Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis.

Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192.

Allele-specific splicing is the production of different RNA isoforms from different alleles of a gene. Altered splicing patterns such as exon skipping can have a dramatic effect on the final protein product yet have traditionally proven difficult to predict. We investigated the splicing effects of a set of nine single nucleotide polymorphisms (SNPs) which are predicted to have a direct impact on mRNA splicing, each in a different gene. Predictions were based on SNP location relative to splice junctions and intronic/exonic splicing elements, combined with an analysis of splice isoform expression data from public sources. Of the nine genes tested, six SNPs led to direct impacts on mRNA splicing as determined by the splicing reporter minigene assay and RT-PCR in human HeLa cells, of which four were allele-specific effects. These included previously unreported alternative splicing patterns in the genes ZNF419 and DKKL1. Notably, the SNP in ZNF419, a transcription factor, leads to the deletion of a DNA-binding domain from the protein and is associated with an expression QTL, while the SNP in DKKL1 leads to shortened transcripts predicted to produce a truncated protein. We conclude that the impact of SNP mutations on mRNA splicing, and its biological relevance, can be predicted by integrating SNP position with available data on relative isoform abundance in human cell lines.

The hybrid pigeonpea (Cajanus cajan) breeding technology based on cytoplasmic male sterility (CMS) is currently unique among legumes and displays major potential for yield increase. CMS is defined as a condition in which a plant is unable to produce functional pollen grains. The novel chimeric open reading frames (ORFs) produced as a results of mitochondrial genome rearrangements are considered to be the main cause of CMS. To identify these CMS-related ORFs in pigeonpea, we sequenced the mitochondrial genomes of three C. cajan lines (the male-sterile line ICPA 2039, the maintainer line ICPB 2039, and the hybrid line ICPH 2433) and of the wild relative (Cajanus cajanifolius ICPW 29). A single, circular-mapping molecule of length 545.7 kb was assembled and annotated for the ICPA 2039 line. Sequence annotation predicted 51 genes, including 34 protein-coding and 17 RNA genes. Comparison of the mitochondrial genomes from different Cajanus genotypes identified 31 ORFs, which differ between lines within which CMS is present or absent. Among these chimeric ORFs, 13 were identified by comparison of the related male-sterile and maintainer lines. These ORFs display features that are known to trigger CMS in other plant species and to represent the most promising candidates for CMS-related mitochondrial rearrangements in pigeonpea.

Synonymous codon usage bias has typically been correlated with, and attributed to translational efficiency. However, there are other pressures on genomic sequence composition that can affect codon usage patterns such as mutational biases. This study provides an analysis of the codon usage patterns in Arabidopsis thaliana in relation to gene expression levels, codon volatility, mutational biases and selective pressures.

Overexpression of the oncomir miR-21 is associated with many cancers, including breast cancer. Elevated levels of Jagged-1 (JAG1), a predicted miR-21 target, are implicated in estrogen receptor negative (ER-) breast cancer. We demonstrate (by ablation of the miR-21 binding site in the JAG1 3'UTR) that miR-21 directly targets and represses JAG1 levels in MCF-7 (ER+) breast cancer cells. MiR-21 targeting of JAG1 in MDA-MB-231 (ER-) breast cancer cells is dependent on miR-21 dosage (levels). In both cell lines, miR-21 and JAG1 expression levels were negatively correlated due to their regulatory relationship. In addition, 17beta-estradiol (E2) increases JAG1 levels by limiting (via downregulating miR-21 levels) the repressive effects of miR-21 on the JAG1 3'UTR. Our results reveal a regulatory interplay between miR-21, JAG1 and E2 that is important for advancing understanding of how the oncogenic potential of miR-21 and JAG1 manifests in different sub-types of breast cancer.

Climate change impacts on food security will involve negative impacts on crop yields, and potentially on the nutritional quality of staple crops. Common bean is the most important grain legume staple crop for human diets and nutrition worldwide. We demonstrate by crop modeling that the majority of current common bean growing areas in southeastern Africa will become unsuitable for bean cultivation by the year 2050. We further demonstrate reductions in yields of available common bean varieties in a field trial that is a climate analogue site for future predicted drought conditions. Little is known regarding the impact of climate change induced abiotic stresses on the nutritional quality of common beans. Our analysis of nutritional and antinutritional compounds reveals that iron levels in common bean grains are reduced under future climate-scenario relevant drought stress conditions. In contrast, the levels of protein, zinc, lead and phytic acid increase in the beans under such drought stress conditions. This indicates that under climate-change induced drought scenarios, future bean servings by 2050 will likely have lower nutritional quality, posing challenges for ongoing climate-proofing of bean production for yields, nutritional quality, human health, and food security.

