FISH- MAPPING AND STANDARD GTG-BANDING KARYOTYPE OF THREE EGYPTIAN SHEEP BREEDS

Mona E. Abd El-Gawad 1 , Hanaiya A. El-Itriby 1 , Hassan M. Sobhy 2 and Ebtissam H. A. Hussein 3 . 1. National Gene Bank, Agriculture Research Center, Giza, Egypt. 2. Department of Natural Resources, Institute of African Research and Studies, Cairo University, Giza, Egypt. 3. Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 06 February 2019 Final Accepted: 08 March 2019 Published: April 2019

Standardized karyotyping by GTG-banding technique and physical chromosome mapping by Fluorescent in Situ Hybridization (FISH) were utilized to characterize the three Egyptian breeds of sheep (Barki, Rahmani and Ossimi). Blood samples were collected from 15 individuals from each breed of sheep. G-banded chromosomes revealed that the karyotype macrostructure was highly conserved and in considerable accordance to the standard karyotype of the Ovis aries. The chromosome diploid number was 54 (2n=54, XX / XY). The karyotype formula was 2n, 54 = L m 6 + M a 22 + S a 24 + sex chromosomes. Physical chromosome mapping of the three breeds (Barki, Rahmani and Ossimi) was carried out by localization of two subtelomeric SSR and two (SPRN) related specific sequences. The two subtelomeric SSR sequences revealed six different loci in five chromosomes (1p37, 1p36 and 17q26 with the EPCDV008 probe) and (2q45, 4q22 and 24q24 with the EPCDV016 probe), respectively. In addition the two (SPRN) related specific sequences were successful in differentiating among the three breeds. The probe OriaBAC273H7 hybridized to a similar locus (20q13) in breeds Rahmani and Ossimi, while, in Barki, it hybridized to a different locus (22q24). However, probe OriaBAC265G4 hybridized to three different loci (17q25, 22q24 and 20q13) in Barki, Rahmani and Ossimi, respectively.

…………………………………………………………………………………………………….... Introduction:-
Conservation of animal genetic resources has become an urgent demand nowadays. This is due to the ever increase in human population, the global climate change and the risk of extinction of some valuable genetic resources. Therefore, conservation of genetic diversity is required to facilitate genetic improvement and selection, and to meet current production needs in various environmental conditions  (Murphy et al., 2004). In addition, the molecular tools have increased the efficiency of germplasm characterization, thus assuring objective criteria for the conservation of genetic resources and establishment of genebanks (Liu et al., 2014). The study of chromosome structure and the precise identification of chromosomes using differential staining techniques such as G-banding During the last two decades, FISH technique has been widely used in domestic animals for different purposes. This has been applied to identify chromosomal rearrangements, gene mapping, comparative mapping and evolutionary chromosome studies (Farhadi et al., 2013).
In Egypt, sheep represent a valuable source of meat and milk production. There are several indigenous breeds (Othman et al., 2015). The three major Egyptian sheep breeds are Barki, Rahmani and Ossimi, representing 65% of the total sheep population (El Shennawy, 1995). These breeds are reared using minimal resources and are well adapted to local environmental conditions. Therefore, they could have acquired valuable alleles and allelic combinations that could be of importance in animal improvement and breeding programs. It is important to characterize a breed for its conservation (Mahmoudi et al., 2010). Yet, a few cytogenetic and molecular studies on sheep have been undertaken. Therefore, in the present investigation, GTG-banding and FISH markers were used in an attempt to provide fine standard karyotypic details and characterize the main Egyptian breeds of domestic sheep (Barki, Rahmani and Ossimi).

Samples collection
Forty five peripheral blood samples were collected from three domestic sheep breeds (Barki, Rahmani and Ossimi). Fifteen samples were taken from each breed. In addition. These materials were kindly provided by the Research and Experimental Station, Faculty of Agriculture, Cairo University. Whole blood samples of the three sheep breeds were collected through jugular venipuncture in heparinized and EDTA vacationers. The blood samples were kept at 4ºC and transported to the Cytogenetics and Molecular Genetics Labs at the National Gene Bank, ARC, Egypt.

