Repeated translocation of a gene cassette drives sex-chromosome turnover in strawberries.

Turnovers of sex-determining systems represent important diversifying forces across eukaryotes. Shifts in sex chromosomes-but conservation of the master sex-determining genes-characterize distantly related animal lineages. Yet in plants, in which separate sexes have evolved repeatedly and sex chromosomes are typically homomorphic, we do not know whether such translocations drive sex-chromosome turnovers within closely related taxonomic groups. This phenomenon can only be demonstrated by identifying sex-associated nucleotide sequences, still largely unknown in plants. The wild North American octoploid strawberries (Fragaria) exhibit separate sexes (dioecy) with homomorphic, female heterogametic (ZW) inheritance, yet sex maps to three different chromosomes in different taxa. To characterize these turnovers, we identified sequences unique to females and assembled their reads into contigs. For most octoploid Fragaria taxa, a short (13 kb) sequence was observed in all females and never in males, implicating it as the sex-determining region (SDR). This female-specific "SDR cassette" contains both a gene with a known role in fruit and pollen production and a novel retrogene absent on Z and autosomal chromosomes. Phylogenetic comparison of SDR cassettes revealed three clades and a history of repeated translocation. Remarkably, the translocations can be ordered temporally due to the capture of adjacent sequence with each successive move. The accumulation of the "souvenir" sequence-and the resultant expansion of the hemizygous SDR over time-could have been adaptive by locking genes into linkage with sex. Terminal inverted repeats at the insertion borders suggest a means of movement. To our knowledge, this is the first plant SDR shown to be translocated, and it suggests a new mechanism ("move-lock-grow") for expansion and diversification of incipient sex chromosomes.

Genetic Mapping and Phylogenetic Analysis Reveal Intraspecific Variation in Sex Chromosomes of the Virginian Strawberry.

With their extraordinary diversity in sexual systems, flowering plants offer unparalleled opportunities to understand sex determination and to reveal generalities in the evolution of sex chromosomes. Comparative genetic mapping of related taxa with good phylogenetic resolution can delineate the extent of sex chromosome diversity within plant groups, and lead the way to understanding the evolutionary drivers of such diversity. The North American octoploid wild strawberries provide such an opportunity. We performed linkage mapping using targeted sequence capture for the subdioecious western Fragaria virginiana ssp. platypetala and compared the location of its sex-determining region (SDR) to those of 2 other (sub)dioecious species, the eastern subspecies, F. virginiana ssp. virginiana (whose SDR is at 0-5.5 Mb on chromosome VI of the B2 subgenome), and the sister species F. chiloensis (whose SDR is at 37 Mb on chromosome VI of the Av subgenome). Male sterility was dominant in F. virginiana ssp. platypetala and mapped to a chromosome also in homeologous group VI. Likewise, one major quantitative trait locus (QTL) for female fertility overlapped the male sterility region. However, the SDR mapped to yet another subgenome (B1), and to a different location (13 Mb), but similar to the location inferred in one population of the naturally occurring hybrid between F. chiloensis and F. virginiana (F. ×ananassa ssp. cuneifolia). Phylogenetic analysis of chromosomes across the octoploid taxa showed consistent subgenomic composition reflecting shared evolutionary history but also reinforced within-species variation in the SDR-carrying chromosome, suggesting either repeated evolution, or recent turnovers in SDR.

Homomorphic ZW chromosomes in a wild strawberry show distinctive recombination heterogeneity but a small sex-determining region.

Recombination in ancient, heteromorphic sex chromosomes is typically suppressed at the sex-determining region (SDR) and proportionally elevated in the pseudoautosomal region (PAR). However, little is known about recombination dynamics of young, homomorphic plant sex chromosomes. We examine male and female function in crosses and unrelated samples of the dioecious octoploid strawberry Fragaria chiloensis in order to map the small and recently evolved SDR controlling both traits and to examine recombination patterns on the incipient ZW chromosome. The SDR of this ZW system is located within a 280 kb window, in which the maternal recombination rate is lower than the paternal one. In contrast to the SDR, the maternal PAR recombination rate is much higher than the rates of the paternal PAR or autosomes, culminating in an elevated chromosome-wide rate. W-specific divergence is elevated within the SDR and a single polymorphism is observed in high species-wide linkage disequilibrium with sex. Selection for recombination suppression within the small SDR may be weak, but fluctuating sex ratios could favor elevated recombination in the PAR to remove deleterious mutations on the W. The recombination dynamics of this nascent sex chromosome with a modestly diverged SDR may be typical of other dioecious plants.

