Method of developing SNP (single nucleotide polymorphism)-SSR (simple sequence repeat) molecular makers closely linked with SNP by utilizing SNP

A molecular marker and labeling technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of no SSR marker positioning method, unfavorable gene fine positioning and cloning, no physical location determination, etc., and shorten the research time. Cycle time, short cycle times, the effect of improving efficiency and quality

Active Publication Date: 2014-08-06
SHANDONG AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, due to the limitation of the difficulty of sequencing the wheat genome, the previous SSR markers could only calculate the genetic position without definite physical position, which is not conducive to the fine mapping and cloning of genes
[0004] However, with the completion of the sequencing of the A genome and the D genome, and the construction of the physical map of the D genome, it has provided assistance for the development of SSR markers whose physical positions are determined, but there is still no effective SSR marker localization method

Method used

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  • Method of developing SNP (single nucleotide polymorphism)-SSR (simple sequence repeat) molecular makers closely linked with SNP by utilizing SNP
  • Method of developing SNP (single nucleotide polymorphism)-SSR (simple sequence repeat) molecular makers closely linked with SNP by utilizing SNP
  • Method of developing SNP (single nucleotide polymorphism)-SSR (simple sequence repeat) molecular makers closely linked with SNP by utilizing SNP

Examples

Experimental program
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Effect test

Embodiment 1

[0050] The method for obtaining a new SNP-SSR molecular marker closely linked to SNP on wheat 5DL includes the following steps:

[0051] (1) According to the position of the molecular marker Xbarc320-Xwmc215 on the 5DL on the genetic map, determine the physical map of this segment in the rough goat grass http: / / probes.pw.usda.gov / WheatDMarker / The general position on it is AT5D4910-AT5D5010, including 90 SNP marker extension sequences;

[0052] (2) The 90 SNP marker extension sequences in step (1) were compared to the wheat D genome sequencing database (Jizeng Jia, Shancenzhao, Xiuying Kong, Yingrui Li, Guangyao Zhao, Weiming He, Rudi Appels. Aegilops tauschiidraft genome sequence reveals a gene repertoire for wheat adaptation. Nature Letter, 2013, 496:91-95.) for local blast or http: / / wheat-urgi.versailles.inra.fr / Seq-Repository / BLAST The provided wheat genome sequence database is compared, and according to the condition of similarity ≥99%, sequence fragments with a length ...

Embodiment 2

[0070] The application of SNP-SSR molecular markers described in Example 1 in detecting wheat varieties, the steps are as follows:

[0071] (1) Extract the genomic DNA of the wheat to be tested:

[0072] ①Take 3-4 pieces of young wheat leaves, put them in a centrifuge tube, put them in a container filled with liquid nitrogen, and grind them for 5-15 minutes after freezing;

[0073] ②Add 900μL of pre-warmed DNA extraction working solution at 65°C, water bath at 65°C for 1 h, shake gently 3 to 5 times during the water bath, and mix thoroughly;

[0074] 3. After cooling at room temperature for 5min, add an equal volume of phenol: chloroform: isoamyl alcohol (volume ratio 25:24:1) and mix well for 30min, centrifuge at 10000g for 20min; take the supernatant and move it into a new centrifuge tube, add an equal volume of chloroform: Isoamyl alcohol (volume ratio 24:1), gently mixed and centrifuged at 10000g for 20min;

[0075] ④ Transfer the supernatant to a new centrifuge tube, ad...

Embodiment 3

[0106] The application of the SNP-SSR molecular marker described in Example 1 in the construction of a molecular genetic map, using Yumai 57 as the male parent and Huapei No. 3 as the female parent for hybridization to obtain F. 1 , F 1 The haploid was induced by hybridization of wheat and maize, and the DH population of 168 families was obtained by chromosome doubling; the application of the new SNP-SSR marker closely linked with SNP on wheat 5DL in the construction of molecular genetic map was carried out. The specific steps are as follows:

[0107] (i) Field planting Yumai 57, Huapei No. 3 and the obtained DH population, using the above-mentioned Triticarte Pty.Ltd (http: / / www.triticarte.com.au) for the leaves of the parent and 168 DH family plants The provided DNA extraction method extracts the DNA of each strain;

[0108] (ii) PCR amplification is carried out with the detection primers of the new SNP-SSR molecular markers Xtdc11, Xtdc31, Xtdc38 and Xtdc44 respectively, a...

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Abstract

The invention relates to a method of developing SNP (single nucleotide polymorphism)-SSR (simple sequence repeat) molecular makers closely linked with SNP by utilizing SNP. The method comprises the following steps of: (1) finding according to a position of a known SNP molecule of a to-be-developed sample on a genetic map to obtain an SNP marker extending sequence; (2) comparing the SNP marker extending sequence in a genome sequencing database of the to-be-developed sample, and selecting a sequence segment; (3) feeding complete type, incomplete type and composite type SSR to obtain SSR locus based on repetitive sequence length not less than 20bp; (4 designing primers according to flanking regions of SSR lotus, selecting the primers with stable and clear banding patterns through PCR (polymerase chain reaction) amplification and sequencing to obtain a to-be-verified molecular marker; and (5) preparing the SNP-SSR molecular marker by utilizing the primer of the to-be-verified molecular marker. The method disclosed by the invention can quickly and greatly screen out SNP-SSR molecular markers closely linked with the SNP.

Description

technical field [0001] The invention relates to a method for utilizing SNP to develop SNP-SSR molecular markers closely linked with SNP, and belongs to the technical field of wheat molecular biotechnology and molecular marker application. Background technique [0002] Wheat is an allohexaploid with a complex genome structure, and the polymorphisms of molecular markers present on each genome are also quite different. Through the molecular genetic map constructed by predecessors, it was found that there are more molecular markers on the A and B genomes, while the D genome has fewer markers on each chromosome, especially the 4D and 5D chromosomes. Cloning brings greater difficulties. Therefore, the development of new molecular markers on 4D and 5D chromosomes can not only encrypt the saturated molecular genetic map, but also lay a good foundation for cloning genes that control important traits (such as dwarf sterility, heading date, etc.). [0003] Among various molecular mar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q2600/156
Inventor 邓志英田纪春陈芳李文静陈建省
Owner SHANDONG AGRICULTURAL UNIVERSITY
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