Студопедия

Главная страница Случайная страница

КАТЕГОРИИ:

АвтомобилиАстрономияБиологияГеографияДом и садДругие языкиДругоеИнформатикаИсторияКультураЛитератураЛогикаМатематикаМедицинаМеталлургияМеханикаОбразованиеОхрана трудаПедагогикаПолитикаПравоПсихологияРелигияРиторикаСоциологияСпортСтроительствоТехнологияТуризмФизикаФилософияФинансыХимияЧерчениеЭкологияЭкономикаЭлектроника






In Situ Hybridization






Gall and Pardue (1969) and John et al. (1969) were the fi rst to localize nucleic acids directly to biological material. They used radioactively labeled RNA to detect rDNA in cytological preparations. Important applications of in situ hybridiza- tion are to display gene expression in a par- ticular organ, tissue, or cell layer of interest (RNA: RNA in situ hybridization), and to localize particular DNA sequences to chro- mosomes, interphase nuclei, or DNA fi bre spreads (DNA: DNA in situ hybridization). The probes used for in situ hybridization can be classifi ed into four categories.

 

1. Chromosome paints (DNA/DNA in situ hybridization): The probes are organized from fl ow-sorted chromosomes and are PCR amplifi ed using degenerate oligonucleotide primers and labeled nucleotides. Chromosome paints are used to label and identify individual chromosomes in a cyto- logical preparation. These probes have been used for phylogenetic studies and medical diagonosis of gross chromosomal abnormal- ities.

2. Total genomic DNA (DNA/DNA in situ hybridization): As total genomic DNA is labeled for hybridization, the technique is called as GISH. It can localize the parental origin of chromatin in hybrid organisms and can used to identify alien chromo- somes and chromosomal segments. The technique can also help to determine the ancestry of natural allopolyploids, and detects intergenomic translocations in hybrids.

3. Cloned DNA fragments (DNA/DNA in situ hybridization): These are the most usu- ally used types of probe, with sequences


 


cloned in bacteria as plasmids, cosmids, or bacterial artifi cial chromosomes (BACs), or in yeast as yeast artifi cial chromosomes (YACs). These probes are normally used to map genes and repetitive sequences on the chromosomes.

4. RNA probes (RNA/RNA in situ hybridization): The sequence of interest is frequently cloned into a vector containing the bacteriophage RNA polymerase promo- tor sequences. By using appropriate RNA polymerases, it is possible to generate single-stranded RNA probes complemen- tary to the coding (sense) or non-coding (antisense) DNA strands.

 

 

Fluorescence In Situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) has considerably contributed to a better understanding of plant genome structure and evolution. Using probes for total genomic DNA, the technique facilitated identification of parental genomes in hybrids and individual chromosomes in chromosome complements (Jiang and Gill, 1994; Taketa et al., 2000), analysis of genomic distribution of mobile genetic ele- ments (Balint-Kurti et al., 2000), integration of genetic and physical maps with marker tagged BAC clones (Yuan et al., 2000), physical mapping of genes on chromo- somes (Dolezelova et al., 1998) and other repetitive DNA sequences (Schmidt and Heslop-Harrison, 1996). The theory is same as for southern hybridization, except that the DNA to which the probe will hybridize is the actual chromosome. The probe is labeled using fluorescently tagged nucleotides, added to a chromosomal preparation from the species of interest and viewed using a fl uorescent microscope. The probe hybridizes to the complementary sequences. Since the technique uses a fl uo- rescent probe, it is called fl uorescence in situ hybridization or FISH. The protocol for FISH analysis is given in Table 2.

Fluorescent in situ hybridization has been used for gene mapping, for integrating genetic and physical maps, and for cytoge-


netic studies of citrus (Roose et al., 1998). The rDNA probes (18S-5.8S-25S) labeled with biotin or rhodamine and 5S rDNA probes labelled with digoxigenin were applied to locate rDNA sites on root-tip metaphase chromosomes of Citrus sinensis L., Poncirus trifoliata L. Raf., and Citrus x Poncirus hybrids. Counterstaining with the fl uorochromes chromomycin A3 and DAPI uniquely identifi ed many but not all chro- mosomes. C. sinensis had five 18S-25S rDNA sites, P. trifoliata had seven, and three different Citrus x Poncirus hybrids had fi ve or six sites. Four 5S rDNA sites were detected as linked to 18S-25S rDNA sites. Karyotype and molecular analysis of Trovita sweet orange chromosomes showed three CMA+/DAPI- heterochromatic regions (Fig. 6.3) positive to the 26S rDNA probe (Matsuyama et al., 1996). Telomere arrays consisting of Tm(A)Gn were detected on the extreme ends of each chromosome and most of the CMA+/DAPI- heterochro- matic regions lied close inside the telom- ere-specific repeated sequences. DNA fi ngerprinting of Citrus genomes using a satellite sequence composed of TGG repeats

