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Somatic mutation site excavation method based on genomic sequencing

A technology of genome sequencing and somatic mutation, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as lack of methods, achieve low price, shorten the time for detecting mutation sites, and low cost.

Inactive Publication Date: 2015-09-30
HUAZHONG AGRI UNIV
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Problems solved by technology

However, there is currently no suitable method for mining somatic mutation sites based on genome sequencing (the fruit tree genome has characteristics such as heterozygosity), and it is necessary to optimize and improve the sequence information analysis process and method

Method used

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  • Somatic mutation site excavation method based on genomic sequencing
  • Somatic mutation site excavation method based on genomic sequencing
  • Somatic mutation site excavation method based on genomic sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Mining of mutation sites of red meat mutants of Guanxi honey pomelo

[0037] 1. Preparation of the genomic DNA of the red meat mutant of Guanxi honey pomelo

[0038] In this example, the modified CTAB method was used to extract the genomic DNA of four tissues (exocarp, mesocarp, sac coat and juice cell) of the wild-type Guanxi pomelo and its red-fleshed mutant.

[0039] 2. Genome sequencing

[0040] 2.1 Construction of DNA sequencing library

[0041] Fragmentation of genomic DNA (fragmentation of genomic DNA into the range of 200-1000 bp with an ultrasonic fragmenter); blunt-end (generates blunt-ended 5' phosphorylated fragments); magnetic bead purification; A addition (adds dAMP to double strands) 3' end of DNA library fragment); adding adapter (connecting a double-stranded DNA adapter with a 3'-dTMP end to the library fragment); magnetic bead purification; cutting gel to recover DNA fragments of about 500bp in size; library amplification (PCR amplification...

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Abstract

The invention discloses a somatic mutation site excavation method based on genomic sequencing. By means of depth re-sequencing on various tissue of a wild type shaddock and a mutation type shaddock, the obtained high-quality sequencing reads and the sweet orange genome are compared, and the single nucleotide polymorphism (SNP) of various sets of materials is obtained. Based on SNP sites distributed on some whole genomes, the case-control type correlation analysis is designed on the wild type shaddock and the mutation type shaddock, potential sites related to mutation are obtained and annotated, the chromosome area where the character correlation variation happens is speculated, finally, the potential sites are verified through a Sanger sequencing method, and the variation site of the mutation type shaddock is obtained. The method has the biggest advantages of being low in cost and short in period. The cost for finishing excavating of one mutant mutation site is about 20 thousand, the excavation can be finished in three months, and combining and innovation exploring of the genome advanced technology and the traditional citrus breeding study are expressed. The invention further discloses development and application of the method.

Description

technical field [0001] The invention relates to the field of citrus genomics and molecular breeding, in particular to a somatic mutation site mining method based on genome sequencing. Background technique [0002] Somatic mutation refers to the mutation that occurs in plants at the level of somatic cells (eg, the meristem of shoots). Bud mutation is a kind of somatic mutation, which is generally manifested in the characteristics and characteristics of branches, leaves, flowers, fruits, phenological stage, and maturity stage. Many excellent varieties have been bred through bud selection. More than 70% of the main citrus cultivars are derived from somatic variation, such as a series of lines of navel orange, mandarin orange, grapefruit and Clementine. Select mutant buds and their growing branches, and through asexual reproduction, new varieties can be created called bud selection. As a method of citrus breeding, bud selection has unique advantages compared with other breedin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q2600/13C12Q2600/156
Inventor 徐强王霞余惠文温少华邓秀新
Owner HUAZHONG AGRI UNIV
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