Genetic imprinting refers to the unequal expression of paternal and maternal alleles of a gene in sexually reproducing organisms, including mammals and flowering plants. Although many imprinted genes have been identified in plants, the functions of these imprinted genes have remained largely uninvestigated. We report genome-wide analysis of gene expression, DNA methylation and small RNAs in the rice endosperm and functional tests of five imprinted genes during seed development using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated gene9 (CRISPR/Cas9) gene editing technology. In the rice endosperm, we identified 162 maternally expressed genes (MEGs) and 95 paternally expressed genes (PEGs), which were associated with miniature inverted-repeat transposable elements, imprinted differentially methylated loci and some 21-22 small interfering RNAs (siRNAs) and long noncoding RNAs (lncRNAs). Remarkably, one-third of MEGs and nearly one-half of PEGs were associated with grain yield quantitative trait loci. Most MEGs and some PEGs were expressed specifically in the endosperm. Disruption of two MEGs increased the amount of small starch granules and reduced grain and embryo size, whereas mutation of three PEGs reduced starch content and seed fertility. Our data indicate that both MEGs and PEGs in rice regulate nutrient metabolism and endosperm development, which optimize seed development and offspring fitness to facilitate parental-offspring coadaptation. These imprinted genes and mechanisms could be used to improve the grain yield of rice and other cereal crops.

Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC. We successfully introduced nucleotide deletions and mutations in the SCD1 gene locus through the NHEJ pathway and disrupted its second exon via insertion of an EGFP-PuroR segment using the HDR pathway. In clones derived from the latter, gene- and protein-expression data indicated that we obtained a monoallelic SCD1 knockout. A T7EN1-mediated assay revealed no off-targets in the surveyed sites. The contents of triacylglycerol and cholesterol and the desaturase index were significantly decreased as a consequence of SCD1 knockout. The deletion of SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including SREBF1 and FASN, as well the fatty acid transporters FABP3 and FABP4. The downregulation of these genes partly explains the decrease of intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat mammary cells using CRISPR-Cas9. The demonstration of the successful use of CRISPR-Cas9 in GMEC is an important step to producing transgenic goats to study mammary biology in vivo.

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Berberine is a natural herbicidal alkaloid from Coptis chinensis Franch. Here we characterized its herbicidal spectrum and absorption and transportation in the plant, along with the possible mechanism. Berberine showed no effect on the germination of the 10 tested plants. The IC values of berberine on the primary root length and fresh weight of the 10 tested plants ranged from 2.91 to 9.79 mg L and 5.76 to 35.07 mg L, respectively. Berberine showed a similar herbicidal effect on Bidens pilosa as the commercial naturally derived herbicide cinmethylin. HPLC and fluorescence analysis revealed that berberine was mainly absorbed by B. pilosa root and transported through vascular bundle acropetally. Enzyme activity studies, GC-MS analysis, and SEM and TEM observations indicated that berberine might first function on the cell membrane indicated by variation of the IUFA percent and then cause POD, PPO, and SOD activity changes and cellular structure deformity, which was eventually expressed as the decrease of cell adaptation ability and abnormal cell function and may even result in cell death. Environmental safety evaluation tests revealed that berberine was low in toxicity to Brachydanio rerio. These indicate that berberine has the potential to be a bioherbicide and/or a lead molecule for new herbicides.

Background Wheat is a powerful genetic model for studying polyploid evolution and crop domestication. Hexaploid bread wheat was formed by two rounds of interspecific hybridization and polyploidization, processes which are often accompanied by genetic and epigenetic changes, including DNA methylation. However, the extent and effect of such changes during wheat evolution, particularly from tetraploid-to-hexaploid wheat, are currently elusive. Results Here we report genome-wide DNA methylation landscapes in extracted tetraploid wheat (ETW, AABB), natural hexaploid wheat (NHW, AABBDD), resynthesized hexaploid wheat (RHW, AABBDD), natural tetraploid wheat (NTW, AABB), and diploid (DD). In the endosperm, levels of DNA methylation, especially in CHG (H=A, T, or C) context, were dramatically decreased in the ETW relative to natural hexaploid wheat; hypo-differentially methylated regions (DMRs) (850,832) were 24-fold more than hyper-DMRs (35,111). Interestingly, those demethylated regions in ETW were remethylated in the resynthesized hexaploid wheat after the addition of the D genome. In ETW, hypo-DMRs correlated with gene expression, and TEs were demethylated and activated, which could be silenced in the hexaploid wheat. In NHW, groups of TEs were dispersed in genic regions of three subgenomes, which may regulate the expression of TE-associated genes. Further, hypo-DMRs in ETW were associated with reduced H3K9me2 levels and increased expression of histone variant genes, suggesting concerted epigenetic changes after separation from the hexaploid. Conclusion Genome merger and separation provoke dynamic and reversible changes in chromatin and DNA methylation. These changes correlate with altered gene expression and TE activity, which may provide insights into polyploid genome and wheat evolution.