Genotype-environment interaction in relation to heat ... - CiteSeerX

Genotype-environment interaction in relation to heat tolerance in chickens. 1. RAPD-PCR analysis for breeds local to the warm regions (Received: 12.04.2005; Accepted: 01.05.2005) E. A. El-Gendy, M. K. Nassar, M. S. Salama* and A. Mostageer Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt *Department of Entomology, Faculty of Science, Ain Shams University, Cairo, Egypt

ABSTRACT The genomes of three breeds local to the warm regions (White Baladi, Fayoumi and Sinai Bedouin) and a commercial broiler strain were screened by 10 random decamer primers for randomly amplified polymorphic DNA bands, using RAPD-PCR analysis. The aim of the study was the assessment of genetic diversity and relationships between the breeds. All primers showed successful amplification of bands, with an average of 4.99 bands. Polymorphism represented 65.19% of the total bands. The heterozygosity estimates between breeds averaged 0.48, 0.45 and 0.44 for unsexed, male and female comparisons, respectively. Several breed-specific, breedpolymorphic and breed-monomorphic alleles were detected in the local breeds, reflecting the genetic specificity for each. The band sharing levels within breeds ranged between 0.60 and 0.81. More variability, less band sharing and more heterozygosity were observed in Fayoumi compared to the other breeds. An average genetic distance index, overall primers, of 0.42 was calculated between White Baladi and each of Fayoumi and Sinai Bedouin. Whereas, the genetic distance between White Baladi and commercial broilers was slightly further with an average of 0.45. Fayoumi was genetically the furthest from Sinai Bedouin with an average genetic distance of 0.53. The phylogenetic tree formed three distinct branches. White Baladi and Fayoumi formed a branch and each of Sinai Bedouin and commercial broilers formed a branch. The branches reflected the genetic relatedness and differentiation between the breeds. They could reflect the genetic-locality interaction, where White Baladi and Fayoumi are locals to the hot climate and Sinai Bedouin is local to the desert climate. Keywords: Local breeds, warm regions, RAPD-PCR, polymorphism, genetic distance. INTRODUCTION

T

he ability of birds to reproduce and produce under severe environments has been found to be a breed-dependent, expressing genetic-environment interaction. Therefore, the permanent and biologicallyfounded genetic-environment interaction can be employed to maximize the efficiency of Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.

poultry production under sub-optimal environments (Horst, 1985; 1989), by means of identifying and exploiting genotypes specifically adapted to these environments. Hence, to achieve ultimate improvement in poultry production, breeding programs must target the genotypes that perform quite well in relevant regions. For this, local breeds currently receive much concern in research.

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E.A.El-Gendy et al.

The researches concentrate on the genetic potential that favorably interacts with particular environments. On the other hand, the molecular techniques allow detecting variation existing among individuals of the same or different populations in specific regions of DNA (Boichard, 2002), thereby, the assessment is at the genotypic rather than the phenotypic level (Siegel et al., 1992). Molecular variation is also influenced by neither environmental conditions nor developmental stages of organisms; hence it is preferable over the genetic variation statistically estimated from phenotypic parameters of the traits. It is, therefore, employed to evince the phylogenetic relationships and genetic diversity among different populations, and is widely used to evaluate the local breeds. Several researchers have targeted the world-wide local breeds. Siegel et al. (1992) studied the genetic diversity among wild jungle fowl and commercial broilers and layers, using DNA fingerprinting technique. The genetic distances between wild jungle fowl and each of broilers and layers were similar; however the genetic diversity within broiler stock was less than within layer stock. The genetic characteristics and relationships of domestic and red jungle fowl populations maintained in Ukraine and Germany were estimated by screening their genomes by microsatellites (Romanov and Weigend, 2001). The information provided has been used to assess the genetic variation and proposed conservation plans for the populations. Zhang et al. (2002) reported genetic diversity estimates between Chinese native chicken breeds, using RAPD procedures, ranging from 0.02 to 0.23. The DNA samples of highly inbred lines of White Leghorn, Fayoumi and Spanish chickens were analyzed, using RAPD-PCR procedures (Plotsky et al., 1995). The band-sharing estimates between lines ranged from 0.66 to Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.

0.99. Polymorphism has also been detected, using RAPD technique, among White Leghorn, Rhode Island Red, Red Cornish, White Plymouth Rock and a native breed in India (Sharma et al., 2001). Twelve random primers yielded distinct polymorphic patterns, representing 25% of the total fragments amplified. The variation in polymorphic DNA in relation to origin and productivity was also assessed, using nine Russian chicken breeds, three European egg breeds, and three Asiatic meat breeds (Semyenova et al., 2002). Ahlawat et al. (2004) studied the genetic relatedness among three strains local to India and White Leghorn as an exotic breed, using RAPD technique. Ninety four bands with molecular size of 200 to 2000 bp were amplified, 32% of them were polymorphic. The genetic similarity within the local strains was high with an average of 0.82, while the genetic similarity between strains ranged from 0.77 to 0.87. The local strains showed the least genetic distances between each other, while White Leghorn appeared most distant from the local strains. The objectives of the present study were the assessment of genetic specificity of the breeds’ native to the subtropics, based on RAPD-PCR analysis and the assessment of phylogenetic relationship between the breeds. MATERIALS AND METHODS Breeds Chicks at 8 weeks old of four genetic stocks, 6 males and 6 females for each, were used. The genetic stocks were of three breeds native to Egypt (White Baladi, WB; Fayoumi, F; and Sinai Bedouin, S) and a commercial broiler (CB) strain. DNA extraction, RAPD-PCR analysis, GEL preparation and electrophoresis DNA extraction: Ten ml of blood were drawn from the wing venous of each chick into

