Heritable epigenetic diversity for conservation and utilization of epigenetic germplasm resources of clonal East African Highland banana (EAHB) accessions.

KEY MESSAGE: Genetically identical East African Highland banana (EAHB) clones are epigenetically diverse with heritable epialleles that can contribute to morphological diversity. Heritable epigenetic variation can contribute to agronomic traits in crops and should be considered in germplasm conservation. Despite the genetic uniformity arising from a genetic bottleneck of one ancestral clone, followed by subsequent vegetative propagation, East African Highland bananas (EAHBs) display significant phenotypic diversity potentially arising from somatic mutations, heritable epialleles and/or genotype-by-environment interactions. Here, we use DNA methylation profiling across EAHB accessions representing most of the primary EAHB genepool to demonstrate that the genetically uniform EAHB genepool harbours significant epigenetic diversity. By analysing 724 polymorphic DNA methylation sites by methylation-sensitive AFLP across 90 EAHB cultivars, we could differentiate the EAHB varieties according to their regions (Kenya and Uganda). In contrast, there was minimal association of DNA methylation variation with the five morphological groups that are used to classify EAHBs. We further analysed DNA methylation patterns in parent-offspring cohort, which were maintained in offspring generated by sexual (seed) and asexual (vegetative) propagation, with higher levels of altered DNA methylation observed in vegetatively generated offspring. Our results indicate that the phenotypic diversity of near-isogenic EAHBs is mirrored by considerable DNA methylation variation, which is transmitted between generations by both vegetative reproduction and seed reproduction. Genetically uniform vegetatively propagated crops such as EAHBs harbour considerable heritable epigenetic variation, where heritable epialleles could arise in offspring and contribute to functional traits. This study provides a basis for developing strategies for conservation of epigenetic resources and for integration of epimarkers into crop breeding programmes.

Imprinted loci in domestic livestock species as epigenomic targets for artificial selection of complex traits.

The phenomenon of genomic imprinting, whereby a subset of mammalian genes display parent-of-origin-specific monoallelic expression, is one of the most active areas of epigenetics research. Over the past two decades, more than 100 imprinted mammalian genes have been identified, while considerable advances have been made in elucidating the molecular mechanisms governing imprinting. These studies have helped to unravel the epigenome--a separate layer of regulatory information contained in eukaryotic chromosomes that influences gene expression and phenotypes without involving changes to the underlying DNA sequence. Although most studies of genomic imprinting in mammals have focussed on mouse models or human biomedical disorders, there is burgeoning interest in the phenotypic effects of imprinted genes in domestic livestock species. In particular, research has focused on imprinted genes influencing foetal growth and development, which are associated with economically important production traits in cattle, sheep and pigs. These findings, when coupled with the data emerging from the various different livestock genome projects, have major implications for the future of animal breeding, health and management. Here, we review current scientific knowledge regarding genomic imprinting in livestock species and evaluate how this information can be used in modern livestock improvement programmes.

CTC-5: A novel digital pathology approach to characterise circulating tumour cell biodiversity.

Metastatic progression and tumor evolution complicates the clinical management of cancer patients. Circulating tumor cell (CTC) characterization is a growing discipline that aims to elucidate tumor metastasis and evolution processes. CTCs offer the clinical potential to monitor cancer patients for therapy response, disease relapse, and screen 'at risk' groups for the onset of malignancy. However, such clinical utility is currently limited to breast, prostate, and colorectal cancer patients. Further understanding of the basic CTC biology of other malignancies is required to progress them towards clinical utility. Unfortunately, such basic clinical research is often limited by restrictive characterization methods and high-cost barrier to entry for CTC isolation and imaging infrastructure. As experimental clinical results on applications of CTC are accumulating, it is becoming clear that a two-tier system of CTC isolation and characterization is required. The first tier is to facilitate basic research into CTC characterization. This basic research then informs a second tier specialised in clinical prognostic and diagnostic testing. This study presented in this manuscript describes the development and application of a low-cost, CTC isolation and characterization pipeline; CTC-5. This approach uses an established 'isolation by size' approach (ScreenCell Cyto) and combines histochemical morphology stains and multiparametric immunofluorescence on the same isolated CTCs. This enables capture and characterization of CTCs independent of biomarker-based pre-selection and accommodates both single CTCs and clusters of CTCs. Additionally, the developed open-source software is provided to facilitate the synchronization of microscopy data from multiple sources (https://github.com/CTC5/). This enables high parameter histochemical and immunofluorescent analysis of CTCs with existing microscopy infrastructure without investment in CTC specific imaging hardware. Our approach confirmed by the number of successful tests represents a potential major advance towards highly accessible low-cost technology aiming at the basic research tier of CTC isolation and characterization. The biomarker independent approach facilitates closing the gap between malignancies with poorly, and well-defined CTC phenotypes. As is currently the case for some of the most commonly occurring breast, prostate and colorectal cancers, such advances will ultimately benefit the patient, as early detection of relapse or onset of malignancy strongly correlates with their prognosis.

