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

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

Analysis of genetic distance between Peruvian Alpaca (Vicugna Pacos) showing two distinct fleece phenotypes, Suri and Huacaya, by means of microsatellite markers

Two coat phenotypes exist in Alpaca, Huacaya and Suri. The two coats show different fleece structure, textile characteristics and prices on the market. Although present scientific knowledge suggests a simple genetic model of inheritance, there is a tendency to manage and consider the two phenotypes as two different breeds. A 13 microsatellite panel was used in this study to assess genetic distance between Suri and Huacaya alpacas in a sample of non-related animals from two phenotypically pure flocks at the Illpa-Puno experimental station in Quimsachata, Peru. The animals are part of a germplasm established approximately 20 years ago and have been bred separately according to their coat type since then. Genetic variability parameters were also calculated. The data were statistically analyzed using the software Genalex 6.3, Phylip 3.69 and Fstat 2.9.3.2. The sample was tested for Hardy-Weinberg equilibrium (HWE) and after strict Bonferroni correction only one locus (LCA37) showed deviation from equilibrium (P<0.05). Linkage disequilibrium (LD) was also tested and 9 loci associations showed significant disequilibrium. Observed heterozygosis (Ho= 0.766; SE=0.044), expected heterozygosis (He=0.769; SE=0.033), number of alleles (Na=9.667, SE=0.772) and Fixation index (F=0.004; SE=0.036) are comparable to data from previous studies. Measures of genetic distance were 0.06 for Nei's and 0.03 for Cavalli-Sforza's. The analysis of molecular variance reported no existing variance between populations. Considering the origin of the animals, their post domestication evolution and the reproductive practices in place, the results do not show genetic differentiation between the two populations for the studied loci

Analysis of genetic distance between Peruvian Alpaca (Vicugna Pacos) showing two distinct fleece phenotypes, Suri and Huacaya, by means of microsatellite markers

Two coat phenotypes exist in Alpaca, Huacaya and Suri. The two coats show different fleece structure, textile characteristics and prices on the market. Although present scientific knowledge suggests a simple genetic model of inheritance, there is a tendency to manage and consider the two phenotypes as two different breeds. A 13 microsatellite panel was used in this study to assess genetic distance between Suri and Huacaya alpacas in a sample of non-related animals from two phenotypically pure flocks at the Illpa-Puno experimental station in Quimsachata, Peru. The animals are part of a germplasm established approximately 20 years ago and have been bred separately according to their coat type since then. Genetic variability parameters were also calculated. The data were statistically analyzed using the software Genalex 6.3, Phylip 3.69 and Fstat 2.9.3.2. The sample was tested for Hardy-Weinberg equilibrium (HWE) and after strict Bonferroni correction only one locus (LCA37) showed deviation from equilibrium (P<0.05). Linkage disequilibrium (LD) was also tested and 9 loci associations showed significant disequilibrium. Observed heterozygosis (Ho= 0.766; SE=0.044), expected heterozygosis (He=0.769; SE=0.033), number of alleles (Na=9.667, SE=0.772) and Fixation index (F=0.004; SE=0.036) are comparable to data from previous studies. Measures of genetic distance were 0.06 for Nei’s and 0.03 for Cavalli-Sforza’s. The analysis of molecular variance reported no existing variance between populations. Considering the origin of the animals, their post domestication evolution and the reproductive practices in place, the results do not show genetic differentiation between the two populations for the studied loci

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals

Alternative splicing of the sheep MITF gene: Novel transcripts detectable in skin

Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor, which regulates the differentiation and development of melanocytes and pigment cell-specific transcription of the melanogenesis enzyme genes. Though multiple splice variants of MITF have been reported in humans, mice and other vertebrate species, in merino sheep (Ovis aries), MITF gene splicing has not yet been investigated until now. To investigate the sheep MITF isoforms, the full length mRNA/cDNAs from the skin of merino sheep were cloned, sequenced and characterized. Reverse transcriptase (RT)-PCR analysis and molecular prediction revealed two basic splice variants with (+) and without (-) an 18 bp insertion viz. CGTGTATTTTCCCCACAG, in the coding region (CDS) for the amino acids 'ACIFPT'. It was further confirmed by the complete nucleotide sequencing of splice junction covering intron-6 (2463 bp), wherein an 18bp intronic sequence is retained into the CDS of MITF (+) isoform. Further, full-length cDNA libraries were enriched by the method of 5' and 3' rapid amplification of cDNA ends (RACE-PCR). A total of seven sheep MITF splice variants, with distinct N-terminus sequences such as MITF-A, B, E, H, and M, the counterparts of human and mouse MITF, were identified by 5' RACE. The other two 5' RACE products were found to be novel splice variants of MITF and represented as 'MITF truncated form (Trn)-1, 2'. These alternative splice (AS) variants were illustrated using comparative genome analysis. By means of 3' RACE three different MITF 3' UTRs (625, 1083, 3167bp) were identified and characterized. We also demonstrated that the MITF gene expression determined at transcript level is mediated via an intron-6 splicing event. Here we summarize for the first time, the expression of seven MITF splice variants with three distinct 3' UTRs in the skin of merino sheep. Our data refine the structure of the MITF gene in sheep beyond what was previously known in humans, mice, dogs and other mammals

Schematic illustration of primer walking strategy for the ovine SCF (oSCF) mRNA/cDNA transcripts from skin. Gel pictures show subsequent RT-PCR and RACE amplification of the resultant full-length structural coverage of s-SCF (+) and m-SCF (−).

