Efficient identification method for excellent alleles of saline-alkaline tolerant genes of rice and application of efficient identification method

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an efficient identification method for excellent alleles of saline-alkaline tolerant genes of rice and application of the efficient identification method. According to the invention, Sanger sequencing verification is carried out aiming at excellent allelic specific SNP sites of five cloned saline-alkaline tolerant major genes, a Hi-TOM primer group is designed, and the Hi-TOM primer group mixed with the five saline-alkaline tolerant genes is used for carrying out two rounds of PCR amplification on DNA of a rice sample to be detected; and carrying out high-throughput sequencing on the second-round PCR amplification product, and analyzing the sequencing result by adopting a Hi-TOM tool, so that the existence condition of the excellent alleles of the five saline-alkaline tolerant genes in the to-be-detected rice sample can be quickly judged at one time. The method is simple, convenient, rapid, efficient and accurate, and can be widely applied to molecular marker-assisted selective breeding and identification of excellent alleles of saline-alkaline-tolerant natural variation genes of rice germplasm resources.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a high-efficiency identification method for an excellent allele of a salt-tolerant gene of rice and application thereof. BACKGROUND

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, and feeds more than half of the world's population. However, large areas of salinization and secondary salinization of soil worldwide have seriously restricted the production and distribution of rice. Salt-alkali stress can cause water imbalance, oxidative damage and nutrient imbalance in rice plants, thereby significantly inhibiting their growth, reducing tiller number, affecting panicle development, and ultimately leading to yield loss and crop quality decline. According to statistics, salt-alkali stress can cause rice yield reduction of up to 30%-50%, posing a continuous threat to food security.

[0003] Traditional methods for improving saline-alkali land, such as water conservancy salt washing and application of soil conditioners, are not only costly, time-consuming and labor-intensive, but also difficult to achieve long-lasting results. Therefore, tapping and utilizing the excellent salt-tolerant genes carried by rice itself to breed new rice varieties with intrinsic salt-tolerant ability is the most economical, environmentally friendly and sustainable fundamental approach to cope with salt-alkali stress. With the rapid development of molecular biology, genetic research on rice salt-tolerant traits has made a series of important progress, and multiple major salt-tolerant genes related to key physiological processes such as ion transport, osmotic regulation and active oxygen scavenging have been successfully cloned. SKC1 encodes a sodium ion transporter OsHKT1;5 of the HKT family, which maintains K⁺ / Na⁺ homeostasis by reducing Na⁺ transport from roots to shoots, thereby enhancing salt tolerance. OsPAO3 encodes a polyamine oxidase, and overexpression of OsPAO3 increases the polyamine content in seed coleoptile, enhances the activity of active oxygen scavenging enzymes, reduces the Na + content in seed coleoptile, thereby maintaining ion homeostasis and reducing Na +Damage. qSE3 encodes HAK family transporter OsHAK21, which regulates Na⁺ and K⁺ homeostasis during rice seed germination, induces abscisic acid biosynthesis and signal pathway gene expression, and inhibits the accumulation of active oxygen in seeds, thereby improving salt tolerance during seed germination. RST1 encodes auxin response factor OsARF18, and loss of function of RST1 increases the expression of OsAS1 (aspartate synthetase 1), improves nitrogen utilization efficiency, and thus enhances salt tolerance. Naturally occurring RST1 activity reduction leads to a 10.5% increase in grain yield under salt conditions. AT1 is the first cloned alkali-tolerant major QTL in the world, which is homologous to the grain shape regulating gene GS3 of rice and is responsible for encoding the G protein gamma subunit. Under high salt and alkali stress, AT1 regulates the activity of aquaporin under stress conditions by regulating the phosphorylation of aquaporin, which is responsible for pumping ROS (reactive oxygen species) generated under stress conditions to the outside of the cell, reducing peroxide stress, and thus improving the alkali tolerance of rice. These genes are important genetic basis for the difference in salt and alkali tolerance between rice varieties. It has been reported that SKC1-G / A Ex1-551 , OsPAO3-G / A Ex9-3474 , OsHAK21-T / C Ex2-807 , RST1-C / G Ex2-1612 and AT1-C / A Ex2-1637 The SNP types at the corresponding sites are closely linked to the salt and alkali tolerance phenotype of rice, and by identifying the SNP types at the corresponding sites of these genes, it can be determined whether the test sample carries the excellent alleles of these salt and alkali tolerance genes, and accordingly, possible salt and alkali tolerant rice resources can be screened, greatly reducing the screening range of possible salt and alkali tolerant rice varieties.

