Comparative Analysis of Flanking Sequence Tags of T-DNA/Transposon Insertional Mutants and Genetic Variations of Fast-neutron Treated Mutants in Rice

Whole genome sequencing analyses of 1,504 fast-neutron (FN)-induced mutants of ‘Kitaake’ rice variety have revealed a new mutant population covering 58.6% of transposable element (TE) genes and 47.6% of non-TE genes throughout the rice genome. Mutation rate for TE gene is much higher in FN-induced mutants (58.6%) than in flanking sequence tag (FST) population (25.7%), implying that the former are more randomly generated than the latter. By adding this resource to FST population, we found that the mutation rate for the rice genome increases from 53.1% to 78.1% and more importantly, the rate with multiple alleles increases from 35.2% to 56.1%. To test the functional significance of mutants produced by both FN-induction and T-DNA/transposon insertions, we analyzed the coverage of functionally characterized genes by using the Overview of functionally characterized Genes in Rice Online database (OGRO, http://qtaro.abr.affrc.go.jp/ogro/table). These combined genetic resources cover the mutations for 90.9% of functionally characterized genes for morphological traits, 91.0% for physiological traits, and 92.6% for resistance or tolerance traits, indicating that a gene-indexed mutant population that includes FN-induced mutants is valuable to future research for improving most of the important agronomic traits.     

Genome-wide association study-based identification genes influencing agronomic traits in rice (Oryza sativa L.)

Rice is one of the most important cereal crops, providing the daily dietary intake for approximately 50% of the global human population. Here, we re-sequenced 259 rice accessions, generating 1371.65 Gb of raw data. Furthermore, we performed genome-wide association studies (GWAS) on 13 agronomic traits using 2.8 million single nucleotide polymorphisms (SNPs) characterized in 259 rice accessions. Phenotypic data and best linear unbiased prediction (BLUP) values of each of the 13 traits over two years of each trait were used for the GWAS. The results showed that 816 SNP signals were significantly associated with the 13 agronomic traits. Then we detected candidate genes related to target traits within 200 kb upstream and downstream of the associated SNP loci, based on linkage disequilibrium (LD) blocks in the whole rice genome. These candidate genes were further identified through haplotype block constructions. This comprehensive study provides a timely and important genomic resource for breeding high yielding rice cultivars.     

Identification and functional analysis of light-responsive unique genes and gene family members in rice

Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families.     

Identification and fine mapping of a new bacterial blight resistance gene,Xa43(t), in Zhangpu wild rice (Oryza rufipogon)

• Bacterial blight (BB) is currently considered one of the most serious rice diseases and is caused by Xanthomonas oryzae pv. oryzae (Xoo). Numerous studies have shown that breeding resistant rice varieties is one of the most effective methods to prevent BB, and it is important to identify and isolate more BB resistance (R) genes from different rice resources.
• Using a map-based approach, we identified a new QTL/gene, Xa43(t), from ZhangPu wild rice, which was highly resistant to the BB isolate PX099. We performed bulked segregant analysis combined with candidate gene prediction to identify the candidate gene.
• The Xa43(t) gene was narrowed down to a 29-kb region containing four putative genes. More importantly, the candidate gene Xa43(t) did not affect the main agronomic traits of rice. We also identified a widely applicable molecular marker, namely Inde1-18, which co-segregates with the Xa43(t) gene.
• The Xa43(t) gene is a new broad-spectrum BB resistance gene without identified alleles and has good application prospects for rice disease resistance breeding.

     

Transmission of important chromosomal regions under selection revealed in rice pedigree breeding programs

Genetic analysis across a whole plant genome based on pedigree information offers considerable potential for enhancing genetic gain from plant breeding programs through quantitative trait loci (QTL) mapping and marker-assisted selection. Here, we report its application for graphically genotyping varieties used in Chinese japonica rice (Oryza sativa L.) pedigree breeding programs. We identified 34 important chromosomal regions from the founder parent that are under selection in the breeding programs, and by comparing donor genomic regions that are under selection with QTL locations of agronomic traits, we found that QTL clustered in important genomic regions, in accordance with association analyses of natural populations and other previous studies. The convergence of genomic regions under selection with QTL locations suggests that donor genomic regions harboring key genes/QTL for important agronomic traits have been selected by plant breeders since the 1950s from the founder rice plants. The results provide better understanding of the effects of selection in breeding programs on the traits of rice cultivars. They also provide potentially valuable information for enhancing rice breeding programs through screening candidate parents for targeted molecular markers, improving crop yield potential and identifying suitable genetic material for use in future breeding programs.     