 
 

Fig. 6.3. FISH doublet signals on a sweet orange (Trovita) chromosome probed with a telomeric repeated sequence (Matsuyama et al., 1996).


 

 

 
 

Table 2. A general protocol for FISH analysis.

1. Treatment to remove substances inhibiting the hybridization

i Wash slide with the mixture of 2% cellulase RS, 1.5% pectolyase, and 0.3% macerozyme R-200 with 1mM EDTA (pH 4.2) for 15 min at 37°C, following washing with twice of 2x SSC for 5 min.

ii Digest proteins by proteinase K (1µg/ml) solution in TE buffer (pH 8.0) for 15 min at 37°C, followed by washing with 2x SSC for 5 min twice.

iii Acidify with 45% acetic acid for 5 min at 37°C.

iv RNase digestion 0.1 mg/ml in 2x SSC for 1 hour at 37°C, then 2x SSC washing, and dehydration with 70%, 95% and 99% EtOH series before fi nal air-drying.

2. Probe preparation (PCR labeling method)

i PCR random labeling with biotynylated dUTP (0.14 mM)+0.06 mM dTTP, 0.2 mM each dATP, dCTP, and dGTP, along with 0.5 µM primer pairs by 2.5 U Ampli TaqGold on 5 µl (20pmol) tem- plate DNA amplifi ed 30 cycles of 94°C for 1 min, 55°C for 2 min and 72°C for 2 min after 3 min denaturation at 94°C, and followed with 7 min extension at 72°C.

ii Denaturation with formamide at 95°C for 10 min and fi nally in 2x SSC.

3. Hybridization

i Denaturation of chromosome specimen in the probe solution at 70°C for 6 min and incubate overnight (10 hr) at 38°C

ii Wash specimen by a series of solution of 2x SSC at 40°C for 10 min, 50% formamide in 2x SSC at 40°C for 10 min, 2x SSC at 40°C for 10 min, then 4x SSC at 40°C for 10 min.

iii Blocking by 5% BSA in BT buffer at 37°C for 5 min

iv Hybridization with avidin-FITC in 1% BSA containing 4x SSC for 60 min in the dark at 37°C, and then wash 3 times with BT buffer at 40°C for each 10 min.

v Blocking with 5% goat serum in BT at 37°C for 5 min.

vi Reaction with 2% Bio-Anti avidin solution in BT at 37°C for 30 min, and then wash 3 times with BT at 40°C of each 5 min.

vii Blocking again with 5% BSA in BT at 37°C for 5 min.

viii Reaction with 2% extra avidin –FITC solution in BT at 37°C for 30 min, and wash twice with BT at 40°C for 10 min.

ix

 
 

Stain with PI added with 1% DABCO in phosphate buffer, and mount for observation.

 


and sequence tagged microsatellite site (STMS) markers has been reported (Matsuyama et al., 1992; Kijas et al., 1995). However, TGG repeat-related sequences were not found in Citrus -specific CMA+/DAPI- heterochromatic regions located at the extreme ends of each chro- mosome (Matsuyama et al., 1999). This sug- gests that the TGG-repeated sequences are evolutionarily conserved and that the CMA+/DAPI- heterochromatic regions are added to the chromosome ends at a recent stage in Citrus evolution.

Multicolor FISH (MCFISH) using 5S and 45S rDNA specifi c probes simultane- ously have provided valuable information on the evolution of rDNA sites and the rela- tionships between wild and cultivated polyploid species (Taketa et al., 1999; Schrader et al., 2000; Mishima et al., 2002). FISH using a 45S rDNA probe was found


useful to elucidate the chromosomal loca- tion and the variation in the number of sites of 45S rDNA in 10 Diospyros species (Choi et al., 2003).

