Blast Resistance Gene Research Articles

Characterization of Blast Resistance in a Diverse Rice Panel from Sub-Saharan Africa.

There is a recent unparalleled increase in demand for rice in sub-Saharan Africa, yet its production is affected by blast disease. Characterization of blast resistance in adapted African rice cultivars can provide important information to guide growers and rice breeders. We used molecular markers for known blast resistance genes (Pi genes; n = 21) to group African rice genotypes (n = 240) into similarity clusters. We then used greenhouse-based assays to challenge representative rice genotypes (n = 56) with African isolates (n = 8) of Magnaporthe oryzae which varied in virulence and genetic lineage. The markers grouped rice cultivars into five blast resistance clusters (BRC) which differed in foliar disease severity. Using stepwise regression, we found that the Pi genes associated with reduced blast severity were Pi50 and Pi65, whereas Pik-p, Piz-t, and Pik were associated with increased susceptibility. All rice genotypes in the most resistant cluster, BRC 4, possessed Pi50 and Pi65, the only genes that were significantly associated with reduced foliar blast severity. Cultivar IRAT109, which contains Piz-t, was resistant against seven African M. oryzae isolates, whereas ARICA 17 was susceptible to eight isolates. The popular Basmati 217 and Basmati 370 were among the most susceptible genotypes. These findings indicate that most tested genes were not effective against African blast pathogen collections. Pyramiding genes in the Pi2/9 multifamily blast resistance cluster on chromosome 6 and Pi65 on chromosome 11 could confer broad-spectrum resistance capabilities. To gain further insights into genomic regions associated with blast resistance, gene mapping could be conducted with resident blast pathogen collections. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pyramiding Rice Blast Resistance Gene Pi2 and Fragrance Gene badh2

Rice is a major food crop across the globe, but the frequent occurrence of rice blast in recent years has seriously affected the yield of rice. In addition, fragrance rice is becoming increasingly popular among consumers. In this study, the fragrant rice variety Wenxiang-1 was used as the donor of the fragrance gene badh2, and the rice variety R1179 was used as the donor of rice blast resistance gene Pi2. Plants that were homozygous for both Pi2 and badh2 were selected using marker-assisted selection (MAS) applied to the Wenxiang-1/R1179 F2 segregation population with the functional markers Pi2-1 and Badh2-1 as well as whole-genome-SNP-genotyping technology. Finally, “elite” rice varieties R365 and R403 that had both high levels of rice blast resistance (level 3 and 4) and fragrance (0.650 and 0.511 mg/kg) were bred. Genetic composition analysis indicated that 40.67% of the whole genome of R365 was inherited from Wenxiang-1, while 59.33% was inherited from R1179. Similarly, 46.26% of the whole genome of R403 was inherited from Wenxiang-1, while 53.74% was inherited from R1179. These new hybrid lines with R365 and R403 as the male parents also exhibit high yield per hectare, especially C815S/R365 and Yu03S/R403 F1, with yields per hectare of 9.93 ± 0.15 and 9.6 ± 0.17 tons. These plants also possess high levels of rice blast resistance (level 3 and 4) and fragrance (0.563 and 0.618 mg/kg).

Identification of Elite R-Gene Combinations against Blast Disease in Geng Rice Varieties.

Rice blast, caused by the Magnaporthe oryzae fungus, is one of the most devastating rice diseases worldwide. Developing resistant varieties by pyramiding different blast resistance (R) genes is an effective approach to control the disease. However, due to complex interactions among R genes and crop genetic backgrounds, different R-gene combinations may have varying effects on resistance. Here, we report the identification of two core R-gene combinations that will benefit the improvement of Geng (Japonica) rice blast resistance. We first evaluated 68 Geng rice cultivars at seedling stage by challenging with 58 M. oryzae isolates. To evaluate panicle blast resistance, we inoculated 190 Geng rice cultivars at boosting stage with five groups of mixed conidial suspensions (MCSs), with each containing 5-6 isolates. More than 60% cultivars displayed moderate or lower levels of susceptibility to panicle blast against the five MCSs. Most cultivars contained two to six R genes detected by the functional markers corresponding to 18 known R genes. Through multinomial logistics regression analysis, we found that Pi-zt, Pita, Pi3/5/I, and Pikh loci contributed significantly to seedling blast resistance, and Pita, Pi3/5/i, Pia, and Pit contributed significantly to panicle blast resistance. For gene combinations, Pita+Pi3/5/i and Pita+Pia yielded more stable pyramiding effects on panicle blast resistance against all five MCSs and were designated as core R-gene combinations. Up to 51.6% Geng cultivars in the Jiangsu area contained Pita, but less than 30% harbored either Pia or Pi3/5/i, leading to less cultivars containing Pita+Pia (15.8%) or Pita+Pi3/5/i (5.8%). Only a few varieties simultaneously contained Pia and Pi3/5/i, implying the opportunity to use hybrid breeding procedures to efficiently generate varieties with either Pita+Pia or Pita+Pi3/5/i. This study provides valuable information for breeders to develop Geng rice cultivars with high resistance to blast, especially panicle blast.

