frequency of rob( 1; 29)

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1 Hvredifus Hereditus 115: (1991) A cattle breed close to 58 diploid number due to high frequency of rob( 1; 29) TERESA RANGEL-FIGUEIREDO and LEOPOLDO IANNUZZI I Departamento de Zootecniu - UTAD - Vila Real, Portugal Consiglio Nazionale delle Ricerche; Istituto di ricerche sull clda~tamento dei Bovini e dei Bufali an Ainbiente del Mezzogiorno (CNR-IABBAM)- Nupoli, Italy RANGEL-FIGUEIREW, T. and IANNUZZI. L A cattle breed close to 58 diploid number due to high frequency of rob( 1; 29). ~ Lund, Sweden. ISSN Received May 14, Accepted July 23, Barrosa cattle, reared in the north of Portugal primarily for meat production, number about 40,000 (about 6 YO of all cattle in Portugal). Their fertility (number of calves per 100 cows) varies from 60 YO to 80 Y, and is lower than that of other Portuguese cattle breeds. 195 animals (28 males and 167 females), randomly selected from a large area, were sampled for cytogenetical investigation. The results were the following: (a) 68 (34.9 YO) animals (7 males and 61 females) had normal karyotypes; (h) 127 (65.1 YO) were found to be carriers of rob( I ; 29), as shown by G- and R-banding; (c) 102 (52.3 YO) animals (17 (8.7 X) males and 85 (43.6%) females) were heterozygous carriers; (d) 25 (12.8 YO) animals (4 (2%) males and 21 (10.8%) females) were homozygous carriers. C-banding patterns revealed one block of constitutive heterochromatin (HC) in the proximal q-arm region of the translocated chromosome. L. Iunnuzzi, CNR-IABBAM, Viu Argine 1085, Ponricelli-Nupolr, Iluly Robertsonian translocations (ROBERTSON 1916) Material and methods are the most common abnormalities in cattle (GUSTAVSSON 1969, 1974; ELDRIDGE 1985). They have also been the most common chromosomal rearrangements involved in the karyotype evolution of Bovidae (WURSTER and BENIRSCHKE 1968; BUCKLAND and EVANS 1978a; BUNCH and NADLER 1980; DI BERARDINO et al. 1981; IAN- NUZZI et al. 1987, 1990). Of all the centric fusions discovered so far in cattle (LONG 1985) rob( 1; 29) has been found to be the most wide-spread, occurring with different frequencies in more than 40 different breeds including In this study 195 cattle (28 males, of which 15 were bulls, and 167 females) reared in the mountains of North Portugal were used. Blood samples were collected on 52 small farms over a large area. Peripheral blood was cultured at 38.5 C in RPMI 1640 (Gibco), with L-Glutamine (1 %I), Pokeweed mitogen (1 YO). and autologous plasma ( 15 %). Six hours before harvesting, 20 pg/ml of 5-Bromodeoxiuridine (BrdU) were added to obtain R-banding patterns. Colcemid treatment (0.05 pg/mi) was for one hour. A hypotonic treatment (KC M) and three fixations some Bos indicus (POPESCU 1984). The first cytoge- (the third overnight) in methanol/acetic acid (3: 1) netic investigation of Portuguese cattle (Maronesa followed. breed) revealed the highest frequency (about 50 %) Additional cultures from 8 heterozygous carriof rob( 1; 29) ever found in a representative cattle ers were set up with early-brdu incorporation to sample ( RANGEL-FIGUEIREDO and IANNUZZI obtain G-banding patterns, as earlier reported 1990). (IANNUZZI et al. 1989). During examination of records of reproductive performance of Portuguese cattle breeds, we found that Barrosa was the poorest. Since the presence of Banding techniques rob( 1; 29) in cattle reduces the fertility of het- CBG. ~ The technique according to SUMNER erozygous carriers ( GUSTAVSSON 1969; DRYEN- (1972) was used. DAHL and GUSTAVSSON 1979), we undertook a cytogenetic investigation to verify the presence and frequency of this translocation in Barrosas. GTG. - The technique according to LIN et al. (1977) was used.