Mitotic metaphase chromosomes are usually selected for FISH but pachytene (meiotic) chromosomes could be better sub- strates. Two homologous chromosomes are present in each pachytene combination that is joined along their entire length by a pro- teinaceous scaffold called the synaptonemal complex (SC) (Moses, 1968). Because each homologue contains two chromatids, there are four closely associated copies of each locus available for hybridization on a biva- lent. In comparison, there are only two nearby copies of each locus available for FISH on a metaphase chromosome. In spreads of pachytene, chromosomes that have been prepared to reveal SCs (SC spreads), chromatin extends as a diffuse


 


cloud around each SC. The loops of DNA extending from the SC appear to be more accessible to FISH probes than the DNA of condensed metaphase chromosomes (Moens and Pearlman, 1989; Heng et al., 1994), and SC spreads can be prepared relatively free of overlying debris. Additionally, pachytene chromosomes are 5–15 times longer than corresponding metaphase chromosomes (Stack, 1984). The closely associated loci that are not resolvable by FISH on metaphase chromosomes should be resolv- able on pachytene chromosomes.

Although the FISH techniques has high potential to identify the specifi c gene or regions of chromosome, the information of genetic linkage map has not been integrated onto the cytological chromosome map. Large insert genomic sequences in BAC have been constructed. The future develop- ment to increase the resolution of FISH sig- nals will contribute to assign each chromosome to the consensus linkage group and also to the development of Citrus genomics to bridge between the linkage map and physical map.

 

 

Genomic In Situ Hybridization (GISH)

The genomic in situ hybridization (GISH) technique provides a direct and visual method for effectively determining the number and position of parental chromo- somes. It has been extensively and success- fully applied to the genetic identifi cation of numerous interspecific and intergeneric plant hybrids (Gavrilenko et al., 2001; Zhou et al., 2001; Xia et al., 2003).

Somatic hybrids can be analyzed by genomic in situ hybridization Fu et al. (2004) analyzed somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange [ Poncirus trifoliata (L.) Raf]. GISH analysis confi rmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant.


Even though GISH is a powerful tool for parental genome analysis of citrus somatic hybrids (Matsuyama et al., 1996; Pedrosa et al., 2000), yet, few GISH studies in citrus somatic hybrids have been reported. Partly, because the citrus chro- mosomes being small and morphologically indistinguishable. Accomplishment of high-quality well-dispersed chromosome preparation need skills and practice. Fu et al. (2004) described an enzyme-macerating- fl ame method to prepare distinct and count- able mitotic chromosomes to avoid intricacy in chromosome preparation.

 

 

Heterochromatin banding

Banding techniques for chromosomes can reveal the detailed structure of karyotype based on molecular features such as consti- tutive heterochromatic region with highly repetitive sequences or nucleolar organiz- ing regions. Amongst such banding tech- niques, C-banding has been applied to Citrus chromosomes by Giemsa staining (Guerra, 1985; Wei et al., 1988). Liang (1988) identifi ed Giemsa C-bands after the treatment with 5% Ba(OH)2 at 50°C for 15

minutes. The heterochromatin blocks in

Poncirus were predominantly telomeric and centromeric bands which were com- posed of highly repeated DNA sequences. They showed the heteromorphism among possible homologous chromosome pairs, including 3 pairs in Fortunella margarita, 4 pairs in C. sinensis, 3 pairs in C. paradisi, 0- 4 pairs in mandarin species.

Compared to such staining as Giemsa and Feulgen methods, fl uorochromes pro- duce clear chromosomes because many of them directly stain DNAs revealed under epi-fluoresent microscope system. The staining of chromosome specimens by a fl u- orochrome 4’, 6-diamidino-2-phenylindole staining (DAPI) revealed clear chromosome fi gure in late prophase to early metaphase as well as typical metaphase cells (Ito et al., 1992). DAPI strongly combines with AT rich sequence region of DNA and allows to visualize chromosomes clearly under UV-


 