Marker-Assisted Pyramiding of Blast-Resistance Genes in a japonica Elite Rice Cultivar through Forward and Background Selection

Rice blast, caused by Pyricularia oryzae, is one of the main rice diseases worldwide. The pyramiding of blast-resistance (Pi) genes, coupled to Marker-Assisted BackCrossing (MABC), provides broad-spectrum and potentially durable resistance while limiting the donor genome in the background of an elite cultivar. In this work, MABC coupled to foreground and background selections based on KASP marker assays has been applied to introgress four Pi genes (Piz, Pib, Pita, and Pik) in a renowned japonica Italian rice variety, highly susceptible to blast. Molecular analyses on the backcross (BC) lines highlighted the presence of an additional blast-resistance gene, the Pita-linked Pita2/Ptr gene, therefore increasing the number of blast-resistance introgressed genes to five. The recurrent genome was recovered up to 95.65%. Several lines carrying four (including Pita2) Pi genes with high recovery percentage levels were also obtained. Phenotypic evaluations confirmed the effectiveness of the pyramided lines against multivirulent strains, which also had broad patterns of resistance in comparison to those expected based on the pyramided Pi genes. The developed blast-resistant japonica lines represent useful donors of multiple blast-resistance genes for future rice-breeding programs related to the japonica group.

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice.

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC1F1 generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC1F1 generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC1F3 generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC1F5 generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC1F5 generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.

Molecular Detection and Analysis of Blast Resistance Genes in Rice Main Varieties in Jiangsu Province, China

Rice blast, caused by Pyricularia oryzae, is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control the disease, and it is crucial to analyze the resistance level and the resistance genes distribution of the main varieties. In this study, we collected 119 rice main varieties in Jiangsu province and evaluated the resistance to leaf and panicle blast and found that indica rice was more resistant to rice blast than japonica rice. Moreover, we detected the distribution of 14 resistance genes (R genes) in the 119 varieties. The distribution frequencies of three R genes, Pish, Pit, and Pia, were higher than 80%, and the Pigm had the lowest distribution frequency (1.68%), followed by Pi2 (15.18%) and Pi5, Piz-t (24.37%). Combined with the multiple stepwise regression and the resistance contribution rate, eight major R genes Pita, Pi5, Pi9, Pib, Pb1, Pikm, Piz-t, and Pi2 significantly affected the resistance of rice, and we also found that six gene combinations with 100% resistance contribution rate could effectively increase the resistance of rice varieties. In summary, monitoring the resistance level of rice varieties and analyzing their resistance genes were beneficial for rice resistance breeding.

Conventional Breeding and Molecular Markers for Blast Disease Resistance in Rice (Oryza sativa L.)

