Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4th lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies.
The Anhui elm Ulmus gaussenii is listed as a critically endangered species by the International Union for Conservation of Nature and is endemic to China, where its only population is restricted to Langya Mountain in Chuzhou, Anhui Province. To better understand the population genetics of U. gaussenii, we developed 12 microsatellite markers using an improved technique. The 12 markers were polymorphic, with the number of alleles per locus ranging from two to nine. Observed and expected heterozygosities ranged from 0.021 to 0.750 and 0.225 to 0.744, respectively. The inbreeding coefficient ranged from –0.157 to 0.960. Significant linkage disequilibrium was detected for two pairs of loci, and significant deviations from Hardy-Weinberg equilibrium were found in nine loci. These microsatellite markers will contribute to the studies of population genetics in U. gaussenii, which in turn will contribute to species conservation and protection.
Gene regulatory mechanisms are often defined in studies performed in the laboratory but are seldom validated for natural habitat conditions, i.e., in natura. Vernalization, the promotion of flowering by winter cold, is a prominent naturally occurring phenomenon, so far best characterized using artificial warm and cold treatments. The floral inhibitor FLOWERING LOCUS C (FLC) gene of Arabidopsis thaliana has been identified as the central regulator of vernalization. FLC shows an idiosyncratic pattern of histone modification at different stages of cold exposure, believed to regulate transcriptional responses of FLC. Chromatin modifications, including H3K4me3 and H3K27me3, are routinely quantified using chromatin immunoprecipitation (ChIP), standardized for laboratory samples. In this report, we modified a ChIP protocol to make it suitable for analysis of field samples. We first validated candidate normalization control genes at two stages of cold exposure in the laboratory and two seasons in the field, also taking into account nucleosome density. We further describe experimental conditions for performing sampling and sample preservation in the field and demonstrate that these conditions give robust results, comparable with those from laboratory samples. The ChIP protocol incorporating these modifications, “Field ChIP”, was used to initiate in natura chromatin analysis of AhgFLC, an FLC orthologue in A. halleri, of which a natural population is already under investigation. Here, we report results on levels of H3K4me3 and H3K27me3 at three representative regions of AhgFLC in controlled cold and field samples, before and during cold exposure. We directly compared the results in the field with those from laboratory samples. These data revealed largely similar trends in histone modification dynamics between laboratory and field samples at AhgFLC, but also identified some possible differences. The Field ChIP method described here will facilitate comprehensive chromatin analysis of AhgFLC in the future to contribute to our understanding of gene regulation in fluctuating natural environments.
A subset of histone genes (H1, H2A, H2B and H4), which are encoded along with H3 within repeating units, were analyzed in Drosophila lutescens, D. takahashii and D. pseudoobscura to investigate the evolutionary mechanisms influencing this multigene family and its GC content. Nucleotide divergence among species was more marked in the less functional regions. A strong inverse relationship was observed between the extent of evolutionary divergence and GC content within the repeating units; this finding indicated that the functional constraint on a region must be associated with both divergence and GC content. The GC content at 3rd codon positions in the histone genes from D. lutescens and D. takahashii was higher than that from D. melanogaster, while that from D. pseudoobscura was similar. These evolutionary patterns were similar to those of H3 gene regions. Based on these findings, we propose that the evolutionary mechanisms governing nucleotide content at 3rd codon positions tend to eliminate A and T nucleotides more frequently than G and C nucleotides. These changes might be the consequence of negative selection and would result in GC-rich 3rd codon positions. In addition, interspecific differences in GC content, which exhibited the same pattern for all histone genes, could be explained by different selection efficiencies that result from changes in population size.
Floricultural cultivars of the Japanese morning glory (Ipomoea nil) carry transposons of the Tpn1 family as active spontaneous mutagens. Half of the characterized mutations related to floricultural traits were caused by insertion of Tpn1 family elements. In addition, mutations comprising insertions of several bp, presumed to be footprints generated by transposon excisions, were also found. Among these, ca-1 and ca-2 are 7-bp insertions at the same position in the InWDR1 gene, which encodes a multifunctional transcription regulator. InWDR1 enhances anthocyanin pigmentation in blue flowers and red stems, and promotes dark brown seed pigmentation as well as seed-trichome formation. The recessive ca mutants show white flowers and whitish seeds. We characterized here a white flower and whitish seed line that is used as a medicinal herb. The mutant line carries a novel ca allele named ca-3, which is the InWDR1 gene carrying an insertion of a Stowaway-like transposon, InSto1. The ca-3 allele is the first example of a mutation induced by transposons other than those in the Tpn1 family in I. nil. Because InSto1 and the 7-bp putative footprints are inserted at identical positions in InWDR1, ca-3 is likely to be the ancestor of ca-1 and ca-2. According to Japanese historical records on whitish seeds of I. nil, putative ca mutants appeared at the end of the 17th century, at the latest. This is around one hundred years before the appearance of many floricultural mutants. This suggests that ca-3 is one of the oldest mutations, and that its origin is different from that of most floricultural mutations in I. nil.
Sakamura (1918) reported the discovery of a polyploid series among eight species of the genus Triticum; this series consisted of 2x, 4x and 6x species with 2n = 14, 28 and 42 chromosomes, respectively. He mentioned in this article that all the materials he used were gifted by T. Minami of the same department of Hokkaido University, Japan. In addition to carrying out an extensive collection of cereal germplasms in the period 1914 to 1916, Minami wrote on October 7, 1915 to K. A. Flaksberger, a wheat taxonomist at the Bureau of Applied Botany, Saint Petersburg, Russia, requesting seeds of Russian wheat and other cereals. He sent Flaksberger a letter of acknowledgement for seed stocks on May 19, 1916; thus, the requested seed package must have arrived from Flaksberger at some time between October 7, 1915 and May 19, 1916. Based on the available documents, there was a considerable period of time between these seed stocks reaching Minami and Sakamura’s publication of the chromosome numbers with the discovery of polyploidy. In fact, the wheat species identified by Flaksberger (1915) and those studied by Sakamura (1918) were identical except for two wild species which appeared only in Flaksberger’s list. The available information supports a proposal that the wheat species used by Sakamura (1918) in his discovery of polyploidy, and later by Kihara (1924, 1930) in his genome analysis, originated from Flaksberger’s collection.