RUDN Journal of Agronomy and Animal IndustriesRUDN Journal of Agronomy and Animal Industries2312-797X2312-7988Peoples’ Friendship University of Russia named after Patrice Lumumba1970010.22363/2312-797X-2021-16-4-326-336Research ArticleScreening of blast resistance genes in rice breeding samplesVozhzhovaNataliya N.<p>Candidate of Agricultural Sciences, senior researcher, Laboratory of marker selection</p>nvozhzh@gmail.comhttps://orcid.org/0000-0002-2046-4000ZhogalevaOlga S.<p>junior researcher, Laboratory of Marker Selection</p>os.zogaleva@mail.ruhttps://orcid.org/0000-0003-1477-3285KupreyshviliNatia T.<p>research technician, Laboratory of Marker Selection</p>kupreyshvilin@mail.ruhttps://orcid.org/0000-0002-1726-4390DubinaAngelina Y.<p>research technician, Laboratory of Marker Selection</p>angel.myshastaja@yandex.ruhttps://orcid.org/0000-0002-1432-7616KostylevPavel I.<p>Doctor of Agricultural Sciences, Head</p>p-kostylev@mail.ruhttps://orcid.org/0000-0002-4371-6848Agrarian Science Center ‘Donskoy’3012202116432633630122021Copyright © 2021, Vozhzhova N.N., Zhogaleva O.S., Kupreyshvili N.T., Dubina A.Y., Kostylev P.I.2021<p style="text-align: justify;">Rice is one of the most widespread and cultivated crops in the world. It is necessary to increase the yield of crops or expand their sown areas to resolve a food security problem in Russia. Current impossibility of expanding rice cultivated areas in the Rostov region and the need to maintain and increase its yield require developing new disease-resistant varieties. Rice genotypes with multiple blast resistance genes avoid significant yield losses. Since pyramiding and selection of resistance genes in the same genotype through traditional selection methods are complicated, it is urgent to search for homozygous samples using marker-assisted selection methods. This study was aimed to identify Pi-1, Pi-2, Pi-33 and Pi-ta blast resistance genes in breeding rice samples by MAS-methods. The study used CTAB-method for DNA-isolation, PCR, electrophoresis on agarose and polyacrylamide gels. The resulting gels were stained in a solution of ethidium bromide and photographed in ultraviolet light. To control the presence of blast resistance genes the following parental cultivars were used: C104LAC for the Pi-1 and Pi-33 genes, C101-A-51 for the Pi-2 gene, IR36 for the Pi-ta gene; Novator and Boyarin as controls of non-functional alleles of all studied genes. The 446 selection samples of the seventh generation were analyzed. As a result of the research, 127 rice samples that combine 2 or 3 different blast resistance genes were identified. The Pi-2 and Pi-33 genes combination was identified in 43 samples (1128/1, 1149/3, 1171/2, 1177/3, 1177/4, 1186/4, et al.). Samples with three resistance genes are the most interesting for selection and further breeding. For developing new blast-resistant varieties, we recommend using rice samples with the following combinations of resistance genes Pi-1+Pi-2+Pi-33 (1197/1, 1226/2, 1271/1, 1272/2), Pi-1+Pi-2+Pi-ta (1197/4, 1304/2, 1304/3, 1482/3, 1482/4, 1486/1) and Pi-2+Pi-33+Pi-ta (1064/1, 1064/3, 1281/2, 1281/3, 1281/4, 1282/2, 1283/1, 1283/2, 1284/3).</p>rice blastPi-1Pi-2Pi-33Pi-taresistance genesriceпирикуляриоз рисаPi-1Pi-2Pi-33Pi-taгены устойчивостирис<p style="text-align: justify;"></p>
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<div class="preview fancybox" style="text-align: center;"><a title="Рис. 1. Электрофореграмма скрининга образцов риса на наличие гена устойчивости к пирикуляриозу Pi-ta на агарозном геле: 1 Маркер молекулярного веса Thermo Scientific GeneRuler 50 bp (501000 п. н.); 2 H2O деионизированная (отрицательный контроль опыта); 3 IR36 (положительный контроль); 4 Д 7787/1; 5 Д 7787/2; 6 Д 7787/3; 7 Д 7787/4; 8 Д 7787/5; 9 Д 7787/6; 10 Д 7787/7; 11 Д 7787/8; 12 Д 7787/9; 13 Д 7787/10; 141064/1; 151064/2; 161064/3; 171064/4; 181065/1" href="/files/journals/1/articles/19700/supp/19700-22026-1-SP.jpg" rel="simplebox"><img style="max-height: 600px; max-width: 600px;" src="/files/journals/1/articles/19700/supp/19700-22026-1-SP.jpg" width="595" height="286" /></a></div>
