Jennifer R. Mandel

SNP Discovery and Development of a High-Density Genotyping Array for Sunflower

Recent advances in next-generation DNA sequencing technologies have made possible the development of high-throughput SNP genotyping platforms that allow for the simultaneous interrogation of thousands of single-nucleotide polymorphisms (SNPs). Such resources have the potential to facilitate the rapid development of high-density genetic maps, and to enable genome-wide association studies as well as molecular breeding approaches in a variety of taxa. Herein, we describe the development of a SNP genotyping resource for use in sunflower (Helianthus annuus L.). This work involved the development of a reference transcriptome assembly for sunflower, the discovery of thousands of high quality SNPs based on the generation and analysis of ca. 6 Gb of transcriptome re-sequencing data derived from multiple genotypes, the selection of 10,640 SNPs for inclusion in the genotyping array, and the use of the resulting array to screen a diverse panel of sunflower accessions as well as related wild species. The results of this work revealed a high frequency of polymorphic SNPs and relatively high level of cross-species transferability. Indeed, greater than 95% of successful SNP assays revealed polymorphism, and more than 90% of these assays could be successfully transferred to related wild species. Analysis of the polymorphism data revealed patterns of genetic differentiation that were largely congruent with the evolutionary history of sunflower, though the large number of markers allowed for finer resolution than has previously been possible.

Genetic diversity and population structure in cultivated sunflower and a comparison to its wild progenitor, Helianthus annuus L

Crop germplasm collections are valuable resources for ongoing plant breeding efforts. To fully utilize such collections, however, researchers need detailed information about the amount and distribution of genetic diversity present within collections. Here, we report the results of a population genetic analysis of the primary gene pool of sunflower (Helianthus annuus L.) based on a broad sampling of 433 cultivated accessions from North America and Europe, as well as a range-wide collection of 24 wild sunflower populations. Gene diversity across the cultivars was 0.47, as compared with 0.70 in the wilds, indicating that cultivated sunflower harbors roughly two-thirds of the total genetic diversity present in wild sunflower. Population structure analyses revealed that wild sunflower can be subdivided into four genetically distinct population clusters throughout its North American range, whereas the cultivated sunflower gene pool could be split into two main clusters separating restorer lines from the balance of the gene pool. Use of a maximum likelihood method to estimate the contribution of the wild gene pool to the cultivated sunflower germplasm revealed that the bulk of the cultivar diversity is derived from two wild sunflower population genetic clusters that are primarily composed of individuals from the east-central United States, the same general region in which sunflower domestication is believed to have occurred. We also identified a nested subset of accessions that capture as much of the allelic diversity present within the sampled cultivated sunflower germplasm collection as possible. At the high end, a core set of 288 captured nearly 90% of the alleles present in the full set of 433, whereas a core set of just 12 accessions was sufficient to capture nearly 50% of the total allelic diversity present within this sample of cultivated sunflower.

The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution

The domesticated sunflower, Helianthus annuus L., is a global oil crop that has promise for climate change adaptation, because it can maintain stable yields across a wide variety of environmental conditions, including drought. Even greater resilience is achievable through the mining of resistance alleles from compatible wild sunflower relatives, including numerous extremophile species. Here we report a high-quality reference for the sunflower genome (3.6 gigabases), together with extensive transcriptomic data from vegetative and floral organs. The genome mostly consists of highly similar, related sequences and required single-molecule real-time sequencing technologies for successful assembly. Genome analyses enabled the reconstruction of the evolutionary history of the Asterids, further establishing the existence of a whole-genome triplication at the base of the Asterids II clade and a sunflower-specific whole-genome duplication around 29 million years ago. An integrative approach combining quantitative genetics, expression and diversity data permitted development of comprehensive gene networks for two major breeding traits, flowering time and oil metabolism, and revealed new candidate genes in these networks. We found that the genomic architecture of flowering time has been shaped by the most recent whole-genome duplication, which suggests that ancient paralogues can remain in the same regulatory networks for dozens of millions of years. This genome represents a cornerstone for future research programs aiming to exploit genetic diversity to improve biotic and abiotic stress resistance and oil production, while also considering agricultural constraints and human nutritional needs.

