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CN-121975969-A - DNA bar code system and method for identifying liquorice medical plants and hybridization complex thereof

CN121975969ACN 121975969 ACN121975969 ACN 121975969ACN-121975969-A

Abstract

The invention discloses a DNA bar code system and a method for identifying liquorice medical plants and hybridization complexes thereof, belonging to the technical fields of molecular biology and traditional Chinese medicine identification. The system is formed by combining three bar codes of a nuclear gene fragment ITS, a chloroplast gene fragment ndhA and a chloroplast gene spacer trnH-psbA. Wherein ndhA is the first screening and verification core bar code of the invention, and the specific ectopic sites can accurately distinguish three medicinal licorice (Glycyrrhiza uralensisFischex DC), glycyrrhiza glabra (Glycyrrhiza glabra L.) and Glycyrrhiza glabra (Glycyrrhiza inflataBatalin). The system fully utilizes the genetic characteristics of the chloroplast male parent of the glycyrrhiza, can identify homozygous species, can efficiently analyze complex hybridization and introgression types, and can trace the male parent source. Compared with the traditional multi-bar code combination, the invention has the advantages of high identification success rate (up to 98.08%), simple and convenient operation and low cost, and is suitable for identifying the germplasm resources of the liquorice, rapidly screening the medicinal material market and breeding improved varieties.

Inventors

  • LU JIAHUI
  • WAN GUIGUI
  • Luo Jiafen
  • LI XIN
  • CHEN CHAONAN

Assignees

  • 石河子大学

Dates

Publication Date
20260505
Application Date
20251205

Claims (8)

  1. 1. A method for identifying a species, hybridization or introgression type of a plant of the genus glycyrrhiza, comprising the steps of: a) Obtaining DNA of a liquorice sample to be detected; b) Determining the sequence of a nuclear gene Internal Transcription Spacer (ITS), a chloroplast gene spacer (trnH-psbA) and a chloroplast gene ndhA in the DNA; c) Comparing the measured ITS, trnH-psbA and ndhA sequences with a haplotype database; d) Based on the haplotype combination of the three, the species identity of the sample is determined, or the hybridization type and the introgression source are identified.
  2. 2. The method of claim 1, wherein the haplotype combination decision in step d) is based on: The ndhA sequences are used to distinguish base species, wherein: haplotype NA-1 corresponds to Glycyrrhiza uralensis; Haplotype NA-2 corresponds to Glycyrrhiza inflata; Haplotype NA-3 corresponds to Glycyrrhiza glabra.
  3. 3. The ITS sequences are used to indicate hybridization events, wherein haplotype I-3 indicates that there is Glycyrrhiza uralensis hybridization to other species.
  4. 4. The trnH-psbA sequences are useful to aid in identifying species and identifying particular introgression types.
  5. 5. The method of claim 2, wherein the haplotypes NA-1, NA-2 and NA-3 are defined by the nucleotide sequences at positions 122-131 and 168 of the ndhA gene, wherein: NA-1 is characterized by the sequence at positions 122-131 being AGCTATCTCC and at position 168 being G; NA-2 is characterized by the sequence at positions 122-131 being AGCTATCTCC and at position 168 being A; NA-3 is characterized by the sequence at positions 122-131 being GGAGATAGCT and at position 168 being A.
  6. 6. A combination of DNA molecular markers for identifying plants of the genus glycyrrhiza, characterized in that said combination consists of a nuclear gene Internal Transcribed Spacer (ITS), a chloroplast gene spacer (trnH-psbA) and a chloroplast gene ndhA.
  7. 7. A primer pair for amplifying the ndhA fragment in the DNA molecular marker combination of claim 4, wherein the nucleotide sequence is: The upstream primer ndhA-F5'-AATCAAGCAATACTCCCC-3' (SEQ ID NO: 1), The downstream primer ndhA-R5'-GAATTATCAATAACCCCAT-3' (SEQ ID NO: 2).
  8. 8. Use of a combination of DNA molecular markers according to claim 4 for the preparation of an identification reagent for identifying glycyrrhiza species, hybridization or introgression types.

