Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes

• Physcomitrella patens is a monoecious moss that is predominantly selfing in the wild. Laboratory crossing techniques have been established and crosses between the sequenced Gransden ecotype and the genetically divergent Villersexel ecotype were used for genetic mapping. The recently introduced ecotype Reute has a high fertility rate and is genetically more closely related to the Gransden ecotype than the Villersexel ecotype. Reute sexual reproduction phenology is similar to Gransden, which should allow successful crossing.
• Using the Reute ecotype and an existing Gransden mutant as a test case, we applied a normalised crossing approach to demonstrate crossing potential between these ecotypes. Also, using a standard transformation approach, we generated Reute fluorescent strains expressing mCherry that allow an easy detection of crossed offspring (sporophyte).
• We show that Reute can be successfully crossed with a self‐infertile DR5:DsRed2 mutant generated in the Gransden background. Using newly established Reute fluorescent strains, we show that they can efficiently fertilise Reute as well as Gransden wild type. The resulting progeny display Mendelian 1:1 segregation of the fluorescent marker(s), demonstrating the suitability of such strains for genetic crossing.
• Overall our results demonstrate that Reute is highly suitable for genetic crossing. The Reute mCherry strain can be used as a suitable background for offspring selection after crossing.

     

Diversity and population structure of northern switchgrass as revealed through exome capture sequencing

Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high‐coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single‐nucleotide polymorphisms (SNPs), of which 1 590 653 high‐confidence SNPs were used in downstream analyses of diversity within and between the populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype‐restricted, non‐synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near‐reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up‐CNVs), 112 630 genes present in fewer copies than in the reference genome (down‐CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland‐specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.     

Alternation of generations – unravelling the underlying molecular mechanism of a 165‐year‐old botanical observation

Characteristically, land plants exhibit a life cycle with an ‘alternation of generations’ and thus alternate between a haploid gametophyte and a diploid sporophyte. At meiosis and fertilisation the transitions between these two ontogenies take place in distinct single stem cells. The evolutionary invention of an embryo, and thus an upright multicellular sporophyte, in the ancestor of land plants formed the basis for the evolution of increasingly complex plant morphologies shaping Earth's ecosystems. Recent research employing the moss Physcomitrella patens revealed the homeotic gene BELL1 as a master regulator of the gametophyte‐to‐sporophyte transition. Here, we discuss these findings in the context of classical botanical observations.     

Biological implications of the occurrence of 32 members of the XTH (xyloglucan endotransglucosylase/hydrolase) family of proteins in the bryophyte Physcomitrella patens

This comprehensive overview of the xyloglucan endotransglucosylase/hydrolase (XTH) family of genes and proteins in bryophytes, based on research using genomic resources that are newly available for the moss Physcomitrella patens, provides new insights into plant evolution. In angiosperms, the XTH genes are found in large multi‐gene families, probably reflecting the diverse roles of individual XTHs in various cell types. As there are fewer cell types in P. patens than in angiosperms such as Arabidopsis and rice, it is tempting to deduce that there are fewer XTH family genes in bryophytes. However, the present study unexpectedly identified as many as 32 genes that potentially encode XTH family proteins in the genome of P. patens, constituting a fairly large multi‐gene family that is comparable in size with those of Arabidopsis and rice. In situ localization of xyloglucan endotransglucosylase activity in this moss indicates that some P. patens XTH proteins exhibit biochemical functions similar to those found in angiosperms, and that their expression profiles are tissue‐dependent. However, comparison of structural features of families of XTH genes between P. patens and angiosperms demonstrated the existence of several bryophyte‐specific XTH genes with distinct structural and functional features that are not found in angiosperms. These bryophyte‐specific XTH genes might have evolved to meet morphological and functional needs specific to the bryophyte. These findings raise interesting questions about the biological implications of the XTH family of proteins in non‐seed plants.     

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio‐lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1‐1 and 1‐2). Both PpAN1 genes complemented the A. thaliana an‐1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1‐promoter– uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1‐1 and PpAN1‐2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip‐growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α‐tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1‐1/1‐2 double‐knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.     

