I. A. Shilov1, Yu. V. Аniskina1, N. S. Velishaeva1, O. S. Kolobova1, Т. V. Shalaeva1, P. I. Kostyilev2, E. V. Dubina3
1All-Russian Research Institute of Agricultural Biotechmology, ul. Timiryazevskaya, 42, Moskva, 127550, Russian Federation
2Agrarian Scientific Center “Donskoj”, pos. Nauchnyi gorodok, 3, Zernograd, 347740, Russian Federation
3All-Russian Research Institute of Rice Breeding, pos. Belozernyi, 3, Krasnodar, 350921, Russian Federation
Abstract. The investigation was carried out with the aim of developing a multilocus microsatellite analysis technology for large-scale screening of rice samples for the presence of three genes of resistance to rice blast Pi-1, Pi-2, Pi-33. The suggested system of multiplex PCR-analysis of microsatellite loci Rm224 (Pi-1), Rm527 (Pi-2), and Rm72 and Rm310 (Pi-33), linked to the resistance genes, enables to identify three resistance genes simultaneously and significantly reduces the cost and duration of genetic analysis. The length of the obtained PCR fragments was determined by the results of the separation with high-resolution electrophoresis in an automated genetic analyzer “Nanofor-05” using the technology of fluorescence detection. Such an approach made it possible to determine the length of the microsatellite fragments under study with an accuracy of one nucleotide. As a result of the genetic analysis of lines-donors of the resistance, the exact lengths of microsatellite markers, determining the presence of the corresponding genes of the resistance to rice blast, were experimentally established: a fragment of locus Rm224 with a length of 137 bp detected in line C104-Lac – the donor of gene Pi-1; a fragment of locus Rm527 with a length of 239 bp, detected in line C101-A-51– the donor of gene Pi-2; a fragment of locus Rm72 with a length of 165 bp and a fragment of locus Rm310 with a length of 95 bp, identified in line C101-Lac – the donor of gene Pi-33. These donor lines were used as positive controls in the examination of rice samples. Genetic analysis of 1200 breeding samples of rice, carried out using the developed technology, revealed plants with resistance gene markers Pi-1, Pi-2 and Pi-33 in the heterozygous and homozygous state. The proposed technology can be introduced into the practice of marker-assisted rice breeding since analysis in the format of a 96-well plate can be automated, which improves accuracy, and also allows for large-scale screening for the presence of genes for resistance to rice blast.
Keywords: rice (Oryza sativa L.); resistance to rice blast; donor; Pi-1, Pi-2, Pi-33 genes; marker-assisted selection; microsatellite markers; multiplex PCR analysis; DNA fragment analysis.
Acknowledgments: The work was carried out within the framework of the State Assignment “Development of New Technologies for High-Throughput Genetic Analysis of Agricultural Crops for Accelerating the Breeding Process” (Project 0574-2018-0007).
Author Details: I. A. Shilov, D. Sc. (Biol.), professor of the RAS, head of laboratory (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); Yu. V. Аniskina, Cand. Sc. (Biol.), senior research fellow; N. S. Velishaeva, Cand. Sc. (Biol.), senior research fellow; O. S. Kolobova, senior research fellow; Т. V. Shalaeva, research trainee; P. I. Kostyilev, D. Sc. (Agr.), prof., 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: Shilov I. A., Аniskina Yu. V., Velishaeva N. S., Kolobova O. S., Shalaeva Т. V., Kostyilev P. I., Dubina E. V. Technology of Mass Screening of Rice Samples for the Presence of Pi-1, Pi-2 and Pi-33 Resistance Genes on the Basis of Multiplex Microsatellite Analysis. Dostizheniya nauki i tekhniki APK. 2018. Vol. 32. No. 11. Pp. 21–25 (in Russ.). DOI: 10.24411/0235-2451-2018-11105.