Blast resistance in rice: a review of conventional breeding to molecular approaches

Blast disease caused by the fungal pathogen Magnaporthe oryzae is the most severe diseases of rice. Using classical plant breeding techniques, breeders have developed a number of blast resistant cultivars adapted to different rice growing regions worldwide. However, the rice industry remains threatened by blast disease due to the instability of blast fungus. Recent advances in rice genomics provide additional tools for plant breeders to improve rice production systems that would be environmentally friendly. This article outlines the application of conventional breeding, tissue culture and DNA-based markers that are used for accelerating the development of blast resistant rice cultivars. The best way for controlling the disease is to incorporate both qualitative and quantitative genes in resistant variety. Through conventional and molecular breeding many blast-resistant varieties have been developed. Conventional breeding for disease resistance is tedious, time consuming and mostly dependent on environment as compare to molecular breeding particularly marker assisted selection, which is easier, highly efficient and precise. For effective management of blast disease, breeding work should be focused on utilizing the broad spectrum of resistance genes and pyramiding genes and quantitative trait loci. Marker assisted selection provides potential solution to some of the problems that conventional breeding cannot resolve. In recent years, blast resistant genes have introgressed into Luhui 17, G46B, Zhenshan 97B, Jin 23B, CO39, IR50, Pusa1602 and Pusa1603 lines through marker assisted selection. Introduction of exotic genes for resistance induced the occurrence of new races of blast fungus, therefore breeding work should be concentrated in local resistance genes. This review focuses on the conventional breeding to the latest molecular progress in blast disease resistance in rice. This update information will be helpful guidance for rice breeders to develop durable blast resistant rice variety through marker assisted selection.     

Early changes of gene activity in developing seedlings of Arabidopsis hybrids relative to parents may contribute to hybrid vigour

Hybrid vigour (heterosis) has been used for decades in crop industries, especially in the production of maize and rice. Hybrid varieties usually exceed their parents in plant biomass and seed yield. But the molecular basis of hybrid vigour is not fully understood. In this project, we studied heterosis at early stages of seedling development in Arabidopsis hybrids derived from crossing Ler and C24 accessions. We found that early heterosis is associated with non‐additive gene expression that resulted from earlier changes in gene expression in the hybrids relative to the parents. The non‐additively expressed genes are involved in metabolic pathways, including photosynthesis, critical for plant growth. The early increased expression levels of genes involved in energy production in hybrids is associated with heterosis in the young seedlings that could be essential for biomass heterosis at later developmental stages of the plant.     

Historical changes in population structure during rice breeding programs in the northern limits of rice cultivation

Key message

The rice local population was clearly differentiated into six groups over the 100-year history of rice breeding programs in the northern limit of rice cultivation over the world.

Abstract

Genetic improvements in plant breeding programs in local regions have led to the development of new cultivars with specific agronomic traits under environmental conditions and generated the unique genetic structures of local populations. Understanding historical changes in genome structures and phenotypic characteristics within local populations may be useful for identifying profitable genes and/or genetic resources and the creation of new gene combinations in plant breeding programs. In the present study, historical changes were elucidated in genome structures and phenotypic characteristics during 100-year rice breeding programs in Hokkaido, the northern limit of rice cultivation in the world. We selected 63 rice cultivars to represent the historical diversity of this local population from landraces to the current breeding lines. The results of the phylogenetic analysis demonstrated that these cultivars clearly differentiated into six groups over the history of rice breeding programs. Significant differences among these groups were detected in five of the seven traits, indicating that the differentiation of the Hokkaido rice population into these groups was correlated with these phenotypic changes. These results demonstrated that breeding practices in Hokkaido have created new genetic structures for adaptability to specific environmental conditions and breeding objectives. They also provide a new strategy for rice breeding programs in which such unique genes in local populations in the world can explore the genetic potentials of the local populations.     

