Method for screening DNA bar code universal sequences of seven major forage grass varieties of gramineous family

Abstract

The invention discloses a method for screening NA bar code universal sequences of seven major forage grass varieties of gramineous family. The method comprises the following steps: (1) firstly, in accordance with nucleotide sequences of gramineous forage grass matk and rbcl genes in GenBank, designing four pairs of universal primers, establishing amplification conditions for target segments of 11 samples of eight forage grasses of seven major forage grass varieties of the gramineous family and conducting amplification; (2) sequencing and analyzing an amplification product, and conducting comparison, screening out 5'-end and 3'-end conserved sequences of four marker sites of the seven major forage grass varieties, and conducting single nucleotide polymorphism haplotype analysis on nucleotide within the conserved regions of the various marker sites, knowing that matk1 has six haplotypes, matk2 has seven haplotypes, matk3 has three haplotypes and the rbcl gene has five haplotypes; and (3) in accordance with four marker points screened from the matk and rbcl genes, establishing specific DNA identification codes corresponding to the eight forage grasses. The method disclosed by the invention is good in primer university and ideal in amplification effect, and meanwhile, the method is high in species identification rate.

Description

technical field [0001] The invention relates to a screening method for the universal sequence of the DNA barcodes of the genus Forage, in particular to a screening method for the universal sequence of the DNA barcodes of the seven major genera of the Gramineae family. Background technique [0002] DNA barcoding (DNA barcoding) technology, also known as DNA barcoding, is often used for species identification due to its rapidity, simplicity, and accuracy. A standard DNA fragment (DNA barcode) is used to construct a biometric database. In the study of 11 different phyla of animals, Hebert et al. found that the interspecific variation of COI (cytochrome coxi-dase subunit 1) gene sequence can better distinguish species, while the variation of COI or other mitochondrial genes in plants is low , not suitable for identification of plant species. According to reports, although the chloroplast genes in plants are relatively conservative, their uniparental inheritance avoids gene rec...

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Problems solved by technology

Relevant studies believe that matK has a fast evolution rate and good inter-species discrimination ability, but the main defect is the poor versatility of the primers. Fu et al. concluded from the experiment on the genus of Vitis vinifera that the success rate of matK amplification reached 93.5%. , the amplification effect is ideal, but when matk is used alone for identification, the species identification rate is not high, only 22.2%

The rbcl sequence has the characteristics of being universal, easy to amplify, and easy to compare, but its variation mainly exists at the level above the species level, and the variation at the species level is usually not large enough

[0004] As the main forage source of livestock, gramineous forages have a wide distribution, including 30 genera with great feeding significance, and most of them are preferred by livestock. However, the application of DNA barcoding technology in forage identification is relatively rare. Therefore, if we can establish the The DNA barcodes of seven genus forage species in this undergraduate course can provide theoretical basis and method reference for further species identification

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