<p>The (+) and (−) product is indicated with two different symbols (see key to symbols). In the above figure, the arrows indicate the corresponding position of fwd and rev primers and the split regions of 5′ UTRs, CDS and 3′ UTRs are labeled with respective positions and base pairs (bp). The start and stop codon is labeled in ‘black’ (ATG) and ‘red’ (TAA) letters repectively. (<b>A</b>) <b>Illustration for the full-length cDNA coverage of ovine s-SCF (+) </b><b><i>isofom-1</i></b>. (<b>a</b>) Amplification of isoform specific coding region (CDS) of ovine s-SCF (+) cDNA fragment (621 bp). Individual animal of merino sheep such as Black, Brown, White and the PCR negative control is indicated as Bl, Br, Wh_1,2 (two individuals) and (−)ve, respectively; (<b>b</b>) 3′ RACE first round amplification of oSCF common region (+/−) showing three different sizes of amplicon ranging from ∼700 bp to 1300 bp; (<b>b₁</b>) Isoform specific second round (Nested 1) 3′ RACE of ovine s-SCF (+) cDNA fragment (855 bp); (<b>b₂</b>) Gel picture shows the purified 3′ RACE product of 793 bp (Nested 2 amplification); (<b>c</b>) 5′ RACE proteolytic site specific amplification (364 bp) of ovine s-SCF (+). (<b>B</b>) <b>Illustration for the full-length cDNA coverage of ovine m-SCF (</b>−<b>) </b><b><i>isoform-2</i></b>. A premature termination codon (PTC) is indicated in red symbol and the resultant alternative open reading frame (ORF) responsible for the shorter truncated product is highlighted in black open box symbol; (<b>b₃</b>) 3′ RACE amplification (Nested-1) from (b) indicates a 597 bp ovine m-SCF (−) amplicon and the other non-specific products (∼0.7/1.2 kb); (<b>b₄</b>) Further, Nested 2 amplification yielded a 389 bp ovine m-SCF (−) amplicon; <b>(b₅</b>) Gel picture shows the Nested 3 amplification of a 336 bp ovine m-SCF (−) product amplified from either (b₃) or (b₄) or (b) or directly from the oligo(dT)₁₈ modified primed cDNA; <b>(d</b>) 5′ RACE amplification of the common region (+/−) showing two oSCF cDNA products (325 and 215 bp) characterized as ovine m-SCF (−) <i>isoform2a/ab</i>, respectively. DNA size markers are indicated as, <b>M₁</b>– λ-DNA EcoRI/HindIII digest; and <b>M₂</b> - 1 kb Gene Ruler. In the above figure, the arrow marks indicate the appropriate size(s) of amplicon of the respective (RT)-PCR amplification. Note: For simplification we removed the tag ‘scf’ from the primer notation (see. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038657#pone.0038657.s008" target="_blank">Table S2</a>).</p

Schematic representation of the topological characteristics of two different ovine SCF (oSCF) protein products in comparison to human SCF (huSCF).

<p>(<b>a</b>) Illustrates the identical topological features for the soluble oSCF (+) and huSCF (+) which corresponds to the 273 aa <i>vs.</i> 274 aa, respectively. The D^174/175G represents the change of aa residue for the alternative natural variant i.e., right at the proteolytic site (28 aa, ‘green line’). The difference in the position is due to sequence divergence of soluble oSCF (+) which has an additional ‘Glu’ residue at ‘E¹⁵⁴’ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038657#pone.0038657.s003" target="_blank">Figure S3d</a>). (<b>b</b>) Demonstrates the difference in topological features of the membrane-bound oSCF (−) and huSCF (−) which corresponds to the 181 aa <i>vs.</i> 245 aa, respectively. This novel ovine m-SCF (−) has a unique C-terminus with an additional uncharacterized 6 aa residue (176–181, see key to symbols) right after D¹⁷⁵G. Given below the diagram (in 5a, b) are the appropriate topological features (see key to symbols) of human and ovine soluble SCF (+) and membrane-bound SCF (−) with referencce to UniProt ID. P79368 and P21583; (<b>c</b>) Schematic representation of ovine SCF gene transcription and translation in skin (hypothetical view). The corresponding oSCF protein products, s-SCF (+) and m-SCF (−) and their topological characteristics are labeled and highlighted respectively.</p