a University of Brescia, Department of Biomedical Sciences and ... and expected heterozygosities were estimated using GENEPOP V3.1 software (Raymond &.
7URSHQWDJ 6WXWWJDUW+RKHQKHLP2FWREHU
&RQIHUHQFHRQ,QWHUQDWLRQDO$JULFXOWXUDO5HVHDUFKIRU'HYHORSPHQW
Comparison Between Bos taurus and Bos indicus by Microsatellites and Casein Haplotypes Carolia Anna, Oliver Jannb, Christina Weimannb and Georg Erhardtb a University of Brescia, Department of Biomedical Sciences and Biotechnologies, Università di Brescia, Viale Europa 11, 25123 Brescia, Italy b Justus-Liebig University of Giessen, Department for Animal Breeding and Genetics, Ludwigstrasse 21b, 35390 Gießen, Germany
Abstract We analysed the genetic diversity of 22 taurine and indicine cattle breeds at three zebu- and African taurine-diagnostic microsatellite loci and three casein genes, and we studied possible indicine and African taurine introgressions into Southern European cattle breeds. This study adds another point of view on the phylogeny of cattle. Results prove a genetic introgression of Bos indicus into South Eastern European cattle. Genetic relationships of South Western European breeds with African taurine breeds, may be assessed as the result of selection effects rather than gene flow. The occurrence of a taurine-indicine hybridisation zone contributes an alternative explanation of the high variability of breeds near the alleged centre of origin. Introduction The need to increase, maintain and conserve genetic diversity in animal and plant species has been recognized (Oldenbrock 2002). Attention has been turned to this direction with different genetic techniques useful to assess genetic diversity. Among them, the analysis of casein haplotypes as well as microsatellite alleles, provides an efficient tool for a discrimination between Bos indicus and Bos taurus populations (MacHugh et al. 1997; Ritz et al. 2000; Hanotte et al. 2002; Ceriotti et al. 2004; Ibeagha-Awemu et al. 2004; Jann et al. 2004). The aim of this study was to analyse the genetic diversity of 22 taurine and indicine cattle breeds at three zebu- and African taurine-diagnostic microsatellite loci and three casein genes, and to evaluate possible indicine and African taurine introgression into Southern European cattle breeds. Material and methods Casein (CSN1S1, CSN2, and CSN3) and three microsatellite loci (ETH 152, HEL1, and BM2113) were typed as indicated in Ibeagha-Awemu et al. (2004) and Jann et al. (2004). Allele frequencies and expected heterozygosities were estimated using GENEPOP V3.1 software (Raymond & Rousset 1995). Haplotype frequency of the linked loci CSN1S1, CSN2, and CSN3 were determined based on the results of combined genotype frequencies using EH software (Xie and Ott 1993). Principal Component Analysis (PCA) of allele and haplotype frequencies was performed using SPSS 8.0.0 Software (SPSS Inc., Chicago, USA). Results and discussion Based on the analysed casein alleles, 84 haplotypes are theoretically possible, but a maximum number of only 16 was found in Turkish Grey Steppe, followed by Asturian Mountain and Istrian (Table 1). Just six haplotypes (NH) were detected in German Yellow, Fighting Bull, and N`dama.
