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CN-121975970-A - Cold-resistant site qCTE3 in rice germination period, molecular marker and application thereof

CN121975970ACN 121975970 ACN121975970 ACN 121975970ACN-121975970-A

Abstract

The invention belongs to agricultural biotechnology engineering, and particularly discloses a cold tolerance site qCTE in a rice germination period, a molecular marker and application thereof. The molecular marker is a site qCTE3 which is positioned at chromosome 28361033 bp and is obviously related to the cold tolerance in the germination period, and the cold tolerance in the germination period of the germplasm carrying the allelic variation of the G nucleotide is extremely stronger than that of the germplasm carrying the allelic variation of the A nucleotide. The cold resistance identification method for the rice germination period is accurate and reliable in verification.

Inventors

  • CUI DI
  • HAN LONGZHI
  • LIANG HUIYUAN
  • Yu Kunchi
  • LIU CHUNHUI
  • MA XIAODING
  • HAN BING

Assignees

  • 中国农业科学院作物科学研究所
  • 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心）

Dates

Publication Date
20260505
Application Date
20251229

Claims (10)

  1. 1. The rice germination cold tolerance molecular marker is characterized in that the rice germination cold tolerance molecular marker is a site qCTE3 which is positioned at chromosome 28361033 bp and is obviously related to germination cold tolerance, particularly a nucleotide difference at a physical position 28361033 bp, and the germination cold tolerance of a germplasm carrying G nucleotide allelic variation is extremely obviously stronger than that of a germplasm carrying A nucleotide allelic variation.
  2. 2. Detection of a molecularly marked primer according to claim 1, preferably it is a KASP primer.
  3. 3. The primer of claim 2, comprising a strong cold tolerance allelic variation specific primer for detecting the G nucleotide allelic variation locus, a weak cold tolerance allelic variation specific primer for detecting the A nucleotide allelic variation, and optionally a universal primer designed based on the conserved sequences flanking the molecular marker locus.
  4. 4. The primer of claim 3, wherein the primer specific for detecting the cold tolerance allelic variation in the germination period carrying the allelic variation site of the G nucleotide is as follows: 5’-GAAGGTCGGAGTCAACGGATTAGGTAATCCAACAACACAGTATACG-3’, The specific primer for detecting the low cold tolerance allelic variation of the A nucleotide in the sprouting period is 5'-GAAGGTGACCAAGTTCATGCTCAGGTAATCCAACAACACAGTATACA-3', The general primers are as follows 5'-GATGTGTAGTCTCTTTAATTCATGCCTG-3'.
  5. 5. The method for detecting cold tolerance molecular markers in rice germination according to claim 1, wherein the method is used for identifying cold tolerance in rice germination or used for cultivating or assisting in cultivating cold tolerance rice in rice germination or rice varieties.
  6. 6. The use according to claim 1, wherein the detection method is gene sequencing, molecular amplification.
  7. 7. A method for identifying cold tolerance in rice germination, characterized in that the result of the molecular marker is obtained by using the method for detecting cold tolerance molecular marker in rice germination according to claim 1, so as to determine cold tolerance in rice germination, wherein the cold tolerance in germination of a germplasm carrying a variation of a nucleotide allele at the locus is extremely significantly stronger than that of a germplasm carrying a variation of a nucleotide allele.
  8. 8. The method according to claim 7, wherein the detection is carried out using the primer according to any one of claims 2 to 4.
  9. 9. The method of claim 8, wherein the specific method comprises extracting genomic DNA of rice to be detected, and performing PCR amplification by using the primer with the genomic DNA as a template to obtain a PCR product; specifically, in the PCR amplification system, the concentration of qCTE-G and qCTE-A is 30-45 mu mol/mu L, the concentration of qCTE3-C is 80-100 mu mol/mu L, the three primers are uniformly mixed according to the volume ratio of 1:1:1 to form KASP primer Mix, the total reaction volume of the reaction system is 10.14 mu L, and the reaction system comprises 5 mu L of DNA, 2x KASP Master Mix 5 mu L and 0.14 mu L of KASP primer Mix; The amplification procedure was (1) 94℃pre-denaturation 15 min, (2) 94℃denaturation 20 s,61℃extension 60 s, drop at 0.6℃per cycle, 10 cycles, and (3) 94℃denaturation 20 s,55℃extension 60 s,26 cycles; The detection step comprises the step of identifying the cold resistance of the rice to be detected in the germination period according to the nucleotide sequence of the PCR product, wherein if the typing of the PCR product is G type, the rice to be detected is or is candidate to be the rice variety with stronger cold resistance in the germination period, and if the typing of the PCR product is A type, the rice to be detected is or is candidate to be the rice variety with weaker cold resistance in the germination period.
  10. 10. A method for cultivating rice or rice varieties with cold tolerance in the germination period of rice, characterized in that the detection method of the molecular marker for cold tolerance in the germination period of rice according to claim 1 or the result of the molecular marker obtained by the identification method according to any one of claims 7 to 9 is adopted to judge the auxiliary standard of the cold tolerance in the germination period of rice in the cultivation process of rice, and more specifically, the primer according to any one of claims 2 to 4 is adopted for detection.

