Construction of Genetic Microsatellite Maps for Some Chromosomes in Chinese Swine Reference Population

In aiming to identify the genes or genetic regions responsible for quantitative traits, a swine reference population had been constructed using three Large White boars and seven Meishan dams as parents. Five F1 males and 23 F1 females were intercrossed to generate 147 F2 offspring. Thirty-one microsatellite markers covering Sus scrofa chromosomes (SSC) 2, 4, 6 and 7 were genotyped for all members. Construction of genetic microsatellite maps was performed using the CRIMAP software package. The lengths of these chromosomes were longer than MARC maps. They were 158.6cM, 180.3cM, 197.3cM and 171.4cM, respectively. A two modified orders of markers were observed for SSC6 and SSC7. The female map on SSC6 was shorter than male map, and the contrary was on SSC 2, 4 and 7. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 10 : 1386-1390)


INTRODUCTION
Most of important economic traits in farm animals are quantitative traits and controlled by quantitative trait loci (QTL).The development of molecular biology techniques and the application of these techniques to farm animals have progressed rapidly and given an opportunity for researchers wishing to identify genes that control quantitative traits.The construction of linkage map is the first step for detection of QTL (Haley et al., 1992).Until now, there were three linkage maps of genetic markers published, i.e. the MARC map (Rohrer et al., 1996), the PiGMaP consortium map (Archibald et al., 1995), and the SCAND map (Marklund et al., 1996).In aiming to identify the genes or genetic regions responsible for quantitative traits, our university has constructed a chinese swine reference family using Large White and Meishan pigs.These two breeds are quite different in traits such as growth and litter size.Recently, we acquired several sets of swine microsatellite primers from Dr. MF Rothschild lab, Iowa State University.Prior to the detection of QTL, in the present study we constructed genetic microsatellite maps for Sus scrofa chromosomes (SSC) 2, 4, 6, and 7 which were chosen as previous work had revealed QTL on these chromosomes affected growth and fat traits (Andersson et al., 1994;de Koning et al., 1999;Knott et al., 1998;Wang et al., 1998).

Animals
The three-generation pedigree used in this study comprised three Large White and seven Meishan founders.Five F 1 males and 23 F 1 females were intercrossed to generate 147 F 2 offspring.

Microsatellite marker genotyping
Thirty-one microsatellite markers were chosen from the genetic markers on linkage map reported by the MARC (Rohrer et al., 1996) in order to cover evenly the genetic maps of SSC 2, 4, 6 and 7.The microsatellite primers provided kindly by Dr. MF Rothschild were used to PCR.PCR performed in a 20 µl volume containing 50 ng template DNA, 30 µmol dNTP, 5pmol of each primer, 1U Taq DNA polymerase in standard Taq DNA polymerase buffer, and MgCl 2 concentration showed in Table 1.The amplification reactions were as follows: 5 min at 94°C, 35 cycles of 94°C for 30 sec, annealing temperature (see Table 1) for 30 sec, and 72°C for 30 sec, finally followed by an extension step of 94°C for 5 min.All the amplification products separated by PAGE in 8% gels and stained with silver (Bassam et al., 1991;Su et al., 2000).These gels were photographed and dried for permanent reservation.The heterozygosity and polymorphic information content were calculated according to Ott (1993).

Linkage analyses
Linkage analyses were performed by CRIMAP version 2.4 (Green et al., 1990).Using the BUILD option, a multipoint linkage map was constructed.The orders of these loci were checked using the FLIPS 2 to 5 procedure to test if the marker order needed to be revised.When all markers were included, the CHROMPIC option was used to identify unlikely crossover events.The recombination rates and marker distances were obtained using the FIXED procedure.

Microsatellite genotyping
The microsatellite marker names selected for genotyping and the number of alleles, size range, informative meioses, heterozygosity, and polymorphic information content (PIC) observed in this study are showed in Table 1.For most of microsatellite primers, annealing temperatures and MgCl 2 concentration were different from the conditions reported by papers (Ellegren et al., 1994;Alexander et al., 1996) or at world-wide web site (http://sol.marc.usda.gov).
Allele sizes of some microsatellites exceeded the size ranges reported before.For example, 7Sw352 was 143 to 191 bp in this study, while it was 149 to 179 bp at USDA site (http://sol.marc.usda.gov).Paszek et al. (1995) found that 6Sw1057 was 150 to 188bp in MARC map, 140 to 191bp in  PiGMaP map.The average informative meiosis for all microsatellite markers was 250.32, average heterozygosity 0.60, and polymorphic information content 0.56.According to these results, microsatellite markers in this study are highly polymorphic.

Microsatellite maps
Figure 1 presents the microsatellite maps of swine chromosomes 2, 4, 6, and 7. Genetic lengths of the sexaveraged maps are 158.6 cM, 180.3 cM, 197.3 cM, and 171.4 cM, respectively.Whereas the female maps were longer than the male maps on SSC 2, 4, and 7, the female map of SSC 6 was shorter than the male map.The difference between sex maps on SSC6 is small and not significant (p=0.2347) with χ 2 test using SAS (SAS, 1989).The orders of markers on chromosomes 2 and 4 were in agreement with maps published by Rohrer et al. (1996).There were differences in two regions between the present maps and the MARC maps on chromosomes 6 and 7.The log likelihood of the two alternative order of the markers were calculated in order to estimate the fidelity of the orders in this study.
The framework and comprehensive genetic linkage maps of porcine chromosome 6 have resulted from the first international effort to integrate genetic maps from multiple laboratories (Paszek et al., 1995).The comprehensive map on SSC6 is 166, 196 and 126 cM (for sex averaged, female and male maps, respectively).In this study, the sexaveraged map of chromosome 6 represented the marker order as S0121-SW607-SW322; whereas the MARC map had S0121-SW322-SW607.The log 10 likelihood for the present and MARC orders were -16.494 and -16.881, respectively.Based on these calculations, the present order of the markers was not indicated to be more likely than the MARC order, it is impossible to conclude that one of the two orders is more likely because of the fact that the likelihood values for the two orders are approximately the same.Mikawa et al. (1999) reported that marker order on chromosome 6 was SWR1130-SW855-RYR1; whereas on the MARC map it was SWR1130-RYR1-SW855.
On SSC7, a comprehensive linkage map is slightly longer than the skeletal map, at 153.3, 215.3 and 183.8 cM for sex averaged, female and male maps, respectively (Rohrer et al., 1997).In our study, the best order was SW2155-SW859-SW1856, which is different from the reported order: SW2155-SW1856-SW859.The log 10 likelihoods of the two different orders were -106.627 and -109.911.Based on these calculations, the present order of the markers was indicated to be more likely than the MARC order at least in our swine reference population of this study.The modified orders of markers for SSC7 were also reported by Rattink et al. (2000), either the p-arm or the q-arm.
The differences in the lengths of maps and marker orders are considered to be the result of differences in swine reference populations used to construction of linkage maps.The linkage maps in this study will lay the foundation for interval mapping of quantitative trait loci in this population.

Figure 1 .
Figure 1.Porcine genetic linkage map based on estimated sex-average and sex-specific maps distances.USDA-MARC sex-averaged, sex-averaged, female-specific, and male-specific maps are shown from left to right in each chromosome.Map distances between two markers, in Kosambi cM, are shown to the left or right of each map.

Table 1 .
PCR condition and characterization of microsatellite markers in this study