There is a need to accelerate crop improvement by introducing alleles conferring host plant resistance, abiotic stress adaptation, and high yield potential. Elite cultivars, landraces and wild relatives harbor useful genetic variation that needs to be more easily utilized in plant breeding. We review genome-wide approaches for assessing and identifying alleles associated with desirable agronomic traits in diverse germplasm pools of cereals and legumes. Major quantitative trait loci and single nucleotide polymorphisms (SNPs) associated with desirable agronomic traits have been deployed to enhance crop productivity and resilience. These include alleles associated with variation conferring enhanced photoperiod and flowering traits. Genetic variants in the florigen pathway can provide both environmental flexibility and improved yields. SNPs associated with length of growing season and tolerance to abiotic stresses (precipitation, high temperature) are valuable resources for accelerating breeding for drought-prone environments. Both genomic selection and genome editing can also harness allelic diversity and increase productivity by improving multiple traits, including phenology, plant architecture, yield potential and adaptation to abiotic stresses. Discovering rare alleles and useful haplotypes also provides opportunities to enhance abiotic stress adaptation, while epigenetic variation has potential to enhance abiotic stress adaptation and productivity in crops. By reviewing current knowledge on specific traits and their genetic basis, we highlight recent developments in the understanding of crop functional diversity and identify potential candidate genes for future use. The storage and integration of genetic, genomic and phenotypic information will play an important role in ensuring broad and rapid application of novel genetic discoveries by the plant breeding community. Exploiting alleles for yield-related traits would allow improvement of selection efficiency and overall genetic gain of multigenic traits. An integrated approach involving multiple stakeholders specializing in management and utilization of genetic resources, crop breeding, molecular biology and genomics, agronomy, stress tolerance, and reproductive/seed biology will help to address the global challenge of ensuring food security in the face of growing resource demands and climate change induced stresses.

The 45S rRNA genes (rDNA) are among the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as 'back-up' copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte, we reduced 45S rDNA copy number (CN) to a plateau of ∼10%. Two independent lines had rDNA CNs reduced by up to 90% at the T7 generation, named low copy number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates, and steady-state levels remained the same as wild-type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. Although overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA CN on genome stability, development, and cellular processes.

Provitamin A enrichment of staple crops through biofortification breeding is a powerful approach to mitigate the public health problem of vitamin A deficiency in developing countries. Twenty-four genetically diverse yellow and orange endosperm maize inbred lines with differing levels of provitamin A content were used for the analysis of their combining ability. Each inbred line was developed from crosses and backcrosses between temperate and tropical germplasm. The inbred lines were grouped into different sets according to their provitamin A levels and were then intercrossed in a factorial mating scheme to generate 80 different single-cross hybrids. The hybrids were evaluated in field trials across a range of agroecological zones in Nigeria. The effect of hybrids was significant on all the measured provitamin A and non-provitamin A carotenoids and agronomic traits. While the effect of genotype-by-environment (GxE) interaction was significant for almost all traits, it was a non-crossover-type interaction for carotenoid content. Partitioning of the variances associated with the carotenoid and agronomic traits into their respective components revealed the presence of significant positive and negative estimates of general combining ability (GCA) and specific combining ability (SCA) effects for both carotenoid content and agronomic traits. The preponderance of GCA effects indicates the importance of additive gene effects in the inheritance of carotenoid content. We found F1 hybrids displaying high parent heterosis for both provitamin A content and agronomic performance. Our study demonstrates that provitamin A biofortification can be effectively implemented in maize breeding programs without adverse effects on important agronomic traits, including grain yield.

