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
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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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