Historical Routes for Diversification of Domesticated Chickpea Inferred from Landrace Genomics.

According to archaeological records, chickpea (Cicer arietinum) was first domesticated in the Fertile Crescent about 10,000 years BP. Its subsequent diversification in Middle East, South Asia, Ethiopia, and the Western Mediterranean, however, remains obscure and cannot be resolved using only archeological and historical evidence. Moreover, chickpea has two market types: "desi" and "kabuli," for which the geographic origin is a matter of debate. To decipher chickpea history, we took the genetic data from 421 chickpea landraces unaffected by the green revolution and tested complex historical hypotheses of chickpea migration and admixture on two hierarchical spatial levels: within and between major regions of cultivation. For chickpea migration within regions, we developed popdisp, a Bayesian model of population dispersal from a regional representative center toward the sampling sites that considers geographical proximities between sites. This method confirmed that chickpea spreads within each geographical region along optimal geographical routes rather than by simple diffusion and estimated representative allele frequencies for each region. For chickpea migration between regions, we developed another model, migadmi, that takes allele frequencies of populations and evaluates multiple and nested admixture events. Applying this model to desi populations, we found both Indian and Middle Eastern traces in Ethiopian chickpea, suggesting the presence of a seaway from South Asia to Ethiopia. As for the origin of kabuli chickpeas, we found significant evidence for its origin from Turkey rather than Central Asia.

Sex-Biased Expression Is Associated With Chromatin State in Drosophila melanogaster and Drosophila simulans.

In Drosophila melanogaster and D. simulans head tissue, 60% of orthologous genes show evidence of sex-biased expression in at least one species. Of these, ∼39% (2,192) are conserved in direction. We hypothesize enrichment of open chromatin in the sex where we see expression bias and closed chromatin in the opposite sex. Male-biased orthologs are significantly enriched for H3K4me3 marks in males of both species (∼89% of male-biased orthologs vs. ∼76% of unbiased orthologs). Similarly, female-biased orthologs are significantly enriched for H3K4me3 marks in females of both species (∼90% of female-biased orthologs vs. ∼73% of unbiased orthologs). The sex-bias ratio in female-biased orthologs was similar in magnitude between the two species, regardless of the closed chromatin (H3K27me2me3) marks in males. However, in male-biased orthologs, the presence of H3K27me2me3 in both species significantly reduced the correlation between D. melanogaster sex-bias ratio and the D. simulans sex-bias ratio. Male-biased orthologs are enriched for evidence of positive selection in the D. melanogaster group. There are more male-biased genes than female-biased genes in both species. For orthologs with gains/losses of sex-bias between the two species, there is an excess of male-bias compared to female-bias, but there is no consistent pattern in the relationship between H3K4me3 or H3K27me2me3 chromatin marks and expression. These data suggest chromatin state is a component of the maintenance of sex-biased expression and divergence of sex-bias between species is reflected in the complexity of the chromatin status.

Demographic history and distinct selection signatures of two domestication genes in mungbean.

Domestication is the long and complex process underlying the evolution of crops, in which artificial directional selection transformed wild progenitors into the desired form, affecting genomic variation and leaving traces of selection at targeted loci. However, whether genes controlling important domestication traits follow the same evolutionary pattern expected under the standard selective sweep model remains unclear. With whole-genome resequencing of mungbean (Vigna radiata), we investigated this issue by resolving its global demographic history and targeted dissection of the molecular footprints of genes underlying 2 key traits representing different stages of domestication. Mungbean originated in Asia, and the Southeast Asian wild population migrated to Australia about 50 thousand generations ago. Later in Asia, the cultivated form diverged from the wild progenitor. We identified the gene associated with the pod shattering resistance trait, VrMYB26a, with lower expression across cultivars and reduced polymorphism in the promoter region, reflecting a hard selective sweep. On the other hand, the stem determinacy trait was associated with VrDet1. We found that 2 ancient haplotypes of this gene have lower gene expression and exhibited intermediate frequencies in cultivars, consistent with selection favoring independent haplotypes in a soft selective sweep. In mungbean, contrasting signatures of selection were identified from the detailed dissection of 2 important domestication traits. The results suggest complex genetic architecture underlying the seemingly simple process of directional artificial selection and highlight the limitations of genome-scan methods relying on hard selective sweeps.

A scaffolded and annotated reference genome of giant kelp (Macrocystis pyrifera).

