The r-X1 deletion induces terminal deficiencies in the maize B chromosome.

In addition to causing the nondisjunction of maize B and normal A chromosomes at the second megaspore division during embryo sac development, the r-X1 deletion results in terminal deficiencies (TDs) in various A chromosomal arms, but whether the r-X1 deletion also induces TDs of the maize B chromosome remains unknown. To answer this question, the chromosomal composition in the r-X1-containing progeny of r-X1/R-r female parents carrying two standard B chromosomes was determined. Nine of 104 (8.7%) examined kernels contained a smaller telocentric B chromosome, and one of these (designated Bdef-1) was further identified as a TD with a breakpoint in the third distal heterochromatic region of the B chromosome. Thus, the results indicated that the r-X1 deletion could also induce TDs of the maize B chromosome during megaspore divisions. The Bdef-1 chromosome lacked nondisjunctional behavior, and this behavior was restored by the presence of the B chromosome in the cell. A transmission analysis of the Bdef-1 chromosome revealed that loss of the distal portion of the B chromosome reduced female but not male transmission of the B chromosome. Furthermore, the Bdef-1 chromosome was used to more finely map B-derived miRNA genes on the B chromosome. Our results indicate that the r-X1 deletion results in TDs of the B chromosome in maize, and the r-X1 deletion system can thus be used to generate a series of terminally truncated B chromosomes that may be used to map features of the B chromosome, including genes and properties related to B chromosome functions.

Studies of rice Hd1 haplotypes worldwide reveal adaptation of flowering time to different environments.

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.

LARGE GRAIN Encodes a Putative RNA-Binding Protein that Regulates Spikelet Hull Length in Rice.

Grain size is a key determiner of grain weight, one of the yield components in rice (Oryza sativa). Therefore, to increase grain yield, it is important to elucidate the detailed mechanisms regulating grain size. The Large grain (Lgg) mutant, found in the nonautonomous DNA-based active rice transposon1 (nDart1)-tagged lines of Koshihikari, is caused by a truncated nDart1-3 and 355 bp deletion in the 5' untranslated region of LGG, which encodes a putative RNA-binding protein, through transposon display and cosegregation analysis between grain length and LGG genotype in F2 and F3. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9-mediated knockout and overexpression of LGG led to longer and shorter grains than wild type, respectively, showing that LGG regulates spikelet hull length. Expression of LGG was highest in the 0.6-mm-long young panicle and gradually decreased as the panicle elongated. LGG was also expressed in roots and leaves. These results show that LGG functions at the very early stage of panicle development. Longitudinal cell numbers of spikelet hulls of Lgg, knockout and overexpressed plants were significantly different from those of the wild type, suggesting that LGG might regulate longitudinal cell proliferation in the spikelet hull. RNA-Seq analysis of 1-mm-long young panicles from LGG knockout and overexpressing plants revealed that the expressions of many cell cycle-related genes were reduced in knockout plants relative to LGG-overexpressing plants and wild type, whereas some genes for cell proliferation were highly expressed in knockout plants. Taken together, these results suggest that LGG might be a regulator of cell cycle and cell division in the rice spikelet hull.

Easy sectioning of whole grain of rice using cryomicrotome.

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.

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

Rice (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.