Sequence Analysis of the Internal Transcribed Spacer ( ITS ) Region of the Nuclear Ribosomal DNA ( nrDNA ) and Chloroplast trn LF Region ( cpDNA ) of Some Lactuca L . ( Asteraceae ) Species in Turkey

In the current study, sequence analysis of some Turkish Lactuca L. species using nrITS DNA and trnL-F cpDNA sequences were performed to elucidate phylogenetic relationships among the taxa under study. Hieracium umbellatum was used as an outgroup. Different plant materials of Lactuca were collected from different parts of Turkey during excursions of summer 2013. Plant materials were either kept in silica gel or kept fresh for immediate DNA isolation. Both phenol chloroform-isoamyl alcohol method and commercial kits were used to extract genomic DNA for PCR reactions. ITS4 and ITS5A primers were utilized for ITS region, while trnLe and trnLf primers were used to amplify the trnL-F region. Obtained DNA sequences were edited both manually and by using BioEdit 7.0.4.1. Sequencing data were aligned via ClustalW program and analyzed using PAUP 4.01b10 software. nrITS sequences varied from 639 nucleotides to 735 nucleotides. Average nucleotide composition for nrITS was 22.1% (T), 27.9% (C), 23.2% (A) and 26.8% (G). It was also found that divergence values differed between 0.0000 and 0.10290. The trnL-F sequences varied from 296 nucleotides to 385 nucleotides. Average nucleotide composition of trnL-F sequences was 34.1% (T), 18.4% (C), 31.6% (A) and 16.0% (G). It was also found that divergence values differed between 0.0000 and 0.09674. Neighbour Joining (NJ) trees were constructed in order to identify the relationships among Lactuca species. Phylogenetic trees based on ITS region were found to be more useful than phylogenetic trees based on trnL-F region. After analysis of the results obtained, the data suggest that Lactuca contains 2 clades, with clade 1 having 2 subclades. These results support the prior phylogenetic studies on Lactuca and hence provide an up to date review of Turkish Lactuca species.


Introduction
The generic name Lactuca L. is derived from a Latin word "lac" means "milk", a common character to all the members of the tribe Cichorieae.The genus Lactuca L. (s.I) was established by Linnaeus (Linnaeus, 1753;Linnaeus, 1754).This genus belong to Asteraceae family which is represented by approximately 100 species and notably temperate and warm regions in Europe, Asia, North America, Australia and Africa widely distrubuted (Dziechciarková et al., 2004).The number of this species is 8 in Turkish flora (Davis, 1975).This number is given as 33 taxa according to the latest arrangements (Güner et al., 2012).Species of this genus have important species for both economics and medicine.For example, L. sativa is used in salads since it contains high levels of vitamins and minerals.L. serriola seeds are used for treating insomnia and coughing.At the same time, they are used in manufacturing varnish, scented soaps and dying materials.Lactucarium, a medication obtained from L. sativa and L. serriola is used for treating bronchitis and asthma (Bano and Qaiser, 2011).L. tatarica (L.) C.A. is a significant forage plant, but when excessively consumed, becomes toxic (Kirpicznikov, 1964).
Internal transcribed spacer (ITS) regions are molecular markers used in molecular systematic classification of many plants (Baldwin et al., 1995;Ogundipe and Chase, 2008).ITS contains noncoding regions that are characterized by sometimes higher nucleotide substitution rates and have proven useful for elucidating phylogenetic relationships at the species and genus levels (Taberlat et al., 1991;Baldwin et al., 1993;Baldwin et al., 1995;Molvray et al., 1999;Ranjbar et al., 2014).
Chloroplast DNA (cpDNA) sequence variation is widely used in investigating the interspecific relation between ddH2O, 2.5 µL of Taq buffer, 1.5 µL of Magnesium chloride (MgCl2), 0.4 µL of dNTP, 2.5 µL for trnLe and 2.5 µL for trnFf primers and 0.3 µL of Taq DNA polymerase.PCR amplification was performed with an initial denaturation step of 94 °C for 5 min, followed by 35 cycles of strand denaturation at 94 °C for 30 sec, annealing at 51 °C for 45 s, and primer extension at 72 °C for 90 s, and a final elongation at 72 °C for 8 min.Purified PCR products were sequenced with the amplifications primers by a commercial biotechnology company.For each sample, forward and reverse sequencing reactions were performed and the sequences checked via GenBank (NCBI) via blast search.Later obtained DNA sequences were edited both manually and by using the Bioedit 7.0.4.1 (Hall, 1999).
Aligment and phylogenetic analysis nrITS and trnL-F sequences were aligned using ClustalW aligment software (Thompson et al., 1994).Ends of the alignment were trimmed to make all the sequences of equal length, which was a total of 744 nucleotide (nt) positions in the final dataset for ITS region and 440 for trnL-F.Aligned DNA sequences were turned to the NEXUS format for the phylogenetic analysis in the PAUP 4.01b10.Sequences distance values and Neighbour Joining trees were using PAUP 4.01b10 (Swofford, 2001).To evaluate the degree of support for given clades the bootstrap analysis (Felsenstein, 1985) was applied.Beside H. umbellatum (for ITS sequence HQ131822.1,as well as for trnL-F sequence KF196061.1)was used outgroup and retrieved from NCBI GenBank.