 

epifl uorescence microscopy. The method is convenient to count chromosome numbers instead of the conventional aceto-carmine or aceto-orcein staining methods. However, the simple staining with DAPI can not show the specifi c banding patterns in Citrus chro- mosome. The chromomycin A3 (CMA) stains strongly GC rich region of chromo- some. To emphasize the fl uoresence by CMA staining, the counter staining is rec- ommended as described in Table 3. By the application of double staining method (Schweizer, 1976; Hizume et al., 1989) with DAPI and CMA, Guerra (1993) identifi ed 6 main CMA positive banding types of Citrus chromosome (Fig. 6.4): A type chromosome has two telomeric and one proximal band, B has one telomeric and one major proxi- mal band, C has two telomeric bands, D has one large telomeric band, E has one small telomeric band, and F has bands absent or only very fine and not always visible. Abkenar et al., (2007) described inter- generic and trigeneric hybrids using CMA


 

banding patterns.

In detailed karyotype analyses of young leaves from various Citrus species, many variations have been identifi ed as summarized in Table 4. Proximal heter- chromatin with CMA+ were conspicuous in Citrus and they were highly variable among species, even distinguishing homoeologous pair in chromosome I. Six pairs of chromo- somes in Citrus and Poncirus showed no or very small heterochromatin regions while all chromosomes have proximal bands in Fortunella (Miranda et al., 1997). The amount of GC rich CMA-positive regions is high in Fortunella compared to other species/genera. The proportion of CMA- positive region to total chromosome length is 24.5% in Fortunella hindsii and 34.2% in

F. crassifolia, and 21.4% in Citrus grandis, 20.5% in C. sinensis, and 22.0% in C. suc- cosa. The similarities between Citrus and Poncirus suggests little heterochromatin diversifi cation among karyotypes of these genera.


 

 

 
 

Table 3. Protocols for DAPI and CMA double staining for fl urochrome banding of Citrus chromosome

A. Chromomycin A3 (CMA) staining

1. Rinse with McIlvaine buffer (pH 7.0) (0.63 g citric acid- 6.19 g Na2HPO4/500 ml) 30 min

2. Pre-treat with 2 or 3 drops of 0.1 mg/ml Distamycin A in buffer and cover with parafi lm

and keep in moistened box 10 min

3. Rinse briefl y with buffer supplemented with 5mM MgSO4 10 min

4. Stain with 2 or 3 drops of CMA staining solution 0.1 mg/ml in 5mM MgSO4 buffer and

covered with parafi lm and keep in moistened box 10 min

5. Rinse with 5mM MgSO4-buffer 10 min

6. Stain with 0.1 µg/ml DAPI in buffer 5 min

7. Wash with buffer 10 min

8. Cover with non-fl uorescent glycerin mounted with glass/cover slip and store overnight in refrigerator

9. Observe with epi-fl uorescent B or BV excitation (420 nm)

 

B. 4’, 6-diamidino-2-phenylindole (DAPI) staining

1. Soaking with MacIlvaine buffer 30 min

2. Drop 0.25 mg/ml Actinomycin D and covered with parafi lm in moistened box 10 min

3. Rinse with buffer 10 min

4. Staining with DAPI solution in buffer 5 min

5. Rinse with buffer 10 min

6.

 
 

Mount with non-fl uorescent glycerin by glass slip and observe with UV excitation (355 nm)

*2% Dabco (1, 4-diazabicyclo[2.2.2] octane) for anti-fading in glycerol: MacIlvaine buffer 1: 1 Modifi ed after Hizume et al., (1989).


 

A B C D E F

Fig. 6.4. Classifi cation of karyotypes in CMA banding pattern in Citrus [after Guerra (1993), modifi ed by Befu et al. (2000)].

 

Guerra et al. (2000) stained citrus chro- mosomes with the fluorochromes chro- momycin A3 (CMA) and 4’-6-diamidino-2-phenylindol (DAPI) and examined variable number of regions that appeared bright or positive with CMA, and faint or negative with DAPI (Fig. 6.5). Following three types of banding patterns were recognized:

 

1. CMA+ heterochromatin associated with nucleolus organizing regions (NORs). Only one pair of CMA+ blocks per monoploid complement is examined in this type of het- erochromatin. Sometimes it appears par- tially decondensed and as a secondary constriction. Generally, it represents a par- ticular type of heterochromatin and the chromatin linked to the nucleolus is CMA+ and GC rich (Schweizer, 1976).

