Gene Pi-ta Research Articles

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.

Detection of Blast Resistant Gene in Rice by Host-pathogen Interaction and DNA-Marker

Rice blast disease caused by Pyricularia oryzae is a major disease in Bangladesh. In the search of resistant source against P. oryzae, resistant gene (R-gene) was assessed by (i) host-pathogen interaction and (ii) PCR-based screening at the laboratory of Bangladesh Rice Research Institute. One hundred and forty four plant materials were classified into 10 cultivar groups (CG) based on their reaction patterns to four distinct blast isolates (SDBI; standard differential blast isolate) H-11-64, H-1-8, H-1-1 and H-11-67. The reaction patterns of plant materials to these four standard blast isolates indicated presence of Pish, Pi9, Pita2 and Pita genes and their combination in the genetic background of tested plant materials. Gene specific primer 195R-1/195F-1, Pita440 (YL153)/Pita440 (YL154), OSM89 and AOL45/AOL48 were used to identify and confirm the presence of Pi9, Pita, Pita2 and Pish genes in plant materials, respectively through PCR-based assay. Plant materials, H13, H23, H25, H35, H47, H49, H136, H138 harbored all three genes, Pita, Pita2 and Pish in their genetic background. The Pi9 gene together with Pita2 was detected in local rice cultivar H100 and H129, which were confirmed by DNA analysis in PCR assay. These materials could be used in gene pyramiding in promising high yielding cultivar for durable blast resistance.

Growth phenotype analysis of heme synthetic enzymes in a halophilic archaeon, Haloferax volcanii.

Halophilic euryarchaea lack many of the genes necessary for the protoporphyrin-dependent heme biosynthesis pathway previously identified in animals and plants. Bioinformatic analysis suggested the presence of two heme biosynthetic processes, an Fe-coproporphyrinogen III (coproheme) decarboxylase (ChdC) pathway and an alternative heme biosynthesis (Ahb) pathway, in Haloferax volcanii. PitA is specific to the halophilic archaea and has a unique molecular structure in which the ChdC domain is joined to the antibiotics biosynthesis monooxygenase (ABM)-like domain by a histidine-rich linker sequence. The pitA gene deletion variant of H. volcanii showed a phenotype with a significant reduction of aerobic growth. Addition of a protoheme complemented the phenotype, supporting the assumption that PitA participates in the aerobic heme biosynthesis. Deletion of the ahbD gene caused a significant reduction of only anaerobic growth by denitrification or dimethylsulfoxide (DMSO) respiration, and the growth was also complemented by addition of a protoheme. The experimental results suggest that the two heme biosynthesis pathways are utilized selectively under aerobic and anaerobic conditions in H. volcanii. The molecular structure and physiological function of PitA are also discussed on the basis of the limited proteolysis and sequence analysis.

Specificity and inheritance of rBE3 and rBE4 endonuclease-induced gene modifications in rice

To gain more insights into the rice base editor (rBE3 and rBE4), we evaluated the mutation efficiency, off-target and inheritance of OsSERK1(D428N) and pi-ta(S918F) genes modified with rBE endonucleases. We predicted and analyzed the putative off-target sites of the sgRNA designed for OsSERK1(D428N) and pi-ta(S918F) by PCR amplification and Sanger sequencing. Then we further characterized the inheritance and stability of targeted base mutations and T-DNA segregation in the progeny of the self-fertilized T0 plants. Analysis of the DNA sequencing data of T0 plants of OsSERK1(D428N) revealed no nucleotide change at any of the four potential off-target sites. For OsSERK1(D428N) and Os08g07774 carry the same sgRNA targeting sites, base substitution at both two loci were detected at a frequency of 41.67%. The targeted base mutations could be transmitted readily to T1 progeny. Furthermore, genetic segregation caused the loss of T-DNA at a frequency between 25.0% and 40.9% in the T1 transgenic plants of OsSERK1(D428N) and pi-ta(S918F). These results demonstrated that the rBE3 and rBE4 systems could mediate specifically targeted base editing in one- or multi-site, and the targeted base editing could be stably inherited to next generation.

