early maturity 7 promotes early flowering by controlling the light input into the circadian clock in barley.

Breeding for variation in photoperiod response is crucial to adapt crop plants to various environments. Plants measure changes in day length by the circadian clock, an endogenous timekeeper that allows plants to anticipate changes in diurnal and seasonal light-dark cycles. Here, we describe the early maturity 7 (eam7) locus in barley (Hordeum vulgare), which interacts with PHOTOPERIOD 1 (Ppd-H1) to cause early flowering under non-inductive short days. We identify LIGHT-REGULATED WD 1 (LWD1) as a putative candidate to underlie the eam7 locus in barley as supported by genetic mapping and CRISPR-Cas9-generated lwd1 mutants. Mutations in eam7 cause a significant phase advance and a misregulation of core clock and clock output genes under diurnal conditions. Early flowering was linked to an upregulation of Ppd-H1 during the night and consequent induction of the florigen FLOWERING LOCUS T1 under short days. We propose that EAM7 controls photoperiodic flowering in barley by controlling the light input into the clock and diurnal expression patterns of the major photoperiod response gene Ppd-H1.

Virus replication in the honey bee parasite, Varroa destructor.

IMPORTANCE: The parasitic mite Varroa destructor is a significant driver of worldwide colony losses of our most important commercial pollinator, the Western honey bee Apis mellifera. Declines in honey bee health are frequently attributed to the viruses that mites vector to honey bees, yet whether mites passively transmit viruses as a mechanical vector or actively participate in viral amplification and facilitate replication of honey bee viruses is debated. Our work investigating the antiviral RNA interference response in V. destructor demonstrates that key viruses associated with honey bee declines actively replicate in mites, indicating that they are biological vectors, and the host range of bee-associated viruses extends to their parasites, which could impact virus evolution, pathogenicity, and spread.

Intergenerational transfer of DNA methylation marks in the honey bee.

The evolutionary significance of epigenetic inheritance is controversial. While epigenetic marks such as DNA methylation can affect gene function and change in response to environmental conditions, their role as carriers of heritable information is often considered anecdotal. Indeed, near-complete DNA methylation reprogramming, as occurs during mammalian embryogenesis, is a major hindrance for the transmission of nongenetic information between generations. Yet it remains unclear how general DNA methylation reprogramming is across the tree of life. Here we investigate the existence of epigenetic inheritance in the honey bee. We studied whether fathers can transfer epigenetic information to their daughters through DNA methylation. We performed instrumental inseminations of queens, each with four different males, retaining half of each male's semen for whole genome bisulfite sequencing. We then compared the methylation profile of each father's somatic tissue and semen with the methylation profile of his daughters. We found that DNA methylation patterns were highly conserved between tissues and generations. There was a much greater similarity of methylomes within patrilines (i.e., father-daughter subfamilies) than between patrilines in each colony. Indeed, the samples' methylomes consistently clustered by patriline within colony. Samples from the same patriline had twice as many shared methylated sites and four times fewer differentially methylated regions compared to samples from different patrilines. Our findings indicate that there is no DNA methylation reprogramming in bees and, consequently, that DNA methylation marks are stably transferred between generations. This points to a greater evolutionary potential of the epigenome in invertebrates than there is in mammals.

Abundant small RNAs in the reproductive tissues and eggs of the honey bee, Apis mellifera.

BACKGROUND: Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. RESULTS: Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in the semen of other animals. Intriguingly we also find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. CONCLUSIONS: We conclude that small RNAs may play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of-origin effects on gene expression and reproductive physiology.

Adaptation to vector-based transmission in a honeybee virus.

Global pollinator declines as a result of emerging infectious diseases are of major concern. Managed honeybees Apis mellifera are susceptible to numerous parasites and pathogens, many of which appear to be transmissible to sympatric non-Apis taxa. The ectoparasitic mite Varroa destructor is considered to be the most significant threat to honeybees due to its role in vectoring RNA viruses, particularly Deformed wing virus (DWV). Vector transmission of DWV has resulted in the accumulation of high viral loads in honeybees and is often associated with colony death. DWV has two main genotypes, A and B. DWV-A was more prevalent during the initial phase of V. destructor establishment. In recent years, the global prevalence of DWV-B has increased, suggesting that DWV-B is better adapted to vector transmission than DWV-A. We aimed to determine the role vector transmission plays in DWV genotype prevalence at a colony level. We experimentally increased or decreased the number of V. destructor mites in honeybee colonies, and tracked DWV-A and DWV-B loads over a period of 10 months. Our results show that the two DWV genotypes differ in their response to mite numbers. DWV-A accumulation in honeybees was positively correlated with mite numbers yet DWV-A was largely undetected in the absence of the mite. In contrast, colonies had high loads of DWV-B even when mite numbers were low. DWV-B loads persisted in miticide-treated colonies, indicating that this genotype has a competitive advantage over DWV-A irrespective of mite numbers. Our findings suggest that the global increase in DWV-B prevalence is not driven by selective pressure by the vector. Rather, DWV-B is able to persist in colonies at higher viral loads relative to DWV-A in the presence and absence of V. destructor. The interplay between V. destructor and DWV genotypes within honeybee colonies may have broad consequences upon viral diversity in sympatric taxa as a result of spillover.