Macrocystis pyrifera (giant kelp), is a brown macroalga of great ecological importance as a primary producer and structure-forming foundational species that provides habitat for hundreds of species. It has many commercial uses (e.g. source of alginate, fertilizer, cosmetics, feedstock). One of the limitations to exploiting giant kelp's economic potential and assisting in giant kelp conservation efforts is a lack of genomic tools like a high quality, contiguous reference genome with accurate gene annotations. Reference genomes attempt to capture the complete genomic sequence of an individual or species, and importantly provide a universal structure for comparison across a multitude of genetic experiments, both within and between species. We assembled the giant kelp genome of a haploid female gametophyte de novo using PacBio reads, then ordered contigs into chromosome level scaffolds using Hi-C. We found the giant kelp genome to be 537 MB, with a total of 35 scaffolds and 188 contigs. The assembly N50 is 13,669,674 with GC content of 50.37%. We assessed the genome completeness using BUSCO, and found giant kelp contained 94% of the BUSCO genes from the stramenopile clade. Annotation of the giant kelp genome revealed 25,919 genes. Additionally, we present genetic variation data based on 48 diploid giant kelp sporophytes from three different Southern California populations that confirms the population structure found in other studies of these populations. This work resulted in a high-quality giant kelp genome that greatly increases the genetic knowledge of this ecologically and economically vital species.

Macroalgal germplasm banking for conservation, food security, and industry.

Ex situ seed banking was first conceptualized and implemented in the early 20th century to maintain and protect crop lines. Today, ex situ seed banking is important for the preservation of heirloom strains, biodiversity conservation and ecosystem restoration, and diverse research applications. However, these efforts primarily target microalgae and terrestrial plants. Although some collections include macroalgae (i.e., seaweeds), they are relatively few and have yet to be connected via any international, coordinated initiative. In this piece, we provide a brief introduction to macroalgal germplasm banking and its application to conservation, industry, and mariculture. We argue that concerted effort should be made globally in germline preservation of marine algal species via germplasm banking with an overview of the technical advances for feasibility and ensured success.

Avalanches during epithelial tissue growth; Uniform Growth and a drosophila eye disc model.

Epithelial tissues constitute an exotic type of active matter with non-linear properties reminiscent of amorphous materials. In the context of a proliferating epithelium, modeled by the quasistatic vertex model, we identify novel discrete tissue scale rearrangements, i.e. cellular rearrangement avalanches, which are a form of collective cell movement. During the avalanches, the vast majority of cells retain their neighbors, and the resulting cellular trajectories are radial in the periphery, a vortex in the core. After the onset of these avalanches, the epithelial area grows discontinuously. The avalanches are found to be stochastic, and their strength is correlated with the density of cells in the tissue. Overall, avalanches redistribute accumulated local spatial pressure along the tissue. Furthermore, the distribution of avalanche magnitudes is found to obey a power law, with an exponent consistent with sheer induced avalanches in amorphous materials. To understand the role of avalanches in organ development, we simulate epithelial growth of the Drosophila eye disc during the third instar using a computational model, which includes both chemical and mechanistic signaling. During the third instar, the morphogenetic furrow (MF), a ~10 cell wide wave of apical area constriction propagates through the epithelium. These simulations are used to understand the details of the growth process, the effect of the MF on the growth dynamics on the tissue scale, and to make predictions for experimental observations. The avalanches are found to depend on the strength of the apical constriction of cells in the MF, with a stronger apical constriction leading to less frequent and more pronounced avalanches. The results herein highlight the dependence of simulated tissue growth dynamics on relaxation timescales, and serve as a guide for in vitro experiments.

Zeta diversity patterns in metabarcoded lotic algal assemblages as a tool for bioassessment.

