Fu-Jin Wei

A rice phenomics study--phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population.

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request.

International Consortium of Rice Mutagenesis: resources and beyond

Rice is one of the most important crops in the world. The rice community needs to cooperate and share efforts and resources so that we can understand the functions of rice genes, especially those with a role in important agronomical traits, for application in agricultural production. Mutation is a major source of genetic variation that can be used for studying gene function. We will present here the status of mutant collections affected in a random manner by physical/chemical and insertion mutageneses.As of early September 2013, a total of 447, 919 flanking sequence tags from rice mutant libraries with T-DNA, Ac/Ds, En/Spm, Tos17, nDART/aDART insertions have been collected and publicly available. From these, 336,262 sequences are precisely positioned on the japonica rice chromosomes, and 67.5% are in gene interval. We discuss the genome coverage and preference of the insertion, issues limiting the exchange and use of the current collections, as well as new and improved resources. We propose a call to renew all mutant populations as soon as possible. We also suggest that a common web portal should be established for ordering seeds.

OsLEA1a, a New Em-Like Protein of Cereal Plants

Proteins abundant in seeds during the late stages of development, late embryogenesis abundant (LEA) proteins, are associated with desiccation tolerance. More than 100 of the group I LEA genes, also termed Em genes, have been identified from plants, bacteria and animals. The wide distribution indicates the functional importance of these genes. In the present study, we characterized a novel Em-like gene, OsLEA1a of rice (Oryza sativa). The encoded OsLEA1a protein has an N-terminal sequence similar to that of other plant Em proteins but lacks a 20-mer motif that is the most significant feature of typical Em proteins. The location of the sole intron indicates that the second exon of OsLEA1a is the mutated product of a typical Em gene. Transcriptome analysis revealed OsLEA1a mainly expressed in embryos, with no or only a few transcripts in osmotic stress-treated vegetative tissues. Structural analysis revealed that the OsLEA1a protein adopts high amounts of disordered conformations in solution and undergoes desiccation-induced conformational changes. Macromolecular interaction studies revealed that OsLEA1a protein interacts with non-reducing sugars and phospholipids but not poly-l-lysine. Thus, although the OsLEA1a protein lost its 20-mer motif, it is still involved in the formation of bioglasses with non-reducing sugars or plasma membrane. However, the protein does not function as a chaperone as do other groups of hydrophilic LEA proteins. The orthologs of the OsLEA1a gene had been identified from various grasses but not in dicot plants. Genetic analysis indicated that rice OsLEA1a locates at a 193 kb segment in chromosome 1 and is conserved in several published cereal genomes. Thus, the ancestor of Em-like genes might have evolved after the divergence of monocot plants.

Comparative analyses of linkage maps and segregation distortion of two F2 populations derived from japonica crossed with indica rice

To facilitate genetic research, we constructed two linkage maps by employing two F₂ populations derived from rice inter-subspecific crosses, japonica Tainung 67 (TNG67)/indica Taichung Sen 10 (TCS10) and japonica TNG67/indica Taichung Sen 17 (TCS17). We established linkage map lengths of 1481.6 cM and 1267.4 cM with average intervals of 13.8 cM and 14.4 cM by using 107 and 88 PCR markers for coverage of 88% of the rice genome in TNG67/TCS10 and TNG67/TCS17, respectively. The discrepancy in genetic maps in the two populations could be due to different cross combinations, crossing-over events, progeny numbers and/or markers. The most plausible explanation was segregation distortion; 18 markers (16.8%) distributed at nine regions of seven chromosomes and 10 markers (11.4%) at four regions of four chromosomes displayed severe segregation distortion (p < 0.01)in TNG67/TCS10 and TNG67/TCS17, respectively. All segregation-distorted markers in these two populations corresponded to reported reproductive barriers, either gametophytic or zygotic genes but not to hybrid breakdown genes. The observed recombination frequency, which was higher or lower than the intrinsic frequency, revealed the association of segregation distortion skewed to the same or different genotypes at the consecutive markers. The segregation distortion, possibly caused by reproductive barriers, affects the evaluation recombination frequencies and consequently the linkage analysis of QTLs and positional cloning.

Somaclonal variation does not preclude the use of rice transformants for genetic screening

Rice (Oryza sativa) is one of the worlds most important crops. Rice researchers make extensive use of insertional mutants for the study of gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype and phenotype is very weak. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. We used high-throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non-transformed and transgenic rice plants for mutations caused by these parameters. For comparison, we also analyzed sequence changes for two additional rice varieties and four T-DNA tagged transformants from the Taiwan Rice Insertional Mutant resource. We identified single-nucleotide polymorphisms, small indels, large deletions, chromosome doubling and chromosome translocations in these lines. Using standard rice regeneration/transformation procedures, the mutation rates of regenerants and transformants were relatively low, with no significant differences among eight tested treatments in the Tainung 67 background and in the cultivars Taikeng 9 and IR64. Thus, we could not conclusively detect sequence changes resulting from Agrobacterium-mediated transformation in addition to those caused by tissue culture-induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10-fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries.