Paternally-biased gene expression follows kin-selected predictions in female honey bee embryos.

The Kinship Theory of Genomic Imprinting (KTGI) posits that, in species where females mate with multiple males, there is selection for a male to enhance the reproductive success of his offspring at the expense of other males and his mating partner. Reciprocal crosses between honey bee subspecies show parent-of-origin effects for reproductive traits, suggesting that males modify the expression of genes related to female function in their female offspring. This effect is likely to be greater in the Cape honey bee (Apis mellifera capensis), because a male's daughters have the unique ability to produce female offspring that can develop into reproductive workers or the next queen without mating. We generated reciprocal crosses between Capensis and another subspecies and used RNA-seq to identify transcripts that are over- or underexpressed in the embryos, depending on the parental origin of the gene. As predicted, 21 genes showed expression bias towards the Capensis father's allele in colonies with a Capensis father, with no such bias in the reciprocal cross. A further six genes showed a consistent bias towards expression of the father's allele across all eight colonies examined, regardless of the direction of the cross. Consistent with predictions of the KTGI, six of the 21 genes are associated with female reproduction. No gene consistently showed overexpression of the maternal allele.

Workers' sons rescue genetic diversity at the sex locus in an invasive honey bee population.

The hallmark of eusociality is the division of labour between reproductive (queen) and nonreproductive (worker) females. Yet in many eusocial insects, workers retain the ability to produce haploid male offspring from unfertilized eggs. The reproductive potential of workers has well-documented consequences for the structure and function of insect colonies, but its implications at the population level are less often considered. We show that worker reproduction in honey bees can have an important role in maintaining genetic diversity at the sex locus in invasive populations. The honey bee sex locus is homozygous-lethal, and, all else being equal, a higher allele number in the population lead to higher mean brood survival. In an invasive population of the honey bee Apis cerana in Australia, workers contribute significantly to male production: 38% of male-producing colonies are queenless, and these contribute one-third of all males at mating congregations. Using a model, we show that such male production by queenless workers will increase the number of sex alleles retained in nascent invasive populations following founder events, relative to a scenario in which only queens reproduce. We conclude that by rescuing sex locus diversity that would otherwise be lost, workers' sons help honey bee populations to minimize the negative effects of inbreeding after founder events and so contribute to their success as invaders.

Evolutionary divergence of the rye Pm17 and Pm8 resistance genes reveals ancient diversity.

KEY MESSAGE: We have isolated a novel powdery mildew resistance gene in wheat that was originally introgressed from rye. Further analysis revealed evolutionary divergent history of wheat and rye orthologous resistance genes. Wheat production is under constant threat from a number of fungal pathogens, among them is wheat powdery mildew (Blumeria graminis f. sp. tritici). Deployment of resistance genes is the most economical and sustainable method for mildew control. However, domestication and selective breeding have narrowed genetic diversity of modern wheat germplasm, and breeders have relied on wheat relatives for enriching its gene pool through introgression. Translocations where the 1RS chromosome arm was introgressed from rye to wheat have improved yield and resistance against various pathogens. Here, we isolated the Pm17 mildew resistance gene located on the 1RS introgression in wheat cultivar 'Amigo' and found that it is an allele or a close paralog of the Pm8 gene isolated earlier from 'Petkus' rye. Functional validation using transient and stable transformation confirmed the identity of Pm17. Analysis of Pm17 and Pm8 coding regions revealed an overall identity of 82.9% at the protein level, with the LRR domains being most divergent. Our analysis also showed that the two rye genes are much more diverse compared to the variants encoded by the Pm3 gene in wheat, which is orthologous to Pm17/Pm8 as concluded from highly conserved upstream sequences in all these genes. Thus, the evolutionary history of these orthologous loci differs in the cereal species rye and wheat and demonstrates that orthologous resistance genes can take different routes towards functionally active genes. These findings suggest that the isolation of Pm3/Pm8/Pm17 orthologs from other grass species, additional alleles from the rye germplasm as well as possibly synthetic variants will result in novel resistance genes useful in wheat breeding.