Developmental behavior of gene expression for brown rice thickness under different environments

The dynamic changes of genetic effects, including main effects, and genotype x environment (GE) interaction effects on brown rice thickness (BRT) across environments were investigated by using the developmental genetic models. Seven cytoplasmic male sterile lines of indica rice (Oryza sativa L.) as females and five restoring lines as males were used in a factorial design to produce grains of F(1)s and F(2)s in two environments (years) for developmental genetic analysis. The results indicate that genetic effects, especially GE interaction effects of triploid endosperm genes, cytoplasm genes, and diploid maternal plant genes were important to the performance of BRT at various filling stages of rice. The BRT was genetically controlled by the net genetic effects of genes expressed at the early and late filling stages (1-7 days and 15-21 days after flowering, respectively). The differences in net genetic effects under different environments for endosperm, cytoplasm, and maternal plant genes were found, and the net GE interaction effects were more important to BRT at the early filling and mature stages of rice. Some net genetic effects, especially for net cytoplasm effects spasmodically expressed, were detected among filling stages. Higher additive and cytoplasm main effects, along with their interaction effects, were found, which would be useful for selection for BRT in breeding programs. The predicated genetic effects at different filling stages show that the parents of V20 and Xieqingzao were better than others for improving BRT of progenies.     

Using the knowns to discover the unknowns: MS‐based dereplication uncovers structural diversity in 17‐hydroxygeranyllinalool diterpene glycoside production in the Solanaceae

Exploring the diversity of plant secondary metabolism requires efficient methods to obtain sufficient structural insights to discriminate previously known from unknown metabolites. De novo structure elucidation and confirmation of known metabolites (dereplication) remain a major bottleneck for mass spectrometry‐based metabolomic workflows, and few systematic dereplication strategies have been developed for the analysis of entire compound classes across plant families, partly due to the complexity of plant metabolic profiles that complicates cross‐species comparisons. 17‐hydroxygeranyllinalool diterpene glycosides (HGL‐DTGs) are abundant defensive secondary metabolites whose malonyl and glycosyl decorations are induced by jasmonate signaling in the ecological model plant Nicotiana attenuata. The multiple labile glycosidic bonds of HGL‐DTGs result in extensive in‐source fragmentation (IS‐CID) during ionization. To reconstruct these IS‐CID clusters from profiling data and identify precursor ions, we applied a deconvolution algorithm and created an MS/MS library from positive‐ion spectra of purified HGL‐DTGs. From this library, 251 non‐redundant fragments were annotated, and a workflow to characterize leaf, flower and fruit extracts of 35 solanaceous species was established. These analyses predicted 105 novel HGL‐DTGs that were restricted to Nicotiana, Capsicum and Lycium species. Interestingly, malonylation is a highly conserved step in HGL‐DTG metabolism, but is differentially affected by jasmonate signaling among Nicotiana species. This MS‐based workflow is readily applicable for cross‐species re‐identification/annotation of other compound classes with sufficient fragmentation knowledge, and therefore has the potential to support hypotheses regarding secondary metabolism diversification.     

Pedigree‐based analysis of derivation of genome segments of an elite rice reveals key regions during its breeding

Analyses of genome variations with high‐throughput assays have improved our understanding of genetic basis of crop domestication and identified the selected genome regions, but little is known about that of modern breeding, which has limited the usefulness of massive elite cultivars in further breeding. Here we deploy pedigree‐based analysis of an elite rice, Huanghuazhan, to exploit key genome regions during its breeding. The cultivars in the pedigree were resequenced with 7.6× depth on average, and 2.1 million high‐quality single nucleotide polymorphisms (SNPs) were obtained. Tracing the derivation of genome blocks with pedigree and information on SNPs revealed the chromosomal recombination during breeding, which showed that 26.22% of Huanghuazhan genome are strictly conserved key regions. These major effect regions were further supported by a QTL mapping of 260 recombinant inbred lines derived from the cross of Huanghuazhan and a very dissimilar cultivar, Shuanggui 36, and by the genome profile of eight cultivars and 36 elite lines derived from Huanghuazhan. Hitting these regions with the cloned genes revealed they include numbers of key genes, which were then applied to demonstrate how Huanghuazhan were bred after 30 years of effort and to dissect the deficiency of artificial selection. We concluded the regions are helpful to the further breeding based on this pedigree and performing breeding by design. Our study provides genetic dissection of modern rice breeding and sheds new light on how to perform genomewide breeding by design.     

