Resistance Gene Pi-ta Research Articles

Using molecular markers to assist in the selection of the rice blast resistance genes Pi1, Pi2, and Pi-ta in rice wide compatibility restorer lines

Hybridization was performed between the rice materials carrying the rice blast resistance genes Pi1, Pi2, and Pi-ta (R283) as the maternal parent and the broad compatibility restorer line R110 as the paternal parent. Molecular marker-assisted breeding techniques were used to detect the target genes in segregating generations. Through field selection and resistance identification, three stable lines with multiple resistance, strong restorer ability, and excellent agronomic traits were bred. Their agronomic and economic traits were analyzed along with their resistance to different physiological races of rice blast. These three stable lines were then crossed with seven different sterile lines (Chunjiang 23A, FA1, FA3, FA4, 81, 57, and 212A) to analyze the agronomic traits and resistance to rice blast pathogens in the hybrid combinations. JR11, JR12, and JR13 were stable, homozygous polymerized lines carrying Pi1, Pi2, and Pi-ta resistance genes and exhibited resistance frequencies ranging from 90.19 to 96.08% against different physiological races of rice blast disease. This indicates the excellent resistance of these three stable lines to rice blast disease. The seed-set frequencies in the hybrid combinations of the seven sterile lines with the three stable lines ranged from 66.4 to 84.1%, showing strong recovery ability. The yield of the selected combination of 23 A/JR11, 81 A/JR11, and 212 A/JR13 was 2.2–2.3% higher than that of the control Yongyou 1540. The research results showed that the three stable aggregated rice blast resistance genes Pi1, Pi2, and Pi-ta rice restorer lines created have good practical value in production.

Pyramiding of bacterial blight resistance genes into promising restorer BRRI31R line through marker-assisted backcross breeding and evaluation of agro-morphological and physiochemical characteristics of developed resistant restorer lines.

BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker-assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh.

Identification of rice blast resistance genes Pi-1, Pi-2, Pi-33, Pi-40, Pi-ta, Pi-b

Rice is a valuable crop used for food throughout the world. To develop modern, productive and disease resistant rice varieties, it is necessary to accelerate the breeding process using molecular biology methods. The purpose of the current study was to identify alleles of six blast resistance genes (Pi-1, Pi-2, Pi-33, Pi-40, Pi-ta and Pi-b) in the selected rice samples using MAS (marker-associated selection method). The objects of the study were 446 breeding rice samples sent to the laboratory of cell breeding for analysis by breeders from the laboratory for rice breeding and seed production of the FSBSI «ARC «Donskoy». Identification of genes in them was carried out differentially and was determined by the pedigree of the samples. In order to determine the alleles of the blast resistance genes Pi-1, Pi-2 and Pi-33, all 446 samples were studied, for the Pi-40 gene there were studied 20 samples, for the Pi-ta gene there were studied 316 samples. For DNA extraction, there was used a Russian-made kit ‘DNA-Extran-3’. PCR was performed using specific primers of the target genes. Identification of reaction products was performed on agarosegels after photographing in ultraviolet light. As a result of the study, there were identified the rice samples which carried from 1 to 5 blast resistance genes in various combinations. There have been identified 14 samples that possess a set of 5 resistance genes, such as 2723, 2724, 2727, 2728, 2729, 2730, 2733, 2735, 2736, 5007, 5671, 5673, 5450/2 and 2450/2. The information obtained from the results of the study could then be used by breeders to use valuable genotypes as donors in crosses, as well as to select promising breeding material resistant to blast disease.

Molecular Marker Assisted Breeding of a New &lt;i&gt;Japonica&lt;/i&gt; Hybrid Rice