A Diverse Range of Novel RNA Viruses in Geographically Distinct Honey Bee Populations.

Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees (Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management.IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator.

The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley.

The wheat gene Lr34 encodes an ABCG-type transporter which provides durable resistance against multiple pathogens. Lr34 is functional as a transgene in barley, but its mode of action has remained largely unknown both in wheat and barley. Here we studied gene expression in uninfected barley lines transgenic for Lr34. Genes from multiple defense pathways contributing to basal and inducible disease resistance were constitutively active in seedlings and mature leaves. In addition, the hormones jasmonic acid and salicylic acid were induced to high levels, and increased levels of lignin as well as hordatines were observed. These results demonstrate a strong, constitutive re-programming of metabolism by Lr34. The resistant Lr34 allele (Lr34res) encodes a protein that differs by two amino acid polymorphisms from the susceptible Lr34sus allele. The deletion of a single phenylalanine residue in Lr34sus was sufficient to induce the characteristic Lr34-based responses. Combination of Lr34res and Lr34sus in the same plant resulted in a reduction of Lr34res expression by 8- to 20-fold when the low-expressing Lr34res line BG8 was used as a parent. Crosses with the high-expressing Lr34res line BG9 resulted in an increase of Lr34sus expression by 13- to 16-fold in progenies that inherited both alleles. These results indicate an interaction of the two Lr34 alleles on the transcriptional level. Reduction of Lr34res expression in BG8 crosses reduced the negative pleiotropic effects of Lr34res on barley growth and vigor without compromising disease resistance, suggesting that transgenic combination of Lr34res and Lr34sus can result in agronomically useful resistance.

Parent-of-origin effects on genome-wide DNA methylation in the Cape honey bee (Apis mellifera capensis) may be confounded by allele-specific methylation.

BACKGROUND: Intersexual genomic conflict sometimes leads to unequal expression of paternal and maternal alleles in offspring, resulting in parent-of-origin effects. In honey bees reciprocal crosses can show strong parent-of-origin effects, supporting theoretical predictions that genomic imprinting occurs in this species. Mechanisms behind imprinting in honey bees are unclear but differential DNA methylation in eggs and sperm suggests that DNA methylation could be involved. Nonetheless, because DNA methylation is multifunctional, it is difficult to separate imprinting from other roles of methylation. Here we use a novel approach to investigate parent-of-origin DNA methylation in honey bees. In the subspecies Apis mellifera capensis, reproduction of females occurs either sexually by fertilization of eggs with sperm, or via thelytokous parthenogenesis, producing female embryos derived from two maternal genomes. RESULTS: We compared genome-wide methylation patterns of sexually-produced, diploid embryos laid by a queen, with parthenogenetically-produced diploid embryos laid by her daughters. Thelytokous embryos inheriting two maternal genomes had fewer hypermethylated genes compared to fertilized embryos, supporting the prediction that fertilized embryos have increased methylation due to inheritance of a paternal genome. However, bisulfite PCR and sequencing of a differentially methylated gene, Stan (GB18207) showed strong allele-specific methylation that was maintained in both fertilized and thelytokous embryos. For this gene, methylation was associated with haplotype, not parent of origin. CONCLUSIONS: The results of our study are consistent with predictions from the kin theory of genomic imprinting. However, our demonstration of allele-specific methylation based on sequence shows that genome-wide differential methylation studies can potentially confound imprinting and allele-specific methylation. It further suggests that methylation patterns are heritable or that specific sequence motifs are targets for methylation in some genes.

Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants.