Single nucleotide polymorphisms in the imprinted bovine insulin-like growth factor 2 receptor gene (IGF2R) are associated with body size traits in Irish Holstein-Friesian cattle.

The regulation of the bioavailability of insulin-like growth factors (IGFs) is critical for normal mammalian growth and development. The imprinted insulin-like growth factor 2 receptor gene (IGF2R) encodes a transmembrane protein receptor that acts to sequester and degrade excess circulating insulin-like growth factor 2 (IGF-II) - a potent foetal mitogen - and is considered an important inhibitor of growth. Consequently, IGF2R may serve as a candidate gene underlying important growth- and body-related quantitative traits in domestic mammalian livestock. In this study, we have quantified genotype-phenotype associations between three previously validated intronic bovine IGF2R single nucleotide polymorphisms (SNPs) (IGF2R:g.64614T>C, IGF2R:g.65037T>C and IGF2R:g.86262C>T) and a range of performance traits in 848 progeny-tested Irish Holstein-Friesian artificial insemination sires. Notably, all three polymorphisms analysed were associated (P ≤ 0.05) with at least one of a number of performance traits related to animal body size: angularity, body depth, chest width, rump width, and animal stature. In addition, the C-to-T transition at the IGF2R:g.65037T>C polymorphism was positively associated with cow carcass weight and angularity. Correction for multiple testing resulted in the retention of two genotype-phenotype associations (animal stature and rump width). None of the SNPs analysed were associated with any of the milk traits examined. Analysis of pairwise r(2) measures of linkage disequilibrium between all three assayed SNPs ranged between 0.41 and 0.79, suggesting that some of the observed SNP associations with performance may be independent. To our knowledge, this is one of the first studies demonstrating associations between IGF2R polymorphisms and growth- and body-related traits in cattle. These results also support the increasing body of evidence that imprinted genes harbour polymorphisms that contribute to heritable variation in phenotypic traits in domestic livestock species.

PHLDA2 is an imprinted gene in cattle.

Genomic imprinting is an epigenetic non-Mendelian phenomenon found predominantly in placental mammals. Imprinted genes display differential expression in the offspring depending on whether the gene is maternally or paternally inherited. Currently, some 100 imprinted genes have been reported in mammals, and while some of these genes are imprinted across most mammalian species, others have been shown to be imprinted in only a few species. The PHLDA2 gene that codes for a pleckstrin homology-like domain, family A (member 2), protein has to date been shown to be a maternally expressed imprinted gene in humans, mice and pigs. Genes subject to imprinting can have major effects on mammalian growth, development and disease. For instance, disruption of imprinted genes can lead to aberrant growth syndromes in cloned domestic mammals, and it has been demonstrated that PHLDA2 mRNA expression levels are aberrant in the placenta of somatic clones of cattle. In this study, we demonstrate that PHLDA2 is expressed across a range of cattle foetal tissues and stages and provide the first evidence that PHLDA2 is a monoallelically expressed imprinted gene in cattle foetal tissues, and also in the bovine placenta.

High concordance of bovine single nucleotide polymorphism genotypes generated using two independent genotyping strategies.

Single nucleotide polymorphisms (SNPs) represent the most common form of DNA sequence variation in mammalian livestock genomes. While the past decade has witnessed major advances in SNP genotyping technologies, genotyping errors caused, in part, by the biochemistry underlying the genotyping platform used, can occur. These errors can distort project results and conclusions and can result in incorrect decisions in animal management and breeding programs; hence, SNP genotype calls must be accurate and reliable. In this study, 263 Bos spp. samples were genotyped commercially for a total of 16 SNPs. Of the total possible 4,208 SNP genotypes, 4,179 SNP genotypes were generated, yielding a genotype call rate of 99.31% (standard deviation ± 0.93%). Between 110 and 263 samples were subsequently re-genotyped by us for all 16 markers using a custom-designed SNP genotyping platform, and of the possible 3,819 genotypes a total of 3,768 genotypes were generated (98.70% genotype call rate, SD ± 1.89%). A total of 3,744 duplicate genotypes were generated for both genotyping platforms, and comparison of the genotype calls for both methods revealed 3,741 concordant SNP genotype call rates (99.92% SNP genotype concordance rate). These data indicate that both genotyping methods used can provide livestock geneticists with reliable, reproducible SNP genotypic data for in-depth statistical analysis.

