Search

CN-121992138-A - EST-SSR marker combination and application thereof in identification of Polygonatum plants

CN121992138ACN 121992138 ACN121992138 ACN 121992138ACN-121992138-A

Abstract

The invention discloses an EST-SSR molecular marker combination and application thereof in sealwort plant identification, belonging to the technical field of plant molecular biology. The invention develops 49 pairs of high polymorphism EST-SSR marker primers based on the rhizome transcriptome combined data of three medicinal species of Polygonatum cyrtonema, polygonatum cyrtonema and Polygonatum cyrtonema, and screens 9 pairs of core marker primers from the primers. The invention also provides a kit comprising the label combination and an identification method, and the kit can accurately distinguish 6 closely related species of rhizoma polygonati, rhizoma polygonati yunnanensis, rhizoma polygonati cyrtomii, rhizoma polygonati of large She Dian, rhizoma polygonati of leaf rolling and rhizoma polygonati of Hubei at one time by adopting a fluorescence capillary electrophoresis detection technology, and effectively analyze the intra-species variation of different geographic communities of rhizoma polygonati of large She Dian. The marker combination has strong universality, high polymorphism, large detection flux and accurate and reliable result, and provides a high-efficiency molecular tool for the identification of Polygonatum germplasm resources, the analysis of genetic diversity and molecular assisted breeding.

Inventors

  • HUANG WENJUAN
  • WANG HUI
  • YANG MAJIN
  • YE CHANGHUA
  • LI ZHEN
  • ZHANG YAN
  • HE FEI

Assignees

  • 四川省农业特色植物研究院

Dates

Publication Date
20260508
Application Date
20260311

Claims (10)

