UTILIZATION OF INTERNAL TRANSCRIBED SPACER (ITS) AS A MOLECULAR MARKER FOR PHYLOGENETIC RELATION- SHIP OF Solanaceae FAMILY

olanaceae family is also called 'nightshades' contains more than two thousand species, and are considered as one of the major plant families providing food (Yadav et al., 2016). Only four genera contain economically significant cultivated food crop-species. The most economically important genus of the family is Solanum, whilst Capsicum, Physalis and Lycium contribute the remainder of cultivated crop species (Samuels, 2015). The last fifty years witnessed a continual relief in horticultural and agricultural biodiversity of nutritionally important plants, including those of the Solanaceae family. So survey of the biodiversity, ethnobotany and taxonomy of subfamily Solanoideae was undertaken and presented as an inventory of food species (Samuels, 2015). Developing new cultivars with significantly increased yield and quality is the main target of plant breeder to play an important role in the improvement of Solanaceae. Bebeli and Mazzucato (2008) checked out the situation of the most economical species: eggplant, pepper and tomato's germplasm resources, breeding methodology and the implementation of plant breeding in these species.

Several molecular markers have been used in recent years to identify and assess the genetic diversity and phylogenetic relationship in plants.Barchi et al. (2011) identified SNP and SSR markers using restriction site associated DNA (RAD) tag sequencing in eggplant.RAPD markers were used to study the molecular diversity of 12 popular potato varieties in Bangladesh (Hoque et al., 2013).Shirasawa and Hirakawa (2013) studied DNA markers benefits and their applications in molecular breeding in tomato, genetic linkage map, QTL and gene mappings, comparative genomics and functional annotations of DNA polymorphism.
Ribosomal DNA (rDNA) consists of one of the largest multigenic families in eukaryotic genomes and is present at one or several locations in arrays of tandem elements.Each unit is composed of three rRNA gene regions (5.8S, 18S, and 28S) that are foregone by an external transcribed spacer, and the two internal transcribed spacers ITS1 and ITS2 separate the genes, respectively (Giudicelli et al., 2016).The rDNA exons are quite conserved across eukaryotic organisms, whereas the ITS regions present length variability due to point mutations and insertions/deletions (indels).ITS sequences have been broadly used in the inference of phylogenetic hypotheses and in molecular S evolution studies of plants at several taxonomic levels (Giudicelli et al., 2016).
The objective of this study was to utilize the ITS as a useful molecular marker to determine the genetic relationship of some Solanaceae species.

Plant materials
Seven different leaf samples of Solanaceae (Two of: Tomato, one from each of: Chili pepper, Eggplant, Potato, Ground cherry and Bell pepper) were kindly obtained from the greenhouse of Horticulture Department, Faculty of Agriculture, Ain-Shams university (Table 1).

DNA extraction and ITS amplification
Total DNA was extracted from fresh leaves of each sample using Spin Column Genomic DNA Minipreps Kit for Plant (Bio Basic INC.) following the manufacturer's protocols.The quantity and quality of DNA samples were checked on agarose gel using 100 bp DNA Ladder.ITS rDNA was amplified using the following primer pair: ITS-4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS-5 (5'-GGAAGTAAAAGTCGTAA CAAGG-3') (Sharma et al., 2002).The reactions were carried out in 50 µl mixture containing 24µl sterile water, 10 µl of 5x PCR reaction buffer (containing MgCl 2 and dNTPs), 5 µl of each primer (0.7 µM), 1 µl (5 U) of Taq polymerase and 5 µl (50 ng) template DNA.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 ITS region according to 100 bp DNA Ladder.The amplified fragments were purified and sequenced in one direction using ITS-4 primer.

