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Method for building an ISSR (inter-simple sequence repeat) fingerprint applicable to distinguishing and identification of Ulmus varieties

A fingerprint and variety differentiation technology, applied in the field of ISSR fingerprint construction, can solve the problems of high cost, seasonal restrictions, long identification cycle, etc., and achieve the effect of simple operation and stable results.

Inactive Publication Date: 2012-07-25
DAQING BRANCH OF HEILONGJIANG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are about 88 representative Ulmus species, varieties and types distributed in China, as well as selected excellent clone varieties, but the identification of these varieties is still based on the traditional morphological identification method and relies on the phenotypic differences of varieties , and the performance of the morphology is greatly affected by the environment, and the workload of the morphology identification method is large, the identification period is long, the cost is high, and it is limited by the season
However, the polymorphisms of isozymes and seed storage proteins are not abundant enough, and the isozymes have tissue and organ specificity and poor stability, which brings difficulties to the identification of Ulmus species and the innovation of Ulmus germplasm resources

Method used

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  • Method for building an ISSR (inter-simple sequence repeat) fingerprint applicable to distinguishing and identification of Ulmus varieties
  • Method for building an ISSR (inter-simple sequence repeat) fingerprint applicable to distinguishing and identification of Ulmus varieties
  • Method for building an ISSR (inter-simple sequence repeat) fingerprint applicable to distinguishing and identification of Ulmus varieties

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Experimental program
Comparison scheme
Effect test

specific Embodiment approach 1

[0030] Embodiment one: the ISSR fingerprint spectrum construction method suitable for Ulmus species distinction and identification is carried out in the following steps:

[0031] 1. DNA extraction:

[0032] Using adult plants of different elm species as materials, take new leaves and use the modified CTAB method suitable for extracting elm DNA to extract DNA, take part of the DNA solution to measure its concentration, dilute to 20ng / uL, and store it at -20°C for later use;

[0033] 2. ISSR-PCR amplification and electrophoresis detection:

[0034] In 25μL reaction system, 10×Buffer (Mg 2+ free) 2.5μL, dNTPs 0.25mmol / L, primer 800pmol / L, DNA template 30ng, Taq enzyme 1.5U, MgCl 2 2.5mmol / L;

[0035] The PCR amplification program conditions are: pre-denaturation at 94°C for 5 minutes, 35 cycles, each cycle includes denaturation at 94°C for 45 seconds, annealing for 1 minute, extension at 72°C for 1.5 minutes, extension at 72°C for 6 minutes after the cycle, and storage at 4°C...

specific Embodiment approach 2

[0056] Specific embodiment two: what this embodiment is different from specific embodiment one is that the concrete kind of elm used in the step 3 is as follows:

[0057]

[0058] Mapping varieties 1 to 9 were collected from Maoer Mountain and Heilongjiang Forest Botanical Garden in Heilongjiang Province; mapping varieties 10 to 18 and identification varieties 1 to 17 were collected from Baiwa Forest Farm in Jinxiang County, Shandong Province. Others are the same as in the first embodiment.

[0059] The elms in this embodiment can be obtained by purchasing.

Embodiment

[0061] A kind of ISSR fingerprint spectrum construction method suitable for Ulmus species distinction and identification is carried out as follows:

[0062] 1. DNA extraction:

[0063] Ulmus species collected in Heilongjiang Province and Shandong Province, a total of 35 adult plants of Ulmus species were used as materials, and DNA was extracted from new leaves using the improved CTAB method suitable for extracting Ulmus DNA, and part of the DNA solution was taken to measure its concentration, and diluted to 20ng / uL , stored at -20°C for later use;

[0064] 2. ISSR-PCR amplification and electrophoresis detection:

[0065] In 25μL reaction system, 10×Buffer (Mg 2+ free) 2.5μL, dNTPs 0.25mmol / L, primer 800pmol / L, DNA template 30ng, Taq enzyme 1.5U, MgCl 2 2.5mmol / L;

[0066] The PCR amplification program conditions are: pre-denaturation at 94°C for 5 minutes, 35 cycles, each cycle includes denaturation at 94°C for 45 seconds, annealing for 1 minute, extension at 72°C for 1.5...

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Abstract

A method for building an ISSR (inter-simple sequence repeat) fingerprint applicable to distinguishing and identification of Ulmus varieties relates to a method for building an ISSR fingerprint, and includes: firstly, extracting DNA; secondly, performing ISSR-PCR (ISSR-polymerase chain reaction) amplification and electrophoresis; thirdly, building a DNA fingerprint; and fourthly, using the built DNA fingerprint to identify Ulmus varieties. The method is convenient for quick and accurate identification of Ulmus varieties. Compared with conventional planting test and biochemical test, the method is insusceptible to crop growth environment and season, and accordingly the results are more stable and reliable, and time and labor are economized. DNA fingerprint building and the identification technology lay a solid basis for wide application of ISSR molecule marker technology to plant variety identification, relationship analysis, plant genetic source innovation and molecular marker assisted selection. The method has the advantages of simplicity in operation, visuality, practicality and the like.

Description

technical field [0001] The invention relates to an ISSR fingerprint spectrum construction method. Background technique [0002] Elm is the general term for plants of the genus Ulmus in the family Ulmae of the family Ulmaceae. There are more than 40 species in the world, distributed in the northern hemisphere. my country's elm is very rich in resources, all over the country, a total of 25 species. Ulmus plants are resistant to drought, salt and alkali, strong adaptability, tough wood, high ecological adaptability and economic value. Most species of Ulmus have strong plasticity. They can be used as solitary trees, street trees, and can also be cut into hedgerows, Bonsai has high ornamental value and application value. Therefore, it has irreplaceable advantages of other tree species and has always been the main tree species in northern my country, especially in saline-alkali landscaping. Therefore, the cultivation of new varieties of Ulmus has always been a research hotspot f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张兴王云云熊燕肖宇高宇孙力关向军唐焕伟
Owner DAQING BRANCH OF HEILONGJIANG ACAD OF SCI
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