-Karyotype Analysis Cell culture
The lymphocytes were cultured using the whole blood microculture technique described by Kenthao et al. (2012) with minor modifications. .

-Unbanded karyotype
Fifteen cells of well-spread mitotic metaphase plates were used for chromosome measurements. The length of short arm (p) and the long arm (q) of each chromosome were measured and the total length (TL) was calculated as (p + q). The relative length (RL %) of each chromosome was estimated in percent of total length of complement according to the formula ((TL/sum TL) x100). While, the formula used for the centromeric index (CI %) was ((p/TL) x100) according to

Probes preparation
Total genomic DNA from the blood samples was used as a template to amplify the desired DNA fragments using specific primer pairs. The PCR products were then employed to prepare the probes by labeling using the DIG-Nick-Translation Kit as described by

Denaturation and hybridization
The labeled probes were precipitated with ethanol. A probe stock solution was prepared by suspending in hybridization solution. The tubes were incubated for 15 min at 37°C with occasional vortexing until the precipitated DNA dissolved. Then, the probe was diluted from the stock solution to the desired concentration (2 ng/μl) in hybridization solution.
A volume of 20-30μl diluted probe was added to the slide, covered with a cover slip (24x50mm) and sealed with rubber cement. The slides were placed on a hotplate at 80°C for 5 min to denaturize the probe. The slides were hybridized in a moist chamber at 37°C overnight. The slides hybridized with subtelomeric SSR probes were washed 3 times for 5 min at 37°C with 2×SSC containing 60% formamide, followed by one time for 5 min with the immunological detection buffer. While, the slides hybridized with the specific (SPRN) probes were washed 3 times for 5 min at 45°C with 2×SSC containing 50% formamide, followed by 5 times for 2 min with 2×SSC and one time for 5 min with the detection buffer.

Microscopic examination karyotyping, idiograming and signals imaging
Chromosomes examination and signals imaging were performed using a vertical fluorescence microscope (Leica DM2500) equipped with a cooled monochrome digital camera (Leica DFC340FX). Twenty cells with clearly observable and well spread chromosomes of each male and female sheep were examined and photographed at 100 × magnification under oil immersion. Chromosome counting and karyotyping were performed using the automated karyotyping & FISH software processing (Leica CW4000) system. Idiograms were constructed from complete chromosomes which showed the maximum possible banding patterns in at least ten different metaphase plates.
Hybridization signals were photographed twice at 100 × magnification under oil immersion, first using a blue filter for DAPI and second using a red filter for rhodamine. Thus, chromosome bands showed up in blue after approximately 300 nm exposure, and probes showed up in red and were exposed for approximately 10 seconds. For each probe, eight to ten metaphases were photographed and images were overlapped using automated karyotyping & FISH software processing (Leica CW4000) system.

Results and Discussion:--Karyotype analyses
In the present investigation the karyotype of the three sheep breeds (Barki, Rahmani and Ossimi) were first investigated to assess their correspondence to the standard karyotype of sheep.

Unbanded chromosomes morphological analysis
The three Egyptian breeds of sheep (Barki, Rahmani and Ossimi) belong to the species (Ovis aries). This species is characterized by a diploid chromosome number of 54 (Hansen, 1973 In the present investigation the karyotype was based on the analysis of twenty well spread metaphase cells of each male and female sheep (Fig. 1). The relative length (RL %), centromeric index (CI %) and centromere position were estimated as displayed in Table (2