[0004] At present, the method for identifying these key salt and alkali tolerance genes mainly relies on sequencing the target fragment or using a single molecular marker for detection. However, the conventional sequencing method has the disadvantages of low throughput, high cost, and difficulty in coping with the screening needs of large-scale breeding populations or germplasm resource banks; and the latter can only detect a single gene or site, which is inefficient and cannot obtain the allele information of multiple genes at one time, which cannot meet the needs of modern rice breeding for multi-gene aggregation and efficient selection. SUMMARY

[0005] Therefore, the present application provides a high-efficiency method for identifying excellent alleles of salt and alkali tolerance genes in rice and its application, and successfully develops a technical method that can simultaneously, rapidly and high-throughput identify the excellent alleles of multiple salt and alkali tolerance major genes. This method can greatly accelerate the precise evaluation of salt and alkali tolerance germplasm resources, efficient mining and aggregation of favorable alleles, and thus significantly improve the breeding efficiency and success rate of salt and alkali tolerant rice new varieties.

[0006] To address the shortcomings of existing technologies, this invention is achieved through the following solution:

[0007] This invention provides a highly efficient method for identifying superior alleles of salt-alkali tolerance genes in rice, the method comprising the following steps:

[0008] (1) Extract DNA from the rice sample to be tested;

[0009] (2) Design amplification primers for SNP sites that are closely linked to the salt-tolerant phenotype. Use multiple (more than 15) rice DNAs with different backgrounds as templates to perform PCR amplification. Perform Sanger sequencing on the amplification products. Compare and analyze the sequencing results. Combine with phenotypic surveys, confirm the specific SNP sites that are closely linked to the salt-tolerant phenotype.

[0010] (3) Hi-TOM primer set I, Hi-TOM primer set II and Hi-TOM primer set III were designed for specific SNP sites closely linked to the salt tolerance phenotype. First, the DNA of the rice sample to be tested was amplified by the first round of PCR using Hi-TOM primer set I, which contains five salt tolerance genes. After verifying that the fragment size was correct and the band was single by running 1% agarose gel, the second round of PCR amplification products were obtained by using the first round of PCR amplification products as templates and Hi-TOM primer set II and Hi-TOM primer set III.

[0011] (4) The second round of PCR amplification products were resequencing to obtain sequencing results;

[0012] (5) The sequencing results were analyzed using the Hi-TOM analysis tool to obtain the identification results.

[0013] In this invention, in step (2), the primers used to verify the SNP type of the target site of the salt tolerance gene include 10 primers as shown in SEQ ID NO:1~10.

[0014] In the actual operation of this invention, the primer nucleotide sequences shown in SEQ ID NO:1~10 are as follows:

[0015] SKC1-gDNA-F: 5'-CGTCAACCCTACCACCACT-3', SEQ ID NO.1;

[0016] SKC1-gDNA-R: 5'-CCCATCACCACGAACATCA-3', SEQ ID NO.2;

[0017] OsPAO3-gDNA-F: 5'-GTGGAAGGAGGAAGCAATA-3', SEQ ID NO.3;

[0018] OsPAO3-gDNA-R: 5'-CGCAGGCATGTAAAACTAGA-3', SEQ ID NO.4;

[0019] OsHAK21-gDNA-F: 5'-AGGATAGCTGGGGGAGGAC-3', SEQ ID NO.5;

[0020] OsHAK21-gDNA-R: 5'-GACGGTGAAGAGCATGAAG-3', SEQ ID NO.6;

[0021] RST1-gDNA-F: 5'-CAGGCATGCTCAATTTGGTA-3', SEQ ID NO.7;

[0022] RST1-gDNA-R: 5'-GACCGATAATTAGACCAGCG-3', SEQ ID NO.8;

[0023] AT1-gDNA-F: 5'-ATGCCCATCTCCCTCGTTTA-3', SEQ ID NO.9;

[0024] AT1-gDNA-R: 5'-ACAAAAAGAAACAGCAGGCT-3', SEQ ID NO. 10.

