In-depth molecular and phenotypic characterization in a rice insertion line library facilitates gene identification through reverse and forward genetics approaches

We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.     

A rice gene activation/knockout mutant resource for high throughput functional genomics

Using transfer DNA (T-DNA) with functions of gene trap and gene knockout and activation tagging, a mutant population containing 55,000 lines was generated. Approximately 81% of this population carries 1–2 T-DNA copies per line, and the retrotransposon Tos17 was mostly inactive in this population during tissue culture. A total of 11,992 flanking sequence tags (FSTs) have been obtained and assigned to the rice genome. T-DNA was preferentially (∼80%) integrated into genic regions. A total of 19,000 FSTs pooled from this and another T-DNA tagged population were analyzed and compared with 18,000 FSTs from a Tos17 tagged population. There was difference in preference for integrations into genic, coding, and flanking regions, as well as repetitive sequences and centromeric regions, between T-DNA and Tos17; however, T-DNA integration was more evenly distributed in the rice genome than Tos17. Our T-DNA contains an enhancer octamer next to the left border, expression of genes within genetics distances of 12.5 kb was enhanced. For example, the normal height of a severe dwarf mutant, with its gibberellin 2-oxidase (GA2ox) gene being activated by T-DNA, was restored upon GA treatment, indicating GA2ox was one of the key enzymes regulating the endogenous level of GA. Our T-DNA also contains a promoterless GUS gene next to the right border. GUS activity screening facilitated identification of genes responsive to various stresses and those regulated temporally and spatially in large scale with high frequency. Our mutant population offers a highly valuable resource for high throughput rice functional analyses using both forward and reverse genetic approaches. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users. Yue-Ie Hsing, Chyr-Guan Chern, and Ming-Jen Fan have contributed equally.     

Mobilized retrotransposon Tos17 of rice by alien DNA introgression transposes into genes and causes structural and methylation alterations of a flanking genomic region

Tos17 is a copia-like endogenous retrotransposon of rice, which can be activated by various stresses such as tissue culture and alien DNA introgression. To confirm element mobilization by introgression and to study possible structural and epigenetic effects of Tos17 insertion on its target sequences, we isolated all flanking regions of Tos17 in an introgressed rice line (Tong35) that contains minute amount of genomic DNA from wild rice (Zizania latifolia). It was found that there has been apparent but limited mobilization of Tos17 in this introgression line, as being reflected by increased but stable copy number of the element in progeny of the line. Three of the five activated copies of the element have transposed into genes. Based on sequence analysis and Southern blot hybridization with several double-enzyme digests, no structural change in Tos17 could be inferred in the introgression line. Cytosine methylation status at all seven CCGG sites within Tos17 was also identical between the introgression line and its rice parent (Matsumae)-all sites being heavily methylated. In contrast, changes in structure and cytosine methylation patterns were detected in one of the three low-copy genomic regions that flank newly transposed Tos17, and all changes are stably inherited through selfed generations.     

Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library

We isolated 13 804 T-DNA flanking sequence tags (FSTs) from a T-DNA insertion library of rice. A comprehensive analysis of the 13 804 FSTs revealed a number of features demonstrating a highly non-random distribution of the T-DNA insertions in the rice genome: T-DNA insertions were biased towards large chromosomes, not only in the absolute number of insertions but also in the relative density; within chromosomes the insertions occurred more densely in the distal ends, and less densely in the centromeric regions; the distribution of the T-DNA insertions was highly correlated with that of full-length cDNAs, but the correlations were highly heterogeneous among the chromosomes; T-DNA insertions strongly disfavored transposable element (TE)-related sequences, but favored genic sequences with a strong bias toward the 5' upstream and 3' downstream regions of the genes; T-DNA insertions preferentially occurred among the various classes of functional genes, such that the numbers of insertions were in excess in certain functional categories but were deficient in other categories. The analysis of DNA sequence compositions around the T-DNA insertion sites also revealed several prominent features, including an elevated bendability from -200 to 200 bp relative to the insertion sites, an inverse relationship between the GC and TA skews, and reversed GC and TA skews in sequences upstream and downstream of the insertion sites, with both GC and TA skews equal to zero at the insertion sites. It was estimated that 365 380 insertions are needed to saturate the genome with P = 0.95, and that the 45 441 FSTs that have been isolated so far by various groups tagged 14 287 of the 42 653 non-TE related genes.     

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.     

Reverse Genetic Approaches for Functional Genomics of Rice

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.     

The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets

The rice retrotransposon Tos17 is highly activated by tissue culture. To evaluate the impact of transposition of Tos17 on the rice genome and examine its utility for insertional mutagenesis, more than 100 sequences flanking newly transposed Tos17 copies were characterised. The 5-bp target-site duplications flanking Tos17 did not show any consensus sequence, and preferred nucleotides, A/T and G/C, were only found at the second and third nucleotides from both ends of the target site duplications, respectively, indicating that Tos17 has relatively low target-site specificity at the nucleotide sequence level. Integration targets were widely distributed over the chromosomes; however, preferential integration into the sucrose synthase 2 gene and into Tos17 itself was demonstrated by PCR screening using pooled DNA prepared from the mutant population. Hybridisation studies indicated that Tos17 preferentially integrates into low-copy-number regions of the genome. In agreement with this result, about 30% of flanking sequences examined showed significant homology to known genes. Taken together, these results show that Tos17 can have a significant impact on the rice genome and can be used as a tool for efficient insertional mutagenesis.     

Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system

We have previously generated a large pool of T-DNA insertional lines in rice. In this study, we screened those T-DNA pools for rice mutants that had defective chlorophylls. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. Among the mutants, 10 lines were beta-glucuronidase (GUS)-positive in the leaves. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in ARABIDOPSIS: This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene. Those phenotypes were co-segregated with Tos17 in the progeny. GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. The chlorophyll content was very low in the OschlH mutants. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes.     

FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.     

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.     

Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency. The 3' end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.     

Computation-assisted SiteFinding- PCR for isolating flanking sequence tags in rice

SiteFinding-PCR is a method for isolating flanking sequence tags (FSTs) of T-DNA insertion lines, but the efficiency needs to be improved. Here we report a computation-assisted design for the random primers used in SiteFinding- PCR. A short sequence, GCATG, was screened from the rice genome and used as the 3' end of the random primer. When applying the optimized primer for isolating FSTs from 168 transgenic rice lines, we obtained 107 specific products, including 64 FSTs. The efficiency of obtaining FSTs using the modified version of SiteFinding-PCR increased by 73.0% compared with the method previously reported (P < 0.01, μ test). We also provide computational results for several other plant species such as maize, sorghum, Arabidopsis, foxtail millet, and Brachypodium based on the available genome data, so that the modified method could be easily adapted to other species.