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CN-122012784-A - Multiplex PCR primer combination for apocarya and application thereof

CN122012784ACN 122012784 ACN122012784 ACN 122012784ACN-122012784-A

Abstract

The invention discloses a apocarya multiplex PCR primer combination and application thereof, and belongs to the technical field of molecular markers. The multiplex PCR primer combination comprises 8 primer combinations, wherein each primer combination comprises 3 pairs of primers, and the sequences of the primers are shown as SEQ ID NO.1-SEQ ID NO. 48. According to the invention, 24 InDel markers with polymorphism are mined based on apocarya transcriptome data, 24 pairs of primers are designed and synthesized, and the 24 pairs of primers are optimized into 8 triple combinations, so that multi-site synchronous detection can be realized, further the analysis of apocarya germplasm resource diversity and the construction work of fingerprint are efficiently developed, and reliable technical support is provided for genetic improvement, variety breeding, fine variety precise propagation and the like of apocarya.

Inventors

  • HOU MENGXIN
  • LI YING
  • LI YONGRONG
  • XUAN JIPING
  • ZUO JILIN
  • ZHAI MIN
  • HU LONGJIAO
  • MO ZHENGHAI

Assignees

  • 江苏省中国科学院植物研究所
  • 宿迁绿宙薄壳山核桃科技有限公司

Dates

Publication Date
20260512
Application Date
20260309

Claims (7)

  1. 1. A multiplex PCR primer combination of apocarya, characterized in that it comprises 8 primer combinations, each comprising 3 pairs of primers; Wherein, the combination 1 comprises a primer shown as SEQ ID NO.1-SEQ ID NO. 6; the combination 2 comprises a primer shown as SEQ ID NO.7-SEQ ID NO. 12; the combination 3 comprises a primer shown as SEQ ID NO.13-SEQ ID NO. 18; The combination 4 comprises a primer shown as SEQ ID NO.19-SEQ ID NO. 24; The combination 5 comprises a primer shown as SEQ ID NO.25-SEQ ID NO. 30; the combination 6 comprises primers shown as SEQ ID NO.31-SEQ ID NO. 36; The combination 7 comprises primers shown as SEQ ID NO.37-SEQ ID NO. 42; the primer shown in SEQ ID NO.43-SEQ ID NO.48 is contained in the combination 8.
  2. 2. A multiplex PCR kit of apocarya, characterized in that the kit comprises the multiplex PCR primer combination of apocarya according to claim 1.
  3. 3. Use of the apocarya multiplex PCR primer combination of claim 1 or the kit of claim 2 in apocarya germplasm genetic diversity analysis, germplasm identification, assisted breeding or fingerprint construction.
  4. 4. The construction method of the apocarya fingerprint is characterized by comprising the following steps: S1, extracting DNA of apocarya; S2, carrying out PCR amplification by using the DNA in the step S1 as a template and utilizing each primer combination in the apocarya multiplex PCR primer combination of claim 1 to obtain a PCR product; and S3, performing electrophoresis detection on the PCR product, and arranging detection results of the primer combinations in series to obtain the apocarya fingerprint.
  5. 5. The method according to claim 4, wherein the PCR amplification reaction system comprises 2X TSINGKE MASTER Mix 30. Mu.L, 1. Mu.L of DNA, 1. Mu.L of 3 pairs of upstream and downstream primers, 13. Mu.L of ddH 2 O, and a total volume of 50. Mu.L.
  6. 6. The method according to claim 4, wherein the PCR amplification is performed by pre-denaturing at 94℃for 5min, denaturing at 95℃for 10 s, annealing at 56℃for 1 min, extending at 72℃for 1 min for 35 cycles, and final extending for 7 min.
  7. 7. Use of a apocarya fingerprint constructed by the construction method according to any one of claims 4-6 in analysis of genetic diversity or germplasm identification of apocarya germplasm.

