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.

The Methylation Patterns and Transcriptional Responses to Chilling Stress at the Seedling Stage in Rice.

Chilling stress is considered the major abiotic stress affecting the growth, development, and yield of rice. To understand the transcriptomic responses and methylation regulation of rice in response to chilling stress, we analyzed a cold-tolerant variety of rice (Oryza sativa L. cv. P427). The physiological properties, transcriptome, and methylation of cold-tolerant P427 seedlings under low-temperature stress (2-3 °C) were investigated. We found that P427 exhibited enhanced tolerance to low temperature, likely via increasing antioxidant enzyme activity and promoting the accumulation of abscisic acid (ABA). The Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) data showed that the number of methylation-altered genes was highest in P427 (5496) and slightly lower in Nipponbare (Nip) and 9311 (4528 and 3341, respectively), and only 2.7% (292) of methylation genes were detected as common differentially methylated genes (DMGs) related to cold tolerance in the three varieties. Transcriptome analyses revealed that 1654 genes had specifically altered expression in P427 under cold stress. These genes mainly belonged to transcription factor families, such as Myeloblastosis (MYB), APETALA2/ethylene-responsive element binding proteins (AP2-EREBP), NAM-ATAF-CUC (NAC) and WRKY. Fifty-one genes showed simultaneous methylation and expression level changes. Quantitative RT-PCR (qRT-PCR) results showed that genes involved in the ICE (inducer of CBF expression)-CBF (C-repeat binding factor)-COR (cold-regulated) pathway were highly expressed under cold stress, including the WRKY genes. The homologous gene Os03g0610900 of the open stomatal 1 (OST1) in rice was obtained by evolutionary tree analysis. Methylation in Os03g0610900 gene promoter region decreased, and the expression level of Os03g0610900 increased, suggesting that cold stress may lead to demethylation and increased gene expression of Os03g0610900. The ICE-CBF-COR pathway plays a vital role in the cold tolerance of the rice cultivar P427. Overall, this study demonstrates the differences in methylation and gene expression levels of P427 in response to low-temperature stress, providing a foundation for further investigations of the relationship between environmental stress, DNA methylation, and gene expression in rice.

OsGRF4 controls grain shape, panicle length and seed shattering in rice.

Traits such as grain shape, panicle length and seed shattering, play important roles in grain yield and harvest. In this study, the cloning and functional analysis of PANICLE TRAITS 2 (PT2), a novel gene from the Indica rice Chuandali (CDL), is reported. PT2 is synonymous with Growth-Regulating Factor 4 (OsGRF4), which encodes a growth-regulating factor that positively regulates grain shape and panicle length and negatively regulates seed shattering. Higher expression of OsGRF4 is correlated with larger grain, longer panicle and lower seed shattering. A unique OsGRF4 mutation, which occurs at the OsmiRNA396 target site of OsGRF4, seems to be associated with high levels of OsGRF4 expression, and results in phenotypic difference. Further research showed that OsGRF4 regulated two cytokinin dehydrogenase precursor genes (CKX5 and CKX1) resulting in increased cytokinin levels, which might affect the panicle traits. High storage capacity and moderate seed shattering of OsGRF4 may be useful in high-yield breeding and mechanized harvesting of rice. Our findings provide additional insight into the molecular basis of panicle growth.

Molecular mapping of the blast resistance genes Pi2-1 and Pi51(t) in the durably resistant rice 'Tianjingyeshengdao'.

Tianjingyeshengdao' (TY) is a rice cultivar with durable resistance to populations of Magnaporthe oryzae (the causal agent of blast) in China. To understand the genetic basis of its resistance to blast, we developed a population of recombinant inbred lines from a cross between TY and the highly susceptible 'CO39' for gene mapping analysis. In total, 22 quantitative trait loci (QTLs) controlling rice blast resistance were identified on chromosomes 1, 3, 4, 5, 6, 9, 11, and 12 from the evaluation of four disease parameters in both greenhouse and blast nursery conditions. Among these QTLs, 19 were contributed by TY and three by CO39. Two QTL clusters on chromosome 6 and 12 were named Pi2-1 and Pi51(t), respectively. Pi2-1 was detected under both growth chamber and natural blast nursery conditions, and explained 31.24 to 59.73% of the phenotypic variation. Pi51(t) was only detected in the natural blast nursery and explained 3.67 to 10.37% of the phenotypic variation. Our results demonstrate that the durable resistance in TY is controlled by two major and seven minor genes. Identification of the markers linked to both Pi2-1 and Pi51(t) in this study should be useful for marker-aided selection in rice breeding programs as well as for molecular cloning of the identified resistance genes.

[Comprehensive evaluation of salt-alkali tolerance of rice germplasms at germination and seedling stages and analysis of salt-tolerant genes].

