IMPROVEMENT OF THE METHOD FOR IDENTIFICATION OF GENES OF RESISTANCE TO RICE BLAST PI-TA AND PI-B

I.A. Shilov¹, O. S. Kolobova¹, Yu.V. Аniskina¹, Т. V. Shalaeva¹, N.S. Velishaeva¹, P.I. Kostyilev², E.V. Dubina^3
1All-Russia Institute of Agricultural Biotechnology, Timiryazevskaya ul., 42, Moskva, 127550, Russian Federation
²All-Russian Research Institute of Grain Crops after I.G. Kalinenko, Nauchny Gorodok, 3, Zernograd, 347740, Russian Federation
³All-Russian Rice Research Institute, pos. Belozernyi, 3, Krasnodar, 350921, Russian Federation

Summary. Polymerase chain reaction (PCR) markers were developed long ago for the analysis of allelic status of blast resistance gene Pi-ta and Pi-b. We developed the technology to analyze the allelic status of these genes by RT-PCR. We studied 17 rice hybrids F2, obtained from the crossing of plants-donors of resistance to blast with Russian varieties in the strategy of pyramiding (300 plants). To design primers and probes for Pi-ta gene the nucleotide sequences of the dominant (variety Yashiro-mochi, AF207842) and recessive (variety Tsuyake, AY196754) alleles were used. To identify the allele state of Pi-b gene known primers (Suprun I.I., Il'nitskaya E.T., Mukhina Zh.M., 2007) were modified; we added one nucleotide on 3’-end to optimize the annealing temperature. To design probes we used nucleotide sequences AB026839 with resistance gene Pi-b and susceptible forms AP004048. PCR was carried out in 25 mcl of solution, containing PCR-buffer, 1.25 EU Taq-polymerase, 100 mcmol dNTP, 10 pmol of each primer, 5 pmol probes, 5 mcl of DNA solution. Amplification was carried out at 95 degrees, 10 min, 40 cycles. In resistant homozygous plants dominant for Pi-ta gene it was registered the growth of fluorescence in the FAM channel; in susceptible homozygous plants – in the R6G channel; in heterozygote – in both channels. The use of different fluorescent labels of probes enabled to use 96-well plates and a robotic pipette station. The developed systems were used in I.G. Kalinenko All-Russian Research Institute of Grain Crops during creation of rice varieties resistant to blast. It was selected 13 plants, homozygous for both genes Pi-ta and Pi-b, and homozygous forms, carrying these genes separately.

Keywords: rice, donor, blast resistance, Pi-ta gene, Pi-b gene, hybrid, marker, RT-PCR.

Author Details: I.A. Shilov, D.Sc. (Biol.), head of laboratory (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); O.S. Kolobova, senior research fellow; Yu.V. Aniskina, Cand. Sc. (Biol.), senior research fellow; T.V. Shalaeva, research trainee; N.S. Velishaeva, Cand. Sc. (Biol.), senior research fellow; P.I. Kostylev, D.Sc. (Agr.), head of laboratory (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); E.V. Dubina, Cand. Sc. (Biol.), leading research fellow (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.).

For citation: Improvement of the Method for Identification of Genes of Resistance to Rice Blast Pi-ta and Pi-b / I.A. Shilov, O. S. Kolobova, Yu.V. Аniskina, Т. V. Shalaeva, N.S. Velishaeva, P.I. Kostyilev, E.V. Dubina. Dostizheniya nauki i tekhniki APK. 2016. V. 30. No. 8. Pp. 45-48 (in Russ.).