Patterns of Volatile Diversity Yield Insights Into the Genetics and Biochemistry of the Date Palm Fruit Volatilome.

Volatile organic compounds are key components of the fruit metabolome that contribute to traits such as aroma and taste. Here we report on the diversity of 90 flavor-related fruit traits in date palms (Phoenix dactylifera L.) including 80 volatile organic compounds, which collectively represent the fruit volatilome, as well as 6 organic acids, and 4 sugars in tree-ripened fruits. We characterize these traits in 148 date palms representing 135 varieties using headspace solid-phase microextraction gas chromatography. We discovered new volatile compounds unknown in date palm including 2-methoxy-4-vinylphenol, an attractant of the red palm weevil (Rhynchophorus ferrugineus Olivier), a key pest that threatens the date palm crop. Associations between volatile composition and sugar and moisture content suggest that differences among fruits in these traits may be characterized by system-wide differences in fruit metabolism. Correlations between volatiles indicate medium chain and long chain fatty acid ester volatiles are regulated independently, possibly reflecting differences in the biochemistry of fatty acid precursors. Finally, we took advantage of date palm clones in our analysis to estimate broad-sense heritabilities of volatiles and demonstrate that at least some of volatile diversity has a genetic basis.

Molecular Clocks and Archeogenomics of a Late Period Egyptian Date Palm Leaf Reveal Introgression from Wild Relatives and Add Timestamps on the Domestication.

The date palm, Phoenix dactylifera, has been a cornerstone of Middle Eastern and North African agriculture for millennia. It was first domesticated in the Persian Gulf, and its evolution appears to have been influenced by gene flow from two wild relatives, P. theophrasti, currently restricted to Crete and Turkey, and P. sylvestris, widespread from Bangladesh to the West Himalayas. Genomes of ancient date palm seeds show that gene flow from P. theophrasti to P. dactylifera may have occurred by ∼2,200 years ago, but traces of P. sylvestris could not be detected. We here integrate archeogenomics of a ∼2,100-year-old P. dactylifera leaf from Saqqara (Egypt), molecular-clock dating, and coalescence approaches with population genomic tests, to probe the hybridization between the date palm and its two closest relatives and provide minimum and maximum timestamps for its reticulated evolution. The Saqqara date palm shares a close genetic affinity with North African date palm populations, and we find clear genomic admixture from both P. theophrasti, and P. sylvestris, indicating that both had contributed to the date palm genome by 2,100 years ago. Molecular-clocks placed the divergence of P. theophrasti from P. dactylifera/P. sylvestris and that of P. dactylifera from P. sylvestris in the Upper Miocene, but strongly supported, conflicting topologies point to older gene flow between P. theophrasti and P. dactylifera, and P. sylvestris and P. dactylifera. Our work highlights the ancient hybrid origin of the date palms, and prompts the investigation of the functional significance of genetic material introgressed from both close relatives, which in turn could prove useful for modern date palm breeding.

The genomes of ancient date palms germinated from 2,000 y old seeds.

Seven date palm seeds (Phoenix dactylifera L.), radiocarbon dated from the fourth century BCE to the second century CE, were recovered from archaeological sites in the Southern Levant and germinated to yield viable plants. We conducted whole-genome sequencing of these germinated ancient samples and used single-nucleotide polymorphism data to examine the genetics of these previously extinct Judean date palms. We find that the oldest seeds from the fourth to first century BCE are related to modern West Asian date varieties, but later material from the second century BCE to second century CE showed increasing genetic affinities to present-day North African date palms. Population genomic analysis reveals that by ∼2,400 to 2,000 y ago, the P. dactylifera gene pool in the Eastern Mediterranean already contained introgressed segments from the Cretan palm Phoenix theophrasti, a crucial genetic feature of the modern North African date palm populations. The P. theophrasti introgression fraction content is generally higher in the later samples, while introgression tracts are longer in these ancient germinated date palms compared to modern North African varieties. These results provide insights into crop evolution arising from an analysis of plants originating from ancient germinated seeds and demonstrate what can be accomplished with the application of a resurrection genomics approach.

Prospects for the Study and Improvement of Abiotic Stress Tolerance in Date Palms in the Post-genomics Era.

