Genetic variation at microsatellite loci in Spanish sheep

Small Ruminant Research 39 (2001) 3±10

Genetic variation at microsatellite loci in Spanish sheep J.J. Arranz, Y. BayoÂn*, F. San Primitivo Departamento de ProduccioÂn Animal, Universidad de LeoÂn E-24071 LeoÂn, Spain Accepted 25 May 2000

Abstract Genetic variation at 18 microsatellite loci was analysed in six indigenous Spanish sheep: Churra; Latxa; Manchega; RasaAragonesa; Castellana and Merino. Merinos had frequently the highest number of alleles per locus, whereas Latxas showed the lowest one at many loci. Markers ordered decreasingly according to the number of variants differentiated in the whole population were: MAF70; TGLA13; CSSM66; BM143, BM6444; MAF36; MAF64; CSSM6; TGLA53; OarFCB11; MAF33; BM4621; MAF48; MAF65; BM1258; ILSTS002; ADCYC and OarCP34. Parameters of variability such as effective number of alleles and gene diversities corroborated the high level of variation frequently displayed by microsatellite markers. Comparison of allele distributions among populations and loci did not reveal consistent shapes. Distributions were centralised in some cases, whereas in others some kind of skewness was evident. Breed-speci®c alleles were detected at most loci, being frequent in Merinos and rare in Churras. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Microsatellite loci; Genetic variation

1. Introduction DNA microsatellite sequences are valuable genetic markers due to their dense distribution in the genome, great variation, co-dominant inheritance and easy genotyping. In recent years, they have been extensively used in parentage testing, linkage analyses, population genetics and other genetic studies (Goldstein and Pollock, 1997). They are very useful to analyse the degree and pattern of genetic variability within and between populations. Their variation is mainly explained by factors such as genetic drift, gene ¯ow and mutation, since they are generally considered non-selective markers (Boyce et al., 1996). Although

* Corresponding author. Fax: ‡34-987-291-311. E-mail address: [email protected] (Y. BayoÂn).

they show some limitations in the analyses of phylogenetically distant organisms, due to their irregular mutation processes involving range constraints and asymmetries (Nauta and Weissing, 1996), they have proved very useful in intra-species population studies. In the particular case of sheep, several investigations have used microsatellite loci to examine and compare genetic variation among different breeds (Buchanan et al., 1994; Forbes et al., 1995). Spain has a variety of indigenous sheep breeds in different locations and for different uses. Merinos are well known for their crimpy and ®ne wool. Other non®ne sheep, also very important numerically, are farmed either for milk, meat or both productions. Microsatellite markers were used to analyse the phylogenetic relationships among Spanish sheep (Arranz et al., 1998). The objective of the study was to describe the variability found at each locus and breed. The differences among breeds and loci in parameters of

0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 0 ) 0 0 1 6 4 - 4

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genetic variation such as number of alleles, gene diversity and allele distributions, are analysed. Data known on the molecular basis of microsatellite variation and their signi®cance in the context of the present work are examined, and differences among breeds discussed in relation to the genetic relationships already established (Arranz et al., 1998). 2. Material and methods Genetic variation at 18 DNA microsatellites was investigated in indigenous Spanish sheep. A sample of unrelated animals was studied in each of the following breeds (the number of animals is indicated in parentheses): Churra (50); Latxa (46); Manchega (52); Rasa-Aragonesa (40); Castellana (48) and Merino (48). The present material includes the breeds studied by Arranz et al. (1998) and Castellana sheep in addition. The loci were chosen on the basis of their location in several chromosomes and a non-linkage criterion for syntenic markers. Microsatellites studied and their chromosomal location (in parentheses) were as follows: MAF64 (Chr 1); BM6444, OarFCB11, TGLA13 (Chr 2); OarCP34 (Chr 3); MAF70 (Chr 4); BM143, BM4621 (Chr 6); CSSM66, MAF33 (Chr 9); TGLA53 (Chr 12); ILSTS002 (Chr 14); ADCYC, MAF65 (Chr 15); CSSM6 (Chr 19); BM1258 (Chr 20); MAF36 (Chr 22) and MAF48 (unassigned) (Maddox et al., 1996; De Gortari et al., 1998). Genomic DNA was obtained either from frozen semen when available (only in the case of Churra sheep) or blood using protease K digestion followed by salting out (Miller et al., 1988). Microsatellites were ampli®ed in 10 ml reaction volumes, from 25 ng DNA template. PCR reaction contained 75 mM KCl, 15 mM Tris±HCl (pH 8.5), 1.75 mM MgCl2 0.02% gelatin 250 mM of each dNTP, 0.25 mM of each primer, 0.8 U Taq DNA polymerase and 0.1 mCi a32-P dATP/ml. Thermal cycling conditions for the ampli®cation reaction are detailed in the references above. After the addition of 1 vol of formamide dye, 3 ml of each reaction was electrophoresed on acrylamide standard sequencing gels. The gels were autoradiographed overnight. Two independent allele identi®cations were performed and discrepancies were resolved.