Assessments of the ecological health of algal assemblages in streams typically focus on measures of their local diversity and classify individuals by morphotaxonomy. Such assemblages are often connected through various ecological processes, such as dispersal, and may be more accurately assessed as components of regional-, rather than local-scale assemblages. With recent declines in the costs of sequencing and computation, it has also become increasingly feasible to use metabarcoding to more accurately classify algal species and perform regional-scale bioassessments. Recently, zeta diversity has been explored as a novel method of constructing regional bioassessments for groups of streams. Here, we model the use of zeta diversity to investigate whether stream health can be determined by the landscape diversity of algal assemblages. We also compare the use of DNA metabarcoding and morphotaxonomy classifications in these zeta diversity-based bioassessments of regional stream health. From 96 stream samples in California, we used various orders of zeta diversity to construct models of biotic integrity for multiple assemblages of diatoms, as well as hybrid assemblages of diatoms in combination with soft-bodied algae, using taxonomy data generated with both DNA sequencing as well as traditional morphotaxonomic approaches. We compared our ability to evaluate the ecological health of streams with the performance of multiple algal indices of biological condition. Our zeta diversity-based models of regional biotic integrity were more strongly correlated with existing indices for algal assemblages classified using metabarcoding compared to morphotaxonomy. Metabarcoding for diatoms and hybrid algal assemblages involved rbcL and 18S V9 primers, respectively. Importantly, we also found that these algal assemblages, independent of the classification method, are more likely to be assembled under a process of niche differentiation rather than stochastically. Taken together, these results suggest the potential for zeta diversity patterns of algal assemblages classified using metabarcoding to inform stream bioassessments.

Natural variation of Macrocystis pyrifera gametophyte germplasm culture microbiomes and applications for improving yield in offshore farms.

With national interest in seaweed-based biofuels as a sustainable alternative to fossil fuels, there is a need for tools that produce high-yield seaweed cultivars and increase the efficiency of offshore farms. Several agricultural studies have demonstrated that the application of microbial inoculants at an early life stage can improve crop yield, and there is an opportunity to use similar techniques in seaweed aquaculture. However, there is a critical knowledge gap regarding host-microbiome associations of macroalgae gametophytes in germplasm cultures. Here, we investigate the microbial community of Macrocystis pyrifera gametophyte germplasm cultures that were used to cultivate an offshore farm in Santa Barbara, California and identify key taxa correlated with increased biomass of mature sporophytes. This work provides a valuable knowledge base for the development of microbial inoculants that produce high-biomass M. pyrifera cultivars to ultimately be used as biofuel feedstocks.

Transcriptomic Analysis of Flowering Time Genes in Cultivated Chickpea and Wild Cicer.

Chickpea (Cicer arietinum L.) is a major grain legume and a good source of plant-based protein. However, comprehensive knowledge of flowering time control in Cicer is lacking. In this study, we acquire high-throughput transcriptome sequencing data and analyze changes in gene expression during floral transition in the early flowering cultivar ICCV 96029, later flowering C. arietinum accessions, and two wild species, C. reticulatum and C. echinospermum. We identify Cicer orthologs of A. thaliana flowering time genes and analyze differential expression of 278 genes between four species/accessions, three tissue types, and two conditions. Our results show that the differences in gene expression between ICCV 96029 and other cultivated chickpea accessions are vernalization-dependent. In addition, we highlight the role of FTa3, an ortholog of FLOWERING LOCUS T in Arabidopsis, in the vernalization response of cultivated chickpea. A common set of differentially expressed genes was found for all comparisons between wild species and cultivars. The direction of expression change for different copies of the FT-INTERACTING PROTEIN 1 gene was variable in different comparisons, which suggests complex mechanisms of FT protein transport. Our study makes a contribution to the understanding of flowering time control in Cicer, and can provide genetic strategies to further improve this important agronomic trait.

Interplay between sex determination cascade and major signaling pathways during Drosophila eye development: Perspectives for future research.

Understanding molecular mechanisms of sexually dimorphic organ growth is a fundamental problem of developmental biology. Recent quantitative studies showed that the Drosophila compound eye is a convenient model to study the determination of the final organ size. In Drosophila, females have larger eyes than males and this is evident even after correction for the larger body size. Moreover, female eyes include more ommatidia (photosensitive units) than male eyes and this difference is specified at the third larval instar in the eye primordia called eye imaginal discs. This may result in different visual capabilities between the two sexes and have behavioral consequences. Despite growing evidence on the genetic bases of eye size variation between different Drosophila species and strains, mechanisms responsible for within-species sexual dimorphism still remain elusive. Here, we discuss a presumptive crosstalk between the sex determination cascade and major signaling pathways during dimorphic eye development. Male- and female-specific isoforms of Doublesex (Dsx) protein are known to control sex-specific differentiation in the somatic tissues. However, no data on Dsx function during eye disc growth and patterning are currently available. Remarkably, Sex lethal (Sxl), the sex determination switch protein, was shown to directly affect Hedgehog (Hh) and Notch (N) signaling in the Drosophila wing disc. The similarity of signaling pathways involved in the wing and eye disc growth suggests that Sxl might be integrated into regulation of eye development. Dsx role in the eye disc requires further investigation. We discuss currently available data on sex-biased gene expression in the Drosophila eye and highlight perspectives for future studies.

