A pan-genome data structure induced by pooled sequencing facilitates variant mining in heterogeneous germplasm.

Valuable genetic variation lies unused in gene banks due to the difficulty of exploiting heterogeneous germplasm accessions. Advances in molecular breeding, including transgenics and genome editing, present the opportunity to exploit hidden sequence variation directly. Here we describe the pan-genome data structure induced by whole-genome sequencing of pooled individuals from wild populations of Patellifolia spp., a source of disease resistance genes for the related crop species sugar beet (Beta vulgaris). We represent the pan-genome as a map of reads from pooled sequencing of a heterogeneous population sample to a reference genome, plus a BLAST data base of the mapped reads. We show that this basic data structure can be queried by reference genome position or homology to identify sequence variants present in the wild relative, at genes of agronomic interest in the crop, a process known as allele or variant mining. Further we demonstrate the possibility of cataloging variants in all Patellifolia genomic regions that have corresponding single copy orthologous regions in sugar beet. The data structure, termed a "pooled read archive," can be produced, altered, and queried using standard tools to facilitate discovery of agronomically-important sequence variation. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-022-01308-6.

Integrating Genomic and Phenomic Approaches to Support Plant Genetic Resources Conservation and Use.

Plant genebanks provide genetic resources for breeding and research programs worldwide. These programs benefit from having access to high-quality, standardized phenotypic and genotypic data. Technological advances have made it possible to collect phenomic and genomic data for genebank collections, which, with the appropriate analytical tools, can directly inform breeding programs. We discuss the importance of considering genebank accession homogeneity and heterogeneity in data collection and documentation. Citing specific examples, we describe how well-documented genomic and phenomic data have met or could meet the needs of plant genetic resource managers and users. We explore future opportunities that may emerge from improved documentation and data integration among plant genetic resource information systems.

Exploring the fate of mRNA in aging seeds: protection, destruction, or slow decay?

Seeds exist in the vulnerable state of being unable to repair the chemical degradation all organisms suffer, which slowly ages seeds and eventually results in death. Proposed seed aging mechanisms involve all classes of biological molecules, and degradation of total RNA has been detected contemporaneously with viability loss in dry-stored seeds. To identify changes specific to mRNA, we examined the soybean (Glycine max) seed transcriptome, using new, whole-molecule sequencing technology. We detected strong evidence of transcript fragmentation in 23-year-old, compared with 2-year-old, seeds. Transcripts were broken non-specifically, and greater fragmentation occurred in longer transcripts, consistent with the proposed mechanism of molecular fission by free radical attack at random bases. Seeds died despite high integrity of short transcripts, indicating that functions encoded by short transcripts are not sufficient to maintain viability. This study provides an approach to probe the asymptomatic phase of seed aging, namely by quantifying transcript degradation as a function of storage time.

Phylogeography of the wild and cultivated stimulant plant qat (Catha edulis, Celastraceae) in areas of historical cultivation.

PREMISE OF THE STUDY: Qat (Catha edulis, Celastraceae) is a woody plant species cultivated for its stimulant alkaloids. Qat is important to the economy and culture in large regions of Ethiopia, Kenya, and Yemen. Despite the importance of this species, the wild origins and dispersal of cultivars have only been described in often contradictory historical documents. We examined the wild origins, human-mediated dispersal, and genetic divergence of cultivated qat compared to wild qat. METHODS: We sampled 17 SSR markers and 1561 wild and cultivated individuals across the historical areas of qat cultivation. KEY RESULTS: On the basis of genetic structure inferred using Bayesian and nonparametric methods, two centers of origin in Kenya and one in Ethiopia were found for cultivated qat. The centers of origin in Ethiopia and northeast of Mt. Kenya are the primary sources of cultivated qat genotypes. Qat cultivated in Yemen is derived from Ethiopian genotypes rather than Yemeni wild populations. Cultivated qat with a wild Kenyan origin has not spread to Ethiopia or Yemen, whereas a small minority of qat cultivated in Kenya originated in Ethiopia. Hybrid genotypes with both Ethiopian and Kenyan parentage are present in northern Kenya. CONCLUSIONS: Ethiopian cultivars have diverged from their wild relatives, whereas Kenyan qat has diverged less. This pattern of divergence could be caused by the extinction of the wild-source qat populations in Ethiopia due to deforestation, undersampling, and/or artificial selection for agronomically important traits.