  1. 1. An EST-SSR molecular marker primer combination for identifying a polygonatum plant, characterized in that the primer combination comprises at least one of the following 9 pairs of primers: (1) Primer pairs with nucleotide sequences shown as SEQ ID NO. 5-6; (2) Primer pairs with nucleotide sequences shown as SEQ ID NO. 9-10; (3) Primer pairs with nucleotide sequences shown as SEQ ID NO. 53-54; (4) Primer pairs with nucleotide sequences shown as SEQ ID NO. 3-4; (5) Primer pairs with nucleotide sequences shown as SEQ ID NO. 7-8; (6) Primer pairs with nucleotide sequences shown as SEQ ID NO. 63-64; (7) Primer pairs with nucleotide sequences shown as SEQ ID NO. 43-44; (8) Primer pairs with nucleotide sequences shown as SEQ ID NO. 67-68; (9) Primer pairs with nucleotide sequences shown as SEQ ID NO. 95-96; Wherein, the nucleotide sequence of each primer pair is developed and obtained based on the rhizome transcriptome combination data of three medicinal species of Polygonatum sibiricum P. kingianum var. Sibiricum P. grandifolium, polygonatum kingianum P. kingianum and Polygonatum cyrtonema P. cyrtonema.
  2. 2. An EST-SSR molecular marker primer combination according to claim 1, wherein said primer combination comprises all 9 pairs of primers.
  3. 3. An EST-SSR molecular marker primer set for identifying a polygonatum plant, characterized in that the primer set comprises the primer combination of claim 1 or 2, and a supplementary primer selected from any one or more of the following pairs of primers: (10) Primer pairs with nucleotide sequences shown as SEQ ID NO. 1-2; (11) Primer pairs with nucleotide sequences shown as SEQ ID NO. 11-12; (12) Primer pairs with nucleotide sequences shown as SEQ ID NO. 13-14; (13) Primer pairs with nucleotide sequences shown as SEQ ID NO. 15-16; (14) Primer pairs with nucleotide sequences shown as SEQ ID NO. 17-18; (15) Primer pairs with nucleotide sequences shown as SEQ ID NO. 19-20; (16) Primer pairs with nucleotide sequences shown as SEQ ID NO. 21-22; (17) Primer pairs with nucleotide sequences shown as SEQ ID NO. 23-24; (18) Primer pairs with nucleotide sequences shown as SEQ ID NO. 25-26; (19) Primer pairs with nucleotide sequences shown as SEQ ID NO. 27-28; (20) Primer pairs with nucleotide sequences shown as SEQ ID NO. 29-30; (21) Primer pairs with nucleotide sequences shown as SEQ ID NO. 31-32; (22) Primer pairs with nucleotide sequences shown as SEQ ID NO. 33-34; (23) Primer pairs with nucleotide sequences shown as SEQ ID NO. 35-36; (24) Primer pairs with nucleotide sequences shown as SEQ ID NO. 37-38; (25) Primer pairs with nucleotide sequences shown as SEQ ID NO. 39-40; (26) Primer pairs with nucleotide sequences shown as SEQ ID NO. 41-42; (27) Primer pairs with nucleotide sequences shown as SEQ ID NO. 45-46; (28) Primer pairs with nucleotide sequences shown as SEQ ID NO. 47-48; (29) A primer pair with a nucleotide sequence shown as SEQ ID NO. 49-50; (30) Primer pairs with nucleotide sequences shown as SEQ ID NO. 51-52; (31) Primer pairs with nucleotide sequences shown as SEQ ID NO. 55-56; (32) Primer pairs with nucleotide sequences shown as SEQ ID NO. 57-58; (33) Primer pairs with nucleotide sequences shown as SEQ ID NO. 59-60; (34) Primer pairs with nucleotide sequences shown as SEQ ID NO. 61-62; (35) Primer pairs with nucleotide sequences shown as SEQ ID NO. 65-66; (36) A primer pair with a nucleotide sequence shown as SEQ ID NO. 69-70; (37) Primer pairs with nucleotide sequences shown as SEQ ID NO. 71-72; (38) Primer pairs with nucleotide sequences shown as SEQ ID NO. 73-74; (39) Primer pairs with nucleotide sequences shown as SEQ ID NO. 75-76; (40) Primer pairs with nucleotide sequences shown as SEQ ID NO. 77-78; (41) A primer pair with a nucleotide sequence shown as SEQ ID NO. 79-80; (42) Primer pairs with nucleotide sequences shown as SEQ ID NO. 81-82; (43) Primer pairs with nucleotide sequences shown as SEQ ID NO. 83-84; (44) Primer pairs with nucleotide sequences shown as SEQ ID NO. 85-86; (45) Primer pairs with nucleotide sequences shown as SEQ ID NO. 87-88; (46) Primer pairs with nucleotide sequences shown as SEQ ID NO. 89-90; (47) Primer pairs with nucleotide sequences shown as SEQ ID NO. 91-92; (48) Primer pairs with nucleotide sequences shown as SEQ ID NO. 93-94; (49) The nucleotide sequence is shown as a primer pair of SEQ ID NO. 97-98.
  4. 4. The EST-SSR molecular marker primer set according to claim 3, wherein the primer set is used for carrying out cluster analysis on Polygonatum plants, so that samples to be detected can be divided into groups corresponding to leaf sequence types, wherein the leaf sequence types comprise irregular leaf sequences, rotifer leaf sequences and inter-leaf sequences.
  5. 5. A kit for identifying plants of the genus polygonatum, characterized in that it comprises an EST-SSR molecular marker primer combination or primer set according to any one of claims 1-4.
  6. 6. A method for identifying a plant of the genus polygonatum, comprising the steps of: (1) Extracting genome DNA of a Polygonatum plant sample to be detected; (2) Performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the EST-SSR molecular marker primer combination or the primer set according to any one of claims 1 to 4; (3) Performing capillary electrophoresis detection on the amplified product in the step (2) to obtain fragment size data of the amplified product; (4) And (3) carrying out species identification and/or genetic diversity analysis on the Polygonatum plants according to the fragment size data in the step (3).
  7. 7. The method according to claim 6, wherein the PCR amplification in step (2) is performed by a procedure of 94℃for 5min, 94℃for 30s, 65℃to 55℃for 30s (1℃drop per cycle), 72℃for 40 s cycles, 94℃for 30s, 55℃for 30s, 72℃for 40 s cycles, 25 cycles, and 72℃for 7 min cycles.
  8. 8. The method of claim 6 or 7, wherein the species identification in step (4) comprises distinguishing at least two of polygonatum sibiricum p. sibiricum, polygonatum kingianum p. kingianum, polygonatum cyrtonema p. cyrtonema, polygonatum cyrtonema p.kingianum, polygonatum sibiricum p. cirrhifolium, and polygonatum hubei p. zanlanscianense.
  9. 9. The method according to claim 6 or 7, wherein the genetic diversity analysis in step (4) comprises analysis of intra-species variation of different geographical constellations of large She Dian sealwort.
  10. 10. Use of the EST-SSR molecular marker primer combination or primer set of any one of claims 1-4, or the kit of claim 5 in the identification of germplasm resources of a polygonatum plant, genetic diversity analysis, genetic map construction, or molecular marker assisted breeding.