The highest mean numbers of alleles (MNA) at microsatellite loci were detected in Anatolian Black, Istrian, and Turkish Grey Steppe, lowest in Pezzata Rossa and Fighting Bull. Gene diversity values were higher in Anatolian Black, Istrian, and Turkish Grey Steppe and lower in Nellore, Fighting Bull, and N`dama. Gene diversity was in tendency lower for microsatellites than for casein haplotypes. Code AB AM AN AV BH BR CH CI CN GV IS MA ME ND NE PI PR PRI SG TG TL WF
Breed Anatolian Black Asturian Mountain Angler Asturian Valley Brahman Bohemian Red Charolais Chianina Casta Navarra German Yellow Istrian Maremmana Menorquina N`dama Nellore Piemontese Polish Red Pezzata Rossa Santa Gertrudis Turkish Grey Steppe Fighting Bull White Fulani
Origin Turkey Spain Germany Spain Paraguay Czech Republic France Italy Spain Germany Croatia Italy Spain Nigeria Brasil Italy Poland Italy Paraguay Turkey Spain Kamerun
N 42 50 50 50 50 54 55 49 50 37 57 51 50 26 50 50 43 50 48 51 46 10
NH 11 13 8 10 9 9 9 11 11 6 13 9 11 6 8 12 10 11 8 16 6 8
MNA 8.00 6.33 7.66 6.33 6.66 8.66 6.00 5.66 6.33 7.00 8.00 7.00 5.33 5.33 6.33 6.66 8.66 5.00 7.33 8.00 5.00 6.33
HH 0.83 0.85 0.78 0.82 0.75 0.81 0.82 0.75 0.81 0.78 0.87 0.73 0.76 0.58 0.69 0.89 0.76 0.81 0.82 0.88 0.76 0.83
MH 0.82 0.74 0.70 0.76 0.70 0.75 0.74 0.64 0.76 0.75 0.79 0.75 0.66 0.66 0.63 0.75 0.73 0.69 0.75 0.79 0.61 0.70
overall 0.82 0.76 0.72 0.77 0.71 0.76 0.76 0.67 0.77 0.76 0.81 0.74 0.68 0.64 0.64 0.79 0.74 0.72 0.76 0.81 0.65 0.73
Table 1 For each breed: code, sample origin, sample number (N), number of found haplotypes (NH), mean number of alleles at the microsatellite loci (MNA), expected casein haplotype heterozygosity (HH), expected microsatellite heterozygosity (MH), and overall expected heterozygosity (overall).
Figure 1 describes the first two Principal Components (PC) respectively from the microsatellite and casein haplotype frequency distribution, summarised in table 2. Both PCA using microsatellite alleles (left) and casein haplotypes (right) indicate an intermediate B. indicus/ B. taurus (BI/BT) hybrid cluster with Anatolian Black, Santa Gertrudis, Turkish Grey Steppe, and White Fulani. This proves that these breeds share genes of indicine origin. The analysis of casein haplotypes results in a noteworthy discrepancy in the assessment of Istrian and Asturian Mountain in comparison to PCA on the basis of microsatellites. The casein cluster is regarded as a QTL region (Freyer et al. 1996), therefore submitted to strong selection pressure. Microsatellites are neutral markers, while caseins are coding genes involved in physiological processes and productive performances, and can give important indications about the functional relationships among breeds. This may also be the explanation of an apparently near relationship between N`dama and Chianina, Maremmana, and Menorquina, which cannot be detected when using microsatellite loci. 20
20
Figure 1 Plotting of the first two princepal components (PC 1 and PC 2) of microsatellite allele (left) and casein haplotype (right) frequencies.
ND
PRI
PC 2
AN AV PI CH PR
CN
10
CH
10
WF
SG
BR PR
0
CN GV BR
IS
AM TL
TG
GV PRI AV
WF
TG
AB
PI
AB
TL
0
ME
AM
IS
SG BH
CI
MA
NE
-10
-10
AN MA
ND ME
BH CI
-20
NE
-20
-10
PC 1
0
10
-20
-10
0
10
20
30
The highest diversity values were found in Turkish and Balkan breeds (Turkish Grey Steppe, Anatolian Black, Istrian), which are originated near the presumed domestication centre, while breeds originated further distant from Anatolia showed lower variability. Indeed, genetic diversity is expected to be higher at the centre of origin of a species or breed group and decreasing with distance (Slatkin and Maddison 1990). On the other hand, Turkish Grey Steppe, Anatolian Black, and Istrian were also subject to a strong genetic introgression of BI, and therefore represent hybrid genomes which maybe an alternative explanation of the high variability. Such theory is supported by the fact that the lowest diversity values were found in breeds which represent mainly ancient genomes like N`dama, Nellore, or Fighting Bull. Table 2 Casein haplotype (CSN1S1/CSN2/CSN3) and microsatellite (HEL1, ETH152, BM2113) allele frequencies in different cattle breeds. For breed abbreviations see Code in table 1.