Alignment and phylogenetic tree:
The obtained sequences were aligned on line using the NCBI (https://www.ncbi.nlm.nih.gov/) web site through Basic Local Alignment Search Tool (BLAST).Then multiple alignment and phylogeny tree of nucleotide were done using the Clustal Omega free web site: (http://www.ebi.ac.uk/Tools/msa/clustalo/) for both phylogenies; one between seven sample's nucleotide sequences and the second between the samples and other related sequences for Solanaceae family from NCBI.

RESULTS AND DISCUSSION
The ITS region has been found to be an effective universal region for molecular identification of plants (Samsuddin et al., 2012).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 Toma-to_1, Chili pepper, Eggplant, Tomato_2, Potato, Ground cherry and Bell pepper, respectively.
Fragments were purified and sequenced; each sequence was aligned individually at BLAST to confirm each species identity and to get the other related sequences as shown in Table (2 Multiple sequence alignments were done using Clustal Omega program for the seven sequences (Fig. 2), which illustrates the orthologous variants that occurred between the species.Small stars under the alignment indicate identical nucleotide sequences, whereas the hyphens refer to the indels (insertion/ deletion) mutation as a gap with multiple alignments.Also, there were a lot of base substitutions as pointed in Fig.
Phylogenetic relationship tree for seven samples of Solanaceae (Fig. 3) showed that Potato was closely related to Tomato as they were grouped in one main cluster.Whereas, the Eggplant was related to both types of pepper (Chili and Bell) and was grouped in another main cluster.The Ground Cherry was separated alone in the third main cluster.Previously, taxonomy of Solanaceae family relied on morphological variability like number of carpels that form the gynoecium, number of locules in the ovary, type of ovules and their number, type of fruit and their reproductive characteristics.However recent studies are based on DNA and RNA molecular markers to obtain the relationships between species is more precise, reliable and powerful even between genera in the same family.
Molecular markers were analyzed in three backcross generations in order to verify the occurrence of recombination between homeologous chromosomes and the efficiency of introgressing useful genes which set up to overcome interspecific barriers existing between the cultivated Solanum tuberosum and the wild species Solanum commersonii (Barone et al., 2001).Bebeli and Mazzucato (2008) reviewed the status of tomato, pepper and eggplant germplasm resources, breeding methodology and proved that markerassisted selection (MAS) is an application that can be efficient for plant breeding, which have been successful in tomato; few examples exist in pepper and still in its infancy in eggplant.Comparative genomics investigations can be used to transfer genome information from tomato to pepper and eggplant.Genetic diversity of 49 accessions of the hot pepper species Capsicum chinensis was estimated through analyses of 12 physicochemical traits of the fruit, with eight multicategorical variables using 32 RAPD primers (Finger et al., 2010).Poczai et al. (2010) illustrated the importance of the sequence-based IT (Intron Targeting) markers within different populations of potato and related species in the genus Solanum, due to their polymorphism and potential for breeding studies in different taxa and their transferability to a related species (Solanum nigrum L.).DNA sequences between Physalis and tomato were compared to analyze genetic diversity in Physalis using tomato markers, where 38 accessions from at least six species of Physalis were subjected to genetic diversity analysis using 97 tomato markers and 25 SSR markers derived from Physalis peruviana (Wei et al., 2012).The results indicated similarity between Physalis and tomato at both molecular markers and DNA sequence levels.Therefore, the molecular markers developed in tomato can be used in genetic study of Physalis.Gramazio et al. (2016)  The obtained results from this study proved the usefulness and the importance of molecular investigation using rDNA regions (ITS) to assess the relationships of some species and genera not only depending on morphological characters as reported by Sharma et al. (2002) who assessed the genetic diversity in species like barley (Hordeum spontaneum) and wheat (Triticum aestivum) through the length and sequence of ITS region of ribosomal DNA.Furthermore, to substantiate our results, 10 related sequences from each alignment obtained from BLAST were picked up (Table 2) to make the multiple alignments for seven samples with other species of Solanaceae family.Figure (4) illustrate the status of seven samples within the big Solanaceae family, that clustered in small groups, one restrain seven sample from this study, followed by another group contain Capsicum sp, while Solanum melongena and Physalis occupied in separate groups.Such studies based on molecular verification between different taxa could lend a valuable reliable and environmental free tool to assess biodiversity and guard against taxonoma errors in classification studies.