Both Hd1 and Ehd1 are important for artificial selection of flowering time in cultivated rice.

Rice is a facultative short-day plant, and it requires a photoperiod shorter than the critical day length to get flowering. Sensitivity to photoperiod has been suggested as a major selection target in cultivated or weedy rice. The modern rice varieties in Taiwan may be cultivated twice a year. These varieties contain loss-of-function of two important flowering-time related genes, Heading date 1 (Hd1) and Early heading date 1 (Ehd1), and are mainly from a mega variety, Taichung 65. However, the parental lines of this variety were sensitive to photoperiod, thus, how Taichung 65 loss its sensitivity is a mystery. In this study, we used accession-specific single nucleotide polymorphism analysis to reveal the gene flow that occurred between different rice accessions decades ago and demonstrate that two landraces introgressed during the breeding process, which led to the loss of photoperiod sensitivity. Both Hd1 and Ehd1 may be important during artificial selection for flowering time, especially in a subtropical region such as Taiwan. This is a good example of introgression playing important roles during rice domestication.

Methods for rice phenomics studies.

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. A systematic phenotypic profiling of mutant collections will provide major insights into gene functions important for crop growth or production. Thus, detailed phenomics analysis is the key to functional genomics. Currently, the two major types of rice mutant collections are insertional mutants and chemical or irradiation-induced mutants. Here we describe how to manipulate a rice mutant population, including conducting phenomics studies and the subsequent propagation and seed storage. We list the phenotypes screened and also describe how to collect data systematically for a database of the qualitative and quantitative phenotypic traits. Thus, data on mutant lines, phenotypes, and segregation rate for all kinds of mutant populations, as well as integration sites for insertional mutant populations, would be searchable, and the collection would be a good resource for rice functional genomics study.

Lack of Genotype and Phenotype Correlation in a Rice T-DNA Tagged Line Is Likely Caused by Introgression in the Seed Source.

Rice (Oryza sativa) is one of the most important crops in the world. Several rice insertional mutant libraries are publicly available for systematic analysis of gene functions. However, the tagging efficiency of these mutant resources–the relationship between genotype and phenotype–is very low. We used whole-genome sequencing to analyze a T-DNA–tagged transformant from the Taiwan Rice Insertional Mutants (TRIM) resource. The phenomics records for M0028590, one of the TRIM lines, revealed three phenotypes–wild type, large grains, and tillering dwarf–in the 12 T1 plants. Using the sequencing data for 7 plants from three generations of this specific line, we demonstrate that introgression from an indica rice variety might occur in one generation before the seed was used for callus generation and transformation of this line. In addition, the large-grain trait came from the GS3 gene of the introgressed region and the tillering dwarf phenotype came from a single nucleotide change in the D17 gene that occurred during the callus induction to regeneration of the transformant. As well, another regenerant showed completely heterozygous single-nucleotide polymorphisms across the whole genome. In addition to the known sequence changes such as T-DNA integration, single nucleotide polymorphism, insertion, deletion, chromosome rearrangement and doubling, spontaneous outcrossing occurred in the rice field may also explain some mutated traits in a tagged mutant population. Thus, the co-segregation of an integration event and the phenotype should be checked when using these mutant populations.

Large-scale phenomics analysis of a T-DNA tagged mutant population

Abstract Rice, Oryza sativa L., is one of the most important crops in the world. With the rising world population, feeding people in a more sustainable and environmentally friendly way becomes increasingly important. Therefore, the rice research community needs to share resources to better understand the functions of rice genes that are the foundation for future agricultural biotechnology development, and one way to achieve this goal is via the extensive study of insertional mutants. We have constructed a large rice insertional mutant population in a japonica rice variety, Tainung 67. The collection contains about 93 000 mutant lines, among them 85% with phenomics data and 65% with flanking sequence data. We screened the phenotypes of 12 individual plants for each line grown under field conditions according to 68 subcategories and 3 quantitative traits. Both phenotypes and integration sites are searchable in the Taiwan Rice Insertional Mutants Database. Detailed analyses of phenomics data, T-DNA flanking sequences, and whole-genome sequencing data for rice insertional mutants can lead to the discovery of novel genes. In addition, studies of mutant phenotypes can reveal relationships among varieties, cultivation locations, and cropping seasons.