Method for obtaining EST-SSR mark

A technology of labeling and simple sequence repetition, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as easy to miss SSR markers, miss SSR short sequences, polymorphism increase, etc. , achieve the effect of shortening R&D time, improving development efficiency and reducing development cost

Inactive Publication Date: 2010-01-06
NORTHEAST AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

SSR markers from 3' ESTs are higher than those from 5' ESTs (Scott K D, Eggler P, Seaton G, et al. 2000. Analysis of SSRs derived from grape ESTs. Theor. Appl. Genet, 100: 723-726) , 3'EST may contain the 3'transcribed untranslated region (3'UTR) of cDNA, the frequency of variation in this region is much higher than that of 5'EST, so most studies start with 3'EST to develop EST-SSR to increase the number of occurrences The possibility of morphic markers, but this method is easy to miss the SSR markers that exist in the 5'EST; it is also reported that with the increase in the number of repetitions of SSR, the possibility of polymorphism will increase, so many studies put the target Lock the sequence with a certain number of SSR repeats, and this method will also miss some short polymorphic SSR sequences
In short, the current development methods of SSR markers have the problems of low efficiency, time-consuming and labor-intensive problems.
At present, there is no relevant report on the efficient development of EST-SSR labeling methods

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  • Method for obtaining EST-SSR mark

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Obtaining the EST-SSR marker of soybean

[0029] 1. Design of primers

[0030] 1. Obtain the EST sequence in the soybean genome

[0031] Soybean EST sequences were downloaded from the NCBI bioinformatics database, and a total of 458,220 soybean EST sequences were obtained.

[0032] 2. Search for EST sequences containing SSRs (ie simple sequence repeats)

[0033]Use SSRIT (Simple Sequence Repeat Identification Tool) software to perform online search on all EST sequences obtained in step 1, and obtain EST sequences containing simple repeat sequences (called SSR-EST sequences); the search criteria are: dinucleotides, trinucleotides The number of repetitions of the nucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeating units is greater than or equal to 6, 5, 4, 4, and 4, respectively.

[0034] 3. Classify SSR-EST sequences

[0035] According to the different types of simple sequence repeating units, all SSR-EST sequences obtained in step 2 ...

Embodiment 2

[0159] Example 2: Comparison between the EST-SSR marker development method of the present invention and conventional methods

[0160] Existing method: From all the EST-SSR sequences obtained in Example 1, 191 sequences were randomly selected for primer development and design, and a pair of primers was obtained for each sequence, and a total of 191 pairs of primers were obtained. Then, the polymorphism of each pair of primers was detected by the method described in Experiment 2 in Example 1, and the ratio of the polymorphic primers was counted. The results are shown in Table 7. Ratio of polymorphic primers=number of polymorphic primers / number of all primers.

[0161] Table 7. EST-SSR polymorphism statistics of existing methods

[0162]

[0163] Comparing the research results in Table 5 and Table 7, it can be seen that the common feature of the two methods is that the ratio of polymorphic primers generated by 3'EST is higher than that of 5'EST and EST from other sourc...

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Abstract

The invention discloses a method for obtaining an EST-SSR mark, comprising the following steps: (1) obtaining an EST sequence containing simple repeat sequence in genome; (2) in the EST sequence which contains the simple repeat sequence and is obtained in the step (1), classifying the EST sequences with the same simple sequence repeat unit into a same type; (3) performing sequence splicing on the EST sequences of the same type obtained in the step (2) to obtain an overlapping group with variable numbers of simple sequence repeat units, an overlapping group without variable numbers of simple sequence repeat units and an EST sequence without overlapping groups; (4) designing primers according to a side-vane conserved sequence of simple repeat sequence in the overlapping group with available numbers of simple sequence repeat units in the step (3), and detecting the polymorphism of the primers to obtain polymorphic primers, i.e. EST-SSR mark. Compared with the conventional method, the invention increases the development efficiency by 2-4 times, and reduces the work capacity and expenditure, thereby shortening the development time, reducing the development cost and simultaneously reducing the possibility of missing the locus of polymorphism SSR.

Description

technical field [0001] The present invention relates to a method for obtaining EST-SSR markers. Background technique [0002] SSR simple sequence repeat (Simple sequence repeat, SSR for short), also known as microsatellite sequence repeat, is a tandem repeat sequence of dozens of nucleotides, and its repeat unit is generally 2-6 nucleotides. It is widely distributed in different positions of various eukaryotic genomes, and the distribution is relatively uniform. On average, there is a microsatellite sequence in every 10kb DNA sequence. SSR markers have co-dominant, highly repetitive, and highly abundant polymorphisms. It has become an ideal tool for constructing genetic linkage maps, studying population genetics, molecular marker-assisted breeding, pedigree analysis, variety fingerprinting, variety purity detection, molecular marker screening for target traits and forensic identification. Expressed Sequence Tag (EST) also contains SSR sequence, which is called EST-SSR. EST-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34C12N15/11
Inventor 李文滨赵雪常玮韩英鹏滕卫丽
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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