Carmen Collada

Cross-species transferability and mapping of genomic and cDNA SSRs in pines

Two unigene datasets of Pinus taeda and Pinus pinaster were screened to detect di-, tri- and tetranucleotide repeated motifs using the SSRIT script. A total of 419 simple sequence repeats (SSRs) were identified, from which only 12.8% overlapped between the two sets. The position of the SSRs within their coding sequences were predicted using FrameD. Trinucleotides appeared to be the most abundant repeated motif (63 and 51% in P. taeda and P. pinaster, respectively) and tended to be found within translated regions (76% in both species), whereas dinucleotide repeats were preferentially found within the 5′- and 3′-untranslated regions (75 and 65%, respectively). Fifty-three primer pairs amplifying a single PCR fragment in the source species (mainly P. taeda), were tested for amplification in six other pine species. The amplification rate with other pine species was high and corresponded with the phylogenetic distance between species, varying from 64.6% in P. canariensis to 94.2% in P. radiata. Genomic SSRs were found to be less transferable; 58 of the 107 primer pairs (i.e., 54%) derived from P. radiata amplified a single fragment in P. pinaster. Nine cDNA-SSRs were located to their chromosomes in two P. pinaster linkage maps. The level of polymorphism of these cDNA-SSRs was compared to that of previously and newly developed genomic-SSRs. Overall, genomic SSRs tend to perform better in terms of heterozygosity and number of alleles. This study suggests that useful SSR markers can be developed from pine ESTs.

Class I chitinases with hevein-like domain, but not class II enzymes, are relevant chestnut and avocado allergens☆☆☆★★★

BACKGROUND Several foods associated with the latex-fruit syndrome present relevant allergens of around 30 kd. Neither these components nor any other responsible for the reported cross-reactions have been identified and purified. OBJECTIVE We sought to isolate and characterize the 30 kd allergens from avocado fruit and chestnut seed, two of the main allergenic foods linked with latex allergy. METHODS Sera from patients allergic to chestnut and avocado were selected according to clinical symptoms, specific IgE levels, and positive skin prick test responses. Class I and II chitinases were purified by affinity and cation-exchange chromatography and characterized by specific IgE and anti-chitinase immunodetection, immunoblot inhibition assays, enzymatic activity tests, and N-terminal sequencing. RESULTS Relevant 32 kd allergens were detected by specific IgE immunodetection in both avocado and chestnut crude extracts. The same bands, together with others of 25 kd, were revealed by a monospecific antiserum against class II chitinases. Purification and characterization of the 32 kd allergens from both plant sources allowed their identification as class I chitinases with an N-terminal hevein-domain. The purified allergens fully inhibited IgE binding by the corresponding crude extract when tested in immunoblot inhibition assays. Highly related 25 kd class II chitinases that lack the hevein-like domain were also isolated from the same protein preparations. No IgE-binding capacity was shown by these class II enzymes. CONCLUSION Class I chitinases are relevant allergens of avocado and chestnut and could be the panallergens responsible for the latex-fruit syndrome. The hevein-like domain seems to be involved in their allergenic reactivity.

Gene flow and hybridisation in a mixed oak forest (Quercus pyrenaica Willd. and Quercus petraea (Matts.) Liebl.) in central Spain.

Oaks are long-standing models for the study of gene flow and hybridisation. Temperate (Quercus petraea) and sub-Mediterranean (Quercus pyrenaica) oaks coexist in central Spain, showing remarkable differences in population size and structure. Q. petraea has a scattered distribution in central Spain, where it is at one of the southernmost limits of its range, and forms low-density stands; in contrast, Q. pyrenaica is widespread in the region. We selected a mixed population of the two species (∼13 ha, 176 adults and 96 saplings) to compare the patterns of gene flow within each species and the extent of introgression between them. Using five nuclear microsatellite markers, we performed a parentage analysis and found considerable immigration from outside the stand (∼38% for Q. petraea and ∼34% for Q. pyrenaica), and estimated average seed-dispersal distances of 42 and 14 m for Q. petraea and Q. pyrenaica, respectively. Introgression between species was also estimated using our microsatellite battery. First, we developed a multivariate discriminant approach and, second, we compared our results with a widely used clustering method (STRUCTURE). Both analyses were consistent with a low level of introgression between Q. petraea and Q. pyrenaica. Indeed, only 15 adult trees, ∼8.5%, were identified as putative hybrids when both methods of analysis were combined. Hybrids may be most common in contact zones due merely to physical proximity.

Phylogeography: English elm is a 2,000-year-old Roman clone

The outbreak of Dutch elm disease in the 1970s ravaged European elm populations, killing more than 25 million trees in Britain alone; the greatest impact was on Ulmus procera, otherwise known as the English elm. Here we use molecular and historical information to show that this elm derives from a single clone that the Romans transported from Italy to the Iberian peninsula, and from there to Britain, for the purpose of supporting and training vines. Its highly efficient vegetative reproduction and its inability to set seeds have preserved this clone unaltered for 2,000 years as the core of the English elm population — and the preponderance of this susceptible variety may have favoured a rapid spread of the disease.

