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

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

Quimica

Pinus subsection Ponderosae includes approximately 17 species distributed from western Canada to Nicaragua. Members of the group are of great ecological and economic importance but phylogenetic relationships among species are poorly understood

Species diversity and plastid DNA haplotype distributions of Pinus subsection Australes (Pinaceae) in Guerrero and Oaxaca

Pinus subsección Australes es un grupo de pinos duros de América del Norte que comprende aproximadamente 29 especies de árboles importantes económicamente y ecológicamente distribuidos a lo largo de toda América del Norte y Central y las Islas Caribeñas. Estudios previos han mostrado que las especies de esta subsección a menudo comparten haplotipos de ADN de plastidio, un patrón que es atribuido a la hibridación introgresiva y la retención de polimorfismos ancestrales. Aquí describimos la diversidad de haplotipos de plastidio y la morfología para este grupo de especies en los estados de Guerrero y Oaxaca, México. Siete especies de Pinus subsección Australes son reconocidas en el área de estudio, una de las cuales, P. patula incluye dos variedades. Siete sitios variables y nueve haplotipos fueron encontrados amplificando un fragmento de 840 p. b. de ADN de la región codificante ycf1. Se encontraron haplotipos compartidos para P. patula var. patula, P. patula var. longipedunculata, P. herrerae y P. tecunumanii. Cuatro de los nueve haplotipos encontrados están restringidos a Oaxaca. Aunque las genealogías de genes son valiosas para estudiar la evolución de este grupo, se requieren mayor muestreo de individuos y más sitios variables para la inferencia de relaciones entre las especies.Pinus subsection Australes is a group of North American hard pines comprising approximately 29 ecologically and economically important tree species distributed throughout North and Central America and the Caribbean Islands. Previous studies have shown that some species of this subsection share plastid DNA haplotypes, a pattern that is attributed to introgressive hybridization or the retention of ancestral polymorphisms. Here we describe the morphological and plastid haplotype diversity for this group of species in the states of Guerrero and Oaxaca, Mexico. Seven species of Pinus subsection Australes are recognized in the study area, one of which, P. patula, includes two varieties. Seven variable sites and nine haplotypes were found in an 840 b.p. fragment of the DNA coding region ycf1. Shared haplotypes were found for P. patula var. patula, P. patula var. longipedunculata, P. herrerae, and P. tecunumanii. Four of the nine haplotypes found were restricted to Oaxaca. Although plastid DNA genealogies are valuable for studying evolution in this group, greater sampling of individuals and the inclusion of more variable sites are needed to more accurately infer species relationships

Species diversity and plastid dna haplotype distributions ofPinus Subsection Australes (pinaceae) in Guerrero and Oaxaca

Pinus subsección Australes es un grupo de pinos duros de América del Norte que comprende aproximadamente 29 especies de árboles importantes económicamente y ecológicamente distribuidos a lo largo de toda América del Norte y Central y las Islas Caribeñas. Estudios previos han mostrado que las especies de esta subsección a menudo comparten haplotipos de ADN de plastidio, un patrón que es atribuido a la hibridación introgresiva y la retención de polimorfismos ancestrales. Aquí describimos la diversidad de haplotipos de plastidio y la morfología para este grupo de especies en los estados de Guerrero y Oaxaca, México. Siete especies de Pinus subsección Australes son reconocidas en el área de estudio, una de las cuales, P. patulaincluye dos variedades. Siete sitios variables y nueve haplotipos fueron encontrados amplificando un fragmento de 840 p. b. de ADN de la región codificante ycf1. Se encontraron haplotipos compartidos para P. patulavar. patula, P. patula var. longipedunculata, P.herrerae y P. tecunumanii. Cuatro de los nueve haplotipos encontrados están restringidos a Oaxaca. Aunque las genealogías de genes son valiosas para estudiar la evolución de este grupo, se requieren mayor muestreo de individuos y más sitios variables para la inferencia de relaciones entre las especies

Complete plastomes of three endemic Mexican pine species (<i>Pinus</i> subsection <i>Australes</i>)

<p>We assembled the plastomes of <i>Pinus greggii</i>, <i>P. jaliscana</i> and <i>P. oocarpa</i> from 100 bp paired-end Illumina reads. We combined <i>de novo</i> (comparing <i>Velvet</i> and <i>SPAdes</i>) with reference-guided assembly and a final step of gap filling. <i>SPAdes</i> performed better than <i>Velvet</i> based on scaffold number (180 vs. 263) and mean length (1886 vs. 560 bp), and number of gaps (2 vs. 4). Annotations were automatically transferred from <i>P. taeda</i> NC_021440 and carefully revised by hand. Phylogenetic analysis with additional plastomes revealed very short branch lengths, supporting a rapid diversification within <i>Australes</i> and close relatedness among pines from Western Mexico.</p

Biodiversidad de Pinophyta (coníferas) en México

Las coníferas (Pinophyta) son árboles o arbustos con hojas simples y estructuras fértiles arregladas en conos polínicos simples y conos ovulados compuestos, excepto en Taxaceae. Las coníferas son los componentes dominantes de diversos tipos de vegetación. En México crecen desde el nivel del mar hasta por encima de los 4 000 m; la mayor diversidad se encuentra en los bosques montañosos de la Sierra Madre Occidental y Sierra Madre Oriental. Están representadas por 4 familias: Pinaceae (4 géneros y 61 especies), Cupressaceae (4 géneros y 29 especies), Podocarpaceae (1 género y 3 especies) y Taxaceae (1 especie). De las 94 especies de coníferas mexicanas, 43 son endémicas del país, de éstas 18 tienen un rango de distribución limitado a 3 o menos estados