Isolation and Characterization of Microsatellite Markers in Tsaiya Duck

An enrichment library of GATA-repeats from genomic DNA was constructed in this study to isolate and characterize microsatellite loci in Tsaiya duck (Anas platyrhynchos). Thirty-three microsatellite markers were developed and used to detect polymorphisms in 30 Tsaiya ducks. A total of 177 alleles were observed and all loci except APT022 were polymorphic. The number of alleles ranged from 2 to 9 with an average of 5.5 per microsatellite locus. The observed and expected heterozygosity of these polymorphic markers ranged from 0.07 to 0.93 with an average number of 0.60 and 0.10 to 0.86 with an average number of 0.61, respectively. Among the polymorphic markers, the observed heterozygosities of 23 loci were higher than 0.50 (69.70%). The polymorphism information content (PIC) in the 32 loci ranged from 0.09 to 0.83 with an average of 0.57. Seven of the 33 duck microsatellite loci had orthologs in the chicken genome, but only APT004 had a similar core repeat to chickens. These microsatellite markers will be useful in constructing a genetic linkage map for the duck and a comparative mapping with the chicken can also provide a valuable tool for studies related to biodiversity and population genetics in this duck species. (


INTRODUCTION
Many important agricultural traits are quantitative traits controlled by multiple genes.The recent development of molecular genetic mapping tools has enabled the identification of quantitative trait loci (QTL) in the genome.The application of marker assisted selection for QTL has the potential to enhance the accuracy of animal breeding programs, particularly for traits that are difficult to improve through traditional selection methods (Meuwissen and Goddard, 1996).Microsatellites, also known as short tandem repeats (STR), are tandem repeated motifs of 1-6 bases.They are found abundantly and at random throughout most eukaryotic genomes (Stallings et al., 1991).Microsatellites are highly polymorphic and have become one of the most useful tools for population genetic studies, linkage mapping, parentage determination and QTL analysis.In chicken, swine and cattle populations a large number of microsatellites have been isolated and widely used for these purposes (Kong et al., 2006;Liu et al., 2006).In contrast, fewer genetic markers have been established in the duck and only a few articles have been published for some species including the Peking duck, eider duck, muscovy duck, mallard, white-headed duck, ruddy duck and musk duck (Maak et al., 2000;Maak et al., 2003;Paulus and Tiedemann, 2003;Stai and Hughes, 2003;Denk et al., 2004;Munoz-Fuentes et al., 2005;Guay and Mulder, 2005;Huang et al., 2005;Huang et al., 2006).Although the first duck genetic linkage map has been developed (Huang et al., 2006), it only spans 1,353.3cM with an average interval distance of 15.04 cM.More microsatellites are needed to establish a complete duck genetic map.We attempted to isolate microsatellite markers for the Tsaiya duck (Anas platyrhynchos) and investigated its polymorphisms.

Collection and extraction of Tsaiya duck genomic DNA
Thirty individuals (15 males and 15 females) were selected from a germplasm preservation population of Tsaiya ducks kept at the Ilan Branch since 1984.In the beginning, the population was reproduced via natural mating.At the 8 th generation, we randomly divided this germplasm preservation duckling population into 15-sire families.The rotational crossbreeding system was then used to avoid inbreeding.We randomly chose one male and one female as samples from each sire family from the 9 th generation.Genomic DNA was extracted from fresh blood using the GenoMaker kit (Watson BioTech, Taiwan) following the manufacturer's instructions.Briefly, 40 μl blood was lysed in 1 ml GenoMaker reagent and extracted with chloroform.DNA was precipitated with isopropanol and quantified using a spectrophotometer.

Construction of genomic DNA libraries enriched for microsatellites
The library was enriched for GATA repeats following a combination of modified procedures according to Hamilton et al. (1999) and Hsu et al. (2003).A pooled genomic DNA of 3 Tsaiya ducks was digested with AluI, HaeIII and RsaI.The fragments were then ligated with SNX linkers (Hamilton et al., 1999).The ligated products were amplified using PCR at 94°C for 5 min, 30 cycles of 94°C for 1 min,  55°C for 1 min and 72°C for 2 min, followed by a final cycle at 72°C for 7 min.The amplified products were used for subtractive hybridization with 3'-biotinylated (GATA) 10 oligonucleotides to select the microsatellite-containing DNA fragments.The biotin-labeled oligonucleotides were eluted using Dynabeads MyOne Streptavidin (Dynal, Norway) according to the manufacturer's protocol.Repeatenriched DNA was made double-stranded and amplified under the above described PCR conditions and then cloned in the pGEM-T Easy Vector (Promega, USA).After transformation into JM109 competent cells, 800 colonies containing inserts were lifted to Nylon membranes (Roche, Germany) and hybridized with 3'-DIG-labeled (GATA) 8 oligonucleotides.The positive colonies were cultured to extract their plasmids and then sequenced using the BigDye Terminator Kit on a 3730xl DNA Analyzer (Applied Biosystems).