High variability of chloroplast DNA in three Mediterranean evergreen oaks indicates complex evolutionary history

Chloroplast DNA variation was studied in three evergreen Quercus species (Q. suber L., Q. ilex L. and Q. coccifera L.) from the Western Mediterranean Basin using PCR-RFLP. We studied five primer pair/enzyme combinations, four of them previously used in other European Quercus, obtaining a large number of haplotypes (81) grouped in three main types (suber type, ilex-coccifera I type and ilex-coccifera II type). Such level of haplotype diversity is higher than previously reported for the genus. Remarkable differences in haplotype richness between species have been found. Q. ilex and Q. coccifera usually share the same haplotypes, while a number of Q. suber populations possesses variants of the ilex-coccifera I type. This fact is interpreted as a result of genetic introgression between Q. suber and Q. ilex. Reproductive factors that could determine this exchange are discussed, as well as the influence of different species histories on the present structure of evergreen Quercus in the Western Mediterranean Basin.

Purification and in vitro chaperone activity of a class I small heat-shock protein abundant in recalcitrant chestnut seeds.

A 20-kD protein has been purified from cotyledons of recalcitrant (desiccation-sensitive) chestnut (Castanea sativa) seeds, where it accumulates at levels comparable to those of major seed storage proteins. This protein, termed Cs smHSP 1, forms homododecameric complexes under nondenaturing conditions and appears to be homologous to cytosolic class I small heat-shock proteins (smHSPs) from plant sources. In vitro evidence has been obtained that the isolated protein can function as a molecular chaperone: it increases, at stoichiometric levels, the renaturation yields of chemically denatured citrate synthase and also prevents the irreversible thermal inactivation of this enzyme. Although a role in desiccation tolerance has been hypothesized for seed smHSPs, this does not seem to be the case for Cs smHSP 1. We have investigated the presence of immunologically related proteins in orthodox and recalcitrant seeds of 13 woody species. Our results indicate that the presence of Cs smHSP 1-like proteins, even at high levels, is not enough to confer desiccation tolerance, and that the amount of these proteins does not furnish a reliable criterion to identify desiccation-sensitive seeds. Additional proteins or mechanisms appear necessary to keep the viability of orthodox seeds upon shedding.

EuroPineDB: a high-coverage web database for maritime pine transcriptome

BackgroundPinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases.DescriptionEuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided.ConclusionsThe EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

Vulnerability to cavitation, hydraulic efficiency, growth and survival in an insular pine (Pinus canariensis)

BACKGROUND AND AIMS It is widely accepted that hydraulic failure due to xylem embolism is a key factor contributing to drought-induced mortality in trees. In the present study, an attempt is made to disentangle phenotypic plasticity from genetic variation in hydraulic traits across the entire distribution area of a tree species to detect adaptation to local environments. METHODS A series of traits related to hydraulics (vulnerability to cavitation and hydraulic conductivity in branches), growth performance and leaf mass per area were assessed in eight Pinus canariensis populations growing in two common gardens under contrasting environments. In addition, the neutral genetic variability (FST) and the genetic differentiation of phenotypic variation (QST) were compared in order to identify the evolutionary forces acting on these traits. KEY RESULTS The variability for hydraulic traits was largely due to phenotypic plasticity. Nevertheless, the vulnerability to cavitation displayed a significant genetic variability (approx. 5 % of the explained variation), and a significant genetic × environment interaction (between 5 and 19 % of the explained variation). The strong correlation between vulnerability to cavitation and survival in the xeric common garden (r = -0·81; P < 0·05) suggests a role for the former in the adaptation to xeric environments. Populations from drier sites and higher temperature seasonality were less vulnerable to cavitation than those growing at mesic sites. No trade-off between xylem safety and efficiency was detected. QST of parameters of the vulnerability curve (0·365 for P50 and the slope of the vulnerability curve and 0·452 for P88) differed substantially from FST (0·091), indicating divergent selection. In contrast, genetic drift alone was found to be sufficient to explain patterns of differentiation for xylem efficiency and growth. CONCLUSIONS The ability of P. canariensis to inhabit a wide range of ecosystems seemed to be associated with high phenotypic plasticity and some degree of local adaptations of xylem and leaf traits. Resistance to cavitation conferred adaptive potential for this species to adapt successfully to xeric conditions.

Epigenetic Variability in the Genetically Uniform Forest Tree Species Pinus pinea L

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Genetic control of functional traits related to photosynthesis and water use efficiency in Pinus pinaster Ait. drought response: integration of genome annotation, allele association and QTL detection for candidate gene identification

BackgroundUnderstanding molecular mechanisms that control photosynthesis and water use efficiency in response to drought is crucial for plant species from dry areas. This study aimed to identify QTL for these traits in a Mediterranean conifer and tested their stability under drought.ResultsHigh density linkage maps for Pinus pinaster were used in the detection of QTL for photosynthesis and water use efficiency at three water irrigation regimes. A total of 28 significant and 27 suggestive QTL were found. QTL detected for photochemical traits accounted for the higher percentage of phenotypic variance. Functional annotation of genes within the QTL suggested 58 candidate genes for the analyzed traits. Allele association analysis in selected candidate genes showed three SNPs located in a MYB transcription factor that were significantly associated with efficiency of energy capture by open PSII reaction centers and specific leaf area.ConclusionsThe integration of QTL mapping of functional traits, genome annotation and allele association yielded several candidate genes involved with molecular control of photosynthesis and water use efficiency in response to drought in a conifer species. The results obtained highlight the importance of maintaining the integrity of the photochemical machinery in P. pinaster drought response.