Description

DNA bar code system and method for identifying liquorice medical plants and hybridization complex thereof Technical Field The invention belongs to the technical field of molecular biology and traditional Chinese medicine identification, in particular relates to a DNA bar code system and a method for identifying medicinal plants of liquorice and hybridization complexes thereof, and particularly relates to a method for species identification, hybridization origin analysis and male parent tracing by utilizing nuclear gene ITS, chloroplast gene spacer trnH-psbA and newly screened chloroplast gene ndhA combinations. Background During the last two decades, the reserves of wild licorice resources have declined dramatically, forcing the major sources of medicinal licorice products to gradually shift to cultivars. However, the quality of the cultivated licorice is not stable, reflecting that it still faces serious challenges in germplasm consistency and genetic stability. The three main medicinal licorice roots have wide hybridization and gene introgression phenomena, which lead to obvious genetic differentiation of seeds and seedlings. The hybridization not only increases the difficulty of standardized planting, but also causes the problem of seed source mixing in the medicinal material harvesting stage. Therefore, it is important to establish reliable morphological discrimination traits suitable for these hybridization complexes. However, since liquorice of different species and producing areas are highly complex in morphological differentiation and genetic structure, hidden species with similar phenotypes and different genetic backgrounds sometimes appear, so that the difficulty of classifying and identifying germplasm resources is further increased, and efficient development and utilization of wild liquorice resources are restricted. Under the background, the system is particularly urgent to elucidate the origin, phylogenetic relationship and accurate identification method of medicinal licorice hybrid complex. Molecular identification techniques, particularly DNA barcode techniques, provide a powerful complement to species identification. In the prior art, the liquorice is often identified by adopting bar codes such as ITS, trnH-psbA, rbcL, matK, trnV-ndhC and the like or combinations thereof. However, these barcodes have the limitations of 1. The resolution is insufficient, the variation sites of the existing barcodes are limited, three medicinal liquorice very close in relatedness cannot be effectively distinguished, and particularly, the filial generation and the complex introgression type of the three medicinal liquorice cannot be easily distinguished. 2. The system is complex, 4-5 bar codes are generally required to be combined to improve the identification rate, so that the operation flow is complicated, the cost is high, the efficiency is low, and the method is not suitable for large-scale germplasm resource screening and quick identification of a medicinal material market. 3. The male parent has difficulty in tracing that the chloroplast is considered to be inherited as a maternal line in the traditional concept, but has been proved to be inherited as a paternal line in the Glycyrrhiza genus. The prior art fails to fully exploit this key genetic trait to trace back the male parent source of the hybrid offspring. Therefore, there is an urgent need in the art to develop a novel DNA barcode system having high resolution, high efficiency and low cost, so as to solve the problem of accurate identification of glycyrrhiza medicinal plants and their hybridization complexes. . Disclosure of Invention The invention aims to overcome the defect of insufficient DNA bar code resolution in the prior liquorice identification technology, provides a method for screening high-resolution and specific DNA bar codes from liquorice chloroplast genome, and establishes a high-efficiency, accurate and convenient molecular identification system so as to solve the classification and identification problems of three medicinal liquorice and hybridization penetration complexes thereof. The invention provides the following technical scheme that the hypervariable regions in the multiple liquorice chloroplast genomes including definite artificial filial generation are systematically screened by sequencing, comparing and analyzing, and specific DNA bar code fragments capable of stably distinguishing three medicinal liquorice are identified. Based on the core conception, the invention provides the following specific schemes: In order to achieve the above purpose, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method of screening for high resolution specific DNA barcodes. The method comprises the steps of sequencing and comparing chloroplast genome of a plurality of liquorice including definite artificial filial generation, systematically screening hypervariable regions