Oryza Rufipogon Research Articles

Impact of osmotic stress on the growth and root architecture of introgression lines derived from a wild ancestor of rice and a modern cultivar.

Many modern rice varieties have been intensively selected for high-yielding performance under irrigated conditions, reducing their genetic diversity and potentially increasing their susceptibility to abiotic stresses such as drought. In this study, we tested benefits for stress tolerance of introducing DNA segments from wild ancestor Oryza rufipogon to the modern cultivar O. sativa cv Curinga (CUR) by applying a gradient of osmotic stress to both parents and seven introgressed lines. Shoot growth of O. rufipogon had a high tolerance to osmotic stress, and the number of total root tips increased under mild osmotic stress. One introgression line showed greater shoot growth, root growth, and higher number of total root tips than the parent line CUR under osmotic stress. Abscisic acid (ABA) is a key hormone mediating plant responses to abiotic stresses. Both root and shoot growth of O. rufipogon were much more sensitive to ABA than CUR. Introgression lines varied in the extent to which the sensitivity of their growth responses to ABA and some lines correlated with their sensitivity to osmotic stress. Our results suggest that rice responses to ABA and osmotic stress are genotype dependent, and growth responses of rice to ABA are not a consistent indicator of resilience to abiotic stress in introgression lines.

Transcriptomic profiling of germinating seeds under cold stress and characterization of the cold-tolerant gene LTG5 in rice

BackgroundLow temperature is a limiting factor of rice productivity and geographical distribution. Wild rice (Oryza rufipogon Griff.) is an important germplasm resource for rice improvement. It has superior tolerance to many abiotic stresses, including cold stress, but little is known about the mechanism underlying its resistance to cold.ResultsThis study elucidated the molecular genetic mechanisms of wild rice in tolerating low temperature. Comprehensive transcriptome profiles of two rice genotypes (cold-sensitive ce 253 and cold-tolerant Y12–4) at the germinating stage under cold stress were comparatively analyzed. A total of 42.44–68.71 million readings were obtained, resulting in the alignment of 29,128 and 30,131 genes in genotypes 253 and Y12–4, respectively. Many common and differentially expressed genes (DEGs) were analyzed in the cold-sensitive and cold-tolerant genotypes. Results showed more upregulated DEGs in the cold-tolerant genotype than in the cold-sensitive genotype at four stages under cold stress. Gene ontology enrichment analyses based on cellular process, metabolic process, response stimulus, membrane part, and catalytic activity indicated more upregulated genes than downregulated ones in the cold-tolerant genotype than in the cold-sensitive genotype. Quantitative real-time polymerase chain reaction was performed on seven randomly selected DEGs to confirm the RNA Sequencing (RNA-seq) data. These genes showed similar expression patterns corresponding with the RNA-Seq method. Weighted gene co-expression network analysis (WGCNA) revealed Y12–4 showed more positive genes than 253 under cold stress. We also explored the cold tolerance gene LTG5 (Low Temperature Growth 5) encoding a UDP-glucosyltransferase. The overexpression of the LTG5 gene conferred cold tolerance to indica rice.ConclusionGene resources related to cold stress from wild rice can be valuable for improving the cold tolerance of crops.

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice.

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence (399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb, respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon (including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.

Some Accessions of Amazonian Wild Rice (Oryza glumaepatula) Constitutively Form a Barrier to Radial Oxygen Loss along Adventitious Roots under Aerated Conditions.

A barrier to radial oxygen loss (ROL), which reduces the loss of oxygen transported via the aerenchyma to the root tips, enables the roots of wetland plants to grow into anoxic/hypoxic waterlogged soil. However, little is known about its genetic regulation. Quantitative trait loci (QTLs) mapping can help to understand the factors that regulate barrier formation. Rice (Oryza sativa) inducibly forms an ROL barrier under stagnant conditions, while a few wetland plants constitutively form one under aerated conditions. Here, we evaluated the formation of a constitutive ROL barrier in a total of four accessions from two wild rice species. Three of the accessions were wetland accessions of O. glumaepatula, and the fourth was a non-wetland species of O. rufipogon. These species have an AA type genome, which allows them to be crossed with cultivated rice. The three O. glumaepatula accessions (W2165, W2149, and W1183) formed an ROL barrier under aerated conditions. The O. rufipogon accession (W1962) did not form a constitutive ROL barrier, but it formed an inducible ROL barrier under stagnant conditions. The three O. glumaepatula accessions should be useful for QTL mapping to understand how a constitutive ROL barrier forms. The constitutive barrier of W2165 was closely associated with suberization and resistance to penetration by an apoplastic tracer (periodic acid) at the exodermis but did not include lignin at the sclerenchyma.