The evolution and diversification of proteins capable of remodeling domains has been critical for transcriptional reprogramming during cell fate determination in multicellular eukaryotes. Chromatin remodeling proteins of the CHD3 family have been shown to have important and antagonistic impacts on seed development in the model plant, Arabidopsis thaliana, yet the basis of this functional divergence remains unknown. In this study, we demonstrate that genes encoding the CHD3 proteins PICKLE (PKL) and PICKLE-RELATED 2 (PKR2) originated from a duplication event during the diversification of crown Brassicaceae, and that these homologs have undergone distinct evolutionary trajectories since this duplication, with PKR2 fast evolving under positive selection, while PKL is subject to purifying selection. We find that the rapid evolution of PKR2 under positive selection reduces the encoded protein's intrinsic disorder, possibly suggesting a tertiary structure configuration which differs from that of PKL. Our whole genome transcriptome analysis in seeds of pkr2 and pkl mutants reveals that they act antagonistically on the expression of specific sets of genes, providing a basis for their differing roles in seed development. Our results provide insights into how gene duplication and neofunctionalization can lead to differing and antagonistic selective pressures on transcriptomes during plant reproduction, as well as on the evolutionary diversification of the CHD3 family within seed plants.

Reproduction in triploid plants is important for understanding polyploid population dynamics. We show that genetically identical reciprocal F1 hybrid triploids can display transgenerational epigenetic effects on viable F2 seed development. The success or failure of reproductive outcomes from intra-species crosses between plants of different ploidy levels is an important factor in flowering plant evolution and crop breeding. However, the effects of inter-ploidy cross directions on F1 hybrid offspring fitness are poorly understood. In Arabidopsis thaliana, hybridization between diploid and tetraploid plants can produce viable F1 triploid plants. When selfed, such F1 triploid plants act as aneuploid gamete production "machines" where the vast majority of gametes generated are aneuploid which, following sexual reproduction, can generate aneuploid swarms of F2 progeny (Henry et al. 2009). There is potential for some aneuploids to cause gametophyte abortion and/or F2 seed abortion (Henry et al. 2009). In this study, we analyse the reproductive success of 178 self-fertilized inter-accession F1 hybrid triploids and demonstrate that the proportions of aborted or normally developed F2 seeds from the selfed F1 triploids depend upon a combination of natural variation and cross direction, with strong interaction between these factors. Single-seed ploidy analysis indicates that the embryonic DNA content of phenotypically normal F2 seeds is highly variable and that these DNA content distributions are also affected by genotype and cross direction. Notably, genetically identical reciprocal F1 hybrid triploids display grandparent-of-origin effects on F2 seed set, and hence on the ability to tolerate aneuploidy in F2 seed. There are differences between reciprocal F1 hybrid triploids regarding the proportions of normal and aborted F2 seeds generated, and also for the DNA content averages and distributions of the F2 seeds. To identify genetic variation for tolerance of aneuploidy in F2 seeds, we carried out a GWAS which identified two SNPs, termed MOT and POT, which represent candidate loci for genetic control of the proportion of normal F2 seeds obtained from selfed F1 triploids. Parental and grandparental effects on F2 seeds obtained from selfed F1 triploids can have transgenerational consequences for asymmetric gene flow, emergence of novel genotypes in polyploid populations, and for control of F2 seed set in triploid crops.

All sequenced genomes contain a proportion of lineage-specific genes, which exhibit no sequence similarity to any genes outside the lineage. Despite their prevalence, the origins and functions of most lineage-specific genes remain largely unknown. As more genomes are sequenced opportunities for understanding evolutionary origins and functions of lineage-specific genes are increasing.

Placental mammals display a huge range of life history traits, including size, longevity, metabolic rate and germ line generation time. Although a number of general trends have been proposed between these traits, there are exceptions that warrant further investigation. Species such as naked mole rat, human and certain bat species all exhibit extreme longevity with respect to body size. It has long been established that telomeres and telomere maintenance have a clear role in ageing but it has not yet been established whether there is evidence for adaptation in telomere maintenance proteins that could account for increased longevity in these species.

Embryonic development requires a correct balancing of maternal and paternal genetic information. This balance is mediated by genomic imprinting, an epigenetic mechanism that leads to parent-of-origin-dependent gene expression. The parental conflict (or kinship) theory proposes that imprinting can evolve due to a conflict between maternal and paternal alleles over resource allocation during seed development. One assumption of this theory is that paternal alleles can regulate seed growth; however, paternal effects on seed size are often very low or non-existent. We demonstrate that there is a pool of cryptic genetic variation in the paternal control of Arabidopsis thaliana seed development. Such cryptic variation can be exposed in seeds that maternally inherit a medea mutation, suggesting that MEA acts as a maternal buffer of paternal effects. Genetic mapping using recombinant inbred lines, and a novel method for the mapping of parent-of-origin effects using whole-genome sequencing of segregant bulks, indicate that there are at least six loci with small, paternal effects on seed development. Together, our analyses reveal the existence of a pool of hidden genetic variation on the paternal control of seed development that is likely shaped by parental conflict.

