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Design method of SSR label primer and wheat SSR label primers

A technology of labeling primers and design methods, which is applied in the field of crop genetic breeding and genetic engineering, can solve the problems of inability to locate genes, no polymorphism, and few polymorphic molecular markers, and achieve high marker density and increase multiple attitude effect

Inactive Publication Date: 2014-02-12
SICHUAN AGRI UNIV
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AI Technical Summary

Problems solved by technology

At present, the use of SSR marker technology to achieve fine gene positioning still has the following technical defects in the following three aspects: 1. The development cost of SSR marker primers is high and the development cycle is long; 2. Although the probability of SSR marker polymorphisms is relatively high, there are still many polymorphism
Especially for the genetic mapping populations derived from sister lines with similar genetic background, the genetic distance between the marked loci is still relatively large. For example, if the wheat chromosome is 0.1cM, more polymorphic molecular markers are needed; When isogenic lines map new wheat genes, because the near isogenic lines themselves are very similar genetically, they will also face the problem of being unable to locate the gene position precisely due to too few polymorphic molecular markers

Method used

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  • Design method of SSR label primer and wheat SSR label primers
  • Design method of SSR label primer and wheat SSR label primers
  • Design method of SSR label primer and wheat SSR label primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This embodiment describes the SSR marker primer design method implemented with wheat materials, figure 1 Shown is the technology roadmap.

[0036] Step S1, select the initial SSR marker primer

[0037] The known SSR marker primer Xgpw1239-1B on wheat chromosome 1B was selected as the initial SSR marker primer, and its upstream and downstream sequences are shown in SEQ ID NO.0511 and SEQ ID NO.0512.

[0038] Xgpw1239-1B is an SSR marker near the chromosomal region determined after the preliminary mapping of the stripe rust resistance gene YrYU25 of L693.

[0039] Step S2, find fragment contigs

[0040]Enter the official website of IWGSC (International Wheat Genome Sequencing Committee) http: / / www.wheatgenome.org / , select the BLAST tool, and select the draft chromosome sequence of wheat 1B chromosome long and short arms (1BL, 1BS) as the BLAST database; put Xgpw1239-1B The sequence was compared with the BLAST database; the comparison result of Identities=100% was selec...

Embodiment 2

[0055] This example describes the design method of SSR marker primers implemented in wheat materials.

[0056] Seven SSR marker primers (Xcfd2-1B, Xcfd65-1B, Xwmc626-1B, Xbarc137-1B, Xwmc597-1B, Xbarc187-1B, Xwmc611-1B) published on the wheat genetic map were selected as initial SSR marker primers , using the same technical scheme as in Example 1 to design new SSR-labeled primers. The initial SSR-labeled primers and the resulting SSR-labeled primers are shown in Table 1. The imaging results after silver staining were as follows: image 3 shown. Figure 3a , Figure 3b , Figure 3c , Figure 3d , Figure 3e , Figure 3f , Figure 3g , Figure 3h , Figure 3i , Figure 3j , Figure 3k Show the results respectively SSR marker primers Xcfd2-1B-1, Xcfd65-1B-1, Xcfd65-1B-2, Xcfd65-1B-3, Xwmc626-1B-1, Xbarc137-1B-1, Xwmc597-1B-1, Xwmc597- 1B-2, Xwmc597-1B-3, Xbarc187-1B-1, Xwmc611-1B-1.

[0057] Embodiment one, embodiment two have selected 8 published SSR marker primers...

Embodiment 3

[0061] This example describes the implementation method of the gene initial mapping step in the design method of wheat materials SSR marker primers.

[0062] Take the precise mapping of the new stripe rust resistance gene of wheat stripe rust resistance new material L693 as an example. The existing technology has located the stripe rust resistance gene YrYU25 of the new stripe rust resistance material L693 on the 1B chromosome of wheat, and there are only 3 pairs of polymorphic markers linked to the resistance gene on the 1B chromosome (Table 3, Table 4) , if the gene is to be accurately positioned, three pairs of polymorphic markers are far from enough. Determine the target gene region according to the location of YrYU25.

[0063] Table 3 Statistics of polymorphic primers linked to stripe rust resistance gene on chromosome 1B

[0064]

[0065] ※——SSR primers with polymorphism

[0066] Table 4 Primer sequences of polymorphisms linked to stripe rust resistance gene on chr...

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Abstract

The invention discloses a design method of an SSR label primer. In order to overcome the defect of insufficient polymorphism of the conventional SSR label primer, the invention provides a method for designing a novel SSR label primer based on a draft sequence of a genome. The design method comprises the following steps: firstly selecting an SSR label primer with a known site as a starting SSR label primer; secondly, comparing the starting SSR label primer with the draft sequence of a chromosome to which the starting SSR label primer belongs, and finding a contig in a comparison result; thirdly, searching an SSR sequence in the contig as a finishing SSR sequence; finally, designing the SSR label primer based on the finishing SSR sequence. The invention provides 14 pairs of novel SSR label primers related to wheat stripe rust resistance, and a method for establishing a wheat genetic map with L693*L661 and L661*L693F2 single plants as a mapping population, wherein five pairs of the SSR label primers generate genetic polymorphism in a genetic test group. According to the design method, the number of the SSR label primers with known sites can be quickly increased and the polymorphism of the SSR label is increased; the genetic map can be quickly encrypted through combination with initial gene localization.

Description

technical field [0001] The invention relates to a method for designing molecular marker primers and wheat molecular marker primers, in particular to a design method for SSR molecular marker primers and wheat SSR marker primers, belonging to the fields of genetic engineering and crop genetic breeding. Background technique [0002] SSR marker is a DNA marker technology with PCR technology as the core. Its technical principle is to use the characteristics that SSR sequences are widely distributed in the genome and have high polymorphism, and most of the sequences at both ends of the SSR sequence are relatively conservative single-copy sequences. Design a pair of specific primers for the single-copy sequence at both ends of the SSR sequence in the region, and then use PCR technology to amplify the SSR sequence at each site, and analyze the length polymorphism of the core sequence by electrophoresis. SSR markers have technical advantages such as high polymorphism and high reliabi...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10G06F19/20C12Q1/68
Inventor 罗培高李鑫陈万权张敏任正隆张怀渝
Owner SICHUAN AGRI UNIV
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