Cytogenetic analysis (GTG, CBG and NOR bands) of a wild boar population (Sus scrofa scrofa) with chromosomal polymorphism in the south-east of Spain

rise to 3 variants in the diploid number [2n = 38 (1 (J’), 2n = 37 (2 (3’, 1 C!! and 2n = 36 (1 !!’, 7 Q )] was observed. By means of GTC-band analysis, this polymorphism proved to be the consequence of a Robertsonian translocation between members of pairs 15 and 17 of karyotypes with 2n = 38, thereby creating a long submetacentric chromosome (15/17) together with 2 free acrocentric chromosomes, (15 and 17) in individuals with 2n = 37, and a pair of long submetacentric chromosomes (15/17) in 2n = 36 specimens. CBG-band analysis revealed 2 types of staining for heterochromatin: a dark staining, characteristic of acrocentric chromosomes and occasionally present in the submetacentric (15/17), and a pale staining, characteristic of the rest of the submetacentric chromosomes. The Y-chromosome presented dark heterochromatin over the entire length of its long arm. The nucleolus organizers were observed on pairs 8 and 10 and showed heteromorphism of the size of the silver block and in the distribution in both pairs. wild board / karyotype / chromosomal polymorphism / nucleolus organizer region / heterochromatin

CBG-band analysis revealed 2 types of staining for heterochromatin: a dark staining, characteristic of acrocentric chromosomes and occasionally present in the submetacentric (15/17), and a pale staining, characteristic of the rest of the submetacentric chromosomes.

MATERIAL AND METHODS
The study comprised 12 specimens (4 d ' , 8 Q ) of a Sus scrofa scrofa population, all native to Sierra Nevada and Sierra de Baza (Almeria, Spain). The karyotype was obtained from whole blood cultures. Blood samples were taken by venous puncture from anaesthesized males (n = 4) and by means of cardiac puncture in the females (n = 8). The cultures consisted of 0.4 ml blood in 7.5 ml RPMI-1640 medium supplemented with 15% fetal bovine serum; L-glutamin, antibiotics and phytohaemaglutinin. The preparations were analysed by the following banding techniques: GTG (Seabright, 1972), CBG (Sumner, 1972) and silver nitrate staining, NOR S , (Bloom and Goodpasture, 1976). Using the same methodology, we analysed the chromosomes of 4 hybrids from a male wild boar and a domestic sow.

RESULTS
Chromosome analysis carried out with conventional Giemsa staining enabled us to verify the existence of numerical variability in the chromosomal sets among the animals analysed. Among the 12 animals analysed, 1 of the males possessed 2n = 38, 2 males and 1 female were 2n = 37, and 7 females and 1 male were 2n = 36. This numeric variability was correlated with a morphological variability in 2 chromosomal paires. However, no phenotypical variability was observed.
Application of different banding techniques allowed us to analyse the cytogenetic mechanisms involved in the numerical and structural variability encountered in the GTG bands The metaphases treated with proteolytic enzymes (GTG bands) showed that the karyotype of the male wild boar with 2n = 38 (Fig 1) consisted of 5 submetacentric chromosomal pairs (1-5), 2 subacrocentric pairs (6-7), 5 metacentric pairs (8-12) and 6 acrocentric pairs (13-18), plus the 2 gonosomes (metacentric X-chromosome, of similar length to the autosomal pair 9, and a small metacentric Y). This karyotype is identical (in number of chromosomes, morphology and banding patterns) to the karyotype of the domestic pig (Halgeltorn and Lin et al, 1980). The metaphases with 2n = 37 (Fig 2) presented structural heteromorphism in pairs 15 and 17, resulting in 2 unpaired acrocentric chromosomes (see arrows in Fig 2) and a long submetacentric chromosome (15/17) with a length intermediate between pairs 1 and 2. The p and q arms of this submecentric chromosome had banding patterns and relative length identical to the 2 acrocentric chromosomes 15 and 17. The other pairs, including the gonosomes, were identical to those of the individuals, with 2n = 38. A Robertsonian translocation (centric fusion) between 1 chromosome, each of pairs 15 and 17, would account for the heteromorphism found in the 2n = 37 specimens, and hence, explain the numerical variation in the diploid chromosome number.

CBG bands
CBG-band analysis (Summer, 1972), revealed 2 types of centromeric heterochromatin in the karyotypes: a darker staining which proved to be alkali resistant and very constant and which was found in all acrocentric chromosomes, and a pale, alkali-labile staining associated with the subtelocentric and meta-submetacentric chromosomes. The heterochromatin blocks of the acrocentric chromosomes varied in size, even for chromosomes of the same pair, but were observed for all chromosomes of this morphological class. In contrast, the heterochromatin of non-acrocentric pairs was pale (at times not even detectable) and showed a graded staining pattern according to the pairs, but was particularly constant in pair 1. The (15/17) chromosome was exceptional in having a double block of dark-staining centromeric similar to that of the unpaired acrocentric chromosomes 15 and 17 (Figs 4 and  5). Frequently, a kind of intercalary heterochromatin was detected in the long arm of pair 10 in the proximity of the secondary constriction, which correspond to the nucleolus organizer of this pair. The long arm of the Y-chromosome was totally heterochromatic. The X-chromosome, however, usually lacked the heterochromatin staining, or was very pale and could not be differentiated from the heterochromatin of the autosomal pairs of similar length and morphology (pairs 8 and 9).