A combined strategy of “in silico” transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies

Traditionally housekeeping genes have been employed as endogenous reference (internal control) genes for normalization in gene expression studies. Since the utilization of single housekeepers cannot assure an unbiased result, new normalization methods involving multiple housekeeping genes and normalizing using their mean expression have been recently proposed. Moreover, since a gold standard gene suitable for every experimental condition does not exist, it is also necessary to validate the expression stability of every putative control gene on the specific requirements of the planned experiment. As a consequence, finding a good set of reference genes is for sure a non-trivial problem requiring quite a lot of lab-based experimental testing. In this work we identified novel candidate barley reference genes suitable for normalization in gene expression studies. An advanced web search approach aimed to collect, from publicly available web resources, the most interesting information regarding the expression profiling of candidate housekeepers on a specific experimental basis has been set up and applied, as an example, on stress conditions. A complementary lab-based analysis has been carried out to verify the expression profile of the selected genes in different tissues and during heat shock response. This combined dry/wet approach can be applied to any species and physiological condition of interest and can be considered very helpful to identify putative reference genes to be shortlisted every time a new experimental design has to be set up.     

Recent Progress on Rice Genetics in China

Through thousands of years of evolution and cultivation, tremendously rich genetic diversity has been accumulated in rice （Oryza sativa L.）, developing a large germplasm pool from which people can select varieties with morphologies of Interest and other important agronomic traits. With the development of modern genetics, scientists have paid more attention to the genetic value of these elite varieties and germplasms, and such rich rice resources provide a good foundation for genetic research in China. Approximately 100 000 accessions of radiation-, chemical- or insertion-induced mutagenesis have been generated since the 1980s, and great progress has been made on rice molecular genetics. So far at least 16 variant/mutant genes Including MOC1, BC1, SKC1, and Rfgenes have been isolated and characterized in China. These achievements greatly promote the research on functional genomics, understanding the mechanism of plant development and molecular design breeding of rice in China. Here we review the progress of three aspects of rice genetics in China： moving forward at the molecular level, genetic research on elite varieties and germplasms, and new gene screening and genetic analysis using mutants. The prospects of rice genetics are also discussed.     

水稻突变体des2和des5的研究和定位

小穗发育是决定水稻产量的主要农艺性状,鉴定控制小穗发育的关键基因对研究和分析调控农艺性状的分子机理是至关重要的.本文中,我们鉴定了一组小穗数目明显减少的突变体,命名为decteased spikelets(des),这里详细研究des2和des5两个突变体.结果显示des2是由单基因隐性位点控制,图位克隆将此位点定位到6号染色体的长臂上,并最终克隆了此基因,发现des2是moc1的一个新的等位突变体.定位克隆和序列分析显示在des5中,LAX基因的编码HLH(螺旋一环一螺旋)结构域的区域发生了一个点突变,暗示des5是lax的一个新的等位突变体.我们的结果暗示小穗和水稻叶腋分枝的发育受相同的遗传途径调控.     

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

水稻(Oryza sativa)籽粒大小是影响其产量的关键农艺性状, 克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义。为深入探究水稻籽粒大小的调控机制, 通过EMS诱变品种宽叶粳(KYJ), 分离了一系列水稻籽粒大小改变的突变体, 其中smg12表现为籽粒变小, 株高变矮, 一级枝梗数和二级枝梗数减少。遗传分析表明, 该小粒突变体受隐性单基因控制。细胞学分析显示, 该突变体颖壳纵向细胞长度显著变短, 表明SMG12主要影响细胞扩展。利用Mutmap方法对候选基因进行克隆, 筛选出SMG12的候选基因OsBRI1, 该基因编码油菜素内酯受体激酶。OsBRI1外显子上的第2 074个碱基发生了由C到T的置换, 产生非同义突变, 使得该位置编码的脯氨酸变为丝氨酸, 从而影响OsBRI1的功能。综上, 该研究鉴定了OsBRI1基因的1个新等位变异, 揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础。     

Genome‐wide association study of callus induction variation to explore the callus formation mechanism of rice

Rice(Oryza sativa) is one of the most widely cultivated food crops, worldwide. Tissue culture is extensively used in rice breeding and functional genome research. The ability to induce callus determines whether a particular rice variety can be subjected to tissue culture and Agrobacterium-mediated transformation. Over the past two decades, many quantitative trait loci(QTLs)related to callus induction traits have been identified;however, individual genes associated with rice callus induction have not been reported. In this study, we characterized three callus-induction traits in a global collection of 510 rice accessions. A genome-wide association study of the rice population in its entirety as well as subpopulations revealed 21 significant loci located in rice callus induction QTLs. We identified three candidate callus induction genes, namely CRL1, Os BMM1, and Os SET1, which Rese are orthologs of Arabidopsis LBD17/LBD29, BBM, and SWN,respectively, which are known to affect callus formation.Furthermore, we predicted that 14 candidate genes might be involved in rice callus induction and showed that RNA interference(RNAi)-mediated disruption of Os IAA10 inhibited callus formation on tissue culture medium.Embryo growth in the Os IAA10 RNAi line was not inhibited by synthetic auxin(2,4-D) treatment, suggesting that Os IAA10 may perceive auxin and activate the expression of downstream genes, such as CRL1, to induce callus formation. The significant loci and candidate genes identified here may provide insight into the mechanism underlying callus formation in rice.     

