Oliver Gailing

A fast and cost-effective approach to develop and map EST-SSR markers: oak as a case study

BackgroundExpressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut).ResultsA catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher.ConclusionWe have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

Patterns of contemporary hybridization inferred from paternity analysis in a four-oak-species forest

BackgroundFew studies address the issue of hybridization in a more than two-species context. The species-rich Quercus complex is one of the systems which can offer such an opportunity. To investigate the contemporary pattern of hybridization we sampled and genotyped 320 offspring from a natural mixed forest comprising four species of the European white oak complex: Quercus robur, Q. petraea, Q. pubescens, and Q. frainetto.ResultsA total of 165 offspring were assigned unambiguously to one of the pollen donors within the study plot. The minimum amount of effective pollen originating from outside the plot varied markedly among the seed parents, ranging from 0.18 to 0.87. The majority of the successful matings (64.1%) occurred between conspecific individuals indicating the existence of reproductive barriers between oak species. However, the isolation was not complete since we found strong evidence for both first-generation (8.4%) and later-generation hybrids (27.5%). Only two out of eight seed parents, belonging to Q. petraea and Q. robur, showed a high propensity to hybridize with Q. pubescens and Q. petraea, respectively. Significant structure of the effective pollen pools (Φpt= 0.069, P = 0.01) was detected in our sample. However, no support was found for the isolation by distance hypothesis. The proportion of hybrids was much higher (79%) in the seed generation when compared to the adult tree generation.ConclusionFirst-generation hybrids were observed only between three out of six possible species combinations. Hybrids between one pair of species preferred to mate with one of their parental species. The observation of first and later-generation hybrids in higher frequency in acorns than in adults might be explained by selection against hybrid genotypes, the history of this uneven-aged forest or past introgression between species.

Genetic and genomic approaches to assess adaptive genetic variation in plants: forest trees as a model.

With the increasing availability of sequence information at putatively important genes or regulatory regions, the characterization of adaptive genetic diversity and their association with phenotypic trait variation becomes feasible for many non-model organisms such as forest trees. Especially in predominantly outcrossing forest tree populations with large effective size, a high genetic variation in relevant genes is maintained, that is the raw material for the adaptation to changing and variable environments, and likewise for plant breeding. Oaks (Quercus spp.) are excellent model species to study the adaptation of forest trees to changing environments. They show a wide geographic distribution in Europe as dominant tree species in many forests and grow under a wide range of climatic and edaphic conditions. With the availability of a growing amount of functional and expressional candidate genes, we are now able to test the functional importance of single nucleotide polymorphisms (SNPs) by associating nucleotide variation in these genes with phenotypic variation in adaptive traits in segregating or natural populations. Here, we report on quantitative trait locus (QTL), candidate gene and association mapping approaches that are applicable to characterize gene markers and SNPs associated with variation in adaptive traits, such as bud burst, drought resistance and other traits showing selective responses to environmental change and stress. Because genome-wide association mapping studies are not feasible because of the enormous amount of SNP markers required in outcrossing trees with high recombination rates, the success of such an approach depends largely on the reasonable selection of candidate genes.

Comparative mapping in the Fagaceae and beyond with EST-SSRs

BackgroundGenetic markers and linkage mapping are basic prerequisites for comparative genetic analyses, QTL detection and map-based cloning. A large number of mapping populations have been developed for oak, but few gene-based markers are available for constructing integrated genetic linkage maps and comparing gene order and QTL location across related species.ResultsWe developed a set of 573 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and located 397 markers (EST-SSRs and genomic SSRs) on the 12 oak chromosomes (2n = 2x = 24) on the basis of Mendelian segregation patterns in 5 full-sib mapping pedigrees of two species: Quercus robur (pedunculate oak) and Quercus petraea (sessile oak). Consensus maps for the two species were constructed and aligned. They showed a high degree of macrosynteny between these two sympatric European oaks. We assessed the transferability of EST-SSRs to other Fagaceae genera and a subset of these markers was mapped in Castanea sativa, the European chestnut. Reasonably high levels of macrosynteny were observed between oak and chestnut. We also obtained diversity statistics for a subset of EST-SSRs, to support further population genetic analyses with gene-based markers. Finally, based on the orthologous relationships between the oak, Arabidopsis, grape, poplar, Medicago, and soybean genomes and the paralogous relationships between the 12 oak chromosomes, we propose an evolutionary scenario of the 12 oak chromosomes from the eudicot ancestral karyotype.ConclusionsThis study provides map locations for a large set of EST-SSRs in two oak species of recognized biological importance in natural ecosystems. This first step toward the construction of a gene-based linkage map will facilitate the assignment of future genome scaffolds to pseudo-chromosomes. This study also provides an indication of the potential utility of new gene-based markers for population genetics and comparative mapping within and beyond the Fagaceae.

Molecular genetic tools to infer the origin of forest plants and wood

Most forest tree species exhibit high levels of genetic diversity that can be used to trace the origin of living plants or their products such as timber and processed wood. Recent progress to isolate DNA not only from living tissue but also from wood and wood products offers new opportunities to test the declared origin of material such as seedlings for plantation establishment or timber. However, since most forest tree populations are weakly differentiated, the identification of genetic markers to differentiate among spatially isolated populations is often difficult and time consuming. Two important fields of “forensic” applications are described: Molecular tools are applied to test the declared origin of forest reproductive material used for plantation establishment and of internationally traded timber and wood products. These applications are illustrated taking examples from Germany, where mechanisms have been developed to improve the control of the trade with forest seeds and seedlings, and from the trade with wood of the important Southeast Asian tree family Dipterocarpaceae. Prospects and limitations of the use of molecular genetic methods to conclude on the origin of forest plants, wood, and wood products are discussed.