Combination of rice blast resistance genes in the genotypes of russian rice varieties with the use of marker assisted selection

Grain productivity of rice is significantly reduced by dangerous disease blast. Therefore, the development of resistant high yielding rice varieties with the Pi group of genes is important. Use of molecular markers linked with the loci of resistance significantly optimizes the breeding process.
 The purpose of the research is to develop rice lines combining 2-6 loci of resistance to blast: Pi-1, Pi-2, Pi-33, Pi-ta, Pi-b, Pi-40 by molecular marking.
 Materials and Methods. As donors of resistance genes foreign samples were used, recipient Russian varieties. In the studies we used micro-satellites markers and PCR analysis.
 Results. In the first stage of the research as a result of hybridization domestic lines with genes Pi-l, Pi-2, Pi-33 were obtained. At the second stage hybrids with all 3 genes were developed. In the third stage genes Pi-b and Pi-ta and on the fourth Pi-40 were introduced.
 Conclusion. As a result, rice genotypes, combining 6 loci of blast resistance were developed with use of marker assisted selection.

Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa.

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.

Characterization and evaluation of rice blast resistance of Chinese indica hybrid rice parental lines

The development of resistant varieties and hybrid combinations has been the most effective and economical strategy to control blast disease caused by Magnaporthe oryzae. However, the distribution of major R genes and blast resistance characterization in hybrid rice parents has not been well investigated, resulting in their limited use in hybrid rice blast-resistance breeding. In the present study, 88 elite indica hybrid rice parental lines were evaluated with 30 isolates of M. oryzae collected from the main planting area of indica hybrid rice in China and were characterized for the presence of 11 major resistance genes using molecular markers. The pathogenicity assays showed that four types of hybrid rice parent line showed some resistance to M. oryzae. However, the proportions of highly resistant lines and the mean resistance frequency (RF) varied among the four types, with resistance in decreasing order shown by three-line restorer lines, three-line maintainer lines, two-line sterile lines, and two-line restorer lines. All 88 hybrid rice parental lines carried more than one R gene, but none carried the R genes Pi1 and Pi2. Although Pid3 and Pi9 were present only in three-line restorer lines and Pigm only in three-line maintainer lines, the remaining six R genes (Pib, Pid2, Pi5, Pia, Pi54, and Pita) were present in the four types of hybrid rice parent with significantly different distribution frequencies. The correlation between R genes and resistance reactions was investigated. The results are expected to provide useful information for rational utilization of major R genes in hybrid rice breeding programs.

Pyramiding Pi-ta , Pi-b , and Wx-mq Genes by Marker-assisted Selection in Rice ( Oryza sativa L.)

Pyramiding <i>Pi-ta</i> , <i>Pi-b</i> , and <i>Wx-mq</i> Genes by Marker-assisted Selection in Rice ( <i>Oryza sativa</i> L.)

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.

Pyramiding of Pi46 and Pita to improve blast resistance and to evaluate the resistance effect of the two R genes

Utilization of R (resistance) genes to develop resistant cultivars is an effective strategy to combat against rice blast disease. In this study, R genes Pi46 and Pita in a resistant accession H4 were introgressed into an elite restorer line Hang-Hui-179 (HH179) using the marker-assisted backcross breeding (MABB) procedure. As a result, three improved lines (e.g., R1791 carrying Pi46 alone, R1792 carrying Pita alone and R1793 carrying both Pi46 and Pita) were developed. The three improved lines had significant genetic similarities with the recurrent parent HH179. Thus, they and HH179 could be recognized as near isogenic lines (NILs). The resistance spectrum of the three improved lines, which was tested at seedling stage, reached 91.1, 64.7 and 97.1%, respectively. This was markedly broader than that of HH179 (23.5%). Interestingly, R1793 showed resistance to panicle blast but neither R1791 nor R1792 exhibited resistance at two natural blast nurseries. The results implied that the stacking of Pi46 and Pita resulted in enhanced resistance, which was unachievable by either R gene alone. Further comparison indicated that the three improved lines were similar to HH179 in multiple agronomic traits; including plant height, tillers per plant, panicle length, spikelet fertility, and 1 000-grain weight. Thus, the three improved lines with different R genes can be used as new sources of resistance for developing variety. There is a complementary effect between the two R genes Pi46 and Pita.

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.

Coevolutionary Dynamics of Rice Blast Resistance Gene Pi-ta and Magnaporthe oryzae Avirulence Gene AVR-Pita 1.

