A robust and cost-effective method for DNA isolation from satureja species (lamiaceae)

Aromatic species of the genus Satureja are rich in secondary metabolites that interfere with DNA isolation procedures. Four protocols based on the standard CTAB DNA extraction protocol of Doyle and Doyle (1987) were tested in six savory taxa. The polyphenol adsorbents activated charcoal and/or polyvinylpyrrolidone 10 were employed in three procedures (B, C and D); for the elimination of polysaccharides, 4M NaCl was applied in the latter two. The highest DNA yield was obtained with Protocol D and averaged 1420.7 +/- 398.3 mu g DNA/g of dry leaf tissue. Optimal values of the absorbance ratio 260/280 of all DNA solutions revealed the absence or only negligible contamination by proteins. Contamination by polysaccharides inferred from the absorbance ratio 260/230 showed that Protocol C provided the least contaminated material (average of 1.7 +/- 0.4). Enzymatic reactions of DNA solutions obtained by Protocol D showed amplification of both loci in all individuals. In conclusion, Protocol D is suitable for the isolation of high quantities of pure DNA from Satureja spp

Cross-species amplification of nuclear est-microsatellites developed for other pinus species in pinus nigra

Due to the current lack of nuclear microsatellites (simple sequence repeats SSRs) specifically developed for Pinus nigra, an important European coniferous species, we cross-species amplified 12 EST-SSRs (expressed sequence tagged SSRs) developed for other Pinus species in P. nigra in order to delineate loci which can be used for assessing levels of genetic diversity and genetic structuring in this species. We amplified these loci in individuals from seven populations from the central Balkans representing four recognized infraspecific taxa of P. nigra (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica). Contrary to expectations on high transferability of EST-SSRs into related species, only three out of 12 tested loci were successfully amplified in P. nigra, but they displayed lack/low levels of polymorphism or generated multilocus amplification products. Thus, our estimates on levels of genetic diversity (HE = 0.183) and genetic differentiation (F-ST = 0.007) were based on variability of a single locus harboring four alleles only and they should be taken with cautions. Our study highlights the need for the development of high-resolution molecular markers, such as co-dominant genic or genomic SSRs or predominantly biallelic SNPs, or utilization of anonymous dominant markers, such as AFLPs, for genotyping in P. nigra

Genetic patterns in Pinus nigra from the central Balkans inferred from plastid and mitochondrial data

Pinus nigra J.F. Arnold, European black pine, is a typical component of Mediterranean and sub-Mediterranean coniferous forests with highly fragmentary distribution. Western Mediterranean populations of this species have been studied genetically to date, while eastern populations from the central Balkans, which are larger and more abundant, are still genetically understudied. We analyzed seven populations of P. nigra representing all infraspecific taxa recognized within the central Balkans (subspecies nigra with varieties nigra and gocensis Dordevic; and subspecies pallasiana (Lamb.) Holmboe with varieties pallasiana and banatica (Endl.) Georgescu et Ionescu), with three chloroplast microsatellites (cpDNA SSRs) and one mitochondrial (mtDNA) locus. Although our molecular data failed to support circumscription of studied infraspecific taxa, we found that genetic patterns at both genomes are in accordance with those found previously in westward populations of this species, that is - exceptionally high levels of genetic diversity (H-T = 0.949) and low genetic differentiation (G(ST) = 0.024) at the cpDNA level, and moderate levels of genetic diversity (H-T = 0.357) and genetic differentiation (G(ST) = 0.358) at the mtDNA level. Based on genealogical relations of mtDNA types currently present in Balkans' and Iberian/African populations, we inferred that the ancestral gene pool of P. nigra already harbored polymorphism at position 328 prior to the divergence to two lineages currently present in westward and eastward parts of the species range distribution. Subsequent occurrence of three mutations, which distinguish these two lineages, suggests their long-term isolation.Ministry of Education, Science, and Technology of the Republic of Serbia {[}173029, 173030

Genetic patterns in Pinus nigra from the central Balkans inferred from plastid and mitochondrial data

Pinus nigra J.F. Arnold, European black pine, is a typical component of Mediterranean and sub-Mediterranean coniferous forests with highly fragmentary distribution. Western Mediterranean populations of this species have been studied genetically to date, while eastern populations from the central Balkans, which are larger and more abundant, are still genetically understudied. We analyzed seven populations of P. nigra representing all infraspecific taxa recognized within the central Balkans (subspecies nigra with varieties nigra and gocensis Dordevic; and subspecies pallasiana (Lamb.) Holmboe with varieties pallasiana and banatica (Endl.) Georgescu et Ionescu), with three chloroplast microsatellites (cpDNA SSRs) and one mitochondrial (mtDNA) locus. Although our molecular data failed to support circumscription of studied infraspecific taxa, we found that genetic patterns at both genomes are in accordance with those found previously in westward populations of this species, that is - exceptionally high levels of genetic diversity (H-T = 0.949) and low genetic differentiation (G(ST) = 0.024) at the cpDNA level, and moderate levels of genetic diversity (H-T = 0.357) and genetic differentiation (G(ST) = 0.358) at the mtDNA level. Based on genealogical relations of mtDNA types currently present in Balkans' and Iberian/African populations, we inferred that the ancestral gene pool of P. nigra already harbored polymorphism at position 328 prior to the divergence to two lineages currently present in westward and eastward parts of the species range distribution. Subsequent occurrence of three mutations, which distinguish these two lineages, suggests their long-term isolation

