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Single-nucleotide polymorphism marker locus, primer pair and kit for identifying single-petal/double-petal traits of peach blossoms and application

A technology of single nucleotide polymorphism and marker loci, applied in the biological field, can solve the problems of long distance, low accuracy, and small number of genomes of target trait genes, and achieve good social and economic benefits and low cost effects

Active Publication Date: 2018-03-30
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Description
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AI Technical Summary

Problems solved by technology

[0005] Since the previous flower type (single petal / double petal) marker is an SSR marker, which has a small number of genomes, there is a problem that it is far from the target trait gene location, and the accuracy rate is low in the early identification of hybrid offspring

Method used

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  • Single-nucleotide polymorphism marker locus, primer pair and kit for identifying single-petal/double-petal traits of peach blossoms and application
  • Single-nucleotide polymorphism marker locus, primer pair and kit for identifying single-petal/double-petal traits of peach blossoms and application
  • Single-nucleotide polymorphism marker locus, primer pair and kit for identifying single-petal/double-petal traits of peach blossoms and application

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Experimental program
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Effect test

Embodiment 1

[0026] The acquisition of embodiment 1 SNPs marker locus

[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 version 2 (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 flower type (single / double) traits, and the SNP that was significantly associated with peach flower type (single / double) was identified at the 25,058,942 bp of chro...

Embodiment 2

[0028] Embodiment 2 Utilizes the method for identifying peach flower type (single petal / double petal) character with SNP marker

[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 150 bp sequences at positions 25, 058, and 942 of chromosome 2 of the peach genome version 2 (see Table 1 for specific nucleotide sequences).

[0033] Table 1 SNP flanking sequence information

[0034]

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

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

Embodiment 3

[0063] Example 3 337 peach natural populations use flower type (single petal / double petal) associated SNP markers to carry out blind test verification of phenotypic traits 1. Selection of experimental materials

[0064] 337 natural populations of common peach germplasm preserved in the resource garden of Zhengzhou Pomology Research Institute were used as experimental materials, including 16 double petals and 321 single petals.

[0065] 2. Identification method using peach-blossom-associated SNP markers

[0066] Using positions 25,058,942 of the second chromosome of the peach genome of the present invention as the nucleotide polymorphism marker site, 337 germplasms were blindly tested and identified. For the specific identification method, refer to the method in Example 2.

[0067] 3. The predictive ability of typing results to phenotype in natural populations (Table 6)

[0068] From the identification results, it can be seen that among the 321 single lobes, only 1 was typed a...

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Abstract

The invention discloses a single-nucleotide polymorphism marker locus, primer pair and kit for identifying the single-petal / double-petal traits of peach blossoms and application. The single-nucleotidepolymorphism marker locus is the 25th-site, 058th-site and 942nd-site nucleotide of the second chromosome of peach genome, and the nucleotide is G or T. By the sequencing comparison of a large numberof peach germ plasm samples, 4063377 SNPs are identified, the SNPs are utilized to perform genome-wide relational analysis on the (single-petal / double-petal) traits of the peach blossom types of 129germ plasm, and the fact that the SNP evidently related to the peach blossom types is located at the 25th bp, the 058th bp and the 942nd bp is identified. The specific PCR primer amplification pair and a single base extension primer are designed to process and analyze products obtained after single base extension reaction so as to acquire the genotyping results of the products according the molecular weights of the different products. A detection method using the single-nucleotide polymorphism marker locus is simple, fast, low in cost, capable of achieving large-scale application in production, and good in social and economic benefits.

Description

technical field [0001] The invention relates to a single nucleotide polymorphism marker site, a primer pair, a kit and an application for identifying single-petal / double-petal traits of peach blossoms, 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...

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 王力荣曹珂孟歌朱更瑞方伟超陈昌文王新卫
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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