Meta-expression analysis of unannotated genes in rice and approaches for network construction to suggest the probable roles

Key message

This work suggests 2020 potential candidates in rice for the functional annotation of unannotated genes using meta-analysis of anatomical samples derived from microarray and RNA-seq technologies and this information will be useful to identify novel morphological agronomic traits.

Abstract

Although the genome of rice (Oryza sativa) has been sequenced, 14,365 genes are considered unannotated because they lack putative annotation information. According to the Rice Genome Annotation Project Database (http://rice.plantbiology.msu.edu/), the proportion of functionally characterized unannotated genes (0.35%) is quite limited when compared with the approximately 3.9% of annotated genes with assigned putative functions. Researchers require additional information to help them investigate the molecular mechanisms associated with those unannotated genes. To determine which of them might regulate morphological or physiological traits in the rice genome, we conducted a meta-analysis of expression data that covered a wide range of tissue/organ samples. Overall, 2020 genes showed cultivar-, tissue-, or organ-preferential patterns of expression. Representative candidates from featured groups were validated by RT-PCR, and the GUS reporter system was used to validate the expression of genes that were clustered according to their leaf or root preference. Taking a molecular and genetics approach, we examined meta-expression data and found that 127 genes were differentially expressed between japonica and indica rice cultivars. This is potentially significant for future agronomic applications. We also used a T-DNA insertional mutant and performed a co-expression network analysis of Sword shape dwarf1 (SSD1), a gene that regulates cell division. This network was refined via RT-PCR analysis. Our results suggested that SSD1 represses the expression of four genes related to the processes of DNA replication or cell division and provides insight into possible molecular mechanisms. Together, these strategies present a valuable tool for in-depth characterization of currently unannotated genes.

     

Rice Expression Database(RED):An integrated RNA-Seq-derived gene expression database for rice

Rice is one of the most important stable food as well as a monocotyledonous model organism for the plant research community.Here,we present RED(Rice Expression Database;http://expression.ic4r.org),an integrated database of rice gene expression profiles derived entirely from RNA-Seq data.RED features a comprehensive collection of 284 high-quality RNA-Seq experiments,integrates a large number of gene expression profiles and covers a wide range of rice growth stages as well as various treatments.Based on massive expression profiles,RED provides a list of housekeeping and tissue-specific genes and dynamically constructs co-expression networks for gene(s) of interest.Besides,it provides user-friendly web interfaces for querying,browsing and visualizing expression profiles of concerned genes.Together,as a core resource in BIG Data Center,RED bears great utility for characterizing the function of rice genes and better understanding important biological processes and mechanisms underlying complex agronomic traits in rice.     

Rice functional genomics research in China

Rice functional genomics is a scientific approach that seeks to identify and define the function of rice genes, and uncover when and how genes work together to produce phenotypic traits. Rapid progress in rice genome sequencing has facilitated research in rice functional genomics in China. The Ministry of Science and Technology of China has funded two major rice functional genomics research programmes for building up the infrastructures of the functional genomics study such as developing rice functional genomics tools and resources. The programmes were also aimed at cloning and functional analyses of a number of genes controlling important agronomic traits from rice. National and international collaborations on rice functional genomics study are accelerating rice gene discovery and application.     

Analysis of genetic architecture and favorable allele usage of agronomic traits in a large collection of Chinese rice accessions

Genotyping and phenotyping large natural populations provide opportunities for population genomic analysis and genome-wide association studies (GWAS). Several rice populations have been re-sequenced in the past decade; however, many major Chinese rice cultivars were not included in these studies. Here, we report large-scale genomic and phenotypic datasets for a collection mainly comprised of 1,275 rice accessions of widely planted cultivars and parental hybrid rice lines from China. The population was divided into three indica/Xian and three japonica/Geng phylogenetic subgroups that correlate strongly with their geographic or breeding origins. We acquired a total of 146 phenotypic datasets for 29 agronomic traits under multi-environments for different subpopulations. With GWAS, we identified a total of 143 significant association loci, including three newly identified candidate genes or alleles that control heading date or amylose content. Our genotypic analysis of agronomically important genes in the population revealed that many favorable alleles are underused in elite accessions, suggesting they may be used to provide improvements in future breeding efforts. Our study provides useful resources for rice genetics research and breeding.