Interaction of the Arabidopsis polycomb group proteins FIE and MEA mediates their common phenotypes.

Genes of the FERTILISATION INDEPENDENT SEED (FIS) class regulate cell proliferation during reproductive development in Arabidopsis [1-5]. The FIS genes FERTILISATION INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) encode homologs of animal Polycomb group (Pc-G) proteins, transcriptional regulators that modify chromatin structure and are thought to form multimeric complexes [3-11]. To test whether similarities in fis mutant phenotypes reflect interactions between their protein products, we characterised FIE RNA and protein localisation in vivo, and FIE protein interactions in yeast and in vitro. Expression of FIE mRNA overlaps with that of MEA during embryo sac and seed development and is unaffected in mea mutants. Results from the yeast two-hybrid system and an in vitro pull-down assay indicate that MEA and FIE proteins interact. The relevance of this interaction in vivo is supported by the finding that FIE and MEA co-localise in the nucleus in transfected plant cells. Interaction of MEA and FIE is mediated by the amino-terminal region of MEA. Despite sequence divergence in this domain, MEA can interact with its corresponding animal partner Extrasexcombs (ESC) in the yeast two-hybrid system. We conclude that FIE and MEA act together as part of a multimeric complex and that this accounts for the similarities in mutant phenotypes. We propose that an ancient mechanism for chromatin modification has been independently recruited to different developmental processes in the two kingdoms.

Genomic imprinting and seed development: endosperm formation with and without sex.

During seed development, coordinated developmental programs lead to the formation of the embryo, endosperm and seed coat. The maternal effects of the genes affected in the fertilisation-independent seed class of mutants play an important role in seed development. The plant Polycomb proteins MEDEA and FERTILIZATION-INDEPENDENT ENDOSPERM physically interact and form a complex, in a manner similar to that of their counterparts in animals. Maternal-effect phenotypes can result from regulation by genomic imprinting, a phenomenon of critical importance for both sexual and apomictic seed development.

Genome-wide identification and in silico characterisation of microRNAs, their targets and processing pathway genes in Phaseolus vulgaris L.

Common bean (Phaseolus vulgaris L., Fabaceae) is a globally important staple crop, which is an important source of calories, protein and essential micronutrients. At the genomic level little is known regarding the small non-coding RNAs within the common bean genome. One of the most important classes of such small non-coding RNAs is microRNAs (miRNAs), which control mRNA and protein expression levels in many eukaryotes. Computational methods have been applied to identify putative miRNAs in the genomes of different organisms. In this study, our objective was to comprehensively identify and characterise miRNAs from the genome and transcriptome of P. vulgaris, including both mature and precursor miRNA forms. We also sought to identify the putative proteins involved in miRNA processing and the likely target genes of common bean miRNAs. We identified 221 mature miRNAs and 136 precursor miRNAs distributed across 52 different miRNA families in the P. vulgaris genome. Amongst these, we distinguished 129 novel mature miRNAs and 123 miRNA precursors belonging to 24 different miRNA families. We also identified 31 proteins predicted to participate in the miRNA-processing pathway in P. vulgaris. Finally, we also identified 483 predicted miRNA targets, including many which corroborate results from other species, suggesting that miRNA regulatory systems are evolutionarily conserved and important for plant development. Our results expand the study of miRNAs and their target genes in common bean, and provide new opportunities to understand their roles in the biology of this important staple crop.

A catalogue of validated single nucleotide polymorphisms in bovine orthologs of mammalian imprinted genes and associations with beef production traits.