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Heat tolerance in chickens

a heperanized tube. The individual blood samples were then stored at -20°C until use. Upon use, the blood samples were thawed and one ml of each sample was used to extract individual genomic DNA according to the procedures of Sambrook et al. (1989). RAPD-PCR analysis: Equal amounts of DNA of the individual samples within breed and sex were drawn and mixed together to get a mixed (pooled) DNA sample. RAPD-PCR analysis was then applied to the pooled samples. Samples were screened with 10 decamer oligonucleotides, arbitrary sequenced primers of Kit C (Operon Technologies, Alameda, CA, USA), with GC contents of 6070% (Table 2). PCR reaction mixture contained 1.5 µl 10X enzyme buffer containing MgCl2, 75 ng genomic DNA, 0.2 µl Taq DNA polymerase (2 units per µl), 2 µl dNTPs (2 mM), 0.5 µl primer (15 pmol) and sdH2O was added to the mix to reach a total volume of 15.0 µl. Amplification of DNA fragments was carried out in a Biometra TGradient thermal cycler (Rudolf-Wissell-Stra Be, Goettingen, Germany). PCR program included three steps. Step 1, was an initial denaturation step at 95°C for 10 minutes. Step 2, was running 40 cycles, each starting with denaturation at 96°C for 30 seconds followed by annealing at 35°C for 30 seconds and lasted by extension at 72°C for 45 seconds. Step 3, was the final extension at 72°C for 5 minutes. Gel preparation and electrophoresis: RAPDPCR products and a standard DNA marker (ØX174 DNA–Hae III Digest) were then loaded on a 2 % agarose gel (Sigma-Aldrich Co., UK) in 1X TAE running buffer and stained with ethidium bromide, to separate the amplified bands by electrophoresis. The standard DNA marker was used to determine the molecular weight of the amplified bands. Electrophoresis was performed in a horizontal apparatus (Biometra, Rudolf-Wissell-Stra Be, Goettingen, Germany), and was run at 90 volts Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.

for 50 minutes. DNA bands were visualized using an ultraviolet (UV) transilluminator (Biometra, Rudolf-Wissell-Stra Be, Goettingen, Germany) and were photographed by an ultraviolet illumination camera, using Polaroid films. The photographs were then developed and used to generate a molecular data set to estimate the genetic parameters. Parameters and statistical analysis 1. Determination of DNA concentration: DNA concentrations in the individual samples were determined by the spectrophotometer using the formula of Sambrook et al. (1989): DNA concentration (µg / µl) = A260 × 400 × 0.05 Where, A260 is the spectrophotometer reading at 260 nm, the value of 400 is the dilution factor (2000 µl double distilled water + 5 µl DNA) and the value of 0.05 is the optical density of dsDNA. 2. Heterozygosity (H): Heterozygosity was calculated according to Ott (1992) as: Where, Pi is the frequency of ith allele among a total of l alleles. H = 1 − ∑ Pi 2

3. Genetic variability (GV): Genetic variability indices were measured, within and between breeds, using the formula of Kuhnlein et al. (1989): Where, vi indicates the frequency of band i in the samples under comparison and N indicates the total number of bands scored. GV = 1 −

1 N

N

∑v i =1

i

4. Band sharing (BS): Band sharing levels were estimated, within and between breeds, according to Wetton et al. (1987) as: BS =

2 ( n ab ) na + nb

Where, nab indicates number of bands shared between samples a and b. na and nb indicate the

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E.A.El-Gendy et al.

total number of bands in samples a and b, respectively. 5. Genetic distance (D): The indices of genetic distance between breeds were calculated, by primer, according to Kuhnlein et al. (1989) as: D = -ln (I) Where, I is the genetic identity index computed according to the equation:

I=

1 N

(1)

by Choi et al. (2000).Analysis of variance was applied to the molecular parameters using the procedures of the Statistical Analysis System (SAS, 1997). The model used was:

Y ij = µ + B i + S j + BS

+ e ij

Where, Y is the dependent variable under study, µ is the overall mean, B and S indicate the effects of breed and sex respectively. Significant breed differences were shown by Duncan’s multiple range test (Duncan, 1955).

( 2)

2 Vi ⋅ Vi ∑ (1) 2, ( 2) 2 i =1 (Vi ) + (Vi ) N

ij

RESULTS AND DISCUSSION

Where, N is the number of different bands in two breeds and Vi(1) and Vi(2) are the frequencies of band i in the two breeds, respectively. Genetic distance indices between breeds were also estimated, by sex, according to Nei and Li, (1979) and Haymer and McInnis (1994) as: D = 1-BS Where, BS indicates the band sharing level between breeds, by sex. 6. Phylogenetic analysis: The genetic distance indices between different breeds, by sex, were used to construct dendograms for the breeds, using PhyloDraw software package established

DNA concentration The breeds differed in the DNA concentration (Table 1), where Sinai Bedouin was significantly the highest. The breed differences appeared only in female comparisons. These differences could be due to the experimental errors. However, average DNA content of males, overall breeds (11.0 ± 1.0 µg/µl) was not significantly different from that of females (9.7 ± 0.7 µg/µl). Klug and Cummings (2003) demonstrated that DNA amount in the diploid red blood cells of chickens was not twice the amount of DNA in the haploid sperms, reporting a difference in DNA contents between male and female gametes.

Table (1): DNA concentration (X ± SE; µg/µl) in red blood cells of chickens, by breed and sex and levels of significance for breed and sex effects. Breed differences Group

WB

F b

10.8± 1.4

S ab

13.1± 1.0

Sex difference

CB a

9.6± 1.0

b

Unsexed

7.9± 1.0

♂♂

8.3± 1.0 a

12.6± 2.8 a

12.2± 1.4 a

11.0± 1.5 a

11.0± 1.0 x

♀♀

7.5± 1.0 b

9.0± 0.4 b

13.9± 1.7 a

8.3± 1.2 b

9.7± 0.7 x

ANOVA Effect

P≤

Breed (B)

0.013

Sex (S)

0.237

B*S

0.333

WB, F, S and CB indicate White Baladi, Fayoumi, Sinai Bedouin and commercial broiler chicks, respectively. a, b , means of different breeds, unsexed and within sex, with different superscripts are significantly different (P ≤ 0.05). x , difference between sexes, overall breeds, was not significant (P ≤ 0.05).