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<p style="text-align: center;"><strong>Fig. 1. Electropherogram of screening rice samples for the presence of the Pi-ta blast resistance gene on agarose gel: 1 Thermo Scientific GeneRuler molecular weight marker 50 bp (501000 bp); 2 deionized H<sub>2</sub>O (negative control); 3 IR36 (positive control); 4 D7787/1; 5 D7787/2; 6 D7787/3; 7 D7787/4; 8 D7787/5; 9 D7787/6; 10 D7787/7; 11 D7787/8; 12 D7787/9; 13 D7787/10; 141064/1; 151064/2; 161064/3; 171064/4; 181065/1</strong></p>
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<div class="preview fancybox" style="text-align: center;"><a title="Рис. 2. Электрофореграмма скрининга образцов риса на наличие гена устойчивости к пирикуляриозу Pi-1 на полиакриламидном геле: 1 Маркер длин ДНК 50+ bp DNA Ladder Евроген (50700 п. н.); 2 C104-LAC (положительный контроль); 3 Д 7787/1; 4 Д 7787/2; 5 Д 7787/3; 6 Д 7787/4; 7 Д 7787/5; 8 Д 7787/6; 9 Д 7787/7; 10 Д 7787/8; 11 Д 7787/9; 12 Д 7787/10; 131141/1; 141141/2; 151141/3" href="/files/journals/1/articles/19700/supp/19700-22027-1-SP.jpg" rel="simplebox"><img style="max-height: 600px; max-width: 600px;" src="/files/journals/1/articles/19700/supp/19700-22027-1-SP.jpg" width="570" height="282" /></a></div>
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<p style="text-align: center;"><strong>Fig. 2. Electropherogram of screening rice samples for the presence of blast resistance gene Pi-1 on a polyacrylamide gel: 1 DNA marker 50+ bp DNA Ladder Evrogen (50700 bp); 2 C104-LAC (positive control); 3 D7787/1; 4 D7787/2; 5 D7787/3; 6 D7787/4; 7 D7787/5; 8 D7787/6; 9 D7787/7; 10 D7787 / 8; 11 D7787/9; 12 D7787/10; 131141/1; 141141/2; 151141/3</strong></p>
<p><em>Table 1. Distribution of alleles of Pi-1, Pi-2, Pi-33 and Pi-ta genes in breeding rice samples</em></p>
<table width="661">
<tbody>
<tr>
<td style="text-align: center;" rowspan="2" width="198">
<p>Alleles of the gene</p>
</td>
<td style="text-align: center;" colspan="4" width="463">
<p>Number of samples carrying the alleles of the gene</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="116">
<p>Pi-1</p>
</td>
<td style="text-align: center;" width="116">
<p>Pi-2</p>
</td>
<td style="text-align: center;" width="116">
<p>Pi-33</p>
</td>
<td style="text-align: center;" width="116">
<p>Pi-ta</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Functional homozygous (dominant)</p>
</td>
<td style="text-align: center;" width="116">
<p><strong>54</strong></p>
</td>
<td style="text-align: center;" width="116">
<p>136</p>
</td>
<td style="text-align: center;" width="116">
<p>192</p>
</td>
<td style="text-align: center;" width="116">
<p>131</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Heterozygous</p>
</td>
<td style="text-align: center;" width="116">
<p>1</p>
</td>
<td style="text-align: center;" width="116">
<p><strong>5</strong></p>
</td>
<td style="text-align: center;" width="116">
<p>16</p>
</td>
<td style="text-align: center;" width="116">
<p><strong>6</strong></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Non-functional homozygous (recessive)</p>
</td>
<td style="text-align: center;" width="116">
<p>204</p>
</td>
<td style="text-align: center;" width="116">
<p>285</p>
</td>
<td style="text-align: center;" width="116">
<p>121</p>
</td>
<td style="text-align: center;" width="116">