Results and Discussion
In this study, nrITS sequences ranged from 639 nt to 735 nt among 12 specimens under study.Average nucleotide composition was 22.1% (T), 27.9% (C), 23.2% (A) and 26.8% (G).The total length of the aligned ITS sequence matrix was of 745 nucleotides.There were a total of 105 variable characters of which 81 were parsimony informative and 559 characters were constant.Genetic distance method based on ITS sequence set was performed with PAUP 4.01b10 (Table 2).The lowest sequence divergence with ingroup taxa was noted between L. kemaliya and L. serriola with a value of 0.0000.The highest sequence divergence with ingroup taxa between L. intricata and L. tatarica was 0.10290 (Table 1).
ITS sequence based on NJ tree comprised of two main clade.Clade 1, consisted of eight taxa, while clade 2 consisted of three taxa.Clade 1 is divided into 2 sub-groups (A and B).Group A consisted of L. viminea, L. orientalis and L. tatarica.A quite high bootstrap value of 100% supported that L. viminea and L. orientalis are sister taxa (Fig. 1).Group B is a monophyletic group consisting of L. aculeata, L. serriola, L. sativa, L. kemaliya and L. saligna.Bootstrap analysis result supported this monophyletic group with a value of 91%.Within the branch, L. aculeate, L. serriola, L. kemaliya and L. sativa are sisters with a bootstrap value of 98% (Fig. 1).Clade 2 consisted of L. undulata, L. incrinata and L. tuberosa.Within the branch, L. undulata and L.incrinata are sister taxa and this relation is supported by a 100% bootstrap value.L. tuberosa was found to be closely related to this sister group with a bootstrap value of 95%.
angiosperm and other plants.However, low evolutionary rate of these molecules is a limitation at interspecific level (Taberlat et al., 1991;Türktaş et al., 2012).
Intergenic space is situated between trnL (UAA) 3'exon and trnF (GAA) gene (Taberlat et al., 1991;Gielly and Taberlat, 1994;Gielly and Taberlat,, 1996;Liu et al., 2005).while trnL gen contains two protected exons.These are divided by a group of introns.This region has a high level of migration.For this reason, they are used in interspecies phylogenetic analyses (Yang and Pak, 2006).
In the hereby study, a molecular phylogenetic analysis of some Turkish Lactuca L. species using nrITS DNA and trnL-F cpDNA sequences were performed to elucidate the phylogenetic relationships using H. umbellatum as an outgroup.

