DACPA, Sezione di Scienze delle Produzioni Animali, Universita degli studi di Catania, Catania, Italy

Genetic polymorphism at CSN1S2 locus in two endangeredsicilian goat breeds

By D. Marletta, S. Bordonaro, A. M. Guastella and G. D’Urso

SummaryIn this study we surveyed two endangered Sicilian goat breeds (Girgentana and Argentata dell’Etna)for genetic polymorphism at the CSN1S2 locus. In a total of 537 goats, we detected CSN1S2A,CSN1S2B, CSN1S2C� (CSN1S2C + CSN1S2E), CSN1S2D, CSN1S2F and CSN1S2O alleles by meansof various polymerase chain reaction (PCR), allele specific-PCR (AS–PCR) and PCR-restrictionfragment length polymorphism (RFLP) reactions with the aim of improving the knowledge of thegenetic resource of these two breeds. Three and five alleles, with six and twelve genotypes, wereidentified at CSN1S2 locus, in Girgentana and Argentata, respectively. Argentata dell’Etna showed ahigher degree of genetic variation. The allelic and genotypic distribution seems to be significantlydifferent (p < 0.001) in the two breeds. In Argentata the rare null allele (CSN1S2O) was found at lowfrequency (0.033); this genetic peculiarity makes its preservation worthwhile.

Zusammenfassung

Genetische Variabilitat am CSN1S2 Genort bei zwei vom Aussterben bedrohten sizilianischenZiegenrassen

In dieser Studie wurden zwei gefahrdete sizilianische Ziegenrassen (Girgentana und Argentana desEtnas) auf Polymorphismen am CSN1S2 Genort untersucht. Insgesamt wurden bei 537 Ziegen dieAllele CSN1S2A, CSN1S2B, CSN1S2C� (CSN1S2C+E), CSN1S2D, CSN1S2F und CSN1S2O anhandvon PCR, AS-PCR und RFLP-PCR-Reaktionen festgestellt mit dem Ziel, die Kenntnisse uber diegenetischen Resourcen dieser beiden Rassen zu verbessern. Drei Allele und sechs Genotypen wurdenbei der Rasse Girgentana festgestellt, wahrend bei Argentata 5 Allele und 12 Genotypen gefundenwerden konnten. Die Verteilung der Allele und Genotypen scheint bei diesen Rassen signifikantunterschiedlich zu sein (p < 0.001). Bei der Rasse Argentata wurde ein seltenes Nullallel (CSN1S2O)mit einer niedrigen Allelfrequenz von 0,033 nachgewiesen; diese genetische Besonderheit isterhaltenswert.

Introduction

In recent years a remarkable genetic polymorphism has been revealed at the CSN1S2 locusin goat. To date, the seven alleles identified at this locus would seem to be associated withthree different expression levels: whereas CSN1S2A, CSN1S2B, CSN1S2C, CSN1S2E andCSN1S2F alleles are associated with a �normal� content of as2-casein (Boulanger et al.1984; Grosclaude et al. 1987; Bouniol et al. 1994; Lagonigro et al. 2001; Ramunno

et al. 2001a), CSN1S2D and CSN1S2O alleles are associated with an �intermediate� amount(Ramunno et al. 2001a) and with the absence of as2-casein in milk (Ramunno et al.2001b). The CSN1S2A, CSN1S2B, CSN1S2C, CSN1S2E and CSN1S2F alleles differ in pointmutations and, as a consequence, in amino acid substitutions at the protein level. TheCSN1S2D allele is characterized by a 106-bp deletion, involving the last 11 bp of the exon11 and the first 95 bp of the following intron, which is responsible for the lack of at leastthree codons. This should result in the loss of the corresponding amino acids in theexpressed protein (Ramunno et al. 2001a).

J. Anim. Breed. Genet. 121 (2004), 52–56� 2004 Blackwell Verlag, BerlinISSN 0931–2668

Ms. received: 12.12.2002Ms. accepted: 25.07.2003

U.S. Copyright Clearance Center Code Statement: 0931–2668/2004/2101–0052$15.00/0 www.blackwell-synergy.com

At molecular level, the mutation that characterizes the null allele is a transition atnucleotide 80 of the exon 11, that determines formation of a premature stop-codon inposition 110. This allele has to be considered as a true null allele: sodium dodecylsulphate (SDS) and urea-poly acrylamide gel electrophoresis (PAGE), reverse-phase-highperformance liquid chromatography (RP-HPLC) and on-line HPLC electrosprayionization mass spectrometry (HPLC/ESI-MS) confirmed that neither as2-casein, nor atruncated form of 109 residues are produced by CSN1S2O allele carriers (Ramunno et al.2001b; Marletta et al. 2002).

Little information is available on the distribution of the genotypes at locus CSN1S2 ingoat breeds. Therefore investigation of this locus is of particular interest as a marker ofbiodiversity and of as2-casein quantity in goat breeds. Sicily has a noteworthy heritage interms of animal biodiversity; Girgentana and Argentata dell’Etna, two endangered goatbreeds, are of particular interest in this context.