Multicentric origin and diversification of atp6-orf79-like structures reveal mitochondrial gene flows in Oryza rufipogon and Oryza sativa.

Cytoplasmic male sterility (CMS) is a widely used genetic tool in modern hybrid rice breeding. Most genes conferring rice gametophytic CMS are homologous to orf79 and co‐transcribe with atp6. However, the origin, differentiation and flow of these mitochondrial genes in wild and cultivated rice species remain unclear. In this study, we performed de novo assembly of the mitochondrial genomes of 221 common wild rice (Oryza rufipogon Griff.) and 369 Asian cultivated rice (Oryza sativa L.) accessions, and identified 16 haplotypes of atp6‐orf79‐like structures and 11 orf79 alleles. These homologous structures were classified into 4 distinct groups (AO‐I, AO‐II, AO‐III and AO‐IV), all of which were observed in O. rufipogon but only AO‐I was detected in O. sativa, causing a decrease in the frequency of atp6‐orf79‐like structures from 19.9% to 8.1%. Phylogenetic and biogeographic analyses revealed that the different groups of these gametophytic CMS‐related genes in O. rufipogon evolved in a multicentric pattern. The geographical origin of the atp6‐orf79‐like structures was further traced back, and a candidate region in north‐east of Gangetic Plain on the Indian Peninsula (South Asia) was identified as the origin centre of AO‐I. The orf79 alleles were detected in all three cytoplasmic types (Or‐CT0, Or‐CT1 and Or‐CT2) of O. rufipogon, but only two alleles (orf79a and orf79b) were observed in Or‐CT0 type of O. sativa, while no orf79 allele was found in other types of O. sativa. Our results also revealed that the orf79 alleles in cultivated rice originated from the wild rice population in South and South‐East Asia. In addition, strong positive selection pressure was detected on the sequence variations of orf79 alleles, and a special evolutionary strategy was noted in these gametophytic CMS‐related genes, suggesting that their divergence could be beneficial to their survival in evolution.

Micro-Evolution Analysis Reveals Diverged Patterns of Polyol Transporters in Seven Gramineae Crops.

Polyol transporters (PLTs), also called polyol/monosaccharide transporters, is of significance in determining plant development and sugar transportation. However, the diverged evolutionary patterns of the PLT gene family in Gramineae crops are still unclear. Here a micro-evolution analysis was performed among the seven Gramineae representative crops using whole-genome sequences, i.e., Brachypodium distachyon (Bd), Hordeum vulgare (Hv), Oryza rufipogon (Or), Oryza sativa (Os), Sorghum bicolor (Sb), Setaria italica (Si), and Zea mays (Zm), leading to the identification of 12, 11, 12, 15, 20, 24, and 20 PLT genes, respectively. In this study, all PLT genes were divided into nine orthogroups (OGs). However, the number of PLT genes and the distribution of PLT OGs were not the same in these seven Gramineae species, and different OGs were also subject to different purification selection pressures. These results indicated that the PLT OGs of the PLT gene family have been expanded or lost unevenly in all tested species. Then, our results of gene duplication events confirmed that gene duplication events promoted the expansion of the PLT gene family in some Gramineous plants, namely, Bd, Or, Os, Si, Sb, and Zm, but the degree of gene family expansion, the type of PLT gene duplication, and the differentiation time of duplicate gene pairs varied greatly among these species. In addition, the sequence alignment and the internal repeat analysis of all PLTs protein sequences implied that the PLT protein sequences may originate from an internal repeat duplication of an ancestral six transmembrane helical units. Besides that, the protein motifs result highlighted that the PLT protein sequences were highly conserved, whereas the functional differentiation of the PLT genes was characterized by different gene structures, upstream elements, as well as co-expression analysis. The gene expression analysis of rice and maize showed that the PLT genes have a wide range of expression patterns, suggesting diverse biological functions. Taken together, our finding provided a perspective on the evolution differences and the functional characterizations of PLT genes in Gramineae representative crops.

