Polyphenolics free DNA isolation and optimization of PCR-RAPD for fennel (Foeniculum vulgare Mill.) from mature and young leaves

Molecular analysis of fennel (Foeniculum vulgare Mill.) strictly relies on high yield, and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide (CTAB) protocol introducing several modifications such as inclusion of variable percentage of polyvinylpyrrolidone (PVP, 2 - 6%), different molarity of sodium chloride (NaCl, 1.5 - 4 M), activated charcoal (1 to 2%), and pre-heated buffer (65°C) with and without liquid N2 extraction for the mature leaves. In contrast, the CTAB protocol without any additional anti-oxidants using liquid N2 extraction was performed for the fresh young leaf tissue. Optimization of polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD) conditions included 10X PCR buffer compositions such as (NH4)2SO4 with 0.1% (v/v) Tween 20 over KCl buffer with and without 0.8% (v/v) Nonidet P40 and an ‘optimized’ buffer which contains KCl and (NH4)2SO4, MgCl2 (2.5 mM), Taq enzyme (1 to 1.5 U), annealing temperature of 37 and 42°C and PCR reaction volume of 10 and 25 μl. The results show that DNA isolated from fresh young leaves were superior in quality and quantity over mature (stored) leaves and was amenable to optimized PCR-RAPD conditions.Keywords: Activated charcoal, cetyl trimethyl ammonium bromide (CTAB)-DNA, Foeniculum vulgare, NaCl, polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD), PCR buffer, polyphenolics, polyvinylpyrrolidone (PVP

Polyphenolics free DNA isolation and optimization of PCR-RAPD for fennel (Foeniculum vulgare Mill.) from mature and young leaves

Molecular analysis of fennel (Foeniculum vulgare Mill.) strictly relies on high yield, and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide (CTAB) protocol introducing several modifications such as inclusion of variable percentage of polyvinylpyrrolidone (PVP, 2 - 6%), different molarity of sodium chloride (NaCl, 1.5 - 4 M), activated charcoal (1 to 2%), and pre-heated buffer (65°C) with and without liquid N2 extraction for the mature leaves. In contrast, the CTAB protocol without any additional anti-oxidants using liquid N2 extraction was performed for the fresh young leaf tissue. Optimization of polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD) conditions included 10X PCR buffer compositions such as (NH4)2SO4 with 0.1% (v/v) Tween 20 over KCl buffer with and without 0.8% (v/v) Nonidet P40 and an ‘optimized’ buffer which contains KCl and (NH4)2SO4, MgCl2 (2.5 mM), Taq enzyme (1 to 1.5 U), annealing temperature of 37 and 42°C and PCR reaction volume of 10 and 25 μl. The results show that DNA isolated from fresh young leaves were superior in quality and quantity over mature (stored) leaves and was amenable to optimized PCR-RAPD conditions

Description and Molecular Phylogeny of a Novel Hypotrich Ciliate from the Soil of Marche Region, Italy; Including Notes on the MOSYSS Project

The morphology and morphogenesis during cell division of a new stylonychine hypotrich, Rigidocortex quadrinucleatus n. sp., were investigated using live observation and protargol staining. The new species was isolated from soil samples collected from an organic farm in the Marche Region, Italy, in framework of the MOSYSS project. Rigidocortex quadrinucleatus is characterized as follows: cell size about 180 9 80 lm in vivo; four ellipsoidal macronuclear nodules; 44 adoral membranelles: 18 fronto-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse, and five transverse cirri; dorsal kinety 3 with multiple fragmentation; resting cyst with hyaline ridges. Rigidocortex quadrinucleatus mainly differs from the type species R. octonucleatus in having four (vs. eight) macronuclear nodules. Rigidocortex quadrinucleatus can be easily confused with Sterkiella cavicola since both have a rather similar ventral ciliature; however, they can be separated by the slightly higher number of cirri in the left marginal row that runs along the posterior cell’s margin in R. quadrinucleatus. Morphogenesis on the ventral surface is highly similar to that of Sterkiella species, but differs significantly on the dorsal surface (multiple vs. simple fragmentation of dorsal kinety 3). Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to S. cavicola

The Alpaca Melanocortin 1 Receptor: Gene Mutations, Transcripts, and Relative Levels of Expression in Ventral Skin Biopsies

The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T) were observed in coding region and three in the 3UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation

Influence of Organic and Conventional Management Systems on Soil Microarthropods in Protected and Non-Protected Areas

Aim: The EU Biodiversity Strategy 2030 aims to increase land-protected areas at 30% and organic farming at 25% of agricultural lands. But which measure could be more effective in preserving soil biodiversity? The aim of the study is, therefore, to assess soil health of arable lands under organic and conventional managements in Non-protected (NPAorg) and Protected (PAcon) areas of Marche region (Italy) and compare the influence of the applied farming practices on soil microarthropods in two seasons, characterized by different intensities of soil management practices: spring (lower) and autumn (higher). Method: Soil health has been assessed through the Biological Quality of Soil index based on arthropods (QBS-ar). Novel approaches (QBS-ab and FAI indices) which consider microarthropods’ abundance in the index calculation, have been also applied. Density (ind/m2), Acari/Collembola ratio, % of Oribatid mites on total mites, biodiversity indices, correlations with chemical-physical parameters, and ordination analysis (nMDS) have been evaluated. Results: In both seasons, different communities have been found according to management and, particularly, PAcon sites showed significantly higher levels of biodiversity compared to NPAorg. However, in autumn, microarthropod communities present higher stability in NPAorg sites, showing an opposite trend and fewer fluctuations of the indices compared to PAcon. Conclusions: PA, even in conventional managed soils, seem to enhance soil biodiversity, while organic farming in NPA, confers a higher resilience to soil, making microarthropod communities more stable. Results showed that agricultural intensity reduction combined with the increased integration of agroecosystems in protected areas may represent an effective, and sustainable measure to preserve soil biodiversity and its ecological services