SUMMERY
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 align-ments for the seven studied samples with other species of Solanaceae family.uality of meat is of concern to researchers, consumers, retailers and governmental control authorities at all steps of the production process.Nevertheless, meat can be attractive target for adulteration in many ways and one very obvious kind is to illegally sell cheaper meat as meat from more profitable and desirable species.

REFERENCE
In Egypt, where population raises the demand for meat products escalates, and, thus, they become the most highlypriced commodities.These encourage economic gain-oriented meat species adulteration especially in ground and comminuted products.One common type of adulteration in Egypt is to add inferior quality meat with superior one (e.g.mixing chevon for mutton, and buffalo and camel meats for cattle meats).Another type is to add deliberately meat from objectionable species with meat commodities commercially available in this country (e.g.mixing pork for mutton, and donkey meat for meats from cattle, buffalo and camel).Laws of Islam prohibit Muslims from eating pork and domesticated donkey meat (Mangar, 2015).The recent donkey meat scandal in Egypt (Beer, 2015) put consumers in red alert vis-à-vis both the presence of 'haram' (not allowed) meat in their food and the indication that the meat they eat has been processed under nonsanitary conditions, thus representing potential risk to their health.
DNA-based techniques are regarded as the most pertinent methods for species identifications.DNA is existent in nearly all tissue types of an individual, stable under different circumstances (e.g.freezing, salting, drying, cooking and manufacturing) and permits for differentiating even very closely-related species due to the diversity afforded by the genetic code.
In spite of their well-known utility in permitting explicit species identification in some Egyptian research laboratories (Abd El-Razik et al., 2007;Abdel Rahman et al., 2009;Abd El-Nasser et al., 2010;Zahran and Hagag, 2015), DNA techniques have not yet been employed by public quality control laboratories and inspection services authorities to verify species composition of traditional meat articles available in this country.

Q
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 (mtCyt-b) gene for the detection of two 'haram' meat species, namely pig and donkey, and five 'halal' (allowed) meat species, namely, goat, sheep, cattle, camel and buffalo.

Raw meat sample collection and preparations
In total, 35 raw meat samples were sampled, representing seven species, viz goats, sheep, pigs, cattle, camels, buffaloes and donkeys (Table 1).All the collected samples were transferred, under ice chilled condition (4C) and stored frozen (-20C) without removing their containers or packages.
The samples used for qualitative detection of meat adulteration included two categories, the first was prepared in single-species and the second included samples made up with equally mixed meat of small species, large species and all small and large species.

DNA extraction and evaluation of its adequacy for PCR amplification
Genomic DNA included mitochondrial DNA was extracted from muscle samples of goats, sheep, pigs, cattle, camels, buffaloes and donkeys using the Thermo Scientific Genejet Genomic DNA Purification Kit, #k0721l (www.thermoscientific.com/onebio)according to the manufacturer manual.Adequacy of extracted DNA for PCR amplification was evaluated by measuring its purity and concentration using NanoDrop 2000c, (Thermo Scientific, Wilmington, USA).

