The short-term effects of the herbicide glyphosate (1.25-10 mM) on the growth, nitrogen fixation, carbohydrate metabolism, and shikimate pathway were investigated in leaves and nodules of nodulated lupine plants. All glyphosate treatments decreased nitrogenase activity rapidly (24 h) after application, even at the lowest and sublethal dose used (1.25 mM). This early effect on nitrogenase could not be related to either damage to nitrogenase components (I and II) or limitation of carbohydrates supplied by the host plant. In fact, further exposure to increasing glyphosate concentrations (5 mM) and greater time after exposure (5 days) decreased nodule starch content and sucrose synthase (SS; EC 2.4.1.13) activity but increased sucrose content within the nodule. These effects were accompanied by a great inhibition of the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31). There were remarkable and rapid effects on the increase of shikimic and protocatechuic (PCA) acids in nodules and leaves after herbicide application. On the basis of the role of shikimic acid and PCA in the regulation of PEPC, as potent competitive inhibitors, this additional effect provoked by glyphosate on 5-enolpyruvylshikimic-3-phosphate synthase enzyme (EPSPS; EC 2.5.1.19) inhibition would divert most PEP into the shikimate pathway, depriving energy substrates to bacteroids to maintain nitrogen fixation. These findings provide a new explanation for the effectiveness of glyphosate as a herbicide in other plant tissues, for the observed differences in tolerance among species or cultivars, and for the transitory effects on glyphosate-resistant transgenic crops under several environmental conditions.

Maritime pine provides essential ecosystem services in the south-western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three-generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene-based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies.

Although recent advances have been gained on genome evolution in angiosperm lineages, virtually nothing is known about karyotype evolution in the other group of seed plants, the gymnosperms. Here, we used high-density gene-based linkage mapping to compare the karyotype structure of two families of conifers (the most abundant group of gymnosperms) separated around 290 Ma: Pinaceae and Cupressaceae. We propose for the first time a model based on the fusion of 20 ancestral chromosomal blocks that may have shaped the modern karyotpes of Pinaceae (with n = 12) and Cupressaceae (with n = 11). The considerable difference in modern genome organization between these two lineages contrasts strongly with the remarkable level of synteny already reported within the Pinaceae. It also suggests a convergent evolutionary mechanism of chromosomal block shuffling that has shaped the genomes of the spermatophytes.

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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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-464) contains supplementary material, which is available to authorized users.

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

BackgroundPinus pinaster Ait. is a major resin producing species in Spain. Genetic linkage mapping can facilitate marker-assisted selection (MAS) through the identification of Quantitative Trait Loci and selection of allelic variants of interest in breeding populations. In this study, we report annotated genetic linkage maps for two individuals (C14 and C15) belonging to a breeding program aiming to increase resin production. We use different types of DNA markers, including last-generation molecular markers.ResultsWe obtained 13 and 14 linkage groups for C14 and C15 maps, respectively. A total of 211 and 215 markers were positioned on each map and estimated genome length was between 1,870 and 2,166 cM respectively, which represents near 65% of genome coverage. Comparative mapping with previously developed genetic linkage maps for P. pinaster based on about 60 common markers enabled aligning linkage groups to this reference map. The comparison of our annotated linkage maps and linkage maps reporting QTL information revealed 11 annotated SNPs in candidate genes that co-localized with previously reported QTLs for wood properties and water use efficiency.ConclusionsThis study provides genetic linkage maps from a Spanish population that shows high levels of genetic divergence with French populations from which segregating progenies have been previously mapped. These genetic maps will be of interest to construct a reliable consensus linkage map for the species. The importance of developing functional genetic linkage maps is highlighted, especially when working with breeding populations for its future application in MAS for traits of interest.