A target enrichment method for gathering phylogenetic information from hundreds of loci: An example from the Compositae

Premise of the study: The Compositae (Asteraceae) are a large and diverse family of plants, and the most comprehensive phylogeny to date is a meta-tree based on 10 chloroplast loci that has several major unresolved nodes. We describe the development of an approach that enables the rapid sequencing of large numbers of orthologous nuclear loci to facilitate efficient phylogenomic analyses. Methods and Results: We designed a set of sequence capture probes that target conserved orthologous sequences in the Compositae. We also developed a bioinformatic and phylogenetic workflow for processing and analyzing the resulting data. Application of our approach to 15 species from across the Compositae resulted in the production of phylogenetically informative sequence data from 763 loci and the successful reconstruction of known phylogenetic relationships across the family. Conclusions: These methods should be of great use to members of the broader Compositae community, and the general approach should also be of use to researchers studying other families.

Association mapping and the genomic consequences of selection in sunflower.

The combination of large-scale population genomic analyses and trait-based mapping approaches has the potential to provide novel insights into the evolutionary history and genome organization of crop plants. Here, we describe the detailed genotypic and phenotypic analysis of a sunflower (Helianthus annuus L.) association mapping population that captures nearly 90% of the allelic diversity present within the cultivated sunflower germplasm collection. We used these data to characterize overall patterns of genomic diversity and to perform association analyses on plant architecture (i.e., branching) and flowering time, successfully identifying numerous associations underlying these agronomically and evolutionarily important traits. Overall, we found variable levels of linkage disequilibrium (LD) across the genome. In general, islands of elevated LD correspond to genomic regions underlying traits that are known to have been targeted by selection during the evolution of cultivated sunflower. In many cases, these regions also showed significantly elevated levels of differentiation between the two major sunflower breeding groups, consistent with the occurrence of divergence due to strong selection. One of these regions, which harbors a major branching locus, spans a surprisingly long genetic interval (ca. 25 cM), indicating the occurrence of an extended selective sweep in an otherwise recombinogenic interval.

Paternal Leakage and Heteroplasmy of Mitochondrial Genomes in Silene vulgaris: Evidence From Experimental Crosses

The inheritance of mitochondrial genetic (mtDNA) markers in the gynodioecious plant Silene vulgaris was studied using a series of controlled crosses between parents of known mtDNA genotype followed by quantitative PCR assays of offspring genotype. Overall, ∼2.5% of offspring derived from crosses between individuals that were homoplasmic for different mtDNA marker genotypes showed evidence of paternal leakage. When the source population of the pollen donor was considered, however, population-specific rates of leakage varied significantly around this value, ranging from 10.3% to zero. When leakage did occur, the paternal contribution ranged from 0.5% in some offspring (i.e., biparental inheritance resulting in a low level of heteroplasmy) to 100% in others. Crosses between mothers known to be heteroplasmic for one of the markers and homoplasmic fathers showed that once heteroplasmy enters a maternal lineage it is retained by ∼17% of offspring in the next generation, but lost from the others. The results are discussed with regard to previous studies of heteroplasmy in open-pollinated natural populations of S. vulgaris and with regard to the potential impact of mitochondrial paternal leakage and heteroplasmy on both the evolution of the mitochondrial genome and the evolution of gynodioecy.

Using phylogenomics to resolve mega‐families: An example from Compositae

Next‐generation sequencing and phylogenomics hold great promise for elucidating complex relationships among large plant families. Here, we performed targeted capture of low copy sequences followed by next‐generation sequencing on the Illumina platform in the large and diverse angiosperm family Compositae (Asteraceae). The family is monophyletic, based on morphology and molecular data, yet many areas of the phylogeny have unresolved polytomies and interpreting phylogenetic patterns has been historically difficult. In order to outline a method and provide a framework and for future phylogenetic studies in the Compositae, we sequenced 23 taxa from across the family in which the relationships were well established as well as a member of the sister family Calyceraceae. We generated nuclear data from 795 loci and assembled chloroplast genomes from off‐target capture reads enabling the comparison of nuclear and chloroplast genomes for phylogenetic analyses. We also analyzed multi‐copy nuclear genes in our data set using a clustering method during orthology detection, and we applied a network approach to these clusters—analyzing all related locus copies. Using these data, we produced hypotheses of phylogenetic relationships employing both a conservative (restricted to only loci with one copy per targeted locus) and a multigene approach (including all copies per targeted locus). The methods and bioinformatics workflow presented here provide a solid foundation for future work aimed at understanding gene family evolution in the Compositae as well as providing a model for phylogenomic analyses in other plant mega‐families.