CROSS-SPECIES AMPLIFICATION OF NUCLEAR EST-MICROSATELLITES DEVELOPED FOR OTHER Pinus SPECIES IN Pinus nigra

Due to the current lack of nuclear microsatellites (simple sequence repeats SSRs) specifically developed for Pinus nigra, an important European coniferous species, we cross-species amplified 12 EST-SSRs (expressed sequence tagged SSRs) developed for other Pinus species in P. nigra in order to delineate loci which can be used for assessing levels of genetic diversity and genetic structuring in this species. We amplified these loci in individuals from seven populations from the central Balkans representing four recognized infraspecific taxa of P. nigra (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica). Contrary to expectations on high transferability of EST-SSRs into related species, only three out of 12 tested loci were successfully amplified in P. nigra, but they displayed lack/low levels of polymorphism or generated multilocus amplification products. Thus, our estimates on levels of genetic diversity (HE = 0.183) and genetic differentiation (F-ST = 0.007) were based on variability of a single locus harboring four alleles only and they should be taken with cautions. Our study highlights the need for the development of high-resolution molecular markers, such as co-dominant genic or genomic SSRs or predominantly biallelic SNPs, or utilization of anonymous dominant markers, such as AFLPs, for genotyping in P. nigra.Ministry of Education, Science, and Technology of the Republic of Serbia {[}173029, 173030

Leaf n-Alkanes as Characters Differentiating Coastal and Continental Juniperus deltoides Populations from the Balkan Peninsula

The composition of the cuticular n-alkanes isolated from the leaves of nine populations of Juniperus deltoides R. P. Adams from continental and coastal areas of the Balkan Peninsula was characterized by GC-FID and GC/MS analyses. In the leaf waxes, 14 n-alkane homologues with chain-lengths ranging from C22 to C35 were identified. n-Tritriacontane (C33) was dominant in the waxes of all populations, but variations between the populations in the contents of all n-alkanes were observed. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses) were used to investigate the diversity and variability of the cuticular-leaf-n-alkane patterns of the nine J. deltoides populations. This is the first report on the n-alkane composition for this species. The multivariate statistical analyses evidenced a high correlation of the leaf-n-alkane pattern with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon.Ministry of Education, Science and Technological Development of Serbia {[}173029, 172053

Leaf n-Alkanes as Characters Differentiating Coastal and Continental Juniperus deltoides Populations from the Balkan Peninsula

The composition of the cuticular n-alkanes isolated from the leaves of nine populations of Juniperus deltoides R. P. Adams from continental and coastal areas of the Balkan Peninsula was characterized by GC-FID and GC/MS analyses. In the leaf waxes, 14 n-alkane homologues with chain-lengths ranging from C22 to C35 were identified. n-Tritriacontane (C33) was dominant in the waxes of all populations, but variations between the populations in the contents of all n-alkanes were observed. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses) were used to investigate the diversity and variability of the cuticular-leaf-n-alkane patterns of the nine J. deltoides populations. This is the first report on the n-alkane composition for this species. The multivariate statistical analyses evidenced a high correlation of the leaf-n-alkane pattern with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon.Ministry of Education, Science and Technological Development of Serbia {[}173029, 172053

Caseload management and outcome of patients with aortic stenosis in primary/secondary versus tertiary care settings-design of the IMPULSE enhanced registry.

Background Severe aortic stenosis (AS) is one of the most common and most serious valve diseases. Without timely intervention with surgical aortic valve replacement or transcatheter aortic valve replacement, patients have an estimated survival of 2-3 years. Guidelines for the treatment of AS have been developed, but studies suggest that as many as 42% of patients with AS are not treated according to these recommendations.The aims of this registry are to delineate the caseload of patients with AS, outline the management of these patients and determine appropriateness of treatments in participating centres with and without onsite access to surgery and percutaneous treatments. Methods/design The IMPULSE enhanced registry is an international, multicentre, prospective, observational cohort registry conducted at four central full access centres (tertiary care hospitals) and at least two satellite centres per hub (primary/secondary care hospitals). An estimated 800 patients will be enrolled in the registry and patient follow-up will last for 12 months. Discussion In addition to the primary aims determining the caseload management and outcome of patients with AS in primary, secondary and tertiary care settings, the registry will also determine a time course for the transition from asymptomatic to symptomatic status and the diagnostic steps, treatment decisions and the identification of decision-makers in tertiary versus primary/secondary care hospitals. The last patient will be enrolled in the registry in 2018 and results of the registry are anticipated in 2019. Registration number NCT03112629