Cultivating salt-alkali tolerant rice varieties is one of the important ways to meet the increasing food demand of growing global population. In this study, twenty-one rice germplasms with different salt-alkali tolerance were treated with six salt-alkali concentrations at germination and seedling stages. The germination potential, germination rate, shoot length, root length, root number, fresh weight of shoot and seedlings were measured. The average value of salt damage rate was used to evaluate the salt-alkali tolerance. As the salt-alkali concentration increases, the inhibition on seed germination and growth became more obvious. Upon treatment with 1% NaCl plus 0.25% NaHCO3, the salt damage rate of germination rate has the largest variation, ranging from 0% to 89.80%. The salt damage rate of each trait shows a similar trend at all concentrations. Four germplasm resources with strong salt-alkali tolerance (Dajiugu, Nippobare, Mowanggu and 02428) and 7 sensitive germplasms were screened. The salt-tolerant gene sequence of 4 salt-alkali tolerant varieties and 3 sensitive germplasms were analyzed. OSHAL3 and OsRR22 were identical among the 7 germplasms, but SKC1 and DST showed clear variations between the salt-alkali tolerant and sensitive germplasms. Besides the salt-alkali tolerant germplasm resources, this study can also serve as a reference for mining of genes involved in salt-alkali tolerance and breeding of salt-alkali tolerant rice varieties.

Molecular mapping of the new blast resistance genes Pi47 and Pi48 in the durably resistant local rice cultivar Xiangzi 3150.

The indica rice cultivar Xiangzi 3150 (XZ3150) confers a high level of resistance to 95% of the isolates of Magnaporthe oryzae (the agent of rice blast disease) collected in Hunan Province, China. To identify the resistance (R) gene(s) controlling the high level of resistance in this cultivar, we developed 286 F(9) recombinant inbred lines (RILs) from a cross between XZ3150 and the highly susceptible cultivar CO39. Inoculation of the RILs and an F(2) population from a cross between the two cultivars with the avirulent isolate 193-1-1 in the growth chamber indicated the presence of two dominant R genes in XZ3150. A linkage map with 134 polymorphic simple sequence repeat and single feature polymorphism markers was constructed with the genotype data of the 286 RILs. Composite interval mapping (CIM) using the results of 193-1-1 inoculation showed that two major R genes, designated Pi47 and Pi48, were located between RM206 and RM224 on chromosome 11, and between RM5364 and RM7102 on chromosome 12, respectively. Interestingly, the CIM analysis of the four resistant components of the RILs to the field blast population revealed that Pi47 and Pi48 were also the major genetic factors responsible for the field resistance in XZ3150. The DNA markers linked to the new R genes identified in this study should be useful for further fine mapping, gene cloning, and marker-aided breeding of blast-resistant rice cultivars.

Transcriptome analysis of near-isogenic line provides novel insights into genes associated with panicle traits regulation in rice.

Panicle traits in rice impact yield and quality. The OsGRF4 gene encodes a growth-regulating factor controlling panicle traits, and was recently cloned. Gene expression profiling analysis can be used to study the molecular mechanisms underlying OsGRF4 regulation. Use of near-isogenic lines (NILs) reduces genetic background noise in omics studies. We compared transcriptome profiling of 7 cm long young panicles of NIL-Osgrf4 and NIL-OsGRF4 using RNAs sequence analyses. Eighty differentially expressed genes (DEGs) were identified. Our target gene OsGRF4 was up-regulated in NIL-OsGRF4 plants, which is consistent with a previous qPCR analysis. Hierarchical cluster analysis showed OsGRF4 is tightly clustered with the up-regulated DEG LOC_Os02g47320. Gene Ontology (GO) and KEGG analysis suggested that DEGs were primarily involved in somatic embryogenesis and chitinase activity. Two up-regulated DEGs, LOC_Os04g41680 and LOC_Os04g41620, were significantly enriched in the top 8 GO terms, and were over_represented in term of seed development, and may play key roles in grain shape regulation. The transcription factor Osmyb1 also exhibited differential expression between NILs, and may be is an important regulator of panicle traits. By searching reported functions of DEGs and by co-localization with previous identified quantitative trait loci (QTL), we determined that the pleiotropic gene OsGRF4 may also be involve in abiotic stress resistance. This study provides new candidates genes for further understanding the molecular mechanisms underlying rice panicle trait regulation.

Molecular mapping of the Pi2/9 allelic gene Pi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson.

BACKGROUND: Utilization of broad-spectrum resistance (R) genes is an effective and economical strategy to control the fungal pathogen Magnaporthe oryzae, the causal agent of the rice blast disease. Among the cloned blast resistance genes, Pi9, Pi2 and Piz-t confer broad-spectrum resistance to diverse M. oryzae isolates and were isolated from the Pi2/9 locus on chromosome 6. Identification and isolation of additional R genes with different resistance spectra from this locus will provide novel genetic resources for better control of this important rice disease. RESULTS: In this study, we identified a dominant R gene, Pi2-2, at the Pi2/9 locus from Jefferson, an elite U.S. rice cultivar, through genetic and physical mapping. Inoculation tests showed that Jefferson has different resistant specificities to M. oryzae isolates compared rice lines with the Pi9, Pi2 and Piz-t genes. Fine mapping delimited Pi2-2 to a 270-kb interval between the markers AP5659-3 and RM19817, and this interval contains three nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes in the Nipponbare genome. Five bacterial artificial chromosome (BAC) clones spanning the region were identified, and a BAC contig covering the Pi2-2 locus was constructed. CONCLUSIONS: We identified a new allelic gene at the Pi2/9 locus and fine-mapped the gene within a 270-kb region. Our results provide essential information for the isolation of the Pi2-2 gene and tightly linked DNA markers for rice blast resistance breeding.