2 74 T RANGEL-FIGUEIREDO AND L IANNULLI GBG, RBA and RBG. -Slides from cultures treated for early- (G-band) and late- (R-band) BrdU incorporation were stained for 10 min with acridine orange (0.01 %, in phosphate buffer, ph = 7.0), mounted as previously reported (IAN- NUZZI et al. 1989) and observed under a fluorescent microscope. Then slides were counterstained for 30 min with Giemsa as previously reported (IAN- NUZZI et al. 1989; IANNUZZI 1990). Nomenclature. ~ G- and R-banded karyotypes were arranged according to the ISCNDA ( 1989) and the G- and R-band comparison of cattle chromosomes ( IANNUZZI 1990). Results and discussion Barrosa, (Fig. 1) is one of the most important breeds reared in the mountains of North Portugal, numbering approximately 40,000 animals (about 6 YO of total Portuguese cattle). It is raised primarily for meat production. The live weight varies between kg in the males and kg in females, with a mean daily gain of kg. Their reproductivity coefficient (number of calves per 100 cows) varies from 60 to 80%), lower than that of other Portuguese cattle breeds, and the females have their first calving at months of age. Fig. 2 shows two conventionally stained metaphases from heterozygous and homozygous carriers of a centric'fusion translocation of the I; 29 type. G- and R-banding patterns (Fig. 3) confirmed that chromosomes 1 and 29 of the cattle karyotype were involved in this translocation, according to previous reported G- and R-banding patterns ( GUSTAVSSON 1974; Dr BERARDINO et al. 1979; IANNUZZI 1990; RANGEL-FIGUETREDO and IANNUZZI 1990). The CBG-technique (Fig. 4) revealed one constitutive heterochromatin (HC) block located in the proximal region of the q-arm, as shown previously ( GUSTAVSSON 1974; PoPEsCu and BOSCHER 1974; DT BERARDINO et al. 1979; MASUDA et al. 1980; CIUPERCESCU et al. 1984; IANNUZZI et al. 1987; RANGEL-FIGUEIREDO and IANNUZZI 1990). IANNUZZI et al. (1987) demonstrated that this translocation involved loss of the centromere of chromosome 29 and retention of that of chromosome I. This was confirmed by synaptonemal complex studies using electron mi- croscope (SWITONSKI et al. 1987). Generally, biarmed chromosome pairs of several species of Bovidae have arisen through Robertsonian translocations during karyotype evolution. These translocations have been accompanied by HC losses, as indicated by small HC blocks seen in the centromeric regions of these chromo- somes ( BUCKLAND and EVANS ; GUPTA and Fig. 1. A Barrosa bull. Notice the typical wide lyre horns.

3 Heredim 11.5 (1991) HIGH FREQUENCY OF ROB( I ; 29) IN CATTLE 75.A B Fig. 2A and B. Conventional stained metaphases from heterozygous (A) and homozygous (B) carriers of centric fusion translocation (large arrows). The sex chromosomes are also indicated. RAY-CHAUDHURI 1978; DI BERARDINO and IANNUZZI 1981; IANNUZZI et al. 1987). Rob( 1; 29) has a large HC block (Fig. 4) but it is possible that this translocation will lose more HC in subsequent evolution. The monocentric nature of this translocation and its diffusion in more than 40 different cattle breeds (POPESCU 1984) have led several authors to postulate an ancient origin for this translocation. However, WILSON ( 1990) examined the pedigree of a British Friesian bull which was a heterozygous carrier of rob( 1; 29) and hypothesized a de novo origin for the translocation, because it had not been seen previously in this breed (LONG 1985; ELDRIDGE 1985). Table 1 shows the incidence of rob( 1; 29) in our sample of Barrosas. Of the 195 animals investigated, only 68 (34.9 %) showed a normal karyotype whereas 127 (65.1 %) were found to carry rob( 1; 29). Of these, 102 (52.3 YO) were heterozygous and 25 (12.8 YO) were homozygous for this translocation. Male carriers (10.8 Yn) were three times as frequent as normal males (3.6 %I), while the percentage of female carriers (54.4 YO) was about twice that of normal females (31.3 YO). Since GUSTAVSSON and ROCKBORN (1964) first found rob( 1; 29) in Swedish cattle, several other breeds have been shown to have this translocation in varying frequencies (POPESCU 1984; LONG 1985; ELDRIDGE 1985). Until now, the highest frequency of this translocation has been found in another Portuguese cattle breed (Maronesa), in which about half of investigated animals were found to be carriers ( RANGEL-FIGUEIREDO and IANNUZZI 1990). However, Barrosas have the highest incidence (65.1 %) of this translocation ever found in cattle. The high frequency of this translocation in Table 1. Karyotype distribution in 195 Barrosa cattle (percentage within parentheses) Karyotype Males Females Males + females Normal karyotype 7 (3.6) 61 (31.3) 68 (34.9) Rob( I; 29) HT 17 (8.7) 85 (43.6) 102 (52.3) HM 4 (2.1) 21 (10.8) 25 (12.8) Total 21 (10.8) 106 (54.4) 127 (65.1) HT, heterorygous carrier; HM. homorygous carrier

4 16 T RANGFLFIGUEIREDO AND L IANNU7ZI Fig. 3A-D. Details of prometaphases showing rob( I; 29) after GTG- (A), GBG- (B), and sequential RBA- (C) and RBG- (D) techniques. Arrows indicate the centromere of translocated chromosome.

5 Herrdiitus J. Nuturr Herrdiitus J Aai Hcrediras Heredim //5 (IYY11 HIGH FREQUENCY OF ROB( I: 29) IN CATTLE 77 possible reason for such a high frequency of the translocation might be the high rate of inbreeding resulting from a small population size. In consideration of the high number of carriers available, measures of meat production parameters will be made to compare normal and carrier animals in regard to their performance. A['~noil.ledRri,ienr.~. The authors thank Mrs. 1. Abobeleira for her excellent technical assistance. Mr. Adelino Bernard0 and Pablo Aguds for the blood collection. and Mrs. L. Keller for her kind cooperation in this paper. References BIJCKI-AND, R. A. and EVANS, H. J. 1978d. Cytogenetic aspects of phylogeny in the Bovidae. I. G-banding. ~ Cyrogmrr. CN/ Gene/. 21: BUCKLAND, R. A. and EVANS, H. J. 1978b. Cytogenetic aspects of phylogeny in the Bovidae. II. C-handing. ~~~ Cyrogener. Cell Gene/. 21: BUNCH, T. D. and NADLER. C. F Giemsa-band patterns 01 the Tahr and chromosomal evolution of the tribe Caprini.. J. Hcred. 71: I10 I16 CIlIPbRCbSClJ, D. D., LIVFSCII. 8. E. and LUNGFANU. A Cylogenetic studies in artificial insemination bulls in Romania. 6/11 Eur. Colioq. Cyrogrnet. Domesr. Anrm. Ziirich. p x2 DI BERARDINO, D. and IANNUZZI, L Chromosome banding homologies in swamp and murrah buffalo. ~~ Hered. 72: Fig. 4A-D. (A) CBG-banded metaphase of a het- DI BmARDiNo, D., IANNUUI, L., FERRARA, L. and erozygous._ male carrier of rob( 1; 29) (large arrows) with MATASSINO, D A new case of Robertsonian transloca- (B-D) details of the translocation taken from different t'on in J. 'O DI BFRARDINO, D., IANNUZ71, L., BETTINI, T. M. and cells and animals. Notice the large HC-block in the MATASSINO. D, 198,, Ag-N~Rs and banding ho. chromosome (arrows). ruurux Cun. J. Grncr. Cytol. 