Phylogenetic relationship of the genus Scorzoneroides (Compositae) inferred from plastid and low-copy nuclear markers, and karyological data

Frühere Untersuchungen der molekularen Phylogenie der Gattung Leontodon mit Markern aus dem Chloroplastengenom und dem nukleären ribosomalen DNA-Marker ITS führten zu einer Neuordnung der Gattung Leontodon. Leontodon subgenus Oporinia wurde durch diese molekularen Informationen zur Gattung Scorzoneroides erhoben. In unserem Projekt sollen die Chloroplasten-DNA Daten zum einen vorhergegangene Ergebnisse bestätigen, und zum anderen eine zusätzliche Informationsquelle darstellen. 11 universelle Marker des Plastidengenoms wurden an je 17 Spezies auf ihre Applizierbarkeit, ihren Sequenzabgleich sowie ihren Informationsgehalt untersucht, daraus wurden die drei besten Regionen verwendet . Sieben nukleäre Genregionen wurden untersucht, um einen möglichen hybriden Ursprung einzelner Arten der Gattung Scorzoneroides aufzuklären. Zwei Regionen des nukleären Genoms, GAPDH und A39 konnten teilweise direkt sequenziert werden. Um die erhaltenen Sequenzen zu verbessern, wurden für beide Marker zusätzliche, interne Primer erzeugt. In beiden Regionen wurden polymorphe Sequenzen gefunden, welche durch Klonierung getrennt werden sollten. 180 Klon-Kolonien (96 für GAPDH, 84 für A39) wurden in weiterer Folge sequenziert, um die unterschiedlichen Fragmente zu erhalten. Eine Chromosomenzahl konnte bei vier annuellen Spezies der Gattung ermittelt werden. Scorzoneroides besitzt eine Basischromosomenzahl von x=6, bei zwei der untersuchten Arten konnten Abweichungen festgestellt werden. Hier beträgt die Chromosomenzahl x=5. Die ermittelte Genomgröße für 8 aus 9 verfügbaren Spezies ist relativ klein und beläuft sich auf Werte zwischen 1C=1.24pg und 1C=1.87pg.Earlier investigations on the molecular phylogeny of the genus Leontodon using chloroplast regions in combination with the nuclear rDNA region ITS, led to a generic status of Leontodon subgenus Oporinia, today Scorzoneroides. To confirm previous results, as well as to get well-resolved phylogenetic trees, more noncoding regions of the chloroplast genome were sequenced. We were investigating the applicability, ease of alignment and informativity of 11 universal markers, the best 3 markers were chosen to work with. Seven low-copy nuclear markers were tested to detect speculated hybridization events in Scorzoneroides. PCR conditions were optimized for all markers. Two of the regions, namely GAPDH and the A39 locus could be directly sequenced from some PCR reactions. Internal primers were designed to increase PCR reliability among the tested accessions from Hypochaeridinae. Both low-copy nuclear genes showed polymorphic sequences in some accessions, which led to cloning. We used 96 colonies from GAPDH and 84 colonies from A39 to get both copies of each individual sequence that showed polymorphic sites. A combination of the two nuclear regions GAPDH and A39 locus with the three plastid markers atpH-atpI, ndhF-rpl32 and rpl16-Intron showed a well-resolved phylogenetic tree. Chromosome counts and genome size measurements from seeds of 9 species of all resulting clades of the phylogenetic analysis were measured. Successful germination was easily obtained for annual species, whereas the germination rate of the included perennial species was very low. Chromosome counts could only be obtained for the annual plants. The basic chromosome number for Scorzoneroides is x=6, two species had a chromosome number of x=5. The genome size of 8 out of 9 available species could be measured by flow cytometry. All species investigated possess a small genome size ranging from 1C=1.24pg to 1C=1.87pg

The UV RESISTANCE LOCUS 8-Mediated UV-B Response Is Required Alongside CRYPTOCHROME 1 For Plant Survival Under Sunlight In Field Conditions