[0025] In this invention, in step (3), the Hi-TOM primer set I is a primer with a bridging sequence, and the Hi-TOM primer set I includes primers as shown in SEQ ID NO:11~20;

[0026] The Hi-TOM primer set II consists of primers with bridging sequences and barcodes, and includes primers as shown in SEQ ID NO:21~40;

[0027] The Hi-TOM primer set III is a Hi-TOM primer set III containing INDEX and sequencing adapter sequences, and the Hi-TOM primer set III includes primers as shown in SEQ ID NO:41~42.

[0028] In the actual operation of this invention, according to the design requirements of the Hi-TOM primer set, the nucleotide sequence of Hi-TOM primer set I, which can specifically amplify the nucleotide sequences near the target SNP sites of each salt tolerance gene (fragment size between 100 and 200 bp) and contains bridging sequences, is shown below:

[0029] Hi-TOM-SKC1-F:5'-ggagtgagtacggtgtgcCGTCAACCCTACCACCACT-3',SEQ IDNO.11;

[0030] Hi-TOM-SKC1-R:5'-gagttggatgctggatggCCCATCACCACGAACATCA-3',SEQ IDNO.12;

[0031] Hi-TOM-OsPAO3-F:5'-ggagtgagtacggtgtgcGTGGAAGGAGGAAGCAATA-3',SEQ IDNO.13;

[0032] Hi-TOM-OsPAO3-R:5'-gagttggatgctggatggCGCAGGCATGTAAACTAGA-3',SEQ IDNO.14;

[0033] Hi-TOM-OsHAK21-F:5'-ggagtgagtacggtgtgcAGGATAGCTGGGGGAGGAC-3',SEQ IDNO.15;

[0034] Hi-TOM-OsHAK21-R:5'-gagttggatgctggatggGACGGTGAAGAGCATGAAG-3',SEQ IDNO.16;

[0035] Hi-TOM-RST1-F:5'-ggagtgagtacggtgtgcCAGGCATGCTCAATTTGGTA-3',SEQ IDNO.17;

[0036] Hi-TOM-RST1-R:5'-gagttggatgctggatggGACCGATAATTAGACCAGCG-3',SEQ IDNO.18;

[0037] Hi-TOM-AT1-F:5'-ggagtgagtacggtgtgcATGCCCATCTCCCTCGTTTA-3',SEQ IDNO.19;

[0038] Hi-TOM-AT1-R:5'-gagttggatgctggatggACAAAAAGAAACAGCAGGCT-3',SEQ IDNO.20;

[0039] In the actual operation of this invention, the nucleotide sequence of Hi-TOM primer set II is as follows:

[0040] F-1: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGCGTtggagtgagtacggtgtgc-3', SEQ ID NO: 21;

[0041] F-2: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGTAGtggagtgagtacggtgtgc-3', SEQ ID NO: 22;

[0042] F-3: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttACGCtggagtgagtacggtgtgc-3', SEQ ID NO: 23;

[0043] F-4: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttCTCGtggagtgagtacggtgtgc-3', SEQ ID NO: 24;

[0044] F-5: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGCTCtggagtgagtacggtgtgc-3', SEQ ID NO: 25;

[0045] F-6: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttAGTCtggagtgagtacggtgtgc-3', SEQ ID NO: 26;

[0046] F-7: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttCGACtggagtgagtacggtgtgc-3', SEQ ID NO: 27;