The development of Japonica &nbsp;hybrid rice is of great significance to ensure supply of good&nbsp;quality Japonica &nbsp;rice and food security in China. Aiming at the problems of late growth period, unstable yield, and the need to improve rice quality and seed production yield of Japonica &nbsp;hybrid rice, a new Japonica &nbsp;hybrid rice combination 'Shenyou28' with early maturity, good&nbsp;quality, blast resistance, stable and high yield and easy seed production was developed by using molecular breeding techniques including high-density rice gene chip. 'Shenyou28', bred by Shanghai Academy of Agricultural Sciences, is a three-line Japonica &nbsp;hybrid rice with 'Shen21A' as sterile line and 'Shenhui26-28' as restorer line. For two-year regional trials&nbsp;from 2018 to 2019, the average output were 10 588.5 kg/hm 2 , which was 11.6% higher than the control variety 'Huayou14'. For the one-year production trial, the output reached 10 473.0 kg/hm 2 , which was 4.8% higher than the control variety 'Huayou14'. 'Shenyou28' has good&nbsp;quality, reaching the second grade of national standard, and has fragrance because it carries fragrance gene badh2-p-5 &rsquo; UTR . It also has good resistance to rice blast and bacterial blight because it aggregates the rice blast resistance genes Pi2 , Pita , Pib , Pi9 , Pi54 , Pikm , Pit , and bacterial blight resistance gene Xa21 . It was approved by the Crop Variety Certification Committee of Shanghai in 2020. The breeding process, variety characteristics and key points of high yield mechanized seed production technology of 'Shenyou28' are introduced in this study.

Rice Blast Resistance Genes Pi54 and Pita Show Single Nucleotide Polymorphisms in an Advanced 4-Way Crossed Segregating Population

Rice Blast Resistance Genes Pi54 and Pita Show Single Nucleotide Polymorphisms in an Advanced 4-Way Crossed Segregating Population

Frequencies and Variations of Magnaporthe oryzae Avirulence Genes in Hunan Province, China.

Rice blast caused by Magnaporthe oryzae poses significant threaten to rice production. For breeding and deploying resistant rice varieties, it is essential to understand the frequencies and genetic variations of avirulence (AVR) genes in the pathogen populations. In this study, 444 isolates were collected from Hunan Province, China in 2012, 2015, and 2016, and their pathogenicity was evaluated by testing them on monogenic rice lines carrying resistance genes Pita, Pizt, Pikm, Pib, or Pi9. The frequencies of corresponding AVR genes AVRPizt, AVRPikm, AVRPib, AVRPi9, and AVRPita were characterized by amplification and sequencing these genes in the isolates. Both Pi9 and Pikm conferred resistance to >75% of the tested isolates, while Pizt, Pita, and Pib were effective against 55.63, 15.31, and 3.15% of the isolates, respectively. AVRPikm and AVRPi9 were detected in 90% of the isolates and AVRPita, AVRPizt, and AVRPib were present in 26.12, 66.22, and 79% of the isolates, respectively. Sequencing of AVR genes showed that most mutations were single nucleotide polymorphisms, transposon insertions, and insertion mutations. The variable sites of AVRPikm and AVRPita were mainly located in the coding sequence regions (CDS), and most were synonymous mutations. A 494-bp Pot2 transposon sequence insertion was found at the 87 bp position upstream of the start codon in AVRPib. Noteworthy, although no mutations were found in CDS of AVRPi9, a GC-rich inserted sequence of ∼200 bp was found at the 1,272 bp position upstream of the start codon in three virulent isolates. As AVRPikm and AVRPi9 were widely distributed with low genetic variation in the pathogen population, Pikm and Pi9 should be promising genes for breeding rice cultivars with blast resistance in Hunan.

Rice Breeding in Russia Using Genetic Markers.

The article concentrates on studying tolerance to soil salinization, water flooding, and blast in Russian and Asian rice varieties, as well as hybrids of the second and third generations from their crossing in order to obtain sustainable paddy crops based on domestic varieties using DNA markers. Samples IR 52713-2B-8-2B-1-2, IR 74099-3R-3-3, and NSIC Rc 106 were used as donors of the SalTol tolerance gene. Varieties with the Sub1A locus were used as donors of the flood resistance gene: Br-11, CR-1009, Inbara-3, TDK-1, and Khan Dan. The lines C101-A-51 (Pi-2), C101-Lac (Pi-1, Pi-33), IR-58 (Pi-ta), and Moroberekan (Pi-b) were used to transfer blast resistance genes. Hybridization of the stress-sensitive domestic varieties Novator, Flagman, Virazh, and Boyarin with donor lines of the genes of interest was carried out. As a result of the studies carried out using molecular marking based on PCR in combination with traditional breeding, early-maturing rice lines with genes for resistance to salinity (SalTol) and flooding (Sub1A), suitable for cultivation in southern Russia, were obtained. Introgression and pyramiding of the blast resistance genes Pi-1, Pi-2, Pi-33, Pi-ta, and Pi-b into the genotypes of domestic rice varieties were carried out. DNA marker analysis revealed disease-resistant rice samples carrying 5 target genes in a homozygous state. The created rice varieties that carry the genes for blast resistance (Pentagen, Magnate, Pirouette, Argamac, Kapitan, and Lenaris) were submitted for state variety testing. The introduction of such varieties into production will allow us to avoid epiphytotic development of the disease, preserving the biological productivity of rice and obtaining environmentally friendly agricultural products.

