Synteny assignment of four genes and two microsatellite markers in river buffalo (Bubalus bubalis L.)
Authors: Othman By O. E. , Nahas S. M. El 
Published: March 2003

IntroductionSyntenic comparisons among related species suggest that large chromosomal regions are conserved among mammals ( Womack & Moll 1986;
Brubacher et al. 1990
;
Copeland & Jenkins 1991;
Levan et al. 1991
). Cattle and buffaloes (NAA = 58) belong to family Bovidae which has a high degree of chromosome conservation (
Buckland & Evans 1978;
Gallagher & Womack 1992). Their diploid chromosome numbers are 60 and 50, respectively. The banding homology between cattle and river buffalo chromosomes suggests that each of the five biarmed pairs of the river buffalo originates by centric fusion between two of ten acrocentric pairs of cattle autosomes (R
eport of the C
ommittee for theStandardization Of Banded Karyotypes Of The River Buffalo 1994). Syntenic conservation between cattle and river buffalo has been reported (
De Hondt et al. 1991
,
1997;
El Nahas et al. 1993
,
1996,
1999;
Oraby et al. 1998
).Materials and methodsForty buffalo/hamster somatic cell hybrids developed from the fusion of buffalo leukocytes and Chinese hamster cell line wg3h ( Echard et al. 1984
) deficient in hypoxanthine phosphoribosyl transferase (
De Hondt et al. 1991
) were used. The six investigated markers comprise four coding loci: prolactin (PRL), fibronectin (FN1), inhibin β‐A (INHBA), and lactoglobulin β (LGB) and two specific DNA segments: CSSM14 and CSSM52. The polymerase chain reaction (PCR) primers were kindly provided by Dr J. E. W
omack, Department of Pathology, College of Veterinary Medicine, Texas A&M University. They were designed from published bovine sequences (
Table 1).

Primer sequences and conditions for amplification reaction

PCR primer

Primer sequence 5′–3′

PCR product size (bp)

Annealing  temperature  (°C)

References

PRL

GGA AAG TGA ACA TGA CTG TCT AG

156

60

Fries et al. (1993)

GCC CTC TCT TCT ACA ATG AAC AC

FN1

TCC AAC CCA GAG AAA CAA GCG T

278

57

Barendse et al. (1994)

GAT CCC AAT CCA AAT CCA AAG T

INHBA

TTC CAT GTA CTA TGA CGA TGG GCA G

177

67

Neibergs et al. (1993)

AAC ATG TCT TCG TGT CAC CAC TGT C

LGB

TGT GCT GGA CAC CGA CTA CAA AAA G

247

55

Medrano & Aguilar‐Cordova (1990)

GCT CCC GGT ATA TGA CCA CCC TCT

CSSM14

AAA TGA CCT CTC AAT GGA AGC TTG

145

60

Moore et al. (1994)

AAT TCT GGC ACT TAA TAG GAT TCA

CSSM52

AAA TGC ACC CGG CTA ATA TGA AAG G

185

60

Moore et al. (1994)

