A dna barcode and its use in identifying muscadine grapes

A muscadine and bar code technology is applied in the field of kits for identifying muscadines, can solve problems such as lack of ITS sequence information of muscadines, and achieve the effects of short identification time, good versatility and high accuracy

Active Publication Date: 2021-01-26
NANJING XIAOZHUANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] For this reason, the technical problem to be solved in the present invention is to solve the lack of ITS sequence information of muscadine grape in the prior art, and it is impossible to use barcode technology to realize the species identification of muscadine grape; thereby provide a kind of DNA barcode, which includes circular The ITS sequence of Vitis vinifera can realize rapid and accurate species identification of Vitis vinifera

Method used

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  • A dna barcode and its use in identifying muscadine grapes
  • A dna barcode and its use in identifying muscadine grapes
  • A dna barcode and its use in identifying muscadine grapes

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Embodiment 1

[0058] This embodiment provides a method for preparing a nucleotide sequence of a DNA barcode, which specifically includes the following steps:

[0059] 1. Design primers

[0060] Using GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) to obtain the ITS sequences of 41 different grape varieties in the genus Vitis, the ITS sequence information of each grape variety is shown in Table 1. These sequences were compared and arranged with the sub-software SeqMan of DNAStar software, so as to obtain the conserved bases and changed bases of the partial or complete sequences of 18S, ITS1, 5.8S, ITS2 and 28S of each grape variety. Sequence design 2 pairs of primers Itc-5, Itc-3 and Itb-5, Itb-3. Among them, primer Itb-5 is designed based on the conserved sequence of 5.8s rDNA, the nucleotide sequence of primer Itb-5 is shown in SEQ ID NO.2, Itb-3 is designed based on the conserved sequence of 28s rDNA, and the nucleotide sequence of primer Itb-3 is The nucleotide sequence is shown in SEQ...

Embodiment 2

[0086] This embodiment provides a molecular identification method of muscadine grape, and constructing a phylogenetic tree based on the nucleotide sequence of the DNA barcode, which specifically includes the following steps:

[0087] 1. Take muscadine Nobel, muscadine Gewell and muscadine Nesbitt respectively as the grape samples to be tested (online purchase from China Guangdong and China Guangxi), according to the extraction method of genomic DNA shown in Example 1 , to extract genomic DNA from three species of muscadine grapes.

[0088] 2. Using the genomic DNA extracted in step 1 as a template, PCR amplification was performed using detection primers Itd-5 and Itd-3, and Ite-5 and Ite-3, respectively. The nucleotide sequence of the detection primer Itd-5 is shown in SEQ ID NO.6, the nucleotide sequence of Itd-3 is shown in SEQ ID NO.7, and the nucleotide sequence of Ite-5 is shown in SEQ ID NO.8 As shown, the nucleotide sequence of Ite-3 is shown in SEQ ID NO.9. The react...

Embodiment 3

[0100] This embodiment provides a kind of kit for identifying muscadine grape, and kit comprises:

[0101] (1) The detection primer pair Itd-5 and Itd-3, or Ite-5 and Ite-3 for amplifying the sequence of grape ITS2 region. The nucleotide sequence of the detection primer Itd-5 is shown in SEQ ID NO.6, the nucleotide sequence of Itd-3 is shown in SEQ ID NO.7, and the nucleotide sequence of Ite-5 is shown in SEQ ID NO.8 As shown, the nucleotide sequence of Ite-3 is shown in SEQ ID NO.9;

[0102] The above-mentioned test kit also includes:

[0103] (2) The reaction system of PCR amplification, each reaction solution in the reaction system is as shown in table 2;

[0104] (3) The DNA barcode standard product of Muscadine grape, the DNA barcode standard product is the pMD18-T vector (pMD18-T purchased from Takara) connected with the sequence shown in SEQ ID NO.1, or a genetically engineered bacterium comprising the above-mentioned vector;

[0105] The kit provided by the present ...

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Abstract

The invention discloses a DNA barcode, the nucleotide sequence of which is shown in SEQ ID NO.1. The DNA barcode disclosed the ITS sequence of Muscadine grape for the first time, which provided an effective molecular marker for the species identification of Muscadine grape. In addition, the DNA barcode also included some conserved sequences, which is universal when applied to the species identification of grapevine. The invention discloses a method for preparing the nucleotide sequence of the DNA barcode and primers, which can be used to obtain the sequence of the ITS region of muscadine grapes. The invention also discloses a molecular identification method of muscadine grapes, detection primers and a kit for identifying muscadine grapes, using the detection primers or kits to carry out PCR amplification to obtain the sequence of the grape ITS2 region, and then combining the amplified sequence with the muscadine The DNA barcode sequences of grapes are compared or applied to construct a phylogenetic tree of grapes to achieve accurate identification of grape varieties, and the identification process is simple, efficient and repeatable.

Description

technical field [0001] The invention belongs to the technical field of species identification, and in particular relates to a DNA barcode, the application of the DNA barcode and a preparation method thereof, a detection primer designed based on the same, a molecular identification method for muscadine grapes, and a kit for identifying muscadine grapes. Background technique [0002] Muscadine grape (Vitis rotundifolia) is native to the southeastern United States and has been commercially grown since the middle of the 18th century. The colors of its varieties are green, copper, red and black, etc. The fruit has a special aroma and flavor and can be eaten fresh , and can be processed into fruit juice, preserved fruit, jam and wine. Muscadine grape is a special grape species with one more pair of chromosomes than other species of Vitis genus. Now it is mainly planted in the United States, with a small amount introduced in China. In recent years, it has been planted on a certain ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 蔡小宁陈守江杜春望陈璐璐钱荣荣葛腊梅赵宇飞王薇
Owner NANJING XIAOZHUANG UNIV
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