Isoform-resolved genome annotation enables mapping of tissue-specific betalain regulation in amaranth.

Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue-specific regulation of the pathway remains mostly unknown. We enhance the high-quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue-specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock-out of red betacyanins in natural accessions of amaranth. We reveal the tissue-specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2. Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co-option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments.

Genetic Incompatibilities and Evolutionary Rescue by Wild Relatives Shaped Grain Amaranth Domestication.

Crop domestication and the subsequent expansion of crops have long been thought of as a linear process from a wild ancestor to a domesticate. However, evidence of gene flow from locally adapted wild relatives that provided adaptive alleles into crops has been identified in multiple species. Yet, little is known about the evolutionary consequences of gene flow during domestication and the interaction of gene flow and genetic load in crop populations. We study the pseudo-cereal grain amaranth that has been domesticated three times in different geographic regions of the Americas. We quantify the amount and distribution of gene flow and genetic load along the genome of the three grain amaranth species and their two wild relatives. Our results show ample gene flow between crop species and between crops and their wild relatives. Gene flow from wild relatives decreased genetic load in the three crop species. This suggests that wild relatives could provide evolutionary rescue by replacing deleterious alleles in crops. We assess experimental hybrids between the three crop species and found genetic incompatibilities between one Central American grain amaranth and the other two crop species. These incompatibilities might have created recent reproductive barriers and maintained species integrity today. Together, our results show that gene flow played an important role in the domestication and expansion of grain amaranth, despite genetic species barriers. The domestication of plants was likely not linear and created a genomic mosaic by multiple contributors with varying fitness effects for today's crops.

Functional analysis of the BEige and Chediak-Higashi domain gene MpSPIRRIG in Marchantia polymorpha.

BEige and Chediak-Higashi domain containing proteins (BDCPs) have been described to function in membrane-dependent processes in eukaryotes. This role was also observed for the BDCP SPIRRIG (SPI) in the model plant Arabidopsis thaliana in the context of cell morphogenesis. Additionally, AtSPI was found to control salt stress resistance by mediating mRNA stability and salt stress-dependent processing body formation. In this work, we utilize an evolutionarily comparative approach to unravel conserved, basal BDCP functions in the liverwort Marchantia polymorpha. Our phenotypic and physiological analyses show that MpSPI is involved in cell morphogenesis and salt resistance regulation, indicating that both functions are evolutionarily conserved between the two species. Co-localization was found with endosomal and P-body markers, suggesting links to membrane-dependent processes and mRNA metabolism. Finally, we present transcriptomics data showing that AtSPI and MpSPI regulate orthologous genes in A. thaliana and M. polymorpha.

Domestication reshaped the genetic basis of inbreeding depression in a maize landrace compared to its wild relative, teosinte.

Inbreeding depression is the reduction in fitness and vigor resulting from mating of close relatives observed in many plant and animal species. The extent to which the genetic load of mutations contributing to inbreeding depression is due to large-effect mutations versus variants with very small individual effects is unknown and may be affected by population history. We compared the effects of outcrossing and self-fertilization on 18 traits in a landrace population of maize, which underwent a population bottleneck during domestication, and a neighboring population of its wild relative teosinte. Inbreeding depression was greater in maize than teosinte for 15 of 18 traits, congruent with the greater segregating genetic load in the maize population that we predicted from sequence data. Parental breeding values were highly consistent between outcross and selfed offspring, indicating that additive effects determine most of the genetic value even in the presence of strong inbreeding depression. We developed a novel linkage scan to identify quantitative trait loci (QTL) representing large-effect rare variants carried by only a single parent, which were more important in teosinte than maize. Teosinte also carried more putative juvenile-acting lethal variants identified by segregation distortion. These results suggest a mixture of mostly polygenic, small-effect partially recessive effects in linkage disequilibrium underlying inbreeding depression, with an additional contribution from rare larger-effect variants that was more important in teosinte but depleted in maize following the domestication bottleneck. Purging associated with the maize domestication bottleneck may have selected against some large effect variants, but polygenic load is harder to purge and overall segregating mutational burden increased in maize compared to teosinte.

PopAmaranth: a population genetic genome browser for grain amaranths and their wild relatives.