     

水稻长日照开花因子Lfm1的图位克隆

开花期是水稻最重要的农艺性状之一,水稻的花期决定着水稻的地区适应性和最终产量。人工选择使水稻从短日照向长日照、低纬度向高纬度扩张,因此水稻已逐渐进化出适应长日照条件下的开花调控机制。目前,虽然鉴定了一些影响水稻长日照的开花基因如SDG724、RFT1、EHD4、DTH2,但是挖掘水稻长日照开花基因还十分有限。本研究通过筛选水稻突变体库,获得一批在长日照下花期有显著差异的突变体材料,其中一份突变体lfm1(late-flowering mutant1),在长日照条件下开花延迟,在短日照条件下开花时间正常。通过图位克隆,将Lfm1基因初定位至第8染色体端粒附近。进一步的精细定位将Lfm1基因定位于分子标记8-0.269和与8-0.283之间,范围为12 kb,该区域包括3个候选基因。经测序分析发现,在突变体lfm1中,LOC_Os08g01420基因的第六外显子2800处缺失9个碱基,突变体lfm1等位于已报道的突变体ehd3。在适度(中日照条件下,~12 h/12 h)的光照条件下,突变体lfm1表现为穗粒数增多,生育期略延长,具有应用于生产的潜力。Lfm1基因的克隆为培育适应不同生态区域的水稻材料提供了重要的基因资源。     

A large-scale screen for artificial selection in maize identifies candidate agronomic loci for domestication and crop improvement

Maize (Zea mays subsp mays) was domesticated from teosinte (Z. mays subsp parviglumis) through a single domestication event in southern Mexico between 6000 and 9000 years ago. This domestication event resulted in the original maize landrace varieties, which were spread throughout the Americas by Native Americans and adapted to a wide range of environmental conditions. Starting with landraces, 20th century plant breeders selected inbred lines of maize for use in hybrid maize production. Both domestication and crop improvement involved selection of specific alleles at genes controlling key morphological and agronomic traits, resulting in reduced genetic diversity relative to unselected genes. Here, we sequenced 1095 maize genes from a sample of 14 inbred lines and chose 35 genes with zero sequence diversity as potential targets of selection. These 35 genes were then sequenced in a sample of diverse maize landraces and teosintes and tested for selection. Using two statistical tests, we identified eight candidate genes. Extended gene sequencing of these eight candidate loci confirmed that six were selected throughout the gene, and the remaining two exhibited evidence of selection in the 3' portion of each gene. The selected genes have functions consistent with agronomic selection for nutritional quality, maturity, and productivity. Our large-scale screen for artificial selection allows identification of genes of potential agronomic importance even when gene function and the phenotype of interest are unknown.     

Structural and functional analysis of rice genome

Rice is an excellent system for plant genomics as it represents a modest size genome of 430 Mb. It feeds more than half the population of the world. Draft sequences of the rice genome, derived by whole-genome shotgun approach at relatively low coverage (4-6 X), were published and the International Rice Genome Sequencing Project (IRGSP) declared high quality (> 10 X), genetically anchored, phase 2 level sequence in 2002. In addition, phase 3 level finished sequence of chromosomes 1, 4 and 10 (out of 12 chromosomes of rice) has already been reported by scientists from IRGSP consortium. Various estimates of genes in rice place the number at >50,000. Already, over 28,000 full-length cDNAs have been sequenced, most of which map to genetically anchored genome sequence. Such information is very useful in revealing novel features of macroand micro-level synteny of rice genome with other cereals. Microarray analysis is unraveling the identity of rice genes expressing in temporal and spatial manner and should help target candidate genes useful for improving traits of agronomic importance. Simultaneously, functional analysis of rice genome has been initiated by marker-based characterization of useful genes and employing functional knock-outs created by mutation or gene tagging. Integration of this enormous information is expected to catalyze tremendous activity on basic and applied aspects of rice genomics.     

Towards the cloning of genes underlying murine QTLs

Many quantitative trait loci (QTLs), including those for ethanol-related traits, have been mapped in the mouse. In light of rapidly developing tools and resources, we briefly review the strategy for identifying the genes underlying these QTLs. We note that positional cloning will soon be a matter of testing candidate genes rather than discovering genes; therefore, we describe a ``congenic test' to support that a candidate gene is indeed a QTL. Considering the rapid development of congenics and mutants, we also identify four areas of investigation—phenotypes, ethanol specificity, environment, and gene interactions—that might be exploited during the course of positional cloning to gain insights into QTL pathways. In particular, we note that multiple mutants of nearly every major neurotransmitter pathway have now been made. These mutants are not only useful for phenotypic tests, but also could be used to conduct ``gene dependence' tests of QTLs. We also consider potential applications for the very recently developed ability to clone mice. Received: 15 September 1998 / Accepted: 8 October 1998