[0047] F-8: 5'-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGATGtggagtgagtacggtgtgc-3', SEQ ID NO: 28;

[0048] F-9:5‘-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttATACtggagtgagtacggtgtgc-3’,SEQ ID NO:29;

[0049] F-10:5‘-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttCACAtggagtgagtacggtgtgc-3’,SEQ ID NO:30;

[0050] F-11:5‘-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGTGCtggagtgagtacggtgtgc-3’,SEQ ID NO:31;

[0051] F-12:5‘-ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttACTAtggagtgagtacggtgtgc-3’,SEQ ID NO:32;

[0052] R-A:5‘-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGCGTtgagttggatgctggatgg-3’,SEQ ID NO:33;

[0053] R-B:5‘-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGTAGtgagttggatgctggatgg-3’,SEQ ID NO:34;

[0054] R-C:5‘-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtACGCtgagttggatgctggatgg-3’,SEQ ID NO:35;

[0055] R-D:5‘-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtCTCGtgagttggatgctggatgg-3’,SEQ ID NO:36;

[0056] R-E:5‘-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGCTCtgagttggatgctggatgg-3’,SEQ ID NO:37;

[0057] RF: 5'-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtAGTCtgagttggatgctggatgg-3', SEQ ID NO: 38;

[0058] RG: 5'-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtCGACtgagttggatgctggatgg-3', SEQ ID NO: 39;

[0059] RH: 5'-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGATGtgagttggatgctggatgg-3', SEQ ID NO: 40;

[0060] In the actual operation of this invention, the nucleotide sequence of Hi-TOM primer set III is as follows:

[0061] 2P-F: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT-3', SEQ IDNO: 41;

[0062] 2P-R:

[0063] 5'-CAAGCAGAAGACGGCATACGAGATCGCTGATCGTGACTGGAGTTCAGACGTGTGCTCTT-3', SEQ ID NO: 42.

[0064] In this invention, in step (5), the analysis is achieved by determining the SNP type of the target site of the salt tolerance gene.

[0065] The target site SNP types of the salt tolerance gene include:

[0066] Gene SKC1-G / A Ex1-551 The base at the site is G or A. When the site is A, it is considered to be an allelic type with excellent salt tolerance.

[0067] Gene OsPAO3-G / A Ex9-3474 The base at the site is G or A. When the site is A, it is considered to be an allelic type with excellent salt tolerance.

[0068] Gene OsHAK21-T / C Ex2-807 The base at the site is T or C. When the site is C, it is considered to be an allelic type with excellent salt tolerance.

[0069] Gene RST1-C / G Ex2-1612The base at the site is C or G. When the site is G, it is considered to be an allelic type with excellent salt tolerance.

[0070] Gene AT1-C / A Ex2-1637 The base at the site is C or A. When the site is A, it is considered to be an allelic type with excellent alkali resistance.

[0071] In this invention, in step (5), the identification result is obtained when the rice sample to be tested is identified as carrying SKC1-G / A. Ex1-551 OsPAO3-G / A Ex9-3474 OsHAK21-T / C Ex2-807 RST1-C / G Ex2-1612 and AT1-C / A Ex2-1637 If at least one superior allele is present in the gene, the sample is considered to be a salt-tolerant rice germplasm resource.

[0072] This invention provides the application of the above-mentioned identification method in molecular marker-assisted selection rice breeding.

[0073] This invention provides the application of the above-mentioned identification method in the rapid identification of salt and alkali tolerance traits in rice germplasm resources.

[0074] The present invention also provides a kit for efficiently identifying salt-tolerant rice germplasm resources, wherein the kit identifies rice germplasm resources using the above-mentioned identification method.

[0075] The present invention also provides a method for breeding salt-tolerant rice germplasm resources, characterized in that the breeding method includes using the above-mentioned identification method to breed salt-tolerant rice.

[0076] This invention also provides the application of the above-mentioned breeding method in the cultivation of new salt-tolerant rice varieties.