The combination of genomic, physiological, and population genetic research has accelerated the understanding and improvement of numerous crops. For nonmodel crops, the lack of interdisciplinary research hinders their improvement. Grain amaranth is an ancient nutritious pseudocereal that has been domesticated three times in different regions of the Americas. We present and employ PopAmaranth, a population genetic genome browser, which provides an accessible representation of the genetic variation of the three-grain amaranth species (Amaranthus hypochondriacus, Amaranthus cruentus, and Amaranthus caudatus) and two wild relatives (Amaranthus hybridus and Amaranthus quitensis) along the A. hypochondriacus reference sequence. We performed population-scale diversity and selection analysis from whole-genome sequencing data of 88 curated genetically and taxonomically unambiguously classified accessions. We employ the platform to show that genetic diversity in the water stress-related MIF1 gene declined during amaranth domestication and provide evidence for convergent saponin reduction between amaranth and quinoa. PopAmaranth is available through amaranthGDB at amaranthgdb.org/popamaranth.html.

Comparative evolutionary genetics of deleterious load in sorghum and maize.

Sorghum and maize share a close evolutionary history that can be explored through comparative genomics1,2. To perform a large-scale comparison of the genomic variation between these two species, we analysed ~13 million variants identified from whole-genome resequencing of 499 sorghum lines together with 25 million variants previously identified in 1,218 maize lines. Deleterious mutations in both species were prevalent in pericentromeric regions, enriched in non-syntenic genes and present at low allele frequencies. A comparison of deleterious burden between sorghum and maize revealed that sorghum, in contrast to maize, departed from the domestication-cost hypothesis that predicts a higher deleterious burden among domesticates compared with wild lines. Additionally, sorghum and maize population genetic summary statistics were used to predict a gene deleterious index with an accuracy greater than 0.5. This research represents a key step towards understanding the evolutionary dynamics of deleterious variants in sorghum and provides a comparative genomics framework to start prioritizing these variants for removal through genome editing and breeding.

Male Linked Genomic Region Determines Sex in Dioecious Amaranthus palmeri.

Dioecy, the separation of reproductive organs on different individuals, has evolved repeatedly in different plant families. Several evolutionary paths to dioecy have been suggested, but the mechanisms behind sex determination is not well understood. The diploid dioecious Amaranthus palmeri represents a well-suited model system to study sex determination in plants. Despite the agricultural importance of the species, the genetic control and evolutionary state of dioecy in A. palmeri is currently unknown. Early cytogenetic experiments did not identify heteromorphic chromosomes. Here, we used whole-genome sequencing of male and female pools from 2 independent populations to elucidate the genetic control of dioecy in A. palmeri. Read alignment to a close monoecious relative and allele frequency comparisons between male and female pools did not reveal significant sex-linked genes. Consequently, we employed an alignment-free k-mer comparison which enabled us to identify a large number of male-specific k-mers. We assembled male-specific contigs comprising a total of almost 2 Mb sequence, proposing a XY sex-determination system in the species. We were able to identify the potential Y chromosome in the A. palmeri draft genome sequence as 90% of our male-specific sequence aligned to a single scaffold. Based on our findings, we suggest an intermediate evolutionary state of dioecy with a young Y chromosome in A. palmeri. Our findings give insight into the evolution of sex chromosomes in plants and may help to develop sustainable strategies for weed management.

Selective Loss of Diversity in Doubled-Haploid Lines from European Maize Landraces.

Maize landraces are well adapted to their local environments and present valuable sources of genetic diversity for breeding and conservation. But the maintenance of open-pollinated landraces in ex-situ programs is challenging, as regeneration of seed can often lead to inbreeding depression and the loss of diversity due to genetic drift. Recent reports suggest that the production of doubled-haploid (DH) lines from landraces may serve as a convenient means to preserve genetic diversity in a homozygous form that is immediately useful for modern breeding. The production of doubled-haploid (DH) lines presents an extreme case of inbreeding which results in instantaneous homozygosity genome-wide. Here, we analyzed the effect of DH production on genetic diversity, using genome-wide SNP data from hundreds of individuals of five European landraces and their related DH lines. In contrast to previous findings, we observe a dramatic loss of diversity at both the haplotype level and that of individual SNPs. We identify thousands of SNPs that exhibit allele frequency differences larger than expected under models of neutral genetic drift and document losses of shared haplotypes. We find evidence consistent with selection at functional sites that are potentially involved in the diversity differences between landrace and DH populations. Although we were unable to uncover more details about the mode of selection, we conclude that landrace DH lines may be a valuable tool for the introduction of variation into maize breeding programs but come at the cost of decreased genetic diversity.

The Temporal Dynamics of Background Selection in Nonequilibrium Populations.

