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SNP molecular marker combination for constructing peach DNA fingerprint spectrum, application and method

A technology of DNA fingerprinting and molecular marking, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of technical stability, complexity and data integration not widely used

Active Publication Date: 2019-09-17
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RFLP, RAPD, and AFLP technologies have been applied to database construction, but they have not been widely used due to technical stability, complexity, and data integration. The International Alliance for the Protection of New Varieties recommends SSR and SNP as the preferred DNA fingerprinting technologies

Method used

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  • SNP molecular marker combination for constructing peach DNA fingerprint spectrum, application and method
  • SNP molecular marker combination for constructing peach DNA fingerprint spectrum, application and method
  • SNP molecular marker combination for constructing peach DNA fingerprint spectrum, application and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Obtaining of SNP marker sites

[0057] In the present invention, 360 peach germplasms obtained from the peach germplasm resource nursery of Zhengzhou Pomology Research Institute, Chinese Academy of Agricultural Sciences are used as samples, and the sample DNA is extracted by the conventional CTAB method, and the 360 ​​peach germplasms are resequenced by an Illumina HiSeq 2000 sequencer , to obtain 151Gb data, with an average sequencing depth of about 8.4×. According to the 50-150bp reads obtained by sequencing, compared with the peach reference genome version 2 (http: / / www.rosaceae.org / node / 355), 16658391 SNPs were identified.

Embodiment 2

[0058] Example 2 Filtering of SNP marker sites

[0059] Use the software vcftools v0.1.13 to filter the SNP marker sites, the specific method is as follows:

[0060] (1) Missing rate filtering

[0061] Use vcftools to calculate the deletion rate of each SNP site, filter out the SNP sites with deletions in 360 samples, and output a vcf file containing 939004 SNPs.

[0062] (2) SNP sequencing depth filtering

[0063] Calculate the sequencing depth of SNP in the vcf file that step (1) outputs, according to statistical result, filter out the SNP site that sequencing depth is lower than 10×, output the vcf file that comprises 310260 SNPs.

[0064] (3) SNP quality value filtering

[0065] Calculate the quality value Q of SNP in the vcf file that step (2) outputs, filter out the SNP locus with quality value lower than 1000, output the vcf file that comprises 141076 SNPs.

[0066] (4) π value filtering

[0067] Calculate the π value of SNP in the vcf file that step (3) outputs, f...

Embodiment 3

[0070] Example 3 Screening of SNP marker sites

[0071] (1) π value sorting

[0072] Carry out π value calculation to the vcf file containing 775 SNPs in step (5) of Example 2, output the txt file, sort the SNPs according to the π value, and use the SNP with the largest π value (Chr1: 9204153) as the first SNP of the set site.

[0073] (2) View sequence specificity

[0074] Use Samtools software to obtain the sequence of 150 bp upstream and downstream of the SNP (Chr1:9204153) molecular marker, and use blast (https: / / www.rosaceae.org / tools / ncbi_blast) to check the sequence specificity (E≥e-157).

[0075] (3) Determine the best SNP combination

[0076] The first SNP molecular marker (Chr1: 9204153) was combined with the remaining 774 SNP molecular markers (Perl program), and the SNP molecular marker (Chr2: 28221961), as the second SNP site.

[0077] Combining the set of the above two SNP molecular markers with the remaining 773 SNP molecular markers (Perl program), the SNP...

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Abstract

The invention discloses an SNP molecular marker combination for constructing a peach DNA fingerprint spectrum, application and a method. The SNP molecular marker combination comprises SNP molecular markers shown as SEQ ID NO.1-17. 16658391 SNPs are identified through sequencing comparison of 360 peach germplasm samples, and the SNPs are filtered and screened to obtain an SNP set for identifying germplasms. 80 bred peach varieties are utilized to predict the distinguishing efficiency of the developed SNP set, and a result shows that the distinguishing rate of the developed SNP set on the bred varieties can reach 100%. It is indicated that the SNP molecular marker combination has the advantages of simplicity, rapidness and low cost in detection by utilizing the single nucleotide marker sites of the combination, and large-scale application in production can be achieved. Due to the fact that the number of original SNPs is large, the polymorphism of the obtained SNPs is guaranteed, and the accuracy of the SNP set identification is effectively improved.

Description

technical field [0001] The invention relates to a SNP molecular marker combination, application and method for constructing peach DNA fingerprints, and belongs to the technical field of peach molecular biology. Background technique [0002] As the origin center of peach, China is rich in peach germplasm resources. Three national peach germplasm resource nurseries (Beijing, Nanjing, Zhengzhou) and many local peach resource nurseries have collected and preserved more than 2000 peach germplasm resources of 6 species. In recent years, many varieties have been preserved by combining local high-quality varieties and foreign varieties. Such a large number of varieties and complex genetic backgrounds have brought great challenges to the identification of peach varieties. According to incomplete statistics, there are more than 5,000 peach varieties cultivated in the world, more than 700 peach varieties have been selected and introduced in the United States, and more than 1,000 varie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 曹珂关利平朱更瑞方伟超王新卫陈昌文李勇
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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