Approaches to functional genomics in filamentous fungi

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.

Functional genomics of Brassica napus:Progresses,challenges,and perspectives

Brassica napus,commonly known as rapeseed or canola,is a major oil crop contributing over 13%to the stable supply of edible vegetable oil worldwide.Identification and understanding the gene functions in the B.napus genome is crucial for genomic breeding.A group of genes controlling agronomic traits have been successfully cloned through functional genomics studies in B.napus.In this review,we present an overview of the progress made in the functional genomics of B.napus,including the availability of germplasm resources,omics databases and cloned functional genes.Based on the current progress,we also highlight the main challenges and perspectives in this field.The advances in the functional genomics of B.napus contribute to a better understanding of the genetic basis underlying the complex agronomic traits in B.napus and will expedite the breeding of high quality,high resistance and high yield in B.napus varieties.

The story of a decade:Genomics,functional genomics,and molecular breeding in Brassica napus

Rapeseed(Brassica napus L.)is one of the major global sources of edible vegetable oil and is also used as a feed and pioneer crop and for sightseeing and industrial purposes.Improvements in genome sequencing and molecular marker technology have fueled a boom in functional genomic studies of major agronomic characters such as yield,quality,flowering time,and stress resistance.Moreover,introgression and pyra-miding of key functional genes have greatly accelerated the genetic improvement of important traits.Here we summarize recent progress in rapeseed genomics and genetics,and we discuss effective molecular breeding strategies by exploring thesefindings in rapeseed.These insights will extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process,ultimately contributing to more sustainable agriculture throughout the world.

Rice Functional Genomics Research： Past Decade and Future

Rice （Oryza sativa） is a major staple food crop for more than 3.5 billion people worldwide. Under- standing the regulatory mechanisms of complex agronomic traits in rice is critical for global food security. Rice is also a model plant for genomics research of monocotyledonso Thanks to the rapid development of functional genomic technologies, over 2000 genes controlling important agronomic traits have been cloned, and their molecular biological mechanisms have also been partially char- acterized. Here, we briefly review the advances in rice functional genomics research during the past 10 years, including a summary of functional genomics platforms, genes and molecular regulatory networks that regulate important agronomic traits, and newly developed tools for gene identification. These achievements made in functional genomics research will greatly facilitate the development of green super rice. We also discuss future challenges and prospects of rice functional genomics research.

From Green Super Rice to green agriculture:Reaping the promise of functional genomics research

Producing sufficient food with finite resources to feed the growing global population while having a smaller impact on the environment has always been a great challenge.Here,we review the concept and practices of Green Super Rice(GSR)that have led to a paradigm shift in goals for crop genetic improvement and models of food production for promoting sustainable agriculture.The momentous achievements and global deliveries of GSR have been fueled by the integration of abundant genetic resources,functional gene discoveries,and innovative breeding techniques with precise gene and whole-genome selection and efficient agronomic management to promote resource-saving,environmentally friendly crop production systems.We also provide perspectives on new horizons in genomic breeding technologies geared toward delivering green and nutritious crop varieties to further enhance the development of green agricul-ture and better nourish the world population.

Graft-accelerated virus-induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS’, where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR, RhA G, and RhNUDXin rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.

Understandings and future challenges in soybean functional genomics and molecular breeding

Soybean(Glycine max)is a major source of plant protein and oil.Soybean breeding has benefited from advances in functional genomics.In particular,the release of soybean reference genomes has advanced our understanding of soybean adaptation to soil nutrient deficiencies,the molecular mechanism of symbiotic nitrogen(N)fixation,biotic and abiotic stress tolerance,and the roles of flowering time in regional adaptation,plant architecture,and seed yield and quality.Nevertheless,many challenges remain for soybean functional genomics and molecular breeding,mainly related to improving grain yield through high-density planting,maize-soybean intercropping,taking advantage of wild resources,utilization of heterosis,genomic prediction and selection breeding,and precise breeding through genome editing.This review summarizes the current progress in soybean functional genomics and directs future challenges for molecular breeding of soybean.