Genetic (or 'genomic') imprinting, a feature of approximately 100 mammalian genes, results in monoallelic expression from one of the two parentally inherited chromosomes. To date, most studies have been directed on imprinted genes in murine or human models; however, there is burgeoning interest in the effects of imprinted genes in domestic livestock species. In particular, attention has focused on imprinted genes that influence foetal growth and development and that are associated with several economically important production traits in cattle, sheep and pigs. We have re-sequenced regions in 20 candidate bovine imprinted genes in order to validate single nucleotide polymorphisms (SNPs) that may influence important production traits in cattle. Putative SNPs detected via re-sequencing were subsequently re-formatted for high-throughput SNP genotyping in 185 cattle samples comprising 138 performance-tested European Bos taurus (all Limousin bulls), 29 African B. taurus and 18 Indian B. indicus samples. Analysis of the resulting genotypic data identified 117 validated SNPs. Preliminary genotype-phenotype association analyses using 83 SNPs that were polymorphic in the Limousin samples with minor allele frequencies ⩾0.05 revealed significant associations between two candidate bovine imprinted genes and a range of important beef production traits: average daily gain, average feed intake, live weight, feed conversion ratio, residual feed intake and residual gain. These genes were the Ras protein-specific guanine nucleotide releasing factor gene (RASGRF1) and the zinc finger, imprinted 2 gene (ZIM2). Despite the relatively small sample size used in these analyses, the observed associations with production traits are supported by the purported biological function of the RASGRF1 and ZIM2 gene products. These results support the hypothesis that imprinted genes contribute significantly to important complex production traits in cattle. Furthermore, these SNPs may be usefully incorporated into future marker-assisted and genomic selection breeding schemes.

Apomixis in agriculture: the quest for clonal seeds.

Apomixis, or asexual reproduction through seeds, is a natural trait that could have an immense positive impact on crop production. Apomictic breeding strategies could allow the fixation and indefinite propagation of any desired genotype, however complex. Apomicts display a wide variety of developmental mechanisms, which can be viewed as a short-circuiting of sexual development. Gametophytic and sporophytic apomixis are distinguished by the developmental origin of apomictically derived embryos. Genetic studies suggest that individual elements of gametophytic apomixis, such as apomeiosis and parthenogenesis, are either controlled by one or two dominant Mendelian factors. As recombination around apomeiosis loci is suppressed, it is currently not known how complex these loci are. Much less is known regarding the genetic control of sporophytic apomixis but initial studies suggest a complex genetic control. Genetic analyses of sexual reproduction in plant model systems have identified genes that, when mutated, display elements of apomixis. Such studies help in the identification of candidate genes and promoters that can be used for the de novo engineering of apomixis through biotechnology. Molecular genetic studies in apomictic and sexual systems will generate the knowledge necessary for the engineering of conditional apomixis technology. Approaches encouraging collaboration and widespread dissemination of the acquired knowledge will constitute the most innovative route to the development, deployment and acceptance of apomixis technology in agriculture.

Concurrent suppression of virus replication and rescue of movement-defective virus in transgenic plants expressing the coat protein of potato virus X.

A line of transgenic tobacco expressing the coat protein (CP) of potato virus X (PVX) was resistant against a broad spectrum of PVX strains. Inoculation of leaves and protoplasts with PVX expressing the jellyfish green fluorescent protein reporter gene revealed that this resistance mechanism suppressed PVX replication in the initially infected cell and systemic spread of the virus. Cell-to-cell movement was also slower in the resistant plants. The resistance at the level of replication was effective against wild-type PVX and also against movement-defective isolates with a frameshift mutation or deletion in the CP ORF. However, the cell-to-cell movement defect of the mutant viruses was rescued on the resistant plants. Based on these results it is proposed that the primary resistance mechanism is at the level of replication.

Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity.

In higher plants, seed development requires maternal gene activity in the haploid (gametophytic) as well as diploid (sporophytic) tissues of the developing ovule. The Arabidopsis thaliana gene MEDEA (MEA) encodes a SET-domain protein of the Polycomb group that regulates cell proliferation by exerting a gametophytic maternal control during seed development. Seeds derived from female gametocytes (embryo sacs) carrying a mutant mea allele abort and exhibit cell proliferation defects in both the embryo and the endosperm. In this study we show that the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte and after fertilization only from maternally inherited MEA alleles. Paternally inherited MEA alleles are transcriptionally silent in both the young embryo and endosperm. Mutations at the decrease in DNA methylation1 (ddm1) locus are able to rescue mea seeds by functionally reactivating paternally inherited MEA alleles during seed development. Rescued seeds are larger than the wild type and exhibit some of the abnormalities found in aborting mea seeds. Our results indicate that the maintenance of the genomic imprint at the mea locus requires zygotic DDM1 activity. Because DDM1 encodes a putative chromatin remodeling factor, chromatin structure is likely to be interrelated with genomic imprinting in Arabidopsis.