Neutral genetic diversity across the genome is determined by the complex interplay of mutation, demographic history, and natural selection. While the direct action of natural selection is limited to functional loci across the genome, its impact can have effects on nearby neutral loci due to genetic linkage. These effects of selection at linked sites, referred to as genetic hitchhiking and background selection (BGS), are pervasive across natural populations. However, only recently has there been a focus on the joint consequences of demography and selection at linked sites, and some empirical studies have come to apparently contradictory conclusions as to their combined effects. To understand the relationship between demography and selection at linked sites, we conducted an extensive forward simulation study of BGS under a range of demographic models. We found that the relative levels of diversity in BGS and neutral regions vary over time and that the initial dynamics after a population size change are often in the opposite direction of the long-term expected trajectory. Our detailed observations of the temporal dynamics of neutral diversity in the context of selection at linked sites in nonequilibrium populations provide new intuition about why patterns of diversity under BGS vary through time in natural populations and help reconcile previously contradictory observations. Most notably, our results highlight that classical models of BGS are poorly suited for predicting diversity in nonequilibrium populations.

Parallel Seed Color Adaptation during Multiple Domestication Attempts of an Ancient New World Grain.

Thousands of plants have been selected as crops; yet, only a few are fully domesticated. The lack of adaptation to agroecological environments of many crop plants with few characteristic domestication traits potentially has genetic causes. Here, we investigate the incomplete domestication of an ancient grain from the Americas, amaranth. Although three grain amaranth species have been cultivated as crop for millennia, all three lack key domestication traits. We sequenced 121 crop and wild individuals to investigate the genomic signature of repeated incomplete adaptation. Our analysis shows that grain amaranth has been domesticated three times from a single wild ancestor. One trait that has been selected during domestication in all three grain species is the seed color, which changed from dark seeds to white seeds. We were able to map the genetic control of the seed color adaptation to two genomic regions on chromosomes 3 and 9, employing three independent mapping populations. Within the locus on chromosome 9, we identify an MYB-like transcription factor gene, a known regulator for seed color variation in other plant species. We identify a soft selective sweep in this genomic region in one of the crop species but not in the other two species. The demographic analysis of wild and domesticated amaranths revealed a population bottleneck predating the domestication of grain amaranth. Our results indicate that a reduced level of ancestral genetic variation did not prevent the selection of traits with a simple genetic architecture but may have limited the adaptation of complex domestication traits.

Genetic architecture and selective sweeps after polygenic adaptation to distant trait optima.

Understanding the genetic basis of phenotypic adaptation to changing environments is an essential goal of population and quantitative genetics. While technological advances now allow interrogation of genome-wide genotyping data in large panels, our understanding of the process of polygenic adaptation is still limited. To address this limitation, we use extensive forward-time simulation to explore the impacts of variation in demography, trait genetics, and selection on the rate and mode of adaptation and the resulting genetic architecture. We simulate a population adapting to an optimum shift, modeling sequence variation for 20 QTL for each of 12 different demographies for 100 different traits varying in the effect size distribution of new mutations, the strength of stabilizing selection, and the contribution of the genomic background. We then use random forest regression approaches to learn the relative importance of input parameters in determining a number of aspects of the process of adaptation, including the speed of adaptation, the relative frequency of hard sweeps and sweeps from standing variation, or the final genetic architecture of the trait. We find that selective sweeps occur even for traits under relatively weak selection and where the genetic background explains most of the variation. Though most sweeps occur from variation segregating in the ancestral population, new mutations can be important for traits under strong stabilizing selection that undergo a large optimum shift. We also show that population bottlenecks and expansion impact overall genetic variation as well as the relative importance of sweeps from standing variation and the speed with which adaptation can occur. We then compare our results to two traits under selection during maize domestication, showing that our simulations qualitatively recapitulate differences between them. Overall, our results underscore the complex population genetics of individual loci in even relatively simple quantitative trait models, but provide a glimpse into the factors that drive this complexity and the potential of these approaches for understanding polygenic adaptation.

From zero to hero: the past, present and future of grain amaranth breeding.