Mature microRNAs (miRNAs) are small, non-coding regulatory RNAs which can elicit post-transcriptional repression of mRNA levels of target genes. Here, we report the identification of 67 mature and 42 precursor miRNAs in the Schistosoma mansoni parasite. The evolutionarily conserved S. mansoni miRNAs consisted of 26 precursor miRNAs and 35 mature miRNAs, while we identified 16 precursor miRNAs and 32 mature miRNAs that displayed no conservation. These S. mansoni miRNAs are located on seven autosomal chromosomes and a sex (W) chromosome. miRNA expansion through gene duplication was suggested for at least two miRNA families miR-71 and mir-2. miRNA target finding analysis identified 389 predicted mRNA targets for the identified miRNAs and suggests that the sma-mir-71 may be involved in female sexual maturation. Given the important roles of miRNAs in animals, the identification and characterization of miRNAs in S. mansoni will facilitate novel approaches towards prevention and treatment of Schistosomiasis.

Parent-of-origin effects arise when a phenotype depends on whether it is inherited maternally or paternally. Parent-of-origin effects can exert a strong influence on F1 seed size in flowering plants, an important agronomic and life-history trait that can contribute to biomass heterosis. Here we investigate the natural variation in the relative contributions of the maternal and paternal genomes to F1 seed size across 71 reciprocal pairs of F1 hybrid diploids and the parental effect on F1 seed size heterosis. We demonstrate that the paternally derived genome influences F1 seed size more significantly than previously appreciated. We further demonstrate (by disruption of parental genome dosage balance in F1 triploid seeds) that hybridity acts as an enhancer of genome dosage effects on F1 seed size, beyond that observed from hybridity or genome dosage effects on their own. Our findings indicate that interactions between genetic hybridity and parental genome dosage can enhance heterosis effects in plants, opening new avenues for boosting heterosis breeding in crop plants.

Plastid ribosomal proteins (PRPs) can play essential roles in plastid ribosome functioning that affect plant function and development. However, the roles of many PRPs remain unknown, including elucidation of which PRPs are essential or display redundancy. Here, we report that the nuclear-encoded PLASTID RIBOSOMAL PROTEIN L5 (PRPL5) is essential for early embryo development in A. thaliana, as homozygous loss-of-function mutations in the PRPL5 gene impairs chloroplast development and leads to embryo failure to develop past the globular stage. We confirmed the prpl5 embryo-lethal phenotype by generating a mutant CRISPR/Cas9 line and by genetic complementation. As PRPL5 underwent transfer to the nuclear genome early in the evolution of Embryophyta, PRPL5 can be expected to have acquired a chloroplast transit peptide. We identify and validate the presence of an N-terminal chloroplast transit peptide, but unexpectedly also confirm the presence of a conserved and functional Nuclear Localization Signal on the protein C-terminal end. This study highlights the fundamental role of the plastid translation machinery during the early stages of embryo development in plants and raises the possibility of additional roles of plastid ribosomal proteins in the nucleus.

The balance between parental genome dosage is critical to offspring development in both animals and plants. In some angiosperm species, despite the imbalance between maternally and paternally inherited chromosome sets, crosses between parental lines of different ploidy may result in viable offspring. However, many plant species, like Arabidopsis thaliana, present a post-zygotic reproductive barrier, known as triploid block which results in the inability of crosses between individuals of different ploidy to generate viable seeds but also, in defective development of the seed. Several paternal regulators have been proposed as active players in establishing the triploid block. Maternal regulators known to be involved in this process are some flavonoid biosynthetic (FB) genes, expressed in the innermost layer of the seed coat. Here we explore the role of selected flavonoid pathway genes in triploid block, including TRANSPARENT TESTA 4 (TT4), TRANSPARENT TESTA 7 (TT7), SEEDSTICK (STK), TRANSPARENT TESTA 16 (TT16), TT8 and TRANSPARENT TESTA 13 (TT13). This approach allowed us to detect that TT8, a bHLH transcription factor, member of this FB pathway is required for the paternal genome dosage, as loss of function tt8, leads to complete rescue of the triploid block to seed development.

Biofortified orange-fleshed sweetpotato (OFSP) varieties are being promoted to reduce vitamin A deficiencies due to their higher beta-carotene content. For OFSP varieties to have impact they need to be accepted and consumed at scale amongst populations suffering from vitamin A deficiencies.