Low-temperature germination resistant rice gene chip in cold region and application thereof

Abstract

The application provides a cold-resistant low-temperature germination rice gene chip and its application, which contains 1016 SNP sites, is distributed in 12 chromosomes, is derived from large-scale GWAS analysis of 216 cold land germplasm, and has the characteristics of high polymorphism, low deletion rate and low heterozygosity. Its preparation method comprises extracting DNA, sequencing, comparing and screening associated sites, and designing specific probes based on this to make a liquid phase chip. The chip shows high precision (genotype consistency ≥98%) and high efficiency in application, and is mainly used for rapid screening of cold-resistant low-temperature rice germplasm resources, whole genome selection breeding and trait association analysis. Using the chip, the breeding period can be shortened from 6-8 generations to 3-4 generations, and the screening efficiency is improved by 4-5 times compared with traditional phenotype identification, which provides a powerful molecular tool and complete solution for precise breeding of cold-resistant low-temperature rice varieties.

Description

Technical Field

[0001] This application relates to the field of molecular plant breeding, specifically to a cold-resistant, low-temperature germination rice gene chip and its application. Background Technology

[0002] As the staple food for more than half of the world's population, the safe production of rice is of paramount importance. In my country's Northeast, Northwest, and high-altitude cold-region rice-growing areas, rice production holds irreplaceable strategic significance for ensuring national food security. However, these regions frequently experience sustained low-temperature stress during the rice planting season, leading to delayed seed germination, low emergence rates, and uneven seedling quality, ultimately resulting in severe yield reduction and quality decline. Therefore, improving the low-temperature germination tolerance of rice, especially cold-region rice varieties, is one of the key bottlenecks in stabilizing and increasing cold-region rice yields.

[0003] Currently, the improvement of low-temperature germination traits in rice mainly relies on the following technical approaches, but all of them have significant limitations: Traditional breeding methods heavily depend on directly investigating the germination rate and seedling survival rate of large batches of breeding materials under low-temperature conditions in early spring. This method has inherent defects: long cycle and low efficiency: limited to a single-crop-per-year planting season, screening can only be conducted once a year, significantly extending the breeding cycle. Significant environmental interference and poor accuracy: the microenvironmental conditions such as field temperature, moisture, and soil are difficult to maintain uniformity, leading to large fluctuations and poor repeatability in phenotypic identification results, resulting in severely insufficient screening accuracy. Germination traits can only be evaluated in the mid-to-late growing season, making effective selection before sowing or in the early seedling stage impossible, wasting considerable human, material, and land resources. Furthermore, systematic research specifically targeting low-temperature tolerance during seed germination is relatively scarce and insufficiently in-depth. Summary of the Invention

[0004] To address the shortcomings in existing technologies regarding the lack of innovation in core germplasm resources and the scarcity of cold-resistant, low-temperature germination rice varieties, and the inability of traditional breeding methods to accurately locate genes in low-temperature germination rice, this application provides a cold-resistant, low-temperature germination rice gene chip and its application, achieving precise labeling through 1016 SNP loci.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] This application proposes a cold-resistant, low-temperature germination rice gene chip, wherein the genotyping sites of the chip include 1016 SNP sites.

[0007] Preferably, the 1016 SNP loci are located at 135 SNP loci on chromosome 1, 95 SNP loci on chromosome 2, 176 SNP loci on chromosome 3, 102 SNP loci on chromosome 4, 51 SNP loci on chromosome 5, 32 SNP loci on chromosome 6, 67 SNP loci on chromosome 7, 87 SNP loci on chromosome 8, 104 SNP loci on chromosome 9, 65 SNP loci on chromosome 10, 38 SNP loci on chromosome 11, and 64 SNP loci on chromosome 12.

[0008] Preferably, the 1016 SNP loci were selected from 62027 initial loci in 216 cold-region rice germplasms.

[0009] This application proposes a method for designing a gene chip for cold-resistant, low-temperature germinating rice, the method comprising the following steps:

[0010] (1) Extract DNA samples from rice samples and sequence them to obtain the original loci of rice;

[0011] (2) The original sites are compared with the target reference genome and duplicates are removed to obtain the filtered sites;

[0012] (3) Screening out low-temperature resistant associated sites from the filter sites, the associated sites include 1016 SNP sites;

[0013] (4) Nucleotide probes were designed based on the 1016 SNP sites selected, and liquid-phase chips were prepared.

[0014] Preferably, the source of the original site in step (1) is the GWAS associated interval related to the low temperature resistance trait.

[0015] Preferably, the screening conditions in step (3) are: MAF≥0.35, uniformly distributed on chromosomes, and deletion rate≤0.1.

[0016] More preferably, the screening criteria also include a heterozygosity rate of ≤0.3.

[0017] Preferably, the chip is used for screening cold-resistant rice germplasm resources.

[0018] Preferably, the chip is used in rice genome selection breeding.

[0019] Another aspect of this application provides an application of a cold-resistant, low-temperature germination rice gene chip, which includes rice genotyping, association analysis of rice low-temperature resistance traits, and rice breeding.

[0020] The beneficial effects of the embodiments in this application are as follows:

[0021] This application obtained 62,027 low-temperature tolerance-related loci through genome-wide association analysis of 216 natural rice populations. Further screening revealed 1,016 SNP loci associated with low-temperature tolerance genes in rice. Based on these 1,016 SNP loci, probe combinations were synthesized according to the principle of base complementarity to prepare a liquid-phase chip. This liquid-phase chip can be used for rice low-temperature tolerance genotyping, marker-assisted breeding of low-temperature germinating rice, and genome-wide selection breeding. Attached Figure Description

[0022] Figure 1 This is a flowchart of the genomic DNA extraction process in Example 1 of this application;

[0023] Figure 2 This is a schematic diagram of 1016 sites in Embodiment 1 of this application. Detailed Implementation

[0024] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0025] In the following examples, the Plant Genomic DNA Kit was purchased from Tiangen Biotech, the purification kit was purchased from Novizan, and the electrophoresis reagent was purchased from Beyotime.

[0026] Example 1

[0027] Obtaining 1016 core SNP loci combinations from rice germplasm resources

[0028] (1) Genomic DNA extraction (e.g.) Figure 1 (As shown)

[0029] 1) Extract DNA using the commercially available Plant Genomic DNA Kit according to the instructions:

[0030] a. Sample processing: Take 216 rice leaves and grind them into fine powder in liquid nitrogen;

[0031] b. Cell lysis: Transfer the powder to a centrifuge tube, add the preheated lysis buffer GP1, mix thoroughly, and heat in a water bath to completely lyse the cells;

[0032] c. Removal of impurities: Add chloroform extraction solution, and after centrifugation, impurities such as proteins and polysaccharides will enter the organic phase, while DNA will remain in the upper aqueous phase.

[0033] d. DNA adsorption: Transfer the supernatant to the CB3 adsorption column and centrifuge; under high salt conditions, DNA will specifically bind to the silica membrane;

[0034] e. Rinsing: Wash the adsorption column twice with rinsing solution PW to remove salt and residual impurities;

[0035] f. Elution: After the adsorption column is emptied and dried, add elution buffer TB to the center of the silica membrane, let stand, and then centrifuge to obtain pure rice genomic DNA.

[0036] 2) The concentration (≥50 ng / μL) and purity (OD260 / 280 of 1.8-2.0) of DNA were determined using NanoDrop, and DNA integrity was detected by 1% agarose gel electrophoresis.

[0037] (2) Genotype data filtering analysis:

[0038] 1) Genomic DNA was extracted from 216 rice germplasm resources in the crop germplasm resource bank for whole-genome scanning, and raw data of whole-genome SNP genotyping of the 216 rice resources were obtained:

[0039] 2) Use the software fastp (version 0.20.0, parameters: n10、 q20、 The raw sequencing data was filtered using u40 to obtain Clean Reads. Based on the alignment results of the Clean Reads with the reference genome, the data was analyzed using GATK software (version v3.5). 0 The UnifiedGenotyper module of g36282e4 was used for variant detection. The variant results were then used to extract genotyping information from SNP loci using a self-developed Perl script. If the coverage depth of a sample at a certain SNP locus was <5X, it indicated insufficient sequencing depth at that locus, and only valid genotyping data (sequencing depth ≥5X) were retained. The minimum allele frequency was ≥0.05, excluding rare variants and retaining SNPs with a frequency >5% in the population. The locus deletion rate was ≤10%. Only diallelic SNPs were retained, excluding all insertions, deletions, and multiallelic loci. This resulted in 62,027 high-information-content rice SNP variant loci. Reference genome: Oryza_sativa.IRGSP-1.0.dna.toplevel.fa.

