SUMMARYRice (Oryza sativa) is one of the world's 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.
Rice domestication/adaptation is a good model for studies of the development and spread of this important crop. Mutations that caused morphological and physiological change, followed by human selection/expansion, finally led to the improvement of phenotypes suitable for different kinds of environments. We used the sequence information for Heading date 1 ( Hd1 ) gene to reveal the association between sequence changes and flowering phenotypes of rice in different regions. Seven loss-of-function hd1 haplotypes had been reported. By data-mining the genome sequencing information in the public domain, we discovered 3 other types. These loss-of-function allele haplotypes are present in subtropical and tropical regions, which indicates human selection. Some of these haplotypes are present locally. However, types 7 and 13 are present in more than one-third of the world’s rice accessions, including landraces and modern varieties. In the present study, phylogenetic, allele network and selection pressure analyses revealed that these two haplotypes might have occurred early in Southeastern Asia and then were introgressed in many local landraces in nearby regions. We also demonstrate that these haplotypes are present in weedy rice populations, which again indicates that these alleles were present in rice cultivation for long time. In comparing the wild rice sequence information, these loss-of-function haplotypes occurred in agro but were not from wild rice.
To obtain a clear intact section of a ripened rice grain, which is suitable for biochemical and histological analysis, the Kawamoto method using a specific adhesive film was applied using a cryomicrotome. The longitudinal and sagittal sections were easily obtained together with the cross-section, and cell characteristics were clearly discerned in the ripened grain. It was demonstrated that the Kawamoto method is readily applicable for intact sectioning of hard tissue, including ripened grain. Intact section sampling may be useful for enzymatic analysis and transcriptomic analysis of plant tissue.
Background Plant domestication/adaptation is a good model for evo-devo studies. Mutations that caused morphological and physiological change, followed by human selection, finally led to improvement of phenotypes suitable for different kinds of environments. Originating from the Yangtze Valley, rice is a facultative short-day plant. Rice spread southward thousands of years ago, but one of the new traits beneficial to crop yield would be loss of sensitivity to photoperiod. That is, if rice could be cultivated 2 or 3 seasons each year, the production would at least double. Results We used the sequence information for Heading date 1 (Hd1) gene to reveal the relationship of sequence changes and flowering phenotypes of rice in different regions. Seven loss-of-function hd1 haplotypes were reported previously. By data mining the genome sequencing information in the public domain, we discovered another 3 types. Allele haplotypes are present in sub-tropical and tropical regions, which indicates human selection. Two of these alleles, types 7 and 13, must have occurred early in southern Asia and then were introgressed in many local landraces. According to the rice 3K database, more than one-third of the world’s rice accessions contain these 2 loss-of-function haplotypes. We also demonstrate that these haplotypes are present in weedy rice populations, again indicating that these alleles were present in rice cultivation for long time. In comparing the wild rice sequence information, these loss-of-function haplotypes occurred in agro but were not from wild rice. Conclusion In the current study, we analyzed how sequence changes in a rice flowering-control gene occurred, were selected and were retained during rice cultivation. Many loss-of-function hd1 alleles have existed in sub-tropical and tropical Asia rice-growing areas for a long time. Some of these haplotypes were present locally, but 2, types 7 and 13, were spread in many regions and are now included in most of the modern varieties in southern Asia.
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