Identification and genetic analysis of qCL1.2, a novel allele of the \u201cgreen revolution\u201d gene SD1 from wild rice (Oryza rufipogon) that enhances plant height

BackgroundThe exploitation of novel alleles from wild rice that were lost during rice cultivation could be very important for rice breeding and evolutionary studies. Plant height (PH) was a target of artificial selection during rice domestication and is still a target of modern breeding. The “green revolution” gene semi-dwarf 1 (SD1) were well documented and used in the past decades, allele from wild rice could provide new insights into the functions and evolution of this gene.ResultsWe identified a PH-related quantitative trait locus, qCL1.2,from wild riceusing a set of chromosome segment substitution lines. qCL1.2encodesa novel allele of SD1 gene. The wild allele of SD1 is a dominant locus that can significantly promote rice internode length by regulating the expression levels of genes involved in gibberellin biosynthesis and signal transduction. Nucleotide diversity and haplotype network analyses of the SD1 gene were performed using 2822 rice landraces. Two previously reported functional nucleotide polymorphisms clearly differentiated japonica and indica rice; however, they were not associated with PH selection. Other new functional nucleotide polymorphisms in the coding, but not promoter, regions were involved in PH selection during rice domestication. Our study increasesunderstanding of the rice SD1 gene and provides additional evidence of this gene’s selection during rice domestication.ConclusionsOur findings provide evidence thatSD1 gene from wild rice enhances plant height and new functional nucleotide polymorphisms of this gene were artificially selected during cultivated rice differentiation.

Morphological characterization and genetic identity of crop wild relatives of rice (Oryza sativa L.) collected from different ecological niches of India

Crop wild relatives of rice (Oryza sativa L.) are an important reservoir of genes for broadening the genetic base of commercial varieties. India is the centre of genetic diversity of some of the wild as-well-as the cultivated rice species. In order to collect genetic diversity, explorations were undertaken in six states located in different agro-ecological zones of India. Forty-four accessions belonging to four wild species (Oryza rufipogon Griff., Oryza nivara Sharma et Shastry, Oryza officinalis Wall. ex Watt and Oryza sativa L. f. spontanea Roshev.) including two accessions of cultivated rice (Oryza sativa L. subsp. indica Kato) were collected from different ecological niches. The rice accessions were characterized using 28 morphological descriptors, which included 15 qualitative and 13 quantitative traits. Wild rice accessions displayed a broad range of phenotypic variation for plant height, main heading days, days to 50% flowering, flag-leaf length and width, panicle shape and size, peduncle length, awning pattern, awn length and colour, seed length and width, seed length/width ratio and 100-seed weight. Cluster analysis based on Jaccard’s similarity coefficients derived from 28 morphological traits grouped 44 accessions into three clades. DNA sequencing revealed that internal transcribed spacer (ITS) region of 18S–5.8S–28S nuclear ribosomal cistron varied from 574 to 577 bp with mean of 576.1 bp in AA-genome species. GC content was high in ITS region, which varied from 69.57 to 70.36% with mean value of 69.99%. Neighbour-joining phylogenetic tree distinguished Oryza sativa–nivara–rufipogon complex from other species of AA-genome. The wild germplasm characterized in this study could be utilized in the genetic enhancement of cultivated rice.

Identification of a novel planthopper resistance gene from wild rice (Oryza rufipogon Griff.)