As sessile, photoautotrophic organisms, plants are subjected to fluctuating sunlight that includes potentially detrimental ultraviolet-B radiation (UV-B). Experiments under controlled conditions have shown that the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) controls acclimation and tolerance to UV-B in Arabidopsis thaliana; however, its long-term impacts on plant fitness remain poorly understood in naturally fluctuating environments. Here we quantified the survival and reproduction of different Arabidopsis mutant genotypes in diverse field and laboratory conditions. We found that uvr8 mutants produced more fruits than wild type when grown in growth chambers under artificial low UV-B conditions but not in natural field conditions, indicating a fitness cost in absence of UV-B stress. Importantly, independent double mutants of UVR8 and the blue-light photoreceptor gene CRYPTOCHROME 1 (CRY1) in two genetic backgrounds showed a drastic reduction in fitness in the field. Experiments with UV-B attenuation experiments in field and with supplemental UV-B in growth chambers demonstrated that UV-B caused the conditional cry1 uvr8 lethality phenotype. Using RNA-seq data of field-grown single and double mutants, we explicitly identified genes showing statistical interaction of UVR8 and CRY1 mutations in the presence of UV-B in the field. They were enriched in Gene Ontology categories related to oxidative stress, photoprotection, and DNA damage repair in addition to UV-B response. Our study demonstrates the functional importance of the UVR8-mediated response across life stages in natura, which is partially redundant with that of cry1. Moreover, these data provide an integral picture of gene expression associated with plant responses under field conditions

Efficient Detection of Novel Nuclear Markers for Brassicaceae by Transcriptome Sequencing

The lack of DNA sequence information for most non-model organisms impairs the design of primers that are universally applicable for the study of molecular polymorphisms in nuclear markers. Next-generation sequencing (NGS) techniques nowadays provide a powerful approach to overcome this limitation. We present a flexible and inexpensive method to identify large numbers of nuclear primer pairs that amplify in most Brassicaceae species. We first obtained and mapped NGS transcriptome sequencing reads from two of the distantly related Brassicaceae species, Cardamine hirsuta and Arabis alpina, onto the Arabidopsis thaliana reference genome, and then identified short conserved sequence motifs among the three species bioinformatically. From these, primer pairs to amplify coding regions (nuclear protein coding loci, NPCL) and exon-primed intron-crossing sequences (EPIC) were developed. We identified 2,334 universally applicable primer pairs, targeting 1,164 genes, which provide a large pool of markers as readily usable genomic resource that will help addressing novel questions in the Brassicaceae family. Testing a subset of the newly designed nuclear primer pairs revealed that a great majority yielded a single amplicon in all of the 30 investigated Brassicaceae taxa. Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies. The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies. Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies.ISSN:1932-620

Efficient detection of novel nuclear markers for brassicaceae by transcriptome sequencing

The lack of DNA sequence information for most non-model organisms impairs the design of primers that are universally applicable for the study of molecular polymorphisms in nuclear markers. Next-generation sequencing (NGS) techniques nowadays provide a powerful approach to overcome this limitation. We present a flexible and inexpensive method to identify large numbers of nuclear primer pairs that amplify in most Brassicaceae species. We first obtained and mapped NGS transcriptome sequencing reads from two of the distantly related Brassicaceae species, Cardamine hirsuta and Arabis alpina, onto the Arabidopsis thaliana reference genome, and then identified short conserved sequence motifs among the three species bioinformatically. From these, primer pairs to amplify coding regions (nuclear protein coding loci, NPCL) and exon-primed intron-crossing sequences (EPIC) were developed. We identified 2,334 universally applicable primer pairs, targeting 1,164 genes, which provide a large pool of markers as readily usable genomic resource that will help addressing novel questions in the Brassicaceae family. Testing a subset of the newly designed nuclear primer pairs revealed that a great majority yielded a single amplicon in all of the 30 investigated Brassicaceae taxa. Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies. The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies. Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies

Data from: Efficient detection of novel nuclear markers for Brassicaceae by transcriptome sequencing

The lack of DNA sequence information for most non-model organisms impairs the design of primers that are universally applicable for the study of molecular polymorphisms in nuclear markers. Next-generation sequencing (NGS) techniques nowadays provide a powerful approach to overcome this limitation. We present a flexible and inexpensive method to identify large numbers of nuclear primer pairs that amplify in most Brassicaceae species. We first obtained and mapped NGS transcriptome sequencing reads from two of the distantly related Brassicaceae species, Cardamine hirsuta and Arabis alpina, onto the Arabidopsis thaliana reference genome, and then identified short conserved sequence motifs among the three species bioinformatically. From these, primer pairs to amplify coding regions (nuclear protein coding loci, NPCL) and exon-primed intron-crossing sequences (EPIC) were developed. We identified 2,334 universally applicable primer pairs, targeting 1,164 genes, which provide a large pool of markers as readily usable genomic resource that will help addressing novel questions in the Brassicaceae family. Testing a subset of the newly designed nuclear primer pairs revealed that a great majority yielded a single amplicon in all of the 30 investigated Brassicaceae taxa. Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies. The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies. Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies

Sequence alignments for Arabis alpina Cardamine hirsuta

We provide here the sequence alignment files of two transcriptome sequencing runs on a Genome Sequencer FLX (Roche, Switzerland) for pooled RNA samples of Arabis alpina and Cardamine hirsuta mapped to Arabidopsis thaliana (TAIR 9). Each sample was sequenced twice on half a picotiter plate. The alignments were used to detect conserved regions among the aforementioned species. The reads have been mapped against the five chromosomes of Arabidopsis thaliana (TAIR9) using samtools (Li et al. 2009)

Concatenated alignments for all three phylogenetic levels tested

Concatenated alignments for all three phylogenetic levels teste

Efficient Detection of Novel Nuclear Markers for Brassicaceae by Transcriptome Sequencing

<div><p>The lack of DNA sequence information for most non-model organisms impairs the design of primers that are universally applicable for the study of molecular polymorphisms in nuclear markers. Next-generation sequencing (NGS) techniques nowadays provide a powerful approach to overcome this limitation. We present a flexible and inexpensive method to identify large numbers of nuclear primer pairs that amplify in most Brassicaceae species. We first obtained and mapped NGS transcriptome sequencing reads from two of the distantly related Brassicaceae species, <i>Cardamine hirsuta</i> and <i>Arabis alpina</i>, onto the <i>Arabidopsis thaliana</i> reference genome, and then identified short conserved sequence motifs among the three species bioinformatically. From these, primer pairs to amplify coding regions (nuclear protein coding loci, NPCL) and exon-primed intron-crossing sequences (EPIC) were developed. We identified 2,334 universally applicable primer pairs, targeting 1,164 genes, which provide a large pool of markers as readily usable genomic resource that will help addressing novel questions in the Brassicaceae family. Testing a subset of the newly designed nuclear primer pairs revealed that a great majority yielded a single amplicon in all of the 30 investigated Brassicaceae taxa. Sequence analysis and phylogenetic reconstruction with a subset of these markers on different levels of phylogenetic divergence in the mustard family were compared with previous studies. The results corroborate the usefulness of the newly developed primer pairs, e.g., for phylogenetic analyses or population genetic studies. Thus, our method provides a cost-effective approach for designing nuclear loci across a broad range of taxa and is compatible with current NGS technologies.</p></div

UV RESISTANCE LOCUS 8-Mediated UV-B Response Is Required Alongside CRYPTOCHROME 1 for Plant Survival in Sunlight under Field Conditions

As sessile, photoautotrophic organisms, plants are subjected to fluctuating sunlight that includes potentially detrimental ultraviolet-B (UV-B) radiation. Experiments under controlled conditions have shown that the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) controls acclimation and tolerance to UV-B in Arabidopsis thaliana; however, its long-term impact on plant fitness under naturally fluctuating environments remain poorly understood. Here, we quantified the survival and reproduction of different Arabidopsis mutant genotypes under diverse field and laboratory conditions. We found that uvr8 mutants produced more fruits than wild type when grown in growth chambers under artificial low-UV-B conditions but not under natural field conditions, indicating a fitness cost in the absence of UV-B stress. Importantly, independent double mutants of UVR8 and the blue light photoreceptor gene CRYPTOCHROME 1 (CRY1) in two genetic backgrounds showed a drastic reduction in fitness in the field. Experiments with UV-B attenuation in the field and with supplemental UV-B in growth chambers demonstrated that UV-B caused the cry1 uvr8 conditional lethal phenotype. Using RNA-seq data of field-grown single and double mutants, we explicitly identified genes showing significant statistical interaction of UVR8 and CRY1 mutations in the presence of UV-B in the field. They were enriched in Gene Ontology categories related to oxidative stress, photoprotection and DNA damage repair in addition to UV-B response. Our study demonstrates the functional importance of the UVR8-mediated response across life stages in natura, which is partially redundant with that of cry1. Moreover, these data provide an integral picture of gene expression associated with plant responses under field conditions.ISSN:0032-0781ISSN:1471-905