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A method for identification of persimmon germplasm using cpdna molecular markers

A molecular marker, germplasm technology, applied in recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of strong randomness, poor stability, high cost, and achieve good reproducibility and high efficiency. , the effect of simple operation

Active Publication Date: 2019-04-23
CHINA PAULOWNIA RES CENT
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Problems solved by technology

[0004] It can be seen that the types of persimmon resources in my country are recorded differently, the statistics of persimmons in local flora and national flora are not consistent, and the classification status of many persimmon resources is not clear, which has great impact on the collection of persimmon resources in my country. , preservation and utilization have a certain impact, which in turn affects the development of hybrid breeding of persimmons
[0005] At present, the identification and classification of plant resources are mainly based on morphology and molecular biology. The traditional morphological identification method relies on phenotypic differences, but the morphological traits of phenotypes are affected by various factors such as climate, environment, nutritional status, and biological period. Moreover, the morphological identification method has a large workload, a long identification cycle, and high costs.
In the identification and classification of woody plants, molecular marker technologies such as RAPD, SSR, and AFLP are mostly used, but most of these markers are developed by technologies without complete genome sequence information. Although they have certain application value, they are highly random and poor stability
Moreover, due to the complex ploidy of Persimmon resources, the structure and sequence of the nuclear genome are also complex and changeable. Compared with nuclear genes, the structure and sequence of the chloroplast genome are very conserved, but so far no research on the use of cpDNA molecular markers for persimmon resources has been found. A report on the study of phylogenetic and genetic relationship among species

Method used

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  • A method for identification of persimmon germplasm using cpdna molecular markers
  • A method for identification of persimmon germplasm using cpdna molecular markers
  • A method for identification of persimmon germplasm using cpdna molecular markers

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no. 1 example

[0034] The first embodiment, the acquisition of persimmon plant material

[0035] Fifteen plant materials of the genus Persimmon were collected from the Persimmon Germplasm Resource Garden of the Economic Forest Research and Development Center of the Chinese Academy of Forestry, and one plant material of the genus Persimmon to be identified—Yunnan Yemao Persimmon (scientific name D.spp.) was collected from the Xishuangbanna Botanical Garden of the Chinese Academy of Sciences. The current literature records and traditional morphological taxonomy cannot identify Yunnan Yemao persimmon as a persimmon species or other persimmon species. The specific information of 15 samples from the Chinese Academy of Forestry is shown in Table 1.

[0036] Table 1. Specific information of 15 persimmon plant materials

[0037]

no. 2 example

[0038] The second embodiment, the extraction of the plant material genome DNA of the genus Persimmon

[0039] Chloroplast genome-wide DNA (cpDNA) was extracted from the leaves of the above-mentioned 16 persimmon plant material samples by the improved CTAB method. The specific method is as follows:

[0040] Step 201, add 800 μl of CTAB DNA extraction buffer in a 1.5ml centrifuge tube, the extraction buffer includes 100mM Tris-HCl (pH 8.0), 1.4M NaCl and 20mM EDTA (pH 8.0), and then add 30 μl of β-mercaptoethanol, After mixing, place in a 65°C water bath to preheat;

[0041] Step 202, take 0.3g leaves, add liquid nitrogen to fully grind to powder, quickly transfer to the centrifuge tube in step 201, crack in a water bath at 65°C for 2 hours, and shake and mix 3-5 times during the period;

[0042]Step 203, after the water bath is over, take out the centrifuge tube to cool down, and centrifuge at room temperature 12000rpm for 12min;

[0043] Step 204. After centrifugation, trans...

no. 3 example

[0051] The third embodiment, screening of primers

[0052] 1. Determine the target gene fragment

[0053] Select the total chloroplast DNA samples of representative persimmon varieties (Mopan persimmon) and other persimmon plants (Jinzao persimmon, Junqianzi, Zhejiang persimmon and oil persimmon);

[0054] Sequence the cpDNA of the above five materials of the genus Persimmon, and determine an insertion / deletion target gene fragment greater than 20 bp through multiple sequence alignment;

[0055] 2. Screening primers

[0056] Primers were designed according to the above-mentioned target gene fragments, and PCR amplification was carried out according to the following system (as shown in Table 2) and conditions:

[0057] Table 2. PCR reaction system

[0058]

[0059] The PCR reaction conditions are:

[0060]

[0061] The PCR products obtained by the above method were detected by electrophoresis on 2.0% agarose gel stained with Gelred. The bands were observed and record...

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Abstract

The invention relates to a method for identifying persimmon germplasm by using a cpDNA (chloroplast deoxyribonucleic acid) molecular marker. The amplification primer of the persimmon cpDNA molecular marker is disclosed as SEQ ID NO:1 or SEQ ID NO:2. The method comprises the following steps: carrying out PCR (polymerase chain reaction) amplification on standard germplasm by using the molecular marker primers; carrying out PCR amplification on the target germplasm by using the molecular marker primers, wherein the target germplasm is a germplasm to be identified; comparing the amplification results obtained in the two steps above, and judging whether the comparison results are consistent to carry out germplasm identification, wherein the forward primer and reverse primer of the molecular marker are respectively disclosed as SEQ ID NO:1 and SEQ ID NO:2. The method is simple to operate, has the advantages of favorable reproducibility and high efficiency, and can effectively identify whether the unknown material is a persimmon species or other Diospyros plants.

Description

technical field [0001] The invention relates to a method for identifying persimmon germplasm, in particular to a method for identifying persimmon germplasm by using cpDNA molecular markers. Background technique [0002] There are about 190 species of persimmon (Diospyros spp.) plants in the world, including trees, shrubs, deciduous and evergreen, most of which are distributed in the tropics and subtropics. Our country is rich in persimmon plant resources, with high economic value, mainly distributed in Southwest and South China. [0003] According to Zuo Daxun (1984) etc., there are 58 species and varieties (types) of Persimmon in my country (excluding species introduced from abroad), including 27 endemic species. According to the Chinese version of "Flora of China" (1987), there are 64 species and varieties (types) in my country, including 57 species, 6 varieties, 1 variant, and 1 cultivated species. Wang Renzi (1988) reported that there are 66 species of persimmons in my...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 傅建敏乌云塔娜韩卫娟孙鹏刁松锋张嘉嘉罗颖张悦
Owner CHINA PAULOWNIA RES CENT
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