GTA TAT ACT GTT GTT TTC TGG CAG

Genomic DNA extractionTo perform PCR analysis, genomic DNA was extracted from buffalo, hamster cell line, and buffalo‐hamster hybrid cells according to established protocols ( Blin & Stafford 1976). Cells were incubated overnight in a shaking water‐bath at 37°C in lysis buffer (10 m
m Tris base, 400 m
m NaCl, 2 m
m sodium EDTA) pH 8.2, 20 % sodium dodecyl sulfate (SDS) and proteinase k (10 mg/ml). Nucleic acids were extracted once with phenol, saturated with Tris‐EDTA (TE) buffer (10 m
m Tris, 10 m
m NaCl, 1 m
m EDTA), followed by extraction in phenol–chloroform–isoamyl alcohol (25 : 24 : 1) until there was no protein at the interface. This was followed by extraction in chloroform–isoamyl alcohol (24 : 1). To each extraction, equal volumes of the solvent were added, followed by thorough mixing and centrifugation for 10 min at 2000 r.p.m. The top layer was carefully transferred to another Falcon tube for the next extraction. To the final aqueous phase, 0.1 volume of 2.5
m Na acetate and 2.5 volume of cold 95 % ethanol were added. The tubes were agitated gently to mix the liquids and a fluffy white ball of DNA was formed. The DNA was picked up with a heat‐sealed Pasteur pipette and washed briefly in 70 % ethanol. The DNA was finally dissolved in an appropriate volume of 1X TE buffer. DNA concentrations were determined and diluted to the working concentration of 50 ng/μl.PCR reactionA PCR cocktail consisted of 1.0 μm upper and lower primers and 0.2 m
m dNTPs, 10 m
m Tris, pH 9.0, 50 m
m KCl, 1.5 m
m MgCl2, 0.01 % gelatin (w/v), 0.1 % Triton X‐100, and 1.25 units of Taq polymerase. The cocktail was aliquoted into tubes with 100 ng DNA of buffalo, hamster or hybrid cells. The reaction mixture was overlaid with sterile mineral oil and was run in a Coy Temp Cycler II (Coy Corporation, MI, USA). The reaction cycled for 1 min at 94°C, 2 min at an optimized annealing temperature which was determined for each primer (
Table 1), and 2 min at 72°C for 30 cycles. The PCR reaction products were electrophoresed on a 3 % agarose gel containing ethidium bromide. The presence or absence of river buffalo‐specific PCR product in the hybrids were scored.Statistical analysisThe segregation profile of buffalo PCR product for each locus was studied in the 40 somatic hybrids. Synteny between markers and assignment to specific syntenic group was tested by calculating the percentage of concordancy and correlation coefficient (ϕ) ( Chevalet & Corpet 1986).Results and discussionThe segregation of six molecular markers in 40 river buffalo‐hamster somatic cell hybrids was studied using PCR primers designed from published bovine sequences. Each hybrid clone was tested for its ability to amplify predicted size fragments. The amplified fragment represents the presence of the buffalo template. Synteny between the investigated markers is shown in Table 2.

. Per cent concordance (%) and correlation coefficient (ϕ) of the cosegregation of the PCR primers

PRL

FN1

INHBA

LGB

CSSM14

CSSM52

PRL

%

–

–

–

–

–

–

ϕ

–

–

–

–

–

–

FN1

%

95.0

–

–

–

–

–

ϕ

0.87

–

–

–

–

–

INHBA

%

82.5

82.5

–

–

–

–

ϕ

0.57

0.57

–

–

–

–

LGB

%

72.5

72.5

75.0

–

–

–

ϕ

0.19

0.19

0.29

–

–

–

CSSM14

%

82.5

82.5

100.0

75.0

–

–

ϕ

0.57

0.57

1.00

0.29

–

–

CSSM52

%

70.0

70.0

72.5

97.5

72.5

–

ϕ

0.14

0.14

0.18

0.92

0.18

–

Two markers are considered syntenic (bold) when ϕ exceeds 0.69 at an error rate Q = 0.025 and a probability for correct decision p = 0.96.

The results show that the six studied markers formed three syntenic groups INHBA and CSSM14 are found to segregate concordantly in all tested hybrids with a 100 % concordance and a correlation coefficient ϕ = 1; LGB and CSSM52 are found to be syntenic (97.5 %ϕ = 0.92). PRL and FN1 were also found to be syntenic (95 %ϕ = 0.87). Figure 1 shows representative ethidium bromide‐stained gels of amplified PCR products of the markers.Representative ethidium bromide‐stained gels of amplified PCR products representing the six molecular makers. L, 123‐bp ladder; B, buffalo control; H, Chinese hamster cell line wg3h; +, positive hybrids; –, negative hybridsimageSynteny between INHBA and CSSM14 has also been reported in cattle. They belong to the syntenic group U13 that is assigned to cattle chromosome 4 (INHBA, Neibergs et al. 1993
and CSSM14,
Moore et al. 1994
). Assignment of U13 to a cattle chromosome has been changed recently. According to
Texas Nomenclature (1996), the position of cattle chromosomes 4 and 6 in the
Reading Conference (1980) and ISCNDA 89 (1990) nomenclature was reversed, taking chromosome 6 as 4 and vice versa. As a result, U13, previously assigned to BTA 4 now maps to BTA 6.Also, LGB and CSSM52, reported in this study to be syntenic in buffalo, are also reported to be syntenic in cattle (U16) and were assigned to cattle chromosome 11 [LGB (T hreadgill & W
omack 1990; H
ayes & P
etit 1993; V
aimanet al. 1994), CSSM52 (
M
ooreet al. 1994
)]. On the other hand, PRL and FN1 that comprise the third syntenic group reported in buffalo are assigned in cattle to two different syntenic groups and consequently to two different chromosomes. PRL is assigned to U20 and BTA23 (
Dietz et al. 1992
;
Lewin et al. 1992
;
Beever et al. 1996
), whereas FN1 is assigned to U17 and BTA2 (
Adkison et al. 1988
;
Beever et al. 1994
).The cosegregation of the six markers in the buffalo/hamster somatic cell hybrids, and those of the autosomal bovine syntenic groups U1–U29 (except U14, no marker is available) and the X chromosome, indicates that INHBA and CSSM14 are assigned to U13 and LGB and CSSM52 are assigned to U16. As for PRL and FN1 they were both found to be syntenic with U17 and U20 ( Table 3). In cattle, synteny between PRL/BoLADR3B (U17) (
Lewin et al. 1992
;
Beever et al. 1996
) and between FN1/INHA (U20) (
Beever et al. 1994
) were reported. In buffalo PRL, BoLADR3B, FN1 and INHA are found to be syntenic (unpublished data) confirming syntenic conservation between cattle and river buffalo and assigning U17 and U20 to a buffalo biarmed chromosome.