The development of high-yielding varieties with broad-spectrum durable disease resistance is the ultimate goal of crop breeding. In plants, immune receptors of the nucleotide-binding-leucine-rich repeat (NB-LRR) class mediate race-specific resistance against pathogen attack. When employed in agriculture this type of resistance is often rapidly overcome by newly adapted pathogen races. The stacking of different resistance genes or alleles in F1 hybrids or in pyramided lines is a promising strategy for achieving more durable resistance. Here, we identify a molecular mechanism which can negatively interfere with the allele-pyramiding approach. We show that pairwise combinations of different alleles of the powdery mildew resistance gene Pm3 in F1 hybrids and stacked transgenic wheat lines can result in suppression of Pm3-based resistance. This effect is independent of the genetic background and solely dependent on the Pm3 alleles. Suppression occurs at the post-translational level, as levels of RNA and protein in the suppressed alleles are unaffected. Using a transient expression system in Nicotiana benthamiana, the LRR domain was identified as the domain conferring suppression. The results of this study suggest that the expression of closely related NB-LRR resistance genes or alleles in the same genotype can lead to dominant-negative interactions. These findings provide a molecular explanation for the frequently observed ineffectiveness of resistance genes introduced from the secondary gene pool into polyploid crop species and mark an important step in overcoming this limitation.

Genetic and molecular characterization of a locus involved in avirulence of Blumeria graminis f. sp. tritici on wheat Pm3 resistance alleles.

Wheat powdery mildew is caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. The allelic series of the wheat Pm3 gene conferring race-specific resistance against powdery mildew has been well characterized functionally, and recently the corresponding avirulence gene AvrPm3a/f triggering the specific recognition by Pm3a and Pm3f alleles was cloned. Here, we describe the genetic and molecular analysis of two additional Blumeria loci involved in the resistance mediated by the Pm3c and Pm3f alleles. We genetically identified the two loci and mapped at high resolution one locus involved in the avirulence towards both Pm3c and Pm3f. The single candidate gene Bcg1 was identified in a physical target interval of 26kb defined by flanking genetic markers. Bcg1 encodes a small secreted protein sharing structural homology with ribonucleases and belongs to a family of clustered putative effector genes under diversifying selection. We found a very good, but not complete, correlation of Bcg1 haplotypes with the phenotypes of natural isolates. Two mutants were generated that were affected in their phenotypes towards Pm3a and Pm3f but did not show any sequence polymorphism in Bcg1. Our results suggest that avirulence to Pm3 in Blumeria is determined by a complex network of genes, in which Bcg1 might have a central role as a modifier of the Pm3/AvrPm3 interactions.

Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew.

The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology-based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single-cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3- and Pm8-like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co-evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.

Serial founder effects slow range expansion in an invasive social insect.

Invasive populations often experience founder effects: a loss of genetic diversity relative to the source population, due to a small number of founders. Even where these founder effects do not impact colonization success, theory predicts they might affect the rate at which invasive populations expand. This is because secondary founder effects are generated at advancing population edges, further reducing local genetic diversity and elevating genetic load. We show that in an expanding invasive population of the Asian honey bee (Apis cerana), genetic diversity is indeed lowest at range edges, including at the complementary sex determiner, csd, a locus that is homozygous-lethal. Consistent with lower local csd diversity, range edge colonies had lower brood viability than colonies in the range centre. Further, simulations of a newly-founded and expanding honey bee population corroborate the spatial patterns in mean colony fitness observed in our empirical data and show that such genetic load at range edges will slow the rate of population expansion.

Accumulation and Competition Amongst Deformed Wing Virus Genotypes in Naïve Australian Honeybees Provides Insight Into the Increasing Global Prevalence of Genotype B.

Honeybee colony deaths are often attributed to the ectoparasitic mite Varroa destructor and deformed wing virus (DWV), vectored by the mite. In the presence of V. destructor both main genotypes (DWV-A and DWV-B) have been correlated with colony loss. Studies show that DWV-B is the most prevalent genotype in the United Kingdom and Europe. More recently DWV-B has increased in prevalence in the United States. The increasing prevalence of DWV-B at the expense of DWV-A suggests that competition exists between the genotypes. Competition may be due to disparities in virulence between genotypes, differences in fitness, such as rate of replication, or a combination of factors. In this study we investigated if DWV genotypes differ in their rate of accumulation in Australian honeybees naïve to both V. destructor and DWV, and if viral load was associated with mortality in honeybee pupae. We singly and co-infected pupae with DWV-A, DWV-B, and a recombinant strain isolated from a V. destructor tolerant bee population. We monitored viral accumulation throughout pupation, up to 192 h post-injection. We found significant differences in accumulation, where DWV-A accumulated to significantly lower loads than DWV-B and the DWV-recombinant. We also found evidence of competition, where DWV-B loads were significantly reduced in the presence of DWV-A, but still accumulated to the highest loads overall. In contrast to previous studies, we found significant differences in virulence between pupae injected with DWV-A and DWV-B. The average mortality associated with DWV-B (0.4% ± 0.33 SE) and DWV-recombinant (2.2% ± 0.83 SE) injection were significantly less than observed for DWV-A (11% ± 1.2 SE). Our results suggest that a higher proportion of DWV-B infected pupae will emerge into adults, compared to DWV-A. Overall, our data suggest that low mortality in pupae and the ability of DWV-B to accumulate to higher loads relative to DWV-A even during co-infection may favor vector transmission by V. destructor, and may thus be contributing factors to the increasing prevalence of DWV-B globally.

