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Construction method of ophiopogon japonicus AFLP molecular fingerprint spectrum library and application of construction method

A technology of molecular fingerprints and construction methods, which is applied in the field of construction of AFLP molecular fingerprint databases, can solve the problems of undiscovered and undiscovered AFLP molecular fingerprint database methods and applications, and improve reliability , shorten the detection cycle, the effect of high resolution

Pending Publication Date: 2020-12-15
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there have been no reports on the molecular detection and identification of the germplasm of Herba genus using AFLP molecular marker technology at home and abroad, let alone the use of the optimized 9 pairs of AFLP primer combinations to construct the AFLP molecular fingerprint of Herba genus Reports on library methods and applications

Method used

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  • Construction method of ophiopogon japonicus AFLP molecular fingerprint spectrum library and application of construction method
  • Construction method of ophiopogon japonicus AFLP molecular fingerprint spectrum library and application of construction method
  • Construction method of ophiopogon japonicus AFLP molecular fingerprint spectrum library and application of construction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Screening of test germplasm and extraction of genomic DNA: The present invention screened 30 germplasms from the planted potted grass germplasm resource garden, of which 19 were collected from different habitats in China, and 11 were cell-induced mutants. The selection principle is that the appearance of the germplasm varies from large to small and difficult to distinguish with naked eyes. During the growing season, fresh young leaves were cut and bagged and brought back to the laboratory for genomic DNA extraction.

[0037] 1. See Table 1 for the number and name of the tested germplasm:

[0038] Table 1: No. and name of germplasm samples of C.

[0039]

[0040]

[0041] 2. Extraction of Genomic DNA of different germplasms:

[0042] The modified CTAB method is preferably used:

[0043] (1) Weigh 50 mg of fresh young and tender Herba pratense leaves and grind them in liquid nitrogen (with 4% PVP) for 3-4 times until powdered;

[0044] (2) Put the powder into a ...

Embodiment 2

[0058] Establishment and optimization of the AFLP reaction system of Cinnamon grass: the corresponding bands or fingerprints of different germplasms were obtained through enzyme digestion, ligation, pre-amplification, selective amplification, electrophoresis, etc., among which the same bands were considered to have the same For the genetic loci, bands with different sizes are considered to have different genetic loci.

[0059] 1. Restriction enzyme digestion and ligation of genomic DNA

[0060] It is preferably carried out by one-step enzyme cleavage and ligation, and the steps of the one-step enzyme cleavage and ligation are as follows:

[0061] (1) First determine the base sequence of the linker used for AFLP analysis, preferably the base sequence of the linker is shown in Table 1.

[0062] Table 1: Base sequence of linker

[0063] EcoR I Adapter 1 5'-CTC GTA GAC TGC GTA CC-3' EcoR I Adapter2 5'-AAT TGG TAC GCA GTC TAC-3' Mse I Adapter 1 5'-GAC GA...

Embodiment 3

[0103] Spectrum analysis of amplification products: After the 30 germplasm selective amplification products obtained in the above steps were subjected to denaturing polyacrylamide gel electrophoresis and silver staining, they were scanned on an ABI 377 sequencer to obtain an AFLP gel electrophoresis separation spectrum library, which was separated by GENESCAN3 .1 Software analysis results, and the size of each fragment of the sample is extracted by Binthere software. After automatic statistical analysis by the software, the electrophoretic bands were recorded according to the method of recording the amplified band at the same migration site as 1, and the absence of the band as 0, and converted it into a "0, 1" original data matrix for statistical analysis of genetic variation information and relatives. Relational polymorphism map analysis. Specific steps are as follows:

[0104] (1) Denaturing polyacrylamide gel electrophoresis steps:

[0105] ①Preferred denaturing solution ...

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Abstract

The invention discloses a primer pair combination, and further discloses application of the primer pair combination in ophiopogon japonicus germplasm identification or variety identification or construction of an AFLP molecular fingerprint spectrum library. The invention further discloses a construction method and application of the ophiopogon japonicus AFLP molecular fingerprint spectrum library.The selective amplification primers and primer combinations capable of effectively identifying different germplasms of ophiopogon japonicus and the high-quality DNA fingerprint spectrum are successfully screened through repeated unloosened tests, so that the method has the advantages of multiple amplification sites, good band type quality, high resolution, proper band density, uniform distribution and easiness in accurately detecting the subtle difference of samples, so that a technical foundation is laid for constructing the ophiopogon japonicus AFLP molecular fingerprint spectrum library; and 30 ophiopogon japonicus with different germplasms can be successfully and effectively identified by nine pairs of primer combinations and the like through a provided reagent method, and a scientific basis is provided for ophiopogon japonicus germplasm identification, new variety registration, registration, property right protection and the like in the future.

Description

technical field [0001] The invention belongs to the field of plant molecular breeding, and relates to a construction method and application of a AFLP molecular fingerprint library of A. Identification techniques for germplasm identification or variety identification and variety rights protection through genetic markers. Background technique [0002] Ophiopogon japonicus, also known as Ophiopogon japonicus, belongs to Liliaceae Ophiopogon japonicus. It is a perennial evergreen grass ground cover or landscape plant. It is also a traditional Chinese medicine. The traditional Chinese medicinal material of lung and heart is an important native plant unique to my country, and it is also distributed in Japan, Vietnam, North Korea and India abroad. Its wild germplasm resources are very rich and diverse; in recent years, the development and utilization of folks have also been vigorously developed, and the germplasm confusion caused by this is also very serious; The discovery and uti...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/156
Inventor 梁慧敏李晨龙梁栋刘艳白玉梅夏阳袁诗宇翟云菲
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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