Date palm (Phoenix dactylifera L.) is a socio-economically important crop in the Middle East and North Africa and a major contributor to food security in arid regions of the world. P. dactylifera is both drought and salt tolerant, but recent water shortages and increases in groundwater and soil salinity have threatened the continued productivity of the crop. Recent studies of date palm have begun to elucidate the physiological mechanisms of abiotic stress tolerance and the genes and biochemical pathways that control the response to these stresses. Here we review recent studies on tolerance of date palm to salinity and drought stress, the role of the soil and root microbiomes in abiotic stress tolerance, and highlight recent findings of omic-type studies. We present a perspective on future research of abiotic stress in date palm that includes improving existing genome resources, application of genetic mapping to determine the genetic basis of variation in tolerances among cultivars, and adoption of gene-editing technologies to the study of abiotic stress in date palms. Development of necessary resources and application of the proposed methods will provide a foundation for future breeders and genetic engineers aiming to develop more stress-tolerant cultivars of date palm.

Genome-wide association mapping of date palm fruit traits.

Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.

Origins and geographic diversification of African rice (Oryza glaberrima).

Rice is a staple food for the majority of the world's population. Whereas Asian rice (Oryza sativa) has been extensively studied, the exact origins of African rice (Oryza glaberrima) are still contested. Previous studies have supported either a centric or a non-centric geographic origin of African rice domestication. Here we review the evidence for both scenarios through a critical reassessment of 206 whole genome sequences of domesticated and wild African rice. While genetic diversity analyses support a severe bottleneck caused by domestication, signatures of recent and strong positive selection do not unequivocally point to candidate domestication genes, suggesting that domestication proceeded differently than in Asian rice-either by selection on different alleles, or different modes of selection. Population structure analysis revealed five genetic clusters localising to different geographic regions. Isolation by distance was identified in the coastal populations, which could account for parallel adaptation in geographically separated demes. Although genome-wide phylogenetic relationships support an origin in the eastern cultivation range followed by diversification along the Atlantic coast, further analysis of domestication genes shows distinct haplotypes in the southwest-suggesting that at least one of several key domestication traits might have originated there. These findings shed new light on an old controversy concerning plant domestication in Africa by highlighting the divergent roots of African rice cultivation, including a separate centre of domestication activity in the Guinea Highlands. We thus suggest that the commonly accepted centric origin of African rice must be reconsidered in favour of a non-centric or polycentric view.

Cross-species hybridization and the origin of North African date palms.

Date palm (Phoenix dactylifera L.) is a major fruit crop of arid regions that were domesticated ∼7,000 y ago in the Near or Middle East. This species is cultivated widely in the Middle East and North Africa, and previous population genetic studies have shown genetic differentiation between these regions. We investigated the evolutionary history of P. dactylifera and its wild relatives by resequencing the genomes of date palm varieties and five of its closest relatives. Our results indicate that the North African population has mixed ancestry with components from Middle Eastern P. dactylifera and Phoenix theophrasti, a wild relative endemic to the Eastern Mediterranean. Introgressive hybridization is supported by tests of admixture, reduced subdivision between North African date palm and P. theophrasti, sharing of haplotypes in introgressed regions, and a population model that incorporates gene flow between these populations. Analysis of ancestry proportions indicates that as much as 18% of the genome of North African varieties can be traced to P. theophrasti and a large percentage of loci in this population are segregating for single-nucleotide polymorphisms (SNPs) that are fixed in P. theophrasti and absent from date palm in the Middle East. We present a survey of Phoenix remains in the archaeobotanical record which supports a late arrival of date palm to North Africa. Our results suggest that hybridization with P. theophrasti was of central importance in the diversification history of the cultivated date palm.

Tracing ancestor rice of Suriname Maroons back to its African origin.