Genome‐wide association study identifies an NLR gene that confers partial resistance to Magnaporthe oryzae in rice

Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C‐RDP‐II), which contains 584 rice accessions and are genotyped with 700 000 single‐nucleotide polymorphism (SNP) markers. The C‐RDP‐II accessions were inoculated with three blast strains collected from different rice‐growing regions in China. Genome‐wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide‐binding site leucine‐rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up‐regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR‐Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non‐strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice.     

A whole‐genome SNP array (RICE6K) for genomic breeding in rice

The advances in genotyping technology provide an opportunity to use genomic tools in crop breeding. As compared to field selections performed in conventional breeding programmes, genomics‐based genotype screen can potentially reduce number of breeding cycles and more precisely integrate target genes for particular traits into an ideal genetic background. We developed a whole‐genome single nucleotide polymorphism (SNP) array, RICE6K, based on Infinium technology, using representative SNPs selected from more than four million SNPs identified from resequencing data of more than 500 rice landraces. RICE6K contains 5102 SNP and insertion–deletion (InDel) markers, about 4500 of which were of high quality in the tested rice lines producing highly repeatable results. Forty‐five functional markers that are located inside 28 characterized genes of important traits can be detected using RICE6K. The SNP markers are evenly distributed on the 12 chromosomes of rice with the average density of 12 SNPs per 1 Mb and can provide information for polymorphisms between indica and japonica subspecies as well as varieties within indica and japonica groups. Application tests of RICE6K showed that the array is suitable for rice germplasm fingerprinting, genotyping bulked segregating pools, seed authenticity check and genetic background selection. These results suggest that RICE6K provides an efficient and reliable genotyping tool for rice genomic breeding.     

Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

Lysine decarboxylase converts l ‐lysine to cadaverine as a branching point for the biosynthesis of plant Lys‐derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys‐derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La‐L/ODC) and identified cadaverine‐derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys‐derived alkaloids in the transgenic lines. Moreover, we found several cadaverine‐derived metabolites specifically detected in the transgenic lines compared with the non‐transformed control. Among these, three specific metabolites were identified and confirmed as 5‐aminopentanal, 5‐aminopentanoate and δ‐valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope‐labeled [α‐¹⁵N]‐ or [ε‐¹⁵N]‐l ‐lysine. Our results show similar ¹⁵N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys‐derived alkaloid biosynthesis.     

RNA‐Seq bulked segregant analysis enables the identification of high‐resolution genetic markers for breeding in hexaploid wheat

The identification of genetic markers linked to genes of agronomic importance is a major aim of crop research and breeding programmes. Here, we identify markers for Yr15, a major disease resistance gene for wheat yellow rust, using a segregating F₂ population. After phenotyping, we implemented RNA sequencing (RNA‐Seq) of bulked pools to identify single‐nucleotide polymorphisms (SNP) associated with Yr15. Over 27 000 genes with SNPs were identified between the parents, and then classified based on the results from the sequenced bulks. We calculated the bulk frequency ratio (BFR) of SNPs between resistant and susceptible bulks, selecting those showing sixfold enrichment/depletion in the corresponding bulks (BFR > 6). Using additional filtering criteria, we reduced the number of genes with a putative SNP to 175. The 35 SNPs with the highest BFR values were converted into genome‐specific KASP assays using an automated bioinformatics pipeline (PolyMarker) which circumvents the limitations associated with the polyploid wheat genome. Twenty‐eight assays were polymorphic of which 22 (63%) mapped in the same linkage group as Yr15. Using these markers, we mapped Yr15 to a 0.77‐cM interval. The three most closely linked SNPs were tested across varieties and breeding lines representing UK elite germplasm. Two flanking markers were diagnostic in over 99% of lines tested, thus providing a reliable haplotype for marker‐assisted selection in these breeding programmes. Our results demonstrate that the proposed methodology can be applied in polyploid F₂ populations to generate high‐resolution genetic maps across target intervals.     

