Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology

Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified ~120 kb plastomes from eight species (seven Pinus, one Picea) in 35 reactions. Pooled products were ligates to modified adapters that included 3 bp indexing tags and samples were multiplexed at four genomes per lane. Tagged microreads were assembled by de novo and reference-guided assembly methods, using previously published Pinus plastomes as surrogate referenes. Assemblies for these eight genomes are estimated at 88-94% complete, with an average sequence depth of 55x to 186x. Mononucleotide repeats interrupt contig assembly with increasing repeat length, and we estimate that the limit for their assembly is 16 bp. Comparisons to 37 kb of Sanger sequence show a validated error rate of 0.056%, and conspicuous errors are evident from the assembly process. This efficient sequencing approach yields high-quality draft genomes and should have immediate applicability to genomes with comparable complexity

Usos y conocimiento tradicional de las gimnospermas en el noreste de Oaxaca, México

Background and Aims: Approximately 156 gymnosperm species divided into six families occur naturally in Mexico. Oaxaca has a high richness of gymnosperms, with 45 species, and its inhabitants have an ancient knowledge about the traditional use of this group. The present study contributes to the documentation of traditional knowledge of the gymnosperms in Oaxaca, to evaluate the importance of plant families as useful resources, to understand the relationships between ethnic groups and richness of useful species, as well as to compare our records of gymnosperms with those at state and national levels.Methods: This study was carried out in 84 municipalities within three Priority Terrestrial Regions in the northeast of Oaxaca. The ethnobotanical data were collected through participant observation, ethnobotanical fieldtrips, content-free interviews of the local guides, and botanical specimens were collected. All data were analyzed qualitatively to integrate ethnofloristic and traditional knowledge, as well as ecological aspects of species studied.Key results: We recorded 30 species belonging to the families Cupressaceae, Pinaceae, Podocarpaceae and Zamiaceae. The largest number of useful species (16) registered belongs to Pinaceae. Among the Zapotecs, uses were found for 16 different species, the Mixes use ten and the Mazatec nine. All taxa registered with any use have a common name, 71% in the local language and 97% in Spanish. The most common uses were timber, construction, ornamental and fuel. The montane cloud forest is the vegetation type with the largest number of useful gymnosperms, with 15 species. Forty-five percent of the registered taxa are endemic to Mexico, six species of Pinaceae and eight of Zamiaceae are considered endemic to Oaxaca.Conclusions: This research contributes to increase records of useful gymnosperms in the state of Oaxaca. It is necessary to reconsider and understand the local value of species within the ethnic worldview in order to preserve traditional uses and promote sustainable management programs.Antecedentes y Objetivos: En México existen alrededor de 156 especies de gimnospermas nativas pertenecientes a seis familias. Para el estado de Oaxaca, se registran 45 especies de gimnospermas y sus habitantes poseen un sobresaliente conocimiento ancestral en el uso tradicional de este grupo taxonómico. El presente estudio contribuye al registro y documentación del conocimiento tradicional de las gimnospermas en Oaxaca, a evaluar la importancia de las familias botánicas como fuente de recursos, analizar las relaciones existentes entre los grupos étnicos y la riqueza de especies útiles, así como a comparar los registros obtenidos con el número de gimnospermas a nivel nacional y estatal.Métodos: El estudio se realizó en 84 municipios en tres Regiones Terrestres Prioritarias al noreste de Oaxaca. Los datos etnobotánicos fueron recopilados mediante observación participante moderada, caminatas etnobotánicas, entrevista libre a guías locales y colecta de ejemplares botánicos. Los datos se analizaron cualitativamente para integrar el conocimiento tradicional, etnoflorístico y los aspectos ecológicos de las especies.Resultados clave: Se registraron 30 especies de las familias Cupressaceae, Pinaceae, Podocarpaceae y Zamiaceae. El mayor número de especies útiles (16) registradas pertenece a Pinaceae. Entre los zapotecos se encontraron usos para 16 diferentes especies, los mixes utilizan diez y los mazatecos nueve. Todos los taxa registrados tienen nombre común, 71% en lengua local y 97% en español. Los usos más frecuentes fueron el maderable, la construcción, el ornamental y el combustible. El bosque mesófilo de montaña es el tipo de vegetación del área estudiada que contiene el mayor número de gimnospermas útiles, con 15 especies. De los taxa registrados 45% son endémicos de México, seis especies de Pinaceae y ocho de Zamiaceae se consideran endémicos de Oaxaca.Conclusiones: Este estudio incrementa los registros sobre gimnospermas útiles en el estado de Oaxaca. Se requiere reconsiderar y entender el valor local de estas especies dentro de la cosmovisión étnica, para preservar los usos tradicionales y promover programas de manejo sostenible

Genetic Diversity of Pinus nigra Arn. Populations in Southern Spain and Northern Morocco Revealed By Inter-Simple Sequence Repeat Profiles †

