Functional specific PCR (Polymerase Chain Reaction) molecular marker of rice blast-resistant gene Pi25 and application of functional specific PCR molecular marker
A rice blast resistance gene and molecular marker technology, applied in the field of agricultural biology, can solve the problems of high detection cost, expensive equipment, misjudgment of Tetep allelic genotype, etc., and achieve the effect of convenient operation and low cost
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Embodiment 1
[0029] Example 1 Screening of functional-specific PCR molecular marker primers for the rice blast resistance gene Pi25
[0030] 1. Design of primers for Pi25 gene function-specific PCR molecular markers
[0031] The basic principle of primer design is derived from the four-primer amplification hindered mutation system PCR marker (Ye et al., 2001), and the specific co-dominant PCR marker (Patent No.: ZL 201610014679.9, for the convenience of description, hereinafter referred to as marker Pi25 -2566) primer design principles are similar, but with the following modifications:
[0032] (1) Outer primer: the full length of Pi25 gene is 2775bp, and SNP775 and SNP2687 are adjacent to both ends of the gene. In order to obtain the ideal amplification effect of the two allele-specific fragments and common fragments of resistance / susceptibility, the genes of Gumei No. 2 (resistance) and Zhong 156 (susceptibility) and their upstream and downstream 200-300 bp were sequenced. Using this a...
Embodiment 2
[0054] Example 2 Accuracy verification of Pi25-2687 detection of Pi25 genotype
[0055] As mentioned above, the Pi25-specific PCR marker Pi25-2566 developed by ZL 201610014679.9 is for the SNP2566 site, and the Pi25-specific PCR marker Pi25-2687 designed and developed by the patent of this application is for the SNP2687 site. The detection effect of the genotype material, the method described in Example 1 is applied, and the primer pairs of Pi25-2687 and Pi25-2566 are used for amplification and electrophoresis detection, wherein:
[0056] For Pi25-2687, the amplification reaction system is as follows: 7.5 μL of 2 × Taq MasterMix (containing dye, Kangwei Century), 0.3 μL of primers (10 μM) each, 1.0 μL of DNA template, ddH 2 O 5.3 μL, total volume 15.0 μL. Reaction conditions: 94°C for 2 minutes; 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 5 minutes.
[0057] For Pi25-2566, the amplification reaction system was as follows: 7.5 μL of 2×Ta...
Embodiment 3
[0060] Example 3 Functional specificity verification of Pi25-2687
[0061] 12 rice restorer lines and 5 hybrid rice combinations cultivated by molecular marker-assisted selection will be identified in the rice blast disease area of Shanghang County, Longyan City, Fujian Province in 2021. The test methods and resistance evaluation and grading standards refer to the national rice Variety area test method.
[0062] The comprehensive index of rice blast of the test susceptible control varieties was 8.5, and the resistance evaluation was high sensitivity, indicating that the test was effective. Among the 12 restorer lines, the Pi25-specific molecular marker Pi25-2687 of materials No. 9 and No. 11 was detected as a homozygous susceptible genotype, the comprehensive index of rice blast was 6.25 and 5.75, respectively, and the resistance evaluation was susceptible and moderate, respectively; the remaining 10 The molecular marker detection of all the materials were all homozygous re...
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