2. CMA+ heterochromatin not associated with the nucleolus organizing regions (NORs). The species having large number of CMA+ blocks show that the most of them are not nucleolus-associated bands. In Citrus sinensis only three of the 16-18 CMA+ blocks corresponded to rDNA sites by in situ hybridization (Matsuyama et al., 1996) indi- cating that the most of the CMA+ blocks were constituted by another DNA sequence.

3. CMA- heterochromatin observed in prox-


 

 
 

 

Fig. 6.5. Banding patterns of some Citrinae and Balsamocitrinae species. (a and b) Metaphase and interphase nucleus of Poncirus trifoliata; (c and d) Citrus reticulata. Arrows show a telomeric CMA+/DAPI- band; (e and f) Fortunella crassifolia. (a), (c) and (e) were stained with CMA, and (b), (d) and (f) were stained with DAPI. Bars = 2.5 µm (Guerra et al., 2000).

 

 

imal regions of some species. After conven- tional staining the proximal region of prophase chromosomes is heteropycnotic and appears to correspond to small, DAPI- brilliant chromocenters (Guerra, 1993) and was identifi ed in a few chromosomes with the C-banding method (Guerra, 1985). However, Ito et al. (1993) examined occur- rence of proximal chromatin in every chro- mosome of Trovita orange.


 

 

Table 4. Karyotypes of Citrus on the CMA banding pattern.

 

Species Cultivar Karyotype Tissue Reference
C. grandis 3A+3C+4D+8E Root Guerra (1993)
  Shadenyu 3A+2C+7D+6E Root Miranda et al. (1997)
  Tosa buntan 1A+1B+5C+2D+9E Young leaf Befu et al. (2000)
  Suisho-buntan 3A+3C+3D+9E Young leaf Befu et al. (2001)
C. medica – 1A+1B+1C+9D+6E(F) Root Guerra et al. (1993)
  Fingered citron 2B+8D+8E Young leaf Befu et al. (2001)
C. sinensis 2B+2C+7D+7E(F) Root Guerra (1993)
  Trovita 2B+2C+7D+7E Root Matsuyama et al.
        (1996)
      Root Miranda et al. (1997)
      Young leaf Befu et al. (2000)
C. aurantifolia 2A+1C+7D+8E(F) Root Guerra (1993)
C. succosa Honchiso 1A+1B+10D+6E Root Miranda et al. (1997)
C. unshiu Okitsu wase 1A+1C+8D+8E Young leaf Befu et al. (2001)
C. leiocarpta Koji 1C+8D+9E Young leaf Befu et al. (2001)
C. paradisi Duncan GF 1A+2B+2C+4D+8E Young leaf Befu et al. (2001)
Poncirus trifoliata 2B+10D+6E Root Miranda et al. (1997)
  4B+8D+6E Young leaf Befu et al. (2000)
Fortunella 2A+2C+14D Root Miranda et al. (1997)
crassifolia        

Type E in this classifi cation includes small telomeric bands and F for absent bands.

 


CMA banded chromosomes proved to be a very useful tool for carrying out a more comprehensive analysis of Citrus phy- logeny. Chromosomes of type A and B were found only in a few mandarin hybrids and in the lime-lemon-citron-pummelo group (Guerra, 1993; Befu et al., 2001). Therefore, it can be hypothesized that all mandarin karyotypes with these chromosome types are hybrids. Chromosomes of type C, pres- ent in Mediterranean mandarins and in most other accessions, may be part of the original mandarin karyotype. On the con- trary, type E chromosome, found only in C. depressa, seems to be restricted to a small group of mandarin species. Cornelio et al. (2003) also revealed highly differentiated banding patterns in mandarin accessions and hybrids, and classifi ed in four groups according to the presence/absence of CMA banded chromosomes. CMA banding pat- terns of chromosomes compared in 17 accessions of mandarins classifi ed chromo- somes into six types (A-F) based on the number and position of CMA positive bands (Yamamoto and Tominaga, 2003). Type F chromosomes are present only in


some mandarins originating in Japan, which could thus be distinguished from mandarins originating in other areas. This is the fi rst report of type F chromosome in citrus. Amounts of relative heterochromatin per karyotype vary largely (Guerra et al., 2000). CMA-banded karyotype estimates a heterochromatin proportion of 20.58- 22.74% in Citrus species (Miranda et al., 1997).