Eight Pinus nigra Arn. populations from Southern Spain and Northern Morocco were examined using inter-simple sequence repeat markers to characterize the genetic variability amongst populations. Pair-wise population genetic distance ranged from 0.031 to 0.283, with a mean of 0.150 between populations. The highest inter-population average distance was between PaCU from Cuenca and YeCA from Cazorla, while the lowest distance was between TaMO from Morocco and MA Sierra Mágina populations. Analysis of molecular variance (AMOVA) and Nei’s genetic diversity analyses revealed higher genetic variation within the same population than among different populations. Genetic differentiation (Gst) was 0.233. Cuenca showed the highest Nei’s genetic diversity followed by the Moroccan region, Sierra Mágina, and Cazorla region. However, clustering of populations was not in accordance with their geographical locations. Principal component analysis showed the presence of two major groups—Group 1 contained all populations from Cuenca while Group 2 contained populations from Cazorla, Sierra Mágina and Morocco—while Bayesian analysis revealed the presence of three clusters. The low genetic diversity observed in PaCU and YeCA is probably a consequence of inappropriate management since no estimation of genetic variability was performed before the silvicultural treatments. Data indicates that the inter-simple sequence repeat (ISSR) method is sufficiently informative and powerful to assess genetic variability among populations of P. nigra

Evolution of Disease Response Genes in Loblolly Pine: Insights from Candidate Genes

BACKGROUND: Host-pathogen interactions that may lead to a competitive co-evolution of virulence and resistance mechanisms present an attractive system to study molecular evolution because strong, recent (or even current) selective pressure is expected at many genomic loci. However, it is unclear whether these selective forces would act to preserve existing diversity, promote novel diversity, or reduce linked neutral diversity during rapid fixation of advantageous alleles. In plants, the lack of adaptive immunity places a larger burden on genetic diversity to ensure survival of plant populations. This burden is even greater if the generation time of the plant is much longer than the generation time of the pathogen. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present nucleotide polymorphism and substitution data for 41 candidate genes from the long-lived forest tree loblolly pine, selected primarily for their prospective influences on host-pathogen interactions. This dataset is analyzed together with 15 drought-tolerance and 13 wood-quality genes from previous studies. A wide range of neutrality tests were performed and tested against expectations from realistic demographic models. CONCLUSIONS/SIGNIFICANCE: Collectively, our analyses found that axr (auxin response factor), caf1 (chromatin assembly factor) and gatabp1 (gata binding protein 1) candidate genes carry patterns consistent with directional selection and erd3 (early response to drought 3) displays patterns suggestive of a selective sweep, both of which are consistent with the arm-race model of disease response evolution. Furthermore, we have identified patterns consistent with diversifying selection at erf1-like (ethylene responsive factor 1), ccoaoemt (caffeoyl-CoA-O-methyltransferase), cyp450-like (cytochrome p450-like) and pr4.3 (pathogen response 4.3), expected under the trench-warfare evolution model. Finally, a drought-tolerance candidate related to the plant cell wall, lp5, displayed patterns consistent with balancing selection. In conclusion, both arms-race and trench-warfare models seem compatible with patterns of polymorphism found in different disease-response candidate genes, indicating a mixed strategy of disease tolerance evolution for loblolly pine, a major tree crop in southeastern United States

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine

<p>Abstract</p> <p>Background</p> <p>Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (<it>Pinus pinaster </it>Ait.), the main conifer used for commercial plantation in southwestern Europe.</p> <p>Results</p> <p>We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 <it>in vitro </it>SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 <it>in silico </it>SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for <it>in silico </it>and <it>in vitro </it>SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a <it>Pinus taeda </it>linkage map, made it possible to align the 12 linkage groups of both species.</p> <p>Conclusions</p> <p>Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.</p

A new look at water transport regulation in plants

Plant function requires effective mechanisms to regulate water transport at a variety of scales. Here, we develop a new theoretical framework describing plant responses to drying soil, based on the relationship between midday and predawn leaf water potentials. The intercept of the relationship (Λ) characterizes the maximum transpiration rate per unit of hydraulic transport capacity, whereas the slope (σ) measures the relative sensitivity of the transpiration rate and plant hydraulic conductance to declining water availability. - This framework was applied to a newly compiled global database of leaf water potentials to estimate the values of Λ and σ for 102 plant species. - Our results show that our characterization of drought responses is largely consistent within species, and that the parameters Λ and σ show meaningful associations with climate across species. Parameter σ was ≤1 in most species, indicating a tight coordination between the gas and liquid phases of water transport, in which canopy transpiration tended to decline faster than hydraulic conductance during drought, thus reducing the pressure drop through the plant. - The quantitative framework presented here offers a new way of characterizing water transport regulation in plants that can be used to assess their vulnerability to drought under current and future climatic conditions