[0077] Compared with the prior art, the present invention has the following beneficial effects:

[0078] (1) A high-throughput technology system for multi-gene collaborative identification was established.

[0079] Based on multi-fragment resequencing technology and the Hi-TOM analysis platform, a one-time simultaneous detection of five key salt-alkali tolerance genes, namely SKC1, OsPAO3, OsHAK21, RST1 and AT1, was achieved. This method breaks through the technical bottleneck of traditional single-gene, low-throughput identification and provides an efficient and integrated molecular tool for the systematic genetic evaluation of rice salt-alkali tolerance.

[0080] (2) It enables precise and rapid identification of superior alleles of salt-tolerant genes.

[0081] By using Hi-TOM primer sets designed for specific SNP sites of five key salt-tolerant genes, combined with two rounds of PCR amplification, high-throughput sequencing, and Hi-TOM analysis tools, the presence of superior alleles of the five salt-tolerant genes in rice materials can be accurately and quickly determined. This provides a reliable basis for accurately screening individuals carrying multiple superior alleles from a large number of germplasm resources.

[0082] (3) Significantly improve the selection efficiency and predictability of salt-tolerant breeding

[0083] Using the primer set and identification method provided by this invention, superior single plants that aggregate multiple salt-tolerant gene alleles can be quickly identified in early breeding populations, significantly shortening the screening cycle and reducing the cost of field phenotypic identification.

[0084] (4) Provide a general platform for germplasm resource evaluation and gene mining

[0085] This invention is not only applicable to the screening of breeding materials, but can also be widely used in the survey of salt-tolerant genotypes and the discovery of superior alleles in rice germplasm resource banks. It has important scientific research and practical value and broad application prospects. Attached Figure Description

[0086] Figure 1 Maps showing the coding regions of the target superior alleles in the SKC1, OsPAO3, OsHAK21, RST1, and AT1 genes;

[0087] in, Figure 1 A represents the salt-tolerant allele of the SKC1 gene, SKC1-G / A. Ex1-551 (A structural diagram of the mutation of base G to A at position 551 of exon 1);

[0088] Figure 1 B represents the salt-tolerant allele of the OsPAO3 gene, OsPAO3-G / A. Ex9-3474 (A structural diagram of the mutation of base G to A at position 3474 of exon 9);

[0089] Figure 1 C represents the salt-tolerant allele of the OsHAK21 gene, OsHAK21-T / C. Ex2-807 (A structural diagram of the mutation of the 807th base in the second exon from T to C);

[0090] Figure 1 D represents the salt-tolerant allele of the RST1 gene, RST1-C / G. Ex2-1612 (A structural diagram of the mutation of base C to G at position 1612 of exon 2);

[0091] Figure 1E represents the AT1-C / A allele, which exhibits superior alkali tolerance in the AT1 gene. Ex2-1637 (A structural diagram of the mutation of base C to A at position 1637 of exon 2).

[0092] Figure 2 To perform the first round of PCR amplification on 18 rice materials using Hi-TOM primer set I containing five salt tolerance genes, 12 amplification products were randomly selected and run on 1% agarose gel to verify fragment size and band uniformity. Detailed Implementation

[0093] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0094] Example 1

[0095] Determination of the superior allelic-specific SNP types of five salt tolerance genes

[0096] 1.1 Selection of Variety Materials

[0097] Eighteen rice varieties were selected as verification materials.

[0098] 1.2 Extraction of DNA from Rice Leaves

[0099] After soaking, germinating, and germinating 18 rice samples, a suitable amount of leaves were cut into 2 mL centrifuge tubes, and DNA was rapidly extracted using 1xTPS extraction solution.