Retention of agronomically important variation in germplasm core collections: implications for allele mining.

The primary targets of allele mining efforts are loci of agronomic importance. Agronomic loci typically exhibit patterns of allelic diversity that are consistent with a history of natural or artificial selection. Natural or artificial selection causes the distribution of genetic diversity at such loci to deviate substantially from the pattern found at neutral loci. The germplasm utilized for allele mining should contain maximum allelic variation at loci of interest, in the smallest possible number of samples. We show that the popular core collection assembly procedure "M" (marker allele richness), which leverages variation at neutral loci, performs worse than random assembly for retaining variation at a locus of agronomic importance in sugar beet (Beta vulgaris L. subsp. vulgaris) that is under selection. We present a corrected procedure ("M+") that outperforms M. An extensive coalescent simulation was performed to demonstrate more generally the retention of neutral versus selected allelic variation in core subsets assembled with M+. A negative correlation in level of allelic diversity between neutral and selected loci was observed in 42% of simulated data sets. When core collection assembly is guided by neutral marker loci, as is the current common practice, enhanced allelic variation at agronomically important loci should not necessarily be expected.

Species delimitation under the general lineage concept: an empirical example using wild North American hops (Cannabaceae: Humulus lupulus).

There is an emerging consensus that the intent of most species concepts is to identify evolutionarily distinct lineages. However, the criteria used to identify lineages differ among concepts depending on the perceived importance of various attributes of evolving populations. We have examined five different species criteria to ask whether the three taxonomic varieties of Humulus lupulus (hops) native to North America are distinct lineages. Three criteria (monophyly, absence of genetic intermediates, and diagnosability) focus on evolutionary patterns and two (intrinsic reproductive isolation and niche specialization) consider evolutionary processes. Phylogenetic analysis of amplified fragment length polymorphism (AFLP) data under a relaxed molecular clock, a stochastic Dollo substitution model, and parsimony identified all varieties as monophyletic, thus they satisfy the monophyly criterion for species delimitation. Principal coordinate analysis and a Bayesian assignment procedure revealed deep genetic subdivisions and little admixture between varieties, indicating an absence of genetic intermediates and compliance with the genotypic cluster species criterion. Diagnostic morphological and AFLP characters were found for all varieties, thus they meet the diagnosability criterion. Natural history information suggests that reproductive isolating barriers may have evolved in var. pubescens, potentially qualifying it as a species under a criterion of intrinsic reproductive isolation. Environmental niche modeling showed that the preferred habitat of var. neomexicanus is climatically unique, suggesting niche specialization and thus compliance with an ecological species criterion. Isolation by distance coupled with imperfect sampling can lead to erroneous lineage identification using some species criteria. Compliance with complementary pattern- and process-oriented criteria provides powerful corroboration for a species hypothesis and mitigates the necessity for comprehensive sampling of the entire species range, a practical impossibility in many systems. We hypothesize that var. pubescens maintains its genetic identity, despite substantial niche overlap with var. lupuloides, via the evolution of partial reproductive isolating mechanisms. Variety neomexicanus, conversely, will likely persist as a distinct lineage, regardless of limited gene flow with vars. lupuloides and pubescens because of ecological isolation--adaptation to the unique conditions of the Rocky Mountain cordillera. Thus, we support recognition of vars. neomexicanus and pubescens as species, but delay making a recommendation for var. lupuloides until sampling of genetic variation is complete or a stable biological process can be identified to explain its observed genetic divergence.

Accurate inference of subtle population structure (and other genetic discontinuities) using principal coordinates.

BACKGROUND: Accurate inference of genetic discontinuities between populations is an essential component of intraspecific biodiversity and evolution studies, as well as associative genetics. The most widely-used methods to infer population structure are model-based, Bayesian MCMC procedures that minimize Hardy-Weinberg and linkage disequilibrium within subpopulations. These methods are useful, but suffer from large computational requirements and a dependence on modeling assumptions that may not be met in real data sets. Here we describe the development of a new approach, PCO-MC, which couples principal coordinate analysis to a clustering procedure for the inference of population structure from multilocus genotype data. METHODOLOGY/PRINCIPAL FINDINGS: PCO-MC uses data from all principal coordinate axes simultaneously to calculate a multidimensional "density landscape", from which the number of subpopulations, and the membership within subpopulations, is determined using a valley-seeking algorithm. Using extensive simulations, we show that this approach outperforms a Bayesian MCMC procedure when many loci (e.g. 100) are sampled, but that the Bayesian procedure is marginally superior with few loci (e.g. 10). When presented with sufficient data, PCO-MC accurately delineated subpopulations with population F(st) values as low as 0.03 (G'(st)>0.2), whereas the limit of resolution of the Bayesian approach was F(st) = 0.05 (G'(st)>0.35). CONCLUSIONS/SIGNIFICANCE: We draw a distinction between population structure inference for describing biodiversity as opposed to Type I error control in associative genetics. We suggest that discrete assignments, like those produced by PCO-MC, are appropriate for circumscribing units of biodiversity whereas expression of population structure as a continuous variable is more useful for case-control correction in structured association studies.