Allele frequencies were calculated by direct count. Estimates of gene diversity (H) were obtained according to Nei (1987). The polymorphism information content (PIC) and the effective number of alleles (ne) were estimated following Botstein et al. (1980) and Hartl and Clark (1989), respectively. 3. Results and discussion Several parameters indicative of the variation degree found at 18 microsatellite loci in six Spanish sheep breeds are summarised in Table 1: number of observed alleles (na), effective number of alleles (ne) and values of gene diversity (H). The distributions of allele frequencies are depicted in Fig. 1A and B. It is to be noted that a general accordance with Hardy±Weinberg equilibrium has been reported at these loci with few exceptions (Arranz et al., 1998). 3.1. Analysis of breeds The analysis of the identi®ed alleles revealed certain differences among breeds. In many cases, Merino sheep showed the highest number of observed alleles per locus. Moreover, the size of variants present only in Merino was frequently out of the range found for the rest of sheep, indicating a wider distribution in this particular breed. These data are in accordance with results from genetic distances estimated between Merino and other Spanish sheep (Arranz et al., 1998) which corroborate the earlier differentiation of Merinos as indicated by historical data. On the contrary, Latxas had the lowest number of variants at many microsatellites. The extreme situation was that of BM1258 marker, with a range from 4 to 7 alleles, except for Latxa sheep with only two different alleles identi®ed. Alleles present exclusively in a particular breed and thus likely to be breed-speci®c were detected at most loci (see Fig. 1A and B). These variants had generally an extreme size (either small or large) and showed low frequencies (average 0.05), representing 15% of the total number of alleles identi®ed. Breed speci®c alleles were described in different sheep breeds by Buchanan et al. (1994) and Forbes et al. (1995). The latter authors found that these results were even more apparent between domestic and bighorn sheep, a large

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Table 1 Number of observed alleles (na), effective number of alleles (ne) (in parentheses) and gene diversity (H) at 18 microsatellite loci in six Spanish sheep

CH LX MA RA CA ME

CH LX MA RA CA ME

na H na H na H na H na H na H

(ne)

na H na H na H na H na H na H

(ne)

(ne) (ne) (ne) (ne) (ne)

(ne) (ne) (ne) (ne) (ne)

BM143

BM4621

BM6444

CSSM6

CSSM66

MAF33

MAF36

MAF48

MAF70

11 (5.6) 0.82 11 (6.9) 0.86 10 (4.8) 0.79 9 (5.7) 0.83 6 (3.0) 0.65 10 (5.6) 0.82 OarCP34

9 (5.0) 0.79 7 (4.5) 0.78 9 (7.0) 0.85 8 (5.0) 0.78 9 (6.7) 0.86 11 (7.8) 0.87 OarFCB11