Analysis of the Relationship between Blast Resistance Genes and Disease Resistance of Rice Germplasm via Functional Molecular Markers

Rice blast disease is one of the most devastating diseases of rice (Oryza sativa L.) caused by the fungus Magnaporthe oryzae (M. oryzae), and neck blast is the most destructive phase of this illness. The underlying molecular mechanisms of rice blast resistance are not well known. Thus, we collected 150 rice varieties from different ecotypes in China and assessed the rice blast resistances under the natural conditions that favoured disease development in Jining, Shandong Province, China in 2017. Results showed that 92 (61.3%) and 58 (38.7%) rice varieties were resistant and susceptible to M. oryzae, respectively. Among the 150 rice varieties screened for the presence of 13 major blast resistance (R) genes against M. oryzae by using functional markers, 147 contained one to eight R genes. The relationship between R genes and disease response was discussed by analysing the phenotype and genotype of functional markers. The results showed that the rice blast resistance gene Pita was significantly correlated with rice blast resistance. Our results provided a basis for the further understanding of the distribution of 13 major R genes of rice blast in the germplasm resources of the tested rice varieties, and were meaningful for rice disease resistance breeding.

Breeding Hybrid Rice with Genes Resistant to Diseases and Insects Using Marker-Assisted Selection and Evaluation of Biological Assay

Developing elite hybrid rice varieties is one important objective of rice breeding programs. Several genes related to male sterilities, restores, and pollinators have been identified through map-based gene cloning within natural variations of rice. These identified genes are good targets for introducing genetic traits in molecular breeding. This study was conducted to breed elite hybrid lines with major genes related to hybrid traits and disease/insect resistance in 240 genetic resources and F1 hybrid combinations of rice. Molecular markers were reset for three major hybrid genes (S5, Rf3, Rf4) and thirteen disease/insect resistant genes (rice bacterial blight resistance genes Xa3, Xa4, xa5, Xa7, xa13, Xa21; blast resistance genes Pita, Pib, Pi5, Pii; brown planthopper resistant genes Bph18(t) and tungro virus resistance gene tsv1). Genotypes were then analyzed using molecular marker-assisted selection (MAS). Biological assay was then performed at the Red River Delta region in Vietnam using eleven F1 hybrid combinations and two control vatieties. Results showed that nine F1 hybrid combinations were highly resistant to rice bacterial blight and blast. Finally, eight F1 hybrid rice varieties with resistance to disease/insect were selected from eleven F1 hybrid combinations. Their characteristics such as agricultural traits and yields were then investigated. These F1 hybrid rice varieties developed with major genes related to hybrid traits and disease/insect resistant genes could be useful for hybrid breeding programs to achieve high yield with biotic and abiotic resistance.

Virulence structure of Magnaporthe oryzae populations from Fujian Province, China