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Number of bands and molecular size All primers showed successful amplification of bands (Table 2), with an average, overall primers, breed and sex, of 4.99 bands/sample. Number of bands varied between breeds and primers, and averaged 5.15 and 5.23 bands in males and females respectively. The amplified products varied in size from 118 to 2262 bp, overall primers. Breeds, by sex, did not significantly differ in the band numbers. Singh and Sharma (2002) screened the genomes of five White Leghorn lines, differing in selection history using 50 random primers in the RAPD technique. The number of bands ranged between 5 and 12 bands with molecular size range of 2503500bp. Ahlawat et al. (2004) applied RAPD technique to four strains of chickens, using 25 primers. The number of bands varied from 7 to 12, among different primers, with molecular size ranged from 200 to 2000 bp. Ponsuksili et al. (1996) also reported in significant differences in number of DNA bands produced for different strains of chickens. Genetic diversity All primers amplified bands with polymorphic patterns between breeds (Table 3). The number of polymorphic loci varied between primers with an average of 5.3 (65.19%) polymorphic loci per primer. The heterozygosity estimates between breeds were in general moderate to high and averaged 0.48, 0.45 and 0.44 for breed unsexed, male and female comparisons, respectively. Primers OPC-08 and OPC-17 were highly polymorphic; they recognized loci with heterozygosity levels of 0.78 and 0.79, respectively. Ott (1992) considered primers with heterozygosity of 0.70 or more highly polymorphic. Among five different chicken breeds, Sharma et al. (2001) detected distinct


5

polymorphic DNA patterns, representing 25% of the total loci amplified by 12 primers. Also, the polymorphic loci among three breeds native to India and White Leghorn, formed 32% of the total bands amplified with an average of 3 polymorphic bands/breed (Ahlawat et al., 2004). Also, Zhang et al. (2002) reported genetic diversity of 0.02 to 0.23 among Chinese breeds of chickens. The polymorphic loci between five White Leghorn lines differing in selection history formed 21.9% of the total amplified bands (Singh and Sharma, 2002). The polymorphic loci have been also detected among chicken breeds differing in the origin, genetic background and breeding history (Wei et al., 1997; Saleh et al., 2002 and Semyenova et al., 2002). Heterozygosity was also estimated between sexes, within breeds (Table 3). The estimates were in general moderate and averaged, overall primers, 0.43, 0.52, 0.35 and 0.35 for White Baladi, Fayoumi, Sinai Bedouin and commercial broilers, respectively. The within-breed genetic diversity based on DNA fingerprints was also revealed in different breeds of chickens, turkeys and ducks (Siegel et al., 1992; Smith et al., 1996; Singh and Sharma, 2002 and ElGendy et al., 2005). Several primers were considered highly polymorphic for different breeds, such as primer OPC-08, for White Baladi, Fayoumi and Sinai Bedouin, primer OPC-10 for Fayoumi, and primer OPC-17 for Fayoumi and commercial broilers, they all showed heterozygosity levels of about 0.70 or more. The results denote to somewhat genetic variation between sexes within breeds, particularly in Fayoumi and to less extent in White Baladi. This could be due to the existence of Fayoumi in a large population, while White Baladi existed in a small closed population.

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E.A.El-Gendy et al.

Table (2): Number of scorable DNA bands amplified by different primers, by breed and sex. ♂♂ Primer

Sequence ( 5' -3' )

Molecular Weight, bp

WB

Mean

♀♀

F

S

CB

WB

F

S

CB

♂♂

♀♀

Overall

OPC-01

TTCGAGCCAG

271 - 700

6

5

4

5

6

6

4

6

5.00

5.50

5.25

OPC-08

TGGACCGGTG

281 - 1078

1

3

3

4

3

1

3

8

2.75

3.75

3.25

OPC-10

TGTCTGGGTG

118 - 872

2

1

3

3

3

3

4

5

2.25

3.75

3.00

OPC-11

AAAGCTGCGG

157 - 2262

5

4

11

12

10

6

9

11

8.00

9.00

8.50

OPC-13

AAGCCTCGTC

194 - 525

3

4

2

4

2

4

4

3

3.25

3.25

3.25

OPC-14

TGCGTGCTTG

234 - 1078

9

8

10

8

7

5

8

9

8.75

7.25

8.00

OPC-16

CACACTCCAG

271 - 744

6

6

6

6

6

7

6

6

6.00

6.25

6.13

OPC-17

TTCCCCCCAG

234 - 1353

4

1

6

5

3

3

6

1

4.00

3.25

3.63

OPC-18

TGAGTGGGTG

342 - 1078

5

5

6

6

4

4

7

7

5.50

5.50

5.50

OPC-19

GTTGCCAGCC

234 - 872

7

4

7

6

6

5

6

2

6.00

4.75

3.38

4.8± 0.8 a

4.1± 0.7 a

5.8± 0.9 a

5.9± 0.8 a

5.0± 0.8 a

4.4± 0.6 a

5.7± 0.6 a

5.8± 1.0 a

5.15± 0.7

5.23± 0.6

4.99± 0.6

X ± SE

Average of probability that individual band information could disappear when individual DNA samples were pooled = 0.0059. WB, F, S and CB indicate White Baladi, Fayoumi, Sinai Bedouin and commercial broiler chicks, a , means of different breeds, within sex, are not significantly different (P ≤ 0.05).

Screening the DNA samples of the breeds by the different primers (Figures 1-10), resulted in detecting several breed-specific alleles. Primer OPC-01 amplified one band of 403 bp specific for White Baladi, and primer OPC-17 primed two bands of 515 and 872 bp specific for Sinai Bedouin. Also, some amplified alleles were breed-polymorphic bands. Primer OPC-11 amplified a band at 1078 bp polymorphic in White Baladi only. Whereas, primer OPC-14 amplified two bands with molecular weights of 310 and 872 bp polymorphic in Fayoumi only. Another band primed by primer OPC-19 at 310 bp was polymorphic in commercial broilers only. Also, several breed-monomorphic bands were recognized in all breeds by different primers. Breed-specific bands were also obtained in different Korean (Yeo et al, 1994) and Indian (Ahlawat et al., 2004) breeds of chickens, and


respectively.

in lines of chickens experienced divergent selection (Dunnington et al., 1990 and ElGendy et al., 2000). Monomorphic alleles specific for different breeds were also detected in White Leghorn, Rhode Island Red, Red Cornish and an Indian breed of chickens (Sharma et al., 2001). Bands specific for local breeds were also detected in different duck breeds (El-Gendy et al., 2005). The presence of a variety of alleles specific, polymorphic and monomorphic to the breeds, mostly in the locals, reflects the genetic specificity for each. It is of interest to denote the probable significance of the existence of these alleles to the adaptation to locality. These alleles can also be used as genetic markers for genotyping and conservation of local breeds. Romanov and Weigend, (2001) assumed that local breeds contain the genes pertinent to their adaptation to particular environments.