B R E E D
CSN1S1 CSN2 CSN3 AB AM AN AV BH BR CH CI CN GV IS MA ME ND NE PI PR PRI SG TG TL WF
B 1 A B 0.02 0.05 0.08 0.08 0.25 0.13 0.40 0.17 0.23 0.08 0.42 0.45 0.61 0.07 0.09 0.06 0.20 0.09 0.33
B 2 A B 0.13 0.13 0.23 0.26 0.02 0.05 0.29 0.22 0.36 0.20 0.24 0.26 0.04 0.09 0.14 0.06 0.11 0.08 0.02 0.27 0.05
B 1 A A
0.33 0.17 0.01 0.26 0.06 0.19 0.05 0.30 0.16 0.07 0.08 0.03 0.16 0.43 0.10 0.27 0.14 0.22
B 2 A A
C C 2 2 A A B H 0.21 0.28 0.11 0.19 0.22 0.14 0.03 0.42 0.22 0.02 0.25 0.10 0.02 0.04 0.14 0.06 0.17 0.07 0.06 0.10 0.11 0.07 0.18 0.12 0.51 0.12 0.12 0.14 0.14 0.04 0.37 0.02 0.19 0.17 0.02 0.20 0.10 0.25 0.25 0.15
C 2 A A 0.13 0.18
B C 2 B A AI A 0.06 0.04 0.06 0.02 0.02 0.15 0.19 0.05 0.09 0.03 0.02 0.07 0.12 0.08 0.02 0.02 0.03 0.03 0.05 0.05 0.10 0.12 0.03 0.13 0.04 0.03 0.05 0.04 0.14 0.07 0.04 0.05 0.10
B B B 0.04
B C C 2 1 1 A A A H A B 0.29 0.08
0.05 0.12 0.03
B I B
C B H
C 1 A H
B 1 A E
B C B
B C 1 1 A A AI H 0.02
B 1 A C
HEL1 HEL1 HEL1 HEL1 ETH152 HEL1 BM2113
101
107
0.12
0.24
0.04
0.02
0.01
0.42
0.03
0.10
0.31 0.07
0.34 0.03
0.06 0.04
0.02 0.06
0.02
0.05 0.03 0.06
0.02 0.01
0.04
0.26 0.07
0.04 0.07
0.26 0.47 0.12
0.02 0.12 0.01 0.02 0.13
0.05 0.02 0.08 0.30
0.02 0.08 0.02 0.04
111 0.18 0.06 0.04 0.07 0.11 0.03 0.08 0.08 0.09 0.09 0.18 0.11 0.22
0.04 0.26 0.09 0.02
0.05 0.14 0.22
0.11 0.04
0.19 0.26
0.16
0.13
0.40
0.02
0.05
117
193 0.36
0.06
0.65 0.01
0.17 0.33 0.10
0.27
0.10 0.81 0.04
0.07 0.03
0.07 0.03
0.08
0.05
0.05
0.01 0.02 0.10
0.09 0.03
0.54 0.21 0.63
109
123
0.18 0.04
0.06
0.05
0.01 0.02 0.02 0.02 0.01 0.05 0.32
0.40
0.01 0.04 0.01 0.01 0.04 0.14 0.08
0.05
It is evident that the high number of observed haplotypes in and around the supposed domestication centre is also caused by hybridisation of BT with migrating BI populations. Cooccurrence of indicine as also taurine haplotypes in the same old hybrid populations gave enough time for recombination events which increased the number of detectable haplotypes. Casein genes are linked in a physical distance of approximately 250 kb (Ferretti et al. 1990; Threadgill and Womack 1990; Rjinkels et al. 1997). According to White et al. (1989) a linkage distance of 1cM corresponds with a physical distance of approximately 1 mb in human. If postulating similar recombination rates in cattle, recombination probability within the casein locus should be approximately about one recombinant in 400 individuals every generation. Measurable proportions of recombinant haplotypes therefore should occur, especially when comparing old breeds, wherein after a hybridisation different ancestral haplotypes were present. Indeed, putative recombined haplotypes like BA2H, CBH, CA1H, BA1AI or CA1H are much more frequent in the mixed taurine-indicine breeds like Anatolian Black, Casta Navarra, Istrian or White Fulani than in the parental population. In contrast in shortly developed synthetic breeds like Santa Gertrudis recombined haplotypes were nearly absent. The short breeding history of this breed (FAO 2002) did not allow recombination events between the casein genes. The predominance of the major casein haplotype BA1B in N´dama and related Southern European breeds, cannot be valuated as evidence for an introgression of African taurine genes to Southern Europe. The same haplotype appears also in breeds where genetic exchanges with the African continent are very unlikely and there is no clear co-occurrence with unique N`dama specific microsatellite alleles. The haplotype frequency gradient might also be interpretated as a product of a selective adaptation of the casein locus which is strongly shaped by selection (Ward et al.
