Abstract
Solanaceae family is considered one of the major plant families providing food. Studies based on DNA and RNA molecular markers are more precise, reliable and powerful to assess the relationships between species also between genera in the same family. ITS sequences have been broadly used in the inference of phylogenetic hypotheses and in molecular evolution studies of plants, because it is variable, represent point mutations and insertions/deletions (indels). Specific ITS fragments were produced using the universal primer through seven sample of Solanaceae. Purified fragments were sequenced and each sequence was aligned individually at BLAST to confirm each species and to determine its relation to other sequence. Multiple Sequence alignment was done using Clustal Omega program for the seven sequences, and phylogenetic relationship showed that Potato was closely related to Tomato as they were grouped in one main cluster. Whereas Eggplant was related to both type of pepper (Chili and Bell) and was grouped in another main cluster. The Ground Cherry was separated alone in the third main cluster. Finally, ten related sequences picked from each alignment were deduced from BLAST to make the multiple alignments for the seven studied samples with other species of Solanaceae family.
Highlights
Techne TC-3000 PCR Thermal Cycler was used with the following PCR steps: an initial denaturation 95 C for 5 min, 35 thermal cycles (95 C for 1 min, 58 C for 1 min and 72 C for 2 min) and a final extension at 72 C for 5 min according to the following diagram: Gel analysis, fragment purification and sequencing: PCR products were run in 2% agarose gel and image was analyzed using the TotalLab TL120 to determine molecular size of the amplified fragments of investigation using rDNA regions (ITS) region according to 100 bp DNA Ladder
Total genomic DNA was extracted from seven different sample species of Solanaceae, and amplified through PCR using the universal primers ITS-4 and ITS-5
Specific single fragment was obtained for each sample after running in 2% agarose gel (Fig. 1), sizes were determined by TotalLab TL120 analysis as follow: 744 bp, 729 bp, 744 bp, 750 bp 759 bp, 759 bp and 798 bp with Tomato_1, Chili pepper, Eggplant, Tomato_2, Potato, Ground cherry and Bell pepper, respectively
Summary
The objective of this study was to utilize the ITS as a useful molecular marker to determine the genetic relationship of some Solanaceae species. As the application of polymerase chain reaction (PCR) seems to give the most satisfactory results (Teletchea et al, 2005), the objective of this study was to use simplex and multiplex PCR assays with species-specific oligonucleotide primers targeting mitochondrial cytochromeb gene for the detection of two 'haram' meat species, namely pig and donkey, and five 'halal' meat species, namely, goat, sheep, cattle, camel and buffalo
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