ID No.157; The specific primer 1 for detecting Osk38 consists of nucleotides 22 and thereafter of SEQ ID No.38, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.98, and the reverse primer consists of nucleotides shown in SEQ ID No.158; The specific primer 1 for detecting Osk39 consists of nucleotides 22 and thereafter of SEQ ID No.39, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.99, and the reverse primer consists of nucleotides shown in SEQ ID No.159; The specific primer 1 for detecting Osk40 consists of nucleotides 22 and thereafter of SEQ ID No.40, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.100, and the reverse primer consists of nucleotides shown in SEQ ID No.160; The specific primer 1 for detecting Osk41 consists of the nucleotides 22 and thereafter of SEQ ID No.41, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.101, and the reverse primer consists of the nucleotides shown in SEQ ID No.161; The specific primer 1 for detecting Osk42 consists of nucleotides 22 and thereafter of SEQ ID No.42, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.102, and the reverse primer consists of nucleotides shown in SEQ ID No.162; The specific primer 1 for detecting Osk43 consists of nucleotides 22 and thereafter of SEQ ID No.43, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.103, and the reverse primer consists of nucleotides shown in SEQ ID No.163; The specific primer 1 for detecting Osk44 consists of nucleotides 22 and thereafter of SEQ ID No.44, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.104, and the reverse primer consists of nucleotides shown in SEQ ID No.164; The specific primer 1 for detecting Osk45 consists of nucleotides 22 and thereafter of SEQ ID No.45, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.105, and the reverse primer consists of nucleotides shown in SEQ ID No.165; The specific primer 1 for detecting Osk46 consists of the nucleotides 22 and thereafter of SEQ ID No.46, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.106, and the reverse primer consists of the nucleotides shown in SEQ ID No.166; The specific primer 1 for detecting Osk47 is composed of nucleotides 22 and thereafter of SEQ ID No.47, the specific primer 2 is composed of nucleotides 22 and thereafter of SEQ ID No.107, and the reverse primer is composed of nucleotides shown in SEQ ID No.167; The specific primer 1 for detecting Osk48 consists of the nucleotides 22 and thereafter of SEQ ID No.48, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.108, and the reverse primer consists of the nucleotides shown in SEQ ID No.168; The specific primer 1 for detecting Osk49 consists of the 22nd and subsequent nucleotides of SEQ ID No.49, the specific primer 2 consists of the 22nd and subsequent nucleotides of SEQ ID No.109, and the reverse primer consists of the nucleotides shown in SEQ ID No.169; The specific primer 1 for detecting Osk50 consists of nucleotides 22 and thereafter of SEQ ID No.50, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.110, and the reverse primer consists of nucleotides shown in SEQ ID No.170; The specific primer 1 for detecting Osk51 consists of nucleotides 22 and thereafter of SEQ ID No.51, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.111, and the reverse primer consists of nucleotides shown in SEQ ID No.171; The specific primer 1 for detecting Osk52 consists of nucleotides 22 and thereafter of SEQ ID No.52, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.112, and the reverse primer consists of nucleotides shown in SEQ ID No.172; The specific primer 1 for detecting Osk53 consists of nucleotides 22 and thereafter of SEQ ID No.53, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.113, and the reverse primer consists of nucleotides shown in SEQ ID No.173; The specific primer 1 for detecting Osk54 consists of the nucleotides 22 and thereafter of SEQ ID No.54, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.114, and the reverse primer consists of the nucleotides shown in SEQ ID No.174; The specific primer 1 for detecting Osk55 consists of nucleotides 22 and thereafter of SEQ ID No.55, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.115, and the reverse primer consists of nucleotides shown in SEQ ID No.175; The specific primer 1 for detecting Osk56 consists of the nucleotides 22 and thereafter of SEQ ID No.56, the specific primer 2 consists of the nucleotides 22 and thereafter of SEQ ID No.116, and the reverse primer consists of the nucleotides shown in SEQ ID No.176; The specific primer 1 for detecting Osk57 consists of nucleotides 22 and thereafter of SEQ ID No.57, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.117, and the reverse primer consists of nucleotides shown in SEQ ID No.177; The specific primer 1 for detecting Osk58 consists of nucleotides 22 and thereafter of SEQ ID No.58, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.118, and the reverse primer consists of nucleotides shown in SEQ ID No.178; The specific primer 1 for detecting Osk59 consists of nucleotides 22 and thereafter of SEQ ID No.59, the specific primer 2 consists of nucleotides 22 and thereafter of SEQ ID No.119, and the reverse primer consists of nucleotides shown in SEQ ID No.179; The specific primer 1 for detecting Osk60 consists of the 22nd and subsequent nucleotides of SEQ ID No.60, the specific primer 2 consists of the 22nd and subsequent nucleotides of SEQ ID No.120, and the reverse primer consists of the nucleotides shown in SEQ ID No.

180.

2. The use according to claim 1, characterized in that: In the forward primer 1, the fluorescent label sequence connected to the 5' end is a FAM fluorescent label sequence; in the forward primer 2, the fluorescent label sequence connected to the 5' end is a HEX fluorescent label sequence.

3. The use according to claim 2, characterized in that: The FAM fluorescent tag sequence is shown as positions 1 to 21 of SEQ ID No. 1; the HEX fluorescent tag sequence is shown as positions 1 to 21 of SEQ ID No.

2.

4. Use of a kit containing the set of KASP primers according to any one of claims 1 to 3 in any of the following: (1) Preparation of a rice SNP site detection kit; (2) Genotyping of rice germplasm resources or genetic populations; (3) Construct a DNA molecular fingerprint database of rice germplasm resources or varieties; (4) Conduct genetic diversity analysis on rice germplasm resources.

5. 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 test rice sample DNA obtained in (a1) according to the 60 SNP sites described in claim 1, and performing PCR amplification respectively using the corresponding primers in the set of KASP primers described in claim 1; (a3) performing fluorescence scanning on the amplified product, reading and analyzing the fluorescence signal, thereby obtaining the genotyping results of the test rice sample DNA at the 60 SNP sites.

6. A method for constructing a rice germplasm resource or variety DNA molecular fingerprint database, characterized in that: The method comprises the following steps: extracting genomic DNA of rice germplasm resources or varieties used for constructing a rice DNA molecular fingerprint database, detecting the genotypes of the 60 SNP sites mentioned in claim 1, and the genotype data of the detected rice germplasm resources or varieties at the 60 SNP sites constitute the rice germplasm resources or varieties DNA molecular fingerprint database.

7. A method for analyzing genetic diversity of rice germplasm resources, characterized in that: The method comprises the following steps: extracting genomic DNA of rice germplasm resources for genetic diversity analysis, detecting the genotypes of the 60 SNP sites mentioned in claim 1, and then constructing a phylogenetic tree of rice resources by using the neighbor-joining method based on the genetic distances between the rice germplasm resources.

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