7


Table (3): Summary of polymorphic information between and within breeds, by primer. Primers of Operon C Parameter

Group

#1

#8

# 10

# 11

# 13 # 14 Between breeds

# 16

# 17

# 18

# 19

X

# Bands

7

8

5

13

5

10

7

9

7

8

7.90

# Polymorphic bands

3

8

4

10

3

6

1

9

3

6

5.30

% Polymorphic bands

42.86

100.00

80.00

76.92

60.00

60.00

14.29

100.00

42.86

75.00

65.19

unsexed

0.35

0.78

0.58

0.57

0.48

0.31

0.14

0.79

0.30

0.46

0.48

♂♂

0.38

0.81

0.64

0.55

0.44

0.19

0.14

0.75

0.25

0.33

0.45

♀♀

0.30

0.74

0.34

0.42

0.48

0.38

0.13

0.81

0.32

0.52

0.44

Breed-specific band

1 (WB)

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

2 (S)

‫ــــ‬

‫ــــ‬

Breed-polymorphic band

‫ــــ‬

‫ــــ‬

‫ــــ‬

1 (WB)

‫ــــ‬

2 (F)

‫ــــ‬

‫ــــ‬

‫ــــ‬

1 (CB)

Breed-monomorphic band

1 (WB)

1 (WB)

1 (CB)

1 (WB)

1 (F)

1 (WB)

‫ــــ‬

1 (WB)

‫ــــ‬

1 (WB)

‫ــــ‬

2 (CB)

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

3 (S)

‫ــــ‬

‫ــــ‬

Sex-specific band

‫ــــ‬

‫ــــ‬

1 (♀♀)

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

‫ــــ‬

Sex-monomorphic band

‫ــــ‬

‫ــــ‬

1 (♀♀)

2 (♀♀)

‫ــــ‬

3 (♂♂)

‫ــــ‬

‫ــــ‬

1 (♂♂)

2 (♂♂)

WB

0.14

0.81

0.65

0.52

0.64

0.39

0.22

0.43

F

0.25

0.88

0.70

0.65

0.30

0.48

0.11

0.89

0.39

0.53

0.52

S

0.43

0.69

0.45

0.31

0.50

0.15

0.14

0.39

0.11

0.28

0.35

CB

0.25

0.47

0.30

0.17

0.45

0.18

0.14

0.83

0.11

0.63

0.35

Mean of heterozygosity

Heterozygsity level

Between sexes, within breeds 0.55 0.25 0.14

WB, F, S and CB indicate White Baladi, Fayoumi, Sinai Bedouin and commercial broiler chicks, respectively.

On the other hand, some alleles were sex-specific. Primer OPC-10 primed a band of 872 bp specific for females, regardless of breed. Such alleles specific for females are perhaps located on the Z chromosome. In addition, primers OPC-14, OPC-18 and OPC19 amplified several alleles monomorphic in males; however primers OPC-10 and OPC-11 primed alleles monomorphic in females only.


Genetic similarity 1- Between sexes, within breeds The genetic similarity within breeds was determined by the estimation of genetic variability and band sharing indices between sexes (Table 4). The genetic variability indices were in general moderate. The averages, overall primers, of the breeds were not significantly different and ranged between 0.29 in Sinai Bedouin and 0.50 in Fayoumi. Also, no significant breed differences were observed in band sharing indices, they ranged

8

E.A.El-Gendy et al.

between 0.60 in Fayoumi and 0.81 in Sinai Bedouin. Much variability between sexes was observed in Fayoumi than in the other breeds. White Baladi and Sinai Bedouin, however, were almost similar to each other. The results may reflect the genetic history of each breed. White Baladi is kept as a small, closed and randomly mated population. Therefore it undergoes effective inbreeding over generations, resulting in diminishing variability between individuals and also between sexes. Sinai Bedouin is a breed originated in Sinai desert long ago and is currently maintained in a small closed population, the high similarity is therefore presumed. Fayoumi is a common breed and exists in a large population. So that,

inbreeding is expected to occur at rate much less than that in White Baladi and Sinai Bedouin. In turn, more variability, less band sharing and more heterozygosity are expected in Fayoumi compared to the other breeds. The commercial broiler chicks are hybrids, so the moderate variability (0.38) is expected. The results are comparable to those reported by Ahlawat et al. (2004), where the genetic similarity indices averaged 0.82 within a chicken breed native to India. Also, inbreeding resulted in genetic variability indices of 0.51 and 0.24 within two White Leghorn strains (Kuhnlein et al., 1989) and of 0.42 to 0.62 within turkey lines (Zhu et al., 1996).

Table (4): Summary of genetic variability and band sharing estimates (X ± SE) within and between breeds. Parameter

Between breeds

Between sexes, within breeds

♂♂

♀♀

WB

F

S

CB

WB* Others

F* Others

S* Others

CB* Others

WB* Others

F* Others

S* Others

CB* Others

Genetic variability

0.31 ± 0.1 a

0.50 ± 0.1 a

0.29 ± 0.1 a

0.38 ± 1.0 a

0.38± 0.1a

0.41± 0.1a

0.32± 0.1a

0.30± 0.1a

0.37± 0.1a

0.38± 0.1a

0.40± 0.1a

0.41± 0.1a

Band sharing

0.78 ± 0.6 a

0.60 ± 0.1a

0.81 ± 0.1 a

0.71 ± 0.1 a

0.71± 0.1a

0.68± 0.1a

0.76± 0.1a

0.77± 0.1a

0.74± 0.1a

0.73± 0.1a

0.71± 0.1a

0.69± 0.1a

WB, F, S and CB indicate White Baladi, Fayoumi, Sinai Bedouin and commercial broiler breeds, respectively. a , means of genetic variability or band sharing, of different breeds, by sex, are not significantly different (P ≤ 0.05)