African rice (Oryza glaberrima) and African cultivation practices are said to have influenced emerging colonial plantation economies in the Americas1,2. However, the level of impact of African rice practices is difficult to establish because of limited written or botanical records2,3. Recent findings of O. glaberrima in rice fields of Suriname Maroons bear evidence of the high level of knowledge about rice among African slaves and their descendants, who consecrate it in ancestor rituals4,5. Here we establish the strong similarity, and hence likely origin, of the first extant New World landrace of O. glaberrima to landraces from the Upper Guinean forests in West Africa. We collected African rice from a Maroon market in Paramaribo, Suriname, propagated it, sequenced its genome6 and compared it with genomes of 109 accessions representing O. glaberrima diversity across West Africa. By analysing 1,649,769 single nucleotide polymorphisms (SNPs) in clustering analyses, the Suriname sample appears sister to an Ivory Coast landrace, and shows no evidence of introgression from Asian rice. Whereas the Dutch took most slaves from Ghana, Benin and Central Africa7, the diaries of slave ship captains record the purchase of food for provisions when sailing along the West African Coast8, offering one possible explanation for the patterns of genetic similarity. This study demonstrates the utility of genomics in understanding the largely unwritten histories of crop cultures of diaspora communities.

Cryptic Genetic Variation for Arabidopsis thaliana Seed Germination Speed in a Novel Salt Stress Environment.

The expansion of species ranges frequently necessitates responses to novel environments. In plants, the ability of seeds to disperse to marginal areas relies in part to its ability to germinate under stressful conditions. Here we examine the genetic architecture of Arabidopsis thaliana germination speed under a novel, saline environment, using an Extreme QTL (X-QTL) mapping platform we previously developed. We find that early germination in normal and salt conditions both rely on a QTL on the distal arm of chromosome 4, but we also find unique QTL on chromosomes 1, 2, 4, and 5 that are specific to salt stress environments. Moreover, different QTLs are responsible for early vs. late germination, suggesting a temporal component to the expression of life history under these stress conditions. Our results indicate that cryptic genetic variation exists for responses to a novel abiotic stress, which may suggest a role of such variation in adaptation to new climactic conditions or growth environments.

Domestication history and geographical adaptation inferred from a SNP map of African rice.

African rice (Oryza glaberrima Steud.) is a cereal crop species closely related to Asian rice (Oryza sativa L.) but was independently domesticated in West Africa ∼3,000 years ago. African rice is rarely grown outside sub-Saharan Africa but is of global interest because of its tolerance to abiotic stresses. Here we describe a map of 2.32 million SNPs of African rice from whole-genome resequencing of 93 landraces. Population genomic analysis shows a population bottleneck in this species that began ∼13,000-15,000 years ago with effective population size reaching its minimum value ∼3,500 years ago, suggesting a protracted period of population size reduction likely commencing with predomestication management and/or cultivation. Genome-wide association studies (GWAS) for six salt tolerance traits identify 11 significant loci, 4 of which are within ∼300 kb of genomic regions that possess signatures of positive selection, suggesting adaptive geographical divergence for salt tolerance in this species.

Extreme QTL mapping of germination speed in Arabidopsis thaliana.

Seed germination is a key life history transition for annual plants and partly determines lifetime performance and fitness. Germination speed, the elapsed time for a nondormant seed to germinate, is a poorly understood trait important for plants' competitiveness and fitness in fluctuating environments. Germination speed varied by 30% among 18 Arabidopsis thaliana populations measured, and exhibited weak negative correlation with flowering time and seed weight, with significant genotype effect (P < 0.005). To dissect the genetic architecture of germination speed, we developed the extreme QTL (X-QTL) mapping method in A. thaliana. The method has been shown in yeast to increase QTL mapping power by integrating selective screening and bulk-segregant analysis in a very large mapping population. By pooled genotyping of top 5% of rapid germinants from ~100 000 F3 individuals, three X-QTL regions were identified on chromosomes 1, 3 and 4. All regions were confirmed as QTL regions by sequencing 192 rapid germinants from an independent F3 selection experiment. Positional overlaps were found between X-QTLs and previously identified seed, life history and fitness QTLs. Our method provides a rapid mapping platform in A. thaliana with potentially greater power. One can also relate identified X-QTLs to the A. thaliana physical map, facilitating candidate gene identification.

Developing maps of fitness consequences for plant genomes.

Predicting the fitness consequences of mutations, and their concomitant impacts on molecular and cellular function as well as organismal phenotypes, is an important challenge in biology that has new relevance in an era when genomic data is readily available. The ability to construct genomewide maps of fitness consequences in plant genomes is a recent development that has profound implications for our ability to predict the fitness effects of mutations and discover functional elements. Here we highlight approaches to building fitness consequence maps to infer regions under selection. We emphasize computational methods applied primarily to the study of human disease that translate physical maps of within-species genome variation into maps of fitness effects of individual natural mutations. Maps of fitness consequences in plants, combined with traditional genetic approaches, could accelerate discovery of functional elements such as regulatory sequences in non-coding DNA and genetic polymorphisms associated with key traits, including agronomically-important traits such as yield and environmental stress responses.

Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop.

Date palms (Phoenix dactylifera) are the most significant perennial crop in arid regions of the Middle East and North Africa. Here, we present a comprehensive catalogue of approximately seven million single nucleotide polymorphisms in date palms based on whole genome re-sequencing of a collection of 62 cultivars. Population structure analysis indicates a major genetic divide between North Africa and the Middle East/South Asian date palms, with evidence of admixture in cultivars from Egypt and Sudan. Genome-wide scans for selection suggest at least 56 genomic regions associated with selective sweeps that may underlie geographic adaptation. We report candidate mutations for trait variation, including nonsense polymorphisms and presence/absence variation in gene content in pathways for key agronomic traits. We also identify a copia-like retrotransposon insertion polymorphism in the R2R3 myb-like orthologue of the oil palm virescens gene associated with fruit colour variation. This analysis documents patterns of post-domestication diversification and provides a genomic resource for this economically important perennial tree crop.

Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii.

We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.

Variation in Arabidopsis flowering time associated with cis-regulatory variation in CONSTANS.

The onset of flowering, the change from vegetative to reproductive development, is a major life history transition in flowering plants. Recent work suggests that mutations in cis-regulatory mutations should play critical roles in the evolution of this (as well as other) important adaptive traits, but thus far there has been little evidence that directly links regulatory mutations to evolutionary change at the species level. While several genes have previously been shown to affect natural variation in flowering time in Arabidopsis thaliana, most either show protein-coding changes and/or are found at low frequency (<5%). Here we identify and characterize natural variation in the cis-regulatory sequence in the transcription factor CONSTANS that underlies flowering time diversity in Arabidopsis. Mutation in this regulatory motif evolved recently and has spread to high frequency in Arabidopsis natural accessions, suggesting a role for these cis-regulatory changes in adaptive variation of flowering time.

Possible loss of the chloroplast genome in the parasitic flowering plant Rafflesia lagascae (Rafflesiaceae).

Rafflesia is a genus of holoparasitic plants endemic to Southeast Asia that has lost the ability to undertake photosynthesis. With short-read sequencing technology, we assembled a draft sequence of the mitochondrial genome of Rafflesia lagascae Blanco, a species endemic to the Philippine island of Luzon, with ∼350× sequencing depth coverage. Using multiple approaches, however, we were only able to identify small fragments of plastid sequences at low coverage depth (<2×) and could not recover any substantial portion of a chloroplast genome. The gene fragments we identified included photosynthesis and energy production genes (atp, ndh, pet, psa, psb, rbcL), ribosomal RNA genes (rrn16, rrn23), ribosomal protein genes (rps7, rps11, rps16), transfer RNA genes, as well as matK, accD, ycf2, and multiple nongenic regions from the inverted repeats. None of the identified plastid gene sequences had intact reading frames. Phylogenetic analysis suggests that ∼33% of these remnant plastid genes may have been horizontally transferred from the host plant genus Tetrastigma with the rest having ambiguous phylogenetic positions (<50% bootstrap support), except for psaB that was strongly allied with the plastid homolog in Nicotiana. Our inability to identify substantial plastid genome sequences from R. lagascae using multiple approaches--despite success in identifying and developing a draft assembly of the much larger mitochondrial genome--suggests that the parasitic plant genus Rafflesia may be the first plant group for which there is no recognizable plastid genome, or if present is found in cryptic form at very low levels.

Molecular relationships between Australian annual wild rice, Oryza meridionalis, and two related perennial forms.