Hybridization,polyploidy and clonality influence geographic patterns of diversity and salt tolerance in the model halophyte seashore paspalum (Paspalum vaginatum)

In plant species, variation in levels of clonality, ploidy and interspecific hybridization can interact to influence geographic patterns of genetic diversity. These factors commonly vary in plants that specialize on saline habitats (halophytes) and may play a role in how they adapt to salinity variation across their range. One such halophyte is the turfgrass and emerging genomic model system seashore paspalum (Paspalum vaginatum Swartz). To investigate how clonal propagation, ploidy variation, and interspecific hybridization vary across ecotypes and local salinity levels in wild P. vaginatum, we employed genotyping‐by‐sequencing, cpDNA sequencing and flow cytometry in 218 accessions representing > 170 wild collections from throughout the coastal southern United States plus USDA germplasm. We found that the two morphologically distinct ecotypes of P. vaginatum differ in their adaptive strategies. The fine‐textured ecotype is diploid and appears to reproduce in the wild both sexually and by clonal propagation; in contrast, the coarse‐textured ecotype consists largely of clonally‐propagating triploid and diploid genotypes. The coarse‐textured ecotype appears to be derived from hybridization between fine‐textured P. vaginatum and an unidentified Paspalum species. These clonally propagating hybrid genotypes are more broadly distributed than clonal fine‐textured genotypes and may represent a transition to a more generalist adaptive strategy. Additionally, the triploid genotypes vary in whether they carry one or two copies of the P. vaginatum subgenome, indicating multiple evolutionary origins. This variation in subgenome composition shows associations with local ocean salinity levels across the sampled populations and may play a role in local adaptation.     

Interplay of robustness and plasticity of life‐history traits drives ecotypic differentiation in thermally distinct habitats

Phenotypic plasticity describes the ability of an individual to alter its phenotype in response to the environment and is potentially adaptive when dealing with environmental variation. However, robustness in the face of a changing environment may often be beneficial for traits that are tightly linked to fitness. We hypothesized that robustness of some traits may depend on specific patterns of plasticity within and among other traits. We used a reaction norm approach to study robustness and phenotypic plasticity of three life‐history traits of the collembolan Orchesella cincta in environments with different thermal regimes. We measured adult mass, age at maturity and growth rate of males and females from heath and forest habitats at two temperatures (12 and 22 °C). We found evidence for ecotype‐specific robustness of female adult mass to temperature, with a higher level of robustness in the heath ecotype. This robustness is facilitated by plastic adjustments of growth rate and age at maturity. Furthermore, female fecundity is strongly influenced by female adult mass, explaining the importance of realizing a high mass across temperatures for females. These findings indicate that different predicted outcomes of life‐history theory can be combined within one species' ontogeny and that models describing life‐history strategies should not assume that traits like growth rate are maximized under all conditions. On a methodological note, we report a systematic inflation of variation when standard deviations and correlation coefficients are calculated from family means as opposed to individual data within a family structure.     

Size selection by a gape‐limited predator of a marine snail: Insights into magic traits for speciation

The intertidal snail Littorina saxatilis has repeatedly evolved two parallel ecotypes assumed to be wave adapted and predatory shore crab adapted, but the magnitude and targets of predator‐driven selection are unknown. In Spain, a small, wave ecotype with a large aperture from the lower shore and a large, thick‐shelled crab ecotype from the upper shore meet in the mid‐shore and show partial size‐assortative mating. We performed complementary field tethering and laboratory predation experiments; the first set compared the survival of two different size‐classes of the crab ecotype while the second compared the same size‐class of the two ecotypes. In the first set, the large size‐class of the crab ecotype survived significantly better than the small size‐class both on the upper shore and in the laboratory. In the second set, the small size‐class of the crab ecotype survived substantially better than that of the wave ecotype both on the upper shore and in the laboratory. Shell‐breaking predation on tethered snails was almost absent within the lower shore. In the laboratory shore crabs (Pachygrapsus marmoratus) with larger claw heights selected most strongly against the small size‐class of the crab ecotype, whereas those with medium claw heights selected most strongly against the thin‐shelled wave ecotype. Sexual maturity occurred at a much larger size in the crab ecotype than in the wave ecotype. Our results showed that selection on the upper shore for rapid attainment of a size refuge from this gape‐limited predator favors large size, thick shells, and late maturity. Model parameterization showed that size‐selective predation restricted to the upper shore resulted in the evolution of the crab ecotype despite gene flow from the wave ecotype snails living on the lower shore. These results on gape‐limited predation and previous ones showing size‐assortative mating between ecotypes suggest that size may represent a magic trait for the thick‐shelled ecotype.     