Description

Multiplex PCR primer combination for apocarya and application thereof Technical Field The invention relates to the technical field of molecular markers, in particular to a apocarya multiplex PCR primer combination and application thereof. Background Apocarya (Carya illinoinensis), also known as hickory nut, pecan, is a plant of the genus hickory, the family Juglandaceae. The oil content of the apocarya kernel is up to 70 percent, which is higher than that of camellia seeds, olives and erythrina seeds, and is one of woody tree seeds with the highest oil content. Conventional methods for analysis of apocarya germplasm resources include leaf morphology, female flower characteristics, fruit characteristics, and the like. The phenotypic characteristics are easily influenced by environmental factors such as plant development stage, seasonal variation, air temperature fluctuation, illumination intensity and the like, so that the data stability is insufficient, meanwhile, the judgment standard is highly dependent on human experience, the subjectivity is high, evaluation deviation is easily introduced, and the reliability of the result is finally reduced. In contrast, the DNA molecular marker technology has the remarkable advantages that the detection result is not interfered by environmental conditions, and the detection result is highly consistent in different development stages and tissue parts (such as roots, stems and leaves) of plants, so that the DNA molecular marker technology becomes a reliable tool for accurately identifying plant varieties, deeply analyzing genetic diversity and efficiently constructing fingerprint patterns. The DNA molecular marking technology has the advantages of no environmental influence, consistent different development periods and parts, and the like, and becomes an effective means for plant variety identification, diversity analysis, fingerprint construction, and the like. Conventional molecular markers such as microsatellite (Simple sequence repeats, SSR), insertion/deletion (InDel) can be subjected to multiplex detection, i.e. 2 or more markers are placed in one polymerase chain reaction (polymerase chain reaction, PCR) for experiment and subsequent detection, which can greatly shorten the experimental period, reduce the reagent cost and increase the detection throughput compared with single marker independent detection. Unfortunately, there is still a lack of a multiple detection technology system in apocarya that is easy to operate and has high stability, and further research and development is needed. Disclosure of Invention The invention aims to provide a apocarya multiplex PCR primer combination and application thereof, so as to solve the problems in the prior art. According to the invention, 24 InDel markers with polymorphism are mined based on apocarya transcriptome data, 24 pairs of primers are designed and synthesized, and the 24 pairs of primers are optimized into 8 triple combinations, so that multi-site synchronous detection can be realized, further the analysis of apocarya germplasm resource diversity and the construction work of fingerprint are efficiently developed, and reliable technical support is provided for genetic improvement, variety breeding, fine variety precise propagation and the like of apocarya. In order to achieve the above object, the present invention provides the following solutions: The invention provides a apocarya multiplex PCR primer combination, which comprises 8 primer combinations, wherein each primer combination comprises 3 pairs of primers; Wherein, the combination 1 comprises a primer shown as SEQ ID NO.1-SEQ ID NO. 6; the combination 2 comprises a primer shown as SEQ ID NO.7-SEQ ID NO. 12; the combination 3 comprises a primer shown as SEQ ID NO.13-SEQ ID NO. 18; The combination 4 comprises a primer shown as SEQ ID NO.19-SEQ ID NO. 24; The combination 5 comprises a primer shown as SEQ ID NO.25-SEQ ID NO. 30; the combination 6 comprises primers shown as SEQ ID NO.31-SEQ ID NO. 36; The combination 7 comprises primers shown as SEQ ID NO.37-SEQ ID NO. 42; the primer shown in SEQ ID NO.43-SEQ ID NO.48 is contained in the combination 8. The invention also provides a apocarya multiplex PCR kit, which comprises the apocarya multiplex PCR primer combination. The invention also provides application of the apocarya multiplex PCR primer combination or the kit in apocarya germplasm genetic diversity analysis, germplasm identification, auxiliary breeding or fingerprint construction. The invention also provides a construction method of the apocarya fingerprint, which comprises the following steps: S1, extracting DNA of apocarya; S2, carrying out PCR amplification by using the DNA in the step S1 as a template and utilizing each primer combination in the apocarya multiplex PCR primer combination to obtain a PCR product; and S3, performing electrophoresis detection on the PCR product, and arranging detection results of the primer combinations in series to