Distinguishing terminal monophyletic groups from reticulate taxa: performance of phenetic, tree-based, and network procedures.

Hybridization is a well-documented, natural phenomenon that is common at low taxonomic levels in the higher plants and other groups. In spite of the obvious potential for gene flow via hybridization to cause reticulation in an evolutionary tree, analytical methods based on a strictly bifurcating model of evolution have frequently been applied to data sets containing taxa known to hybridize in nature. Using simulated data, we evaluated the relative performance of phenetic, tree-based, and network approaches for distinguishing between taxa with known reticulate history and taxa that were true terminal monophyletic groups. In all methods examined, type I error (the erroneous rejection of the null hypothesis that a taxon of interest is not monophyletic) was likely during the early stages of introgressive hybridization. We used the gradual erosion of type I error with continued gene flow as a metric for assessing relative performance. Bifurcating tree-based methods performed poorly, with highly supported, incorrect topologies appearing during some phases of the simulation. Based on our model, we estimate that many thousands of gene flow events may be required in natural systems before reticulate taxa will be reliably detected using tree-based methods of phylogeny reconstruction. We conclude that the use of standard bifurcating tree-based methods to identify terminal monophyletic groups for the purposes of defining or delimiting phylogenetic species, or for prioritizing populations for conservation purposes, is difficult to justify when gene flow between sampled taxa is possible. As an alternative, we explored the use of two network methods. Minimum spanning networks performed worse than most tree-based methods and did not yield topologies that were easily interpretable as phylogenies. The performance of NeighborNet was comparable to parsimony bootstrap analysis. NeighborNet and reverse successive weighting were capable of identifying an ephemeral signature of reticulate evolution during the early stages of introgression by revealing conflicting phylogenetic signal. However, when gene flow was topologically complex, the conflicting phylogenetic signal revealed by these methods resulted in a high probability of type II error (inferring that a monophyletic taxon has a reticulate history). Lastly, we present a novel application of an existing nonparametric clustering procedure that, when used against a density landscape derived from principal coordinate data, showed superior performance to the tree-based and network procedures tested.

Evolutionary conservation of the FLOWERING LOCUS C-mediated vernalization response: evidence from the sugar beet (Beta vulgaris).

In many plant species, exposure to a prolonged period of cold during the winter promotes flowering in the spring, a process termed vernalization. In Arabidopsis thaliana, the vernalization requirement of winter-annual ecotypes is caused by the MADS-box gene FLOWERING LOCUS C (FLC), which is a repressor of flowering. During the vernalization process, FLC is downregulated by alteration of its chromatin structure, thereby permitting flowering to occur. In wheat, a vernalization requirement is imposed by a different repressor of flowering, suggesting that some components of the regulatory network controlling the vernalization response differ between monocots and dicots. The extent to which the molecular mechanisms underlying vernalization have been conserved during the diversification of the angiosperms is not well understood. Using phylogenetic analysis, we identified homologs of FLC in species representing the three major eudicot lineages. FLC homologs have not previously been documented outside the plant family Brassicaceae. We show that the sugar beet FLC homolog BvFL1 functions as a repressor of flowering in transgenic Arabidopsis and is downregulated in response to cold in sugar beet. Cold-induced downregulation of an FLC-like floral repressor may be a central feature of the vernalization response in at least half of eudicot species.

wolfPAC: building a high-performance distributed computing network for phylogenetic analysis using 'obsolete' computational resources.

wolfPAC is an AppleScript-based software package that facilitates the use of numerous, remotely located Macintosh computers to perform computationally-intensive phylogenetic analyses using the popular application PAUP* (Phylogenetic Analysis Using Parsimony). It has been designed to utilise readily available, inexpensive processors and to encourage sharing of computational resources within the worldwide phylogenetics community.