).
In general, the morphological features of the chromosome complement of the three sheep breeds were in accordance with the basic sheep karyotype revealing the karyotype formula 2n = 54 = L m 6 + M a 22 + S a 24 + sex chromosomes. For each chromosome pair, slight differences in the measured parameters were detected among the three breeds as shown in Table (2), and illustrated in the histograms (Figs. 2 and 3). The centromeric indices for the chromosomes of the three sheep breeds are presented in Table (2) and illustrated as histogram in (Fig.3) aries (1985).
The G-banding method using the photolytic enzyme trypsin affects the interaction that stabilises the structure of different proteins and nucleic acid components of the chromatin. Therefore, the G-band mechanism is based mainly upon differences in protein composition and organization (Holmquit, 1988 and Ali et al., 2011). It has also been suggested that trypsin treatment leads to the unfolding of protein loops and permits the protein structure associated with the alignment of AT-rich sequences, as reported by Popescu et al. (2000). Moreover, examination of the G-banded metaphase cells revealed variation in the number of bands in each chromosome depending on the degree of the chromosome contraction. Therefore, the analysis of G-banding has been conducted on early metaphase chromosomes as they displayed the highest number of G-bands and thus had the greatest utility for detailed comparative analysis.
The G-band idiogram of the chromosomes was developed based on twenty selected metaphases using automated karyotyping & FISH software processing (Leica CW4000).
GTG-banding karyotype of sheep (Ovis aries) confirmed that, the chromosome diploid number was 54 (2n=54, XX / XY) and the karyotype formula was 2n=54 = L m 6 + M a 22 + S a 24 + sex chromosomes (Figs. 4 and 5). The G-banded idiogram of each of the three Egyptian sheep breeds consisted of 345 bands in one set of haploid chromosome complement, including sex chromosomes. The number of G-positive (heterochromatic) bands was 159, whereas the total number of G-negative (euchromatic) bands was 186 (Table 3).
At the cytogenetic level, the identification of chromosomes 4, 5 and 6 was difficult, due to their indistinct band organization. Similarly, chromosomes 8, 9 and 10 were often difficult to differentiate. The precise identification of chromosomes 19 to 26 also required great care due to their small size. These results are in agreement with Ansari et al.