Monogenic lines, which carried 23 genes for blast resistance were tested and used donors to transfer resistance genes by crossing method. The results under blast nursery revealed that 9 genes from 23 genes were susceptible to highly susceptible under the three locations (Sakha, Gemmeza, and Zarzoura in Egypt); Pia, Pik, Pik-p, Piz-t, Pita, Pi b, Pi, Pi 19 and Pi 20. While, the genes Pii, Pik-s, Pik-h, Pi z, Piz-5, Pi sh, Pi 3, Pi 1, Pi 5, Pi 7, Pi 9, Pi 12, Pikm and Pita-2 were highly resistant at the same locations. Clustering analysis confirmed the results, which divided into two groups; the first one included all the susceptible genes, while the second one included the resistance genes. In the greenhouse test, the reaction pattern of five races produced 100% resistance under artificial inoculation with eight genes showing complete resistance to all isolates. The completely resistant genes: Pii, Pik-s, Piz, Piz-5 (=bi2) (t), Pita (=Pi4) (t), Pita, Pi b and Pi1 as well as clustering analysis confirmed the results. In the F1 crosses, the results showed all the 25 crosses were resistant for leaf blast disease under field conditions. While, the results in F2 population showed seven crosses with segregation ratio of 15 (R):1 (S), two cross gave segregated ratio of 3 R:1 S and one gave 13:3. For the identi- fication of blast resistance genes in the parental lines, the marker K3959, linked to Pik-s gene and the variety IRBLKS-F5 carry this gene, which was from the monogenic line. The results showed that four genotypes; Sakha 105, Sakha 103, Sakha 106 and IRBLKS-F5 were carrying Pik-s gene, while was absent in the Sakha 101, Sakha 104, IRBL5-M, IRBL9-W, IRBLTACP1 and IRBL9-W(R) genotypes. As for Pi 5 gene, the results showed that it was present in Sakha 103 and Sakha 104 varieties and absent in the rest of the genotypes. In addition, Pita-Pita- 2 gene was found in the three Egyptian genotypes (Sakha 105, Sakha 101 and Sakha 104) plus IRBLTACP1 monogenetic. In F2 generation, six populations were used to study the inheritance of blast resistance and specific primers to confirm the ratio and identify the resistance genes. However, the ratios in molecular markers were the same of the ratio under field evaluation in the most population studies. These findings would facilitate in breeding programs for gene pyramiding and gene accumulation to produce durable resistance for blast using those genotypes.

Molecular Marker Assisted Breeding of a New <i>Japonica</i> Hybrid Rice

The development of Japonica  hybrid rice is of great significance to ensure supply of good quality Japonica  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  hybrid rice, a new Japonica  hybrid rice combination 'Shenyou28' with early maturity, good 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  hybrid rice with 'Shen21A' as sterile line and 'Shenhui26-28' as restorer line. For two-year regional trials 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 quality, reaching the second grade of national standard, and has fragrance because it carries fragrance gene badh2-p-5 ’ 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.

Screening microsatellite markers for establishing parental polymorphism in Indian rice (Oryza sativa L.)

The experiment was conducted to investigate the parental diversity along the rice genome and to understand and screen out the SSR markers-indicated polymorphism between two indica rice (Oryza sativa L.) cultivars. Namely K343, the most well-liked rice variety in the hill zone of the Jammu Region, and RML22, a rice line created at IRRI, Philippines. The study is to select polymorphic markers (Simple Sequence Repeat- SSR) associated with hill ecologies rice cultivars and additional research projects like gene pyramiding and background selection to recover the recurrent parent genome (RPG) in blast gene introgression in elite lines. 450 SSR markers, evenly distributed throughout the rice genome, were used to assess the parental polymorphism between these genotypes. Of these two cultivars, 51 markers (11.33%) showed polymorphism with bands in different spectrums throughout the genome. The study has been used to Marker Assisted Backcross (MAB) breeding to integrate rice blast resistance genes in the parental genotype. The pool of polymorphic markers has the potential to use in similar studies and work, with a high probability of polymorphism for the cultivars of hill ecologies, and thus increase the chance of selection of probability in marker selection.

Screening of rice blast resistance in Thai upland rice using pathogenicity assays and molecular markers

ABSTRACT Rice blast disease caused by the fungus Pyricularia oryzae is one of the most devastating diseases of rice (Oryza sativa L.). Therefore, the use of resistant rice varieties would be the most effective way to control this disease. Based on disease evaluation, upland rice varieties were classified into two groups: resistant (35%) and moderately resistant (65%). Forty upland rice varieties and two lowland rice varieties were genotyped for seven major rice blast resistance genes Pi37, Pid2, Pi9, Pi36(t), Pi5, Pik-m, and Pi54. The gene frequencies of the seven major R genes ranged from 2.38% to 100%. The 42 varieties contained one to five R genes. Two varieties had five blast resistance genes, whereas 21 varieties contained four R genes, 15 varieties contained three R genes, 3 varieties contained two R genes, and only one variety contained one R gene. Furthermore, the relationship between the presence of different R genes and disease reactions was investigated using a multiple stepwise regression model. Three markers, Pi5, Pi54MAS, and Ckm2, for three R genes (Pi5, Pi54, and Pik-m) were moderately correlated with blast disease with partial correlation coefficients of 0.35 to 0.47. These results provide new sources of resistance genes for designing future breeding program to develop leaf blast-resistant rice varieties.

Evaluation of Molecular Markers Linked to Rice Blast Resistance Gene in Korean Rice Varieties

Evaluation of Molecular Markers Linked to Rice Blast Resistance Gene in Korean Rice Varieties

Surveillance of Rice Blast Resistance Effectiveness and Emerging Virulent Isolates in Taiwan.

