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CN-120796560-B - KASP primer group for distinguishing Pinus sylvestris and white bark pine, and detection method and application thereof

CN120796560BCN 120796560 BCN120796560 BCN 120796560BCN-120796560-B

Abstract

The invention provides a KASP primer group for distinguishing Pinus sylvestris from Pinus bungeana, and a detection method and application thereof, and belongs to the technical field of gene detection. According to the invention, through resequencing of the mass samples of the Pinus sylvestris and the Pinus bungeana, mutation condition analysis is carried out on the sequences subjected to quality control, two SNP loci with differentiation are obtained through screening, and specific KASP primer groups are designed for the two loci, and are respectively shown as SEQ ID NO. 1-3 and SEQ ID NO. 4-6. The invention determines that the detected genotype has 100% consistency with the genotype of a known variety when the KASP primer group is used for distinguishing the Pinus pentadactyla and the white bark pine through verification and comparison of known materials. The primer pair of the two SNP loci can be singly or in combination applied to identification of varieties of Pinus sylvestris and Pinus bungeana.

Inventors

  • LI RUOFEI
  • ZHANG RENGANG
  • MA YONGPENG
  • LIU DETUAN
  • LIU ZHENGJIANG
  • Liu Xiongfang
  • ZHANG TICAO
  • GAO XUESONG

Assignees

  • 中国科学院昆明植物研究所

Dates

Publication Date
20260505
Application Date
20250805

Claims (8)

  1. 1. A KASP primer set for distinguishing between pinus sylvestris and pinus sylvestris, wherein the KASP primer set comprises at least one of the following primer sets: A first SNP primer set for amplifying a first SNP site; a second SNP primer set for amplifying a second SNP site; The first upstream primer of the first SNP primer set is shown as SEQ ID NO.1, the second upstream primer is shown as SEQ ID NO.2, and the downstream primer is shown as SEQ ID NO. 3; the first upstream primer of the second SNP primer set is shown as SEQ ID NO.4, the second upstream primer is shown as SEQ ID NO.5, and the downstream primer is shown as SEQ ID NO. 6; The first SNP locus is in the chromosome position of > chr04:434506971, and the second SNP locus is in the chromosome position of > chr10:1961560286.
  2. 2. A kit for distinguishing pinus sylvestris from pinus sylvestris comprising the KASP primer set of claim 1.
  3. 3. A method for detecting the distinction between the five-needle pine and the white bark pine of the Qiaojia, which is characterized by comprising the following steps: S1, extracting genome DNA of a sample to be detected; S2, using the extracted genome DNA as a template, carrying out competitive allele by using the SNP primer set as set forth in claim 1 Carrying out gene specificity PCR amplification to obtain a PCR product; s3, distinguishing the Pinus sylvestris from the white pinus sylvestris according to genotype determination results of fluorescence or amplification products read in the competitive allele specific PCR process: Judging as Pinus sylvestris if the fluorescent signal of the FAM group is read, and judging as white pinus sylvestris if the fluorescent signal of the HEX group is read; When the first SNP primer set is used for amplification, the amplification product is judged to be the Pinus sylvestris if the genotype of the amplification product is G; when the second SNP primer set is used for amplification, the Pinus sylvestris is judged to be the Pinus sylvestris if the genotype of the amplified fragment is G, and the Pinus sylvestris is judged to be the Pinus sylvestris if the genotype of the amplified fragment is T.
  4. 4. The method according to claim 3, wherein the competitive allele-specific PCR amplification reaction system is 10. Mu.L, 2 Xv 4 Flu-Arms Mix is 5. Mu.L, the genomic DNA of the sample to be tested is 4.5. Mu.L, and the mixed primer is 0.5. Mu.L, and the mixed primer consists of a first upstream primer, a second upstream primer and a downstream primer.
  5. 5. The method according to claim 4, wherein the initial concentration of the mixed primer is 10. Mu.M, and the molar ratio of the first upstream primer to the second upstream primer to the downstream primer is 1:1:3.
  6. 6. The method of claim 5, wherein the competitive allele-specific PCR amplification is performed by the following reaction procedure: (1) Pre-denaturation at 95 ℃ for 10min; (2) Drop PCR cycle: (2.1)95°C,15s; (2.2)61°C,45s; (2.3) repeating steps (2.1) and (2.2) for 10 cycles, each cycle at 61 ℃ being reduced by 0.6 ℃; (3) Routine PCR cycles: (3.1)95°C,15s; (3.2)55°C,45s; (3.3) repeating steps (3.1) and (3.2) for 36 cycles; (4) Fluorescence reading at 30 ℃ for 30s.
  7. 7. Use of the KASP primer set of claim 1, in at least one of: (1) Application in distinguishing or assisting in distinguishing the pinus sylvestris and the white bark pine; (2) Application in preparing a kit for distinguishing the pinus sylvestris from the pinus bungeana; (3) The application in the protection of the germplasm resources of the Pinus sylvestris.
  8. 8. Use of the kit of claim 2 in at least one of: (1) Application in distinguishing or assisting in distinguishing the pinus sylvestris and the white bark pine; (2) Application in preparing a kit for distinguishing the pinus sylvestris from the pinus bungeana; (3) The application in the protection of the germplasm resources of the Pinus sylvestris.