Conventional PCR amplification assays
PCR mix components and its performing steps were performed according to the manufacturer manual.Amplimers were resolved by electrophoresis on 2% agarose gel (Promega, Giza, Egypt) run in TBE buffer (5.5 gm boric acid, 10.89 tris and 4ml EDTA (0.5 H) adjusted to PH 8, with the final volume is made up to 1L with distilled water).After being stained with 0.2 µg/ml ethidium bromide, the PCR product gel was densitometrically scanned and analyzed using Genus3, (Syngene, Berlin, Germany) and following the manufacturer's Quantity One Software Package.
In order to check the specificity of the oligonucleotide primers using the simplex PCR assay with raw meats, each set of oligonucleotide reverse primers was performed in simplex PCR with a noninvestigated species to trace any case of cross-reaction.To confirm the specificity of the oligonucleotide reverse primers, the simplex PCR was carried out on the DNA samples extracted from one sample of each of the seven investigated species, and then from five samples representing each of these species.
The specificity of the oligonucleotide primers using the multiplex PCR with raw meats was tested analogous samples, mixed meats were used in a triplex PCR for meats of goats, sheep and pigs, a quadruplex PCR for meats of cattle, camels, buffaloes and donkeys and, finally, a heptuplex PCR to detect meats of goats, sheep, pigs, cattle, camels, buffaloes and donkeys.The three assays were performed in Techne Thermal Cycler TC-512 (Bibby Scientific Ltd, Staffordshire, United Kingdom) with the same steps and cycling parameters already given in Tables (3 and   4).

RESULTS AND DISCUSSION
Optimal primal sequence and appropriate primer concentration are essential for maximal specificity and efficiency of PCR.A poorly designed primer can result in little or no product due to nonfulfillment of a number of conditions among which (i) Primer length: In the present study (Table 2) oligonucleotide primers were at least 18 nucleotides in length to reduce the probabilities of confronting problems with a second hybridization site on the vector or insert.Oligonucleotide primers did not exceed 28 nucleotides as it is especially imperative to avoid 4 or more G's or C's in a row.Dieffenbach et al. (1995) indicated those oligonucleotide primers between 18 and 24 bp long can be very sequence specific if the PCR reaction annealing temperature is assigned within a few degrees of the melting temperature (T m ) of the primer (Table 2).The PCR reaction temperature used in the current work (Table 2) was set within degrees not exceeding 5C of the primers T m .(ii) Melting temperature: the melting temperatures for the primers used in the present work (Table 2) were in the range 55-60C.Primers with lower T m were sidestepped because of potential unclear results.Primers with higher T m were also avoided because of potential PCR secondary annealing.(iii) GCcontent: oligonucleotide primers had a GC-content between 44 and 60 percent with the exception of pig primers (42 percent) for which the primer sequence was extended to 28 bp to keep the T m above the recommended lower limit of 50C.according to Rychlik et al. (1990), GCcontent and T m are strictly dependent on each other.(iv) 3'-End sequence: Care has been taken to ensure that oligonucleotide primers had higher GC-content on their 5' ends than on their 3' ends.This does not exclude the fact that a "G" or "C" is desirable on the 3' end.Kwok et al. (1990) stressed the importance of the 3' terminal position for the control of mis-priming.(v) Dimers: caution has also been taken in such a way that oligonucleotide primers do not contain sequences of nucleotides that would allow one primer to anneal to itself or to the other primer used in PCR reactions (primer dimer formation).This would result, according to Breslauer et al. (1986), in an unproductive priming event that reduces the overall signal obtained.
(vi) specificity: oligonucleotide primers were chosen so that they have a unique sequence within the template DNA that is to be amplified.Oligonucleotide primers designed with a highly repetitive sequence were avoided as they result in a smear when amplifying DNA.(vii) Complementary oligonucleotide primer sequence: In the present work (Table 2) oligonucleotide primers were designed with absolutely no intra-primer homology belong 3 basepairs.It was also avoided to permit interprimer homology as this can interfere with hybridization.(viii) Suitability of primers to the size of the designed PCR products: As the specifics of the size of the desired PCR products often depend on the application, Dieffenbach et al. (1995) indicated that PCR products of 150-1000 bp are generally produced for the purpose of detecting DNA sequence.The species specific oligonucleotide primers in the present study were designed to amplify amplicons within this range (290-1000 bp) (Figs 1a, 1b and 2).