Genotyping
Sequences were aligned with SeqWeb Version 2.1 (Wisconsin Package).The Primer Express software (Applied Biosystems) was used to design PCR primers.The primer pairs showing the expected PCR products were selected for polymorphism screening.The forward primers of these primer pairs were labeled with FAM or HEX fluorescent dye.PCR reaction mixtures (10 μl) containing 10 ng genomic DNA, 2.5 mM or 3 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl pH 8.3, 200 μM of each dNTP, 0.4 μM of forward and reverse primer and 0.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems/Roche) were prepared.After initial incubation at 95°C for 10 min, each PCR amplification was performed for 30 cycles of denaturing at 95°C for 20 s, annealing for 30 s at an appropriate annealing temperature (50 or 55°C) for each primer pair and extension at 72°C for 30 s.This was followed by a final cycle at 72°C for 1 h.The PCR products were analyzed in a MegaBACE 1000 auto sequencer (Amersham Biosciences).The sizes of the DNA fragments were investigated using the Genetic Profiler Version 2.2 software (Amersham Biosciences).

Statistics and similarity searching
The observed and expected heterozygosities and polymorphism information content (PIC) were calculated using the CERVUS 2.0 program (Marshall et al., 1998).
Hardy-Weinberg expectation and linkage disequilibrium tests were performed with the FSTAT 2.9.3.2 software (http://www2.unil.ch/popgen/softwares/fstat.htm).The sequences were analyzed using the BLAST program (NCBI) to identify the orthologous microsatellite duck DNA in the chicken genome.The unique match sequences with E-values smaller than e-20 from the chicken were used as orthologs to the duck microsatellite DNA.

RESULTS AND DISCUSSION
Eighty positive clones out of 800 colonies screened from the GATA-enriched genomic library were sequenced.There were 47 different loci from 75 sequences containing GATA repeats and 33 of these loci were chosen for further polymorphism tests in 30 Tsaiya ducks.After searching further with the BLAST program, we found that these 33 microsatellite Tsaiya duck loci were novel.The characteristics of the 33 microsatellite loci are summarized in Table 1.A total of 177 alleles were observed and all loci except APT022 were polymorphic.The number of alleles ranged from 2 to 9 with an average of 5.5 per microsatellite locus.The observed and expected heterozygosity of these polymorphic markers ranged from 0.07 to 0.93, with an average number of 0.60, and 0.10 to 0.86, with an average number of 0.61, respectively.Among the polymorphic markers, the observed heterozygosities of 23 loci were higher than 0.50 (69.70%).The polymorphism information content (PIC) of the 32 loci ranged from 0.09 to 0.83, with an average number of 0.57.Based on the classification of Botstein et al. (1980), twenty-one (65.63%) polymorphic markers were highly informative (PIC>0.50),seven (21.88%) were reasonably informative (0.50>PIC>0.25),and four (12.50%) were slightly informative (PIC<0.25).No locus showed significant deviation from Hardy-Weinberg equilibrium and there was no linkage disequilibrium among the loci (p>0.05).The orthologous microsatellite duck DNA in the chicken genome search results are presented in Table 2.Only APT004 out of the seven duck microsatellite loci with orthologs in the chicken genome had a similar core repeat.The others had different core repeats or were absent from the orthologous loci in the chicken genome.Values of 94.44% (Maak et al., 2003), 80.00% (Huang et al., 2005) and 77.89% (Huang et al., 2006) polymorphisms have been reported for duck-specific microsatellite markers tested in the duck genome.The higher (96.97%) polymorphism seen in this study could be a reflection of the test population genetic constitution, which was derived from a germplasm preservation population without artificial selection.Based on the PIC values, most polymorphic markers were highly or reasonably informative and only a few were slightly informative.Therefore, these markers will be very useful for mapping the duck genome.As the Chicken Genome Project moves toward functional genomics studies, the availability of the chicken genome sequence has proven to be an invaluable tool in studying the genomes of other avian species, including the duck.A good comparative genetic map based on the orthologous microsatellite markers will provide the substrates for major gene identification (Reed et al., 2005).After a similar BLAST search, 21.21% of the microsatellite loci were conserved between the duck and the chicken.This result was similar to a previous report (20.42%,Huang et al., 2006).In conclusion, these microsatellite markers will be useful in construction of a duck genetic linkage map.Comparative mapping with the chicken can provide a valuable tool for studies related to duck biodiversity and population genetics.
T a = Annealing temperature, H O = Observed heterozygosity, H E = Expected heterozygosity, PIC = Polymorphism information content.

Table 1 .
Characteristics of 33 novel microsatellite loci in the Tsaiya duck

Table 2 .
The orthologous microsatellite duck DNA in the chicken genome