Isolation of microsatellite markers for the endemic Phlomis lychnitis (Lamiaceae).

BACKGROUND: Phlomis lychnitis is a mostly endemic species of the Iberian Peninsula that frequently hybridizes with the narrow endemic P. crinita subsp. malacitana in southern Spain. Whenever they coexist they form homoploid hybrid zones. Unlike hybridization at the polyploid level, the process of hybridization at the homoploid level is much less well known. METHODS AND RESULTS: In this study we report the development of 22 microsatellite markers through next-generation sequencing technologies for P. lychnitis. We characterize the genetic diversity for two populations of this species for the 10 markers that resulted to be polymorphic. Further, we check the transferability of these polymorphic markers to one population of P. crinita subsp. malacitana to verify the potential use of these markers for hybridization studies. The values of expected heterozygosity for P. lychnitis were higher than in P. crinita subsp. malacitana, and the three analyzed populations displayed negative values for the inbreeding coefficient which is compatible with the frequent instances of hybridization and introgression between species. CONCLUSIONS: This set of polymorphic markers are useful for further studies aiming at a deeper understanding of the homoploid hybrid process between these species. Additionally, this is the first panel of microsatellite markers developed for the genus Phlomis, a genus very rich in endemic species and with medicinal properties that could benefit from the use of these new markers.

De novo assembly of Phlomis purpurea after challenging with Phytophthora cinnamomi.

BACKGROUND: Phlomis plants are a source of biological active substances with potential applications in the control of phytopathogens. Phlomis purpurea (Lamiaceae) is autochthonous of southern Iberian Peninsula and Morocco and was found to be resistant to Phytophthora cinnamomi. Phlomis purpurea has revealed antagonistic effect in the rhizosphere of Quercus suber and Q. ilex against P. cinnamomi. Phlomis purpurea roots produce bioactive compounds exhibiting antitumor and anti-Phytophthora activities with potential to protect susceptible plants. Although these important capacities of P. purpurea have been demonstrated, there is no transcriptomic or genomic information available in public databases that could bring insights on the genes underlying this anti-oomycete activity. RESULTS: Using Illumina technology we obtained a de novo assembly of P. purpurea transcriptome and differential transcript abundance to identify putative defence related genes in challenged versus non-challenged plants. A total of 1,272,600,000 reads from 18 cDNA libraries were merged and assembled into 215,739 transcript contigs. BLASTX alignment to Nr NCBI database identified 124,386 unique annotated transcripts (57.7%) with significant hits. Functional annotation identified 83,550 out of 124,386 unique transcripts, which were mapped to 141 pathways. 39% of unigenes were assigned GO terms. Their functions cover biological processes, cellular component and molecular functions. Genes associated with response to stimuli, cellular and primary metabolic processes, catalytic and transporter functions were among those identified. Differential transcript abundance analysis using DESeq revealed significant differences among libraries depending on post-challenge times. Comparative cyto-histological studies of P. purpurea roots challenged with P. cinnamomi zoospores and controls revealed specific morphological features (exodermal strips and epi-cuticular layer), that may provide a constitutive efficient barrier against pathogen penetration. Genes involved in cutin biosynthesis and in exodermal Casparian strips formation were up-regulated. CONCLUSIONS: The de novo assembly of transcriptome using short reads for a non-model plant, P. purpurea, revealed many unique transcripts useful for further gene expression, biological function, genomics and functional genomics studies. The data presented suggest a combination of a constitutive resistance and an increased transcriptional response from P. purpurea when challenged with the pathogen. This knowledge opens new perspectives for the understanding of defence responses underlying pathogenic oomycete/plant interaction upon challenge with P. cinnamomi.

Estimation of phylogenetic relationships of Phlomis species based on seed protein polymorphism

In this study, phylogenetic relationships among 39 Phlomis taxa were investigated based on seed protein profiles produced by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). A total of 21 polypeptide bands were scored, of which, 19 were polymorphic among the taxa of the genus Phlomis. A distance matrix was generated from the similarity matrix which was computed by using Jaccard’s similarity coefficients, based on polymorphic bands and then an UPGMA tree was established through cluster analysis performed on the distance matrix. Genetic distances ranged from 0.00 to 0.50 within subsection Dendrophlomis; from 0.00 to 0.625 within subsection Gymnophlomis and from 0.00 to 0.769 within subsection Oxyphlomis. The UPGMA tree formed four groups. The topology of the tree is in agreement with the taxonomic view regarding the section Phlomis as it is divided into three subsections as Dendrophlomis, Gymnophlomis and Oxyphlomis based on morphological characters. The grouping pattern of the tree also indicated that subsection Dendrophlomis is more closely related to subsection Gymnophlomis than subsection Oxyphlomis.

Phylogenetic relationships among species of the subsection Dendrophlomis Bentham

This study used randomly amplified polymorphic DNA markers to determine genetic relationships among species of the subsection Dendrophlomis. Twenty accessions of the eleven Phlomis taxa were evaluated to determine genetic variability using fourteen ten mer primers selected from a 125 random oligonucleotide set. These 14 selected primers generated 85 RAPD bands that ranged in size from 200 to 1200 base pairs. Of the total bands, 88 percent (75) were polymorphic among the samples. Genetic distances among accessions were computed to produce a dendrogram based on UPGMA. Genetic distances ranged from 0.133 (between P. amanica and P. monocephala) to 0.494 (between P. chimerae and P. lunariifolia). The UPGMA tree based on distances has two major groups. The first comprised 9 taxa that were clustered into two subgroups. The first subgroup consisted of P. viscosa, P. lycia, P. amanica and P. monocephala while the second comprised P. lunariifolia, P. bourgaei, P. longifolia var. longifolia, P. grandiflora var. grandiflora and P. grandiflora var. fimbrilligera. The second group comprised 2 species, P. leucophracta and P. chimerae. Species-specific bands were observed for P. lycia, P. leucophracta, P. lunariifolia, P. bourgaei, P. chimerae and P. longifolia var longifolia.