The Pi-ta gene in rice is effective in preventing infections by Magnaporthe oryzae strains that contain the corresponding avirulence gene, AVR-Pita1. Diverse haplotypes of AVR-Pita1 have been identified from isolates of M. oryzae from rice production areas in the United States and worldwide. DNA sequencing and mapping studies have revealed that AVR-Pita1 is highly unstable, while expression analysis and quantitative resistance loci mapping of the Pi-ta locus revealed complex evolutionary mechanisms of Pi-ta-mediated resistance. Among these studies, several Pi-ta transcripts were identified, most of which are probably derived from alternative splicing and exon skipping, which could produce functional resistance proteins that support a new concept of coevolution of Pi-ta and AVR-Pita1. User-friendly DNA markers for Pi-ta have been developed to support marker-assisted selection, and development of new rice varieties with the Pi-ta markers. Genome-wide association studies revealed a link between Pi-ta-mediated resistance and yield components suggesting that rice has evolved a complicated defense mechanism against the blast fungus. In this review, we detail the current understanding of Pi-ta allelic variation, its linkage with rice productivity, AVR-Pita allelic variation, and the coevolution of Pi-ta and AVR-Pita in Oryza species and M. oryzae populations, respectively. We also review the genetic and molecular basis of Pi-ta and AVR-Pita interaction, and its value in marker-assisted selection and engineering resistance.

Evaluation of DNA markers linked to blast resistant genes, Pikh, Pit (p), and Pita, for parental selection in Sri Lankan rice breeding

Rice blast, is a serious disease affecting rice, caused by a fungal pathogen Magnaporthe grisea (anarmorph - Pyricularia grisea). This paper summarizes the progress and the achievements made in detection of blast resistance genes in phenotypically evaluated, selected rice cultivars in Sri Lanka based on simple sequence repeats (SSR) DNA markers. In the present study blast pathogen was identified by morphological characters, molecular techniques which confirmed the identity as M. grisea. Total of 34 rice varieties including several Sri Lankan rice varieties and IRRI recommended rice varieties were evaluated for blast resistance. Total of 17 varieties were susceptible to the fungal infection and 17 varieties remained resistant. Tetep, Bg359, Bg304, IR64, Bg352, Bg357 were found to be highly resistant to the rice blast disease, while LD125, Bg94/1, Bw267-3, Bw451, Bw400, H7, Bg34-6, H10, IRRI119 were highly susceptible. In marker genotyping, most of the highly resistant and resistant varieties demonstrated the presence of blast resistant Pita gene of variety Tetep and Pita gene was absent in the highly susceptible varieties. But Pita gene was absent in some of the resistant varieties like Bg300, Bg348 and Ptb33 and present in susceptible varieties H10, Bg276-5 and IR07F291. For polymorphism of Pikh gene most of the resistant varieties depicted the band lengths of 161-171bp. Susceptible varieties gave the band length of 151bp. According to results of marker genotyping with Pit(p) blast resistant gene, the resistant varieties gave a band length of 117bp while susceptible varieties produced the band length of 111bp. Hence, resulted polymorphism findings can be used in future studies to differentiate the resistant and susceptible varieties. Tropical Agricultural Research Vol. 26 (1): 82 – 93 (2014)

A Magnaporthe Avr-pita gene orthologous in Rhizoctonia solani AG1-IA shows characteristics of an effector protein

Sheath blight is one of the most destructive diseases of rice that is caused by Rhizoctonia solani. Therefore, understanding molecular mechanisms of rice–Rhizoctonia interaction is necessary for adoption of effective approaches of disease management. Here for the first time, we report an ortholog of Magnaporthe oryzae Avr−Pita1 gene, designated as Rhiz_Pita in R. solani AG1-IA. Rhiz_Pita showed multiple characteristics of effector proteins; First, more than 98 % identity was calculated between Rhiz_Pita deduced protein sequence and Mag-Pita complete protein sequence. SignalP and PrediSi programs predicted a secretion signal upstream of deduced ORF of Rhiz−Pita gene. Also, Rhiz_Pita secretion potential was approved by using yeast secretion trap system (YST). Detected by Real-time PCR, Rhiz−Pita gene expression level was increased following the application of rice leaf extraction in R. solani culture medium. Also, Rhiz_Pita gene expression was significantly elevated in the infected rice plants since 24 h post infection. Overall, these results suggest that novel Rhiz−Pita gene might encode a bona fide effector protein that its expression is associated to the fungal penetration in the early stages of infection.

Creation of blast disease-resistant rice varieties with modern DNA-markers

Based on modern technologies of molecular DNA-markers, blast disease-resistance genes (Pi-ta, Pi-b, Pi-1, Pi-2, and Pi-33) were introgressed and pyramided into domestic rice varieties to give them long-term disease resistance. For that purpose, this case study uses SSR-markers closely linked to these genes, as well as intragenic markers of genes Pi-ta and Pi-b. Multiplex PCR systems were created for simultaneous identification of two resistance genes in the hybrid progeny for the following combinations: Pi-1 + Pi-2, Pi-ta + Pi-b, Pi-ta + Pi-33.