Comparison of nuclear, plastid, and mitochondrial phylogenies and the origin of wild octoploid strawberry species.

PREMISE OF THE STUDY: Molecular phylogenies derived from all three plant genomes can provide insight into the evolutionary history of plant groups influenced by reticulation. We sought to reconstruct mitochondrial exome, chloroplast, and nuclear genome phylogenies for octoploid Fragaria and their diploid ancestors and to document patterns of incongruence between and within the cytoplasmic genomes and interpret these in the context of evolutionary origin of the octoploid strawberries. METHODS: Using a genome-skimming approach, we assembled chloroplast genomes and mitochondrial exomes, and we used the POLiMAPS method to assemble nuclear sequence for octoploid species and constructed phylogenies from all three genomes. We assessed incongruence between and within cytoplasmic genomes using topology-based phylogenetic incongruence tests. KEY RESULTS: The incongruent cytoplasmic genome phylogeny with respect to the placement of octoploids suggests potential breakage in linkage disequilibrium of cytoplasmic genomes during allopolyploid origin of the octoploids. Furthermore, a single mitochondrial chimeric gene with a putative role in cytoplasmic male sterility yields a phylogeny that is inconsistent with the rest of the mitochondrial genome but consistent with the chloroplast phylogeny, suggesting intracellular gene transfer between heteroplasmic mitochondria, possibly driven by selection to overcome the effects of mito-nuclear incompatibility in octoploid origins. CONCLUSIONS: This work expands on the current understanding of evolutionary history of the octoploid ancestors of cultivated strawberry. It demonstrates phylogenetic incongruence between cytoplasmic genomes in octoploids with respect to diploid ancestors, indicating breakage in linkage disequilibrium of cytoplasmic genomes. We discuss potential organism-level processes that may have contributed to the observed incongruence in Fragaria.

Bioclimatic, ecological, and phenotypic intermediacy and high genetic admixture in a natural hybrid of octoploid strawberries.

PREMISE OF THE STUDY: Hybrid zones provide "natural laboratories" for understanding the processes of selection, reinforcement, and speciation. We sought to gain insight into the degree of introgression and the extent of ecological-phenotypic intermediacy in the natural hybrid strawberry, Fragaria × ananassa subsp. cuneifolia. • METHODS: We used whole-plastome sequencing to identify parental species-specific (Fragaria chiloensis and F. virginiana) chloroplast single-nucleotide polymorphisms and combined the use of these with nuclear microsatellite markers to genetically characterize the hybrid zone. We assessed the potential role of selection in the observed geographic patterns by bioclimatically characterizing the niche of the hybrid populations and phenotypically characterizing hybrid individuals of known genomic constitution. • KEY RESULTS: Significant admixture and little overall maternal bias in chloroplast or nuclear genomes suggest a high degree of interfertility among the parental and hybrid species and point to a long history of backcrossing and genetic mixing in the hybrid zone. Even though hybrids were phenotypically intermediate to the parental species, there was a discernible fingerprint of the parental genotype within hybrid individuals. Thus, although the pattern of introgression observed suggests geographic limitations to gene flow, it may be reinforced by selection for specific parental traits in the bioclimatically intermediate habitat occupied by the hybrid. • CONCLUSIONS: This work uncovered the genetic complexity underlying the hybrid zone of the wild relatives of the cultivated strawberry. It lays the foundation for experimental dissection of the causes of genomic introgression and nuclear-cytoplasmic disassociation, and for understanding other parts of Fragaria evolutionary history.

Phylogenetic and population genetic analyses of diploid Leucaena (Leguminosae; Mimosoideae) reveal cryptic species diversity and patterns of divergent allopatric speciation.

