Y Chromosome Introns

Five primer sets were chosen from Y chromosome introns which amplified in the common shrew, Sorex araneus (Hellborg and Ellegren 2003). The introns chosen were DBY1, DBY3, DBY7, UTY11, and ZFY4 generating products totaling approximately 3.2 kbp. Two other introns (DBY8, DBY14), which amplified in S. araneus (Hellborg and Ellegren 2003) were not chosen due to a lack of polymorphism in several races of the common shrew (G. Yannic, unpublished data). Amplification of the Y chromosome introns was based on the protocol used by Hellborg and Ellegren (2003). Briefly, PCR reactions consisted of 3 ml DNA (60-300 ng/ml), 5.0 ml 10 x Buffer (Invitrogen), 2.5 ml of MgCl2 (2.5 mM; Invitrogen), 1.0 ml dNTPs (0.2 mM; Invitrogen), 1.0 ml (0.25 mM; Sigma Aldrich) of forward and reverse primer for each locus and 0.3 ml of Platinum Taq Polymerase (5 U/ml; Invitrogen) to make up a 50 ml reaction.

PCR amplification was carried out using an initial denaturation at 95°C for 10 min followed by 20 cycles of 95°C for 30 s, a touchdown annealing temperature

Table 1 List of samples and haplotypes (both mtDNA and concatenated Y chromosome introns) present in each locality. The number of individuals per locality is represented by n. The coordinates of each sampling locality (numbered from 1 to 38) is plotted in Figs. 3 and 4

Map

mtDNA

Location

Country

Coordinates

No.

n

haplotype

Y haplotype

Rascafria

Spain

40°54'

N 03°53'

W

1

3

Spa1,2

Spa1,2

Encamp

Andorra

42°35'

N 01°35'

E

2

2

And1,2

Fra3

Le Mas-d'Artige

France

45°42'

N 02°08'

E

3

1

Fra1

-

Nexon

France

45°45'

N 01°15'

E

4

1

Fra2

Fra1

Belle Île

France

47°20'

N 03°11'

W

5

10

BI1,2,3,4,5

BI1

Fresseneville

France

50°05'

N 01°34'

E

6

1

Fra3

-

Broualan

France

48°28'

N 01°37'

W

7

2

Fra4,5

Fra2,3

Plancoet

France

48°30'

N 02°15'

W

8

1

Fra6

Fra4

Morlaix

France

48°35'

N 03°50'

W

9

2

Fra6

Fra3

Boxtel

Belgium

51°36'

N 05°20'

E

10

1

Bel1

-

Wageningen

Holland

51°58'

N 05°40'

E

11

1

Hol1

-

Hartz Mountains

Germany

51°45'

N 10°40'

E

12

2

Ger1

Ger1,2

Eberswalde

Germany

52°10'

N 13°45'

E

13

1

Ger2

Ger3

Kobbero

Denmark

56°43'

N 08°20'

E

14

1

Den1

North1

Askland

Norway

58°40'

N 08°35'

E

15

3

Nor1,2,3

Nor1

Klockkarvik

Norway

60°07'

N 05°00'

E

16

2

Nor4

-

Steinkjer

Norway

64°00'

N 13°53'

E

17

1

Nor5

North1

Tromso

Norway

68°50'

N 19°00'

E

18

1

-

North1

Karlskrona

Sweden

56°09'

N 15°15'

E

19

1

Swe1

Swe1

Vehkalahti

Finland

60°15'

N 27°00'

E

20

1

Fin1

-

Val d'Illiez

Switzerland

46°16'

N 06°43'

E

21

1

Swz1

Swz1

Bretolet

Switzerland

46°13'

N 06°40'

E

22

1

Swz2

Swz1

Bassin

Switzerland

46°32'

N 06°39'

E

23

1

Swz3

-

Trento

Italy

46°15'

N 11°50'

E

24

2

Ita1,2

-

Abruzzo

Italy

42°00'

N 14°00'

E

25

4

Ita3,4,5,6

Ita1

Calabre

Italy

37°07'

N 16°38'

E

26

1

Ita7

Ita2

Reggio

Italy

44°32'

N 11°21'

E

27

1

Ita8

Ita3

Republic

50°41'

N 13°34'

E

28

2

Cze1,2

North1, Cze1

Donnerskirchen

Austria

47°56'

N 16°40'

E

29

1

Aut1

Aut1

Bratislava

Slovakia

48°07'

N 17°00'

E

30

5

Svk1,2,3,4

North1, Svk1

Blizocin

Poland

51°36'

N 22°16'

E

31

2

Pol1,Pol2

North1,Pol1

Vilnius

Lithuania

54°40'

N 25°19'

E

32

2

Lit1

North1

Kanev

Ukraine

49°41'

N 32°00'

E

33

1

Ukr1

North1

Brjansk

Russia

52°20'

N 34°00'

E

34

1

-

North1

Novosibirsk

Siberia

54°49'

N 83°06'

E

35

1

Sib1

North1

Lake Baikal

Siberia

53°40'

N 108°00' E

36

1

-

Sib1

Pelister Mountain

Macedonia

41°00'

N 21°10'

E

37

1

Mac1

-

Strandzha

Turkey

41°45'

N 27°41'

E

38

2

Tur1,2

Tur1

Mountains

from either 60-50°C (DBY1, DBY7) or 55-45°C (DBY3, UTY11) for 1 min, decreasing by 0.5°C per cycle and then an extension of 72°C for 90 s. This was followed by 20 cycles of 95°C for 30 s, 50°C (DBY1, DBY7) or 45°C (DBY3, UTY11) for 1 min, and 72°C for 90 s and a final extension step at 72°C for 10 min. All PCR reactions included a known female sample as a negative control. Amplification of a fragment in the female sample was used as evidence that the gene fragment was not specific to the Y chromosome. For this reason, intron ZFY4 was disregarded after amplification in several female samples.

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