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Method for obtaining plant introne sequence amplification polymorphism and its special primer
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A sequence amplification and intron technology, applied in the field of plant intron sequence amplification polymorphism, can solve the problem of low reliability and repetition rate of RAPD test results, difficult statistical analysis of AFLP bands, and development of SSR markers Problems such as high cost, to achieve the effect of low synthesis cost, simple and convenient workload, and reduced synthesis cost
Inactive Publication Date: 2006-12-27
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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RFLP requires a large amount of DNA, low polymorphism frequency, requires radioactive probes, and is time-consuming; the reliability and repetition rate of RAPD test results are low, and the resolution is also low; AFLP band analysis is difficult to analyze statistically, and it is difficult for operators. The level of experimental technology is high; the development cost of SSR markers is high, and the frequency of polymorphism is low; and the currently used markers cannot be linked with the expression sequence, and there is a certain blindness in their application, especially in the process of QTL mapping. Molecular The marker is too far away from the QTL, making it less useful
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[0025] 1. Primer design for ISAP molecular markers
[0026] Screen the BAC library of cotton to find introns that meet the following rules: the front end is GT and before GT is AAG or CAG, after GT is AA; the back end is AG, before AG is TTGC and the 7 bases before this 4 bases The base contains at least 4 pyridines. Such as: CGAACAAGTCTCAG GTAATG………AGATTCATTGATTTGCAG (Underlined is an intron), intercept 11 bases CGAACAAGTCT together with CAGGTAA as a front primer before CAGGTAA, intercept 12 bases AGATTCATTGAT together with TTGCAG before TTGCAG and reverse transcribe it into CTGCAAATCAATGAATCT and reverse transcribe 18 bases bases CTGCAAATCAATGAATCT as the back-end primer.
[0027] According to this design scheme, a total of 9 ISAP forward primers F1 to F9 and 8 reverse primers R1 to R8 were designed:
[0028] F1: 5'-CGATATAAGCAAAGGTAA-3' (SEQ ID NO: 1),
[0029] R1: 5'-CTGCAATTAAGCAAGAAC...
Embodiment 2
[0078] Embodiment 2, the application of ISAP molecular markers in various plants
[0079] The primer combinations F1R2, F4R1, and F3R5 were used to amplify DNA from 8 kinds of plants, including wheat, corn, pepper, tobacco, peanut, sorghum, Chlorophytum, and Yichuanhong. In each plant, ISAP primers F1R2, F4R1, and F3R5 could amplify bands, and showed better polymorphisms in different varieties ( Figure 3A , Figure 3B and Figure 3C ). Figure 3A and Figure 3B and Figure 3C Among them, 1-8 are wheat (1-8 are Kenong 199, Liangji 99, Han 7086, Zhou 98100, Wennong 6, Yan 2070, Heng 4338, Kemai 1), 9-12 are corn (in order Dongnong 250, Zhongdan 9409, Nongda 108, Jingyu No. 7), 13-15 are peppers (sheep horn pepper, Tianying pepper, Nonglei No. 2), 16 is tobacco, 17 is peanut, 18 is sorghum, 19 For Chlorophytum, 20 for a bunch of red. Figure 3A and Figure 3B and Figure 3C In , the unit of the marked band size is bp.
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Abstract
The invention discloses a method for obtaining plant subsequence augmentation polymorphism and its special-purpose primer. The special-purpose primer comprises positive primer and reversed primer, in which the said positive primer and reversed primer are evenly constituted of nucleotides, the nucleotide sequence of positive primer is 5'-NNNNNNNNNNNMAGGTAA-3',the nucleotide sequence of reversed primer is 5'-CTGCAANNNNNNNNNNNN-3', in which N can be any one base of A, T, C and G and M can be A or C. The special-purpose primer of this invention can produce polymorphism site, the composite and development cost is low, the design procedure is very simple, and the positive primer can mutually combine with the reversed primer, which can largely increase the special-purpose primer number, reduce the synthesized positive primer or reversed primer number and composite cost; the method for obtaining plant subsequence augmentation polymorphism is simple and easy and adopts PCR augmentation product electrophoresis without fussy step and radioactivity probe, which is simple and has lightens the quantity of work.
Description
technical field [0001] The invention relates to a method for obtaining plantintron sequence amplification polymorphism and special primers thereof. Background technique [0002] In the development and use of molecular markers, all kinds of molecular markers have certain defects and deficiencies. RFLP requires a large amount of DNA, low polymorphism frequency, requires radioactive probes, and is time-consuming; the reliability and repetition rate of RAPD test results are low, and the resolution is also low; AFLP band analysis is difficult to analyze statistically, and it is difficult for operators. The level of experimental technology is high; the development cost of SSR markers is high, and the frequency of polymorphism is low; and the currently used markers cannot be linked with the expression sequence, and there is a certain blindness in their application, especially in the process of QTL mapping. Molecular The distance between the marker and the QTL is too far, making i...
Claims
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