Ancestral Gene Flow and Parallel Organellar Genome Capture Result in Extreme Phylogenomic Discord in a Lineage of Angiosperms.

Abstract While hybridization has recently received a resurgence of attention from systematists and evolutionary biologists, there remains a dearth of case studies on ancient, diversified hybrid lineages—clades of organisms that originated through reticulation. Studies on these groups are valuable in that they would speak to the long‐term phylogenetic success of lineages following gene flow between species. We present a phylogenomic view of Heuchera, long known for frequent hybridization, incorporating all three independent genomes: targeted nuclear (˜400,000 bp), plastid (˜160,000 bp), and mitochondrial (˜470,000 bp) data. We analyze these data using multiple concatenation and coalescence strategies. The nuclear phylogeny is consistent with previous work and with morphology, confidently suggesting a monophyletic Heuchera. By contrast, analyses of both organellar genomes recover a grossly polyphyletic Heuchera,consisting of three primary clades with relationships extensively rearranged within these as well. A minority of nuclear loci also exhibit phylogenetic discord; yet these topologies remarkably never resemble the pattern of organellar loci and largely present low levels of discord inter alia. Two independent estimates of the coalescent branch length of the ancestor of Heuchera using nuclear data suggest rare or nonexistent incomplete lineage sorting with related clades, inconsistent with the observed gross polyphyly of organellar genomes (confirmed by simulation of gene trees under the coalescent). These observations, in combination with previous work, strongly suggest hybridization as the cause of this phylogenetic discord.

A Protocol for Targeted Enrichment of Intron-Containing Sequence Markers for Recent Radiations: A Phylogenomic Example from Heuchera (Saxifragaceae)

Premise of the study: Phylogenetic inference is moving to large multilocus data sets, yet there remains uncertainty in the choice of marker and sequencing method at low taxonomic levels. To address this gap, we present a method for enriching long loci spanning intron-exon boundaries in the genus Heuchera. Methods: Two hundred seventy-eight loci were designed using a splice-site prediction method combining transcriptomic and genomic data. Biotinylated probes were designed for enrichment of these loci. Reference-based assembly was performed using genomic references; additionally, chloroplast and mitochondrial genomes were used as references for off-target reads. The data were aligned and subjected to coalescent and concatenated phylogenetic analyses to demonstrate support for major relationships. Results: Complete or nearly complete (>99%) sequences were assembled from essentially all loci from all taxa. Aligned introns showed a fourfold increase in divergence as opposed to exons. Concatenated analysis gave decisive support to all nodes, and support was also high and relationships mostly similar in the coalescent analysis. Organellar phylogenies were also well-supported and conflicted with the nuclear signal. Discussion: Our approach shows promise for resolving a recent radiation. Enrichment for introns is highly successful with little or no sequencing dropout at low taxonomic levels despite higher substitution and indel frequencies, and should be exploited in studies of species complexes.

High Genetic Diversity and Low Population Structure in Porter’s Sunflower (Helianthus porteri)

Granite outcrops in the southeastern United States are rare and isolated habitats that support edaphically controlled communities dominated by herbaceous plants. They harbor rare and endemic species that are expected to have low genetic variability and high population structure due to small population sizes and their disjunct habitat. We test this expectation for an annual outcrop endemic, Helianthus porteri (Porters sunflower). Contrary to expectation, H. porteri has relatively high genetic diversity (H e = 0.681) and relatively low genetic structure among the native populations (F ST = 0.077) when compared to 5 other Helianthus species (N = 288; 18 expressed sequence tag-SSR markers). These findings suggest greater gene flow than expected. The potential for gene flow is supported by the analysis of transplant populations established with propagules from a common source in 1959. One population established close to a native population (1.5 km) at the edge of the natural range is genetically similar to and shares rare alleles with the adjacent native population and is distinct from the central source population. In contrast, a transplant population established north of the native range has remained similar to the source population. The relatively high genetic diversity and low population structure of this species, combined with the long-term success of transplanted populations, bode well for its persistence as long as the habitat persists.