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. The aim of the present study was to elucidate the virulence structure of M. oryzae populations in Fujian Province, China, over the period 2006–2015. For this purpose, 456 M. oryzae isolates collected from diverse cultivars from eight rice cropping regions in Fujian were screened for the presence of 11 known avirulence genes: Avr-Pik, Avr-Pita1, Avr-Pita2, Avr-Pita3, PWL2, Avr1-CO39, ACE1, Avr-Piz-t, Avr-Pia, Avr-Pii and Avr-Pikm, with gene-specific molecular markers. The results showed that Avr-Pik and Avr-Pita3 occurred at the highest frequency (94.5% and 91.7%, respectively), while Avr1-CO39 and Avr-Pii were not detected. Further, the remaining avirulence genes occurred at frequencies ranging from 5.9% to 89.0%. Temporal population dynamics revealed that Avr-Pik was uniformly distributed in all 10 years, at frequencies of more than 87.5%. Spatial distribution analysis showed avirulence genes were present at different frequencies among the geographic regions. In addition, 24 rice monogenic lines of IRRI-Japan with known blast resistance genes were inoculated to assess the virulence of 60 isolates. The results revealed that the resistance gene Pik showed the broadest resistance spectrum to the isolates tested and therefore would be the most useful in rice blast resistance breeding. The resistance genes Pi-z5, Pi-1(1), Pi-kp, Pi-9(t), Pi-ta(1) and Pi-kh also were effective and would therefore also be of value to resistance breeding programmes. The present study provides information that should be useful for the development and deployment of rice blast resistant cultivars in Fujian Province.

Marker-assisted introgression of three dominant blast resistance genes into an aromatic rice cultivar Mushk Budji

Modern high yielding rice varieties have replaced most of the traditional cultivars in recent past. Mushk Budji, is one such short grained landrace known for its aroma and exquisite quality, however, is highly susceptible to blast disease that has led to considerable decline in its area. Mushk Budji was crossed to a triple-gene donor line, DHMAS 70Q 164-1b and followed through marker-assisted foreground and background selection in first and second backcross generations that helped to incorporate blast resistance genes Pi54, Pi1 and Pita. Marker-assisted background selection was carried out using 78 SSR and STS markers that helped to reduce linkage drag around the genes Pi54, Pi1 and Pita to 2.74, 4.60 and 2.03 Mb, respectively. The three-gene lines in BC2F2:3 were genotyped using 50 K SNP chip and revealed more than 92% genome similarity to the RP. 2-D gel assay detected differentially expressing 171 protein spots among a set of backcross derived lines, of which 38 spots showing match score of 4 helped us to calculate the proteome recovery. MALDI-TOF analysis helped to detect four significant proteins that were linked to quality and disease resistance. The improved lines expressed resistance to blast under artificial and natural field conditions.

Insights on Diversity of Leucine-Rich Repeat (LRR) Domain among Major Blast Resistance Genes of Rice

Rice blast is a continuous threat in rice ecosystems across the globe; its dynamics is becoming complex in the changing climatic conditions. Host resistance is still a viable option; hence, the exploration of resistance genes and their novel alleles is indispensable. The majority of the blast resistance genes belong to nucleotide-binding site and leucine-rich repeat (LRR) domain. In the present study, diverse LRR alleles of five major blast resistance genes (Pi2, Pi9, Pib, Pita, and Pi37) were cloned from 13 different Oryza species to determine the nucleotide diversity as well as to identify the single nucleotide polymorphisms, InDels, conserved domains, and protein functional sites. Although Pi9 and Pi2 are homologous genes, significant nucleotide variations and variants in the motifs distribution were observed. Among the five genes, Pi37 showed the highest nucleotide diversity and Pita showed the least diversity. The phylogenetic groups of alleles were correlated with the identified haplotypes. The motif (xxLxLxx) was present among all the alleles of blast R gene sequences across various Oryza species, indicating its importance. The appearance of post-translational modification sites in the protein sequences of these alleles also indicates its nature of involvement in host–pathogen interactions. The present study offers clues in further understanding the molecular evolution of the LRR domain of resistance genes, which is a key determinant of host–pathogen interactions.

Analysis of the diversity and function of the alleles of the rice blast resistance genes Piz-t, Pita and Pik in 24 rice cultivars

Understanding the sequence diversity of rice blast resistance genes is important for breeding new resistant rice cultivars against the rice blast fungus Magnaporthe oryzae. In this study, we selected 24 rice cultivars with different genetic backgrounds to study the allelic diversity of rice blast resistance genes Piz-t, Pita and Pik. For Piz-t, a total of 17 allelic types were found within the 24 cultivars. Blast inoculations showed that most of the mutations can affect the function of the resistance gene. For Pita, except for the difference at the 918th amino acid, a majority of the 21 mutations were detected among the cultivars. Inoculations with blast isolates carrying Avr-Pita revealed that cultivars with mutations in other sites except for the 918th amino acid did not affect the function of the Pita gene. For Pik, a total of six allelic types were found within the 24 cultivars, but five of them lost the function of the resistance gene. In addition, we found that Piz-t, Pita and Pik were expressed constitutively in the 24 rice cultivars and the expression level was not related to resistance. Our results have provided the sequence diversity information of the resistance genes Piz-t, Pita and Pik among the popular rice cultivars grown in the northeast region of China.