[0100] 1.3 Target gene SKC1-G / A Ex1-551 OsPAO3-G / A Ex9-3474 OsHAK21-T / C Ex2-807 RST1-C / G Ex2 -1612 and AT1-C / A Ex2-1637 Comparison of fragment amplification and sequencing results

[0101] Based on the SKC1, OsPAO3, OsHAK21, RST1, and AT1 genome sequences provided on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), amplification primers were designed for the genome sequences near the target SNP sites as follows:

[0102] SKC1-gDNA-F: 5'-CGTCAACCCTACCACCACT-3', SEQ ID NO.1;

[0103] SKC1-gDNA-R: 5'-CCCATCACCACGAACATCA-3', SEQ ID NO.2;

[0104] OsPAO3-gDNA-F: 5'-GTGGAAGGAGGAAGCAATA-3', SEQ ID NO.3;

[0105] OsPAO3-gDNA-R: 5'-CGCAGGCATGTAAAACTAGA-3', SEQ ID NO.4;

[0106] OsHAK21-gDNA-F: 5'-AGGATAGCTGGGGGAGGAC-3', SEQ ID NO.5;

[0107] OsHAK21-gDNA-R: 5'-GACGGTGAAGAGCATGAAG-3', SEQ ID NO.6;

[0108] RST1-gDNA-F: 5'-CAGGCATGCTCAATTTGGTA-3', SEQ ID NO.7;

[0109] RST1-gDNA-R: 5'-GACCGATAATTAGACCAGCG-3', SEQ ID NO.8;

[0110] AT1-gDNA-F: 5'-ATGCCCATCTCCCTCGTTTA-3', SEQ ID NO.9;

[0111] AT1-gDNA-R: 5'-ACAAAAAGAAACAGCAGGCT-3', SEQ ID NO. 10.

[0112] Using the primers described above, DNA extracted from rice leaves was amplified by PCR (using Vazyme, 2 × Rapid Taq Master Mix). Ten amplification products were randomly selected and run on 1% agarose gel to verify fragment size and band uniformity. After confirming that the fragment size was correct, the PCR products were sent to a biotechnology company for Sanger sequencing. The sequencing results were analyzed by sequence alignment to determine the base types of the five salt tolerance gene target sites SNPs in each rice material (Table 1).

[0113] Table 1. Sanger sequencing identification of superior allele-specific SNPs for salt-alkali tolerance genes in 18 rice varietal materials.

[0114]

[0115] Note: The bolded bases represent the specific SNP types corresponding to the superior alleles of the salt-tolerant gene.

[0116] Example 2

[0117] Hi-TOM primer set design for superior allelic-specific SNP sites

[0118] 2. Based on the design requirements of the Hi-TOM primer set, the following is the sequence of Hi-TOM primer set I, which can specifically amplify nucleotide sequences (fragment size between 100 and 200 bp) near the target SNP sites of various salt tolerance genes and contains bridging sequences:

[0119] Hi-TOM-SKC1-F: 5'-ggagtgagtacggtgtgcCGTCAACCCTACCACCACT-3', SEQ IDNO.11;

[0120] Hi-TOM-SKC1-R: 5'-gagttggatgctggatggCCCATCACCACGAACATCA-3', SEQ IDNO.12;

[0121] Hi-TOM-OsPAO3-F: 5'-ggagtgagtacggtgtgcGTGGAAGGAGGAAGCAATA-3', SEQ IDNO.13;

[0122] Hi-TOM-OsPAO3-R: 5'-gagttggatgctggatggCGCAGGCATGTAAAACTAGA-3', SEQ IDNO.14;

[0123] Hi-TOM-OsHAK21-F: 5'-ggagtgagtacggtgtgcAGGATAGCTGGGGGAGGAC-3', SEQ IDNO.15;

[0124] Hi-TOM-OsHAK21-R: 5'-gagttggatgctggatggGACGGTGAAGAGCATGAAG-3', SEQ IDNO.16;

[0125] Hi-TOM-RST1-F: 5'-ggagtgagtacggtgtgcCAGGCATGCTCAATTTGGTA-3', SEQ IDNO.17;

[0126] Hi-TOM-RST1-R: 5'-gagttggatgctggatggGACCGATAATTAGACCAGCG-3', SEQ IDNO.18;

[0127] Hi-TOM-AT1-F: 5'-ggagtgagtacggtgtgcATGCCCATCTCCCTCGTTTA-3', SEQ IDNO.19;

[0128] Hi-TOM-AT1-R: 5'-gagttggatgctggatggACAAAAAGAAACAGCAGGCT-3', SEQ ID NO. 20.