2-Between breeds The genetic variability and band sharing estimates between breeds, by sex, are presented in Table (4). Males of different breeds showed different levels of variability. The genetic variability estimates between males of Fayoumi and males of other breeds were the highest and averaged 0.41, overall primers. Also, males of White Baladi genetically varied from males of other breeds by an average of 0.38. The variability estimates of Sinai Bedouin males from the other breeds were considerably less and averaged 0.32. The least genetic variability Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.

estimates were between commercial broiler males and those of local breeds and averaged 0.30. The genetic variability among females of different breeds was of different trend. The commercial broiler females tended to vary from those of the other breeds with an average of 0.41. Similarly, Sinai Bedouin females varied from those of the other breeds with an average of 0.40. However, White Baladi or Fayoumi females showed variability of 0.37 and 0.38, respectively. The band sharing estimates in general coincided with the estimates of genetic variability. It is noticeable that the genetic variability and band sharing

9


estimates within breeds are not, in general, different from those obtained between breeds within sexes. Since the estimates within breeds are between sexes, the variation between breeds is attributed to the sex differences. It seems that because of the non-intentional inbreeding within the local breeds, each breed has been segregated by sex into two distinct groups with high similarity within each. High band sharing levels were also found between strains of chickens local to India and ranged from 0.77 to 0.87 (Ahlawat et al., 2004), between highly inbred lines of White Leghorn, Fayoumi and Spanish chickens and ranged between 0.66 and 0.99 (Plotsky et al., 1995), and between five lines of White Leghorn chickens and ranged from 0.76 to 0.96 (Singh and Sharma, 2002). Also, band sharing levels ranging from 0.78 to 0.95 were found among

White Leghorn, Rhode Island Red, Red Cornish and White Plymouth Rock and a local Indian breed (Sharma et al., 2001), and ranging from 0.39 to 0.67 among several standard, commercial and local chicken breeds (Ponsuksili et al., 1996). Genetic distance and phylogenetic analysis Estimates of the genetic distance (Table 5) indicate that the breeds are obviously distant from each other. An average genetic distance index, overall primers, of 0.42 was calculated between White Baladi and Fayoumi and also between White Baladi and Sinai Bedouin. The genetic distance between White Baladi and commercial broilers was slightly further with an average of 0.45. Fayoumi was genetically the furthest from Sinai Bedouin with an average genetic distance of 0.53, although both are natives to Egypt.

Table (5): Summary of the genetic distance indices (X ± SE) between White Baladi (WB), Fayoumi(F), Sinai Bedouin (S) and commercial broilers (CB) breeds. Group Unsexed ♂♂ ♀♀

WB ↔ F 0.42± 0.09 0.34± 0.12 0.18± 0.05

WB ↔ S 0.42± 0.11 0.26± 0.10 0.28± 0.06

WB ↔ CB 0.45± 0.10 0.27± 0.09 0.31± 0.09

F↔S 0.53± 0.12 0.32± 0.09 0.31± 0.09

F ↔ CB 0.33±0.10 0.28± 0.10 0.35± 0.10

S ↔ CB 0.33± 0.09 0.13± 0.05 0.29± 0.10

# 08

Fig. (1): RAPD profile screened by primer OPC01 for White Baladi (WB), Fayoumi (F), Sinai Bedouin (S) and commercial broilers (CB). Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.

Fig. (2): RAPD profile screened by primer OPC-08 for White Baladi (WB), Fayoumi (F), Sinai Bedouin (S) and commercial broilers (CB).

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11


Fig. (8): RAPD profile screened by primer OPC17 for White Baladi (WB), Fayoumi (F), Sinai Bedouin (S) and commercial broilers (CB).




12

E.A.El-Gendy et al.

The genetic distance indices were also estimated, by sex. A distance index averaging 0.34, overall primers, was between White Baladi and Fayoumi males. However, an average genetic distance of 0.13 was between Sinai Bedouin and commercial broiler males. Also, the distances between Fayoumi and Sinai Bedouin males were large and averaged 0.32. The genetic distance estimates between breed females were, in general similar in the trend to those between males, but different in the extent. White Baladi and Fayoumi females were the closest to each other with an average distance of 0.18, whereas Fayoumi and commercial broiler females were most distant to each other with an average distance of 0.35. Such results agree with the results reported for chickens (Dunnington et al., 1991 and Ahlawat et al., 2004) and for ducks (El-Gendy et al., 2005), that local breeds or lines within breed are genetically less distant from each other compared to the distance between any of them and any of the exotic breeds or lines. Singh and Sharma (2002) reported genetic distance estimates with a range between 0.05 and 0.28 among five lines of White Leghorn. El-Gendy et al. (2000) reported a genetic distance of 0.29 between two lines of chickens divergently selected from their parental population for four generations. The results herein support the assumption that males and females within each breed, particularly the locals, form two separate sub-populations, with different variability for each. Thereby, males and females showed different distances between breeds. The results indicate that the local breeds partially share similar genetic composition, although each breed still reflects the genetic background peculiar to it. Fayoumi and White Baladi are much closer to each


other than the closeness between any of them and Sinai Bedouin. However the commercial broiler strain remarkably differs from the local breeds. The dendogram of the phylogenetic tree for the breeds was constructed, by sex (Figure 11). The male and female trees (Figure 11a, b) were exactly similar in the topology, but different in the distances between breeds. The tree topology formed three distinct branches. White Baladi and Fayoumi formed a branch, and each of Sinai Bedouin and commercial broilers formed a branch. The branches reflect the genetic relatedness and differentiation between the breeds. The branch of White Baladi and Fayoumi reflects their much similarity in allele distribution; however Sinai Bedouin showed less similarity to them. Although White Baladi, Fayoumi and Sinai Bedouin are locals to Egypt, they belong to different climate conditions. White Baladi and Fayoumi belong to the hot climate most of the year. However Sinai Bedouin belongs to the desert (continental) climate, yet much diurnal fluctuation in temperature occurs. Therefore, to attain the genetic homeostasis related to desert, the alleles of Sinai Bedouin have been distributed to interact favorably with the desert diurnal fluctuation. Arad et al. (1981) have reported the ability of Sinai Bedouin, compared to White Leghorn, in withstanding the desert conditions. However, White Baladi and Fayoumi have the genetic compositions that interact favorably with the hot climate. The adaptation of White Baladi to the hot conditions was earlier reported by El-Gendy et al. (1995). Similar patterns of the relationships between the topology of the genetic tree and the genetic relatedness and differentiation between different breeds of chickens were also obtained by Romanov and Weigend (2001).