Adequacy of extracted DNA for species-specific conventional PCR amplification
The results given in Table (3) indicate that extracted DNA was adequate for species-specific conventional PCR amplification.The overall range was 12.35-46.29(ng/µl), for concentration and 1.12-1.46(OD 260 /OD 280 ratio), for purity.The goat extracted DNA showed the highest purity and concentration values (1.45 and 35.96, respectively) while the pig DNA showed the lowest values (1.31 and 12.56).

Simplex PCR specificity isolated from raw meats
PCR products amplified from meats of goats, sheep, pigs, cattle, camels, buffaloes and donkeys (Figs 1a and 1b) were single DNA fragments of 290, 370, 480, 580, 700, 800 and 1000 bp, respectively.The results indicate no case of cross-reaction was traced when a set of reverse oligonucleotide primers was performed in simplex PCR with a noninvestigated species.

Multiplex PCR specificity of DNA isolated from raw meats
In a triplex PCR for meats of goats, sheep and pigs (Fig. 2, lane 9), a quadruplex PCR for meats of cattle, camels, buffaloes and donkeys (Fig. 2, lane 11) and a heptuplex PCR for meats of goats, sheep, pigs, cattle, camels, buffaloes and donkeys (Fig. 2, lane 13), products amplified, with species-specific oligonucleotide primers, retained the same specificity observed with simplex PCR (Fig. 2, lanes 1 through 7).A single band of target size was shown from one meat species without producing any fragment of non-specific amplification.
This work described a simplex and a multiplex polymerase chain reaction (PCR) assays for the accurate identification of two meat kinds forbidden in Islamic foods (pig and donkey meats) and five meat kinds commonly marketed in Egypt (goat, sheep, cattle, camel and buffalo meats).Meat samples from the seven investigated species were used for molecular analysis of each species as per standard method.Cytochrome-b gene was amplified by PCR using a common forward oligonucleotide primer.By mixing species specific reverse oligonucleotide primers in the appropriate ratio, DNA-fragments could be identified by only one multiplex PCR.PCR products were resolved by agarose gel electrophoresis and characteristic band pattern was observed for each species.The PCR products showed amplicons of 290,370,480,580,700,800 and 1000 bp from goat, sheep, pig, cattle, camel, buffalo and donkey meats, respectively.The sequel of this study suggests that the method of detection used can be applied by quality control laboratories and inspection services to determine adulteration different kinds of meats and meat products.
The multiplex PCR (M-PCR) technique is a variant of PCR in which two or more DNA loci are synchronously amplified in the aforesaid reaction within a sole PCR mixture to produce amplicons of inconstant sizes specific to different DNA sequences.Henegariu et al. (1997) described critical parameters and step-bystep protocol of the M-PCR technique.M-PCR was used successfully in the present study and several previous works (e.g.Abd El-Razik et al., 2007;Bai et al., 2009;Sakalar and Abasiyanik, 2011).M-PCR proved to offer the following advantages: (i) PCR internal control: false negatives that pose potential problems in PCR are revealed in multiplex amplification since each amplicon allows internal control for the other amplified fragments.(ii) PCR efficiency of DNA template quality determination: with M-PCR technique degraded DNA templates show weaker signals for long bands than for short ones and for fall in amplification efficiency.(iii) PCR economic efficiency: by spotting multiple genes simultaneously, further information may be acquired from a single test run than would require more PCR reagents and preparation time.(iv) PCR rapidity: with M-PCR many meat products belonging to ruminants and non-ruminants can simultaneously analyzed in the same reaction for example, in the present work 100 meat samples could have been analyzed in 25 reaction tubes at the same time by using seven primer sets belonging to goat, sheep, pig, cattle, camel, buffalo and donkey.The present study depicts (Fig. 2 lanes 9, 11 and 13) the consequences of an optimized M-PCR, which resulted in a single band of target from one species (goat, sheep, pig, cattle, camel, buffalo and donkey) and no fragment was produced by non-specific amplicon.
The results of the present study (Fig. 2) agreed with those of many works in obtaining different amplicon molecular length for animal species and, thus, capable to be used to differentiate between mixed-species meats lawfully or unlawfully produced.In few reports, however, cattle and buffalo (Jain et al., 2007), goat and sheep (Mafra et al., 2007), donkey and horse (Abdel-Rahman et al., 2009) showed the same amplicon molecular length.It was recommended to develop specific oligonucleotide primers for buffalo (Jain et al., 2007) and sheep (Mafra et al., 2007) and to use specific restriction enzyme following RFLP-PCR to distinguish between donkey and horse.
Finally, this study suggests an accurate, sensitive and rapid analytical technique for goat, sheep, pig, cattle, camel, buffalo and donkey meats and traceability identification based on PCR analysis of Cytochrome-b gene of mitochondrial DNA for enforcement of labeling regulations and ensuring that meats comply with religion regulations ('halal' authentication).