. Percentage of concordancy and correlation coefficient (ϕ) of cosegregation of tested markers and markers representing 29 cattle syntenic groups (except U14) and X chromosome

INHBA

CSSM14

LGB

CSSM52

PRL

FN1

Syntenic group

%

ϕ

%

ϕ

%

ϕ

%

ϕ

%

ϕ

%

ϕ

U1

60.0

−0.11

60.0

−0.11

71.0

0.23

76.9

0.26

79.5

0.38

69.0

0.18

U2

76.0

0.46

76.0

0.46

61.9

−0.04

68.4

0.15

68.9

0.22

68.9

0.22

U3

73.0

0.23

73.0

0.23

61.0

−0.12

56.8

−0.16

76.3

0.53

80.0

0.48

U4

75.8

0.38

75.8

0.38

65.5

0.07

62.1

0.01

75.9

0.42

66.7

0.17

U5

77.0

0.49

77.0

0.49

72.0

0.39

70.4

0.24

61.5

0.02

63.0

0.17

U6

82.0

−0.08

82.0

−0.08

79.0

−0.09

80.6

−0.08

80.6

0.34

82.4

0.34

U7

70.0

−0.03

70.0

−0.03

70.0

0.09

74.4

0.21

82.9

0.50

83.7

0.53

U8

78.0

0.08

78.0

0.08

70.0

−0.16

74.4

0.04

86.8

0.61

86.7

0.58

U9

72.0

0.14

72.0

0.14

75.0

0.14

69.2

0.06

68.4

0.13

65.0

0.00

U10

68.0

0.13

68.0

0.13

61.9

−0.04

64.1

0.07

62.2

0.01

60.0

−0.01

U11

56.0

−0.13

56.0

−0.13

61.9

−0.13

61.5

−0.13

75.6

0.32

75.6

0.32

U12

78.0

0.20

78.0

0.20

79.0

0.33

66.7

−0.07

74.4

0.22

75.6

0.27

U13

100.0

1.00

100.0

1.00

78.0

0.30

75.9

0.29

74.0

0.03

80.0

0.42

U15

79.4

0.24

79.4

0.24

73.5

0.03

73.5

0.03

76.5

0.02

81.3

0.46

U16

75.9

0.29

75.0

0.29

100.0

1.00

97.5

0.92

72.5

0.19

73.8

0.19

U17

82.5

0.57

82.5

0.57

72.5

0.19

70.0

0.14

95.0

0.87

100.0

1.00

U18

82.1

0.32

82.1

0.32

79.5

−0.10

76.9

−0.08

74.4

−0.09

75.0

−0.08

U19

85.0

0.38

85.0

0.38

75.0

−0.10

79.5

0.16

76.3

0.14

84.6

0.35

U20

82.5

0.57

82.5

0.57

72.5

0.19

70.0

0.14

100.0

1.00

95.0

0.87

U21

79.5

0.00

79.5

0.00

82.6

0.00

79.5

0.00

75.0

0.00

75.0

0.00

U22

56.0

−0.28

56.0

−0.28

95.5

−0.15

64.1

−0.15

66.7

0.13

64.4

0.04

U23

69.0

0.08

69.0

0.08

58.0

−0.13

68.4

0.05

72.9

0.24

69.0

0.12

U24

68.0

0.36

68.0

0.36

59.5

0.02

56.4

−0.09

68.9

0.27

64.4

0.16

U25

66.7

0.00

66.7

0.00

70.8

0.18

70.3

0.25

79.2

0.48

62.5

0.05

U26

88.2

0.55

88.2

0.55

88.2

0.55

88.2

0.55

85.3

0.49

73.7

−0.14

U27

74.0

0.15

74.0

0.15

71.0

0.13

59.0

−0.19

77.0

0.36

78.6

0.34

U28

83.0

0.42

83.0

0.42

66.0

−0.02

67.9

0.00

88.0

0.51

84.2

0.47

U29

77.0

0.36

77.0

0.36

65.0

−0.16

66.7

−0.07

76.0

0.42

84.1

0.49

X

55.0

0.00

55.0

0.00

61.0

0.19

52.2

−0.04

58.3

0.11

57.7

0.05

Syntenic conservation between cattle and river buffalo is confirmed as reported from studies using buffalo/hamster somatic cell hybrids ( De Hondt et al. 1991
,
1997;
El Nahas et al. 1996
,
1999;
Oraby et al. 1998
). Also chromosomal conservation between the two species has been proved by loci assignment to homologous chromosomes using somatic cell hybrids (