Girgentana is traditionally reared in the Province of Agrigento, for its good productionof drinking milk. Over the last 20 years this old breed has been really close to extinction(http://dad.fao.org). The severe reduction of Girgentana, to about 500 head in 1997, wasalso the result of the marked decrease in fresh goat milk consumption. Today, Girgentanais mainly reared in the area of origin by aged breeders in small herds.

Argentata dell’Etna is an autochthonous endangered goat breed of about 5000 head(http://dad.fao.org), reared in quite sizeable groups of some hundreds, in the mountainousareas of north-east Sicily. With regards to its difficult habitat and the extensive rearingsystem adopted, it displays a reasonable propensity towards milk production, destinedexclusively for cheese-making (mixed with cow or sheep milk) and/or for the production of�ricotta�. The milk produced by Girgentana and Argentata dell’Etna goats is not sufficientlyappreciated, neither for drinking nor for processing into goat cheese.

The aim of this work was to characterize, by genomic analysis, the CSN1S2 locus inthese two Sicilian endangered goat breeds.

Material and methods

Blood samples were collected from 537 goats (323 Girgentana and 214 Argentata dell’Etna)reared in 11 and 10 herds, respectively. The genomic DNA was extracted from leucocytesaccording to Goossens and Kan (1981). The molecular analysis at CSN1S2 locus wasperformed to identify the CSN1S2A, CSN1S2B, CSN1S2C� (CSN1S2C + CSN1S2E),CSN1S2D, CSN1S2F and CSN1S2O alleles by either polymerase chain reaction (PCR),AS-PCR and PCR–restriction fragment length polymorphism (RFLP) reactions (Fig. 1)followed by analysis of the products on agarose gel (Ramunno et al. 2000 and 2001a).Allelic and genotypic frequencies were assessed in the two breeds. Genic and genotypicdifferentiation were estimated by Genepop 3.3 statistic package.

Results

The results of the genomic analysis are reported in Tables 1 and 2 and for Girgentana andArgentata dell’Etna, respectively. Six and 12 genotypes were observed in these two breeds.In Girgentana (323) the genomic analysis did not identify the CSN1S2B, CSN1S2D andCSN1S2O alleles. At the time of the trial our sample consisted of nearly the wholepopulation of the Girgentana breed in Italy, so we can state that these alleles are almostcertainly absent in this breed. In Girgentana the frequency of CSN1S2AA genotype (0.595)and, as a consequence, of the CSN1S2A allele (0.744) was particularly high. The CSN1S2C�

allele was identified only in 10% of the population and showed a frequency of 0.056.Girgentana was not at Hardy–Weinberg genetic equilibrium at this locus, probably due tothe severe population bottleneck and high degree of inbreeding.

53Genetic polymorphism at CSN1S2 locus

In the tested sample of Argentata dell’Etna (214) the most common genotype wasCSN1S2AF (0.285). CSN1S2A allele was again the most common (0.413), followed byCSN1S2F (0.369). The genomic analysis revealed that the CSN1S2D allele is absent in thesample tested for Argentata, whereas the CSN1S2B one, identified only in twoheterozygotes, has shown a very low frequency (0.005). The most important finding wasthe identification, in Argentata goat breed, of 13 carriers (one homozygote and 12heterozygotes) of the null allele CSN1S2O, that in the tested sample showed a frequency of0.033. Argentata dell’Etna is at genetic equilibrium at the analysed locus.

Discussion

With regards to the genetic variability at CSN1S2 locus, Girgentana showed a minorgenetic variability in terms of number of alleles (3 versus 5) and heterozygosity or gene

M 21 43

301 bp

168 bp133 bp

62 bp

Fig. 1. DNA restriction patterns produced by digesting with NcoI endonuclease the 301 bppolymerase chain reaction fragment containing the 11th exon of the goat CSN1S2 gene. M, marker100 bp; lane 1: CSN1S2O/O; lane 2: CSN1S2D/O; lane 3: CSN1S2N*/D; lane 4: CSN1S2N*/N*.

N* ¼ CSN1S2 A, B, C, F or E

Table 1. Observed genotypes and allelic frequencies at the CSN1S2 locus in Girgentana breed

Genotypes AA C�C� FF AC� AF C�F

Heads 192 3 26 25 72 5Allelic frequencies A ¼ 0.744 C� ¼ 0.056 F ¼ 0.200

C� ¼ C + E undistinguishable.

Table 2. Observed genotypes and allelic frequencies at the CSN1S2 locus in Argentata dell’Etna

Genotypes AA C�C� FF OO AB AC� AF AO BC� C�F C�O FO

Heads 40 8 33 1 1 30 61 5 1 27 3 4Allelic frequencies A ¼ 0.413 B ¼ 0.005 C� ¼ 0.180 F ¼ 0.369 0 ¼ 0.033

C� ¼ C + E undistinguishable.