NOR bands
The nucleolus organizer regions, detected by means of silver staining (Bloom and Goodpasture, 1976) are located in the paracentrometric region of the short arm of pair 8 and the long arm of pair 10 (Fig 6). Polymorphism, both in the size of the silver blocks and in the distribution within the pair, was observed. Block size was larger in the chromosomes of pair 10, and even within this pair there existed size variability of the block in the homologous chromosomes. Analysis of nucleolus organizer distribution on 347 metaphases of 4 animals (2 C3', 2 !) is summarized in Table I. Table I shows that 84% of the metaphases contained some active nucleolus organizer regions. Of these, 80% were from metaphases with at least 1 active chromosome in pair 10, while 72% of metaphases showed staining in both chromosome of pair 10. Only 4% presented staining in a chromosome of pair 8, and only 1.15% of the metaphases showed 4 active chromosome.
In addition, chromosomal associations were observed in pair 10, giving rise to tetraradial structures (Fig 6b), a phenomenon which was not recorded for pair 8, or between pairs 8 and 10.
In fact, the wild boar and domestic pig karyotypes are very similar. In specimens with 2n = 38, it proved to be impossible cytogenetically to differentiate between subspecies, as they showed identical features in the number of chromosomes, pair morphology and even banding patterns for both gonosomes and autosomes. In addition, the Spanish wild boar population presents a numerical polymorphism with 3 variants in their chromosomal sets (2n = 36, 37 and 38) as a consequence of a translocation involving pairs 15 and 17. A similar polymorphism has also been observed in German wild boar populations studied by McFee et al (1986), in the Dutch specimens (Bosma, 1976), as well as in the Austrian population reported by Mayr et al, (1984). However, the German specimens analysed by Gropp et al (1969) as well as the French studies (Mauget et al, 1977;Popescu et al, 1980) and the Swiss population (Jotterand-Bellomo and Beattig, 1981), did not present this polymorphism and are homogenous populations with 2n = 36. Finally, Zivkovic et al (1971) discovered 2n = 38 sets in certain specimens native to different regions in Yugoslavia, and Popescu et al, (1980) in animals from the island of Corsica. These studies confirm the variability in the wild boar karyotype which contrasts with the constancy found for domestic pig karyotype, independent of origin (Hansen, 1972;Gustavsson et al, 1972;Berger, 1972; Hageltorn and Hansen-Melander and Melander, 1974;Ford et al, 1980;Lin et al, 1980;Ronne et al, 1987).
In the light of the analogies between the wild boar and domestic pig karyotypes differentiated only by a Robertsonian translocation, one can only speculate about the ancestral karyotype of these species. Are the 15 and 17 acrocentric chromosomes, or the 15/17 submetacentric chromosomes the ancestral morphology? The involvement of chromosome 17 in 2 Robertsonian translocations [(15/17) in the European wild boar and (16/17) in the Asian species (Tikhonov and Troshina, 1975)] suggests fusion as the most likely process during evolution, and hence, the ancestral karyotype most likely contained the acrocentric chromosomes. On the other hand, the centrometric heterochromatin of the submetacentric pair 15/17, unlike the other meta-submetacentric chromosomes, showed dark staining similar to acrocentric chromosomes. This exception, in turn, allows for speculation about the ancestral character of the acrocentric chromosomes. It is legitimate to assume the dark heterochromatin to be ancestral heterochromatin and the pale heterochromatin to be a derivative of the former.
This assumption is indirectly supported by evidence provided by Arroyo Nombela and Rodriguez Murcia (1977), who reported a spontaneous double Robertsonian translocation which appeared in a colony of NMRI mice, showing that the resulting biarmed chromosomes (2,3) and (X,3) had dark-stained double heterochromatin block and even retained the relative lengths of the acrocentric chromosome blocks from which they had originated. Hence, the translocation does not alter the nature of the heterochromatin. Moreover, its differentiation would occur at a later stage as Furthermore, we suggest that the ancestral karyotype with 2n = 38 gave rise to European wild boar species by means of a 15/17 Robertsonian translocation, and to the Asian wild boar variety by means of another 16/17 translocation.
The Robertsonian translocations differentiating the domestic pig and the wild boar karyotypes do not inhibit hybridization of the subspecies. We have examined a litter of 4 young wild boars which resulted from the breeding of a male wild boar and a domestic sow. The descendants showed sets of 2n = 38 and 37. In this context, the work of Troshina and Tikhonov (1980) on Landrace pig and and European wild boar (15/17), as well as Asian wild boar (16/17) hybrids, is highly conclusive, demonstrating that neither of the 2 translocations constitute an isolating reproductive barrier, and that viable and fertile hybrids were obtained from crosses of these subspecies, resulting in 2n = 36 (34 + 15/17 + 16/17). In this light, it seems legitimate to consider Sus scrofa domestica, Sus scrofa scrofa and Sus scrofa nigri P ens as 3 karyotypic races.
It is not clear whether the present numerical polymorphism observed is due to a recent translocation or to interbreeding of domestic pig and wild boar. The size and general presence of the silver blocks on the pair 10 in all analysed individuals cause us to think that this pair must be considered as the main site of r-RNA production.
No phenotypic variability was observed in any of the analysed specimens, which have the appearance of true typical wild boars. Usually Robertsonian polymorphisms have no phenotypic expression.