13


WB

F (a) between males

CB S

WB

F

(b) between females CB S Fig. (11): Dendogram of the phylogenetic tree for the relationships between males (a) and females (b) of White Baldai (WB), Fayoumi (F), Sinai Bedouin (S) and commercial broilers (CB).


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E.A.El-Gendy et al.

REFERENCES Ahlawat, S.P.S., Sunder, J. Kundu, A. Chatterjee, R.N. Rai, R.B. Kumar, B. Senani, S. Saha, S.K. and Yadav, S.P. (2004). Use of RAPD-PCR for genetic analysis of Nicobari fowl of Andamans. British Poultry Science 45:194-200. Arad, Z., Marder, J. and Soller, M. (1981). Effect of gradual acclimatization to temperatures up to 44 °C on productive performance of the desert Bedouin fowl, the commercial White Leghorn and the two reciprocal crossbreds. British Poultry Science 22:511-520. Boichard, M. T. (2002). From phenotype to genotype: major genes in chickens. World’s Poultry Science Journal 58:65-75. Choi, J., Jung, H. Sun Kim, H. and Cho, H. (2000). PhyloDraw: A phylogenetic tree drawing system. Bioinformatics 16:10561058. Duncan, D.B. (1955). Multiple ranged multiple F test. Biometrics 11:1-42. Dunnington, E.A., Gal, O. Siegel, P.B. Haberfeld, A. Cahaner, A. Lavi, U. Plotsky Y. and Hillel, J. (1991). Deoxyribonucleic acid fingerprint comparisons between selected populations of chickens. Poultry Science 70:463-467. Dunnington, E.A., Gal, O. Plotsky, Y. Haberfeld, A. Kirk, T. Goldberg, A. Lavi, U. Cahaner, A. Siegel, P.B. and Hillel, J. (1990). DNA fingerprints of chicken selected for high and low body weight for 31 generations. Animal Genetics 21:247-257. El-Gendy, E.A., Atallah, A.A. Mohamed F.R., Atta, A.M. and Goher, N.E. (1995). Strain variation in young chicken in response to chronic heat stress conditions. Egyptian Journal of Animal Production 32:237-251. El-Gendy, E.A., Dean, R.G. and Washburn, W. (2000). Genetic analysis of DNA fingerprints for two lines of chickens divergently selected for resistance and Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.

susceptibility to a heat stress environment. Archiv für Geflügelkunde 64: 237-243. El-Gendy, E.A., Helal, M.A. Goher, N.H. and Mostageer, A. (2005). Molecular characterization of genetic biodiversity in ducks, using RAPD-PCR analysis. Arab J. Biotech. 8:253-264. Haymer, D.S. and McInnis, D. (1994). Resolution of populations of the Mediterranean fruit fly at the DNA level using random primers for the polymerase chain reaction. Genome 37:244-248. Horst, P. (1985). Effects of genotype × environment interactions on efficiency of improvement of egg production. In: Poultry Genetics and Breeding. Edited by W.G. Hill, J.M. Manson and D. Hewitt. British Poultry Science Ltd., Longman Group, Harlow, UK, pp. 147-156. Horst, P. (1989). Native fowl as a reservoir for genomes and major genes with direct and indirect effects on the inadaptability and their potential for tropically oriented breeding plans. Archiv für Geflügelkunde 53:93-101. Klug, W.S. and Cummings, M.R. (2003). Concepts of genetics, 7th edition. Published by: Pealson Education, Inc., Upper Saddle River, New Jersey 07458, USA. Kuhnlein, U., Zadworny, D. Dawe, Y. Firfull, R.W. and Gavora, J.S. (1989). DNA fingerprinting: a tool for determining genetic distance between strains in poultry. Theoretical and Applied Genetics 77:669672. Nei, M. and Li, W.H. (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Science of the USA 76:5269-5273. Ott, J. (1992). Strategies for characterizing polymorphic markers in human gene mapping. American Journal of Human Genetics 51:283-290.


Plotsky, Y., Kaiser, M.G. and Lamont, S.J. (1995). Genetic characterization of highly inbred chicken lines by two DNA methods :DNA fingerprinting and polymerase chain reaction using arbitrary primers. Animal Genetics 26:163-170. Ponsuksili, S., Wimmers, K. and Horst, P. (1996). Valuation of different combinations of oligonecletide probes and restriction enzymes to generate DNA fingerprints reflecting genetic variability in different strains of chicken. Archiv für Geflügelkunde 60:227-235. Romanov, M.N. and Weigend, S. (2001). Analysis of genetic relationships between various populations of domestic and jungle fowl using microsatellite markers. Poultry Science 80:1057-1063. Saleh, K. M., Younis, H.H. El-Sayed, T.M. and Bakhaty, A.M. (2002). Some genetic aspects for antibody response to Newcastle disease virus in chickens: 3-Multiple molecular forms of DNA and isozymes. Egyptian Poultry Science 22:73-93. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press, NY, USA. SAS Institute (1997). SAS/STAT User's Guide: Statistics. SAS Institute Inc, Cary, NC, USA. Semyenova, S.K., Moiseev, I.G. Vasil'ev, V.A. Filenko, A.L. Nikiforov, A.A. Sevast'yanova A.A. and Ryskov, A.P. (2002). Genetic polymorphism of Russian, European, and Asian chicken breeds as revealed with DNA and protein markers. Russian Journal of Genetics. 38:1109-1112. Sharma, D., Appa Rao, K.B.C. Singh, R.V. and Totey, S.M. (2001). Genetic diversity among chicken breeds estimated through randomly amplified polymorphic DNA. Animal Biotechnology 12:111-120. Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.