1997). On the other hand predominance of BA1A and BA2A in North-Central European breeds and CA2H in zebu and the co-occurrence of these haplotypes with BI-diagnostic microsatellite alleles suggest their ancestry. Our results do not necessarily support the theory of an exclusive NearEastern origin of European cattle which was proven only for female lines by using mtDNA (Troy et al. 2001). Recently, evidence has been shown for two distinct cattle migrations in Europe from the Near East, and for more recent input in Mediterranean cattle breeds from both the Near East and Africa (Cymbron et al. 2005). The complete absence of the predominant haplotypes BA1A and BA2A of Northern and Central European breeds in Anatolian Black and low frequency of such haplotypes in South and South Eastern European cattle may indicate that Northern and Central European breeds are at least in part of independent origin. For a confirmation of such a conclusion it would be necessary to analyse a higher number of Near Eastern breeds or to include archaeological material. The high variability within the centre of origin is mainly due to a secondary introgression, and cannot be seen as evidence of a domestication centre, but as evidence of an extense hybridisation zone, wherein cattle breeds carry haplotypes of indicine like also taurine origin. Acknowledgement The authors thank the German breeder associations, E.M. Ibeagha-Awemu, J.Citek, R.Zieminski, and the RESGEN Consortium for providing samples. References Ceriotti G, Marletta D, Caroli A, Erhardt G (2004). J. Anim. Breed. Genet. 121: 404-415. Cymbron T, Freeman AR, A1, Malheiro MI, Vigne JD, Bradley DG (2005). Proc. R. Soc. B 272: 1837 - 1843. FAO (2002). http://dad.fao.org. Ferretti L, Leone P, Sgaramella V (1990). Nucleic Acids Res. 18: 6829-6833. Freyer G, Liu Z, Erhardt G, Panicke L (1996). Arch. Tierz. Dummerstorf 39: 369-385. Hanotte O, Bradley DG, Ochieng JW, Verjee Y, Hill EW, Rege JEO (2002). Science 296: 336339. Ibeagha-Awemu EM, Jann OC, Weimann C, Erhardt G (2004). Genet. Sel. Evol. 36: 673-690. Jann OC, Ibeagha-Awemu EM, Ozbeyaz C, Zaragoza P, Williams JL, Ajmone-Marsan P, Lenstra JA, Moazami-Goudarzi K, Erhardt G (2004). Genet. Sel. Evol. 36: 243-257. MacHugh DE, Shriver MD, Loftus RT, Cunningham P, Bradley DG (1997). Genetics 146: 10711086. Oldenbrock JK (2002). in: Oldenbrock JK (Ed.). Genebanks and the Conservation of Farm Animal Genetic Resources, ID-Lelystad, The Netherlands, pp. 1–31. Raymond M, Rousset F (1995). J. Hered. 86: 248-249. Ritz LR, Glowatzki-Mullis M-L, Machugh DE, Gaillard C (2000). Anim. Genet. 31: 178-185. Rjinkels M, DeBoer HA, Pieper FR (1997). Mamm. Genome 8: 148-152. Slatkin M, Maddison WP (1990). Genetics 126: 249-260. Threadgill DW, Womack JE (1990). Nucleic Acids Res. 18: 6935-6942. Troy CS, MacHugh DE, Bailey JF, Magee DA, Loftus RT, Cunningham P, Chamberlain AT, Sykes BC, Bradley DG (2001). Nature 410: 1088-1091. Ward TJ, Honeycutt RL, Derr JN (1997). Genetics 147: 1863-1872. White R, Lalouel JM, Leppert M, Lathrop M, Nakamura Y, O'Connell P (1989). Genome 31: 1066-1072. Xie X, Ott J (1993). Am. J. Hum. Genet. 53: 1107.
