A method using SNPs to develop SNP-SSR molecular markers tightly linked to SNPs

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

Active Publication Date: 2015-09-30
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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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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  • A method using SNPs to develop SNP-SSR molecular markers tightly linked to SNPs
  • A method using SNPs to develop SNP-SSR molecular markers tightly linked to SNPs
  • A method using SNPs to develop SNP-SSR molecular markers tightly linked to SNPs

Examples

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Embodiment 1

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

[0051] (1) According to the position of the molecular markers Xbarc320-Xwmc215 on the 5DL on the genetic map, it is determined that this segment is in the physical map of A. roughus http: / / probes.pw.usda.gov / WheatDMarker / The approximate position on the SNAP is AT5D4910-AT5D5010, which contains 90 SNP marker extension sequences;

[0052] (2) The extended sequences of the 90 SNP markers in step (1) were compared to the wheat D genome sequencing database (Jizeng Jia, Shancen zhao, Xiuying Kong, Yingrui Li, Guangyao Zhao, Weiming He, Rudi Appels. Aegilops tauschii draft 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 Compare the provided wheat genome sequence database, and select sequence fragments with a length ranging from 5...

Embodiment 2

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

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

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

[0073] ② Add 900 μL DNA extraction working solution preheated at 65°C, bathe in water at 65°C for 1 hour, shake gently 3 to 5 times during the water bath, and mix thoroughly;

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

[0075] ④ Transfer the supernatant ...

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 3 as the female parent to obtain F 1 , F 1 The haploid was induced by crossing wheat and maize, and the DH population containing 168 families was obtained through chromosome doubling; the application of new SNP-SSR markers closely linked to SNPs on wheat 5DL in the construction of molecular genetic maps was carried out, and the specific steps were as follows:

[0107] (ⅰ) Plant Yumai 57, Huapei 3 and the obtained DH populations in the field, and use the above-mentioned Triticarte Pty.Ltd (http: / / www.triticarte.com.au) on the leaves of the parents and 168 DH family plants The provided DNA extraction method extracts the DNA of each strain;

[0108] (ii) Perform PCR amplification with the detection primers of the new SNP-SSR molecular markers Xtdc11, Xtdc31, Xtdc38, and Xtdc44 respectively, and the reaction sys...

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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 using SNP to develop a SNP-SSR molecular marker closely linked with the 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 on each genome are also quite different. According to the molecular genetic map constructed by the predecessors, it is found that there are more molecular markers on the A and B genomes, while there are fewer markers on each chromosome of the D genome, especially the 4D and 5D chromosomes, which is also the reason for the presence of genes controlling important traits on these chromosomes Cloning poses greater difficulties. Therefore, the development of new molecular markers on chromosome 4D and 5D can not only encrypt the saturated molecular genetic map, but also lay a good foundation for cloning genes contr...

Claims

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Application Information

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Patent Type & Authority Patents(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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