Rapid speciation and the evolution of hummingbird pollination in neotropical Costus subgenus Costus (Costaceae): evidence from nrDNA ITS and ETS sequences.

We estimate phylogenetic relationships and the biogeographic and pollination history of Costus subgenus Costus (Costaceae) using sequence data from the internal and external transcribed spacer (ITS and ETS) regions of 18S-26S nuclear ribosomal DNA. The African members of the subgenus form a series of lineages basal to a monophyletic neotropical species radiation. The neotropical species have large, showy flowers visited by either euglossine bees or hummingbirds. The hummingbird pollination syndrome is supported as a derived character state from the bee pollination syndrome, and we estimate that it has evolved independently seven or more times in the neotropics. A molecular clock approach suggests that diversification of the neotropical clade has been recent and rapid and that it coincides with dramatic climatic and geologic changes, Andean orogeny, and the closing of the Panama isthmus that occurred in the Pliocene and Pleistocene epochs. We propose a scenario for the diversification of Costus, in which rapid floral adaptation in geographic isolation and range shifts in response to environmental changes contribute to reproductive isolation among close relatives. We suggest that these processes may be common in other recently diversified plant lineages centered in Central America or the Northern Andean phytogeographic region.

Inference of higher-order relationships in the cycads from a large chloroplast data set.

We investigated higher-order relationships in the cycads, an ancient group of seed-bearing plants, by examining a large portion of the chloroplast genome from seven species chosen to exemplify our current understanding of taxonomic diversity in the order. The regions considered span approximately 13.5 kb of unaligned data per taxon, and comprise a diverse range of coding sequences, introns and intergenic spacers dispersed throughout the plastid genome. Our results provide substantial support for most of the inferred backbone of cycad phylogeny, and weak evidence that the sister-group of the cycads among living seed plants is Ginkgo biloba. Cycas (representing Cycadaceae) is the sister-group of the remaining cycads; Dioon is part of the next most basal split. Two of the three commonly recognized families of cycads (Zamiaceae and Stangeriaceae) are not monophyletic; Stangeria is embedded within Zamiaceae, close to Zamia and Ceratozamia, and not closely allied to the other genus of Stangeriaceae, Bowenia. In contrast to the other seed plants, cycad chloroplast genomes share two features with Ginkgo: a reduced rate of evolution and an elevated transition:transversion ratio. We demonstrate that the latter aspect of their molecular evolution is unlikely to have affected inference of cycad relationships in the context of seed-plant wide analyses.

Evolution of the TCP gene family in Asteridae: cladistic and network approaches to understanding regulatory gene family diversification and its impact on morphological evolution.

In the plant subclass Asteridae, bilaterally symmetrical flowers have evolved from a radially symmetrical ancestral phenotype on at least three independent occasions: in the Boraginaceae, Solanaceae, and Lamiales. Development of bilateral flower symmetry has been shown to be determined by the early-acting cycloidea (cyc) and dichotoma (dich) genes in Antirrhinum, a member of the Lamiales. cyc and dich belong to the TCP gene family of putative transcription factors. TCP gene sequences were isolated from 11 Asteridae taxa using an array of degenerate PCR primers. Closely related species exhibiting either ancestral actinomorphic or derived zygomorphic flowers were sampled for each independent origin of bilateral flower symmetry. Cladistic and network-based analyses were performed to establish viable hypotheses regarding the evolution of bilateral symmetry in Asteridae. For the TCP gene family, the use of cladistic phylogenetic analysis to identify orthologous genes is complicated by a paucity of alignable data, frequent gene duplication and extinction, and the possibility of reticulate evolution via intergenic recombination. These complicating factors can be generalized to many regulatory gene families. As an alternative to cladistic analysis, we propose the use of network analysis for the reconstruction of regulatory gene family phylogenetic and functional relationships. Results of analyses support the hypothesis that the origin of bilaterally symmetrical flowers in the Boraginaceae and Solanaceae did not require orthologs or functional analogs of cyc or dich. This suggests that the genetic mechanism that determines bilateral flower symmetry in these taxa is not homologous to that of the Lamiales. Results of analyses are consistent with the hypothesis that the evolution of bilateral floral symmetry in the Lamiales required the origin of a novel gene function subsequent to gene duplication.