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Siegel, P.B, Haberfeld, A. Mukherjee, T.K. Stallard, L.S Marks, H.L. Anthony, N.B. and Dunnington E.A (1992). Jungle fowldomestic fowl relationship a use of DNA fingerprint. World's Poultry Science 48:147155. Singh, R.V. and Sharma, D. (2002). Withinand between-strain genetic variability in White Leghorn population detected through RAPD markers. British Poultry Science 43:33-37. Smith, E.J., Jones, C.P. Bartlett, J. and Nestor, K.E. (1996). Use of randomly amplified polymorphic DNA markers for the genetic analysis of relatedness and diversity in chickens and turkeys. Poultry Science 75:579-584. Wei, R., Dentine, M.R. and Bitgood, J.J. (1997). Random amplified polymorphic DNA markers in crosses between inbred lines of Rhode Island Red and White Leghorn chickens. Animal Genetics 28:291294. Wetton, J.H., Carter, R.E. Parkin, D.T. and Walters, D. (1987). Demographic study of a wide house sparrow population by DNA fingerprinting. Nature 327:147-149. Yeo, J.S., Kim, J.W. and Chol, C.B. (1994). Identification of Korean native fowl using DNA fingerprinting. Animal Breeding Abstracts 62:930. Zhang, X., Leung, F.C. Chan, D.K.O. Yang, G. and Wu, C. (2002). Genetic diversity of Chinese native chicken breeds based on protein polymorphism, random amplified polymorphic DNA, and microsatellite polymorphism. Poultry Science 81:1463-1472. Zhu, J., Nestor, K.E. and Moritsu, Y. (1996). Relationship between band sharing levels of DNA fingerprints and inbreeding coefficients and estimation of true inbreeding in turkey lines. Poultry Science 75:25-28.

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‫ﻋﺼﺎﻡ ﻋﺒﺎﺱ ﺍﻟﺠﻨﺩﻱ‪ ،‬ﻤﺼﻁﻔﻰ ﻜﻤﺎل ﻨﺼﺎﺭ‪ ،‬ﻤﺤﻤﺩ ﺴﻴﺩ ﺴﻼﻤﺔ* ﻭ ﺃﺤﻤﺩ ﻤﺴﺘﺠﻴﺭ‬ ‫ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻟﺤﻴﻭﺍﻨﻲ‪ -‬ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ‪ -‬ﺠﺎﻤﻌﺔ ﺍﻟﻘﺎﻫﺭﺓ‬ ‫* ﻗﺴﻡ ﺍﻟﺤﺸﺭﺍﺕ‪ -‬ﻜﻠﻴﺔ ﺍﻟﻌﻠﻭﻡ‪ -‬ﺠﺎﻤﻌﺔ ﻋﻴﻥ ﺸﻤﺱ‬