Genotyping and lipid profiling of 601 cultivated sunflower lines reveals novel genetic determinants of oil fatty acid content.

BACKGROUND: Sunflower is an important oilseed crop domesticated in North America approximately 4000 years ago. During the last century, oil content in sunflower was under strong selection. Further improvement of oil properties achieved by modulating its fatty acid composition is one of the main directions in modern oilseed crop breeding. RESULTS: We searched for the genetic basis of fatty acid content variation by genotyping 601 inbred sunflower lines and assessing their lipid and fatty acid composition. Our genome-wide association analysis based on the genotypes for 15,483 SNPs and the concentrations of 23 fatty acids, including minor fatty acids, revealed significant genetic associations for eleven of them. Identified genomic regions included the loci involved in rare fatty acids variation on chromosomes 3 and 14, explaining up to 34.5% of the total variation of docosanoic acid (22:0) in sunflower oil. CONCLUSIONS: This is the first large scale implementation of high-throughput lipidomic profiling to sunflower germplasm characterization. This study contributes to the genetic characterization of Russian sunflower collections, which made a substantial contribution to the development of sunflower as the oilseed crop worldwide, and provides new insights into the genetic control of oil composition that can be implemented in future studies.

The Evolution of Gene Expression in cis and trans.

There is abundant variation in gene expression between individuals, populations, and species. The evolution of gene regulation and expression within and between species is thought to frequently contribute to adaptation. Yet considerable evidence suggests that the primary evolutionary force acting on variation in gene expression is stabilizing selection. We review here the results of recent studies characterizing the evolution of gene expression occurring in cis (via linked polymorphisms) or in trans (through diffusible products of other genes) and their contribution to adaptation and response to the environment. We review the evidence for buffering of variation in gene expression at the level of both transcription and translation, and the possible mechanisms for this buffering. Lastly, we summarize unresolved questions about the evolution of gene regulation.

Quantitative analysis reveals genotype- and domain- specific differences between mRNA and protein expression of segmentation genes in Drosophila.

In many biological systems gene expression at mRNA and protein levels is not identical. Rigorous comparison of such differences on a spatio-temporal scale is still not feasible by high-throughput transcriptomic and proteomic analyses of early embryo development. Here, we characterize differences between mRNA and protein expression of Drosophila segmentation genes at the level of individual gene expression domains. We obtained quantitative imaging data on expression of gap genes gt and hb and pair-rule gene eve for Drosophila wild type embryos, Kr null mutants and Kr+/Kr- heterozygotes. To compare mRNA and protein expression we use several criteria including difference in amplitude and positions of expression domains, pattern shape and positional variability. For a number of gene expression domains we show examples where protein expression does not repeat mRNA expression even after a temporal delay. We calculated time delays between eve pattern formation at the level of mRNA and protein for wild type embryos, Kr mutants and Kr+/Kr- heterozygotes. We detect that in wild type embryos, the amplitudes of eve stripes 3 and 7 do not differ significantly at the level of mRNA, however, stripe 3 is higher than stripe 7 at the protein level. We further show that hb mRNA and protein expression in both anterior and posterior domains significantly differs at specific time points. The formation of hb PS4 stripe at the mRNA level proceeds five times faster than at the level of protein. With regard to spatial expression, we show that the offset between posterior gt mRNA and protein domains is much larger in Kr mutants than in wild type embryos and heterozygotes. Finally, we analyze differences in positional variability of eve stripe 7 expression in Kr mutants and Kr+/Kr- heterozygotes at the level of mRNA and protein. These results enable further perspectives to uncover mechanisms underlying discrepancies between mRNA and protein expression in early embryo.

Multi-trait multi-locus SEM model discriminates SNPs of different effects.

BACKGROUND: There is a plethora of methods for genome-wide association studies. However, only a few of them may be classified as multi-trait and multi-locus, i.e. consider the influence of multiple genetic variants to several correlated phenotypes. RESULTS: We propose a multi-trait multi-locus model which employs structural equation modeling (SEM) to describe complex associations between SNPs and traits - multi-trait multi-locus SEM (mtmlSEM). The structure of our model makes it possible to discriminate pleiotropic and single-trait SNPs of direct and indirect effect. We also propose an automatic procedure to construct the model using factor analysis and the maximum likelihood method. For estimating a large number of parameters in the model, we performed Bayesian inference and implemented Gibbs sampling. An important feature of the model is that it correctly copes with non-normally distributed variables, such as some traits and variants. CONCLUSIONS: We applied the model to Vavilov's collection of 404 chickpea (Cicer arietinum L.) accessions with 20-fold cross-validation. We analyzed 16 phenotypic traits which we organized into five groups and found around 230 SNPs associated with traits, 60 of which were of pleiotropic effect. The model demonstrated high accuracy in predicting trait values.