PREMISE OF THE STUDY: Leucaena comprises 17 diploid species, five tetraploid species, and a complex series of hybrids whose evolutionary histories have been influenced by human seed translocation, cultivation, and subsequent spontaneous hybridization. Here we investigated patterns of evolutionary divergence among diploid Leucaena through comprehensively sampled multilocus phylogenetic and population genetic approaches to address species delimitation, interspecific relationships, hybridization, and the predominant mode of speciation among diploids. METHODS: Parsimony- and maximum-likelihood-based phylogenetic approaches were applied to 59 accessions sequenced for six SCAR-based nuclear loci, nrDNA ITS, and four cpDNA regions. Population genetic comparisons included 1215 AFLP loci representing 42 populations and 424 individuals. RESULTS: Phylogenetic results provided a well-resolved hypothesis of divergent species relationships, recovering previously recognized clades of diploids as well as newly resolved relationships. Phylogenetic and population genetic assessments identified two cryptic species that are consistent with geography and morphology. CONCLUSIONS: Findings from this study highlight the importance and utility of multilocus data in the recovery of complex evolutionary histories. The results are consistent with allopatric divergence representing the predominant mode of speciation among diploid Leucaena. These findings contrast with the potential hybrid origin of several tetraploid species and highlight the importance of human translocation of seed to the origin of these tetraploids. The recognition of one previously unrecognized species (L. cruziana) and the elevation of another taxon (L. collinsii subsp. zacapana) to specific status (L. zacapana) is consistent with a growing number of newly diagnosed species from neotropical seasonally dry forests, suggesting these communities harbor greater species diversity than previously recognized.

The complex evolutionary dynamics of ancient and recent polyploidy in Leucaena (Leguminosae; Mimosoideae).

PREMISE OF THE STUDY: The evolutionary history of Leucaena has been impacted by polyploidy, hybridization, and divergent allopatric species diversification, suggesting that this is an ideal group to investigate the evolutionary tempo of polyploidy and the complexities of reticulation and divergence in plant diversification. METHODS: Parsimony- and ML-based phylogenetic approaches were applied to 105 accessions sequenced for six sequence characterized amplified region-based nuclear encoded loci, nrDNA ITS, and four cpDNA regions. Hypotheses for the origin of tetraploid species were inferred using results derived from a novel species tree and established gene tree methods and from data on genome sizes and geographic distributions. RESULTS: The combination of comprehensively sampled multilocus DNA sequence data sets and a novel methodology provide strong resolution and support for the origins of all five tetraploid species. A minimum of four allopolyploidization events are required to explain the origins of these species. The origin(s) of one tetraploid pair (L. involucrata/L. pallida) can be equally explained by two unique allopolyploidizations or a single event followed by divergent speciation. CONCLUSIONS: Alongside other recent findings, a comprehensive picture of the complex evolutionary dynamics of polyploidy in Leucaena is emerging that includes paleotetraploidization, diploidization of the last common ancestor to Leucaena, allopatric divergence among diploids, and recent allopolyploid origins for tetraploid species likely associated with human translocation of seed. These results provide insights into the role of divergence and reticulation in a well-characterized angiosperm lineage and into traits of diploid parents and derived tetraploids (particularly self-compatibility and year-round flowering) favoring the formation and establishment of novel tetraploids combinations.

Integration of high-density genetic mapping with transcriptome analysis uncovers numerous agronomic QTL and reveals candidate genes for the control of tillering in sorghum.