Rice planthoppers, including brown planthopper (BPH) and white-backed planthopper (WBPH), are the most destructive pests in Asian rice cultivation regions. Planthopper resistance genes that have been mapped and characterized advance our understanding of underlying resistance mechanisms and facilitate the breeding of resistant varieties, thereby contributing to an efficient pest management strategy. In this study, a novel resistance gene Bph38 derived from the wild rice species Oryza rufipogon Griff. was found to confer high resistance to BPH and WBPH. Conventional mapping was performed to identify regions associated with BPH and WBPH resistance, and two mapping efforts led to the same region on chromosome 4 flanked by markers RM16563 and RM16763. Bulked-segregant analysis and next-generation sequencing were performed using the same population to detect the resistance gene. Conventional mapping narrowed the region to a 12.3-Mb segment, and fine mapping using BC1F2 recombinants identified a 79-kb segment flanked by markers YM112 and YM190. Near-isogenic lines (NILs) carrying Bph38 in the 9311 (indica) and BR54 (japonica) genetic backgrounds were developed by crossing and backcrossing with marker-assisted selection. The agronomic traits and BPH and WBPH resistance of the NILs were similar to those of the recurrent parents. Mandatory feeding and host-choice tests revealed that Bph38 showed both antibiotic and antixenotic effects in both insects, with stronger effects in indica-background lines. Further fine mapping and characterization of the major gene may result in map-based cloning of the gene and allow its application in breeding insect-resistant rice varieties.

Draft genomes of two outcrossing wild rice, Oryza rufipogon and O. longistaminata, reveal genomic features associated with mating-system evolution.

Oryza rufipogon and O. longistaminata are important wild relatives of cultivated rice, harboring a promising source of novel genes for rice breeding programs. Here, we present de novo assembled draft genomes and annotation of O. rufipogon and O. longistaminata. Our analysis reveals a considerable number of lineage‐specific gene families associated with the self‐incompatibility (SI) and formation of reproductive separation. We show how lineage‐specific expansion or contraction of gene families with functional enrichment of the recognition of pollen, thus enlightening their reproductive diversification. We documented a large number of lineage‐specific gene families enriched in salt stress, antifungal response, and disease resistance. Our comparative analysis further shows a genome‐wide expansion of genes encoding NBS‐LRR proteins in these two outcrossing wild species in contrast to six other selfing rice species. Conserved noncoding sequences (CNSs) in the two wild rice genomes rapidly evolve relative to selfing rice species, resulting in the reduction of genomic variation owing to shifts of mating systems. We find that numerous genes related to these rapidly evolving CNSs are enriched in reproductive structure development, flower development, and postembryonic development, which may associate with SI in O. rufipogon and O. longistaminata.

Genome-Wide Analysis of WRKY Genes and Their Response to Salt Stress in the Wild Progenitor of Asian Cultivated Rice, Oryza rufipogon

WRKY gene family is widespread in plants, which is of significance in determining plant development and stress response. Although WRKY transcription factors have been widely characterized in many plants, a genome-wide analysis of the WRKY gene family is still lacking in Oryza rufipogon. In this study, we identified 101 O. rufipogon WRKY (OrWRKY) transcription factors, which were further classified into eight subgroups. Phylogenetic analysis showed that OrWRKY transcription factors were supported by highly conserved motifs and gene structures. Chromosomal distribution of OrWRKYs indicated that most genes were dispersed on all twelve chromosomes, especially enriched on Chromosome 1. Syntenic analysis revealed that 69 (68.3%) genes were derived from either segmental (49) or tandem duplication events (20), suggesting a essential role of segmental duplications. We characterized a total of 39 orthologous gene pairs between O. sativa ssp. japonica WRKY (OsjWRKY) and OrWRKY. We then performed quantitative real-time polymerase chain reaction (qPCR) experiments to validate tissue-specific and differential expression of the OrWRKYs. We also investigated corresponsive expression of the OrWRKYs in response to salt stresses in leaves and roots. This study gains a new insight on the evolution of the OrWRKYs and will help further functional characterization of candidate genes towards wild rice germplasm exploration for rice genetic improvement.

Association Mapping of Quantitative Trait Loci for Grain Size in Introgression Line Derived from Oryza Rufipogon

Grain size is one of the critical agronomic traits governing grain yield and quality in rice. However, the underlying genetic mechanisms that control grain size in rice are poorly understood. We used an introgression line derived from Zhonghui 8015 and Oryza rufipogon Griff. This introgression line was evaluated under two different environmental conditions to dissect the quantitative trait loci controlling grain size. Genome-wide association study (GWAS) was performed using 28 193 SNPs through a general linear model, and 56 significant SNPs on different loci associated with the 4 grain size traits were detected. Cloned genes including GS3 and qGL3 showed substantial effects on grain length and size. Seven new stable loci were identified with pleiotropic effects on grain size. Haplotype, gene expression analyses, combined gene-based associations, and functional annotations permitted the shortlisting of important dominant genes including GS3 and qGL3.

Identification of Major Locus Bph35 Resistance to Brown Planthopper in Rice

An introgression line RBPH660, derived from wild rice Oryza rufipogon, showed stable resistance to brown planthopper (BPH). Segregation analysis indicated BPH resistance of RBPH660 was controlled by multiple genes/QTLs. By using the bulked segregant analysis (BSA)-seq method, two genomic regions harboring QTLs resistance to BPH were identified from 1.20 to 16.70 Mb on chromosome 4 and from 10.20 to 12.60 Mb on chromosome 9 in RBPH660, respectively. A major resistance locus, designated as Bph35 accounting for 51.27% of the phenotypic variation with a LOD score of 42.51, was mapped to the candidate region of chromosome 4 between InDel (insertion-deletion) markers PSM16 and R4M13. For fine mapping of Bph35, one simple sequence repeat and three newly developed InDel markers were used to screen the recombinants. Finally, the Bph35 locus was delimited in the region from 6.28 to 6.93 Mb and there were 18 predicted protein-encoding genes with a total of 114 non-synonymous single nucleotide polymorphism (SNP) variant sites between the resistant and susceptible parents. Out of these genes, Os04g0193950, encoding a putative NB-ARC (nucleotide- binding adaptor shared by APAF-1, R proteins and CED-4) and LRR (leucine-rich repeat) domain protein with nine non-synonymous SNP substitutions in its coding sequence regions, might be the candidate gene for Bph35. These findings would facilitate the map-based cloning of the Bph35 gene and development of resistant varieties against BPH in rice.

Genotypic Variation in Spatial Distribution of Fe in Rice Grains in Relation to Phytic Acid Content and Ferritin Gene Expression

Rice varieties having high Fe concentration in the endospermic region can be used as a good source for Fe deficit population. In this study, 303 Oryza sativa varieties and 1 Oryza rufipogon accession were assessed for spatial Fe accumulation in grains by Prussian blue staining method. Spatial ferritin protein distribution in grains was visualized by immunohistochemistry, and ferritin expression was assessed in selected rice varieties using semi-quantitative reverse transcription PCR. Three popular rice varieties, namely Sarjoo 52, Madhukar and Jalmagna, and the O. rufipogon variety showed Fe in all the regions of grains, and the highest Fe concentration was observed in the embryo region. Some high-yielding varieties like Swarna, Swarna Sub 1, CSR13 and NDRR359 had lower Fe concentration in the embryo region. The highest Fe concentration was detected in O. rufipogon (49.8μg/g), followed by Sarjoo 52 (26.1μg/g) and Madhukar (25.7μg/g). Phytic acid concentration was the minimum in O. rufipogon (5.75mg/g) followed by Sarjoo 52 (5.83mg/g). Western blot and semi-quantitative reverse transcription PCR showed higher expression of ferritin gene in O. rufipogon, Sarjoo 52 and Madhukar. In conclusion, O. rufipogon and Sarjoo 52 had higher Fe concentration in the embryo regions as well as endosperm and aleurone layer, whereas the other varieties had lower Fe concentration in the endosperm. Sarjoo 52 could be used as a donor in the rice breeding program for the generation of new varieties with elevated grain Fe concentration.

Divergence in flowering time is a major component contributing to reproductive isolation between two wild rice species (Oryza rufipogon and O. nivara).

It is of critical importance for our understanding of speciation process to determine the forms of reproductive isolation and their relative importance in species divergence. Oryza nivara and O. rufipogon are direct ancestors of Asian cultivated rice and a progenitor-daughter species pair. Investigating the reproductive isolation between them provides insights into plant speciation and helps understanding of the rice domestication. Here, we quantitatively measured the major components of reproductive isolation between the two species based on common garden and crossing experiments for three pairs of sympatric populations in Nepal, Cambodia and Laos. We revealed significant differences in the flowering times between species pairs, with O. nivara flowering much earlier than O. rufipogon. A very weak reduction in seed set but no reduction in F1 viability and fertility were detected for the crosses between species relative to those within species. Moreover, we detected asymmetrical compatibility between species and found that emasculation significantly decreased pollination success in O. nivara but not in O. rufipogon. Our study demonstrates that the divergence between O. nivara and O. rufipogon is maintained almost entirely by the difference in flowering times and suggests that differential flowering times contribute to both habitat preferences and reproductive isolation between species.

SMRT sequencing of the Oryza rufipogon genome reveals the genomic basis of rice adaptation

Asian cultivated rice is believed to have been domesticated from a wild progenitor, Oryza rufipogon, offering promising sources of alleles for world rice improvement. Here we first present a high-quality chromosome-scale genome of the typical O. rufipogon. Comparative genomic analyses of O. sativa and its two wild progenitors, O. nivara and O. rufipogon, identified many dispensable genes functionally enriched in the reproductive process. We detected millions of genomic variants, of which large-effect mutations could affect agronomically relevant traits. We demonstrate how lineage-specific expansion of gene families may have contributed to the formation of reproduction isolation. We document thousands of genes with signatures of positive selection that are mainly involved in the reproduction and response to biotic- and abiotic stresses. We show that selection pressures may serve as forces to govern substantial genomic alterations that form the genetic basis of rapid evolution of mating and reproductive systems under diverse habitats.

Genome-wide transcriptome profile of rice hybrids with and without Oryza rufipogon introgression reveals candidate genes for yield

In this study, we compared genome-wide transcriptome profile of two rice hybrids, one with (test hybrid IR79156A/IL50-13) and the other without (control hybrid IR79156A/KMR3) O. rufipogon introgressions to identify candidate genes related to grain yield in the test hybrid. IL50-13 (Chinsurah Nona2 IET21943) the male parent (restorer) used in the test hybrid, is an elite BC4F8 introgression line of KMR3 with O. rufipogon introgressions. We identified 2798 differentially expressed genes (DEGs) in flag leaf and 3706 DEGs in panicle. Overall, 78 DEGs were within the major yield QTL qyld2.1 and 25 within minor QTL qyld8.2. The DEGs were significantly (p < 0.05) enriched in starch synthesis, phenyl propanoid pathway, ubiquitin degradation and phytohormone related pathways in test hybrid compared to control hybrid. Sequence analysis of 136 DEGs from KMR3 and IL50-13 revealed 19 DEGs with SNP/InDel variations. Of the 19 DEGs only 6 showed both SNP and InDel variations in exon regions. Of these, two DEGs within qyld2.1, Phenylalanine ammonia- lyase (PAL) (Os02t0626400-01, OsPAL2) showed 184 SNPs and 11 InDel variations and Similar to phenylalanine ammonia- lyase (Os02t0627100-01, OsPAL4) showed 205 SNPs and 13 InDel variations. Both PAL genes within qyld2.1 and derived from O. rufipogon are high priority candidate genes for increasing grain yield in rice.