‫ﻓﻲ ﺩﺭﺍﺴﺔ ﻟﺘﻘﻴﻴﻡ ﺍﻟﺘﻨﻭﻉ ﺍﻟﻭﺭﺍﺜﻲ ﺒﻴﻥ ﺴﻼﻻﺕ ﺍﻟﺩﺠﺎﺝ ﻭﻋﻼﻗﺘﻬﺎ ﺒﺎﻟﺒﻴﺌﺔ ﺍﻟﻤﺤﻴﻁﺔ‪ ،‬ﺍﺴﺘﺨﺩﻡ ﺠﻴﻨﻭﻡ ﺜﻼﺙ ﺴﻼﻻﺕ ﻤـﻥ‬ ‫ﺍﻟﻤﻨﺎﻁﻕ ﺍﻟﺩﺍﻓﺌﺔ ﻭﻫﻲ ﺍﻟﺒﻠﺩﻱ ﺍﻷﺒﻴﺽ‪ ،‬ﺍﻟﻔﻴﻭﻤﻲ ﻭﺍﻟﺴﻴﻨﺎﻭﻱ ﻟﻠﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺠﻴﻨﻭﻡ ﺴﻼﻟﺔ ﺇﻨﺘﺎﺝ ﻟﺤﻡ ﺘﺠﺎﺭﻴﺔ‪ .‬ﺃﺠﺭﻴﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ‬ ‫ﻋﺸﺭﺓ ﺒﺎﺩﺌﺎﺕ ﻋﺸﻭﺍﺌﻴﺔ ﻟﺘﻀﺨﻴﻡ ﻤﻘﺎﻁﻊ ﻤﻥ ﺍﻟﻤﺎﺩﺓ ﺍﻟﻭﺭﺍﺜﻴﺔ ﻟﻜل ﺴﻼﻟﺔ ﺒﻁﺭﻴﻘﺔ ‪ .RAPD-PCR‬ﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻋﺩﺩ ﺍﻟﻤﻘﺎﻁﻊ ‪٤,٩٩‬‬ ‫ﻤﻘﻁﻌﹰﺎ‪ .‬ﻭﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻋﺩﺩ ﺍﻟﻤﻘﺎﻁﻊ ﺍﻟﺘﻲ ﺃﻅﻬﺭﺕ ﺘﻌﺩﺩ ﺍﻟﻤﻅﻬﺭ )‪ (Polymorphism‬ﺘﻤﺜل ‪ %٦٥,١٩‬ﻤﻥ ﺠﻤﻠﺔ ﺍﻟﻤﻘﺎﻁﻊ ﺍﻟﺘﻲ ﺘﻡ‬ ‫ﺍﻟﺘﻌﺭﻑ ﻋﻠﻴﻬﺎ‪ .‬ﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻨﺴﺏ ﺍﻟﺨﻠﻁ )‪ (Heterozygosity‬ﺒﻴﻥ ﺍﻟﺴﻼﻻﺕ ﻫﻭ ‪ ٠,٤٥‬ﺒﻴﻥ ﺍﻟـﺫﻜﻭﺭ‪ ٠,٤٤ ،‬ﺒـﻴﻥ ﺍﻹﻨـﺎﺙ‪.‬‬ ‫ﺃﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﻭﺠﻭﺩ ﺃﻟﻴﻼﺕ ﻤﺤﺩﺩﺓ ﻭﺃﻟﻴﻼﺕ ﻤﺘﻌﺩﺩﺓ ﺍﻟﻤﻅﻬﺭ ﻭﺃﻟﻴﻼﺕ ﺃﺤﺎﺩﻴﺔ ﺍﻟﻤﻅﻬﺭ ﻟﻜل ﺴﻼﻟﺔ‪ ،‬ﻭﺃﻴﻀﹰﺎ ﻟﻜل ﺠﻨﺱ ﺒﻐﺽ ﺍﻟﻨﻅﺭ‬ ‫ﻋﻥ ﺍﻟﺴﻼﻟﺔ‪ .‬ﻜﺎﻨﺕ ﻨﺴﺒﺔ ﺍﻟﻤﺸﺎﺭﻜﺔ ﺒﻴﻥ ﻤﻘﺎﻁﻊ ﺍﻟﻤﺎﺩﺓ ﺍﻟﻭﺭﺍﺜﻴﺔ ﺒﻴﻥ ﺫﻜﻭﺭ ﻭﺇﻨﺎﺙ ﻜل ﺴﻼﻟﺔ ﺘﺘﺭﺍﻭﺡ ﺒﻴﻥ ‪ ٠,٦٠‬ﺇﻟﻰ ‪ .٠,٨١‬ﻭﻜـﺎﻥ‬ ‫ﺍﻟﺘﺒﺎﻴﻥ ﺍﻟﻭﺭﺍﺜﻲ ﺃﻜﺜﺭ ﻭﻨﺴﺒﺔ ﺍﻟﻤﺸﺎﺭﻜﺔ ﺒﻴﻥ ﺍﻟﻤﻘﺎﻁﻊ ﺍﻟﻭﺭﺍﺜﻴﺔ ﺃﻗل‪ ،‬ﻭﻨﺴﺒﺔ ﺍﻟﺨﻠﻁ ﺃﻜﺜﺭ ﻓﻲ ﺍﻟﻔﻴﻭﻤﻲ ﻋﻥ ﺒـﺎﻗﻲ ﺍﻟـﺴﻼﻻﺕ‪ .‬ﻜﺎﻨـﺕ‬ ‫ﺍﻟﻤﺴﺎﻓﺔ ﺍﻟﻭﺭﺍﺜﻴﺔ ﺒﻴﻥ ﺍﻟﺒﻠﺩﻱ ﺍﻷﺒﻴﺽ ﻭﻜل ﻤﻥ ﺍﻟﻔﻴﻭﻤﻲ ﻭﺍﻟﺴﻴﻨﺎﻭﻱ ﻫﻲ ‪ ،٠,٤٢‬ﺒﻴﻨﻤﺎ ﻜﺎﻨﺕ ﺒﻴﻥ ﺍﻟﺒﻠﺩﻱ ﺍﻷﺒﻴﺽ ﻭﺴﻼﻟﺔ ﺍﻟﺘـﺴﻤﻴﻥ‬ ‫ﻫﻲ ‪ ٠,٤٥‬ﻭﻜﺎﻨﺕ ﺒﻴﻥ ﺍﻟﺩﺠﺎﺝ ﺍﻟﻔﻴﻭﻤﻲ ﻭﺍﻟﺴﻴﻨﺎﻭﻱ ﻫﻲ ‪ .٠,٥٣‬ﻋﻜﺴﺕ ﻗﻴﻡ ﺍﻟﻤﺴﺎﻓﺔ ﺍﻟﻭﺭﺍﺜﻴﺔ ﻟﺤ ‪‬ﺩ ﻜﺒﻴﺭ ﺍﻻﺨﺘﻼﻓﺎﺕ ﺍﻟﻭﺭﺍﺜﻴـﺔ ﺒـﻴﻥ‬ ‫ﺍﻟﺴﻼﻻﺕ ﻭﺍﻟﺘﻲ ﻜﺎﻨﺕ ﺃﻗل ﺒﻴﻥ ﻜل ﻤﻥ ﺍﻟﺒﻠﺩﻱ ﺍﻷﺒﻴﺽ ﻭﺍﻟﻔﻴﻭﻤﻲ ﻭﺒﺩﺭﺠﺔ ﺃﻜﺒﺭ ﺒﻴﻥ ﺃ ﹴ‬ ‫ﻱ ﻤﻨﻬﻤﺎ ﻭﺍﻟﺩﺠﺎﺝ ﺍﻟﺴﻴﻨﺎﻭﻱ‪ ،‬ﻤﻤﺎ ﻴﺩل ﻋﻠﻰ ﺃﻥ‬ ‫ﺍﻟﺩﺠﺎﺝ ﺍﻟﺴﻴﻨﺎﻭﻱ ﻴﻘﻊ ﻋﻠﻰ ﻓﺭﻉ ﻭﺭﺍﺜﻲ ﻤﺴﺘﻘل ﻋﻥ ﻓﺭﻉ ﺍﻟﺒﻠﺩﻱ ﺍﻷﺒﻴﺽ ﻭﺍﻟﻔﻴﻭﻤﻲ‪ .‬ﺍﻷﻤﺭ ﺍﻟﺫﻱ ﻗﺩ ﻴ‪‬ﺸﻴﺭ ﺇﻟﻰ ﺍﺤﺘﻤﺎل ﺍﺭﺘﺒﺎﻁ ﺘﻠﻙ‬ ‫ﻼ ﻟﻠﻤﻨﺎﻁﻕ ﺍﻟﺤﺎﺭﺓ ﺃﻤﺎ ﺍﻟﺴﻴﻨﺎﻭﻱ ﻓﻴﺘﺤﻤل‬ ‫ﺍﻻﺨﺘﻼﻓﺎﺕ ﺍﻟﻭﺭﺍﺜﻴﺔ ﺒﺎﻟﺒﻴﺌﺔ ﺍﻟﻤﺤﻴﻁﺔ ﻟﻜل ﺴﻼﻟﺔ‪ ،‬ﻓﺎﻟﺒﻠﺩﻱ ﺍﻷﺒﻴﺽ ﻭﺍﻟﻔﻴﻭﻤﻲ ﻴ‪‬ﻅﻬﹺﺭﺍﻥ ﺘﺤﻤ ﹰ‬ ‫ﺘﻘﻠﺒﺎﺕ ﺍﻟﺒﻴﺌﺔ ﺍﻟﺼﺤﺭﺍﻭﻴﺔ ﺃﻴﻀ ﹰﺎ‪.‬‬

‫‪Arab J. Biotech., Vol. 9, No. (1) Jan. (2006):1-16.‬‬

Genotype-environment interaction in relation to heat ... - CiteSeerX

Genotype-environment interaction in relation to heat ... - CiteSeerX

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