Dynamical climatic model for time to flowering in Vigna radiata.

BACKGROUND: Phenology data collected recently for about 300 accessions of Vigna radiata (mungbean) is an invaluable resource for investigation of impacts of climatic factors on plant development. RESULTS: We developed a new mathematical model that describes the dynamic control of time to flowering by daily values of maximal and minimal temperature, precipitation, day length and solar radiation. We obtained model parameters by adaptation to the available experimental data. The models were validated by cross-validation and used to demonstrate that the phenology of adaptive traits, like flowering time, is strongly predicted not only by local environmental factors but also by plant geographic origin and genotype. CONCLUSIONS: Of local environmental factors maximal temperature appeared to be the most critical factor determining how faithfully the model describes the data. The models were applied to forecast time to flowering of accessions grown in Taiwan in future years 2020-2030.

Genome-wide association study in accessions of the mini-core collection of mungbean (Vigna radiata) from the World Vegetable Gene Bank (Taiwan).

BACKGROUND: Mungbean (Vigna radiata (L.) R. Wilczek, or green gram) is important tropical and sub-tropical legume and a rich source of dietary protein and micronutrients. In this study we employ GWAS to examine the genetic basis of variation in several important traits in mungbean, using the mini-core collection established by the World Vegetable Center, which includes 296 accessions that represent the major market classes. This collection has been grown in a common field plot in southern European part of Russia in 2018. RESULTS: We used 5041 SNPs in 293 accessions that passed strict filtering for genetic diversity, linkage disequilibrium, population structure and GWAS analysis. Polymorphisms were distributed among all chromosomes, but with variable density. Linkage disequilibrium decayed in approximately 105 kb. Four distinct subgroups were identified within 293 accessions with 70% of accessions attributed to one of the four populations. By performing GWAS on the mini-core collection we have found several loci significantly associated with two important agronomical traits. Four SNPs associated with possibility of maturation in Kuban territory of Southern Russia in 2018 were identified within a region of strong linkage which contains genes encoding zinc finger A20 and an AN1 domain stress-associated protein. CONCLUSIONS: The core collection of mungbean established by the World Vegetable Center is a valuable resource for mungbean breeding. The collection has been grown in southern European part of Russia in 2018 under incidental stresses caused by abnormally hot weather and different photoperiod. We have found several loci significantly associated with color of hypocotyl and possibility of maturation under these stressful conditions. SNPs associated with possibility of maturation localize to a region on chromosome 2 with strong linkage, in which genes encoding zinc finger A20 and AN1 domain stress associated protein (SAP) are located. Phenotyping of WorldVeg collection for maturation traits in temperate climatic locations is important as phenology remains a critical breeding target for mungbean. As demand rises for mungbean, production in temperate regions with shorter growing seasons becomes crucial to keep up with needs. Uncovering SNPs for phenology traits will speed breeding efforts.

Genomic Analysis of Vavilov's Historic Chickpea Landraces Reveals Footprints of Environmental and Human Selection.

A defining challenge of the 21st century is meeting the nutritional demands of the growing human population, under a scenario of limited land and water resources and under the specter of climate change. The Vavilov seed bank contains numerous landraces collected nearly a hundred years ago, and thus may contain 'genetic gems' with the potential to enhance modern breeding efforts. Here, we analyze 407 landraces, sampled from major historic centers of chickpea cultivation and secondary diversification. Genome-Wide Association Studies (GWAS) conducted on both phenotypic traits and bioclimatic variables at landraces sampling sites as extended phenotypes resulted in 84 GWAS hits associated to various regions. The novel haploblock-based test identified haploblocks enriched for single nucleotide polymorphisms (SNPs) associated with phenotypes and bioclimatic variables. Subsequent bi-clustering of traits sharing enriched haploblocks underscored both non-random distribution of SNPs among several haploblocks and their association with multiple traits. We hypothesize that these clusters of pleiotropic SNPs represent co-adapted genetic complexes to a range of environmental conditions that chickpea experienced during domestication and subsequent geographic radiation. Linking genetic variation to phenotypic data and a wealth of historic information preserved in historic seed banks are the keys for genome-based and environment-informed breeding intensification.

