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A method for identification of hazelnut germplasm using nuclear its sequence

A germplasm and sequence technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as strong randomness, poor stability, limited sites, and high cost

Inactive Publication Date: 2020-06-02
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SNP marker information sites are limited, expensive, etc.
Most of the above-mentioned markers are developed based on technologies without full genome sequence information, which have certain application value, but are highly random and less stable

Method used

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  • A method for identification of hazelnut germplasm using nuclear its sequence
  • A method for identification of hazelnut germplasm using nuclear its sequence
  • A method for identification of hazelnut germplasm using nuclear its sequence

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specific Embodiment approach

[0023] The method of using the nuclear ITS sequence of the present invention to carry out the identification of hazel germplasm, its preferred embodiment is:

[0024] Amplification primers including a pair of Hazel ITS sequences:

[0025] Forward primer: 5'-AGTCGTAACAAGGTTTCCGT-3';

[0026] Reverse primer: 5'-TCCTCCGCTTATTTATATGC-3';

[0027] The method for carrying out the germplasm identification of hazelnut through the amplification primers of the above-mentioned pair of hazel ITS sequences, comprising the following steps:

[0028] Use forward primer and reverse primer to carry out PCR amplification on standard germplasm;

[0029] Use forward primer and reverse primer to carry out PCR amplification of the target germplasm to be identified;

[0030] The amplified sequences obtained in the above two steps were compared, and the hazel germplasm identification was carried out according to whether the comparison results were consistent.

[0031] Before using the above-mentio...

no. 1 example

[0054] The first embodiment, the acquisition of Corylus plant material.

[0055] According to the actual survey, 14 Corylus plant materials were collected from different regions in my country, and the information of the 14 standard germplasm materials is shown in Table 1. A part of Corylus plant material A to be identified was collected from Taibai County, Shaanxi Province. The Qinling Mountains in Shaanxi Province are located in the cross-distribution area of ​​Chuanzhen and Pingzhen. According to literature records, both Pingzhen and Chuanzhen are distributed in this area. According to traditional morphology, it is impossible to accurately identify which kind of Corylus the material to be identified is. In addition, there is another material collected from Chifeng, Inner Mongolia, whose shape is similar to that of hazel and tiger hazel, which is material B to be identified.

[0056] Table 1 Specific information of 14 Corylus plant materials

[0057]

no. 2 example

[0058] The second embodiment, the extraction of Corylus plant material genomic DNA

[0059] The optimized CTAB method was used to extract the whole genome DNA from the above 16 material samples, the specific method is as follows:

[0060] Step 201, preheating 2% CTAB extract and 2% β-mercaptoethanol in a 65°C water bath;

[0061] Step 202, take 0.2 g of leaves, add liquid nitrogen to fully grind, then quickly transfer to a 2 mL centrifuge tube, add 1 ml of preheated CTAB extract, bathe in 65 °C water for 60 min, and mix gently by inversion every 10 min;

[0062] Step 203, after the water bath is over, take out the centrifuge tube and cool to room temperature; centrifuge at 13000 rpm for 5 minutes, take 800 μL of the supernatant and transfer it to a new centrifuge tube;

[0063] Step 204, add 800 μL of chloroform / isoamyl alcohol (V:V=24:1), mix thoroughly, centrifuge at 13000 rpm for 5 min, and transfer the supernatant into a new 2 mL centrifuge tube;

[0064] Step 205, repea...

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Abstract

The invention discloses a method for identifying hazelnut germplasm through a nuclear ITS sequence. The method utilizes a pair of amplification primers of a hazelnut ITS sequence. The amplification primers comprise a forward primer of 5'-AGTCGTAACAAGGTTTCCGT-3' and a reverse primer of 5'-TCCTCCGCTTATTTATATGC-3'. Through the forward and reverse primers, a standard germplasm is subjected to PCR amplification, through the forward and reverse primers, a target germplasm to be identified is subjected to PCR amplification, the amplification sequences obtained by the above two steps are compared, and hazelnut germplasm is identified according to the comparison result. The ITS sequence used by the method has a strong identifying capability and good repeatability, can effectively identify easily-confused germplasm resources or plants belonging to other species, provides an important technical support for specie classification, genetic relationship analysis and phylogenetic analysis of hazelnut plant resources and has a great meaning for hazelnut plant wild resource investigation and screening and crossbreeding.

Description

technical field [0001] The invention relates to a method for germplasm identification of hazelnut, in particular to a method for germplasm identification of hazelnut by using nuclear ITS sequence. Background technique [0002] Hazel is a plant of the genus Corylus in the family Corylaceae, and these hazel species include both tree types and shrub types (Zhang Yuhe et al., 2005). There are about 20 species of Corylus in the world, which are widely distributed in the temperate regions of Asia, Europe, and North America in the northern hemisphere. Corylus plant resources are abundant in my country. There are 8 species and 2 varieties naturally distributed in China. The distribution range is also very wide and the economic utilization value is high. [0003] Since the 1950s, the classification of Corylus plants in my country is still based entirely on morphological characteristics. Different taxonomists have different classification results due to different morphological scales...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895
CPCC12Q1/6895C12Q2600/156
Inventor 赵天田王贵禧马庆华梁丽松
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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