Phenotypes such as branching, photoperiod sensitivity, and height were modified during plant domestication and crop improvement. Here, we perform quantitative trait locus (QTL) mapping of these and other agronomic traits in a recombinant inbred line (RIL) population derived from an interspecific cross between Sorghum propinquum and Sorghum bicolor inbred Tx7000. Using low-coverage Illumina sequencing and a bin-mapping approach, we generated ∼1920 bin markers spanning ∼875 cM. Phenotyping data were collected and analyzed from two field locations and one greenhouse experiment for six agronomic traits, thereby identifying a total of 30 QTL. Many of these QTL were penetrant across environments and co-mapped with major QTL identified in other studies. Other QTL uncovered new genomic regions associated with these traits, and some of these were environment-specific in their action. To further dissect the genetic underpinnings of tillering, we complemented QTL analysis with transcriptomics, identifying 6189 genes that were differentially expressed during tiller bud elongation. We identified genes such as Dormancy Associated Protein 1 (DRM1) in addition to various transcription factors that are differentially expressed in comparisons of dormant to elongating tiller buds and lie within tillering QTL, suggesting that these genes are key regulators of tiller elongation in sorghum. Our study demonstrates the usefulness of this RIL population in detecting domestication and improvement-associated genes in sorghum, thus providing a valuable resource for genetic investigation and improvement to the sorghum community.

The regulatory landscape of a core maize domestication module controlling bud dormancy and growth repression.

Many domesticated crop plants have been bred for increased apical dominance, displaying greatly reduced axillary branching compared to their wild ancestors. In maize, this was achieved through selection for a gain-of-function allele of the TCP transcription factor teosinte branched1 (tb1). The mechanism for how a dominant Tb1 allele increased apical dominance, is unknown. Through ChIP seq, RNA seq, hormone and sugar measurements on 1 mm axillary bud tissue, we identify the genetic pathways putatively regulated by TB1. These include pathways regulating phytohormones such as gibberellins, abscisic acid and jasmonic acid, but surprisingly, not auxin. In addition, metabolites involved in sugar sensing such as trehalose 6-phosphate were increased. This suggests that TB1 induces bud suppression through the production of inhibitory phytohormones and by reducing sugar levels and energy balance. Interestingly, TB1 also putatively targets several other domestication loci, including teosinte glume architecture1, prol1.1/grassy tillers1, as well as itself. This places tb1 on top of the domestication hierarchy, demonstrating its critical importance during the domestication of maize from teosinte.

Bulked-Segregant Analysis Coupled to Whole Genome Sequencing (BSA-Seq) for Rapid Gene Cloning in Maize.

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1 Our method provides a quick, simple way to clone genes in maize.

Multilocus Sex Determination Revealed in Two Populations of Gynodioecious Wild Strawberry, Fragaria vesca subsp. bracteata.

Gynodioecy, the coexistence of females and hermaphrodites, occurs in 20% of angiosperm families and often enables transitions between hermaphroditism and dioecy. Clarifying mechanisms of sex determination in gynodioecious species can thus illuminate sexual system evolution. Genetic determination of gynodioecy, however, can be complex and is not fully characterized in any wild species. We used targeted sequence capture to genetically map a novel nuclear contributor to male sterility in a self-pollinated hermaphrodite of Fragaria vesca subsp. bracteata from the southern portion of its range. To understand its interaction with another identified locus and possibly additional loci, we performed crosses within and between two populations separated by 2000 km, phenotyped the progeny and sequenced candidate markers at both sex-determining loci. The newly mapped locus contains a high density of pentatricopeptide repeat genes, a class commonly involved in restoration of fertility caused by cytoplasmic male sterility. Examination of all crosses revealed three unlinked epistatically interacting loci that determine sexual phenotype and vary in frequency between populations. Fragaria vesca subsp. bracteata represents the first wild gynodioecious species with genomic evidence of both cytoplasmic and nuclear genes in sex determination. We propose a model for the interactions between these loci and new hypotheses for the evolution of sex determining chromosomes in the subdioecious and dioecious Fragaria.

Evolutionary origins and dynamics of octoploid strawberry subgenomes revealed by dense targeted capture linkage maps.