Bayesian model selection for the Drosophila gap gene network.

BACKGROUND: The gap gene system controls the early cascade of the segmentation pathway in Drosophila melanogaster as well as other insects. Owing to its tractability and key role in embryo patterning, this system has been the focus for both computational modelers and experimentalists. The gap gene expression dynamics can be considered strictly as a one-dimensional process and modeled as a system of reaction-diffusion equations. While substantial progress has been made in modeling this phenomenon, there still remains a deficit of approaches to evaluate competing hypotheses. Most of the model development has happened in isolation and there has been little attempt to compare candidate models. RESULTS: The Bayesian framework offers a means of doing formal model evaluation. Here, we demonstrate how this framework can be used to compare different models of gene expression. We focus on the Papatsenko-Levine formalism, which exploits a fractional occupancy based approach to incorporate activation of the gap genes by the maternal genes and cross-regulation by the gap genes themselves. The Bayesian approach provides insight about relationship between system parameters. In the regulatory pathway of segmentation, the parameters for number of binding sites and binding affinity have a negative correlation. The model selection analysis supports a stronger binding affinity for Bicoid compared to other regulatory edges, as shown by a larger posterior mean. The procedure doesn't show support for activation of Kruppel by Bicoid. CONCLUSIONS: We provide an efficient solver for the general representation of the Papatsenko-Levine model. We also demonstrate the utility of Bayes factor for evaluating candidate models for spatial pattering models. In addition, by using the parallel tempering sampler, the convergence of Markov chains can be remarkably improved and robust estimates of Bayes factors obtained.

Analysis of Genetic Variation Indicates DNA Shape Involvement in Purifying Selection.

Noncoding DNA sequences, which play various roles in gene expression and regulation, are under evolutionary pressure. Gene regulation requires specific protein-DNA binding events, and our previous studies showed that both DNA sequence and shape readout are employed by transcription factors (TFs) to achieve DNA binding specificity. By investigating the shape-disrupting properties of single nucleotide polymorphisms (SNPs) in human regulatory regions, we established a link between disruptive local DNA shape changes and loss of specific TF binding. Furthermore, we described cases where disease-associated SNPs may alter TF binding through DNA shape changes. This link led us to hypothesize that local DNA shape within and around TF binding sites is under selection pressure. To verify this hypothesis, we analyzed SNP data derived from 216 natural strains of Drosophila melanogaster. Comparing SNPs located in functional and nonfunctional regions within experimentally validated cis-regulatory modules (CRMs) from D. melanogaster that are active in the blastoderm stage of development, we found that SNPs within functional regions tended to cause smaller DNA shape variations. Furthermore, SNPs with higher minor allele frequency were more likely to result in smaller DNA shape variations. The same analysis based on a large number of SNPs in putative CRMs of the D. melanogaster genome derived from DNase I accessibility data confirmed these observations. Taken together, our results indicate that common SNPs in functional regions tend to maintain DNA shape, whereas shape-disrupting SNPs are more likely to be eliminated through purifying selection.

Novel approach to quantitative spatial gene expression uncovers genetic stochasticity in the developing Drosophila eye.

Robustness in development allows for the accumulation of genetically based variation in expression. However, this variation is usually examined in response to large perturbations, and examination of this variation has been limited to being spatial, or quantitative, but because of technical restrictions not both. Here we bridge these gaps by investigating replicated quantitative spatial gene expression using rigorous statistical models, in different genotypes, sexes, and species (Drosophila melanogaster and D. simulans). Using this type of quantitative approach with molecular developmental data allows for comparison among conditions, such as different genetic backgrounds. We apply this approach to the morphogenetic furrow, a wave of differentiation that patterns the developing eye disc. Within the morphogenetic furrow, we focus on four genes, hairy, atonal, hedgehog, and Delta. Hybridization chain reaction quantitatively measures spatial gene expression, co-staining for all four genes simultaneously. We find considerable variation in the spatial expression pattern of these genes in the eye between species, genotypes, and sexes. We also find that there has been evolution of the regulatory relationship between these genes, and that their spatial interrelationships have evolved between species. This variation has no phenotypic effect, and could be buffered by network thresholds or compensation from other genes. Both of these mechanisms could potentially be contributing to long term developmental systems drift.