Rice blast caused by Magnaporthe oryzae is a dangerous threat to rice production and food security worldwide. Breeding and proper deployment of resistant varieties are effective and environmentally friendly strategies to manage this notorious disease. However, a highly dynamic and quickly evolved rice blast pathogen population in the field has made disease control with resistance germplasms more challenging. Therefore, continued monitoring of pathogen dynamics and application of effective resistance varieties are critical tasks to prolong or sustain field resistance. Here, we report a team project that involved evaluation of rice blast resistance genes and surveillance of M. oryzae field populations in Taiwan. A set of International Rice Research Institute-bred blast-resistant lines (IRBLs) carrying single blast resistance genes was utilized to monitor the field effectiveness of rice blast resistance. Resistance genes such as Ptr (formerly Pita2) and Pi9 exhibited the best and most durable resistance against the rice blast fungus population in Taiwan. Interestingly, line IRBLb-B harboring the Pib gene with good field protection has recently shown susceptible lesions in some locations. To dissect the genotypic features of virulent isolates against the Pib resistance gene, M. oryzae isolates were collected and analyzed. Screening of the AvrPib locus revealed that the majority of field isolates still maintained the wild-type AvrPib status but eight virulent genotypes were found. Pot3 insertion appeared to be a major way to disrupt the AvrPib avirulence function. Interestingly, a novel AvrPib double-allele genotype among virulent isolates was first identified. Pot2 repetitive element-based polymerase chain reaction (rep-PCR) fingerprinting analysis indicated that mutation events may occur independently among different lineages in different geographic locations of Taiwan. This study provides our surveillance experience of rice blast disease and serves as the foundation to sustain rice production.

Genome-Wide Association Study Identifies a Rice Panicle Blast Resistance Gene Pb3 Encoding NLR Protein.

Rice blast is a worldwide fungal disease that seriously affects the yield and quality of rice. Identification of resistance genes against rice blast disease is one of the effective ways to control this disease. However, panicle blast resistance genes, which are useful in the fields, have rarely been studied due to the difficulty in phenotypic identification and the environmental influences. Here, panicle blast resistance-3 (Pb3) was identified by a genome-wide association study (GWAS) based on the panicle blast resistance phenotypes of 230 Rice Diversity Panel I (RDP-I) accessions with 700,000 single-nucleotide polymorphism (SNP) markers. A total of 16 panicle blast resistance loci (PBRLs) within three years including one repeated locus PBRL3 located in chromosome 11 were identified. In addition, 7 genes in PBRL3 were identified as candidate genes by haplotype analysis, which showed significant differences between resistant and susceptible varieties. Among them, one nucleotide-binding domain and Leucine-rich Repeat (NLR) gene Pb3 was highly conserved in multiple resistant rice cultivars, and its expression was significantly induced after rice blast inoculation. Evolutionary analysis showed that Pb3 was a typical disease resistance gene containing coiled-coil, NB-ARC, and LRR domains. T-DNA insertion mutants and CRISPR lines of Pb3 showed significantly reduced panicle blast resistance. These results indicate that Pb3 is a panicle blast resistance gene and GWAS is a rapid method for identifying panicle blast resistance in rice.

Association analysis of rice resistance genes and blast fungal avirulence genes for effective breeding resistance cultivars

Utilization of rice blast-resistance (R) genes is the most economical and environmentally friendly method to control blast disease. However, rice varieties with R genes influence the outcome of genetic architectures of Magnaporthe oryzae (M. oryzae), and mutations in avirulence (AVR) genes of M. oryzae may cause dysfunction of the corresponding R genes in rice varieties. Although monitoring and characterizing rice R genes and pathogen AVR genes in field populations may facilitate the implementation of effective R genes, little is known about the changes of R genes over time and their ultimate impact on pathogen AVR genes. In this study, 117 main cultivated rice varieties over the past five decades and 35 M. oryzae isolates collected from those diseased plants were analyzed by PCR using gene-specific markers of the nine R genes and six primer pairs targeting the coding sequence or promoter of AVR genes, respectively. The R genes Pigm, Pi9, Pi2, Piz-t, Pi-ta, Pik, Pi1, Pikp, and Pikm were identified in 5, 0, 1, 4, 18, 0, 2, 1, and 0 cultivars, respectively. Significantly, none of these R genes had significant changes that correlated to their application periods of time. Among the four identified AVR genes, AVR-Pik had the highest amplification frequency (97.14%) followed by AVR-Pita (51.43%) and AVR-Pi9 (48.57%); AVR-Piz-t had the lowest frequency (28.57%). All these AVR genes except AVR-Pi9 had 1-2 variants. Inoculation mono-genic lines contained functional genes of Pi2/9 and Pik loci with 14 representative isolates from those 35 ones revealed that the presence of certain AVR-Piz-t, AVR-Pita variants, and AVR-Pik-E + AVR-Pik-D in M. oryzae populations, and these variants negated the ability of the corresponding R genes to confer resistance. Importantly, Pi2, Pi9, and Pigm conferred broad-spectrum resistance to these local isolates. These findings reveal that the complex genetic basis of M. oryzae and some effective blast R genes should be considered in future rice blast-resistance breeding programs.