Description

EST-SSR marker combination and application thereof in identification of Polygonatum plants Technical Field The invention belongs to the technical field of plant molecular biology, and particularly relates to an EST-SSR molecular marker primer combination developed based on a medicinal Polygonatum transcriptome sequence and application thereof in Polygonatum plant germplasm resource identification, genetic diversity analysis and molecular assisted breeding. Background Rhizoma Polygonati (Polygonati Rhizoma) is a traditional Chinese medicinal material with homology of medicine and food, and is derived from various plants of Polygonatum (Polygonatum) of Liliaceae, and mainly comprises rhizoma Polygonati (P. sibiricum), polygonatum kingianum (P. kingianum), polygonatum cyrtonema (P. cyrtonema), polygonatum grandiflorum (P. kingianum var. grandifolium), polygonatum sibiricum (P. cirrhifolium), etc. The Polygonatum plants are widely distributed, contain a plurality of active ingredients such as polysaccharide, saponin, flavonoid and the like, have the traditional effects of tonifying middle-jiao and Qi, strengthening spleen and moistening lung, tonifying kidney and the like, have modern pharmacological effects of reducing blood sugar, regulating immunity, resisting fatigue and the like, and have increasingly demanded market. However, the morphological identification characteristics of Polygonatum plants are complex, the phenotypic characteristics such as leaf sequence, rhizome morphology and the like are unstable, the inter-species overlap is serious, and the morphological identification is extremely easy to confuse especially in non-growing seasons or on the basis of the medical material morphology. The phenomenon of 'homonymy' or 'foreign matter homonymy' seriously hinders standardization of the sealwort industry and breeding of high-quality seed sources. Therefore, the development of molecular marker technology capable of accurately and efficiently identifying Polygonatum species and intraspecies variation thereof has important significance for germplasm resource protection, variety identification and genetic breeding work. Simple repeated sequence (Simple Sequence Repeat, SSR) markers have become ideal tools for genetic analysis because of the advantages of high polymorphism, co-dominance, good repeatability and the like. Wherein EST-SSR markers derived from expressed sequence tags are located in the gene transcription region, possibly associated with functional traits, and have higher versatility and conservation among closely related species. The existing SSR marking technology has the defects of limited marking quantity, incomplete species coverage, low intra-species variation resolution, lagged detection technology and the like, and is difficult to meet the requirements of precise identification, genetic diversity evaluation and molecular auxiliary breeding of Polygonatum germplasm resources. Particularly for species with abundant internal variation and important economic value, such as large She Dian rhizoma polygonati, a molecular marking tool capable of effectively distinguishing different geographic communities or excellent types is lacking. Disclosure of Invention Aiming at the technical problems, 49 pairs of high polymorphism EST-SSR markers are developed based on the rhizome transcriptome combined data of three medicinal species of Polygonatum cyrtonema, polygonatum cyrtonema and Polygonatum cyrtonema, and 9 pairs of core marker combinations are screened from the high polymorphism EST-SSR markers. The marker combination can accurately distinguish 6 closely related species of Polygonatum sibiricum, polygonatum kingianum, polygonatum cyrtonema, polygonatum sibiricum and Polygonatum hupehensis, effectively distinguish the intraspecies geographic population of Polygonatum sibiricum, and meanwhile, a fluorescent marker capillary electrophoresis detection technology is adopted, so that high-throughput, high-precision and automatic genotyping is realized, and a high-efficiency molecular tool is provided for researching the germplasm resources of Polygonatum. The inventor continuously reforms and innovates through long-term exploration and trial and repeated experiments and efforts, and in order to solve the technical problems, the technical scheme provided by the invention is that an EST-SSR molecular marker primer combination for identifying Polygonatum plants is provided, and the primer combination comprises at least one of the following 9 pairs of primers: (1) Primer pairs with nucleotide sequences shown as SEQ ID NO. 5-6; (2) Primer pairs with nucleotide sequences shown as SEQ ID NO. 9-10; (3) Primer pairs with nucleotide sequences shown as SEQ ID NO. 53-54; (4) Primer pairs with nucleotide sequences shown as SEQ ID NO. 3-4; (5) Primer pairs with nucleotide sequences shown as SEQ ID NO. 7-8; (6) Primer pairs with nucleotide sequences shown as SEQ ID NO. 63-64; (7) Primer pairs with nucleotide sequence