Clone and molecular markers of rice broad-spectrum blast-resistance genes
A molecular marker, rice technology, applied in genetic engineering, plant genetic improvement, microbial determination/inspection, etc.
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preparation example Construction
[0241] a. Preparation of Gumei 4 Genomic DNA
[0242] (1) After two days of germination, the 4 seeds of rice Gumei are evenly sprinkled on a porcelain plate covered with absorbent paper, and cultivated in the dark at a suitable temperature of 28°C for 2 weeks in an incubator, and 2-3 yellow leaves grow. seedling.
[0243] (2) Weigh about 50 grams of etiolated seedlings after root removal, grind them into powder with liquid nitrogen, and add pre-cooled 1×HB solution (0.01mol / L Tris-HCl, 0.08mol / L Tris-HCl, 0.08mol / L KCl, 0.01mol / L EDTA, 1mmol / L spermine, 1mmol / L spermidine, 0.5mol / L sucrose, 0.15% β-mercaptoethanol, 0.5% TritonX-100, PH=9.4~9.5), stirring on ice About 15 minutes until smooth.
[0244] (3) Filter with two layers of gauze and one layer of Mirocloth, collect the filtrate, centrifuge at 1800×g, 4°C for 20 minutes, suspend the precipitate with 30 mL of 1×HB solution, centrifuge at 60×g, 4°C for 5 minutes, and transfer the supernatant to a new Centrifuge at 1800×g...
Embodiment 1
[0450] Embodiment 1, construct Pigm fine positioning group
[0451] The strain CH109 was used to inoculate the disease-resistant rice variety Gumei 4 (indica rice) and the susceptible parent Cpslo 17 (japonica rice) to construct F 2 Population, the resistance-sensitivity phenotypes were clearly separated, including 1021 resistant plants and 306 susceptible plants, and the resistance-sensitivity segregation ratio was 3:1. ) of BC 1 f 1 In the population, 3842 resistant strains and 1250 susceptible strains were isolated, and the same resistance-sensitivity conformed to the segregation ratio of 3:1, which further verified the previous genetic analysis results. A total of 1556 recessively infected individuals were obtained as a finely mapped population.
Embodiment 2
[0452] Example 2, SSR marker screening and linkage analysis between parents
[0453] Search for SSR markers between the initially positioned SSR markers SRF5 and SRF52, search and design SSR markers in the SSRIT software of WWW.GRAMENE.ORG / MARKER, and there are polymorphic SSR markers between the parent Gumei 4 and Cpslo17 and Maratelli, which are used to Detect recombination exchange single plants in the mapping population, and carry out linkage analysis. Pigm was further located in the 4.3em region between C26348 and S47656. See image 3 , Pi9, Pi2 / Piz have been located near the area where Pigm locates t , Piz, Pi25 and Pi26, according to the position of these disease resistance genes on the chromosome, Pi9, Pi2 / Piz can be determined t , Piz, Pi26 and Pigm are allelic or closely linked. However, the sources of these disease resistance genes are different, among which Pi9 is from the tetraploid O.mmuta, Pi2 is from a resistant line 5137 in South America, and Piz is the di...
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