MNP marker site, primer composition and kit for mango variety identification and application of MNP marker site, primer composition and kit

A primer composition and marker site technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve problems such as backwardness, and achieve high discrimination, high throughput, and high accuracy. Effect

Active Publication Date: 2022-05-13
JIANGHAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, our country lags behind in the protection of intellectual property rights in the seed industry, especially tropical crops are mostly long-term cultivated crops (trees, shrubs). According to the conventional DUS test method, a new variety will take 3 to 5 years or even longer from the application to the grant of the new variety right. For a long time, most tropical crops have not applied modern biotechnology such as molecular markers and DNA fingerprinting to the testing of new varieties of tropical crops

Method used

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  • MNP marker site, primer composition and kit for mango variety identification and application of MNP marker site, primer composition and kit
  • MNP marker site, primer composition and kit for mango variety identification and application of MNP marker site, primer composition and kit
  • MNP marker site, primer composition and kit for mango variety identification and application of MNP marker site, primer composition and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Screening of MNP marker loci for identification of mango varieties and design of multiplex PCR amplification primers

[0038] S1. Screening of MNP marker loci for identification of mango varieties

[0039] Genome sequencing was performed on 53 mango varieties collected, including Guifei Mango, Kate Mango, Three Years Mango, and Tommy Mango. Each sample obtained more than 20 times the coverage of the sequencing data. The publicly released mango genome sequence GCA_011075055.1 was used as the reference genome , first use Samtools (Version 1.2) and BCFtools (Version: 1.2) for sequence analysis to obtain the SNP sites on the mango genome, and compare and analyze with the NT library of NCBI, and carry out the screening of MNP markers according to the following principles: (1) markers The sequence is only unique to mango and does not appear in other species; (2) The sequence has a single copy in the genome; (3) There are at least three or more discontinuous SNP dif...

Embodiment 2

[0057] Example 2, Evaluation of MNP markers, primer compositions and kits for mango variety identification

[0058] After 654 pairs of primers were synthesized, an equal amount of 5ul per primer was mixed to form a 1:1 equal amount primer mix of F and R-terminal primers. The 60 mango varieties collected by the unit were used to evaluate the developed MNP markers, primers and kits to test the detection rate, accuracy and discrimination of MNP marker loci.

[0059] (1) MNP marker detection rate

[0060] According to the kit of the present invention, multiple PCR amplification and construction of sequencing library were carried out, and these 60 mango DNA samples were subjected to multiple amplification, second-generation high-throughput sequencing and data analysis, and one experiment reached 654×60= 39,240 markers were detected, and the average sequencing coverage of each sample was more than 700 times, showing the high efficiency of MNP marker detection.

[0061] The distrib...

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Abstract

The invention discloses an MNP marker site, a primer composition and a kit for mango variety identification and application of the MNP marker site, the primer composition and the kit, and the MNP marker site is a genome region which is screened on a mango genome and has a plurality of nucleotide polymorphisms in a mango population, and comprises marker sites of MNP-1-MNP-654 on the mango genome GCA011075055.1. The primers are as shown in SEQ ID NO. 1 to SEQ ID NO. 1308. The invention also discloses a kit for detecting the content of the primers The MNP marker is large in number of sites, high in polymorphism and strong in variety distinguishing ability, and meets the requirements of mango variety authenticity identification and substantive derived variety identification; the primers do not interfere with one another, the identification accuracy is high, the result reproducibility is high, and the requirement for DNA fingerprint database construction is met; the method can be applied to large-scale mango variety identification, and has great application value in the aspects of market supervision, intellectual property protection, new variety cultivation and the like of crop products.

Description

technical field [0001] The embodiment of the present invention relates to the field of biotechnology, in particular to a MNP marker site, a primer composition, a kit and applications thereof for identification of mango varieties. Background technique [0002] Mango (Mangifera indica L.), known as the "King of Tropical Fruits", is a famous tropical fruit in the world. According to the statistics of the World Food and Agriculture Organization (FAO), the actual harvested area and output of mango in the world in 2016 were 96.166 million mu and 51.279 million tons; mango is also one of the important tropical fruits in my country, ranking fourth in area after bananas, lychees and longan. According to the data from the South Asia Office of the Ministry of Agriculture, the national mango planting area reached 4.842 million mu in 2019. The output is 2.782 million tons, and the output value is 19.03 billion yuan. However, our country lags behind in the protection of intellectual proper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/122C12Q2537/165Y02A50/30
Inventor 彭海陈亚渊李甜甜高爱平黄建峰李琼何云方治伟周俊飞
Owner JIANGHAN UNIVERSITY
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