23: DRYF.NIMHI, 1. and GUSTAVSSON, I Sexual functions, semen characteristics and fertility of bulls carrying 1;29 chromosome translocation. ~ 90: Barrosas indicates that this translocation in this ELDRIDGE, E. F Cytogenetics of livestock. ~ Publishing Company, INC, Westporr, Conneclicur, USA, p. I23 breed is probably of ancient origin. GUPTA. P. and RAY-CHAUDHURI, S. P Robertsonian The low fertility in this breed may, in part, be changes in chromosomes of lndian Murrah butfalo. BuhuluJ huhii.y. The Nucleus 21 : explained by the high frequency rob( 29) GUSTAVSSON, I Cytogenetics. distribution and phenotypic effects of a translocation in Swedish cattle. Hereditus 63: G~JSTAVSSON, Distribution and effects of the 1/29 Robertsonian translocation in cattle. ~ Dairy Sci. 62: G~JSTAVSSON, 1. and ROCKBORN, G Chromosome abnormality in 3 cases of lymphatic leukaemia. ~~ 20.3: 990 IANNUZZI, L An improved characterization of cattle chromosomes by means of high resolution G- and R-band comparison. -~ J. Hered. 81: [ANNUI%I. L., DI BERARDINO, 0.. GUSTAVSSON, I., FERRARA, L. and DI MEO, G. P Centromeric loss in translocations of centric fusion type in cattle and water butfalo. ~ 106: IANNUZZI. L., GUSTAVSSON, I., DI Mco, G. P. and FCRRARA, L High-resolution studies on late-replicating segments (G + C-hands) in mammalian chromosomes. ~ I10: IANNUZZI, L., DI MEO, G. P., PtHUCAlTI, A. and FEKKARA. L A comparison of G- and R-banding in cattle and river which is expected to give rise to chromosomally unbalanced gametes resulting from segregation at meiosis of trivalents in heterozygous carriers. Such unbalanced gametes in turn result in the death of embryos (GUSTAVSSON 1969, 1974). When the animals are homozygous carriers, both meiosis and embryonic development are expected to be normal. The low fertility of heterozygous carriers could be the price the breed must pay to obtain possible new advantage derived from new genetic linkages of translocated chromosomes. In fact if this abnormality is present in such a high frequency in this breed, it is reasonable to suppose that the breeders select carriers because they are phenotypically superior to karyologically normal animals. Another

6 Curyologia 78 T. RANGEL-FIGUEIREDO AND L IANNUZZI Heredim 115 (1991) buffalo prometaphase chromosomes. ~ 43: ISCNDA International System for Cytogenetic Nomenclature of Domestic Animals (eh D. DI BERARDINO. H. HAYES, R. FRIES and S. LONG). - Cyrogenet. re// Germ 53: LIN, C. C., NEWTON, D. R, and CHURCH, K. B Identification and nomenclature Ibr G-banded bovm chromosomes. ~ Can. J. Genet. Cytol. 19: LONG, S Centric fusion translocations in cattle: A review. - Vet. Rec. 116: MASUDA, H., SHIOYA, Y. and FUKIJHARA, R Robertsonian translocation in Japanese Black cattle. - Jap. J. Zootech. Sci. 51: POPFSCLI, C. P The 1/29 translocation twenty years after. 6th Eur. Col/oy. Cytogenet. Domesr. Anim. Zurich, p POPESCIJ, C. P. and BOSCHER, J Etude du caryotype bovin par une nouvelle methode cytogenetique: les bandes C. ~ 1st World Congr. Genet. Appl. Livest. Prod., 3 Symposia. Madrid. p RANGEL-FIGU~IREDO, T. and IANNUZZI, L High incidence of 1/29 centric fusion translocation in a Portuguese cattle breed. -~ J. Genet. Breed. 44: ROBERTSON, W. R. B Chromosome studies. I. Taxonomic relationships shown in the chromosomes of Tettigidae and Acrididae, Locustidae and Gryllidae: chromosomes and variation. --J Morphoi. 17: SUMNER, A. T A simple technique for demonstrating centromeric heterochromatin. ~- Exp. Cell Res. 7s: SWITONSKI, M., GUSTAVSSON, 1. and PLOEN, L The nature of the 1;29 translocation in cattle as revealed by synaptonemal complex analysis using electron microscopy. ~~ Cytogenet. Cell Gener. 44: 103 I1 I WILSON, T. D Identification of the 1/29 Robertsonian translocation chromosome in British Friesian cattle. - Vet. Rer. 1 /2: WURSTER, D. H. and BENIRSCHKE, K Chromosome studies in the superfamily Bovidae. - - Chromosoma 25: