The Microbial Connection to Sustainable Agriculture.

Microorganisms are an important element in modeling sustainable agriculture. Their role in soil fertility and health is crucial in maintaining plants' growth, development, and yield. Further, microorganisms impact agriculture negatively through disease and emerging diseases. Deciphering the extensive functionality and structural diversity within the plant-soil microbiome is necessary to effectively deploy these organisms in sustainable agriculture. Although both the plant and soil microbiome have been studied over the decades, the efficiency of translating the laboratory and greenhouse findings to the field is largely dependent on the ability of the inoculants or beneficial microorganisms to colonize the soil and maintain stability in the ecosystem. Further, the plant and its environment are two variables that influence the plant and soil microbiome's diversity and structure. Thus, in recent years, researchers have looked into microbiome engineering that would enable them to modify the microbial communities in order to increase the efficiency and effectiveness of the inoculants. The engineering of environments is believed to support resistance to biotic and abiotic stressors, plant fitness, and productivity. Population characterization is crucial in microbiome manipulation, as well as in the identification of potential biofertilizers and biocontrol agents. Next-generation sequencing approaches that identify both culturable and non-culturable microbes associated with the soil and plant microbiome have expanded our knowledge in this area. Additionally, genome editing and multidisciplinary omics methods have provided scientists with a framework to engineer dependable and sustainable microbial communities that support high yield, disease resistance, nutrient cycling, and management of stressors. In this review, we present an overview of the role of beneficial microbes in sustainable agriculture, microbiome engineering, translation of this technology to the field, and the main approaches used by laboratories worldwide to study the plant-soil microbiome. These initiatives are important to the advancement of green technologies in agriculture.

Microbe Related Chemical Signalling and Its Application in Agriculture.

The agriculture sector has been put under tremendous strain by the world's growing population. The use of fertilizers and pesticides in conventional farming has had a negative impact on the environment and human health. Sustainable agriculture attempts to maintain productivity, while protecting the environment and feeding the global population. The importance of soil-dwelling microbial populations in overcoming these issues cannot be overstated. Various processes such as rhizospheric competence, antibiosis, release of enzymes, and induction of systemic resistance in host plants are all used by microbes to influence plant-microbe interactions. These processes are largely founded on chemical signalling. Producing, releasing, detecting, and responding to chemicals are all part of chemical signalling. Different microbes released distinct sorts of chemical signal molecules which interacts with the environment and hosts. Microbial chemicals affect symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm growth, to name a few. We present an in-depth overview of chemical signalling between bacteria-bacteria, bacteria-fungi, and plant-microbe and the diverse roles played by these compounds in plant microbe interactions. These compounds' current and potential uses and significance in agriculture have been highlighted.

QTL and Candidate Genes: Techniques and Advancement in Abiotic Stress Resistance Breeding of Major Cereals.

At least 75% of the world's grain production comes from the three most important cereal crops: rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays). However, abiotic stressors such as heavy metal toxicity, salinity, low temperatures, and drought are all significant hazards to the growth and development of these grains. Quantitative trait locus (QTL) discovery and mapping have enhanced agricultural production and output by enabling plant breeders to better comprehend abiotic stress tolerance processes in cereals. Molecular markers and stable QTL are important for molecular breeding and candidate gene discovery, which may be utilized in transgenic or molecular introgression. Researchers can now study synteny between rice, maize, and wheat to gain a better understanding of the relationships between the QTL or genes that are important for a particular stress adaptation and phenotypic improvement in these cereals from analyzing reports on QTL and candidate genes. An overview of constitutive QTL, adaptive QTL, and significant stable multi-environment and multi-trait QTL is provided in this article as a solid framework for use and knowledge in genetic enhancement. Several QTL, such as DRO1 and Saltol, and other significant success cases are discussed in this review. We have highlighted techniques and advancements for abiotic stress tolerance breeding programs in cereals, the challenges encountered in introgressing beneficial QTL using traditional breeding techniques such as mutation breeding and marker-assisted selection (MAS), and the in roads made by new breeding methods such as genome-wide association studies (GWASs), the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and meta-QTL (MQTL) analysis. A combination of these conventional and modern breeding approaches can be used to apply the QTL and candidate gene information in genetic improvement of cereals against abiotic stresses.

Plant-Microbe Interaction: Aboveground to Belowground, from the Good to the Bad.

Soil health and fertility issues are constantly addressed in the agricultural industry. Through the continuous and prolonged use of chemical heavy agricultural systems, most agricultural lands have been impacted, resulting in plateaued or reduced productivity. As such, to invigorate the agricultural industry, we would have to resort to alternative practices that will restore soil health and fertility. Therefore, in recent decades, studies have been directed towards taking a Magellan voyage of the soil rhizosphere region, to identify the diversity, density, and microbial population structure of the soil, and predict possible ways to restore soil health. Microbes that inhabit this region possess niche functions, such as the stimulation or promotion of plant growth, disease suppression, management of toxicity, and the cycling and utilization of nutrients. Therefore, studies should be conducted to identify microbes or groups of organisms that have assigned niche functions. Based on the above, this article reviews the aboveground and below-ground microbiomes, their roles in plant immunity, physiological functions, and challenges and tools available in studying these organisms. The information collected over the years may contribute toward future applications, and in designing sustainable agriculture.

Effects of Abiotic Stress on Soil Microbiome.

Rhizospheric organisms have a unique manner of existence since many factors can influence the shape of the microbiome. As we all know, harnessing the interaction between soil microbes and plants is critical for sustainable agriculture and ecosystems. We can achieve sustainable agricultural practice by incorporating plant-microbiome interaction as a positive technology. The contribution of this interaction has piqued the interest of experts, who plan to do more research using beneficial microorganism in order to accomplish this vision. Plants engage in a wide range of interrelationship with soil microorganism, spanning the entire spectrum of ecological potential which can be mutualistic, commensal, neutral, exploitative, or competitive. Mutualistic microorganism found in plant-associated microbial communities assist their host in a number of ways. Many studies have demonstrated that the soil microbiome may provide significant advantages to the host plant. However, various soil conditions (pH, temperature, oxygen, physics-chemistry and moisture), soil environments (drought, submergence, metal toxicity and salinity), plant types/genotype, and agricultural practices may result in distinct microbial composition and characteristics, as well as its mechanism to promote plant development and defence against all these stressors. In this paper, we provide an in-depth overview of how the above factors are able to affect the soil microbial structure and communities and change above and below ground interactions. Future prospects will also be discussed.

SA-Mediated Regulation and Control of Abiotic Stress Tolerance in Rice.

Environmental or abiotic stresses are a common threat that remains a constant and common challenge to all plants. These threats whether singular or in combination can have devastating effects on plants. As a semiaquatic plant, rice succumbs to the same threats. Here we systematically look into the involvement of salicylic acid (SA) in the regulation of abiotic stress in rice. Studies have shown that the level of endogenous salicylic acid (SA) is high in rice compared to any other plant species. The reason behind this elevated level and the contribution of this molecule towards abiotic stress management and other underlying mechanisms remains poorly understood in rice. In this review we will address various abiotic stresses that affect the biochemistry and physiology of rice and the role played by SA in its regulation. Further, this review will elucidate the potential mechanisms that control SA-mediated stress tolerance in rice, leading to future prospects and direction for investigation.

Meta-Analysis of Quantitative Traits Loci (QTL) Identified in Drought Response in Rice (Oryza sativa L.).

Rice is an important grain that is the staple food for most of the world's population. Drought is one of the major stresses that negatively affects rice yield. The nature of drought tolerance in rice is complex as it is determined by various components and has low heritability. Therefore, to ensure success in breeding programs for drought tolerant rice, QTLs (quantitative trait loci) of interest must be stable in a variety of plant genotypes and environments. This study identified stable QTLs in rice chromosomes in a variety of backgrounds and environments and conducted a meta-QTL analysis of stable QTLs that have been reported by previous research for use in breeding programs. A total of 653 QTLs for drought tolerance in rice from 27 genetic maps were recorded for analysis. The QTLs recorded were related to 13 traits in rice that respond to drought. Through the use of BioMercartor V4.2, a consensus map containing QTLs and molecular markers were generated using 27 genetic maps that were extracted from the previous 20 studies and meta-QTL analysis was conducted on the consensus map. A total of 70 MQTLs were identified and a total of 453 QTLs were mapped into the meta-QTL areas. Five meta-QTLs from chromosome 1 (MQTL 1.5 and MQTL 1.6), chromosome 2 (MQTL2.1 and MQTL 2.2) and chromosome 3 (MQTL 3.1) were selected for functional annotation as these regions have high number of QTLs and include many traits in rice that respond to drought. A number of genes in MQTL1.5 (268 genes), MQTL1.6 (640 genes), MQTL 2.1 (319 genes), MQTL 2.2 (19 genes) and MQTL 3.1 (787 genes) were annotated through Blast2GO. Few major proteins that respond to drought stress were identified in the meta-QTL areas which are Abscisic Acid-Insensitive Protein 5 (ABI5), the G-box binding factor 4 (GBF4), protein kinase PINOID (PID), histidine kinase 2 (AHK2), protein related to autophagy 18A (ATG18A), mitochondrial transcription termination factor (MTERF), aquaporin PIP 1-2, protein detoxification 48 (DTX48) and inositol-tetrakisphosphate 1-kinase 2 (ITPK2). These proteins are regulatory proteins involved in the regulation of signal transduction and gene expression that respond to drought stress. The meta-QTLs derived from this study and the genes that have been identified can be used effectively in molecular breeding and in genetic engineering for drought resistance/tolerance in rice.

Comparative Genomics: Insights on the Pathogenicity and Lifestyle of Rhizoctonia solani.

Proper management of agricultural disease is important to ensure sustainable food security. Staple food crops like rice, wheat, cereals, and other cash crops hold great export value for countries. Ensuring proper supply is critical; hence any biotic or abiotic factors contributing to the shortfall in yield of these crops should be alleviated. Rhizoctonia solani is a major biotic factor that results in yield losses in many agriculturally important crops. This paper focuses on genome informatics of our Malaysian Draft R. solani AG1-IA, and the comparative genomics (inter- and intra- AG) with four AGs including China AG1-IA (AG1-IA_KB317705.1), AG1-IB, AG3, and AG8. The genomic content of repeat elements, transposable elements (TEs), syntenic genomic blocks, functions of protein-coding genes as well as core orthologous genic information that underlies R. solani's pathogenicity strategy were investigated. Our analyses show that all studied AGs have low content and varying profiles of TEs. All AGs were dominant for Class I TE, much like other basidiomycete pathogens. All AGs demonstrate dominance in Glycoside Hydrolase protein-coding gene assignments suggesting its importance in infiltration and infection of host. Our profiling also provides a basis for further investigation on lack of correlation observed between number of pathogenicity and enzyme-related genes with host range. Despite being grouped within the same AG with China AG1-IA, our Draft AG1-IA exhibits differences in terms of protein-coding gene proportions and classifications. This implies that strains from similar AG do not necessarily have to retain similar proportions and classification of TE but must have the necessary arsenal to enable successful infiltration and colonization of host. In a larger perspective, all the studied AGs essentially share core genes that are generally involved in adhesion, penetration, and host colonization. However, the different infiltration strategies will depend on the level of host resilience where this is clearly exhibited by the gene sets encoded for the process of infiltration, infection, and protection from host.

A Meta-Analysis of Quantitative Trait Loci Associated with Multiple Disease Resistance in Rice (Oryza sativa L.).

Rice blast, sheath blight and bacterial leaf blight are major rice diseases found worldwide. The development of resistant cultivars is generally perceived as the most effective way to combat these diseases. Plant disease resistance is a polygenic trait where a combinatorial effect of major and minor genes affects this trait. To locate the source of this trait, various quantitative trait loci (QTL) mapping studies have been performed in the past two decades. However, investigating the congruency between the reported QTL is a daunting task due to the heterogeneity amongst the QTLs studied. Hence, the aim of our study is to integrate the reported QTLs for resistance against rice blast, sheath blight and bacterial leaf blight and objectively analyze and consolidate the location of QTL clusters in the chromosomes, reducing the QTL intervals and thus identifying candidate genes within the selected meta-QTL. A total of twenty-seven studies for resistance QTLs to rice blast (8), sheath blight (15) and bacterial leaf blight (4) was compiled for QTL projection and analyses. Cumulatively, 333 QTLs associated with rice blast (114), sheath blight (151) and bacterial leaf blight (68) resistance were compiled, where 303 QTLs could be projected onto a consensus map saturated with 7633 loci. Meta-QTL analysis on 294 QTLs yielded 48 meta-QTLs, where QTLs with membership probability lower than 60% were excluded, reducing the number of QTLs within the meta-QTL to 274. Further, three meta-QTL regions (MQTL2.5, MQTL8.1 and MQTL9.1) were selected for functional analysis on the basis that MQTL2.5 harbors the highest number of QTLs; meanwhile, MQTL8.1 and MQTL9.1 have QTLs associated with all three diseases mentioned above. The functional analysis allows for determination of enriched gene ontology and resistance gene analogs (RGAs) and other defense-related genes. To summarize, MQTL2.5, MQTL8.1 and MQTL9.1 have a considerable number of R-genes that account for 10.21%, 4.08% and 6.42% of the total genes found in these meta-QTLs, respectively. Defense genes constitute around 3.70%, 8.16% and 6.42% of the total number of genes in MQTL2.5, MQTL8.1 and MQTL9.1, respectively. This frequency is higher than the total frequency of defense genes in the rice genome, which is 0.0096% (167 defense genes/17,272 total genes). The integration of the QTLs facilitates the identification of QTL hotspots for rice blast, sheath blight and bacterial blight resistance with reduced intervals, which helps to reduce linkage drag in breeding. The candidate genes within the promising regions could be utilized for improvement through genetical engineering.

Fatty Acid Synthase Beta Dehydratase in the Lipid Biosynthesis Pathway Is Required for Conidiogenesis, Pigmentation and Appressorium Formation in Magnaporthe oryzae S6.

Lipid biosynthesis produces glycerol, which is important in fueling turgor pressure necessary for germination and penetration of plant host by fungi. As the relationship between pathogenicity and the lipid biosynthetic pathway is not fully understood, we have elucidated the role of the fatty acid synthase beta subunit dehydratase (FAS1) gene in lipid biosynthesis. The FAS1 gene was silenced through homologous double crossover in Magnaporthe oryzae strain S6 to study the effect on lipid biosynthesis. The vegetative growth of Δfas1 mutants show the highest drop on oleic acid (between 10 and 50%), while the mycelial dry weight of mutants dropped significantly on all media. Conidiation of FAS1 mutants show a ~10- and ~5-fold reduction on oatmeal and Potato Dextrose Agar (PDA), respectively. Mutants formed mycelium that were mildly pigmented, indicating that the deletion of FAS1 may have affected melanin biosynthesis. Biochemical and gene expression studies concluded that the fatty acid degradation pathway might have been interrupted by FAS1 deletion. FAS1 mutants showed no enzyme activity on glucose or olive oil, suggesting that the mutants may lack functional peroxisomes and be defective in ß-oxidation of fatty acids, hence explaining the reduced lipid deposits in the spores.

ROS Homeostasis in Abiotic Stress Tolerance in Plants.

Climate change-induced abiotic stress results in crop yield and production losses. These stresses result in changes at the physiological and molecular level that affect the development and growth of the plant. Reactive oxygen species (ROS) is formed at high levels due to abiotic stress within different organelles, leading to cellular damage. Plants have evolved mechanisms to control the production and scavenging of ROS through enzymatic and non-enzymatic antioxidative processes. However, ROS has a dual function in abiotic stresses where, at high levels, they are toxic to cells while the same molecule can function as a signal transducer that activates a local and systemic plant defense response against stress. The effects, perception, signaling, and activation of ROS and their antioxidative responses are elaborated in this review. This review aims to provide a purview of processes involved in ROS homeostasis in plants and to identify genes that are triggered in response to abiotic-induced oxidative stress. This review articulates the importance of these genes and pathways in understanding the mechanism of resistance in plants and the importance of this information in breeding and genetically developing crops for resistance against abiotic stress in plants.

Elicitor and Receptor Molecules: Orchestrators of Plant Defense and Immunity.

Pathogen-associated molecular patterns (PAMPs), microbe-associated molecular patterns (MAMPs), herbivore-associated molecular patterns (HAMPs), and damage-associated molecular patterns (DAMPs) are molecules produced by microorganisms and insects in the event of infection, microbial priming, and insect predation. These molecules are then recognized by receptor molecules on or within the plant, which activates the defense signaling pathways, resulting in plant's ability to overcome pathogenic invasion, induce systemic resistance, and protect against insect predation and damage. These small molecular motifs are conserved in all organisms. Fungi, bacteria, and insects have their own specific molecular patterns that induce defenses in plants. Most of the molecular patterns are either present as part of the pathogen's structure or exudates (in bacteria and fungi), or insect saliva and honeydew. Since biotic stresses such as pathogens and insects can impair crop yield and production, understanding the interaction between these organisms and the host via the elicitor-receptor interaction is essential to equip us with the knowledge necessary to design durable resistance in plants. In addition, it is also important to look into the role played by beneficial microbes and synthetic elicitors in activating plants' defense and protection against disease and predation. This review addresses receptors, elicitors, and the receptor-elicitor interactions where these components in fungi, bacteria, and insects will be elaborated, giving special emphasis to the molecules, responses, and mechanisms at play, variations between organisms where applicable, and applications and prospects.

Drought Response in Rice: The miRNA Story.

As a semi-aquatic plant, rice requires water for proper growth, development, and orientation of physiological processes. Stress is induced at the cellular and molecular level when rice is exposed to drought or periods of low water availability. Plants have existing defense mechanisms in planta that respond to stress. In this review we examine the role played by miRNAs in the regulation and control of drought stress in rice through a summary of molecular studies conducted on miRNAs with emphasis on their contribution to drought regulatory networks in comparison to other plant systems. The interaction between miRNAs, target genes, transcription factors and their respective roles in drought-induced stresses is elaborated. The cross talk involved in controlling drought stress responses through the up and down regulation of targets encoding regulatory and functional proteins is highlighted. The information contained herein can further be explored to identify targets for crop improvement in the future.

Transposable Elements Adaptive Role in Genome Plasticity, Pathogenicity and Evolution in Fungal Phytopathogens.

Transposable elements (TEs) are agents of genetic variability in phytopathogens as they are a source of adaptive evolution through genome diversification. Although many studies have uncovered information on TEs, the exact mechanism behind TE-induced changes within the genome remains poorly understood. Furthermore, convergent trends towards bigger genomes, emergence of novel genes and gain or loss of genes implicate a TE-regulated genome plasticity of fungal phytopathogens. TEs are able to alter gene expression by revamping the cis-regulatory elements or recruiting epigenetic control. Recent findings show that TEs recruit epigenetic control on the expression of effector genes as part of the coordinated infection strategy. In addition to genome plasticity and diversity, fungal pathogenicity is an area of economic concern. A survey of TE distribution suggests that their proximity to pathogenicity genes TEs may act as sites for emergence of novel pathogenicity factors via nucleotide changes and expansion or reduction of the gene family. Through a systematic survey of literature, we were able to conclude that the role of TEs in fungi is wide: ranging from genome plasticity, pathogenicity to adaptive behavior in evolution. This review also identifies the gaps in knowledge that requires further elucidation for a better understanding of TEs' contribution to genome architecture and versatility.

Draft Genome Sequence of Rhizoctonia solani Anastomosis Group 1 Subgroup 1A Strain 1802/KB Isolated from Rice.

Sheath blight, caused by Rhizoctonia solani anastomosis group 1 subgroup 1A (AG1-1A), is one of the most devastating rice diseases worldwide. Here, we report the draft genome sequence of R. solani AG1-1A strain 1802/KB isolated from a popular Malaysian rice variety. To the best of our knowledge, this is the second reported representative genome from AG1-1A.

Identification of functionally important microRNAs from rice inflorescence at heading stage of a qDTY4.1-QTL bearing Near Isogenic Line under drought conditions.

A cross between IR64 (high-yielding but drought-susceptible) and Aday Sel (drought-tolerant) rice cultivars yielded a stable line with enhanced grain yield under drought screening field trials at International Rice Research Institute. The major effect qDTY4.1 drought tolerance and yield QTL was detected in the IR77298-14-1-2-10 Backcrossed Inbred Line (BIL) and its IR87705-7-15-B Near Isogenic Line (NIL) with 93.9% genetic similarity to IR64. Although rice yield is extremely susceptible to water stress at reproductive stage, currently, there is only one report on the detection of drought-responsive microRNAs in inflorescence tissue of a Japonica rice line. In this study, more drought-responsive microRNAs were identified in the inflorescence tissues of IR64, IR77298-14-1-2-10 and IR87705-7-15-B via next-generation sequencing. Among the 32 families of inflorescence-specific non-conserved microRNAs that were identified, 22 families were up-regulated in IR87705-7-15-B. Overall 9 conserved and 34 non-conserved microRNA families were found as drought-responsive in rice inflorescence with 5 conserved and 30 non-conserved families induced in the IR87705-7-15-B. The observation of more drought-responsive non-conserved microRNAs may imply their prominence over conserved microRNAs in drought response mechanisms of rice inflorescence. Gene Ontology annotation analysis on the target genes of drought-responsive microRNAs identified in IR87705-7-15-B revealed over-representation of biological processes including development, signalling and response to stimulus. Particularly, four inflorescence-specific microRNAs viz. osa-miR5485, osa-miR5487, osa-miR5492 and osa-miR5517, and two non-inflorescence specific microRNAs viz. osa-miR169d and osa-miR169f.2 target genes that are involved in flower or embryonic development. Among them, osa-miR169d, osa-miR5492 and osa-miR5517 are related to flowering time control. It is also worth mentioning that osa-miR2118 and osa-miR2275, which are implicated in the biosynthesis of rice inflorescence-specific small interfering RNAs, were induced in IR87705-7-15-B but repressed in IR77298-14-1-2-10. Further, gene search within qDTY4.1 QTL region had identified multiple copies of NBS-LRR resistance genes (potential target of osa-miR2118), subtilisins and genes implicated in stomatal movement, ABA metabolism and cuticular wax biosynthesis.

Identification of four functionally important microRNA families with contrasting differential expression profiles between drought-tolerant and susceptible rice leaf at vegetative stage.