[0129] Using Hi-TOM primer set I, which mixes the five genes mentioned above, the DNA extracted from 18 rice samples in Example 1 was subjected to the first round of PCR amplification (using Vazyme, 2 × Rapid Taq Master Mix). Twelve PCR products were randomly selected and electrophoresed with 1% agarose gel. The results showed that the amplified fragments were of the correct size and the bands were single and bright. Figure 2 Preliminary results indicate that the Hi-TOM primer set I is usable.

[0130] Example 3

[0131] Hi-TOM sequencing identification of specific SNP loci corresponding to superior alleles of rice salt-alkali tolerance genes

[0132] 3.1 Selection of Variety Materials

[0133] Same as 1.1 in Example 1.

[0134] 3.2 Extraction of DNA from rice population materials

[0135] Same as 1.2 in Example 1.

[0136] 3.3 The first round of PCR amplification was performed using Hi-TOM primer set I, which contains a mixture of the above five genes.

[0137] Taking a 20 μL amplification system (using Vazyme, 2 × Rapid Taq Master Mix as an example), the amplification system is shown in Table 2.

[0138] Table 2 First-round PCR amplification system

[0139]

[0140] Amplification program: 95℃ for 3 min, 95℃ for 15 s, 56℃ for 15 s, 72℃ for 5 s (2 to 4 steps, 40 cycles), 72℃ for 5 min, 16℃ for 5 min.

[0141] 3.4 Hi-TOM Second Round PCR Amplification

[0142] The product from the first round of PCR amplification was used as a template for the second round of amplification. Taking a 20 μL amplification system (using Vazyme, 2 × Rapid Taq Master Mix as the enzyme) as an example, the amplification system is shown in Table 3.

[0143] Table 3 Second-round PCR amplification system

[0144]

[0145] Table 3 lists the primer sequences and concentrations involved: primer sets with bridging sequences and barcodes are shown in Table 4, and primer sets with INDEX and sequencing adapter sequences are shown in Table 5.

[0146] Table 4 Primer sets with bridging sequences and barcodes

[0147]

[0148] Table 5 Primer sets containing INDEX and sequencing adapter sequences:

[0149]

[0150] In the amplification program, the number of cycles is reduced compared to the general program, as follows: 95℃ 3 min, 95℃ 15 s, 60℃ 15 s, 72℃ 15 s (2 to 4 steps, 15 cycles), 72℃ 5 min, 18℃ 2 min.

[0151] After the second round of amplification, next-generation sequencing was performed, and the obtained data was used for subsequent mutation identification and analysis.

[0152] 3.5 Processing and Application of Hi-TOM Analysis Results

[0153] After obtaining the raw data from the second round of amplification using next-generation sequencing, the Hi-TOM tool (http: / / www.hi-tom.net / hi-tom / ) was used for analysis. For details, please refer to the paper published by Professor Liu Qing of the China National Rice Research Institute (Liu et al., 2019). After classifying and organizing the Hi-TOM results, it was determined that SKC1-G / A was the most important of the five salt-tolerant genes mentioned above. Ex1-551 OsPAO3-G / AEx9-3474 OsHAK21-T / C Ex2-807 RST1-C / G Ex2-1612 and AT1-C / A Ex2-1637 The base types of the corresponding SNP sites are shown in Table 6. When a superior allelic type of at least one of the five salt-tolerant genes mentioned above is present in the rice population to be tested, the sample is considered to be a salt-tolerant rice germplasm resource. Phenotypic examination of the sample can then be conducted to screen for potential salt-tolerant rice resources, significantly narrowing down the range of possible salt-tolerant rice varieties.