Twenty years of rice genomics research: From sequencing and functional genomics to quantitative genomics

Since the completion of the rice genome sequencing project in 2005,we have entered the era of rice genomics,which is still in its ascendancy.Rice genomics studies can be classified into three stages:structural genomics,functional genomics,and quantitative genomics.Structural genomics refers primarily to genome sequencing for the construction of a complete map of rice genome sequence.This is fundamental for rice genetics and molecular biology research.Functional genomics aims to decode the functions of rice genes.Quantitative genomics is large-scale sequence-and statistics-based research to define the quantitative traits and genetic features of rice populations.Rice genomics has been a transformative influence on rice biological research and contributes significantly to rice breeding,making rice a good model plant for studying crop sciences.

Effort and Contribution of T-DNA Insertion Mutant Library for Rice Functional Genomics Research in China:Review and Perspective

With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes（TILLING）, which combines traditional chemical mutagenesis with high‐throughput genome‐wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or evenallohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small‐scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2（or M3） plants.

Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

Common wheat(Triticum aestivum L.)is one of the three major food crops in the world;thus,wheat breeding programs are important for world food security.Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding.Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools,especially CRISPR/Cas9 genome editing technology,which allows multiplex genome engineering.Here,we describe the development of CRISPR/Cas9,which has revo-lutionized the field of genome editing.In addition,we emphasize technological breakthroughs(e.g.base editing and prime editing)based on CRISPR/Cas9.We also summarize recent applications and advances in the functional annotation and breeding of wheat,and we introduce the production of CRISPR-edited DNA-free wheat.Combined with other achievements,CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol,a next-generation biofuel,in Saccharomyces cerevisiae.Here,we explore how two of these strategies,pathway re-localization and redox cofactor-balancing,affect the performance and physiology of isobutanol producing strains.We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced(NADPH-dependent)or redox-balanced(NADH-dependent)ketol-acid reductoisomerase enzyme.We then conducted transcriptomic,proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains.Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes.Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version,albeit at low overall pathway flux.Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters,which are required cofactors for the dihydroxyacid dehydratase enzyme.We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation,thereby increasing cellular iron levels.The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold,demonstrating that both localizations can support flux to isobutanol.

Boosting wheat functional genomics via an indexed EMS mutant library of KN9204

A better understanding of wheat functional genomics can improve targeted breeding for better agronomic traits and environmental adaptation.However,the lack of gene-indexed mutants and the low transformation efficiency of wheat limit in-depth gene functional studies and genetic manipulation for breeding.In this study,we created a library for KN9204,a popular wheat variety in northern China,with a reference genome,transcriptome,and epigenome of different tissues,using ethyl methyl sulfonate(EMS)mutagenesis.This library contains a vast developmental diversity of critical tissues and transition stages.Exome capture sequencing of 2090 mutant lines using KN9204 genome-designed probes revealed that 98.79%of coding genes had mutations,and each line had an average of 1383 EMS-type SNPs.We identified new allelic variations for crucial agronomic trait-related genes such as Rht-D1,Q,TaTB1,and WFZP.We tested 100 lines with severemutations in 80 NAC transcription factors(TFs)under drought and salinity stress and identified 13 lines with altered sensitivity.Further analysis of three lines using transcriptome and chromatin accessibility data revealed hundreds of direct NAC targets with altered transcription patterns under salt or drought stress,including SNAC1,DREB2B,CML16,and ZFP182,factors known to respond to abiotic stress.Thus,we have generated and indexed a KN9204 EMS mutant library that can facilitate functional genomics research and offer resources for genetic manipulation of wheat.

Cotton functional genomics reveals global insight into genome evolution and fiber development

Due to the economic value of natural textile fiber, cotton has attracted much research attention, which has led to the publication of two diploid genomes and two tetraploid genomes. These big data facilitate functional genomic study in cotton, and allow researchers to investigate cotton genome structure, gene expression, and protein function on the global scale using high-throughput methods. In this review, we summarized recent studies of cotton genomes. Population genomic analyses revealed the domestication history of cultivated upland cotton and the roles of transposable elements in cotton genome evolution.Alternative splicing of cotton transcriptomes was evaluated genome-widely. Several important gene families like MYC, NAC, Sus and GhPLDal were systematically identified and classified based on genetic structure and biological function. High-throughput proteomics also unraveled the key functional proteins correlated with fiber development. Functional genomic studies have provided unprecedented insights into global-scale methods for cotton research.