BACKGROUND: Developing drought-tolerant rice varieties with higher yield under water stressed conditions provides a viable solution to serious yield-reduction impact of drought. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success of rice molecular breeding programmes. microRNAs have received tremendous attention recently due to its importance in negative regulation. In plants, apart from regulating developmental and physiological processes, microRNAs have also been associated with different biotic and abiotic stresses. Hence here we chose to analyze the differential expression profiles of microRNAs in three drought treated rice varieties: Vandana (drought-tolerant), Aday Sel (drought-tolerant) and IR64 (drought-susceptible) in greenhouse conditions via high-throughput sequencing. RESULTS: Twenty-six novel microRNA candidates involved in the regulation of diverse biological processes were identified based on the detection of miRNA*. Out of their 110 predicted targets, we confirmed 16 targets from 5 novel microRNA candidates. In the differential expression analysis, mature microRNA members from 49 families of known Oryza sativa microRNA were differentially expressed in leaf and stem respectively with over 28 families having at least a similar mature microRNA member commonly found to be differentially expressed between both tissues. Via the sequence profiling data of leaf samples, we identified osa-miR397a/b, osa-miR398b, osa-miR408-5p and osa-miR528-5p as being down-regulated in two drought-tolerant rice varieties and up-regulated in the drought-susceptible variety. These microRNAs are known to be involved in regulating starch metabolism, antioxidant defence, respiration and photosynthesis. A wide range of biological processes were found to be regulated by the target genes of all the identified differentially expressed microRNAs between both tissues, namely root development (5.3-5.7 %), cell transport (13.2-18.4 %), response to stress (10.5-11.3 %), lignin catabolic process (3.8-5.3 %), metabolic processes (32.1-39.5 %), oxidation-reduction process (9.4-13.2 %) and DNA replication (5.7-7.9 %). The predicted target genes of osa-miR166e-3p, osa-miR166h-5p*, osa-miR169r-3p* and osa-miR397a/b were found to be annotated to several of the aforementioned biological processes. CONCLUSIONS: The experimental design of this study, which features rice varieties with different drought tolerance and tissue specificity (leaf and stem), has provided new microRNA profiling information. The potentially regulatory importance of the microRNA genes mentioned above and their target genes would require further functional analyses.

Molecular characterization and screening for sheath blight resistance using Malaysian isolates of Rhizoctonia solani.

Two field isolates of Rhizoctonia solani were isolated from infected paddy plants in Malaysia. These isolates were verified via ITS-rDNA analysis that yielded ~720 bp products of the ITS1-5.8S-ITS4 region, respectively. The sequenced products showed insertion and substitution incidences which may result in strain diversity and possible variation in disease severity. These strains showed some regional and host-specific relatedness via Maximum Likelihood and further phylogenetic analysis via Maximum Parsimony showed that these strains were closely related to R. solani AG1-1A (with 99-100% identity). Subsequent to strain verification and analysis, these isolates were used in the screening of twenty rice varieties for tolerance or resistance to sheath blight via mycelial plug method where both isolates (1801 and 1802) showed resistance or moderate resistance to Teqing, TETEP, and Jasmine 85. Isolate 1802 was more virulent based on the disease severity index values. This study also showed that the mycelial plug techniques were efficient in providing uniform inoculum and humidity for screening. In addition this study shows that the disease severity index is a better mode of scoring for resistance compared to lesion length. These findings will provide a solid basis for our future breeding and screening activities at the institution.

The 172-kb genomic DNA region of the O. rufipogon yld1.1 locus: comparative sequence analysis with O. sativa ssp. japonica and O. sativa ssp. indica.

Common wild rice (Oryza rufipogon) plays an important role by contributing to modern rice breeding. In this paper, we report the sequence and analysis of a 172-kb genomic DNA region of wild rice around the RM5 locus, which is associated with the yield QTL yld1.1. Comparative sequence analysis between orthologous RM5 regions from Oryza sativa ssp. japonica, O. sativa ssp. indica and O. rufipogon revealed a high level of conserved synteny in the content, homology, structure, orientation, and physical distance of all 14 predicted genes. Twelve of the putative genes were supported by matches to proteins with known function, whereas two were predicted by homology to rice and other plant expressed sequence tags or complementary DNAs. The remarkably high level of conservation found in coding, intronic and intergenic regions may indicate high evolutionary selection on the RM5 region. Although our analysis has not defined which gene(s) determine the yld1.1 phenotype, allelic variation and the insertion of transposable elements, among other nucleotide changes, represent potential variation responsible for the yield QTL. However, as suggested previously, two putative receptor-like protein kinase genes remain the key suspects for yld1.1.

Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon.

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.