Genetic divergence and phenotypic plasticity contribute to variation in cuticular hydrocarbons in the seaweed fly Coelopa frigida

Cuticular hydrocarbons (CHCs) form the boundary between insects and their environments and often act as essential cues for species, mate, and kin recognition. This complex polygenic trait can be highly variable both among and within species, but the causes of this variation, especially the genetic basis, are largely unknown. In this study, we investigated phenotypic and genetic variation of CHCs in the seaweed fly, Coelopa frigida, and found that composition was affected by both genetic (sex and population) and environmental (larval diet) factors. We subsequently conducted behavioral trials that show CHCs are likely used as a sexual signal. We identified general shifts in CHC chemistry as well as individual compounds and found that the methylated compounds, mean chain length, proportion of alkenes, and normalized total CHCs differed between sexes and populations. We combined these data with whole genome resequencing data to examine the genetic underpinnings of these differences. We identified 11 genes related to CHC synthesis and found population‐level outlier SNPs in 5 that are concordant with phenotypic differences. Together these results reveal that the CHC composition of C. frigida is dynamic, strongly affected by the larval environment, and likely under natural and sexual selection.     

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families.     

Host‐associated genomic differentiation in congeneric butterflies: now you see it,now you do not

Ecotypic variation among populations may become associated with widespread genomic differentiation, but theory predicts that this should happen only under particular conditions of gene flow, selection and population size. In closely related species, we might expect the strength of host‐associated genomic differentiation (HAD) to be correlated with the degree of phenotypic differentiation in host‐adaptive traits. Using microsatellite and Amplified Fragment Length Polymorphism (AFLP) markers, and controlling for isolation by distance between populations, we sought HAD in two congeneric species of butterflies with different degrees of host plant specialization. Prior work on Euphydryas editha had shown strong interpopulation differentiation in host‐adapted traits, resulting in incipient reproductive isolation among host‐associated ecotypes. We show here that Euphydryas aurinia had much weaker host‐associated phenotypic differentiation. Contrary to our expectations, we detected HAD in Euphydryas aurinia, but not in E. editha. Even within an E. aurinia population that fed on both hosts, we found weak but significant sympatric HAD that persisted in samples taken 9 years apart. The finding of significantly stronger HAD in the system with less phenotypic differentiation may seem paradoxical. Our findings can be explained by multiple factors, ranging from differences in dispersal or effective population size, to spatial variation in genomic or phenotypic traits and to structure induced by past histories of host‐adapted populations. Other infrequently measured factors, such as differences in recombination rates, may also play a role. Our result adds to recent work as a further caution against assumptions of simple relationships between genomic and adaptive phenotypic differentiation.     

Local adaptation,genetic divergence,and experimental selection in a foundation grass across the US Great Plains’ climate gradient

Many prior studies have uncovered evidence for local adaptation using reciprocal transplant experiments. However, these studies are rarely conducted for a long enough time to observe succession and competitive dynamics in a community context, limiting inferences for long‐lived species. Furthermore, the genetic basis of local adaptation and genetic associations with climate has rarely been identified. Here, we report on a long‐term (6‐year) experiment conducted under natural conditions focused on Andropogon gerardii, the dominant grass of the North American Great Plains tallgrass ecosystem. We focus on this foundation grass that comprises 80% of tallgrass prairie biomass and is widely used in 20,000 km² of restoration. Specifically, we asked the following questions: (a) Whether ecotypes are locally adapted to regional climate in realistic ecological communities. (b) Does adaptive genetic variation underpin divergent phenotypes across the climate gradient? (c) Is there evidence of local adaptation if the plants are exposed to competition among ecotypes in mixed ecotype plots? Finally, (d) are local adaptation and genetic divergence related to climate? Reciprocal gardens were planted with 3 regional ecotypes (originating from dry, mesic, wet climate sources) of Andropogon gerardii across a precipitation gradient (500–1,200 mm/year) in the US Great Plains. We demonstrate local adaptation and differentiation of ecotypes in wet and dry environments. Surprisingly, the apparent generalist mesic ecotype performed comparably under all rainfall conditions. Ecotype performance was underpinned by differences in neutral diversity and candidate genes corroborating strong differences among ecotypes. Ecotype differentiation was related to climate, primarily rainfall. Without long‐term studies, wrong conclusions would have been reached based on the first two years. Further, restoring prairies with climate‐matched ecotypes is critical to future ecology, conservation, and sustainability under climate change.     