Marker assisted pyramiding of major blast resistance genesPi9andPitain the genetic background of an elite Basmati rice variety, Pusa Basmati 1

Basmati is a premium quality rice of India which is highly priced in the international market. Pusa Basmati 1, an elite Basmati rice variety is highly susceptible to rice blast caused by Magnaporthe oryzae. Therefore, pyramiding blast resistance genes is essential to effectively combat the blast disease and increase the durability of resistance genes. The blast resistance genes Pi9 and Pita have been earlier demonstrated to be effective in Basmati growing regions of the country. Therefore, in the present study, monogenicnear isogenic lines Pusa 1637-18-7-6-20 and Pusa 1633-3-88-16-1 carrying Pi9 and Pita, respectively, were intercrossed to generate pyramided lines through marker assisted foreground, background and phenotypic selection for recurrent parent phenotype. The pyramided lines carrying Pi9+Pita were found to be either at par or superior to the recurrent parent Pusa Basmati 1 for agro-morphological, grain and cooking quality traits. Further, these pyramided lines were also found to show resistance against three virulent pathotypes of M. oryzae namely, Mo-nwi-kash 1, Mo-nwi-lon2 and Mo-ei-ran1, when evaluated under artificial inoculation conditions as well as in the natural epiphytotic conditions of uniform blast nursery at two locations. The developed pyramided lines are the potential sources of blast resistance genes in the Basmati improvement program and can also be released for commercial cultivation after required testing.

Statistical inference of selection and divergence of the rice blast resistance gene Pi-ta.

The resistance gene Pi-ta has been effectively used to control rice blast disease, but some populations of cultivated and wild rice have evolved resistance. Insights into the evolutionary processes that led to this resistance during crop domestication may be inferred from the population history of domesticated and wild rice strains. In this study, we applied a recently developed statistical method, time-dependent Poisson random field model, to examine the evolution of the Pi-ta gene in cultivated and weedy rice. Our study suggests that the Pi-ta gene may have more recently introgressed into cultivated rice, indica and japonica, and U.S. weedy rice from the wild species, O. rufipogon. In addition, the Pi-ta gene is under positive selection in japonica, tropical japonica, U.S. cultivars and U.S. weedy rice. We also found that sequences of two domains of the Pi-ta gene, the nucleotide binding site and leucine-rich repeat domain, are highly conserved among all rice accessions examined. Our results provide a valuable analytical tool for understanding the evolution of disease resistance genes in crop plants.

Effectiveness and Durability of the Rice Pi-ta Gene in Yunnan Province of China

Rice blast is one of the most damaging diseases of rice worldwide. In the present study, we analyzed DNA sequence variation of avirulence (AVR) genes of AVR-Pita1 in field isolates of Magnaporthe oryzae in order to understand the effectiveness of the resistance gene Pi-ta in China. Genomic DNA of 366 isolates of M. oryzae collected from Yunnan province of China were used for polymerase chain reaction (PCR) amplification to examine the existence of AVR-Pita1 using gene-specific PCR markers. Results of PCR products revealed that 218 isolates of M. oryzae carry AVR-Pita1. Among of them, 62.5, 56.3, 58.5, 46.7, 72.4, and 57.4% of M. oryzae carry AVR-Pita1 from northeastern, southeast, western, northwest, southwestern, and central Yunnan province, respectively. The detection rate of AVR-Pita1 was, in order: southwestern &gt; northeastern &gt; western &gt; central &gt; southeastern &gt; northwestern Yunnan province. Moreover, in total, 18 AVR-Pita1 haplotypes encoding 13 novel AVR-Pita1 variants were identified among 60 isolates. Most DNA sequence variation was found to occur in the exon region, resulting in amino acid substitution. Six virulent haplotypes of AVR-Pita1 to Pita were identified among 60 field isolates. The AVR-Pita1 has evolved to virulence from avirulent origins via base substitution. These findings demonstrate that AVR-Pita1 is under positive selection and mutations of AVR-Pita1 are responsible for defeating race-specific resistance in nature.