Whole-genome duplications are radical evolutionary events that have driven speciation and adaptation in many taxa. Higher-order polyploids have complex histories often including interspecific hybridization and dynamic genomic changes. This chromosomal reshuffling is poorly understood for most polyploid species, despite their evolutionary and agricultural importance, due to the challenge of distinguishing homologous sequences from each other. Here, we use dense linkage maps generated with targeted sequence capture to improve the diploid strawberry (Fragaria vesca) reference genome and to disentangle the subgenomes of the wild octoploid progenitors of cultivated strawberry, Fragaria virginiana and Fragaria chiloensis. Our novel approach, POLiMAPS (Phylogenetics Of Linkage-Map-Anchored Polyploid Subgenomes), leverages sequence reads to associate informative interhomeolog phylogenetic markers with linkage groups and reference genome positions. In contrast to a widely accepted model, we find that one of the four subgenomes originates with the diploid cytoplasm donor F. vesca, one with the diploid Fragaria iinumae, and two with an unknown ancestor close to F. iinumae. Extensive unidirectional introgression has converted F. iinumae-like subgenomes to be more F. vesca-like, but never the reverse, due either to homoploid hybridization in the F. iinumae-like diploid ancestors or else strong selection spreading F. vesca-like sequence among subgenomes through homeologous exchange. In addition, divergence between homeologous chromosomes has been substantially augmented by interchromosomal rearrangements. Our phylogenetic approach reveals novel aspects of the complicated web of genetic exchanges that occur during polyploid evolution and suggests a path forward for unraveling other agriculturally and ecologically important polyploid genomes.

Targeted sequence capture provides insight into genome structure and genetics of male sterility in a gynodioecious diploid strawberry, Fragaria vesca ssp. bracteata (Rosaceae).

Gynodioecy is a sexual system wherein females coexist with hermaphrodites. It is of interest not only because male-sterile plants are advantageous in plant breeding but also because it can be a crucial step in the evolutionary transition to entirely separate sexes (dioecy) from a hermaphroditic ancestor. The gynodioecious diploid wild strawberry, Fragaria vesca ssp. bracteata (Rosaceae), is a member of a clade with both dioecious and cultivated species, making it an ideal model in which to study the genetics of male sterility. To create a genetic map of F. v. ssp. bracteata, we identified informative polymorphisms from genomic sequencing (3-5x coverage) of two outbred plants from the same population. Using targeted enrichment, we sequenced 200 bp surrounding each of 6575 polymorphisms in 48 F1 offspring, yielding genotypes at 98% of targeted sites with mean coverage >100x, plus more than 600-kb high-coverage nontargeted sequence. With the resulting linkage map of 7802 stringently filtered markers (5417 targeted), we assessed recombination rates and genomic incongruities. Consistent with past work in strawberries, male sterility is dominant, segregates 1:1, and maps to a single location in the female. Further mapping an additional 55 offspring places male sterility in a gene-dense, 338-kb region of chromosome 4. The region is not syntenic with the sex-determining regions in the closely related octoploids, F. chiloensis and F. virginiana, suggesting either independent origins or translocation. The 57 genes in this region do not include protein families known to control male sterility and thus suggest alternate mechanisms for the suppression of male function.

Serendipitous backyard hybridization and the origin of crops.

Backyard gardens, dump heaps, and kitchen middens are thought to have provided important venues for early crop domestication via generation of hybrids between otherwise isolated plant species. However, this process has rarely been demonstrated empirically. For the majority of polyploid crops, it remains uncertain to what extent hybridization and polyploidization preceded domestication or were precipitated by human activities. Using archaeological, ethnobotanical, geographical, and genetic data, we investigate the extent and significance of predomestication cultivation, backyard sympatry, and spontaneous hybridization for the Mimosoid legume tree Leucaena, which is used as a food crop throughout south-central Mexico. We show that predomestication cultivation was widespread, involved numerous independent transitions from the wild to cultivation, and resulted in extensive artificial sympatry of 2-6 species locally and 13 species in total. Using chloroplast and rapidly evolving nuclear-encoded DNA sequences, we demonstrate that hybridization in Leucaena has been extensive and complex, spawning a diverse set of novel hybrids as a result of juxtaposition of species in cultivation. The scale and complexity of hybridization in Leucaena is significantly greater than that documented for any other Mexican plant domesticates so far. However, there are striking parallels between Leucaena and the other major Mexican perennial domesticates Agave and Opuntia, which show very similar domestication via backyard hybridization pathways. Our results suggest that backyard hybridization has played a central role in Mesoamerican crop domestication and demonstrate that the simple step of bringing species together in cultivation can provide a potent trigger for domestication.