El Nahas et al. 1993
) and in situ hybridization using radio‐labelled probes (
Hassanane et al. 1993
,
1994) and FISH (
Iannuzzi 1997;
Iannuzzi 1997) .Based on the chromosome homology (R eport of the C
ommittee for theStandardization Of Banded Karyotypes Of The River Buffalo 1994) and genetic conservation between cattle and river buffalo, speculation on the assignment of the two syntenic groups INHBA‐CSSM14 and LGB‐CSSM52 can be made. The syntenic group INHBA‐CSSM14 (U13) can be assigned to BBU 8 which is homologous to BTA 6, whereas LGB‐CSSM52 (U16) may be located on BBU 12 which is homologous to BTA 11.According to Iannuzzi et al. (1990a,
b) and R
eport of the C
ommittee for theStandardization Of Banded Karyotypes Of The River Buffalo (1994), the first five biarmed river buffalo chromosomes were thought to develop from fusion of ten acrocentric cattle chromosomes, where buffalo chromosome 2 results from fusion of cattle chromosomes 23 and 2. Synteny between PRL and FN1 in buffalo, which are assigned to chromosomes 23 and 2 in cattle, assigns PRL to the short arm and FN1 to the long arm of the river buffalo chromosome 2.The use of molecular markers to confirm the origin of the buffalo biarmed chromosomes proved to be very important. In a previous study ( El Nahas et al. 1997
) using molecular markers, synteny between markers representing U10 (BTA 1,
Ryan et al. 1994
) and U25 (BTA 27,
Gallagher et al. 1995
) was reported. This indicated that buffalo chromosome 1 results from fusion between cattle chromosomes 1/27 and not 1/25 as was reported in the standard karyotype of the river buffalo (R
eport of the C
ommittee for theStandardization Of Banded Karyotypes Of The River Buffalo 1994). The latter was based on chromosome banding homology.Syntenic and chromosomal assignment of six PCR markers: prolactin (PRL), fibronectin (FN1), inhibin β‐A (INHBA), lactoglobulin β (LGB), and two microsatellites CSSM14 and CSSM52 have been investigated. Their segregation in forty buffalo/hamster somatic cell hybrids were recorded. INHBA‐CSSM14, LGB‐CSSM52 and PRL‐FN1 formed three syntenic groups. In cattle, the first two syntenic groups were also syntenic but not the third group, where the markers PRL and FN1 are assigned to two separate syntenic groups, U20 and U17 and consequently to two different chromosomes BTA 23 and BTA 2, respectively. Synteny of PRL and FN1 in river buffalo confirms the cytological speculation that river buffalo chromosome 2 results from fusion of cattle chromosomes 2 and 23. Based on the chromosome conservation and genetic homology between cattle and river buffalo, the two syntenic groups: INHBA‐CSSM14 (U13) and LGB‐CSSM52 (U16) may be assigned to two river buffalo chromosomes: BBU 8 and BBU 12, respectively.