Description

Cold-resistant site qCTE3 in rice germination period, molecular marker and application thereof Technical Field The invention belongs to agricultural biotechnology engineering, and particularly relates to a cold tolerance site qCTE in a rice germination period, a molecular marker and application thereof. Background The greatest difference between direct seeding (water direct seeding and dry direct seeding) and seedling raising and transplanting is that seeds are germinated directly in a field, and the heat preservation and moisture preservation protection of a seedling raising shed is lacking, so that the direct seeding (water direct seeding and dry direct seeding) and seedling raising and transplanting are more directly influenced by low temperature and temperature change in the field, and the requirement on the cold resistance of rice varieties in the germination period is higher. In the northern japonica rice area, a strong cold-resistant japonica rice variety needs to be selected for direct seeding in early spring, and in the southern indica rice area, if the indica rice variety is subjected to cold in the reverse spring, the indica rice variety with poor cold resistance is difficult to emerge, the seedling rate is low, and finally the yield is influenced. Therefore, the cultivation of the rice variety with strong cold resistance in the germination period is the guarantee of successful planting and stable yield of the direct-seeding rice. The cold tolerance of rice in germination period is a typical quantitative character, is controlled by multiple genes, and is characterized by continuous variation and is easily influenced by environmental conditions. Therefore, it is difficult to efficiently cultivate rice varieties having high cold resistance by only using a conventional rice breeding method using phenotypic selection. With the continuous development of quantitative genetics and molecular biology technologies, the genetic mechanism of the cold tolerance of the rice in the germination period is analyzed, the molecular markers related to the cold tolerance of the rice are developed, accurate auxiliary breeding is realized, and technical support is provided for improving the cold tolerance breeding efficiency of the rice. Disclosure of Invention According to the invention, research shows that a site qCTE3 which is obviously related to cold tolerance in the germination period exists in a chromosome 28361033 bp of rice, and the cold tolerance in the germination period of the germplasm carrying G nucleotide allelic variation is extremely obviously stronger than that of the germplasm carrying A nucleotide allelic variation, so that the invention is completed. The invention provides a rice germination cold tolerance molecular marker, which is a site qCTE which is positioned at chromosome 28361033 bp and is obviously related to germination cold tolerance, particularly a nucleotide difference at a physical position 28361033 bp, wherein the germination cold tolerance of a germplasm carrying G nucleotide allelic variation is extremely higher than that of a germplasm carrying A nucleotide allelic variation. The present invention provides a primer for detecting said molecular marker, preferably a KASP primer. Specifically, the primer comprises a strong cold tolerance allelic variation specific primer qCTE-G for detecting the G nucleotide allelic variation locus, a weak cold tolerance allelic variation specific primer qCTE-A for detecting the A nucleotide allelic variation, and optionally a universal primer qCTE3-C. The universal primer takes the SNP locus as the center, the conserved flanking sequences of 50-100 bp on two sides are obtained, and the universal primer needs to target the conserved region so as to avoid covering the polymorphic locus. The universal primer is matched with the specific primer, so that the length of the PCR product is controlled to be 60-120bp (70-100 bp is commonly used), and the amplification efficiency and the fluorescent signal interpretation accuracy are ensured. More specifically, the molecular marker qCTE-G primer is 5'-GAAGGTCGGAGTCAACGGATTAGGTAATCCAACAACACAGTATACG-3', the qCTE3-A primer is 5'-GAAGGTGACCAAGTTCATGCTCAGGTAATCCAACAACACAGTATACA-3', and the qCTE3-C primer is 5'-GATGTGTAGTCTCTTTAATTCATGCCTG-3'. The invention also provides application of the detection method of the rice germination cold tolerance molecular marker in identifying the rice germination cold tolerance. Specifically, the detection method is genetic sequencing or molecular amplification. The invention provides a method for identifying cold resistance of rice in germination, which adopts the detection method of the cold resistance molecular marker in the germination to obtain the result of the molecular marker so as to judge the cold resistance of the rice in germination, wherein the cold resistance of the rice in germination of a germplasm carrying G nucleotide allelic variation at the locus is extremely obvio