Tissue-culture-responsive and autotetraploidy-responsive changes in metabolic profiles of cucumber (Cucumis sativus L.)

Somaclonal variation commonly occurs during in vitro plant regeneration and may introduce unintended changes in numerous plant characters. In order to assess the range of tissue-culture-responsive changes on the biochemical level, the metabolic profiles of diploid and tetraploid cucumber R1 plants regenerated from leaf-derived callus were determined. Gas chromatography and mass spectrometry were used for monitoring of 48 metabolites and many significant changes were found in metabolic profiles of these plants as compared to a seed-derived control. Most of the changes were common to diploids and tetraploids and were effects of tissue culture. However, tetraploids showed quantitative changes in 14 metabolites, as compared to regenerated diploids. These changes include increases in serine, glucose-6P, fructose-6P, oleic acid and shikimic acid levels. Basing on this study we conclude that the variation in metabolic profiles does not correlate directly with the range of genome changes in tetraploids.     

Plastic responses in the metabolome and functional traits of maize plants to temperature variations

Environmentally inducible phenotypic plasticity is a major player in plant responses to climate change. However, metabolic responses and their role in determining the phenotypic plasticity of plants that are subjected to temperature variations remain poorly understood. The metabolomic profiles and metabolite levels in the leaves of three maize inbred lines grown in different temperature conditions were examined with a nuclear magnetic resonance metabolomic technique. The relationship of functional traits to metabolome profiles and the metabolic mechanism underlying temperature variations were then explored. A comparative analysis showed that during heat and cold stress, maize plants shared common plastic responses in biomass accumulation, carbon, nitrogen, sugars, some amino acids and compatible solutes. We also found that the plastic response of maize plants to heat stress was different from that under cold stress, mainly involving biomass allocation, shikimate and its aromatic amino acid derivatives, and other non‐polar metabolites. The plastic responsiveness of functional traits of maize lines to temperature variations was low, while the metabolic responsiveness in plasticity was high, indicating that functional and metabolic plasticity may play different roles in maize plant adaptation to temperature variations. A linear regression analysis revealed that the maize lines could adapt to growth temperature variations through the interrelation of plastic responses in the metabolomes and functional traits, such as biomass allocation and the status of carbon and nitrogen. We provide valuable insight into the plastic response strategy of maize plants to temperature variations that will permit the optimisation of crop cultivation in an increasingly variable environment.     

Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5‐hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1‐knockdown rice lines, which were produced via an RNA‐interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss‐of‐function mutants of OsCAld5H1 using the CRISPR/Cas9‐mediated genome editing system. Homozygous OsCAld5H1‐knockout lines harboring anticipated frame‐shift mutations in OsCAld5H1 were successfully obtained. A series of wet‐chemical and two‐dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ‐p‐coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non‐γ‐p‐coumaroylated units, whereas grass‐specific γ‐p‐coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non‐γ‐p‐coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass‐specific γ‐p‐coumaroylated S units in rice.     

