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Single nucleotide polymorphism marker site, primer and kit for identifying coloring property of peach epidermis and application

A technology of single nucleotide polymorphism and marking sites, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problem of low accuracy, inability to guarantee SNPs, and target gene positioning distance Distant and other problems, to achieve the effect of high accuracy

Active Publication Date: 2016-09-07
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0005] However, the existing SSR markers linked to peach fruit skin coloring traits are far away from the target gene location, and the accuracy rate is low in the early identification of hybrid offspring; while the existing SNPs markers linked to target traits are mostly from microarray identification Due to the small number of chip sites (less than 9000), it cannot be guaranteed that the identified SNPs are the most associated or linked sites with the target traits

Method used

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  • Single nucleotide polymorphism marker site, primer and kit for identifying coloring property of peach epidermis and application
  • Single nucleotide polymorphism marker site, primer and kit for identifying coloring property of peach epidermis and application
  • Single nucleotide polymorphism marker site, primer and kit for identifying coloring property of peach epidermis and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Obtaining of SNPs marker sites

[0027] In the present invention, 129 peach germplasms randomly obtained from the peach germplasm resource garden of Zhengzhou Institute of Pomology, Chinese Academy of Agricultural Sciences were used as samples, and the sample DNA was extracted by conventional CTAB method, and the 129 peach germplasms were reconstructed by Illumina HiSeq 2000 sequencer. Sequencing obtained 121Gb data, covering an average of 89.28% of the peach genome, and the average sequencing depth was about 4.21×. According to the 50-150bp reads obtained by sequencing, compared with the peach reference genome v.1.0 (http: / / www.rosaceae.org / node / 355), 4,063,377 SNPs were identified, and these SNPs were used to analyze 129 germplasm Genome-wide association analysis was performed on the phenotypic traits, and the SNPs significantly associated with peach peel coloring were identified at the 3473250bp of chromosome 3.

Embodiment 2

[0028] Example 2 The method for identifying peach fruit epidermis coloring (red) traits using SNP markers

[0029] 1. DNA extraction

[0030] The DNA of the peach sample tissue to be tested was extracted by the conventional CTAB method, and the RNA was removed. The total volume of the DNA sample was not less than 15 μl. Measure the OD value of the DNA sample at 260nm and 280nm with a UV photometer, and calculate the DNA content and OD 260 / 280 ratio. DNA sample purity OD 260 / 280 The value should be between 1.8-2.0 and the concentration should be diluted to 10ng / μl.

[0031] 2. Design primers

[0032] Primers were designed according to the 200 bp sequences at the left and right positions at position 3473250 of the third chromosome of the peach genome (see Table 1 for the specific nucleotide sequence).

[0033] Table 1 SNPs flanking sequence information

[0034]

[0035] Among them, K represents T or G.

[0036]After the primers were synthesized by the biotechnology com...

Embodiment 3

[0062] Example 3 Blind test verification of phenotypic traits in 15 hybrid populations using pericarp coloring-associated SNP markers

[0063] 1. Selection of experimental materials

[0064] Taking the conventional peach varieties planted in the resource garden of Zhengzhou Pomology Research Institute as experimental materials, 15 hybrid populations with investigated phenotypic traits were selected from which a total of 221 individual plants were selected. See Table 6 for details.

[0065] Table 6 The name and population size information of the tested hybrid population

[0066]

[0067]

[0068] 2. Identification method using pericarp coloring-associated SNP markers

[0069] Using the 3473250th position of the third chromosome of the peach genome of the present invention as a nucleotide polymorphism marker site, a total of 221 peach single plant peel colorings from 15 hybrid populations were blindly tested and identified. For the specific identification method, refer to...

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Abstract

The invention discloses a single nucleotide polymorphism marker site, a primer and a kit for identifying the coloring property of peach epidermis and application. The single nucleotide polymorphism marker site is the 3473250th nucleotide of a third chromosome of a peach genome, and the nucleotide is T or G. The 3473250bp of the third chromosome of the peach genome is taken as a nucleotide polymorphism maker site which can be used for identification or assisting identification of the coloring property of peach epidermis, and is relatively high in accuracy rate when identifying the coloring property of peach epidermis. The SNPs accuracy rates of 221 single peach plants of 15 hybridized colonies to be tested are verified, the result shows that when the phenotypic characters of the hybridized colonies are predicted, and the accuracy rates of dominant phenotypes are relatively high, that is, the accuracy rates are increased to 61.29% from 8.06% when being compared with those of other research reported lately. The results show that when being used for detection, the single nucleotide polymorphism marker site disclosed by the invention has the advantages of simplicity, rapidness and low cost, and large-scale application on practical production can be achieved.

Description

technical field [0001] The invention relates to a single nucleotide polymorphism marker site, a primer, a kit and an application for identifying the coloring (red) trait of peach fruit epidermis, and belongs to the field of biotechnology. Background technique [0002] Selection is one of the most important links in breeding. It refers to selecting genotypes that meet the requirements in a population for subsequent breeding. However, in traditional breeding, because it is difficult to know the genotype of the offspring, the basis of selection is usually phenotype rather than genotype. This selection method is generally effective for qualitative traits, but for quantitative traits, because The lack of a clear correspondence between its phenotype and genotype makes it inefficient. In addition, for fruit trees with fruit traits as the target, these traits have their specific expression period, usually need to spend 3-5 years or even longer childhood, so the selection time is re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 曹珂王力荣朱更瑞方伟超陈昌文王新卫王琪
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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