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Single nucleotide polymorphism marker sites, primer pair and kit for identification of peach flower shape (bell shape or rose shape) characters and applications thereof

A technology of single nucleotide polymorphism and marker loci, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of unguaranteed SNPs, long linkage distance of traits, and low accuracy And other issues

Active Publication Date: 2018-04-10
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

However, the existing SSR markers have a long linkage distance with the peach-shaped (bell-shaped / rose-shaped) traits, and the accuracy rate in the early identification of hybrid offspring is low; while the existing SNPs markers linked to the target traits come from the results of 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 sites, primer pair and kit for identification of peach flower shape (bell shape or rose shape) characters and applications thereof
  • Single nucleotide polymorphism marker sites, primer pair and kit for identification of peach flower shape (bell shape or rose shape) characters and applications thereof
  • Single nucleotide polymorphism marker sites, primer pair and kit for identification of peach flower shape (bell shape or rose shape) characters and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The acquisition of embodiment 1 SNPs marker locus

[0026] 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 version 2 (http: / / www.rosaceae.org / node / 355), 4,063,377 SNPs were identified. Using these SNPs to conduct genome-wide association analysis on the phenotypic traits of 129 germplasms, it was identified that the SNP significantly associated with peach blossom (bell / rose) traits was located at 14,484,624 bp of chromosome 8.

Embodiment 2

[0027] Embodiment 2 Utilizes the method for identifying peach blossom type (bell type / rose type) traits with SNP markers

[0028] 1. DNA extraction

[0029] 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.

[0030] 2. Design primers

[0031] Primers were designed according to the 150 bp sequences on the left and right sides of chromosome 14, 484, and 624 of the second edition of the peach genome (see Table 1 for the specific nucleotide sequence).

[0032] Table 1 SNP flanking sequence information

[0033]

[0034] Among them, M represents A or C.

[0035] After the primers were synthesized by t...

Embodiment 3

[0063] Example 3 Blind test verification of phenotypic traits in 7 hybrid populations using peach blossom (bell shape / rose shape) trait SNP markers

[0064] 1. Selection of experimental materials

[0065] The conventional peach varieties planted in the resource garden of Zhengzhou Pomology Research Institute were used as experimental materials, and a total of 170 individual plants were selected from 7 hybrid populations whose phenotypic traits had been investigated. See Table 6 for details.

[0066] Table 6 The name and population size information of the tested hybrid population

[0067]

[0068] 2. Identification method using peach-blossom-associated SNP markers

[0069] Using the 14th, 484, 624th position of the 8th chromosome of the peach genome of the present invention as a nucleotide polymorphism marker site, a total of 170 flower type (bell type / rose type) traits of peach single plants in 7 hybrid populations were blindly tested and identified. The identification me...

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Abstract

Single nucleotide polymorphism marker sites, a primer pair and a kit which are used for identification of peach flower shape (bell shape or rose shape) characters and applications thereof are disclosed. The single nucleotide polymorphism marker sites are number 14, number 484 and number 624 nucleotides of the number 8 chromosome in genome of amygdalus persica L., and the nucleotides are A or C. Through sequencing and comparing a large number of germplasm samples of the amygdalus persica L., 4063377 SNPs are identified, the SNPs are utilized to perform genome-wide association study on flower shape (bell shape or rose shape) characters of 129 germplasms, and an identification result shows that SNPs significantly associated with the peach flower shape (bell shape or rose shape) are at the number 14, number 484 and number 624 base pairs of the number 8 chromosome. The specific PCR primer amplification pair and single-base extension primers are designed. Products after single base extensionreactions are finished are processed and analyzed, and a genotype result can be obtained according to molecular weights of different products. A detection method provided by the invention has advantages of capability of being simple, rapid and cheap, can achieve large-scale application in production, and has good social and economic benefit.

Description

technical field [0001] The invention relates to a single nucleotide polymorphism marker site, a primer pair, a kit and an application for identifying peach-blossom (bell-shaped / rose-shaped) traits, 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2533/101C12Q2565/627
Inventor 曹珂王力荣朱更瑞
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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