KEY MESSAGE: Grain amaranth is an underutilized crop with high nutritional quality from the Americas. Emerging genomic and biotechnological tools are becoming available that allow the integration of novel breeding techniques for rapid improvement of amaranth and other underutilized crops. Out of thousands of edible plants, only three cereals-maize, wheat and rice-are the major food sources for a majority of people worldwide. While these crops provide high amounts of calories, they are low in protein and other essential nutrients. The dependence on only few crops, with often narrow genetic basis, leads to a high vulnerability of modern cropping systems to the predicted climate change and accompanying weather extremes. Broadening our food sources through the integration of so-called orphan crops can help to mitigate the effects of environmental change and improve qualitative food security. Thousands of traditional crops are known, but have received little attention in the last century and breeding efforts were limited. Amaranth is such an underutilized pseudocereal that is of particular interest because of its balanced amino acid and micronutrient profiles. Additionally, the C4 photosynthetic pathway and ability to withstand environmental stress make the crop a suitable choice for future agricultural systems. Despite the potential of amaranth, efforts of genetic improvement lag considerably behind those of major crops. The progress in novel breeding methods and molecular techniques developed in model plants and major crops allow a rapid improvement of underutilized crops. Here, we review the history of amaranth and recent advances in genomic tools and give a concrete perspective how novel breeding techniques can be implemented into breeding programs. Our perspectives are transferable to many underutilized crops. The implementation of these could improve the nutritional quality and climate resilience of future cropping systems.

How to make a domesticate.

The Neolithic Revolution brought about the transition from hunting and gathering to sedentary societies, laying the foundation for the development of modern civilizations. The primary innovation that facilitated these changes was the domestication of plants and animals. In the case of plants, this involved the cultivation and selection of individuals with larger edible parts, easier harvesting, and decreased defenses, traits that allowed for the production of a food surplus and occupational specialization. Plant domestication is a process which started approximately 10,000 years ago and has thereafter been repeated independently in many locales around the world. Here, we offer a perspective that seeks to predict what factors influence the success of domestication, how many genes contributed to the process, where these genes originated and the implications for de novo domestication.

Increased root hair density by loss of WRKY6 in Arabidopsis thaliana.

Root hairs are unicellular elongations of certain rhizodermal cells that improve the uptake of sparingly soluble and immobile soil nutrients. Among different Arabidopsis thaliana genotypes, root hair density, length and the local acclimation to low inorganic phosphate (Pi) differs considerably, when analyzed on split agar plates. Here, genome-wide association fine mapping identified significant single nucleotide polymorphisms associated with the increased root hair density in the absence of local phosphate on chromosome 1. A loss-of-functionmutant of the candidate transcription factor gene WRKY6, which is involved in the acclimation of plants to low phosphorus, had increased root hair density. This is partially explained by a reduced cortical cell diameter in wrky6-3, reducing the rhizodermal cell numbers adjacent to the cortical cells. As a consequence, rhizodermal cells in positions that are in contact with two cortical cells are found more often, leading to higher hair density. Distinct cortical cell diameters and epidermal cell lengths distinguish other Arabidopsis accessions with distinct root hair density and -Pi response from diploid Col-0, while tetraploid Col-0 had generally larger root cell sizes, which explain longer hairs. A distinct radial root morphology within Arabidopsis accessions and wrky6-3explains some, but not all, differences in the root hair acclimation to -Pi.

Analysis of phylogenetic relationships and genome size evolution of the Amaranthus genus using GBS indicates the ancestors of an ancient crop.

The genus Amaranthus consists of 50-70 species and harbors several cultivated and weedy species of great economic importance. A small number of suitable traits, phenotypic plasticity, gene flow and hybridization made it difficult to establish the taxonomy and phylogeny of the whole genus despite various studies using molecular markers. We inferred the phylogeny of the Amaranthus genus using genotyping by sequencing (GBS) of 94 genebank accessions representing 35 Amaranthus species and measured their genome sizes. SNPs were called by de novo and reference-based methods, for which we used the distant sugarbeet Beta vulgaris and the closely related Amaranthus hypochondriacus as references. SNP counts and proportions of missing data differed between methods, but the resulting phylogenetic trees were highly similar. A distance-based neighbor joining tree of individual accessions and a species tree calculated with the multispecies coalescent supported a previous taxonomic classification into three subgenera although the subgenus A. Acnida consists of two highly differentiated clades. The analysis of the Hybridus complex within the A. Amaranthus subgenus revealed insights on the history of cultivated grain amaranths. The complex includes the three cultivated grain amaranths and their wild relatives and was well separated from other species in the subgenus. Wild and cultivated amaranth accessions did not differentiate according to the species assignment but clustered by their geographic origin from South and Central America. Different geographically separated populations of Amaranthus hybridus appear to be the common ancestors of the three cultivated grain species and A. quitensis might be additionally be involved in the evolution of South American grain amaranth (A. caudatus). We also measured genome sizes of the species and observed little variation with the exception of two lineages that showed evidence for a recent polyploidization. With the exception of two lineages, genome sizes are quite similar and indicate that polyploidization did not play a major role in the history of the genus.