PCR amplifications and sequencing
Double-stranded DNA of the complete ITS and trnL-F regions were amplified in each genomic DNA.Amplification of the whole ITS region ( ITS1 + 5.8S + ITS2) was performed with primers ITS5A (Standford et al., 2000) and ITS4 (White et al., 1990) (Table 1).The amplification process was performed in 25 µL of PCR reaction volume.Each PCR reaction contained 2.5 µL of Taq buffer, 1.5 µL of magnesium chloride (MgCl2), 0.4 µL of dNTP, 2.5 µL for ITS4 and 2.5 µL for ITS5A primers, 0.3 µL of Taq DNA polymerase (Fermentas), 2,5 µL of total genomic DNA and 10.8 µL of ddH2O.PCR amplification was performed with an initial denaturation step of 94 °C for 5 min, followed by 35 cycles of strand denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s, and primer extension at 72 °C for 2 min, and a final elongation at 72 °C for 10 min.trnL-F moleculer marker analyzed in this study belongs to the chloroplast genome (cpDNA).Polymerase Chain Reaction (PCR) amplifications of trnL-F cpDNA were performed using the primers designed by Taberlet et al. (1991) (Table 1).The amplification process was performed in 25 µL of PCR reaction volume.Each PCR reaction contained 2.5 µL of total genomic DNA, 10.8 µL of   For trnL-F sequences obtained a base lenght that ranged from 296 to 385 among the 12 specimens.Average nucleotide composition was of 34.1% (T), 18.4% (C), 31.6%(A) and 16% (G).The total length of the aligned trnL-F sequence matrix were 440 nucleotides.There were a total of 28 variable characters of which 7 were parsimony informative and 405 characters were constant.Genetic distance method based on trnL-F set was performed with PAUP 4.01b10 (Table 3).The lowest sequence divergence ingroup taxa was noted between L. saligna and L. intricata, between L. sativa and L. aculeata, between L. serriola and L. aculeata, between L. serriola and L. sativa respectivley 0,0000.The highest sequence divergence ingroup taxa between L. intricata and L. kemaliya was 0.09674.trnL-F sequence based on NJ tree comprised of two main clade (Fig. 2).Clade 1 consisted of ten taxa, while clade 2 consisted of one taxa.
NJ tree created based on trnL-F sequences was found to be slightly different than the NJ tree created based on ITS sequences.Clade 1 was divided into 2 sub-groups (A and B) within itself.Group A consists of only L. undulata.Group B is made up of L. viminea, L. orientalis, L. kemaliya, L. aculeata, L. serriola, L. sativa, L. saligna and L. intricata, and is supported by an 87% bootstrap value.Within the group, L. viminea and L. orientalis are sister taxa and were supported by bootstrap value of 97%.Similarly, L. saligna and L. intricata are sister taxa and were supported by a 66% bootstrap value.Group C is only made up of L. tatarica (Fig. 2).Clade 2, consist of only L. tuberosa (Fig. 2).
There are many phylogenetics and genetic variety studies conducted on Lactuca species (Landry et al., 1987;Kesseli et al., 1991;Kesseli et al., 1994;Vermeulen et al., 1994;Koopman et al., 2001).Koopman et al. (1998) carried out phylogenetic analysis of among Lactuca species based on ITS1 region.The results of their study were similar to ours.In the current study, ITS and trnL-F analysis indicated that L. saligna, L. serriola, L. aculeata and L. sativa were in the same clade.The results of a study by Koopman et al. (1998) were also similar to the hereby obtained data.In the present study, in ITS sequence, L. tuberosa was within clade 2 and was distinguished from L. sativa, L. saligna, L. serriola L. viminea and L. tatarica.A similar result was obtained in the study by Koopman et al., (1998).The only difference noted between the two experiments was sequenced at ITS1 region.Whereas in the current study were sequenced all of ITS1, 5.8S and ITS2 regions.Therefore, it was obtained a longer base pair.

Conclusions
Recently, phylogenetic analyses based on DNA have been used for the purpose of revealing taxonomic data.These analyses are used in rebuilding phylogenetics of many taxonomically complex species, types or groups that contain a great number of taxons.Because of the fact that phylogeny is widely used, many methods were developed for reconstructing these.In the current study, sequence analysis of several Turkish Lactuca species, using ITS and trnL-F sequences, were performed to elucidate phylogenetic relationships.Thus, the study can be illustrated as a supportive molecular and phylogenetic research carried out on some Lactuca species of Anatolia.

Fig. 1 .
Fig. 1.The Neighbour Joining tree generated using ITS DNA sequences of genus Lactuca specimens and the related sequences retrieved from NCBI GenBank Fig. 2. The Neighbour Joining tree generated using trnL-F DNA sequences of genus Lactuca specimens and the related sequences retrieved from NCBI GenBank

Table 1 .
ITS and trnL-F primers used for the study

Table 2 .
Pairwise sequence distances among some Lactuca species for ITS nrDNA data using PAUP distance matrix (except outgroup)

Table 3 .
Pairwise sequence distances among some Lactuca species for trnL-F cpDNA data using PAUP distance matrix (except outgroup)