A Single Gene Causes Thelytokous Parthenogenesis, the Defining Feature of the Cape Honeybee Apis mellifera capensis.

In honeybees, the ability of workers to produce daughters asexually, i.e., thelytokous parthenogenesis, is restricted to a single subspecies inhabiting the Cape region of South Africa, Apis mellifera capensis. Thelytoky has unleashed new selective pressures and the evolution of traits such as social parasitism, invasiveness, and social cancer. Thelytoky arises from an abnormal meiosis that results in the fusion of two maternal pronuclei, restoring diploidy in newly laid eggs. The genetic basis underlying thelytoky is disputed. To resolve this controversy, we generated a backcross between thelytokous A. m. capensis and non-thelytokous A. m. scutellata from the neighboring population and looked for evidence of genetic markers that co-segregated with thelytokous reproduction in 49 backcross females. We found that markers associated with the gene GB45239 on chromosome 11, including non-synonymous variants, showed consistent co-segregation with thelytoky, whereas no other region did so. Alleles associated with thelytoky were present in all A. m. capensis genomes examined but were absent from all other honeybees worldwide including A. m. scutellata. GB45239 is derived in A. m. capensis and has a putative role in chromosome segregation. It is expressed in ovaries and is downregulated in thelytokous bees, likely because of polymorphisms in the promoter region. Our study reveals how mutations affecting the sequence and/or expression of a single gene can change the reproductive mode of a population.

Cross-Kingdom RNAi of Pathogen Effectors Leads to Quantitative Adult Plant Resistance in Wheat.

Cross-kingdom RNA interference (RNAi) is a biological process allowing plants to transfer small regulatory RNAs to invading pathogens to trigger the silencing of target virulence genes. Transient assays in cereal powdery mildews suggest that silencing of one or two effectors could lead to near loss of virulence, but evidence from stable RNAi lines is lacking. We established transient host-induced gene silencing (HIGS) in wheat, and demonstrate that targeting an essential housekeeping gene in the wheat powdery mildew pathogen (Blumeria graminis f. sp. tritici) results in significant reduction of virulence at an early stage of infection. We generated stable transgenic RNAi wheat lines encoding a HIGS construct simultaneously silencing three B.g. tritici effectors including SvrPm3 a1/f1 , a virulence factor involved in the suppression of the Pm3 powdery mildew resistance gene. We show that all targeted effectors are effectively downregulated by HIGS, resulting in reduced fungal virulence on adult wheat plants. Our findings demonstrate that stable HIGS of effector genes can lead to quantitative gain of resistance without major pleiotropic effects in wheat.

Unique DNA Methylation Profiles Are Associated with cis-Variation in Honey Bees.

DNA methylation is an important epigenetic modification that mediates diverse processes such as cellular differentiation, phenotypic plasticity, and genomic imprinting. Mounting evidence suggests that local DNA sequence variation can be associated with particular DNA methylation states, indicating that the interplay between genetic and epigenetic factors may contribute synergistically to the phenotypic complexity of organisms. Social insects such as ants, bees, and wasps have extensive phenotypic plasticity manifested in their different castes, and this plasticity has been associated with variation in DNA methylation. Yet, the influence of genetic variation on DNA methylation state remains mostly unknown. Here we examine the importance of sequence-specific methylation at the genome-wide level, using whole-genome bisulfite sequencing of the semen of individual honey bee males. We find that individual males harbor unique DNA methylation patterns in their semen, and that genes that are more variable at the epigenetic level are also more likely to be variable at the genetic level. DNA sequence variation can affect DNA methylation by modifying CG sites directly, but can also be associated with local variation in cis that is not CG-site specific. We show that covariation in sequence polymorphism and DNA methylation state contributes to the individual-specificity of epigenetic marks in social insects, which likely promotes their retention across generations, and their capacity to influence evolutionary adaptation.