Functional genomic approach to the study of biodiversitywithin Trichoderma

Trichoderma is a fungal genus of great and demonstrable biotechnological value, but its genome is poorly surveyed compared with other model microorganisms. Due to their ubiquity and rapid substrate colonization, Trichoderma species have been widely used as biocontrol organisms for agriculture, and their enzyme systems are widely used in industry. Therefore, there is a clear interest to explore beyond the phenotype to exploit the underlying genetic systems using functional genomics tools. The great diversity of species within the Trichoderma genus, the absence of optimized systems for its exploration, and the great variety of genes expressed under a wide range of ambient conditions are the main challenges to consider when starting a comprehensive functional genomics study. An initial project started by three Spanish groups has been extended into the project TRICHOEST, funded by the EU (FP5, QLRT-2001-02032) to target the transcriptome analysis of selected Trichoderma strains with biocontrol potential, in conditions related to antagonism, nutrient stress and plant interactions. Once specific conditions were defined, cDNA libraries were produced and used for EST sequencing. Nine strains from seven Trichoderma species have been considered in this study and an important amount of gene sequence data has been generated, analyzed and used to compare the gene expression in different strains. In parallel to sequencing, genomic expression studies were carried out by means of macro-arrays to identify genes expressed in specific conditions. In silico analysis of DNA sequencing data together with macro-array expression results have lead to a selection based on the potential use of the gene sequences. The selected clone sequences were completed and cloned in appropriate vectors to initiate functional analysis by means of expression studies in homologous and heterologous systems.

The function genomics study

Genomics is a biology term appeared ten years ago, used todescribe the researches of genomic mapping, sequencing,and structure analysis, etc. Genomics, the first journal forpublishing papers on genomics research was born in 1986.In the past decade, the concept of genomics has beenwidely accepted by scientists who are engaging in biologyresearch. Meanwhile, the research scope of genomics hasbeen extended continuously, from simple gene mappingand sequencing to function genomics study. To reflect thechange, genomics is divided into two parts now, thestructure genomics and the function genomics.Structure genomics retains the primary research con-tent of genomics, such as constructing high density genetic

Multiomics approaches to explore drought tolerance in cotton

The situation of global warming imparts negative impacts on crop growth and development.Cotton is the most important fiber crop around the globe.However,frequent drought episodes pose serious threats to cotton production worldwide.Due to the complex genetic structure of drought tolerance,the development of a tolerant cultivar is cumbersome via conventional breeding.Multiple omics techniques have appeared as successful tool for cotton improvement in drought tolerance.Advanced omics-based biotechniques have paved the way for generation of omics data like transcriptomics,genomics,metabolomics and proteomics,which greatly expand the knowledge of cotton response to drought stress.Omics methodologies and have provided ways for the identification of quantitative trait loci(QTLs),gene regulatory networks,and other regulatory pathways against drought stress in cotton.These resources could speed up the discovery and incorporation of drought tolerant traits in the elite genotypes.The genome wide association study(GWAS),gene-editing system CRISPER/Cas9,gene silencing through RNAi are efficient tools to explore the molecular mechanism of drought tolerance and facilitate the identification of mechanisms and candidate genes for the improvement of drought tolerance in cotton.

Development of Studies on RNA Interference

RNA interference （RNAi）, caused by endogenous or exogenous double- stranded RNA （dsRNA） homologous with target genes, refers to gene silencing widely existing in animals and plants. It was first found in plants, and now it has developed into a kind of biotechnology as well as an important approach in post- genome era. This paper is to summarize the achievements of studies on RNAi tech- nology in basic biology, medicine, pharmacy, botany and other fields.

The Past, Present, and Future of Maize Improvement: Domestication, Genomics, and Functional Genomic Routes toward Crop Enhancement

After being domesticated from teosinte,cultivated maize(Zea mays ssp.mays)spread worldwide and now is one of the most important staple crops.Due to its tremendous phenotypic and genotypic diversity,maize also becomes to be one of the most widely used model plant species for fundamental research,with many important discoveries reported by maize researchers.Here,we provide an overview of the history of maize domestication and key genes controlling major domestication-related traits,review the currently available resources for functional genomics studies in maize,and discuss the functions of most of the maize genes that have been positionally cloned and can be used for crop improvement.Finally,we provide some perspectives on future directions regarding functional genomics research and the breeding of maize and other crops.