(1999), Lopez and Arruga (1996) and Stone and Stephens (1993).
The G-banded idiogram of the three Egyptian sheep breeds could be identified as four chromosomal groups.
The first group was consisted of three large metacentric pairs, chromosomes no. 1, 2, and 3. In this group the Gbanding pattern revealed three arms (1p, 1q and 2q) with two regions, while, in the other three arms (2p, 3p and 3q) the G-bands constituted one region. The number of bands in the chromosomes 1, 2 and 3 was 30, 34 and 26, respectively. The percentage of band length to the total length of the chromosome ranged from 1.3 for the dark band 1q21 to 15.87 for the light band 3p13. While, the relative lengths of these three chromosomes to the chromosome complement were 9.8, 9.32 and 7.81 for chromosome 1, 2, and 3, respectively.
The second group was consisted of eleven medium acrocentric pairs, from chromosome no. (4) to chromosome no. (14). The G-banding pattern of these eleven chromosomes was comprised of one region. Fifteen bands were observed in the three chromosomes no. 4, 5 and 8, thirteen bands were detected in the chromosomes no. 11 and 13, and also twelve bands were observed in the chromosomes no. 7 and 9. While, the number of bands in the four chromosomes no. 6, 10, 12 and 14 were 14, 11, 10 and 9, respectively. The percentage of band length to the total length of the chromosome ranged from 1.43 observed as a light band in chromosome 4 (4q111) to 25.00 for the light band 9q13. While, the relative length of this group of chromosomes ranged from 3.0 for chromosome 14 to 4.0 for chromosome 4.
The third group was consisted of twelve small acrocentric pairs, from chromosome no. (15) to chromosome no. (26), where all the bands were in one region. Eleven bands were detected in two chromosomes no. 15 and 17, nine bands 379 were observed in the five chromosomes no. 16,18,20,24, and 25, seven bands were observed in two chromosomes no. 19 and 21, and five bands were observed in two chromosomes no. 22 and 26. While, six bands were detected in the chromosome no. 23. The percentage of band length to the total length of the chromosome ranged from 2.86 for the light band observed in chromosome 20 (20q17) to 27.27 for the dark band in chromosome 22 (22q12). While, the relative length of this group of chromosomes ranged from 2.02 for chromosome 26 to 2.89 for chromosome 15.
The fourth group comprised the two sex chromosomes (X and Y). The X chromosome was submetacentric where the long arm (q) was divided into two G-banded regions, while, the short arm had one region. The short arm comprised three bands, while, in the long arm, eleven bands were present in the two regions. The percentage of band length to the total length of the chromosome X ranged from 1.96 for the light band (Xq13) to 57.89 for the dark band (Xp12). The acrocentric Y chromosome comprised five bands represented in one region. The percentage of band length to the total length of the chromosome Y ranged from 12.50 for the light band (Yq15) to 31.25 for the light band (Yq11). While, the relative lengths of the X and Y chromosomes were 5.42 and 1.43, respectively. In the present investigation two subtelomeric microsatellite probes (EPCDV008 and EPCDV016) and two specific SPRN related probes (OriaBAC273H7 and OriaBAC265G4) were hybridized to the metaphase chromosomes. The use of DAPI-stained chromosomes with the rhodamine-labeled probes was useful to accurately assigning each of the probes to its cytological location. Polymorphic hybridization sites were observed among the investigated breeds.
In chromosome spreads with telomeric signals, two signals were visible with TRITC -filter as two spots at both ends of each chromosome, regardless of the age of the slides. However, there were variations in signal intensities among chromosomes, which appeared to be random.
The EPCDV008 probe hybridized to the short arm (p) of chromosomes No.1 in Barki and Rahmani breeds (1p37 and 1p36) (Fig. 6) and to chromosome No.17 (17q26) (Fig.6) in the Ossimi breed. These three loci were reported by Vaiman et al. (2000) for the same probe in sheep. While, the EPCDV016 probe revealed three different loci, two subtelomeric loci in Barki and Ossimi (2q45 and 24q24) (Fig.7), and a unique interstitial band on chromosome No.4 (4q22) (Fig.7) in Rahmani. These results are in good agreement with Vaiman et al. (2000). Therefore, the results proved that the two microsatellites subtelomeric probes (EPCDV008 and EPCDV016) were successful in differentiating among the three breeds.
In situ hybridization of the specific SPRN related probes (OriaBAC273H7 and OriaBAC265G4) with the three sheep breeds revealed three different loci on three small acrocentric chromosomes (No. 17,20 and 22). The signals were visible as two spots on each chromosome, except in the sheep Barki where only one spot was detected per chromosome. Similarly, Sera et al. (1995) found one spot at each end of the chromosome when hybridizing the sheep chromosomes with the telomeric (TTAGGG) sequence. The probe OriaBAC273H7 hybridized to a similar locus (20q13) in breeds Rahmani and Ossimi (Fig.8), while, in Barki, it hybridized to a different locus (22q24) (Fig.8). However, probe OriaBAC265G4 hybridized to three different loci (17q25, 22q24 and 20q13) in Barki, Rahmani and Ossimi, (Fig.9).
This demonstrates that the probe OriaBAC265G4 was successful in differentiating among the three breeds, while, OriaBAC273H7 was only successful in characterizing the Barki breed. In this context The present results assured that the FISH-mapping technique is a powerful tool in cytogenetic investigations. In this context, FISH has been reported previously as the best and fast method to (a) physically map loci in specific chromosome regions (Di Meo et al., 2007 and Goldammer et al., 2009), (b) identify correctly chromosomes and chromosome regions involved in chromosome abnormalities (Iannuzzi et al., 2001a and Molteni et al., 2007), (c) anchor radiation hybrid maps to specific chromosome regions (Perucatti et al., 2009) and (d) clarify the chromosome evolution of species by analyzing the gene order among homologous chromosomes of species (Iannuzzi et al., 2009).
It is worth noting that the variation observed in FISH analysis among the three sheep breeds could be due to their different origin and / or the occurrence of rearrangements in their genomes. The sheep Barki breed originated in North Africa in the Coastal Mediterranean Zone. While, the origin of the Ossimi breed is Giza and it is the most popular among the Nile Valley and Delta breeds. Rahmani originated in Northern Syria and Southern Turkey and was introduced into Egypt in the 19 th century (El Shennawy, 1995).
In conclusion, in the present cytogenetic study the first standard karyotype for the three main Egyptian sheep breeds was established by GTG-banding. In addition, the results revealed the adequacy of the FISH technique for physically mapping two subtelomeric and two specific SPRN related loci to the Egyptian sheep breed chromosomes. All hybridization signals were different, indicating high specificity and reproducibility of the technique used and at the same time proving that these subtelomeric and specific SPRN related loci are suitable markers for accurate identification of Egyptian breed chromosomes or specific regions within them.