<p>285</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Allele was not identified</p>
</td>
<td style="text-align: center;" width="116">
<p>247</p>
</td>
<td style="text-align: center;" width="116">
<p>80</p>
</td>
<td style="text-align: center;" width="116">
<p>177</p>
</td>
<td style="text-align: center;" width="116">
<p>24</p>
</td>
</tr>
</tbody>
</table>
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<div class="preview fancybox" style="text-align: center;"><a title="Рис. 3. Распределение образцов риса по количеству сочетаемых генов устойчивости к пирикуляриозу" href="/files/journals/1/articles/19700/supp/19700-22028-1-SP.jpg" rel="simplebox"><img style="max-height: 600px; max-width: 600px;" src="/files/journals/1/articles/19700/supp/19700-22028-1-SP.jpg" /></a></div>
</center>
<p style="text-align: center;"><strong>Fig. 3. Distribution of rice samples by the number of combined blast resistance genes</strong></p>
<p><em>Table 2. Identified rice samples having multiple blast resistance genes</em></p>
<table width="659">
<tbody>
<tr>
<td style="text-align: center;" width="198">
<p>Combination of resistance genes</p>
</td>
<td style="text-align: center;" width="141">
<p>Number of samples</p>
</td>
<td style="text-align: center;" width="321">
<p>Name of samples</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi1+2</p>
</td>
<td style="text-align: center;" width="141">
<p><strong>5</strong></p>
</td>
<td style="text-align: center;" width="321">
<p>1270/4, 1271/2, 1293/3, 1318/3, 1371/1</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi1+33</p>
</td>
<td style="text-align: center;" width="141">
<p>12</p>
</td>
<td style="text-align: center;" width="321">
<p>1231/4, 1263/3, 1268/3, 1277/1, 1277/2, 1277/3, 1322/1, 1323/1, 1323/2, 1323/3, 1323/4, 1457/2</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi1+ta</p>
</td>
<td style="text-align: center;" width="141">
<p>9</p>
</td>
<td style="text-align: center;" width="321">
<p>1141/1, 1304/4, 1468/1, 1468/2, 1476/1, 1476/2, 1486/2, 1486/3, 1486/4</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi2+33</p>
</td>
<td style="text-align: center;" width="141">
<p><strong>43</strong></p>
</td>
<td style="text-align: center;" width="321">
<p>1128/1, 1149/3, 1171/2, 1177/3, 1177/4, 1186/4 et al.</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi2+ta</p>
</td>
<td style="text-align: center;" width="141">
<p>22</p>
</td>
<td style="text-align: center;" width="321">
<p>1064/2, 1064/4, 1135/4, 1141/3, 1141/4, 1151/1 et al.</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi33+ta</p>
</td>
<td style="text-align: center;" width="141">
<p>16</p>
</td>
<td style="text-align: center;" width="321">
<p>1065/2, 1242/2, 1242/3, 1242/4, 1281/1, 1281/3, 1281/4 et al.</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi33+b</p>
</td>
<td style="text-align: center;" width="141">
<p>1</p>
</td>
<td style="text-align: center;" width="321">
<p>1127/2</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi1+2+33</p>
</td>
<td style="text-align: center;" width="141">
<p><strong>4</strong></p>
</td>
<td style="text-align: center;" width="321">
<p>1197/1, 1226/2, 1271/1, 1272/2</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi1+2+ta</p>
</td>
<td style="text-align: center;" width="141">
<p><strong>6</strong></p>
</td>
<td style="text-align: center;" width="321">
<p>1197/4, 1304/2, 1304/3, 1482/3, 1482/4, 1486/1</p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="198">
<p>Pi2+Pi33+Pi-ta</p>
</td>
<td style="text-align: center;" width="141">
<p>9</p>
</td>
<td style="text-align: center;" width="321">
<p>1064/1, 1064/3, 1281/2, 1281/3, 1281/4, 1282/2, 1283/1, 1283/2, 1284/3</p>
</td>
</tr>
</tbody>
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