Genetic improvement of traditional Basmati rice Ranbir Basmati for semi-dwarfism and blast resistance through molecular breeding

The traditional Basmati rices grown in north-western foothills of Himalayas in India are known world-wide for their exquisite aroma and exceptional eating and cooking qualities. These varieties are tall and suffer heavy losses due to their susceptibility to lodging as well as blast disease. In present study, we improved a popular traditional Basmati rice variety ‘Ranbir Basmati’ for semi-dwarfism and resistance to blast by incorporating the semi-dwarfing gene, sd1 and two blast resistance genes, Pi9 and Pi54 through marker-assisted backcross breeding. Combining marker-assisted background selection with stringent phenotypic selection for aroma and seed quality traits during backcrossing enabled faster recovery of recurrent parent genome and reconstitution of the genomic regions associated with Basmati quality traits in the gene pyramided lines. All pyramided lines were significantly short statured with a reduction of 23.46 to 40.26 cm in height, as compared to recurrent parent, with many of them exhibiting higher grain yield and superior quality attributes than Ranbir Basmati. The yield superiority of the pyramided lines primarily resulted from the improvement in panicle number and semi-dwarf stature conferred by sd1 gene. The gene pyramided lines showed complete resistance to five highly virulent blast races that collectively showed compatibility with 16 different resistance genes, suggesting the broad-spectrum resistance in these lines. The semi-dwarf, high yielding and blast resistant lines generated herein are potential candidates for release as cultivars for overcoming losses in Ranbir Basmati due to lodging and blast disease.

Improving Broad Spectrum Blast Resistance by Introduction of the Pita2 Gene: Encoding the NB-ARC Domain of Blast-Resistant Proteins into Upland Rice Breeding Programs

Blast disease is generally more important in upland rather than lowland rice cultivation, representing one of the biotic obstacles in the development of upland rice. The objective of this study was to detect broad-spectrum blast resistance gene Pita2 encoding the NB-ARC (nucleotide-binding adaptor common in APAF-1, R proteins, and CED-4) domain of blast-resistant proteins in new upland rice lines from the breeding program for landrace rice varieties, with the goal of providing a novel source of blast-resistant germplasm for application in future upland rice breeding programs. In this study, we screened 19 inbred lines of landrace rice varieties challenged using local virulent isolates in greenhouse conditions and performed field evaluations to confirm blast resistance. Molecular analysis was conducted using six specific primers to detect broad-spectrum blast resistance, and sequence analysis was performed to detect the NB-ARC domain of blast-resistant proteins in the lines. Consistent results were observed between greenhouse screening and field evaluations, although there was variance in the level of resistance. The PCR assay showed that there were eight positive lines (G7, G8, G9, G11, G13, G14, G15, and G18) containing the Pita2 gene. Conserved domain analysis revealed that eight blast-resistant rice lines encode NB-ARC at sequence lengths ranging between 300 and 870 (450 bp). Using these sequences in BLASTX searching revealed 15 gene homologs of the eight rice lines, which were detected as Pita2 genes, with a similarity level of 81–99%. Further comprehensive studies should be performed to confirm the performance and resistance of candidate lines in field trials in various blast-endemic areas before being released as new upland rice varieties able to overcome the problem of blast disease in the field. In addition, the lines can also be used as a novel genetic resource in the blast-resistant upland rice breeding program on various rice cultivars.