54 D. Marletta et al.

diversity (Hexp 0.403 versus 0.661). Both breeds present a significant deficit in the numberof heterozygotes observed at the locus investigated (Hobs 0.316 versus Hexp 0.403,p < 0.001 in Girgentana; Hobs 0.617 versus Hexp 0.661, p < 0.05 in Argentata dell’Etna).Comparison of the molecular data of these two goat breeds showed that the allelic andgenotypic differentiations at CSN1S2 locus were both highly significant (p < 0.001). Inparticular, with reference to the genotypes observed in both breeds, differences ingenotypes AA and CF were highly significant (Table 3); moreover, Argentata goatsshowed higher frequencies of FF (p < 0.01) and C�C� and AC� genotypes (p < 0.05).

The results of the trial allow us to presume that the genetic polymorphism in theGirgentana breed does not affect the as2-casein quantity, whereas the minor presence ofthe null allele in the Argentata dell’Etna reduces its quantity in some animals. The minoramount of this casein in milk, as a consequence, reduces the allergenic potency of thecasein fraction of goat’s milk in an in vitro immunological assay (Marletta et al. 2003,in press).

An �in situ� preservation strategy of the Argentata dell’Etna breed should thereforeevidently adopt this locus as genetic marker for milk quality in a marked assisted �selection�,to re-establish its commercial value and preservation.

References

Boulanger, A.; Grosclaude, F.; Mahe, M. F., 1984: Polymorphisme des caseines as1 et as2 de lachevre (Capra hircus). Genet. Sel. Evol. 16: 157–175.

Bouniol, C.; Brignon, G.; Mahe, M. F.; Printz, C., 1994: Biochemical and genetic analysis ofvariant C of caprine as2-casein (Capra hircus). Anim. Genet. 25: 173–177.

Goossens, M.; Kan, Y. W., 1981: DNA analysis in the diagnosis of hemoglobin disorders. Meth. Enz.76: 805–817.

Grosclaude, F.; Mahe, M. F.; Brignon, G.; Di Stasio, L.; Jeunet, R., 1987: A Mendelianpolymorphism underlying quantitative variations of goat as1-casein. Genet. Sel. Evol. 19: 399–412.

Lagonigro, R.; Pietrola, E.; D’Andrea, M.; Veltri, C.; Pilla, F., 2001: Molecular geneticcharacterization of the goat as2-casein E allele. Anim. Genet. 32: 391–393.

Marletta, D.; Bordonaro, S.; Galliano, F.; Cunsolo, V.; Saletti, R.; Pastore, N.; D’Urso, G.,2002: Identification of CSN1S20 allele in a Sicilian goat breed and characterization of as2 caseinfraction by HPLC/ESI-MS. In: Proceedings of the Seventh WCGALP Communication N. 9–32,12–23 August, Montpellier, France, pp. 19–23.

Table 3. Genotypic distribution and frequencies at the CSN1S2 locus in Girgentana andArgentata dell’Etna goats

Genotypes

Girgentana Argentata

v2 p(1)n Frequency n Frequency

AA 192 0.595 40 0.187 ***C�C� 3 0.009 8 0.037 *FF 26 0.081 33 0.154 **OO – – 1 0.005 NSAB – – 1 0.005 NSAC� 25 0.077 30 0.140 *AF 72 0.223 61 0.285 NSAO – – 5 0.023 **BC� – – 1 0.005 NSC�F 5 0.015 27 0.126 ***C�O – – 3 0.014 NSFO – – 4 0.019 *

NS, not significant.C� ¼ C+E undistinguishable.(1)Significance at v2 (or at Fisher’s test) ***p < 0.001; **p < 0.01; *p < 0.05.

55Genetic polymorphism at CSN1S2 locus

Marletta, D.; Bordonaro, S.; Guastella, A. M.; Cosenza, G.; Falagiani, P.; Crimi, N.; D’Urso,G., 2003: Goat milk with different as2-casein content: analysis of allergenic potency by REAST-inhibition assay. Small Rumin. Res. (in press).

Ramunno, L.; Pappalardo, M.; Cosenza, G.; Pastore, N.; Gallo, D.; Grimaldi, G.; Rubino, R.;Calandrelli, M.; Rando, A., 2000: Genetic characterization at as1, b and as2 casein loci of a goatpopulation reared in the peninsula sorrentina. Proceedings of the XIV Congress Naz. SIPaOC.Vietri sul mare (SA), Italy, pp. 321–324.

Ramunno, L.; Cosenza, G.; Pappalardo, M.; Longobardi, E.; Gallo, D.; Pastore, N.; Di

Gregorio, P.; Rando, A., 2001a: Characterization of two new alleles at the goat CSN1S2 locus.Anim. Genet. 32: 264–268.

Ramunno, L.; Longobardi, E.; Pappalardo, M.; Rando, A.; Di Gregorio, P.; Cosenza, G.;Mariani, P.; Pastore, N.; Masina, P., 2001b: An allele associated with a non detectable amountof as2 casein in goat milk. Anim. Genet. 32: 19–26.

Author’s address: Dr Donata Marletta (for correspondence), DACPA: sezione di Scienze delleProduzioni Animali, Facolta di Agraria, Universita degli studi di Catania, viaValdisavoia, 5-95123 Catania, Italy, Tel.: +39-95-234331; Fax: +39-95-234345;E-mail: d.marletta@unict.it

56 D. Marletta et al.