BACKGROUND: The perennial, Oryza rufipogon distributed from Asia to Australia and the annual O. meridionalis indigenous to Australia are AA genome species in the Oryza. However, recent research has demonstrated that the Australian AA genome perennial populations have maternal genomes more closely related to those of O. meridionalis than to those found in Asian populations of O. rufipogon suggesting that the Australian perennials may represent a new distinct gene pool for rice. RESULTS: Analysis of an Oryza core collection covering AA genome species from Asia to Oceania revealed that some Oceania perennials had organellar genomes closely related to that of O meridionalis (meridionalis-type). O. rufipogon accessions from New Guinea carried either the meridionalis-type or rufirpogon-type (like O. rufipogon) organellar genomes. Australian perennials carried only the meridionalis-type organellar genomes when accompanied by the rufipogon-type nuclear genome. New accessions were collected to better characterize the Australian perennials, and their life histories (annual or perennial) were confirmed by field observations. All of the material collected carried only meridionalis-type organellar genomes. However, there were two distinct perennial groups. One of them carried an rufipogon-type nuclear genome similar to the Australian O. rufipogon in the core collection and the other carried an meridionalis-type nuclear genome not represented in the existing collection. Morphologically the rufipogon-type shared similarity with Asian O. rufipogon. The meridionalis-type showed some similarities to O. meridionalis such as the short anthers usually characteristic of annual populations. However, the meridionalis-type perennial was readily distinguished from O. meridionalis by the presence of a larger lemma and higher number of spikelets. CONCLUSION: Analysis of current accessions clearly indicated that there are two distinct types of Australian perennials. Both of them differed genetically from Asian O. rufipogon. One lineage is closely related to O. meridionalis and another to Asian O. rufipogon. The first was presumed to have evolved by divergence from O. meridionalis becoming differentiated as a perennial species in Australia indicating that it represents a new gene pool. The second, apparently derived from Asian O. rufipogon, possibly arrived in Australia later.

Regional modulation of a stochastically expressed factor determines photoreceptor subtypes in the Drosophila retina.

Stochastic mechanisms are sometimes utilized to diversify cell fates, especially in nervous systems. In the Drosophila retina, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) controls photoreceptor subtype choice. In one randomly distributed subset of R7 photoreceptors, Ss activates Rhodopsin4 (Rh4) and represses Rhodopsin3 (Rh3); counterparts lacking Ss express Rh3 and repress Rh4. In the dorsal third region of the retina, the Iroquois Complex transcription factors induce Rh3 in Rh4-expressing R7s. Here, we show that Ss levels are controlled in a binary on/off manner throughout the retina yet are attenuated in the dorsal third region to allow Rh3 coexpression with Rh4. Whereas the sensitivity of rh3 repression to differences in Ss levels generates stochastic and regionalized patterns, the robustness of rh4 activation ensures its stochastic expression throughout the retina. Our findings show how stochastic and regional inputs are integrated to control photoreceptor subtype specification in the Drosophila retina.

Diversifying selection underlies the origin of allozyme polymorphism at the phosphoglucose isomerase locus in Tigriopus californicus.

The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.

Phylogeography of Asian wild rice, Oryza rufipogon: a genome-wide view.

Asian wild rice (Oryza rufipogon) that ranges widely across the eastern and southern part of Asia is recognized as the direct ancestor of cultivated Asian rice (O. sativa). Studies of the geographic structure of O. rufipogon, based on chloroplast and low-copy nuclear markers, reveal a possible phylogeographic signal of subdivision in O. rufipogon. However, this signal of geographic differentiation is not consistently observed among different markers and studies, with often conflicting results. To more precisely characterize the phylogeography of O. rufipogon populations, a genome-wide survey of unlinked markers, intensively sampled from across the entire range of O. rufipogon is critical. In this study, we surveyed sequence variation at 42 genome-wide sequence tagged sites (STS) in 108 O. rufipogon accessions from throughout the native range of the species. Using Bayesian clustering, principal component analysis and amova, we conclude that there are two genetically distinct O. rufipogon groups, Ruf-I and Ruf-II. The two groups exhibit a clinal variation pattern generally from north-east to south-west. Different from many earlier studies, Ruf-I, which is found mainly in China and the Indochinese Peninsula, shows genetic similarity with one major cultivated rice variety, O. satvia indica, whereas Ruf-II, mainly from South Asia and the Indochinese Peninsula, is not found to be closely related to cultivated rice varieties. The other major cultivated rice variety, O. sativa japonica, is not found to be similar to either O. rufipogon groups. Our results support the hypothesis of a single origin of the domesticated O. sativa in China. The possible role of palaeoclimate, introgression and migration-drift balance in creating this clinal variation pattern is also discussed.