[0040] (3) SNP site screening:

[0041] From 62,027 high-information-content rice SNP variant sites, core sites were further selected. The screening criteria were MAF ≥ 0.35, uniform distribution on chromosomes, deletion rate ≤ 0.1%, and heterozygosity ≤ 0.3%. Stable variant sites with high frequency in the population were selected. Using Oligo7 software, probes were designed with a length of 110 nt, GC content of 30%–70%, and homology ≤ 5. Finally, 1016 specific probes were synthesized, and 1016 core sites were selected as follows: Figure 2 As shown, probe specificity verification: through qPCR amplification and Sanger sequencing, 98.3% of the probes specifically bound to the target sites without cross-hybridization. The 1016 sites are shown below: Site - Physical Location - Before / After Mutation:

[0042] 1-17263-A / C;1-31071-A / G;1-80943-C / T;1-170814-A / G;1-174826-A / T;1-181426-G / A;1-195934-G / A;1-201819-A / C;1-210196-C / A;1-213267-C / T;1-214775-G / T;1-215833-C / T;1-219433-A / G;1-219457-A / G;1-219473-G / A;1-219551-C / T;1-219576-T / C;1-219610-A / G;1-222246-C / G;1-222271-C / T;1-222299-A / G;1-222344-T / A;1-227099-T / G;1-232583-T / C;1-241900-T / C;1-248817-C / A;1-252367-A / G;1-256314-A / G;1-256966-G / A;1-268360-G / T;1-289085-G / T;1-292235-G / A;1-294834-G / T;1-303146-T / G;1-304168-A / T;1-306583-C / T;1-311351-A / G;1-322667-G / T;1-328854-G / A;1-329071-C / A;1-329402-G / A;1-332832-T / C;1-334976-C / T;1-348351-G / A;1-349360-G / A;1-353617-A / G;1-361010-G / A;1-364352-A / G;1-371149-C / T;1-374236-T / C;1-376675-C / T;1-380686-T / C;1-390604-C / T;1-396291-T / G;1-397655-C / T;1-401157-A / G;1-401732-G / T;1-405217-C / A;1-408697-C / T;1-417416-G / T;1-421243-T / C;1-423109-C / A;1-442974-C / T;1-443105-A / T;1-448952-G / A;1-460576-T / G;1-480914-T / G;1-502007-A / G;1-504555-G / A;1-505607-A / C;1-514225-A / G;1-521357-G / A;1-534357-C / T;1-543196-T / C;1-551300-C / T;1-553036-T / C;1-555916-C / T;1-571465-T / C;1-576553-T / G;1-582591-C / T;1-586510-C / T;1-593355-A / G;1-601216-G / A;1-627145-G / C;1-645644-C / T;1-648321-G / A;1-671536-T / A;1-689925-A / G;1-720399-G / A;1-720425-A / G;1-723307-G / A;1-728942-T / C;1-730611-C / T;1-732381-A / T;1-733541-C / T;1-737648-G / T;1-743838-G / A;1-748653-G / A;1-750856-A / G;1-846255-T / C;1-854371-G / T;1-912913-T / C;1-919972-A / C;1-925637-G / A;1-931331-A / G;1-935988-A / G;1-955269-C / G;1-1004660-T / C;1-1019463-G / A;1-1019532-G / A;1-1022215-A / G;1-1024084-C / T;1-1027391-T / A;1-1028922-G / A;1-1032971-C / A;1-1034952-T / C;1-1036408-A / C;1-1036850-C / T;1-1040689-T / C;1-1044946-G / A;1-1045896-T / G;1-1046238-T / C;1-1050995-T / C;1-1051618-A / G;1-1053269-A / G;1-1056261-G / T;1-1063106-C / T;1-1067873-A / G;1-1081724-G / A;1-1116499-C / T;1-1121581-C / A;1-1121740-G / A;1-1126726-C / T;1-1155479-C / T;1-1168376-G / A;2-60863-T / G;2-133676-C / G;2-272684-G / A;2-421684-A / G;2-463652-A / G;2-783225-G / T;2-857371-A / G;2-891366-T / C;2-1000481-T / G;2-1247915-A / G;2-1332461-A / G;2-1389486-C / G;2-1464369-C / T;2-1614380-A / C;2-1734488-A / C;2-1750390-A / G;2-1778296-G / T;2-1968779-A / G;2-2027122-T / A;2-2153815-T / C;2-2287246-T / C;2-2338784-A / G;2-2368074-G / A;2-2373406-C / T;2-2399467-A / G;2-2408885-C / A;2-2414268-C / A;2-2424723-A / G;2-2487326-G / A;2-2494080-C / T;2-2524758-T / C;2-2536421-C / T;2-2556922-A / G;2-2558275-C / T;2-2571068-G / A;2-2608447-A / G;2-2645419-A / G;2-2655316-T / A;2-2666340-T / A;2-2672932-G / A;2-2699874-C / T;2-2742744-G / C;2-2757151-A / C;2-2836008-C / T;2-2878228-G / A;2-2878570-A / G;2-2880308-A / G;2-2881031-G / A;2-2922603-G / A;2-2929520-T / C;2-3103373-G / A;2-3125818-C / T;2-3126572-G / A;2-3169470-C / T;2-3173701-C / T;2-3174256-G / T;2-3178843-C / A;2-3203508-G / A;2-3218538-C / T;2-3229077-T / C;2-3249197-G / A;2-3256759-G / A;2-3276317-A / G;2-3278960-A / G;2-3285548-C / T;2-3292003-T / A;2-3293759-A / G;2-3296459-C / T;2-3304088-G / T;2-3317603-A / G;2-3339289-C / T;2-3340405-G / A;2-3340607-C / T;2-3416075-G / T;2-3440590-G / T;2-3463117-G / A;2-3481572-C / T;2-3487281-C / T;2-3512392-C / A;2-3532375-G / A;2-3551343-G / C;2-3583204-G / A;2-3594053-G / A;2-3600254-A / C;2-3622167-T / C;2-3658953-G / A;2-3688518-T / G;2-3691252-C / T;2-3707113-G / A;2-3708720-T / C;2-3709795-T / C;2-3728838-G / T;2-3752117-G / A;2-3761354-G / A;2-3766637-C / T;3-186520-G / A;3-461723-T / C;3-497197-T / C;3-504885-G / A;3-795389-A / C;3-795570-C / T;3-842747-T / C;3-848430-C / T;3-856612-C / T;3-857834-G / A;3-907508-A / G;3-922752-C / T;3-1248025-T / A;3-1249665-G / A;3-1250685-G / T;3-1252016-A / T;3-1254187-T / C;3-1256994-A / G;3-1258318-T / G;3-1270709-G / A;3-1272786-C / T;3-1273981-A / T;3-1275298-A / G;3-1276357-T / C;3-1278952-T / G;3-1279955-A / G;3-1283500-G / T;3-1286390-G / A;3-1288360-T / C;3-1289489-G / A;3-1293432-C / A;3-1294645-C / G;3-1296209-T / C;3-1298125-T / C;3-1300254-C / T;3-1301571-C / T;3-1304869-C / T;3-1305058-G / A;3-1305123-A / G;3-1306541-G / A;3-1322371-G / T;3-1360019-A / T;3-1377228-G / A;3-1380055-G / T;3-1393220-G / A;3-1394134-A / G;3-1399943-C / T;3-1400129-G / T;3-1406973-G / A;3-1420851-G / T;3-1425668-T / C;3-1427948-G / A;3-1430394-C / A;3-1430488-C / T;3-1434871-A / G;3-1436173-A / G;3-1436503-A / G;3-1436616-T / C;3-1437014-C / T;3-1437578-C / A;3-1438591-C / G;3-1444960-T / G;3-1446101-A / G;3-1448643-G / T;3-1448973-A / G;3-1451460-A / T;3-1451798-T / C;3-1455952-G / T;3-1457311-C / T;3-1459282-A / G;3-1462924-T / C;3-1463821-C / T;3-1476420-A / G;3-1481616-G / T;3-1486092-A / C;3-1494364-T / C;3-1500066-C / T;3-1505344-T / C;3-1515028-G / A;3-1529116-G / A;3-1532172-A / G;3-1541326-T / C;3-1553530-A / G;3-1558346-A / G;3-1561296-G / C;3-1563831-A / G;3-1565071-C / T;3-1574149-G / A;3-1579073-T / C;3-1580192-C / T;3-1581050-T / C;3-1593583-G / A;3-1601569-A / C;3-1605159-C / T;3-1610119-C / A;3-1614950-T / C;3-1620253-C / A;3-1627957-G / A;3-1628447-G / A;3-1629494-A / C;3-1636046-C / T;3-1645427-A / G;3-1649929-A / C;3-1650615-T / G;3-1653350-A / G;3-1661691-C / T;3-1662089-G / A;3-1663199-A / G;3-1663206-A / G;3-1663292-A / G;3-1663314-C / T;3-1663320-A / C;3-1670761-A / G;3-1672634-A / G;3-1673420-T / C;3-1673966-T / C;3-1678038-A / G;3-1688638-G / A;3-1690052-C / T;3-1701502-G / A;3-1715154-A / G;3-1720432-A / G;3-1742135-C / T;3-1742398-T / C;3-1756796-C / A;3-1760646-A / G;3-1770343-A / G;3-1789894-A / G;3-1790627-G / A;3-1791686-A / G;3-1791875-A / G;3-1801407-G / A;3-1803592-A / C;3-1816191-A / G;3-1817536-T / C;3-1821654-C / T;3-1827167-A / C;3-1878844-G / A;3-1953172-A / C;3-2173253-A / G;3-2261232-A / G;3-2420174-G / T;3-2573901-A / G;3-2984246-A / G;3-3134555-C / A;3-3172258-A / T;3-3313047-T / C;3-3329976-C / A;3-3531349-G / A;3-3565195-A / G;3-3597749-T / C;3-3648863-T / G;3-3698114-A / G;3-3699501-C / A;3-3705655-G / A;3-3743800-C / T;3-3800635-T / C;3-3802846-C / A;3-3818708-C / A;3-3883563-T / C;3-3912447-C / T;3-3940304-A / C;3-3970237-T / C;3-3980429-T / C;3-4007709-C / G;3-4010852-A / C;3-4028493-G / A;3-4060435-C / T;3-4079512-T / C;3-4094527-G / A;3-4108212-T / C;3-4133596-G / C;3-4137113-G / A;3-4181529-A / C;3-4193314-T / C;3-4202677-T / C;4-6958-C / T;4-9382-C / T;4-11832-A / G;4-59046-A / G;4-61722-T / C;4-83050-G / A;4-90596-A / G;4-94653-T / C;4-97682-A / C;4-99927-G / T;4-109600-A / G;4-115465-C / T;4-124528-G / T;4-125461-A / G;4-138244-G / A;4-152524-C / T;4-158541-G / A;4-171915-T / C;4-177092-T / C;4-180737-C / T;4-189477-A / G;4-196952-G / A;4-204409-G / A;4-204512-T / C;4-209855-G / A;4-211143-C / A;4-217146-G / A;4-219526-T / C;4-222357-T / C;4-230061-T / C;4-243214-T / C;4-246631-G / A;4-259253-T / C;4-262887-T / C;4-268277-A / G;4-279744-G / T;4-294003-C / T;4-297348-G / A;4-301532-G / A;4-315653-G / A;4-321287-T / C;4-329167-G / T;4-332258-G / A;4-332314-G / A;4-332338-T / C;4-332704-G / A;4-336779-A / G;4-337388-A / C;4-346652-T / C;4-355211-C / A;4-355976-G / A;4-364240-G / A;4-370652-G / A;4-370778-C / T;4-387357-G / C;4-395996-C / T;4-396177-G / A;4-404963-C / T;4-412454-A / C;4-415435-G / T;4-441404-A / G;4-454563-G / A;4-456956-G / A;4-457032-A / G;4-458660-A / G;4-462758-T / C;4-464453-G / A;4-465991-C / T;4-471311-A / G;4-474253-T / C;4-477866-G / A;4-483338-A / G;4-485897-A / G;4-492973-C / T;4-500837-T / C;4-512356-C / T;4-522093-G / T;4-525199-C / A;4-539229-T / C;4-556665-C / T;4-561346-T / C;4-570286-G / A;4-574000-A / G;4-585535-G / A;4-594521-G / A;4-594935-A / G;4-600821-G / A;4-614313-C / T;4-616201-G / T;4-638005-A / C;4-643168-T / G;4-657395-A / G;4-666759-A / G;4-667473-G / T;4-676117-T / C;4-687006-T / C;4-691599-T / C;4-693830-G / A;4-696991-T / C;4-713094-G / C;4-717737-G / T;4-721279-G / A;5-4264-G / A;5-7169-G / T;5-13406-T / C;5-33760-A / G;5-44262-T / G;5-44370-C / T;5-44806-C / G;5-51658-G / T;5-61799-G / A;5-65465-C / T;5-75367-T / G;5-82638-G / T;5-91700-T / C;5-101129-C / T;5-103866-T / C;5-112002-C / A;5-120590-T / C;5-139420-A / C;5-141209-C / A;5-150234-C / T;5-158839-T / C;5-167655-G / A;5-170536-T / C;5-170550-C / G;5-170683-A / G;5-179062-T / C;5-180044-C / T;5-185685-T / C;5-187666-C / A;5-188276-T / C;5-189871-A / C;5-194007-T / C;5-202581-C / T;5-207291-T / C;5-207831-G / A;5-225917-T / C;5-230239-C / T;5-232598-C / T;5-257046-A / C;5-259241-G / A;5-263950-A / G;5-273159-A / G;5-277725-T / C;5-277784-G / A;5-295458-A / C;5-309569-A / G;5-312763-T / C;5-320221-C / A;5-328358-C / T;5-328758-G / A;5-329835-C / T;6-122412-T / C;6-122415-T / C;6-122426-G / A;6-122439-A / G;6-122581-G / A;6-122582-G / A;6-122590-A / G;6-122591-G / C;6-122595-A / T;6-122619-C / T;6-192409-T / C;6-213602-T / C;6-294450-C / A;6-399302-T / G;6-520361-A / G;6-547345-A / G;6-555780-A / G;6-600149-T / G;6-838869-C / T;6-841495-A / G;6-865932-G / A;6-867643-C / T;6-873447-C / A;6-875023-A / G;6-881325-A / G;6-887478-A / G;6-891877-T / G;6-909123-A / C;6-1044890-A / G;6-1053193-G / A;6-1063432-G / A;6-1149287-G / A;7-10802-G / A;7-16195-T / C;7-16220-C / T;7-16238-A / C;7-16263-T / C;7-39045-A / G;7-44501-A / C;7-48868-T / C;7-52331-C / A;7-68275-T / G;7-70372-G / A;7-74892-T / C;7-82549-G / A;7-92980-A / G;7-97239-T / C;7-101173-G / A;7-102273-C / A;7-110396-T / C;7-117506-C / A;7-126370-G / A;7-126393-T / G;7-127781-T / A;7-128769-G / A;7-134554-C / A;7-135579-G / A;7-143731-C / A;7-146665-T / C;7-147861-C / T;7-150000-A / G;7-150259-C / T;7-151411-G / A;7-156650-T / G;7-158556-T / C;7-178325-A / G;7-180829-A / G;7-181607-C / T;7-182813-T / C;7-183592-C / A;7-188388-G / A;7-190999-C / A;7-192515-T / C;7-196813-A / C;7-200264-G / A;7-200526-G / A;7-201420-T / C;7-205086-G / C;7-205561-T / C;7-206187-C / A;7-213662-T / C;7-214725-A / G;7-218245-A / G;7-218893-A / G;7-219326-A / G;7-222206-C / T;7-234494-C / T;7-235993-A / G;7-238044-C / T;7-240401-G / A;7-251951-C / T;7-253886-T / G;7-256259-G / T;7-258395-T / G;7-260479-T / G;7-261601-G / A;7-262481-G / T;7-264254-C / T;7-265708-C / T;8-16866-A / G;8-17010-A / G;8-17661-G / A;8-17663-G / A;8-36320-G / A;8-40302-G / A;8-45402-G / A;8-62082-C / T;8-65738-G / A;8-67412-A / G;8-71799-G / A;8-86771-G / T;8-109516-A / G;8-110934-G / A;8-119152-A / C;8-120338-T / C;8-138417-T / G;8-140547-C / T;8-167858-A / C;8-173466-G / C;8-178346-G / A;8-182538-A / G;8-200234-T / G;8-212149-T / C;8-213505-A / G;8-231614-T / C;8-232010-A / C;8-240019-C / T;8-252358-G / A;8-256526-T / G;8-268833-T / G;8-272949-C / T;8-276743-G / A;8-289260-T / C;8-341501-G / A;8-342972-C / T;8-345519-C / T;8-366660-G / T;8-370754-T / G;8-376048-G / A;8-384817-G / T;8-388050-A / G;8-389278-C / A;8-401373-T / C;8-409956-C / T;8-410904-T / G;8-419143-G / T;8-432353-T / C;8-433759-C / A;8-437373-G / A;8-445667-A / G;8-449253-A / G;8-457935-G / T;8-461067-C / T;8-461299-G / A;8-470124-G / T;8-500511-C / T;8-502745-T / C;8-502818-G / A;8-509610-A / G;8-512142-A / G;8-519519-G / A;8-520963-T / C;8-528441-A / G;8-529389-G / T;8-531744-T / C;8-544269-T / C;8-552815-T / C;8-557390-T / C;8-558615-T / G;8-561057-G / A;8-565398-T / C;8-566669-G / A;8-573143-A / G;8-591946-A / G;8-597007-C / A;8-598215-T / C;8-648647-T / C;8-655881-A / C;8-662509-G / A;8-667319-G / A;8-670192-A / G;8-674656-T / C;8-680887-T / C;8-681230-C / T;8-683994-A / C;8-686810-C / A;9-144038-A / C;9-146070-G / A;9-152998-T / A;9-259900-C / T;9-286434-C / T;9-311002-T / G;9-412246-T / C;9-421304-T / C;9-422185-C / T;9-425879-G / T;9-444945-C / A;9-465596-C / T;9-498016-A / G;9-499186-T / C;9-501720-T / C;9-617347-T / C;9-625057-T / G;9-632657-G / A;9-646892-G / C;9-654168-G / A;9-735417-A / G;9-737140-T / C;9-916467-A / G;9-945171-A / C;9-961738-A / G;9-1004710-A / G;9-1027960-T / A;9-1052543-A / G;9-1170861-C / T;9-1291741-T / C;9-1313070-C / A;9-1317284-G / A;9-1402810-G / T;9-1436413-G / T;9-1443786-C / A;9-1568456-T / C;9-1638777-T / C;9-1764339-T / A;9-1797083-T / A;9-1854333-T / G;9-1855764-A / G;9-1934202-T / C;9-2084105-G / C;9-2222992-C / T;9-2296697-C / A;9-2370923-T / C;9-2469648-T / C;9-2535785-T / C;9-2581089-A / G;9-2641881-T / C;9-2645073-C / A;9-2666584-T / C;9-2667806-T / C;9-2707549-G / A;9-2918349-T / C;9-3132386-G / A;9-3211043-T / C;9-3345496-T / C;9-3423063-A / G;9-3479246-T / C;9-3507920-T / C;9-3603266-G / A;9-3604352-C / T;9-3635355-A / G;9-3708404-T / C;9-3746627-T / C;9-3830762-T / C;9-3838704-T / C;9-3844624-C / T;9-3854628-C / T;9-3948049-C / G;9-3973083-G / A;9-4163424-A / C;9-4213116-G / A;9-4350506-G / C;9-4387935-T / C;9-4424330-A / G;9-4469351-T / A;9-4473539-A / G;9-4478326-T / C;9-4481200-C / T;9-4485136-G / A;9-4489410-A / C;9-4502785-T / G;9-4504082-G / A;9-4534492-G / A;9-4536486-A / G;9-4539494-G / T;9-4546896-G / A;9-4555751-G / T;9-4562212-C / T;9-4564117-C / T;9-4570776-T / C;9-4580415-T / C;9-4586972-A / G;9-4591567-G / A;9-4593756-T / C;9-4598919-T / C;9-4602409-C / G;9-4624812-G / A;9-4626076-G / A;9-4628297-T / C;9-4630349-G / T;9-4641583-C / G;10-46485-A / G;10-46523-C / T;10-49245-G / A;10-58063-A / G;10-58481-G / A;10-62315-A / C;10-67342-G / A;10-70306-A / C;10-76415-T / C;10-82215-G / A;10-95229-G / A;10-100057-T / C;10-104882-A / G;10-105039-G / A;10-105999-A / G;10-109811-C / G;10-116301-C / T;10-116895-T / C;10-121669-G / A;10-139849-G / A;10-149079-C / T;10-151784-A / C;10-153213-G / A;10-160235-G / A;10-173538-C / T;10-176002-T / G;10-179526-T / C;10-187309-T / C;10-206491-G / A;10-223588-C / T;10-279392-G / A;10-283563-T / G;10-285086-C / T;10-288275-C / G;10-299285-C / T;10-303826-T / A;10-305596-G / A;10-305732-T / C;10-306113-C / T;10-315805-G / A;10-321500-C / A;10-322558-G / C;10-326421-C / T;10-339584-A / G;10-340963-T / C;10-342812-A / G;10-345576-T / C;10-356482-A / T;10-357816-C / A;10-364776-C / T;10-367747-G / A;10-377203-A / G;10-378275-G / A;10-383037-A / G;10-407364-T / C;10-408093-A / C;10-413299-C / T;10-416515-A / G;10-417431-A / C;10-417455-C / A;10-417462-G / A;10-417533-C / T;10-417539-G / A;10-417565-A / G;10-417631-C / T;11-130090-G / A;11-140228-T / C;11-155439-G / T;11-162793-A / G;11-175636-A / G;11-188237-G / A;11-192690-G / T;11-193403-A / C;11-226280-G / A;11-231839-G / A;11-257538-C / T;11-262501-A / G;11-270182-G / A;11-276646-G / A;11-291218-T / C;11-292789-A / T;11-303931-C / A;11-309473-C / T;11-328920-A / G;11-335548-G / A;11-357437-G / A;11-406993-C / T;11-442387-G / A;11-510996-A / T;11-548820-A / G;11-550322-G / A;11-551567-A / T;11-573517-T / C;11-573610-T / G;11-632791-A / G;11-682530-C / T;11-682737-C / A;11-702318-T / C;11-712633-G / T;11-741739-C / T;11-765476-T / G;11-768964-A / G;11-780611-A / G;12-63361-C / T;12-104896-T / C;12-120396-G / T;12-124273-T / C;12-130488-G / A;12-133529-A / G;12-141714-G / A;12-148272-G / A;12-161500-A / C;12-206618-A / G;12-212308-A / G;12-225104-T / C;12-231166-T / C;12-239186-A / G;12-249844-G / T;12-264366-T / C;12-273623-G / T;12-279439-C / T;12-2815 24-C / A;12-307387-A / G;12-309820-G / A;12-310769-C / T;12-318897-C / T;12-324278-A / C;12-324610-C / T; 12-328010-A / G;12-330564-G / A;12-341764-T / G;12-343927-G / A;12-343960-A / T;12-343984-G / A;12-3439 95-A / G;12-344002-C / A;12-346594-C / T;12-354737-C / T;12-365296-G / A;12-379909-G / A;12-380698-G / A; 12-423866-G / A;12-448212-T / C;12-457395-C / T;12-463581-T / C;12-469450-T / C;12-479088-C / T;12-4819 14-C / T;12-485600-G / T;12-493219-A / G;12-499980-A / G;12-516664-C / A;12-520445-C / T;12-528702-T / C; 12-530223-G / C;12-536346-A / G;12-557616-C / T;12-558117-G / T;12-558125-A / G;12-558289-A / G;12-5624 68-A / C;12-563688-T / C;12-563735-A / C;12-563747-T / C;12-565573-T / G;12-595746-G / A;12-609304-C / T. ;

