The Identification of Small RNAs Differentially Expressed in Apple Buds Reveals a Potential Role of the Mir159-MYB Regulatory Module during Dormancy.

Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.

Structure of multilocus genetic diversity in predominantly selfing populations.

Predominantly selfing populations are expected to have reduced effective population sizes due to nonrandom sampling of gametes, demographic stochasticity (bottlenecks or extinction-recolonization), and large scale hitchhiking (reduced effective recombination). Thus, they are expected to display low genetic diversity, which was confirmed by empirical studies. The structure of genetic diversity in predominantly selfing species is dramatically different from outcrossing ones, with populations often dominated by one or a few multilocus genotypes (MLGs) coexisting with several rare genotypes. Therefore, multilocus diversity indices are relevant to describe diversity in selfing populations. Here, we use simulations to provide analytical expectations for multilocus indices and examine whether selfing alone can be responsible for the high-frequency MLGs persistent through time in the absence of selection. We then examine how combining single and multilocus indices of diversity may be insightful to distinguish the effects of selfing, population size, and more complex demographic events (bottlenecks, migration, admixture, or extinction-recolonization). Finally, we examine how temporal changes in MLG frequencies can be insightful to understand the evolutionary trajectory of a given population. We show that combinations of selfing and small demographic sizes can result in high-frequency MLGs, as observed in natural populations. We also show how different demographic scenarios can be distinguished by the parallel analysis of single and multilocus indices of diversity, and we emphasize the importance of temporal data for the study of predominantly selfing populations. Finally, the comparison of our simulations with empirical data on populations of Medicago truncatula confirms the pertinence of our simulation framework.

How to optimize the precision of allele and haplotype frequency estimates using pooled-sequencing data.

Sequencing pools of individuals rather than individuals separately reduces the costs of estimating allele frequencies at many loci in many populations. Theoretical and empirical studies show that sequencing pools comprising a limited number of individuals (typically fewer than 50) provides reliable allele frequency estimates, provided that the DNA pooling and DNA sequencing steps are carefully controlled. Unequal contributions of different individuals to the DNA pool and the mean and variance in sequencing depth both can affect the standard error of allele frequency estimates. To our knowledge, no study separately investigated the effect of these two factors on allele frequency estimates; so that there is currently no method to a priori estimate the relative importance of unequal individual DNA contributions independently of sequencing depth. We develop a new analytical model for allele frequency estimation that explicitly distinguishes these two effects. Our model shows that the DNA pooling variance in a pooled sequencing experiment depends solely on two factors: the number of individuals within the pool and the coefficient of variation of individual DNA contributions to the pool. We present a new method to experimentally estimate this coefficient of variation when planning a pooled sequencing design where samples are either pooled before or after DNA extraction. Using this analytical and experimental framework, we provide guidelines to optimize the design of pooled sequencing experiments. Finally, we sequence replicated pools of inbred lines of the plant Medicago truncatula and show that the predictions from our model generally hold true when estimating the frequency of known multilocus haplotypes using pooled sequencing.

Single-nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula.

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single-nucleotide polymorphism (SNP) markers in M. truncatula through large-scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.

Molecular adaptation in flowering and symbiotic recognition pathways: insights from patterns of polymorphism in the legume Medicago truncatula.

BACKGROUND: We studied patterns of molecular adaptation in the wild Mediterranean legume Medicago truncatula. We focused on two phenotypic traits that are not functionally linked: flowering time and perception of symbiotic microbes. Phenology is an important fitness component, especially for annual plants, and many instances of molecular adaptation have been reported for genes involved in flowering pathways. While perception of symbiotic microbes is also integral to adaptation in many plant species, very few reports of molecular adaptation exist for symbiotic genes. Here we used data from 57 individuals and 53 gene fragments to quantify the overall strength of both positive and purifying selection in M. truncatula and asked if footprints of positive selection can be detected at key genes of rhizobia recognition pathways. RESULTS: We examined nucleotide variation among 57 accessions from natural populations in 53 gene fragments: 5 genes involved in nitrogen-fixing bacteria recognition, 11 genes involved in flowering, and 37 genes used as control loci. We detected 1757 polymorphic sites yielding an average nucleotide diversity (pi) of 0.003 per site. Non-synonymous variation is under sizable purifying selection with 90% of amino-acid changing mutations being strongly selected against. Accessions were structured in two groups consistent with geographical origins. Each of these two groups harboured an excess of rare alleles, relative to expectations of a constant-sized population, suggesting recent population expansion. Using coalescent simulations and an approximate Bayesian computation framework we detected several instances of genes departing from selective neutrality within each group and showed that the polymorphism of two nodulation and four flowering genes has probably been shaped by recent positive selection. CONCLUSION: We quantify the intensity of purifying selection in the M. truncatula genome and show that putative footprints of natural selection can be detected at different time scales in both flowering and symbiotic pathways.

Iron homeostasis and fire blight susceptibility in transgenic pear plants overexpressing a pea ferritin gene.

The bacterial pathogen Erwinia amylovora causes the devastating disease known as fire blight in some rosaceous plants including apple and pear. One of the pathogenicity factors affecting fire blight development is the production of a siderophore, desferrioxamine, which overcomes the limiting conditions in plant tissues and also protects bacteria against active oxygen species. In this paper we examine the effect of an iron chelator protein encoded by the pea ferritin gene on the fire blight susceptibility of pear (Pyrus communis). Transgenic pear clones expressing this gene controlled either by the constitutive promoter CaMV 35S or by the inducible promoter sgd24 promoter were produced. The transgenic clones produced were analysed by Q-RT-PCR to determine the level of expression of the pea transgene. A pathogen-inducible pattern of expression of the pea transgene was observed in sgd24-promoter transformants. Adaptation to iron deficiency in vitro was tested in some transgenic clones and different iron metabolism parameters were measured. No strong effect on iron and chlorophyll content, root reductase activity and fire blight susceptibility was detected in the transgenic lines tested. No transformants showed a significant reduction in susceptibility to fire blight in greenhouse conditions when inoculated with E. amylovora.

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium x hortorum, 'Panaché Sud'.

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium x hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-microF capacitor in a 250-V cm(-1) electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33-36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 microS cm(-1) allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l(-1) kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l(-1) kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.