11 (5.1) 0.80 8 (5.3) 0.81 11 (5.8) 0.83 9 (6.6) 0.85 9 (6.9) 0.85 12 (6.7) 0.85 TGLA13

9 (6.3) 0.83 7 (4.1) 0.71 9 (6.8)) 0.85 9 (4.6) 0.76 8 (4.6) 0.77 10 (6.9) 0.84 TGLA53

11 (7.7) 0.87 8 (5.5) 0.81 12 (7.5) 0.86 11 (7.5) 0.86 10 (6.2) 0.84 13 (8.3) 0.88 ADCYC

7 (3.6) 0.71 6 (4.6) 0.78 5 (3.8) 0.72 7 (3.4) 0.69 7 (3.6) 0.69 8 (3.8) 0.70 ILSTS002

10 (8.9) 0.87 8 (4.7) 0.79 9 (5.0) 0.80 10 (5.3) 0.80 7 (3.8) 0.72 12 (5.8) 0.84 MAF64

6 (3.5) 0.71 6 (3.1) 0.69 11 (5.9) 0.84 8 (4.0) 0.74 6 (3.9) 0.75 7 (3.8) 0.75 MAF65

14 (7.3) 0.86 11 (6.7) 0.84 13 (9.5) 0.90 11 (7.2) 0.86 13 (6.7) 0.85 16 (9.5) 0.89 BM1258

6 (5.1) 0.80 7 (5.3) 0.81 8 (5.4) 0.81 8 (4.4) 0.76 10 (6.4) 0.84 7 (5.3) 0.81

10 (5.4) 0.82 10 (4.4) 0.77 10 (5.5) 0.81 9 (5.9) 0.83 8 (6.3) 0.84 10 (5.5) 0.81

10 (4.1) 0.75 10 (6.8) 0.85 9 (4.0) 0.75 8 (4.4) 0.76 9 (5.2) 0.80 11 (7.4) 0.86

5 (3.1) 0.66 5 (4.4) 0.77 5 (3.6) 0.71 5 (2.5) 0.57 5 (3.0) 0.65 6 (3.6) 0.73

5 (2.9) 0.62 5 (4.0) 0.75 6 (4.1) 0.75 7 (3.8) 0.72 7 (3.3) 0.68 7 (4.9) 0.79

8 (3.9) 0.73 8 (5.0) 0.80 7 (4.0) 0.74 6 (3.2) 0.72 7 (3.5) 0.72 12 (5.1) 0.80

7 (5.4) 0.81 5 (4.7) 0.79 7 (2.9) 0.61 8 (4.3) 0.76 5 (3.3) 0.73 6 (3.3) 0.73

5 (3.2) 0.68 2 (1.8) 0.36 6 (2.6) 0.57 4 (2.8) 0.62 7 (4.0) 0.75 7 (4.3) 0.76

6 (3.6) 0.71 6 (3.1) 0.70 6 (3.9) 0.73 6 (4.3) 0.74 7 (3.3) 0.70 6 (4.0) 0.75

number of alleles (71%) being speci®c of a particular species. In the present study, Merinos showed a markedly higher number of speci®c alleles (15 variants concerning 12 loci) when compared with the other breeds (ranging from 1 to 5 alleles). The latter alleles might derive from relative recent mutations during the divergence process, according to the genetic relationships already indicated. The effective number of alleles (ne) represents the number of equally frequent variants that would give a PIC value. The comparison of this parameter with the number of observed alleles at each locus gives information about the predominance of certain alleles in each breed. Contrasts na/ne reveal some interesting aspects. For instance, Latxas displayed a low number of observed alleles at many loci, but they showed a different situation at some markers when ne was considered (sequences MAF65, ADCYC, MAF33 and MAF64). On the other hand Merinos, which

frequently exhibited the largest number of observed alleles, had sometimes considerably reduced ne values (loci MAF33, MAF36 and MAF64). Variation at microsatellite loci is assumed to be mainly determined by non-selective processes (with a few exceptions) since they are generally considered as evolutionary neutral. Thus, differences in allele frequencies among the breeds could be mainly attributable to founder effect, genetic drift, gene ¯ow and mutations that may originate new or already existent variants. Sample size (from 40 to 52 individuals) is considered representative in the analysis of microsatellite markers (Takezaki and Nei, 1996) and thus it probably does not account for allele frequency differences. 3.2. Analysis of loci All microsatellites were highly polymorphic as indicated by the total number of different alleles which was, decreasingly, as follows: 19 (MAF70); 15

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J.J. Arranz et al. / Small Ruminant Research 39 (2001) 3±10

Fig. 1. (A) Distributions of allele frequencies at microsatellite loci in six Spanish sheep: Churra (CH); Latxa (LX); Manchega (MA); RasaAragonesa (RA); Castellana (CA) and Merino (ME). *Alleles present exclusively in one breed.