Wide hybridization in Citrus affects karyotype stability. Chromosome organiza- tion at prophase show heteropycnotic blocks with Giemsa or Feulgen staining in many chromosomes at the proximal region but at terminal region of few (Guerra, 1985, 1987). Heteropycnotic regions show varia- tion (Guerra et al., 2000) and commonly maintained in species with a low DNA amount and small chromosome size (Guerra, 1987). Diploid genomes of Citrus appear relatively small (between 0.73 and

0.82 pg/2C for 2n=2x=18) and variations were observed within species as C. reticu- lata has the smallest nuclear genome while

C. medica has the largest (Ollitrault et al., 1994).


 


Cyto-taxonomy

Polyploid levels, chromosome number, their size and shape etc., are given due weightage for the classifi cation in addition to morphological characters in cyto-taxo- nomic investigations.

Lapin (1937) confi rmed polyploidy and noted peculiar configurations (quadriva- lents) at metaphase attached with slender threads and rods, particularly the triva- lents. Moreover, Kandelaki (1938) noted characteristic differences in citrus chromo- some morphology. Cytological studies with respect to mitosis and meiosis of different species and biotypes of Citrus plants revealed variable chromosome size and shape (Rao et al., 1992). Therefore, group- ing all the species into Citrus according to Reece (1969) and Tanaka (1969) needs to be debated in view of their salient cyto-taxo- nomic characteristics.

 

 

Flow Karyotyping

A quick and accurate approach to look at changes in genome size during evolution and differentiation is flow cytometry (FCM). Polyploidy often accompanies dif- ferentiation, an important part of plant development, with different cell types having characteristic ploidies (Galbraith et al., 1991; Bino et al., 1993) where fl ow cytometry can be used as a rapid screening tool. The genome size of citrus is small with 382 Mb (Arumuganathan and Earle, 1991). FCM has shown that mean nuclear DNA content of the species of the genera Fortunella and Citrus, was 0.81pg/2C (Kayim et al., 1998).

Flow cytogenetics can be used for detection of aberrant cell cycles, changes in nuclear DNA amounts and sorting of chro- mosomes for gene mapping and library con- struction (Heslop-Harrison, 1995). The


sensitivity of the flow karyotyping can detect numerical and structural chromo- some changes in plants, including chromo- some polymorphism (Dolezel et al., 1994). The numerical changes in chromosome have been demonstrated in barley, where trisomy of chromosome 6 was identifi ed (Lee et al., 2000). An alien chromosome presence was detected in an oat–maize addition line (Li et al., 2001) and six wheat- rye lines, where fl ow karyotyping moni- tored the frequency of alien chromosomes in the population (Kubalakova et al., 2003). Recognition of chromosome translocations and deletions has also been known in fi eld bean, garden pea, barley, rye and wheat (Dolezel and Lucretti, 1995; Neumann et al., 1998; Gill et al., 1999; Lysak et al., 1999; Vrana et al., 2000; Kubalakova et al., 2002, 2003). Detection of chromosomes is gener- ally dependent on change in chromosome size and difference from the remaining chromosomes. Kubalakova et al. (2002) reported that a weakly differentiated chro- mosome can be detected by a characteristic change in the fl ow karyotype. Similarly, fl ow karyotyping is sensitive enough to detect polymorphism in relative DNA con- tent of chromosomes in some agronomic crops, and the fi ngerprint patterns of fl ow karyotypes characteristic for certain culti- vars are heritable (Lee et al., 2000, 2002; Kubalakova et al., 2002, 2003).

The results obtained todate mainly focus on several agronomic and vegetable crop species and confi rmed the usefulness of fl ow cytogenetics. About 17 species have been fl ow karyotyped (Dolezel et al., 2004). Flow cytometry is routinely used in identi- fi cation of triploids and other polyploids of citrus at the Citrus Research and Education Center-University of Florida, Lake Alfred, USA. It is expected that the technology will play a greater role in the study of nuclear genome, gene isolation and mapping in citrus.


 

 


Поделиться с друзьями:

mylektsii.su - Мои Лекции - 2015-2024 год. (0.027 сек.)Все материалы представленные на сайте исключительно с целью ознакомления читателями и не преследуют коммерческих целей или нарушение авторских прав Пожаловаться на материал