Screening of blast resistance genes in rice breeding samples

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).

Mapping Blast Resistance Genes in Rice Varieties ‘Minghui 63’ and ‘M-202’

Rice blast disease caused by the fungus Magnaporthe oryzae (syn. M. grisea) is one of the most lethal diseases for sustainable rice production worldwide. Blast resistance mediated by major resistance genes are often broken-down after a short period of deployment, while minor blast resistance genes, each providing a small effect on disease reactions, are more durable. In the present study, we first evaluated disease reactions of two rice breeding parents ‘Minghui 63’ and ‘M-202’ with 11 US blast races, IA45, IB1, IB45, IB49, IB54, IC1, IC17, ID1, IE1, IG1, and IH1 commonly found under greenhouse conditions using a category disease rating resembling infection types under field conditions. ‘Minghui 63’ exhibited differential resistance responses in comparison with that of ‘M-202’ to the tested blast races. A recombinant inbred line (RIL) population of 275 lines from a cross between ‘Minghui 63’ and ‘M-202’ was also evaluated with the above mentioned blast races. The population was genotyped with 156 simple sequence repeat (SSR) and insertion and deletion (Indel) markers. A linkage map with a genetic distance of 1022.84 cM was constructed using inclusive composite interval mapping (ICIM) software. A total of 10 resistance QTLs, eight from ‘Minghui 63’ and two from ‘M-202’, were identified. One major QTL, qBLAST2 on chr 2, was identified by seven races/isolates. The remaining nine minor resistance QTLs were mapped on chromosome 1, 3, 6, 9, 10, 11 and 12. These findings provide useful genetic markers and resources to tag minor blast resistance genes for marker assisted selection in rice breeding program and for further studies of underlying genes.

Predicting Protein–Protein Interactions Between Rice and Blast Fungus Using Structure-Based Approaches

Rice blast, caused by the fungus Magnaporthe oryzae, is the most devastating disease affecting rice production. Identification of protein–protein interactions (PPIs) is a critical step toward understanding the molecular mechanisms underlying resistance to blast fungus in rice. In this study, we presented a computational framework for predicting plant–pathogen PPIs based on structural information. Compared with the sequence-based methods, the structure-based approach showed to be more powerful in discovering new PPIs between plants and pathogens. Using the structure-based method, we generated a global PPI network consisted of 2,018 interacting protein pairs involving 1,344 rice proteins and 418 blast fungus proteins. The network analysis showed that blast resistance genes were enriched in the PPI network. The network-based prediction enabled systematic discovery of new blast resistance genes in rice. The network provided a global map to help accelerate the identification of blast resistance genes and advance our understanding of plant–pathogen interactions.

Rice Blast Disease in India: Present Status and Future Challenges

Rice (Oryza sativa L.) is the staple food of the majority of Indians, and India is both the major producer and consumer of rice. Rice cultivation in India is confronted with diverse agro-climatic conditions, varying soil types, and several biotic and abiotic constraints. Among major fungal diseases of Rice in India, the blast caused by Magnaporthe oryzae is the most devastating disease, with the neck blast being the most destructive form. Most of the blast epidemic areas in India have been identified with a mixture of races blast fungus resulting in the resistance breakdown in a short period. At present, a more significant number of the rice varieties cultivated in India were bred by conventional breeding methods with blast resistance conferred by a single resistance gene. Therefore, the blast disease in India is predominantly addressed by the use of ecologically toxic fungicides. In line with the rest of the world, the Indian scientific community has proven its role by identifying several blast resistance genes and successfully pyramiding multiple blast resistance genes. Despite the wealth of information on resistance genes and the availability of biotechnology tools, not a great number of rice varieties in India harbor multiple resistance genes. In the recent past, a shift in the management of blast disease in India has been witnessed with a greater focus on basic research and modern breeding tools such as marker-assisted selection, marker-assisted backcross breeding, and gene pyramiding.

Marker-assisted development and evaluation of monogenic lines of rice cv. Kaohsiung 145 carrying blast resistance genes

Rice blast is a serious threat to global rice production. Large-scale and long-term cultivation of rice varieties with a single blast resistance gene usually leads to breakdown of resistance. To effectively control rice blast in Taiwan, marker-assisted backcrossing was conducted to develop monogenic lines carrying different blast resistance genes in the genetic background of an elite japonica rice cultivar, ‘Kaohsiung 145’ (KH145). Eleven International Rice Research Institute (IRRI)-bred blast-resistant lines (IRBLs) showing broad-spectrum resistance to local Pyricularia oryzae isolates were used as resistance donors. Sequencing analysis revealed that the recurrent parent, KH145, does not carry known resistance alleles at the target Pi2/9, Pik, Pita, and Ptr loci. For each IRBL x KH145 cross, we screened 21–370 (average of 108) plants per generation from the BC1F1 to BC3F1/BC4F1 generation. A total of 1499 BC3F2/BC4F2 lines carrying homozygous resistance alleles were selected and self-crossed for 4–6 successive generations. The derived lines were also evaluated for background genotype using genotyping by sequencing, for blast resistance under artificial inoculation and natural infection conditions, and for agronomic performance in multiple field trials. In Chiayi and Taitung blast nurseries in 2018–2020, Pi2, Pi9, and Ptr conferred high resistance, Pi20 and Pik-h moderate resistance, and Pi1, Pi7, Pik-p, and Pik susceptibility to leaf blast; only Pi2, Pi9, and Ptr conferred effective resistance against panicle blast. The monogenic lines showed similar agronomic traits, yield, and grain quality as KH145, suggesting the potential of growing a mixture of lines to achieve durable resistance in the field.

Phenotypic evaluation of DH-lines developed on the base of rice samples with blast resistance genes

Gametoclonal changes are an additional source of genetic variation suitable for use in crop improvement programs for a range of agronomic traits. The aim of the study was to accelerate development of genetically stable DH rice lines with high morphological and genetic uniformity by the method of experimental haploidy in vitro based on breeding valuable samples with specified characteristics and carrying genes of a wide spectrum of blast resistance, as well as samples with racespecific genes for resistance to the pathogen, and subsequent assessment of agronomic traits of androgenic lines. The data on the callusogenic and regenerative capacity of 30 genotypes of domestic rice and samples of Chinese introduction with genes for blast resistance in anther culture in vitro are presented. Genetically stable (homozygous) androgenic lines were developed on the basis of these breeding valuable samples. The characteristics of economic and biological traits and elements of panicle productivity of 30 DH-line developed from three samples with blast resistance genes are given. The variability of traits arising as a result of gametoclonal variability in androgenic lines was noted for such traits as plant height, panicle length, blast resistance, and weight of 1000 grains.

Development of rice variety with blast resistance genes Pi-b and Pi-z basing on DNA-technologies

The article presents stages of development and testing new rice variety Nautilus with blast resistance genes Pi-b and Pi-z. Basing on the use of DNA-marking methods, breeding material carrying the target genes was developed. The obtained material was studied in the links of breeding process in the field. Based on the results of field testing, 6 lines were identified, of which VNIIR 6016 turned out to be the best in terms of a complex of economically valuable traits. In 2016, this sample was transferred to the State variety testing under the name Nautilus. In 2019, a new rice variety Nautilus was included in the register of breeding achievements and approved for use in the Russian Federation in Krasnodar region. The study of variety Nautilus in 2019-2020 in an ecological test confirmed its high resistance to blast, in comparison with other released varieties. It took 10 - 12 years to develop the variety.