Misregulation of Genomic Imprinting Drives Abnormal Seed Development in Hybrid Monkeyflowers (Mimulus)

Genomic imprinting is the preferential expression of one allele over the other. It is an epigenetic phenomenon that occurs in the placentas of mammals and the endosperm of angiosperms. Endosperm, like placentas, is a nutrient rich tissue that supports the growing embryo within the seed. All grains are predominantly composed of this tissue. It is the product of a second fertilization event, resulting in both maternal and paternal alleles. Some alleles are regulated differentially, resulting in imprinted genes. There are both paternally expressed imprinted genes (PEGs) and maternally expressed imprinted genes (MEGs) in the endosperm. In general PEGs tend to have functions that induce the proliferation of endosperm (and the placenta in mammals) and MEGs tend to regulate or limit proliferation. There are many theories on the evolution of imprinting and parent-specific functions in such diverged taxa. Interploidy hybridization systems are often used to study these parent-specific effects. Such systems occur when a diploid is crossed with a polyploid, typically a tetraploid. By switching the parentage, parent specific genome dosage can be altered: if the tetraploid is the mother, then the offspring, or endosperm, has maternal genomic excess, and if the tetraploid is the father, then the endosperm has paternal excess. Maternal excess is typically characterized by endosperm underproliferation and paternal excess is characterized by endosperm overproliferation, as predicted by MEG and PEG functions. While significant progress has been made in genomic imprinting, there is still much unknown. For example, in plants, maternal excess is predicted to be more stable for evolutionary and functional reasons, yet there are many cases where the opposite occurs. By using a system in Mimulus that is both interploidy and interspecies (M. guttatus is diploid and M. luteus is tetraploid) and where paternal excess is favored in offspring viability, we aim to uncover further clues behind the mechanisms and evolutionary drivers of genomic imprinting. Here we show that the paternal excess hybrid suffers from endosperm underproliferation, opposite of what is predicted, and the maternal excess hybrid suffers from complete endosperm and embryo failure. We show that smaller endosperm results in failed or delayed germination. Furthermore, using genomic techniques, we show that M. luteus is genomically dominant in the hybrids regardless of crossing direction, likely interfering with imprinting patterns. We identify new PEGs involved in cellular proliferation. We show an overall paternal bias in M. luteus, which is unexpected and uncommon – potentially suggesting other adaptive drivers in imprinting. We suggest that abnormalities in the hybrids may be due to this genomic dominance and potentially other genetic and developmental differences between the two species that interferes with MEG and PEG roles

A Likelihood Ratio Test for Changes in Homeolog Expression Bias

Background Gene duplications are a major source of raw material for evolution and a likely contributor to the diversity of life on earth. Duplicate genes (i.e., homeologs, in the case of a whole genome duplication) may retain their ancestral function, sub- or neofunctionalize, or be lost entirely. A primary way that duplicate genes evolve new functions is by altering their expression patterns. Comparing the expression patterns of duplicate genes gives clues as to whether any of these evolutionary processes have occurred. Results We develop a likelihood ratio test for the analysis of the expression ratios of duplicate genes across two conditions (e.g., tissues). We demonstrate an application of this test by comparing homeolog expression patterns of 1448 homeologous gene pairs using RNA-seq data generated from leaves and petals of an allotetraploid monkeyflower (Mimulus luteus). We assess the sensitivity of this test to different levels of homeolog expression bias and compare the method to several alternatives. Conclusions The likelihood ratio test derived here is a direct, transparent, and easily implemented method for detecting changes in homeolog expression bias that outperforms alternative approaches. While our method was derived with homeolog analysis in mind, this method can be used to analyze changes in the ratio of expression levels between any two genes in any two conditions

A Whole-Transcriptome Approach to Evaluating Reference Genes for Quantitative Gene Expression Studies: A Case Study in Mimulus

While quantitative PCR (qPCR) is widely recognized as being among the most accurate methods for quantifying gene expression, it is highly dependent on the use of reliable, stably expressed reference genes. With the increased availability of high-throughput methods for measuring gene expression, whole-transcriptome approaches may be increasingly utilized for reference gene selection and validation. In this study, RNA-seq was used to identify a set of novel qPCR reference genes and evaluate a panel of traditional housekeeping reference genes in two species of the evolutionary model plant genus Mimulus. More broadly, the methods proposed in this study can be used to harness the power of transcriptomes to identify appropriate reference genes for qPCR in any study organism, including emerging and nonmodel systems. We find that RNA-seq accurately estimates gene expression means in comparison to qPCR, and that expression means are robust to moderate environmental and genetic variation. However, measures of expression variability were only in agreement with qPCR for samples obtained from a shared environment. This result, along with transcriptome-wide comparisons, suggests that environmental changes have greater impacts on expression variability than on expression means. We discuss how this issue can be addressed through experimental design, and suggest that the ever-expanding pool of published transcriptomes represents a rich and low-cost resource for developing better reference genes for qPCR

Ml_T1_RPKM

M. l. luteus inbred line EY7 - transcriptome T1 - RPKM expression report plus raw count

Endosperm-based incompatibilities in hybrid monkeyflowers

Endosperm is an angiosperm innovation central to their reproduction whose development, and thus seed viability, is controlled by genomic imprinting, where expression from certain genes is parent-specific. Unsuccessful imprinting has been linked to failed inter-specific and inter-ploidy hybridization. Despite their importance in plant speciation, the underlying mechanisms behind these endosperm-based barriers remain poorly understood. Here, we describe one such barrier between diploid Mimulus guttatus and tetraploid M. luteus. The two parents differ in endosperm DNA methylation, expression dynamics, and imprinted genes. Hybrid seeds suffer from underdeveloped endosperm, reducing viability, or arrested endosperm and seed abortion when M. guttatus or M. luteus is seed parent, respectively, and transgressive methylation and expression patterns emerge. The two inherited M. luteus subgenomes, genetically distinct but epigenetically similar, are expressionally dominant over the M. guttatus genome in hybrid embryos and especially their endosperm, where paternal imprints are perturbed. In aborted seeds, de novo methylation is inhibited, potentially owing to incompatible paternal instructions of imbalanced dosage from M. guttatus imprints. We suggest that diverged epigenetic/regulatory landscapes between parental genomes induce epigenetic repatterning and global shifts in expression, which, in endosperm, may uniquely facilitate incompatible interactions between divergent imprinting schemes, potentially driving rapid barriers

Mg_T1_RPKM

M. guttatus inbred line CG - transcriptome T1 - RPKM expression report plus raw count

Mg_T1

Mimulus guttatus inbred line CG - transcriptome T1 - complete annotated transcriptom

Mg_T1

Mimulus guttatus inbred line CG - transcriptome T1 - complete annotated transcriptom

Ml_T1_RPKM

M. l. luteus inbred line EY7 - transcriptome T1 - RPKM expression report plus raw count