Genetic structure of Quercus rubra L. and Quercus ellipsoidalis E. J. Hill populations at gene-based EST-SSR and nuclear SSR markers

Sympatric hybridizing oak species provide a model system for studying local adaptation. Disjunct populations of Quercus rubra L. and Quercus ellipsoidalis E. J. Hill at the northern edge of their distribution may harbor important reservoirs of adaptive genetic variation. Genic (expressed sequence tag- simple sequence repeat = EST-SSR) and non-genic nuclear microsatellite (nuclear SSR = nSSR) markers were used to estimate neutral and potentially adaptive genetic variation in these two supposedly interfertile oak species showing different adaptations to drought. Eleven populations of putative Q. rubra and Q. ellipsoidalis located in the Western Upper Peninsula of Michigan were characterized using seven EST-SSRs and eight nSSRs. Bayesian cluster analysis revealed two distinct groups corresponding to each species with evidence of low levels of potential introgression. A comparison of the genetic structure of adult trees and seedlings revealed no evidence for selection against hybrids. Overall, similar levels of genetic variation and differentiation between populations and species were found at both EST-SSRs and nSSRs indicating that most EST-SSRs chosen reflect neutral variation. Two loci, 3A05 (nSSR) and GOT021 (EST-SSR, putative histidine kinase 4-like), were identified as putative outlier loci between species showing largely reduced variation in Q. ellipsoidalis. Future analyses of an increased number of EST-SSRs located in functional genes will allow the identification of genes involved in the reproductive isolation between both species.

Development and Characterization of Genomic and Gene-Based Microsatellite Markers in North American Red Oak Species

Oaks (Quercus: Fagaceae) are ecological and economic keystones of many forested ecosystems but effective genetic management strategies are hindered by high levels of phenotypic plasticity within species and frequent hybridization among them. These same features, however, make oak communities suited for the study of speciation, hybridization, and genetic adaptation. Efforts to develop new and to adapt existing genomic resources to less-studied members of this genus should not only improve oak conservation and management but also aid the study of fundamental evolutionary processes. Here, we present a suite of 27 highly polymorphic simple sequence repeat (SSR) markers tested in four North American red oak (Quercus section Lobatae) species: Q. rubra, Q. ellipsoidalis, Q. coccinea, and Q. velutina. Five markers are genomic SSRs (gSSRs) — four novel and one previously transferred from Q. petraea — and 22 are gene-based SSRs derived from Q. robur and Q. petraea expressed sequence tags (EST-SSRs). Overall, levels of polymorphism detected with these primer pairs were high, with gene diversity (He) averaging 0.66 across all loci in natural populations. In addition, we show that EST-SSR markers may have the potential to detect divergent selection at stress-resistance candidate genes among closely related oak species.

Transferability of Simple Sequence Repeat (SSR) Markers Developed in Litchi chinensis to Blighia sapida (Sapindaceae)

Ackee (Blighia sapida, Sapindaceae) is a multipurpose fruit tree species of high economic importance, native to the Guinean forests of West Africa, and belongs to the same family as that of lychee (Litchi chinensis). In this study, a set of 12 primer pairs for simple sequence repeats (SSRs) previously developed for lychee has been evaluated for polymorphism in 16 ackee trees from different populations. Seven primer pairs have been found to be transferable, and four have revealed polymorphisms. However, the average number of alleles per locus has dropped from 4.9 for lychee to 3.7 for ackee. Characterization of the four polymorphic markers in 279 individuals belonging to14 different ackee populations from Benin has revealed that the numbers of alleles per locus range from two to 14 with a mean number of 5.8. The observed and expected heterozygosities range between 0.020 to 0.359 and 0.020 to 0.396, respectively.

Genetical genomics of Populus leaf shape variation

BackgroundLeaf morphology varies extensively among plant species and is under strong genetic control. Mutagenic screens in model systems have identified genes and established molecular mechanisms regulating leaf initiation, development, and shape. However, it is not known whether this diversity across plant species is related to naturally occurring variation at these genes. Quantitative trait locus (QTL) analysis has revealed a polygenic control for leaf shape variation in different species suggesting that loci discovered by mutagenesis may only explain part of the naturally occurring variation in leaf shape. Here we undertook a genetical genomics study in a poplar intersectional pseudo-backcross pedigree to identify genetic factors controlling leaf shape. The approach combined QTL discovery in a genetic linkage map anchored to the Populus trichocarpa reference genome sequence and transcriptome analysis.ResultsA major QTL for leaf lamina width and length:width ratio was identified in multiple experiments that confirmed its stability. A transcriptome analysis of expanding leaf tissue contrasted gene expression between individuals with alternative QTL alleles, and identified an ADP-ribosylation factor (ARF) GTPase (PtARF1) as a candidate gene for regulating leaf morphology in this pedigree. ARF GTPases are critical elements in the vesicular trafficking machinery. Disruption of the vesicular trafficking function of ARF by the pharmacological agent Brefeldin A (BFA) altered leaf lateral growth in the narrow-leaf P. trichocarpa suggesting a molecular mechanism of leaf shape determination. Inhibition of the vesicular trafficking processes by BFA interferes with cycling of PIN proteins and causes their accumulation in intercellular compartments abolishing polar localization and disrupting normal auxin flux with potential effects on leaf expansion.ConclusionsIn other model systems, ARF proteins have been shown to control the localization of auxin efflux carriers, which function to establish auxin gradients and apical-basal cell polarity in developing plant organs. Our results support a model where PtARF1 transcript abundance changes the dynamics of endocytosis-mediated PIN localization in leaf cells, thus affecting lateral auxin flux and subsequently lamina leaf expansion. This suggests that evolution of differential cellular polarity plays a significant role in leaf morphological variation observed in subgenera of genus Populus.

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.