Ecotype differences in aggression,neural activity and behaviorally relevant gene expression in cichlid fish

In Lake Malawi, two ecologically distinct lineages of cichlid fishes (rock‐ vs sand‐dwelling ecotypes, each comprised of over 200 species) evolved within the last million years. The rock‐dwelling species (Mbuna) are aggressively territorial year‐round and males court and spawn with females over rocky substrate. In contrast, males of sand‐dwelling species are not territorial and instead aggregate on seasonal breeding leks in which males construct courtship “bowers” in the sand. However, little is known about how phenotypic variation in aggression is produced by the genome. In this study, we first quantify and compare behavior in seven cichlid species, demonstrating substantial ecotype and species differences in unconditioned mirror‐elicited aggression. Second, we compare neural activity in mirror‐elicited aggression in two representative species, Mchenga conophoros (sand‐dwelling) and Petrotilapia chitimba (rock‐dwelling). Finally, we compare gene expression patterns between these two species, specifically within neurons activated during mirror aggression. We identified a large number of genes showing differential expression in mirror‐elicited aggression, as well as many genes that differ between ecotypes. These genes, which may underly species differences in behavior, include several neuropeptides, genes involved in the synthesis of steroid hormones and neurotransmitter activity. This work lays the foundation for future experiments using this emerging genetic model system to investigate the genomic basis of evolved species differences in both brain and behavior.     

Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase‐like (CUS) proteins that are conserved among land plants

The aerial epidermis of all land plants is covered with a hydrophobic cuticle that provides essential protection from desiccation, and so its evolution is believed to have been prerequisite for terrestrial colonization. A major structural component of apparently all plant cuticles is cutin, a polyester of hydroxy fatty acids; however, despite its ubiquity, the details of cutin polymeric structure and the mechanisms of its formation and remodeling are not well understood. We recently reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL‐motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Here, we present additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, which represent a distinct clade of cutin synthases within the large GDSL superfamily. We demonstrate that members of this ancient and conserved family of cutin synthase‐like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution‐state NMR analysis indicates that CD1 catalyzes the formation of primarily linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross‐linking, may require additional, as yet unknown, factors.     

A collection of enhancer trap insertional mutants for functional genomics in tomato

With the completion of genome sequencing projects, the next challenge is to close the gap between gene annotation and gene functional assignment. Genomic tools to identify gene functions are based on the analysis of phenotypic variations between a wild type and its mutant; hence, mutant collections are a valuable resource. In this sense, T‐DNA collections allow for an easy and straightforward identification of the tagged gene, serving as the basis of both forward and reverse genetic strategies. This study reports on the phenotypic and molecular characterization of an enhancer trap T‐DNA collection in tomato (Solanum lycopersicum L.), which has been produced by Agrobacterium‐mediated transformation using a binary vector bearing a minimal promoter fused to the uidA reporter gene. Two genes have been isolated from different T‐DNA mutants, one of these genes codes for a UTP‐glucose‐1‐phosphate uridylyltransferase involved in programmed cell death and leaf development, which means a novel gene function reported in tomato. Together, our results support that enhancer trapping is a powerful tool to identify novel genes and regulatory elements in tomato and that this T‐DNA mutant collection represents a highly valuable resource for functional analyses in this fleshy‐fruited model species.     

Arabidopsis thaliana IRX10 and two related proteins from psyllium and Physcomitrella patens are xylan xylosyltransferases

The enzymatic mechanism that governs the synthesis of the xylan backbone polymer, a linear chain of xylose residues connected by β‐1,4 glycosidic linkages, has remained elusive. Xylan is a major constituent of many kinds of plant cell walls, and genetic studies have identified multiple genes that affect xylan formation. In this study, we investigate several homologs of one of these previously identified xylan‐related genes, IRX10 from Arabidopsis thaliana, by heterologous expression and in vitro xylan xylosyltransferase assay. We find that an IRX10 homolog from the moss Physcomitrella patens displays robust activity, and we show that the xylosidic linkage formed is a β‐1,4 linkage, establishing this protein as a xylan β‐1,4‐xylosyltransferase. We also find lower but reproducible xylan xylosyltransferase activity with A. thaliana IRX10 and with a homolog from the dicot plant Plantago ovata, showing that xylan xylosyltransferase activity is conserved over large evolutionary distance for these proteins.