Genotypic and Phenotypic Selection of Newly Improved Putra Rice and the Correlations among Quantitative Traits

This experiment was conducted to genotypically and phenotypically select new rice lines pyramided with blast and bacterial leaf blight disease resistance genes after a marker-assisted backcross breeding programme. The inter-relationship among agro-morphological characteristics and their effect on yield was also studied. The polymorphic functional and microsatellite markers Xa21FR and pTA248 (Xa21), Xa13prom (xa13), RM21 (xa5), MP (Xa4), RM6836 (Pi2, Pi9 & Piz) and RM8225 (Piz) were first confirmed for the target genes. The selected markers were used for foreground selection of BC2F2 homozygous progenies with the target genes. Plants that had homozygous IRBB60 alleles for these markers were evaluated for their recovery of the recurrent parent genome. IRBB60 was used as the donor parent for bacterial blight resistance genes while Putra-1 served as a recipient/recurrent parent with background blast-resistance genes and high yield. After the foreground selection, 79 polymorphic simple sequence repeat markers identified from the marker polymorphism survey were used for marker-assisted background selection to determine the percentage recovery of the recurrent parent genome. In order to make a selection on a phenotypic basis, 14 agro-morphological traits were measured and recorded. The result obtained from the study showed that 16 lines received the seven resistance genes in sufficiently varied numbers and were selected. The distribution of yield per hectare showed that about 50% of the selected lines had yields as high as 5 t/ha and above. Some of the lines produced as high as 8.4 t/ha. These lines demonstrated the potential of recording uniform 8t/ha upon recombination at BC2F2. The study also indicated that the number of panicles per hill correlated strongly, significantly and positively with the number of tillers (r = 0.962 **), total grain weight per hill (r = 0.928 **) and yield per hectare (r = 0.927 **). It was concluded that the newly improved resistant lines which were selected have the capability to compete with Putra-1 in terms of its productivity and yield. The newly developed lines would be useful in future breeding programmes as donors for bacterial leaf blight and blast resistance genes. These lines are recommended for release to farmers in Malaysia and other rice-growing agro-ecologies for commercial cultivation.

Molecular evolution of the Pi-d2 gene conferring resistance to rice blast in Oryza.

The exploitation of plant disease resistance (R) genes in breeding programs is an effective strategy for coping with pathogens. An understanding of R gene variation is the basis for this strategy. Rice blast disease, caused by the Magnaporthe oryzae fungus, is a destructive disease of rice. The rice blast resistance gene Pi-d2 represents a new class of plant R gene because of its novel extracellular domain. We investigated the nucleotide polymorphism, phylogenetic topology and evolution patterns of the Pi-d2 gene among 67 cultivated and wild rice relatives. The Pi-d2 gene originated early in the basal Poales and has remained as a single gene without expansion. The striking finding is that susceptible Pi-d2 alleles might be derived from a single nucleotide substitution of the resistant alleles after the split of Oryza subspecies. Functional pleiotropy and linkage effects are proposed for the evolution and retention of the disease-susceptible alleles in rice populations. One set of DNA primers was developed from the polymorphic position to detect the functional nucleotide polymorphism for disease resistance of the Pi-d2 gene based on conventional Polymerase Chain Reaction. The nucleotide diversity level varied between different domains of the Pi-d2 gene, which might be related to distinct functions of each domain in the disease defense response. Directional (or purifying) selection appears dominant in the molecular evolution of the Pi-d2 gene and has shaped its conserved variation pattern.

Analysis of a rice blast resistance gene Pita-Fuhui2663 and development of selection marker

Rice blast is a detrimental rice disease caused by the fungus Magnaporthe oryzae. Here, we identified a resistance gene from the rice cultivar Fuhui 2663 which is resistant to the rice blast isolate KJ201. Through isolated population analyses and sequencing approaches, the candidate gene was traced to chromosome 12. With the use of a map-based cloning strategy, the resistance gene was ultimately mapped to an 80-kb resistance locus region containing the Pita gene. Candidate gene prediction and cDNA sequencing indicated that the target resistance gene in Fuhui 2663 was allelic to Pita, thus being referred to as Pita-Fuhui2663 hereafter. Further analysis showed that the Fuhui 2663 protein had one amino acid change: Ala (A) residue 918 in Pita-Fuhui2663 was replaced by Ser (S) in Pita-S, leading to a significant change in the 3D structure of the Pita-S protein. CRISPR/Cas9 knockout experiments confirmed that Pita-Fuhui2663 is responsible for the resistance phenotype of Fuhui 2663. Importantly, Pita-Fuhui2663 did not affect the main agronomic traits of the variety compared to the Pita gene as verified by knockout experiments, indicative of potential applications of Pita-Fuhui2663 in broader breeding programs. Furthermore, a Pita-Fuhui2663-dCAPS molecular marker with good specificity and high efficiency was developed to facilitate rice breeding for resistance to this devastating disease.

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