[0043] Example 2

[0044] GBTS Marking Method Establishment

[0045] (1) The GBTS labeling method mainly includes the following steps: primer design, multiplex PCR, PCR fragment recovery and high-throughput sequencing, and sequencing result analysis.

[0046] (2) Extract 200 bp sequences upstream and downstream of the mutation site and design primers according to the following primer design principles: primer length 18~36 bp; TM value 59℃~64℃; GC content 15%~70%. Further evaluation and selection of the designed primers: perform non-specific evaluation on the designed primers, and then select non-specific primers; perform dimerization evaluation between primers to avoid dimerization; perform pairwise non-specific evaluation again on the primers selected in the above two steps; select the primers that finally meet the requirements. Mix equal volumes of the qualified primers to a final primer concentration of 0.2 μmol / LM to prepare a functional marker primer mixture for detecting rice target genes. Multiplex PCR reaction system: 18 μL functional marker primer mixture, template DNA (≥50 ng), 10 μL 3×T Master Mix, and make up to 30 μL with ultrapure water. PCR reaction program: 95℃ hot start for 3 min; 95℃ denaturation for 30 s, 60℃ annealing and extension for 4 min, 15 cycles; 72℃ extension for 4 min.

[0047] (3) After the multiplex PCR reaction, the PCR products were purified and recovered using a DNA purification kit. Sequencing primers, adapters, and barcodes were added to the purified and recovered PCR fragments for a second round of PCR amplification. The reaction mixture consisted of: I5 barcode (10 μmol / L)-MGI 1 μL, I7 barcode (2 μmol / L)-MGI 5 μL, 3×T Master Mix 10 μL, and ultrapure water 14 μL. The PCR amplification program was: 95℃ hot start for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 7 cycles; extension at 72℃ for 4 min. The PCR products were then purified and DNA concentration determined using a purification kit. The products were directly used for sequencing.

[0048] (4) Data Analysis Methods: High-throughput sequencing data were analyzed using GATK software to obtain vcf files containing SNP data. The reference genome was the 'Nipponbare' genome irgsp1.0 (Kawahara et al., 2013). Vcf files from different varieties were merged using NGSEP software (Tello et al., 2019), and heterozygous sites in the vcf files were filtered out. Genotypic analysis was performed graphically using Flapjack software (Milne et al., 2010). Cluster analysis was performed using the hclust function in R system, and a phylogenetic tree was constructed using GGTREE (Yu et al., 2017).

[0049] Example 3

[0050] Liquid chip fabrication

[0051] (1) Probe immobilization and liquid phase capture: 1016 specific probes (110nt) synthesized were conjugated with biotin to prepare a liquid phase probe library; rice genomic DNA from 130 cases in Example 1 was randomly selected, and fragmented into 300-500bp fragments by sonication. The fragments were hybridized with the liquid phase probe library at 65℃ for 48h (oscillation rate 1000rpm), and the hybridized DNA fragments were captured by streptavidin magnetic beads.