A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus.

Recalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters, and also planktonic cells and is associated with chronic or relapsing infections with pathogens such as Staphylococcus aureus. Here we report the in vitro evolution of an S. aureus strain that exhibits a high degree of nonsusceptibility to daptomycin as a result of cyclic challenges with bactericidal concentrations of the drug. This phenotype was attributed to stationary growth phase-dependent drug tolerance and was clearly distinguished from resistance. The underlying genetic basis was revealed to be an adaptive point mutation in the putative inorganic phosphate (Pi) transporter gene pitA. Drug tolerance caused by this allele, termed pitA6, was abrogated when the upstream gene pitR was inactivated. Enhanced tolerance toward daptomycin, as well as the acyldepsipeptide antibiotic ADEP4 and various combinations of other drugs, was accompanied by elevated intracellular concentrations of Pi and polyphosphate, which may reversibly interfere with critical cellular functions. The evolved strain displayed increased rates of survival within human endothelial cells, demonstrating the correlation of intracellular persistence and drug tolerance. These findings will be useful for further investigations of S. aureus drug tolerance, toward the development of additional antipersister compounds and strategies.

The ActP acetate transporter acts prior to the PitA phosphate carrier in tellurite uptake by Escherichia coli

The tellurium oxyanion tellurite is harmful for most microorganisms. Since its toxicity occurs chiefly once the toxicant reaches the intracellular compartment, unveiling the toxicant uptake process is crucial for understanding the whole phenomenon of tellurium toxicity. While the PitA phosphate transporter is thought to be one of the main paths responsible for toxicant entry into Escherichia coli, genetic and physiological evidence have identified the ActP acetate carrier as the main tellurite importer in Rhodobacter capsulatus. In this work, new background on the role of these transporters in tellurite uptake by E. coli is presented. It was found that, similar to what occurs in R. capsulatus, ActP is able to mediate toxicant entry to this bacterium. Lower reactive oxygen species levels were observed in E. coli lacking the actP gene.Antioxidant enzyme catalase and fumarase C activity was almost unchanged after short exposure of E. coli ΔactP to sublethal tellurite concentrations, suggesting a low antioxidant response. In this strain, tellurite uptake decreased significantly during the first 5min of exposure and inductively coupled plasma optical emission spectroscopy assays using an actP-overexpressing strain confirmed that this carrier mediates toxicant uptake. Relative gene expression experiments by qPCR showed that actP expression is enhanced at short times of tellurite exposure, while pitA and pitB genes are induced later. Summarizing, the results show that ActP is involved in tellurite entry to E. coli and that its participation occurs mainly at early stages of toxicant exposure.

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.

Blast Resistant Genes Distribution and Resistance Reaction to Blast in Korean Landraces of Rice (Oryza sativa L.)

Rice blast (Magnaporthe oryza B.) is one of the most important diseases in rice that causing great yield losses every year around the world. It is important to screen valuable genetic resources for improving blast resistance. This study was conducted to identify the blast resistance in 279 Korean rice landraces using blast nursery tests and isolate inoculum screening. The results showed that 11 landrace accessions found to be resistant to rice blast in blast nursery and inoculation screening tests and the degree of lesions in most accessions showed that they were susceptible to reactions. In order to find the distribution of blast resistant genes, a molecular survey was conducted to identify the presence of major blast resistance (R) gene in 279 Korean landraces. The results revealed that their frequency distribution was Pik-m (36.2%), Piz (25.4%), Pit (13.6%), and Pik (10%). Besides, the frequency distribution of Piz-t, Pii, Pik-m/Pik-p, Pi-39(t), Pib, Pi-d(t)2, Pita/Pita-2 and Pi-ta genes were identified as less than 10%. The results did not consist with the reactions against blast diseases between genotypes and phenotypic part of the nursery tests and isolate inoculation. For concluding these results, we used genome-wide SSR markers that have closely been located with resistance genes. The PCoA analysis showed that the landrace accessions formed largely two distinct groups according to their degree of blast resistance. By comparing genetic diversities using polymorphic information contents (PIC) value among the resistant, total and susceptible landraces, we found that PIC values decreased in four SSR markers and increased in six markers in the resistant accessions, which showed contrary to total and susceptible groups. These regions might be linked to resistance alleles. In this study, we evaluated the degree of blast resistance and the information about the distribution of rice blast resistant genes in Korean rice landraces. This study might be the basis for association analysis of blast resistance in rice.

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.