[0154] Table 6. Hi-TOM sequencing identification of superior alleles of salt-alkali tolerance genes in 18 rice materials.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for efficiently identifying an excellent allele of a salt-tolerant gene in rice, characterized in that, The identification method comprises the following steps: (1) extracting DNA of the to-be-tested rice sample; (2) designing primers for target SNP sites of five salt-tolerant main-effect genes, using the DNA of the to-be-tested rice sample as a template to perform PCR amplification to obtain an amplification product, performing Sanger sequencing on the amplification product, and performing alignment analysis on the sequencing result to confirm the base type of the specific SNP site closely linked to the salt-tolerant phenotype; (3) designing Hi-TOM primer group I, Hi-TOM primer group II and Hi-TOM primer group III according to the specific SNP site closely linked to the salt-tolerant phenotype that has been confirmed; using Hi-TOM primer group I to perform first-round PCR amplification on the DNA of the to-be-tested rice sample to obtain a first-round PCR amplification product, and using the first-round PCR amplification product as a template to perform amplification with Hi-TOM primer group II and Hi-TOM primer group III to obtain a second-round PCR amplification product; (4) performing resequencing on the second-round PCR amplification product to obtain a sequencing result; (5) using a Hi-TOM analysis tool to analyze the sequencing result to obtain an identification result.

2. The method of claim 1, wherein, In step (2), the primers for verifying the SNP type of the target site of the salt-tolerant gene comprise 10 primers as shown in SEQ ID NOs: 1-10.

3. The method of claim 1, wherein, In step (3), the Hi-TOM primer group I is a primer with a bridging sequence, and the Hi-TOM primer group I comprises 10 primers as shown in SEQ ID NOs: 11-20; the Hi-TOM primer group II is a primer with a bridging sequence and a barcode, and the Hi-TOM primer group II comprises 20 primers as shown in SEQ ID NOs: 21-40; the Hi-TOM primer group III is a Hi-TOM primer group III with an INDEX and a sequencing adapter sequence, and the Hi-TOM primer group III comprises 2 primers as shown in SEQ ID NOs: 41-42.

4. The method of claim 1, wherein, In step (5), the analysis is achieved by determining the SNP type of the target site of the salt-tolerant gene; the SNP type of the target site of the salt-tolerant gene comprises: Gene SKC1- G / A Ex1-551 The base at the site is G or A, and when the site is A, it is considered an excellent salt-tolerant allele. Gene OsPAO3 G / A Ex9-3474 the base at the site is G or A, and when the site is A, it is considered an excellent salt-tolerant allele; Gene OsHAK21 T / C Ex2-807 the base at the site is T or C, and when the site is C, it is considered an excellent salt-tolerant allele; Gene RST1 C / G Ex2-1612 the base at the site is C or G, and when the site is G, it is considered an excellent salt-tolerant allele; Gene AT1 C / A Ex2-1637 The base at the site is C or A, and when the site is A, it is considered an excellent alkali-tolerant allele.

5. The method of claim 1, wherein, In step (5), the identification result, when the sample to be tested is identified as carrying at least one excellent allele of the genes of SKC1- G / A Ex1-551 , OsPAO3 -G / A Ex9-3474 , OsHAK21 -T / C Ex2-807 , RST1 -C / G Ex2 -1612 and AT1 -C / A Ex2-1637 , it is considered that the sample may be a saline-alkali tolerant rice germplasm resource.

6. Use of the identification method of any one of claims 1-5 in molecular marker-assisted selection of rice breeding.

7. Use of the identification method of any one of claims 1-5 in rapid identification of salt-tolerant traits of rice germplasm resources.

8. A kit for efficiently identifying saline-alkaline tolerant rice germplasm resources, characterized in that, The kit is used for identifying rice germplasm resources by the identification method of any one of claims 1-5.

9. A method for breeding a saline-alkaline water-tolerant rice germplasm resource, characterized in that, The breeding method comprises breeding salt-tolerant rice by using the identification method of any one of claims 1-5.

10. Use of the breeding method of claim 9 in cultivating new varieties of salt-tolerant rice.

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