[0052] (2) Library construction and sequencing verification: The DNA fragments on the magnetic beads were washed off and amplified by PCR (KAPA HiFi enzyme, 15 cycles) to construct the sequencing library. The Illumina NovaSeq 6000 was used for sequencing to verify the probe capture efficiency (the target site capture rate must be ≥95%). The chip can stably detect 1016 core SNPs; the genotypic consistency (compared with Sanger sequencing) is ≥98%, and the inter-batch / intra-batch coefficient of variation (CV) is ≤5%, forming a standardized "low-temperature resistant rice breeding liquid phase chip" product.

[0053] Example 4

[0054] Screening of cold-resistant germination rice germplasm resources

[0055] (1) 120 cold-region rice germplasm resources were selected, covering the main varieties, local farm varieties and intermediate breeding materials in the Northeast rice region. All materials were from the National Cold-Region Rice Germplasm Resource Bank, with seed purity ≥98% and germination rate ≥85%;

[0056] (2) Genotyping of 120 materials was performed using the cold-resistant low-temperature germination rice liquid phase chip prepared in this application: Genomic DNA was extracted from each material according to the method in Example 1, ensuring that the DNA concentration was ≥50ng / μL and the OD260 / 280 was 1.8-2.0. The DNA integrity was verified by 1% agarose gel electrophoresis to ensure no degradation. Probe hybridization, DNA fragment capture, PCR amplification and high-throughput sequencing were performed to obtain genotypic data of 1016 SNP loci.

[0057] (3) Use GATK software to filter low-quality data, retain sites with sequencing depth ≥5X and clear genotypes, and finally obtain complete genotype maps of each material;

[0058] (4) Phenotypic identification: The low-temperature tolerance phenotype was identified by artificial low-temperature stress germination test: Seed pretreatment: Select plump and uniform seeds, disinfect with 1% sodium hypochlorite solution for 10 min, rinse with distilled water 3 times, and soak for 24 h;

[0059] 1) Low temperature stress treatment: After soaking, the seeds were evenly placed in petri dishes lined with double-layered filter paper, 50 seeds per dish, 10 mL of distilled water was added, and the dishes were placed in an artificial climate chamber. The low temperature stress conditions were set as follows: constant temperature of 10℃, light intensity of 3000 lux for 12 h / d, and humidity of 80%.

[0060] 2) Control treatment: A room temperature control group was set up, with culture conditions at a constant temperature of 25℃, and other conditions were the same as those of the low temperature group;

[0061] (5) Index determination: After 7 days of low temperature treatment, the germination rate (number of germinated seeds / number of tested seeds × 100%) was calculated; after 10 days of treatment, the shoot length (length from the seed radicle sheath to the shoot tip) and root length (length from the radicle tip to the seed base) were measured; the relative germination rate (germination rate of low temperature group / germination rate of normal temperature group × 100%), relative shoot length (shoot length of low temperature group / shoot length of normal temperature group × 100%), and relative root length (root length of low temperature group / root length of normal temperature group × 100%) were calculated, and each material was replicated 3 times;

[0062] (6) The association analysis between 1016 SNP loci and low-temperature tolerance phenotypic indicators (relative germination rate, relative shoot length, and relative root length) was performed using TASSEL software, and the phenotypic contribution rate of each locus was calculated. Screening criteria: Based on the three phenotypic indicators, materials with a relative germination rate ≥70%, a relative shoot length ≥60%, and a relative root length ≥55% were defined as low-temperature resistant germination materials. Based on the association analysis results, materials carrying ≥80% of low-temperature tolerance dominant alleles were screened.

[0063] (7) Genotyping results: The success rate of detecting 1016 SNP sites in 120 rice materials was 98.7%, of which 96.2% of the sites had genotypic consistency (verified with Sanger sequencing results) ≥98%, indicating that the chip detection data is reliable;

[0064] (8) Phenotypic identification results: Under low temperature stress, the germination ability of different materials varied significantly, with relative germination rate ranging from 32.4% to 89.7%, relative shoot length ranging from 28.6% to 75.3%, and relative root length ranging from 23.5% to 71.2%.

[0065] (9) Screening results: Through combined genotypic and phenotypic analysis, 18 low-temperature germination rice germplasm resources were finally screened, accounting for 15.0% of the tested materials. These materials all carry 82%~95% of the low-temperature resistant dominant alleles, and under low-temperature stress, the relative germination rate is ≥72%, the relative shoot length is ≥63%, and the relative root length is ≥58%, showing stable low-temperature germination characteristics;

[0066] (10) The cold-resistant low-temperature germination rice gene chip of this application can quickly and accurately screen out low-temperature germination rice germplasm resources. The screening efficiency is 4-5 times higher than that of traditional phenotypic identification methods, and it is not affected by environmental factors, providing a high-quality germplasm basis for cold-resistant rice breeding.

[0067] Example 5

[0068] Application of whole-genome selection breeding of cold-resistant low-temperature germinating rice

[0069] (1) Using 10 high low-temperature germination germplasm samples (numbered N1-N10) selected in Example 4 as male parents and 3 local main high-yield varieties (numbered M1-M3, with weak low-temperature germination ability) as female parents, 10×3=30 hybrid combinations were configured to obtain F1 generation seeds;

[0070] (2) F1 generation authenticity identification: F1 generation seeds and parent seeds were sown. When the seedlings grew to the 3-leaf stage, genomic DNA was extracted from the leaves. The 20 core SNP sites in the chip of this application (evenly distributed on 12 chromosomes, with a phenotypic contribution rate of ≥5%) were used for PCR detection to verify the hybridization authenticity of the F1 generation and eliminate false hybrids.

[0071] (3) F2 generation genotype selection: plant real F1 generation plants, self-pollinate to obtain F2 generation seeds, plant 200 plants in each combination to form an F2 segregating population;

[0072] (4) DNA was extracted from the seedlings at the 3-leaf stage. Genotyping of 1016 SNP loci was performed using the gene chip of this application. Based on the association analysis results, the low-temperature resistance breeding value of each individual plant was calculated (weighted calculation based on the number of dominant alleles and phenotypic contribution rate). The top 20% of individual plants with the highest breeding value (40 plants per combination) were selected and transplanted to the experimental field for routine field management.

[0073] (5) F3 representative type verification and screening: F2 generation single plants were selected and self-pollinated to obtain F3 generation. 50 plants were planted for each line, with 3 replicates. Parents were planted as controls. The low temperature stress germination test method in Example 4 was used to identify the low temperature germination phenotype of the F3 generation lines. Yield-related traits (number of effective spikes, number of grains per spike, seed setting rate, and thousand-grain weight) were investigated at maturity.

[0074] (6) Screening criteria: Low-temperature phenotype indicators (relative germination rate, relative shoot length, relative root length) reach or exceed the level of the male parent, and the yield is ≥8% higher than that of the female parent;

[0075] (7) Breeding results:

[0076] 1) F1 generation authenticity identification: Through the detection of 20 core SNP loci, 32 false hybrids were eliminated, and the true hybrid rate was 94.7%, ensuring the purity of the breeding population;

[0077] 2) F2 generation genotype selection: From the F2 population of 30 combinations, a total of 1200 high-breeding-value individual plants were selected. The number of cold-resistant alleles carried by these individual plants increased by an average of 35% compared with the randomly selected population;

[0078] 3) F3 representative type verification: 15 superior lines were finally obtained, accounting for 12.5% ​​of the tested lines. These lines had a relative germination rate of 75%~88%, a relative shoot length of 65%~78%, and a relative root length of 60%~73%, all significantly better than the maternal parent; the yield was on average 10.3%~15.6% higher than the maternal parent, and the overall field traits (plant height, disease resistance, maturity) were stable.

[0079] 4) Conclusion: The cold-resistant low-temperature germination rice gene chip of this application can be applied to whole-genome selection breeding. Through early genotype screening, the breeding cycle can be significantly shortened (from 6-8 generations in traditional breeding to 3-4 generations), improving the efficiency and accuracy of breeding selection, and rapidly cultivating new cold-resistant rice varieties that combine low-temperature germination characteristics and high yield.

[0080] Example 6

[0081] Application of gene chips in association analysis of low-temperature tolerance trait in rice

[0082] (1) 216 natural populations of cold-region rice were selected (consistent with the populations used for SNP site screening in Example 1), covering different ecological types and genetic backgrounds to ensure the genetic diversity of the populations;

[0083] (2) Genotype detection: The gene chip prepared in this application was used to perform genotype detection on 1016 SNP sites in 216 materials. The specific process is as described in Example 3, and a complete genotype data matrix was obtained.