J.J. Arranz et al. / Small Ruminant Research 39 (2001) 3±10

Fig. 1. (Continued ).

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(TGLA13, CSSM66); 14 (BM143, BM6444); 12 (MAF36, MAF64, CSSM6, TGLA53); 11 (OarFCB11, MAF33, BM4621, MAF48); 10 (MAF65, BM1258); 9 (ILSTS002) and 7 (ADCYC, OarCP34). High mutation rates account for the large variability microsatellites display. Their instability is partially explained by polymerase slippage mechanisms that tend to produce non-identical copies of repeated DNA sequences (SchloÈtterer and Tautz, 1992). Moreover, unequal recombination exchange between homologous chromosomes during meiosis seems also to contribute to the high microsatellite mutation rate. However, this high mutation rate is combined with a limited range of alleles. Thus, mutation may lead to the reappearance of lost alleles and as a consequence mutation could be a factor counteracting the diversifying effect of random drift (Nauta and Weissing, 1996). Other parameters also indicative of the genetic variation were estimated such as H and PIC. Values of H are included in Table 1 whereas PIC estimates, which showed a marked relationship with them, are not shown. Estimates of gene diversity (H) indicated that marker MAF70 showed the greatest variability (from 0.84 to 0.90) and BM1258 the lowest one (from 0.36 to 0.76). With respect to the polymorphism information content (PIC), all the loci may be considered as highly informative. Only in the case of microsatellite BM1258 in Latxas the PIC value was <0.5 (PICˆ0.30). Apart from this particular case PIC estimates ranged from 0.52 (at ADCYC in RasaAragonesas) to 0.89 (at MAF70 in Manchegas). The high gene diversities estimated are in accordance with expectations for microsatellite loci and particularly for sheep. In this regard, Crawford et al. (1998) compared sheep and cattle using microsatellites originated from both species, and found that markers polymorphic in both species had higher mean gene diversities and larger fragment sizes in sheep regardless of origin. They suggested that this result is consistent with the genetic diversity of sheep being probably higher than that of cattle. The complexity of the molecular basis of microsatellite evolution leads to large differences among loci and alleles regarding their degree of variation. Differences have been detected in mutation rates related to their motif sizes (di-, tri- or tetranucleotides) although a general pattern was not found (Weber and

Wong, 1993; Chakraborty et al., 1997). The loci analysed in our study were dinucleotide sequences, which according to Chakraborty et al. (1997) have mutation rates higher than the tri- and tetranucleotides (except for the disease causing tri-nucleotides). Moreover, several data in the literature suggest a correlation between mutation rate and the number of homogeneous repeats indicating a higher susceptibility of long homogeneous stretches to mutation events (Wierdl et al., 1997; Brinkmann et al., 1998). Goldstein and Clark (1995) found in Drosophila that perfect repeats are signi®cantly more variable than imperfect repeats, and Jin et al. (1996) concluded, using a phylogenetic approach in humans, that certain alleles lost their ability to mutate because of nucleotide substitutions that shorten the length of the uninterrupted repeat arrays. In the present study, most of loci analysed were perfect repeats and examination of measures of variability such as number of alleles and gene diversities did not allow a separation between both types of sequences. When a range of variation was established among all the loci analysed, the imperfect repeats were found among the loci showing largest (BM6444), intermediate (TGLA13, TGLA53 and CSSM6) or lowest variation (OarCP34). 3.3. Allele frequency distributions Fig. 1A and B reveal that a few markers (e.g. OarCP34, ADCYC and CSSM6) had distributions that did not markedly differ between breeds. For the cited loci a narrow range of variation was found in the number of observed alleles and no great difference was evident between populations regarding the predominant alleles. When this situation is found the most frequent alleles are likely to be the oldest, the others being the result of mutation process through insertion±deletion mechanisms (Chakraborty et al., 1991). On the other hand, MAF64 allele distribution is to be mentioned with an opposite pattern. For this particular marker (with a wide range of observed variants), the predominant alleles varied between breeds. The distribution of sequence BM1258 is also to be noted, with large differences between breeds with respect to the alleles identi®ed, but with the same allele showing a large frequency in all populations.