SUMMARY
The current study aimed to use the mitochondrial cytochrome b gene for authentication of meats from different animal sources, with special relevance to hala-related meat sources.In total, 35 raw meat samples were sampled, representing seven species, viz goats, sheep, pigs, cattle, camels, buffaloes and donkeys.Mitochondrial Cytochrome-b (mtCyt-b) gene sequences for the seven-investigated species were retrieved from GenBank database and aligned.Common universal forward oligonucleotide primer and specificreverse primer for each species were designed.DNA was extracted, PCR was performed using conventional assay and amplicons were resolved using standard gel electrophoresis.M-PCR proved to offer the following advantages: (i) PCR internal control: false negatives that pose potential problems in PCR are revealed in multiplex amplification since each amplicon allows internal control for the other amplified fragments.(ii) PCR efficiency of DNA template quality determination: with M-PCR technique degraded DNA templates show weaker signals for long bands than for short ones and for fall in amplification efficiency.(iii) PCR economic efficiency: by spotting multiple genes simultaneously, further information may be acquired from a single test run than would require more PCR reagents and preparation time.(iv) PCR rapidity: with M-PCR many meat products belong-ing to ruminants and non-ruminants can simultaneously analyzed in the same reaction for example, in the present work 100 meat samples could have been analyzed in 25 reaction tubes at the same time by using seven primer sets belonging to goat, sheep, pig, cattle, camel, buffalo and donkey.The present study depicts the consequences of an optimized M-PCR, which resulted in a single band of target from one species and no fragment was produced by non-specific amplicon.This study suggests an accurate, sensitive and rapid analytical technique for goat, sheep, pig, cattle, camel, buffalo and donkey meats based on PCR analysis of Cytochrome-b gene of mitochondrial DNA for discovery of meat-adulteration and mixed processed meat.

Fig. ( 2
Fig. (2): Multiple alignment of seven ITS nucleotide sequences for the different samples of Solanaceae family using Clustal Omega program .

Fig
Fig. (1a): Agarose gel electrophoreses of simplex PCR products from five samples taken for meats of goats (G), sheep (S) and pigs (P).M: DNA ladder marker (one microliter contains 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp).The bold-faced letter refers to species-specific reverse oligonucleotide primer; the light-faced letter refers to species DNA.Lane 1 result when the set of reverse primers was performed in simplex PCR without template DNA.(negative control).Lanes 2 through 6 results when the set of reverse primers was performed in simplex PCR with a target species.* = with; the underlined (primer * DNA) refers to the same species.

Table ( 1
): List of the seven different samples, species and genus of Solanaceae family

Table ( 1
): Number and source of meat samples collected from the seven-investigated species in this study.

Table ( 2
): Sequence, length, melting temperature and GC-content of the oligonucleotide primers designed for the seven-investigated species in this study.

Table ( 3
): Ranges of concentration and purity of DNA of samples from the seven-investigated species (OD = Optical density).