[0084] (3) Phenotypic identification: Low temperature germination test: According to the method of Example 4, a low temperature stress treatment of 10℃ was set, and three phenotypic indicators, namely relative germination rate, relative shoot length and relative root length, were measured; Low temperature seedling stage stress test: At the 3-leaf stage of seedlings, potted seedlings were placed in an artificial climate chamber and subjected to a low temperature stress treatment of 8℃ for 5 days. After restoring normal temperature culture for 7 days, the seedling survival rate (number of surviving plants / number of tested plants × 100%) and chlorophyll content were measured. All phenotypic indicators were set up in 3 replicates, and the average value was taken as the final phenotypic data;

[0085] (4) Association analysis: Genome-wide association analysis (GWAS) was performed using GAPIT software. Population structure (Q matrix) and kinship (K matrix) were used as covariates to control for false positive associations. The significant association threshold was set as P<1e-4 to screen SNP sites that were significantly associated with the low-temperature resistance trait.

[0086] (5) The correlation analysis of the experimental results detected a total of 42 SNP sites that were significantly associated with the cold resistance trait of cold-resistant rice, as shown in Table 1;

[0087] Table 1. Statistical analysis of SNP loci significantly associated with low-temperature tolerance traits in cold-region rice.

[0088]

[0089] (6) Conclusion: Table 1 shows that 42 SNP loci were significantly associated with the cold tolerance trait of cold-resistant rice. The SNP loci associated with different cold tolerance traits differed in number, chromosome distribution, and phenotypic contribution rate: 15 loci were associated with relative germination rate, distributed on chromosomes 1, 3, 4, and 9, with a phenotypic contribution rate of 6.2%–12.5% ​​and no concentrated hotspots; 11 loci were associated with relative shoot length, distributed on chromosomes 2, 3, 7, and 8, with a phenotypic contribution rate of 5.8%–11.3%, and chromosome 3 accounted for 27.3%; 9 loci were associated with relative root length, distributed on chromosomes 3, 5, and 10, with a phenotypic contribution rate of 5.5%–10.8%; and 7 loci were associated with seedling survival rate, distributed on chromosomes 3, 6, and 12, with a phenotypic contribution rate of 6.0%–11.7%, and chromosome 3 accounted for 42.9%. It is worth noting that a hotspot region exists at the 1.2-1.8Mb physical location on chromosome 3, containing 8 associated loci with a cumulative phenotypic contribution rate of 35.2%. This region contains a key gene cluster that regulates the low-temperature germination tolerance and seedling cold tolerance of rice.

[0090] In summary, this application developed a liquid-phase gene chip for cold-resistant, low-temperature germinating rice based on 1016 core SNPs, which features high precision and high stability. It enables rapid and accurate screening of cold-resistant rice germplasm and has been successfully applied to whole-genome selection breeding, significantly improving breeding efficiency and shortening the breeding cycle. The association analysis conducted through this chip not only verified its effectiveness but also identified key chromosomal regions that regulate cold-resistant traits, providing important targets for subsequent gene function research.

[0091] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A gene chip for cold-resistant, low-temperature germination rice, characterized in that, The genotyping loci of the chip include 1016 SNP loci, which were selected from 62,027 initial loci in 216 cold-region rice germplasms. The physical location information of the genotyping loci was determined based on the rice reference genome Oryza_sativa.IRGSP-1.