Scale effect on rice pollen‐mediated gene flow: implications in assessing transgene flow from genetically engineered plants

Transgene flow from genetically engineered (GE) crops to non‐GE varieties and wild relatives via pollen‐mediated gene flow (PMGF) may create food and environmental biosafety concerns. Assessing the level of PMGF from GE crops is required before commercialization. Whether the level of PMGF estimated at relatively small scales can sufficiently represent the actual scenario at large production scales remains unresolved. Here, we estimated average PMGF frequencies from three insect‐resistant GE rice lines to their non‐GE counterparts at four scales ranging from 9 to 576 m², having the number of GE to non‐GE plants constantly at the ratio of 8:1. Based on nearly 1.3 million examined seedlings from non‐GE rice plots, very low frequencies (<0.1%) of transgene flow were detected. The highest frequencies were found in plots at the smallest scales. Scale had a significantly negative effect on the frequency of PMGF in rice, with decreased gene flow at increased scale. An extended PMGF model could well represent the experimental data. Field experiments at relatively small scales combined with mathematical modelling could provide useful prediction on the level of rice transgene flow at large production scales. This is probably applicable for other crop species with wind‐ and self‐pollination.     

Extreme genetic similarity does not predict non‐breeding distribution of two closely related warblers

Detailed knowledge of migratory connectivity can facilitate effective conservation of Neotropical migrants by helping biologists understand where and when populations may be most limited. We studied the migratory behavior and non‐breeding distribution of two closely related species of conservation concern, the Golden‐winged Warbler (Vermivora chrysoptera) and Blue‐winged Warbler (Vermivora cyanoptera). Although both species have undergone dynamic range shifts and population changes attributed to habitat loss and social interactions promoting competition and hybridization, full life‐cycle conservation planning has been limited by a lack of information about their non‐breeding ecology. Because recent work has demonstrated that the two species are nearly identical genetically, we predicted that individuals from a single breeding population would have similar migratory timing and overwintering locations. In 2015, we placed light‐level geolocators on 25 males of both species and hybrids in an area of breeding sympatry at the Fort Drum Military Installation in Jefferson and Lewis counties, New York. Despite extreme genetic similarity, non‐breeding locations and duration of migration differed among genotypes. Golden‐winged Warblers (N = 2) overwintered > 1900 km southeast of the nearest Blue‐winged Warbler (N = 3) and spent nearly twice as many days in migration; hybrids (N = 2) had intermediate wintering distributions and migratory timing. Spring migration departure dates were staggered based on distance from the breeding area, and all birds arrived at the breeding site within 8 days of each other. Our results show that Golden‐winged Warblers and Blue‐winged Warblers in our study area retain species‐specific non‐breeding locations despite extreme genetic similarity, and suggest that non‐breeding locations and migratory timing vary along a genetic gradient. If the migratory period is limiting for these species, our results also suggest that Golden‐winged Warblers in our study population may be more vulnerable to population decline than Blue‐winged Warblers because they spend almost twice as many days migrating.     

Enhanced yield performance of <Emphasis Type="Italic">Bt</Emphasis> rice under target-insect attacks: implications for field insect management

The rapid development of transgenic biotechnology has greatly promoted the breeding of genetically engineered (GE) rice in China, and many GE rice lines are in the pipeline for commercialization. To understand field performances of GE rice, key agronomic traits of two insect-resistant Bt rice lines that have been granted biosafety certificates for commercial production in China were evaluated together with their nontransgenic counterparts under environmental conditions with significant differences in insect pressure. Results from the experiments showed enhanced field performances of the Bt GE rice lines compared with the non-GE counterparts for yield-related traits such as number of panicles and filled seeds per plant, under environmental conditions with no insecticide application. No detectable underlying cost of the Bt transgene was observed in the two insect-resistant GE rice lines, particularly in the GE hybrid rice line. Results further indicated significantly greater yield performances of the two insect-resistant GE rice lines under environmental conditions with non-target insect control compared with no insect control. It is concluded from this study that insect-resistant Bt GE rice, particularly the hybrid line, has great potential to maintain its high yield when ambient insect pressure is high. In addition, proper application of insecticides to control non-target insects will guarantee optimal performance of insect-resistant Bt GE rice.