Identification and Characterization of a Large Effect QTL from Oryza glumaepatula Revealed Pi68(t) as Putative Candidate Gene for Rice Blast Resistance

BackgroundField resistance is often effective and durable as compared to vertical resistance. The introgression line (INGR15002) derived from O. glumaepatula has proven broad spectrum field resistance for both leaf and neck blast.ResultsQuantitative Trait Loci (QTL) analysis of INGR15002, led to the identification of two major QTL - qBL3 contributing about 34% and 32% phenotypic variance towards leaf and neck blast resistance, respectively and qBL7 contributing about 25% of phenotypic variance for leaf blast. Further, qBL3 was fine mapped, narrowed down to 300 kb region and a linked SNP maker was identified. By combining mapping with microarray analysis, a candidate gene, Os03g0281466 (malectin-serine threonine kinase), was identified in the fine mapped region and named as Pi68(t). The nucleotide variations in the coding as well as upstream region of the gene was identified through cloning and sequence analysis of Pi68(t) alleles. These significant variations led to the non-synonymous changes in the protein as well as variations (presence/absence) in four important motifs (W-box element; MYC element; TCP element; BIHD1OS) at promoter region those are associated with resistance and susceptible reactions. The effect of qBL3 was validated by its introgression into BPT5204 (susceptible variety) through marker-assisted selection and progeny exhibiting resistance to both leaf and neck blast was identified. Further, the utility of linked markers of Pi68(t) in the blast breeding programs was demonstrated in elite germplasm lines.ConclusionsThis is the first report on the identification and characterization of major effect QTL from O. glumaepatula, which led to the identification of a putative candidate gene, Pi68(t), which confers field resistance to leaf as well as neck blast in rice.

Identification of the blast resistance gene Picl(t) from Chaling common wild rice (Oryza rufipogon Griff.)

AbstractRice blast, caused by the fungal pathogen Magnaporthe oryzae (M. oryzae), is one of the most destructive and widespread plant diseases in the world. Utilization of resistance genes in rice breeding is considered to be an effective and economical method to control this disease. To identify new sources of blast resistance, a set of 1160 introgression lines (ILs) containing chromosome segments of Chaling common wild rice (Oryza rufipogon Griff.) in the genetic background of an elite indica rice variety 93‐11 were developed and phenotyped in the blast nursery. Thirty‐three ILs displaying stable blast resistance in three consecutive years were obtained. Among them, one line, IL1043, was subsequently found to be resistant to all of the 28 M. oryzae isolates from different regions through artificial inoculation in greenhouse. By combining bulk segregant analysis coupled with next‐generation sequencing (BSA‐seq) and recessive class analysis (RCA), a major blast resistance gene in IL1043, designated Picl(t), was mapped on rice chromosome 6 flanked by the markers RM527 and Indel6 with an interval of approximately 925 kb, which covers the Pi2/9 locus. These results will facilitate fine mapping and cloning of Picl(t), and the linked markers will further provide a useful tool for rice blast resistance breeding.

Natural Sequence Variations and Combinations of GNP1 and NAL1 Determine the Grain Number per Panicle in Rice

BackgroundThe grain number per panicle (GNP), which is one of three grain yield components, is an important trait for the genetic improvement of rice. Although the NAL1 and GNP1 genes regulating the rice GNP and grain yield have been cloned, their allelic diversity, functional differences in rice germplasms, and effects of their combination on GNP and grain yield remain unclear.ResultsBased on DNA sequences of these two genes in 198 cultivated rice (Oryza sativa) and 8–10 wild rice (Oryza rufipogon) germplasms, 16 and 14 haplotypes were identified for NAL1 and GNP1, respectively. The NAL1 gene had the strongest effects on GNP in indica (xian) and japonica (geng) subpopulations. In contrast, GNP1 had no significant effects in the geng subpopulation and was rare in the xian background, in which the superior GNP1 allele (GNP1–6) was detected in only 4.0% of the 198 germplasms. Compared with the transgenic lines with GNP1 or NAL1, the transgenic lines with both genes had a higher GNP (15.5%–25.4% and 11.6%–15.9% higher, respectively) and grain yield (5.7%–9.0% and 8.3%–12.3% higher, respectively) across 3 years. The two genes combined in the introgression lines in Lemont background resulted in especially favorable effects on the GNP.ConclusionsOur results indicated that the GNP1 and NAL1 exhibited obvious differentiation and their combinations can significantly increase the grain yield in geng rice cultivars. These observations provide insights into the molecular basis of the GNP and may be useful for rice breeding of high yield potential by pyramiding GNP1 and NAL1.