Description

KASP primer group for distinguishing Pinus sylvestris and white bark pine, and detection method and application thereof Technical Field The invention relates to the technical field of gene detection, in particular to a KASP primer group for distinguishing Pinus thunbergii and Pinus bungeana, and a detection method and application thereof. Background The Pinus sylvestris (Pinus squarata) is a very small population plant of Pinus sylvestris group white bark pine subgroup of pinaceae (Pinaceae) Pinus, and currently only 35 wild individuals worldwide exist and all grow in Zhaojia county of Zhaotong city in Yunnan province. This species is the key object of biodiversity protection, and its protection work is of paramount importance. The protection of the pinus sylvestris has achieved remarkable results in thirty years through means of artificial sowing and breeding, land-to-land protection, original land regression and the like. White bark pine (Pinus bungeana) is a special tree species in China, and is a plant of the Pinaceae (Pinacea) Pinus genus single-vascular bundle Pinus subgenera white bark pine group. The species is widely distributed and cultivated in most areas of the country as an important garden tree species. In the study of the skillful jersey, the tree species closest to the skillful jersey are mainly white bark pine and Tibetan white bark pine. Of note, white bark pine as a broad seed has been artificially planted in provinces of Sichuan, yunnan, guizhou, etc., with a distribution area geographically adjacent to or overlapping with the original land and the protected-from-land population of the Pinus sylvestris, e.g., the protected-from-land population of the Pinus sylvestris has been in the same city as the white bark pine population cultivated in gardens—Kunming. The present situation of close-range coexistence is that two tree species have high similarity in morphology, and confusion is easy to occur only by naked eye observation or a traditional plant identification method, so that potential source confusion risks are brought, and influence is formed on genetic resource protection, germplasm resource management and long-term protection of the rare endangered species of the Pinus maritima. In view of the above morphological and traditional identification limitations, it is important to develop a highly specific identification method based on the genetic level. At present, although the gene sequencing technology can provide species identification at the gene level, the high cost and relatively long turnover time of the technology make it difficult to provide a solution which is efficient, economical and quick in the scenes of large-scale population investigation, daily monitoring, rapid field identification and the like, and a simple, economical and rapid accurate identification approach is still lacking at present. Disclosure of Invention In view of this, the present invention provides a KASP primer set for distinguishing pinus sylvestris from pinus bungeana, and a detection method and application thereof. In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a KASP primer set for distinguishing between pinus skillful and pinus bungeana, said KASP primer set comprising at least one of the following primer sets: A first primer set for amplifying a first SNP site; a second primer set for amplifying a second SNP site; The first upstream primer of the first SNP primer set is shown as SEQ ID NO.1, the second upstream primer is shown as SEQ ID NO.2, and the downstream primer is shown as SEQ ID NO. 3; the first upstream primer of the second SNP primer set is shown as SEQ ID NO.4, the second upstream primer is shown as SEQ ID NO.5, and the downstream primer is shown as SEQ ID NO. 6; The first SNP locus is in the chromosome position of > chr04:434506971, and the second SNP locus is in the chromosome position of > chr10:1961560286. Preferably, the 5 'end of the first upstream primer is connected with a FAM fluorescent group tag sequence, and the 5' end of the second upstream primer is connected with a HEX fluorescent group tag sequence. The invention also provides a kit for distinguishing the pinus sylvestris from the pinus bungeana, which comprises the KASP primer group. The invention also provides a detection method for distinguishing the Pinus sylvestris from the white bark pine, which comprises the following steps: S1, extracting genome DNA of a sample to be detected; s2, performing competitive allele-specific PCR amplification by using the extracted genome DNA as a template and utilizing the SNP primer set to obtain a PCR product; s3, distinguishing the Pinus sylvestris from the white pinus sylvestris according to genotype determination results of fluorescence or amplification products read in the competitive allele specific PCR process: Judging as Pinus sylvestris if the fluorescent signal of the FAM group is read, an