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DNA (deoxyribonucleic acid) bar code and application of bar code to identification of muscadine

A muscadine grape and barcode technology, which is applied in the field of identification kits for muscadine grapes, can solve problems such as the lack of ITS sequence information of muscadine grapes, and achieve the effects of short identification time, fast evolution rate, and good repeatability

Active Publication Date: 2018-03-09
NANJING XIAOZHUANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • DNA (deoxyribonucleic acid) bar code and application of bar code to identification of muscadine
  • DNA (deoxyribonucleic acid) bar code and application of bar code to identification of muscadine
  • DNA (deoxyribonucleic acid) bar code and application of bar code to identification of muscadine

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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 (deoxyribonucleic acid) bar code. The nucleotide sequence of the DNA bar code is as shown in SEQ ID NO.1. According to the DNA bar code, an ITS sequence of a muscadine isfirstly disclosed, available molecular markers are provided for species identification of the muscadine, and the DNA bar code further comprises parts of conserved sequences and has universality whenbeing used for species identification of grapes. The invention discloses a method and a primer for preparing the nucleotide sequence of the DNA bar code and can acquire sequences of an ITS area of themuscadine. The invention further discloses a molecular identification method of the muscadine and a detection primer and a kit for identifying the muscadine. PCR (polymerase chain reaction) amplification is performed by the aid of the detection primer or the kit to obtain sequences of ITS areas of the grapes, an amplification sequence and the sequence of the DNA bar code of the muscadine are compared or applied to construction of phylogenetic trees of the grapes, so that grape varieties are accurately identified, and identifying processes are simple to operate, high in efficiency and good inrepeatability.

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 Applications(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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