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Allele distributions also differ in the possible presence of predominant alleles. In this regard, we can point out loci such as MAF70 with a large number of alleles with low frequencies leading to ¯at distributions. In contrast, one or a few alleles were clearly predominant at the sequence BM1258. Another aspect to analyse is if the distribution shows some kind of asymmetry. In several cases, the most frequent alleles showed an intermediate size in most breeds (e.g. BM1258 and TGLA13), producing a centralised distribution. In other markers (e.g. MAF64, TGLA53, ILSTS002 and OarCP34) the variants with the greatest frequencies showed an extreme size. It is also to be noted that when the latter is the case, this effect is less important in Merino sheep, probably due to the larger number of alleles that this breed generally displays. However, in most cases a common shape was not found. Even when some kind of skewness was evident it did not follow a consistent pattern, either to the right or to the left, in all the populations. These results are in accordance with data previously reported for microsatellites. As an example, Forbes et al. (1995) analysed different domestic and bighorn sheep and found that distributions at some loci were highly centralised, while others were skewed, ¯at, or multimodal. Asymmetries of microsatellite mutation distributions have been found in different studies. Goldstein and Pollock (1997) reviewed data in the literature and indicated that a positive asymmetry (a tendency to mutate to alleles of larger size) was ®rst observed for large alleles at trinucleotide-expansion loci, but further investigations revealed asymmetry towards mutations that decrease size. They concluded that apart from differences among loci, allele size is an important factor in the determination of asymmetry effects. Another aspect that contributes to the irregularity of microsatellite distributions is the complex evolution at these loci. Although a simple stepwise mutation model (SMM) was primarily put forward to explain microsatellite evolution (Shriver et al., 1993; Valdes et al., 1993), an SMM which also includes multistep changes seems to be more suitable for microsatellites. Particularly single step mutations (those involving one repeat unit) seem to account for 90% of STR mutation events, followed by double-step mutations

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and a very limited number of multistep mutations (Weber and Wong, 1993; Di Rienzo et al., 1994). The latter authors found that this extended SMM produces a much larger variance of allele size when compared to the simple SMM. Moreover, Samadi et al. (1998) used a simulation approach to compare mutation including slippage only vs. slippage and unequal exchange of homologous chromosomes and found much wider distributions in the latter case. Finally, results of several studies suggest a different mutation model (either single-step or two-phase model) for distinct loci within the same organism (Di Rienzo et al., 1994; Estoup et al., 1995). All these data reveal the complexity of the molecular basis of microsatellite variation and explain the dif®culties in the establishment of consistent patterns when analysing different populations. Such was the case in our study, which revealed great inter-loci variability. The high level of genetic variation was evident in all the breeds studied, although Merino seemed to display larger variability and Latxa lower than the other Spanish sheep. Acknowledgements This work was supported by CICYT project grant No. AGF96-0819-CP. References Arranz, J.J., BayoÂn, Y., San Primitivo, F., 1998. Genetic relationships among Spanish sheep using microsatellites. Anim. Genet. 29, 435±440. Botstein, D., White, R.L., Skolnick, M., Davis, R.W., 1980. Construction of a genetic linkage map in human using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314±331. Boyce, W.M., Hedrick, P.W., Muggli-Cockett, N.E., Kalinowski, S., Penedo, M.C., Ramey, R.R., 1996. Genetic variation of major histocompatibility complex and microsatellite loci: a comparison in Bighorn sheep. Genetics 145, 421±433. Brinkmann, B., Klintschar, M., Neuhuber, F., HuÈhne, J., Rolf, B., 1998. Mutation rate in human microsatellites: in¯uence of the structure and length of the tandem repeat. Am. J. Hum. Genet. 62, 1408±1415. Buchanan, F.C., Adams, L.J., Littlejohn, R.P., Maddox, J.F., Crawford, A., 1994. Determination of evolutionary relationships among sheep breeds using microsatellites. Genomics 22, 397±403.

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