Detection method and application of rice blast resistance gene Pigm
A detection method and genetic technology, applied in the field of agricultural biology, can solve the problems of poor marker stability and poor repeatability of experimental results, and achieve the effects of reducing labor costs and time costs, low cost, and high stability
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Embodiment 1
[0043] Example 1: Analysis of specific single base polymorphism (SNP) near both ends of Pigm
[0044] Since the Pigm gene has not been cloned yet, its detailed DNA sequence cannot be known, and specific primers cannot be designed for its functional variation to detect its presence or absence. Using the rice 60K gene chip to scan the whole genome of Gumei 4, Wuyunjing 7 and 9311, and select the mutation sites that are different from Gumei 4, Wuyunjing 7 and 9311 near both ends of the Pigm gene (such as Table 1) to design HRM primers so that a molecular marker that is completely co-segregated with Pigm is developed near both ends, and the two specific mutation sites are respectively T→C and A→C, specifically as the following SEQ ID NO: 5 and shown in SEQID NO:6.
[0045] 5'-
[0046] GCTTAATCAAGCCCATTTCCTCTATAACCACAGAGGATGAGAGGGATTCCTACCTAGAAGATGCT T /
[0047] CGCAATCGATCAGGTAGCAACACTGACGAGTCAGAACTTGTGGGCT-3' (SEQ ID NO: 5);
[0048] 5'-
[0049] AAGCTGTCACGACCTGAGAAGGGAT...
Embodiment 2
[0054] Example 2: Pigm co-segregation molecular marker primer design and HRM detection
[0055] (1) Primer design
[0056] According to the principle of HRM primer design, a pair of gene-specific molecular marker primers were designed near the two ends of the Pigm gene. The base sequences of the primer pairs are as follows:
[0057] Pigm-HRM-1F: 5'-GCTTAATCAAGCCCATTTCC-3' (SEQ ID NO: 1);
[0058] Pigm-HRM-1R: 5'-AGCCCACAAGTTCTGACTCG-3' (SEQ ID NO: 2);
[0059] Pigm-HRM-2F: 5'-AAGCTGTCACGACCTGAGAAG-3' (SEQ ID NO: 3);
[0060] Pigm-HRM-2R: 5'-CGTGCTCACCTGTGAGTTCTA-3' (SEQ ID NO: 4);
[0061] (2) HRM detection
[0062] The rice material "Gumei 4", rice two-line male sterile line, The DNA templates of the three-line maintainer line, restorer line, conventional rice, and other rice disease-resistant parents (as shown in Table 2) were amplified by PCR, and the amplified products were collected by HRM instrument LightScanner96 with high-resolution melting curves to obtain the po...
Embodiment 3
[0082] Example 3: Application analysis of Pigm co-segregation molecular markers in assisted breeding
[0083] The rice material containing the rice blast resistance gene Pigm was used as the parent of "Gumei No. 4", and it was crossed with other rice varieties that did not contain the rice blast resistance gene Pigm, and the genomic DNA of the hybrid offspring and the two parents were extracted, and the primer pair Pigm- HRM-1F / 1R and Pigm-HRM-2F / 2R were amplified by PCR respectively, and the high-resolution melting curves of the amplified products showed three types, among which the melting curve of the positive control "Gumei No. 4" was red, The other control parent is blue, and the hybrid offspring of the two parents is gray, and the peak is significantly lower than that of the two parents (such as image 3 and Figure 4 ). The F of two parents containing Pigm gene and not containing the gene 2 The 46 samples of isolated individuals were subjected to molecular detection ...
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