0. The 1016 SNP loci are represented as: locus-physical location-before / after mutation. 1-17263-A / C;1-31071-A / G;1-80943-C / T;1-170814-A / G;1-174826-A / T;1-181426-G / A;1-195934-G / A;1-201819-A / C;1-210196-C / A;1-213267-C / T;1-214775-G / T;1-215833-C / T;1-219433-A / G;1-219457-A / G;1-219473-G / A;1-219551-C / T;1-219576-T / C;1-219610-A / G;1-222246-C / G;1-222271-C / T;1-222299-A / G;1-222344-T / A;1-227099-T / G;1-232583-T / C;1-241900-T / C;1-248817-C / A;1-252367-A / G;1-256314-A / G;1-256966-G / A;1-268360-G / T;1-289085-G / T;1-292235-G / A;1-294834-G / T;1-303146-T / G;1-304168-A / T;1-306583-C / T;1-311351-A / G;1-322667-G / T;1-328854-G / A;1-329071-C / A;1-329402-G / A;1-332832-T / C;1-334976-C / T;1-348351-G / A;1-349360-G / A;1-353617-A / G;1-361010-G / A;1-364352-A / G;1-371149-C / T;1-374236-T / C;1-376675-C / T;1-380686-T / C;1-390604-C / T;1-396291-T / G;1-397655-C / T;1-401157-A / G;1-401732-G / T;1-405217-C / A;1-408697-C / T;1-417416-G / T;1-421243-T / C;1-423109-C / A;1-442974-C / T;1-443105-A / T;1-448952-G / A;1-460576-T / G;1-480914-T / G;1-502007-A / G;1-504555-G / A;1-505607-A / C;1-514225-A / G;1-521357-G / A;1-534357-C / T;1-543196-T / C;1-551300-C / T;1-553036-T / C;1-555916-C / T;1-571465-T / C;1-576553-T / G;1-582591-C / T;1-586510-C / T;1-593355-A / G;1-601216-G / A;1-627145-G / C;1-645644-C / T;1-648321-G / A;1-671536-T / A;1-689925-A / G;1-720399-G / A;1-720425-A / G;1-723307-G / A;1-728942-T / C;1-730611-C / T;1-732381-A / T;1-733541-C / T;1-737648-G / T;1-743838-G / A;1-748653-G / A;1-750856-A / G;1-846255-T / C;1-854371-G / T;1-912913-T / C;1-919972-A / C;1-925637-G / A;1-931331-A / G;1-935988-A / G;1-955269-C / G;1-1004660-T / C;1-1019463-G / A;1-1019532-G / A;1-1022215-A / G;1-1024084-C / T;1-1027391-T / A;1-1028922-G / A;1-1032971-C / A;1-1034952-T / C;1-1036408-A / C;1-1036850-C / T;1-1040689-T / C;1-1044946-G / A;1-1045896-T / G;1-1046238-T / C;1-1050995-T / C;1-1051618-A / G;1-1053269-A / G;1-1056261-G / T;1-1063106-C / T;1-1067873-A / G;1-1081724-G / A;1-1116499-C / T;1-1121581-C / A;1-1121740-G / A;1-1126726-C / T;1-1155479-C / T;1-1168376-G / A;2-60863-T / G;2-133676-C / G;2-272684-G / A;2-421684-A / G;2-463652-A / G;2-783225-G / T;2-857371-A / G;2-891366-T / C;2-1000481-T / G;2-1247915-A / G;2-1332461-A / G;2-1389486-C / G;2-1464369-C / T;2-1614380-A / C;2-1734488-A / C;2-1750390-A / G;2-1778296-G / T;2-1968779-A / G;2-2027122-T / A;2-2153815-T / C;2-2287246-T / C;2-2338784-A / G;2-2368074-G / A;2-2373406-C / T;2-2399467-A / G;2-2408885-C / A;2-2414268-C / A;2-2424723-A / G;2-2487326-G / A;2-2494080-C / T;2-2524758-T / C;2-2536421-C / T;2-2556922-A / G;2-2558275-C / T;2-2571068-G / A;2-2608447-A / G;2-2645419-A / G;2-2655316-T / A;2-2666340-T / A;2-2672932-G / A;2-2699874-C / T;2-2742744-G / C;2-2757151-A / C;2-2836008-C / T;2-2878228-G / A;2-2878570-A / G;2-2880308-A / G;2-2881031-G / A;2-2922603-G / A;2-2929520-T / C;2-3103373-G / A;2-3125818-C / T;2-3126572-G / A;2-3169470-C / T;2-3173701-C / T;2-3174256-G / T;2-3178843-C / A;2-3203508-G / A;2-3218538-C / T;2-3229077-T / C;2-3249197-G / A;2-3256759-G / A;2-3276317-A / G;2-3278960-A / G;2-3285548-C / T;2-3292003-T / A;2-3293759-A / G;2-3296459-C / T;2-3304088-G / T;2-3317603-A / G;2-3339289-C / T;2-3340405-G / A;2-3340607-C / T;2-3416075-G / T;2-3440590-G / T;2-3463117-G / A;2-3481572-C / T;2-3487281-C / T;2-3512392-C / A;2-3532375-G / A;2-3551343-G / C;2-3583204-G / A;2-3594053-G / A;2-3600254-A / C;2-3622167-T / C;2-3658953-G / A;2-3688518-T / G;2-3691252-C / T;2-3707113-G / A;2-3708720-T / C;2-3709795-T / C;2-3728838-G / T;2-3752117-G / A;2-3761354-G / A;2-3766637-C / T;3-186520-G / A;3-461723-T / C;3-497197-T / C;3-504885-G / A;3-795389-A / C;3-795570-C / T;3-842747-T / C;3-848430-C / T;3-856612-C / T;3-857834-G / A;3-907508-A / G;3-922752-C / T;3-1248025-T / A;3-1249665-G / A;3-1250685-G / T;3-1252016-A / T;3-1254187-T / C;3-1256994-A / G;3-1258318-T / G;3-1270709-G / A;3-1272786-C / T;3-1273981-A / T;3-1275298-A / G;3-1276357-T / C;3-1278952-T / G;3-1279955-A / G;3-1283500-G / T;3-1286390-G / A;3-1288360-T / C;3-1289489-G / A;3-1293432-C / A;3-1294645-C / G;3-1296209-T / C;3-1298125-T / C;3-1300254-C / T;3-1301571-C / T;3-1304869-C / T;3-1305058-G / A;3-1305123-A / G;3-1306541-G / A;3-1322371-G / T;3-1360019-A / T;3-1377228-G / A;3-1380055-G / T;3-1393220-G / A;3-1394134-A / G;3-1399943-C / T;3-1400129-G / T;3-1406973-G / A;3-1420851-G / T;3-1425668-T / C;3-1427948-G / A;3-1430394-C / A;3-1430488-C / T;3-1434871-A / G;3-1436173-A / G;3-1436503-A / G;3-1436616-T / C;3-1437014-C / T;3-1437578-C / A;3-1438591-C / G;3-1444960-T / G;3-1446101-A / G;3-1448643-G / T;3-1448973-A / G;3-1451460-A / T;3-1451798-T / C;3-1455952-G / T;3-1457311-C / T;3-1459282-A / G;3-1462924-T / C;3-1463821-C / T;3-1476420-A / G;3-1481616-G / T;3-1486092-A / C;3-1494364-T / C;3-1500066-C / T;3-1505344-T / C;3-1515028-G / A;3-1529116-G / A;3-1532172-A / G;3-1541326-T / C;3-1553530-A / G;3-1558346-A / G;3-1561296-G / C;3-1563831-A / G;3-1565071-C / T;3-1574149-G / A;3-1579073-T / C;3-1580192-C / T;3-1581050-T / C;3-1593583-G / A;3-1601569-A / C;3-1605159-C / T;3-1610119-C / A;3-1614950-T / C;3-1620253-C / A;3-1627957-G / A;3-1628447-G / A;3-1629494-A / C;3-1636046-C / T;3-1645427-A / G;3-1649929-A / C;3-1650615-T / G;3-1653350-A / G;3-1661691-C / T;3-1662089-G / A;3-1663199-A / G;3-1663206-A / G;3-1663292-A / G;3-1663314-C / T;3-1663320-A / C;3-1670761-A / G;3-1672634-A / G;3-1673420-T / C;3-1673966-T / C;3-1678038-A / G;3-1688638-G / A;3-1690052-C / T;3-1701502-G / A;3-1715154-A / G;3-1720432-A / G;3-1742135-C / T;3-1742398-T / C;3-1756796-C / A;3-1760646-A / G;3-1770343-A / G;3-1789894-A / G;3-1790627-G / A;3-1791686-A / G;3-1791875-A / G;3-1801407-G / A;3-1803592-A / C;3-1816191-A / G;3-1817536-T / C;3-1821654-C / T;3-1827167-A / C;3-1878844-G / A;3-1953172-A / C;3-2173253-A / G;3-2261232-A / G;3-2420174-G / T;3-2573901-A / G;3-2984246-A / G;3-3134555-C / A;3-3172258-A / T;3-3313047-T / C;3-3329976-C / A;3-3531349-G / A;3-3565195-A / G;3-3597749-T / C;3-3648863-T / G;3-3698114-A / G;3-3699501-C / A;3-3705655-G / A;3-3743800-C / T;3-3800635-T / C;3-3802846-C / A;3-3818708-C / A;3-3883563-T / C;3-3912447-C / T;3-3940304-A / C;3-3970237-T / C;3-3980429-T / C;3-4007709-C / G;3-4010852-A / C;3-4028493-G / A;3-4060435-C / T;3-4079512-T / C;3-4094527-G / A;3-4108212-T / C;3-4133596-G / C;3-4137113-G / A;3-4181529-A / C;3-4193314-T / C;3-4202677-T / C;4-6958-C / T;4-9382-C / T;4-11832-A / G;4-59046-A / G;4-61722-T / C;4-83050-G / A;4-90596-A / G;4-94653-T / C;4-97682-A / C;4-99927-G / T;4-109600-A / G;4-115465-C / T;4-124528-G / T;4-125461-A / G;4-138244-G / A;4-152524-C / T;4-158541-G / A;4-171915-T / C;4-177092-T / C;4-180737-C / T;4-189477-A / G;4-196952-G / A;4-204409-G / A;4-204512-T / C;4-209855-G / A;4-211143-C / A;4-217146-G / A;4-219526-T / C;4-222357-T / C;4-230061-T / C;4-243214-T / C;4-246631-G / A;4-259253-T / C;4-262887-T / C;4-268277-A / G;4-279744-G / T;4-294003-C / T;4-297348-G / A;4-301532-G / A;4-315653-G / A;4-321287-T / C;4-329167-G / T;4-332258-G / A;4-332314-G / A;4-332338-T / C;4-332704-G / A;4-336779-A / G;4-337388-A / C;4-346652-T / C;4-355211-C / A;4-355976-G / A;4-364240-G / A;4-370652-G / A;4-370778-C / T;4-387357-G / C;4-395996-C / T;4-396177-G / A;4-404963-C / T;4-412454-A / C;4-415435-G / T;4-441404-A / G;4-454563-G / A;4-456956-G / A;4-457032-A / G;4-458660-A / G;4-462758-T / C;4-464453-G / A;4-465991-C / T;4-471311-A / G;4-474253-T / C;4-477866-G / A;4-483338-A / G;4-485897-A / G;4-492973-C / T;4-500837-T / C;4-512356-C / T;4-522093-G / T;4-525199-C / A;4-539229-T / C;4-556665-C / T;4-561346-T / C;4-570286-G / A;4-574000-A / G;4-585535-G / A;4-594521-G / A;4-594935-A / G;4-600821-G / A;4-614313-C / T;4-616201-G / T;4-638005-A / C;4-643168-T / G;4-657395-A / G;4-666759-A / G;4-667473-G / T;4-676117-T / C;4-687006-T / C;4-691599-T / C;4-693830-G / A;4-696991-T / C;4-713094-G / C;4-717737-G / T;4-721279-G / A;5-4264-G / A;5-7169-G / T;5-13406-T / C;5-33760-A / G;5-44262-T / G;5-44370-C / T;5-44806-C / G;5-51658-G / T;5-61799-G / A;5-65465-C / T;5-75367-T / G;5-82638-G / T;5-91700-T / C;5-101129-C / T;5-103866-T / C;5-112002-C / A;5-120590-T / C;5-139420-A / C;5-141209-C / A;5-150234-C / T;5-158839-T / C;5-167655-G / A;5-170536-T / C;5-170550-C / G;5-170683-A / G;5-179062-T / C;5-180044-C / T;5-185685-T / C;5-187666-C / A;5-188276-T / C;5-189871-A / C;5-194007-T / C;5-202581-C / T;5-207291-T / C;5-207831-G / A;5-225917-T / C;5-230239-C / T;5-232598-C / T;5-257046-A / C;5-259241-G / A;5-263950-A / G;5-273159-A / G;5-277725-T / C;5-277784-G / A;5-295458-A / C;5-309569-A / G;5-312763-T / C;5-320221-C / A;5-328358-C / T;5-328758-G / A;5-329835-C / T;6-122412-T / C;6-122415-T / C;6-122426-G / A;6-122439-A / G;6-122581-G / A;6-122582-G / A;6-122590-A / G;6-122591-G / C;6-122595-A / T;6-122619-C / T;6-192409-T / C;6-213602-T / C;6-294450-C / A;6-399302-T / G;6-520361-A / G;6-547345-A / G;6-555780-A / G;6-600149-T / G;6-838869-C / T;6-841495-A / G;6-865932-G / A;6-867643-C / T;6-873447-C / A;6-875023-A / G;6-881325-A / G;6-887478-A / G;6-891877-T / G;6-909123-A / C;6-1044890-A / G;6-1053193-G / A;6-1063432-G / A;6-1149287-G / A;7-10802-G / A;7-16195-T / C;7-16220-C / T;7-16238-A / C;7-16263-T / C;7-39045-A / G;7-44501-A / C;7-48868-T / C;7-52331-C / A;7-68275-T / G;7-70372-G / A;7-74892-T / C;7-82549-G / A;7-92980-A / G;7-97239-T / C;7-101173-G / A;7-102273-C / A;7-110396-T / C;7-117506-C / A;7-126370-G / A;7-126393-T / G;7-127781-T / A;7-128769-G / A;7-134554-C / A;7-135579-G / A;7-143731-C / A;7-146665-T / C;7-147861-C / T;7-150000-A / G;7-150259-C / T;7-151411-G / A;7-156650-T / G;7-158556-T / C;7-178325-A / G;7-180829-A / G;7-181607-C / T;7-182813-T / C;7-183592-C / A;7-188388-G / A;7-190999-C / A;7-192515-T / C;7-196813-A / C;7-200264-G / A;7-200526-G / A;7-201420-T / C;7-205086-G / C;7-205561-T / C;7-206187-C / A;7-213662-T / C;7-214725-A / G;7-218245-A / G;7-218893-A / G;7-219326-A / G;7-222206-C / T;7-234494-C / T;7-235993-A / G;7-238044-C / T;7-240401-G / A;7-251951-C / T;7-253886-T / G;7-256259-G / T;7-258395-T / G;7-260479-T / G;7-261601-G / A;7-262481-G / T;7-264254-C / T;7-265708-C / T;8-16866-A / G;8-17010-A / G;8-17661-G / A;8-17663-G / A;8-36320-G / A;8-40302-G / A;8-45402-G / A;8-62082-C / T;8-65738-G / A;8-67412-A / G;8-71799-G / A;8-86771-G / T;8-109516-A / G;8-110934-G / A;8-119152-A / C;8-120338-T / C;8-138417-T / G;8-140547-C / T;8-167858-A / C;8-173466-G / C;8-178346-G / A;8-182538-A / G;8-200234-T / G;8-212149-T / C;8-213505-A / G;8-231614-T / C;8-232010-A / C;8-240019-C / T;8-252358-G / A;8-256526-T / G;8-268833-T / G;8-272949-C / T;8-276743-G / A;8-289260-T / C;8-341501-G / A;8-342972-C / T;8-345519-C / T;8-366660-G / T;8-370754-T / G;8-376048-G / A;8-384817-G / T;8-388050-A / G;8-389278-C / A;8-401373-T / C;8-409956-C / T;8-410904-T / G;8-419143-G / T;8-432353-T / C;8-433759-C / A;8-437373-G / A;8-445667-A / G;8-449253-A / G;8-457935-G / T;8-461067-C / T;8-461299-G / A;8-470124-G / T;8-500511-C / T;8-502745-T / C;8-502818-G / A;8-509610-A / G;8-512142-A / G;8-519519-G / A;8-520963-T / C;8-528441-A / G;8-529389-G / T;8-531744-T / C;8-544269-T / C;8-552815-T / C;8-557390-T / C;8-558615-T / G;8-561057-G / A;8-565398-T / C;8-566669-G / A;8-573143-A / G;8-591946-A / G;8-597007-C / A;8-598215-T / C;8-648647-T / C;8-655881-A / C;8-662509-G / A;8-667319-G / A;8-670192-A / G;8-674656-T / C;8-680887-T / C;8-681230-C / T;8-683994-A / C;8-686810-C / A;9-144038-A / C;9-146070-G / A;9-152998-T / A;9-259900-C / T;9-286434-C / T;9-311002-T / G;9-412246-T / C;9-421304-T / C;9-422185-C / T;9-425879-G / T;9-444945-C / A;9-465596-C / T;9-498016-A / G;9-499186-T / C;9-501720-T / C;9-617347-T / C;9-625057-T / G;9-632657-G / A;9-646892-G / C;9-654168-G / A;9-735417-A / G;9-737140-T / C;9-916467-A / G;9-945171-A / C;9-961738-A / G;9-1004710-A / G;9-1027960-T / A;9-1052543-A / G;9-1170861-C / T;9-1291741-T / C;9-1313070-C / A;9-1317284-G / A;9-1402810-G / T;9-1436413-G / T;9-1443786-C / A;9-1568456-T / C;9-1638777-T / C;9-1764339-T / A;9-1797083-T / A;9-1854333-T / G;9-1855764-A / G;9-1934202-T / C;9-2084105-G / C;9-2222992-C / T;9-2296697-C / A;9-2370923-T / C;9-2469648-T / C;9-2535785-T / C;9-2581089-A / G;9-2641881-T / C;9-2645073-C / A;9-2666584-T / C;9-2667806-T / C;9-2707549-G / A;9-2918349-T / C;9-3132386-G / A;9-3211043-T / C;9-3345496-T / C;9-3423063-A / G;9-3479246-T / C;9-3507920-T / C;9-3603266-G / A;9-3604352-C / T;9-3635355-A / G;9-3708404-T / C;9-3746627-T / C;9-3830762-T / C;9-3838704-T / C;9-3844624-C / T;9-3854628-C / T;9-3948049-C / G;9-3973083-G / A;9-4163424-A / C;9-4213116-G / A;9-4350506-G / C;9-4387935-T / C;9-4424330-A / G;9-4469351-T / A;9-4473539-A / G;9-4478326-T / C;9-4481200-C / T;9-4485136-G / A;9-4489410-A / C;9-4502785-T / G;9-4504082-G / A;9-4534492-G / A;9-4536486-A / G;9-4539494-G / T;9-4546896-G / A;9-4555751-G / T;9-4562212-C / T;9-4564117-C / T;9-4570776-T / C;9-4580415-T / C;9-4586972-A / G;9-4591567-G / A;9-4593756-T / C;9-4598919-T / C;9-4602409-C / G;9-4624812-G / A;9-4626076-G / A;9-4628297-T / C;9-4630349-G / T;9-4641583-C / G;10-46485-A / G;10-46523-C / T;10-49245-G / A;10-58063-A / G;10-58481-G / A;10-62315-A / C;10-67342-G / A;10-70306-A / C;10-76415-T / C;10-82215-G / A;10-95229-G / A;10-100057-T / C;10-104882-A / G;10-105039-G / A;10-105999-A / G;10-109811-C / G;10-116301-C / T;10-116895-T / C;10-121669-G / A;10-139849-G / A;10-149079-C / T;10-151784-A / C;10-153213-G / A;10-160235-G / A;10-173538-C / T;10-176002-T / G;10-179526-T / C;10-187309-T / C;10-206491-G / A;10-223588-C / T;10-279392-G / A;10-283563-T / G;10-285086-C / T;10-288275-C / G;10-299285-C / T;10-303826-T / A;10-305596-G / A;10-305732-T / C;10-306113-C / T;10-315805-G / A;10-321500-C / A;10-322558-G / C;10-326421-C / T;10-339584-A / G;10-340963-T / C;10-342812-A / G;10-345576-T / C;10-356482-A / T;10-357816-C / A;10-364776-C / T;10-367747-G / A;10-377203-A / G;10-378275-G / A;10-383037-A / G;10-407364-T / C;10-408093-A / C;10-413299-C / T;10-416515-A / G;10-417431-A / C;10-417455-C / A;10-417462-G / A;10-417533-C / T;10-417539-G / A;10-417565-A / G;10-417631-C / T;11-130090-G / A;11-140228-T / C;11-155439-G / T;11-162793-A / G;11-175636-A / G;11-188237-G / A;11-192690-G / T;11-193403-A / C;11-226280-G / A;11-231839-G / A;11-257538-C / T;11-262501-A / G;11-270182-G / A;11-276646-G / A;11-291218-T / C;11-292789-A / T;11-303931-C / A;11-309473-C / T;11-328920-A / G;11-335548-G / A;11-357437-G / A;11-406993-C / T;11-442387-G / A;11-510996-A / T;11-548820-A / G;11-550322-G / A;11-551567-A / T;11-573517-T / C;11-573610-T / G;11-632791-A / G;11-682530-C / T;11-682737-C / A;11-702318-T / C;11-712633-G / T;11-741739-C / T;11-765476-T / G;11-768964-A / G;11-780611-A / G;12-63361-C / T;12-104896-T / C;12-120396-G / T;12-124273-T / C;12-130488-G / A;12-133529-A / G;12-141714-G / A;12-148272-G / A;12-161500-A / C;12-206618-A / G;12-212308-A / G;12-225104-T / C;12-231166-T / C;12-239186-A / G;12-249844-G / T;12-264366-T / C;12-273623-G / T;12-279439-C / T;12-281524-C / A;12-307387-A / G;12-309820-G / A;12-310769-C / T;12-318897-C / T;12-324278-A / C;12-324610-C / T;12-328010-A / G;12-330564-G / A;12-341764-T / G;12-343927-G / A;12-343960-A / T;12-343984-G / A;12-343995-A / G;12-344002-C / A;12-346594-C / T;12-354737-C / T;12-365296-G / A;12-379909-G / A;12-380698-G / A;12-423866-G / A;12-448212-T / C;12-457395-C / T;12-463581-T / C;12-469450-T / C;12-479088-C / T;12-481914-C / T;12-485600-G / T;12-493219-A / G;12-499980-A / G;12-516664-C / A;12-520445-C / T;12-528702-T / C;12-530223-G / C;12-536346-A / G;12-557616-C / T;12-558117-G / T;12-558125-A / G;12-558289-A / G;12-562468-A / C;12-563688-T / C;12-563735-A / C;12-563747-T / C;12-565573-T / G;12-595746-G / A;12-609304-C / T。; 2. The use of the cold-resistant low-temperature germination rice gene chip as described in claim 1 in the screening of cold-resistant low-temperature rice germplasm resources.

3. The use of the cold-resistant low-temperature germinating rice gene chip as described in claim 1 in the genomic selection breeding of cold-resistant low-temperature germinating rice.

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