Genomic and phenotypic evidence for an incomplete domestication of South American grain amaranth (Amaranthus caudatus).

The domestication syndrome comprises phenotypic changes that differentiate crops from their wild ancestors. We compared the genomic variation and phenotypic differentiation of the two putative domestication traits seed size and seed colour of the grain amaranth Amaranthus caudatus, which is an ancient crop of South America, and its two close wild relatives and putative ancestors A. hybridus and A. quitensis. Genotyping 119 accessions of the three species from the Andean region using genotyping by sequencing (GBS) resulted in 9485 SNPs that revealed a strong genetic differentiation of cultivated A. caudatus from its two relatives. A. quitensis and A. hybridus accessions did not cluster by their species assignment but formed mixed groups according to their geographic origin in Ecuador and Peru, respectively. A. caudatus had a higher genetic diversity than its close relatives and shared a high proportion of polymorphisms with their wild relatives consistent with the absence of a strong bottleneck or a high level of recent gene flow. Genome sizes and seed sizes were not significantly different between A. caudatus and its relatives, although a genetically distinct group of A. caudatus from Bolivia had significantly larger seeds. We conclude that despite a long history of human cultivation and selection for white grain colour, A. caudatus shows a weak genomic and phenotypic domestication syndrome and proposes that it is an incompletely domesticated crop species either because of weak selection or high levels of gene flow from its sympatric close undomesticated relatives that counteracted the fixation of key domestication traits.

Crossing Methods and Cultivation Conditions for Rapid Production of Segregating Populations in Three Grain Amaranth Species.

Grain amaranths (Amaranthus spp.) have been cultivated for thousands of years in Central and South America. Their grains are of high nutritional value, but the low yield needs to be increased by selection of superior genotypes from genetically diverse breeding populations. Amaranths are adapted to harsh conditions and can be cultivated on marginal lands although little is known about their physiology. The development of controlled growing conditions and efficient crossing methods is important for research on and improvement of this ancient crop. Grain amaranth was domesticated in the Americas and is highly self-fertilizing with a large inflorescence consisting of thousands of very small flowers. We evaluated three different crossing methods (open pollination, hot water emasculation and hand emasculation) for their efficiency in amaranth and validated them with genetic markers. We identified cultivation conditions that allow an easy control of flowering time by day length manipulation and achieved flowering times of 4 weeks and generation times of 2 months. All three different crossing methods successfully produced hybrid F1 offspring, but with different success rates. Open pollination had the lowest (10%) and hand emasculation the highest success rate (74%). Hot water emasculation showed an intermediate success rate (26%) with a maximum of 94% success. It is simple to perform and suitable for a more large-scale production of hybrids. We further evaluated 11 single nucleotide polymorphism (SNP) markers and found that they were sufficient to validate all crosses of the genotypes used in this study for intra- and interspecific hybridizations. Despite its very small flowers, crosses in amaranth can be carried out efficiently and evaluated with inexpensive SNP markers. Suitable growth conditions strongly reduce the generation time and allow the control of plant height, flowering time, and seed production. In combination, this enables the rapid production of segregating populations which makes amaranth an attractive model for basic plant research but also facilitates further the improvement of this ancient crop by plant breeding.

Uncovering genes and ploidy involved in the high diversity in root hair density, length and response to local scarce phosphate in Arabidopsis thaliana.

Plant root hairs increase the root surface to enhance the uptake of sparingly soluble and immobile nutrients, such as the essential nutrient phosphorus, from the soil. Here, root hair traits and the response to scarce local phosphorus concentration were studied in 166 accessions of Arabidopsis thaliana using split plates. Root hair density and length were correlated, but highly variable among accessions. Surprisingly, the well-known increase in root hair density under low phosphorus was mostly restricted to genotypes that had less and shorter root hairs under P sufficient conditions. By contrast, several accessions with dense and long root hairs even had lower hair density or shorter hairs in local scarce phosphorus. Furthermore, accessions with whole-genome duplications developed more dense but phosphorus-insensitive root hairs. The impact of genome duplication on root hair density was confirmed by comparing tetraploid accessions with their diploid ancestors. Genome-wide association mapping identified candidate genes potentially involved in root hair responses tp scarce local phosphate. Knock-out mutants in identified candidate genes (CYR1, At1g32360 and RLP48) were isolated and differences in root hair traits in the mutants were confirmed. The large diversity in root hair traits among accessions and the diverse response when local phosphorus is scarce is a rich resource for further functional analyses.