Stacking of five favorable alleles for amylase content, fragrance and disease resistance into elite lines in rice (Oryza sativa) by using four HRM-based markers and a linked gel-based marker

Marker-assisted selection (MAS) for qualitative traits such as grain quality and resistance to certain diseases has proven to be highly effective. Multiple genes responsible for various quality components and disease resistances can be simultaneously stacked to boost the performance and to lengthen the commercial lifespan of high-yield varieties. Grain quality genes (fgr and Wx) and three disease resistance genes (Pita, Pik and Xa23) have been well characterized and used in MAS breeding. However, stacking all of them together into a single variety has not been reported. We reported here the stacking of the five genes into elite lines in rice. We achieved this through the development of functional markers from causal mutations at fgr, Wx and Pita, a gene-targeted marker at Pik and the use of a linked marker for Xa23. We employed and optimized the high-resolution melting (HRM) analysis method for use as the genotyping platform of fgr, Wx, Pik and Pita. By combining high-throughput DNA isolation, multiplex and nested-PCR methods, we showed that HRM could serve as cost-effective, highly automated, moderate-throughput and reliable non-gel genotyping platform for a small-scale MAS program.

Studies on mechanisms of adaptation of the blast fungus to rice resistance genes

The genus Pyricularia is the causal agent of blast diseases of monocot species. The most familiar species are P. grisea pathogenic on crabgrass and P. oryzae pathogenic on cultivated gramineous plants. Pyricularia oryzae is composed of several host-specific subgroups such as Triticum isolates pathogenic on wheat, Panicum isolates pathogenic on common millet, Setaria isolates pathogenic on foxteil millet, Oryza isolates pathogenic on rice, and so on (Hirata et al. 2007; Kato et al. 2000). Rice-blast-resistant cultivars were first developed by introducing resistance genes and began to be cultivated widely in Japanese farmer’s fields in the early 1960s. Within a few years, however, the resistance broke down due to rapid adaptation of the pathogen; that is, new races evolved that overcame the introduced resistance genes. The question is, how do plant pathogens adapt to new resistance genes so quickly? To obtain clues toward answering this question, we focused on evolutionary processes of a subtelomeric avirulence effector gene, AVR-Pita (Orbach et al. 2000), which is involved in race-cultivar specificity and corresponds to the rice resistance gene Pita. Chromosomal location of AVR-Pita

Resistance spectrum assay and fine mapping of the blast resistance gene from a rice experimental line, IRBLta2-Re

In the present study, we performed the resistance assessment by rice blast inoculation on IRBLta2-Re and IRBLta-CP1, the experimental lines supposed to carry rice blast resistance genes Pita2 and Pita, respectively. The analysis by using 196 rice blast isolates derived from China indicated that the resistance spectrum of IRBLta2-Re was broader than that of IRBLta-CP1. Both IRBLta2-Re and IRBLta-CP1 have the Pita gene by analyzing the functional single amino acid difference of Pita/pita locus. To identify the additional gene in IRBLta2-Re, 1250 F2 individuals from the cross between CO39 and IRBLta2-Re were used as the mapping population. The F2 population was inoculated with the blast isolate 08-T4 which was incompatible to IRBLta2-Re, but compatible to CO39 and IRBLta-CP1. In the phenotypic data analysis, the F2 population segregated in a 3:1 ratio for resistant and susceptible plants, respectively, suggesting that IRBLta2-Re has an additional resistance gene other than Pita, which was tentatively designated Pita3(t) (supposed to be Pita2). To identify the Pita3(t), a total of 50 microsatellite and 12 position specific microsatellite markers distributed by two sides of the Pita gene were selected in the parent polymorphism screening. The results showed that PT4 and PT5 were co-segregated with the target gene. A contig map corresponding to the resistance gene and Pita genes was constructed based on the fine mapping and bioinformatics assay. The resistance gene, Pita3(t), was, thus, assumed to be in an interval of approximately 178 kb which containing a total of 5 NBS–LRR genes, and was about 500 kb away from the Pita gene.

Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo-devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion-deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi(non/syn) was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.