Post-translational modifications of nucleosomal core histones play important roles in biological processes via altering chromatin structure and creating target sites for proteins acting on chromatin. Molecular genetic studies with Arabidopsis have verified several epigenetic factors that regulate flowering time. However, the roles of chromatin remodeling factors have not been well explored in rice. Here, we identified chromatin remodeling factors, OsVIL1, 2, and 4 (Oryza sativa VIN3-LIKE) genes, that regulate grain yield. OsVIL proteins contain a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. We showed that plant height and number of spikelets per panicle were increased in the OsVIL2-overexpression (OsVIL2-OX) and osvil4 plants, respectively. Each mutants (OsVIL2-OX and osvil4) exhibited longer internodes and thicker stems than wild type controls. Histochemical analysis revealed that cells are smaller in OsVIL2-OX and osvil4 plants. We performed an RNA-seq using 1st internodes of WT and OsVIL2-OX stems and got the suppressed target genes in the OsVIL2-OX. OsCKX2, which encodes cytokinin oxidase/dehydrogenase is one of the suppressed genes in the OX plants and we verified decrease of that gene using qRT-PCR and closed chromatins of OsCKX2 were enriched in the OX plants by using ChIP. As results of these, cytokinins were enriched in the OX plants. These demonstrate that OsVIL2 and OsVIL4 antagonistically regulate plant height and number of spikelets by controlling cytokinin contents. Like OsVIL2-OX and osvil4 plants, besides, OsVIL1-OX plants were also shown increased plant height and biomass. We propose that OsVILs may be used for improving grain yield by increasing biomass.

Polymerase chain reaction (PCR) is highly utilized for QTL analysis, positional cloning of valuable genes, and molecular breeding in crop science. Usually those experiments handle DNA samples of many genotypes (up to several thousands). However, many DNA extraction protocols require longer time using harmful chemicals such as chloroform, phenol, and liquid nitrogen. Here, we introduce a new DNA extraction method for PCR with agarose/PAGE analysis from a diversity panel of rice genotypes identified with yield enhancing traits. This protocol consists of four steps including injection of extraction buffer (20 mM Tris-HCl pH9.5, 200 mM KCl, 2 mM EDTA) into the tubes containing leaf tissues and steel balls, and crushing tissues using Geno-Grinder without liquid nitrogen, sample incubation at 65°C, and then centrifugation for removing cell debris. After centrifugation the crude extracts directly used as template DNA for PCR. Through this protocol we could complete F1 hybridity test from approximately 2,100 plants that come from 96 cross combinations with 13 SSR markers. In addition, we tested the DNA quality by PCR amplification of high GC-rich region and large target size (-2kb). From these results our DNA extraction method produces enough DNA quality for PCR and is suitable for large scale molecular analysis from rice plants.

Flowering is exquisitely regulated by both promotive and inhibitory factors. Molecular genetic studies with Arabidopsis have verified several epigenetic repressors that regulate flowering time. However, the roles of chromatin remodeling factors in developmental processes have not been well explored in rice. We identified a chromatin remodeling factor OsVIL2 (O. sativa VIN3-LIKE 2) that promotes flowering. OsVIL2 contains a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. Insertion mutations in OsVIL2 caused late flowering under both long and short days. In osvil2 mutants OsLFL1 expression was increased, but that of Ehd1, Hd3a and RFT1 was reduced. We demonstrated that OsVIL2 is bound to native histone H3 in vitro. Chromatin immunoprecipitation analyses showed that OsVIL2 was directly associated with OsLFL1 chromatin. We also observed that H3K27me3 was significantly enriched by OsLFL1 chromatin in the wild type, but that this enrichment was diminished in the osvil2 mutants. These results indicated that OsVIL2 epigenetically represses OsLFL1 expression. We showed that OsVIL2 physically interacts with OsEMF2b, a component of polycomb repression complex 2. As observed from osvil2, a null mutation of OsEMF2b caused late flowering by increasing OsLFL1 expression and decreasing Ehd1 expression. Thus, we conclude that OsVIL2 functions together with PRC2 to induce flowering by repressing OsLFL1. Transgenic plants over-expressing OsVIL2 flowered early. In addition, they were taller and ticker due to increased in cell number, resulting in yield increase. The same phenotypes were observed from OsVIL4 knockout mutants. These indicate that OsVIL4 represses OsVIL2 function by directly binding to the protein.

With the development of diverse agricultures worldwide, biofortified rice noted for its preferable marketability and palatability plays an important role in the world's agricultural economics and rice breeding programs. In this report, several M5 of T-DNA inserted lines derived from the donor cultivars, 'Hwayong' and 'Dongjin', were selected for high or low protein, high lipid and low amylose content, respectively. The coefficients and ranges of variation for the chemical constituents between M4 and M5 T-DNA inserted lines were evaluated in comparison with those of the donor varieties. Results indicated that T-DNA insertion might be an effective way to generate useful variations for chemical composition of rice grains which could be used for the development of biofortified rice cultivars.