C. W. Huang¹, Y. S. Cheng², R. Rouvier³, K..T.Yang¹, C. P. Wu^1,4, and M. C. Huang^1,*

¹ Department of Animal Science, National Chung Hsing University, 250 Kuo-Kung Road, Taichung 40227, Taiwan

² Livestock Research Institute, Council of Agriculture, Hsin-Hua, Tainan 71210, Taiwan

³ Institut National de la Recherche Agronomique, Station d’Amélioration Génétique des Animaux, Centre de Recherches de Toulouse, BP 27, F31326 Castanet-Tolosan Cedex, France

⁴ Department of Animal Science, National Chiayi University. 300 Syuefu Road, Chiayi 60004, Taiwan

* Corresponding author, e-mail: mchuang@mail.nchu.edu.tw

ABSTRACT

The purpose of this study was to investigate the allele and genotype frequencies of ducks. Multicolour fluorescent microsatellite analysis in APH01, APH07, APH09 and APH14 DNA microsatellite loci was performed. A total of two hundred and five Brown Tsaiya ducks of a selected line (S) for the duration of fertility and one hundred and sixty eight ducks of the unselected control line(C), coming from the same base population, at the 10^th generation of the selection experiment, were used for the present study. The polymorphic PCR products were detected by capillary electrophoresis in ABI PRISM 3100 DNA genetic analyzer. The data were collected and analyzed by using GeneScan and Genotyper softwares. The frequencies of alleles and genotypes showed noticeable differences for APH01 except for APH07, APH09 and APH14 between lines. Five alleles and twelve genotypes of APH01 loci were all revealed in both lines. The frequencies of 198 alleles in S line were higher than the control in 0.46. The frequencies of 227 and 235 alleles in S line were lower than the control in 0.24 and 0.24, respectively. The genotype frequencies of 198/198 and 227/235 in S line were increased of 0.35 and decreased of 0.27, respectively. These results demonstrated that the frequencies of gene and genotype in S line of Brown Tsaiya ducks were changed after selection for the duration of fertility.

KEY WORDS: Brown Tsaiya duck, Genotype, Microsatellite, Multicolour fluorescent.

INTRODUCTION

In 1992, a selection experiment on the maximum duration of fertility in mule ducks was established by the Taiwan Livestock Research Institute (TLRI), under a cooperative research program with Station d’Amélioration Génétique des Animaux, Institut National de la Recherche Agronomique (INRA). A ten-generation selection experiment comprising of a selected (S) and a unselected control (C) line was conducted with the objective of increasing the number of fertile eggs in female Brown Tsaiya ducks after a single AI with pooled Muscovy semen. Both lines were consisted of about 20 males and 60 females as parents The purpose of this experiment was to measure the selection response on the number of fertile eggs in female Brown Tsaiya ducks following a single artificial insemination with pooled Muscovy semen (Cheng et al., 2002; Tai et al., 1994). The selected and control lines were built from the same Brown Tsaiya population (TLRI Line 105).

Microsatellites assignment has become a technique for molecular genetic selection (Vicente et al., 2004), evaluation (Huang et al., 2005) and linkage mapping (Jacobsson et al., 2004). By developing a polymerase chain reaction (PCR) utilizing primers designed to bind to regions flanking a microsatellite repeat, development of suitable and a sufficient number of microsatellite primers requires considerable skill (Matsumoto et al., 2004). The purpose of this study was to investigate the genetic characterization within and between the S and C lines of female Brown Tsaiya duck breeding populations using microsatellites to identify markers that could be used to genetically improve the maximum duration of fertility.

MATERIALS AND METHODS

The selection on the maximum duration of fertility for mule duck production was carried out by Cheng et al. (2002). Genomic DNA was extracted according to the methods described by Huang et al. (2003). Based on an earlier report on a microsatellite markers developed for the Pekin duck (Maak et al., 2000), four microsatellite loci, APH01, APH07, APH09 and APH14, related with polymorphisms were selected for this study (Huang et al., 2004). All amplifications were resolved and separated on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The data were collected and analyzed using ABI PRISM GeneScan 3.7 and Genotyper 3.7 software. All amplified DNA fragments between 102 and 235 bp were selected for data analysis. The gene and genotype frequencies were estimated.

RESULTS AND DISCUSSION

The detected polymorphisms were automatically labeled and then checked manually on electropherograms. The polymorphic peaks seen on the electopherograms are due to different numbers of repeats in the loci (Shimizu et al., 2002). Differences of allele and genotype frequencies between the two lines were observed for APH01 except for APH07, APH09 and APH14 loci. The distribution of different genotypes frequency with APH01 microsatellite locus in the different lines of female Brown Tsaiya ducks was shown in Figure 1.

The 196, 198, 200, 227 and 235 alleles of APH01 microsatellite DNA loci were detected in S and C lines of Brown Tsaiya ducks. All of the twelve genotypes revealed in two lines of ducks, embracing five genotypes of homozygosity 196/196, 198/198, 200/200, 227/227 and 235/235. The frequencies of 196/198, 196/235, 198/198, 198/200, 198/227 and 200/200 genotypes of APH01 locus were greater in the S line. Lower frequencies of 196/196, 196/200, 198/235, 227/227, 227/235 and 235/235 were found in the S line. The frequencies of 198 and 200 alleles in S line were higher than the control in 0.46 and 0.04, respectively. The frequencies of 227 and 235 alleles in S line were lower than the control in 0.24 and 0.24, respectively. The frequency of 198/198 genotype in S line was increased 0.35. The frequency of 227/235 genotype in S line was decreased 0.27. It was indicated that the frequencies of gene and genotype in S line of Brown Tsaiya ducks were changed after selection for the duration of fertility.

These results demonstrated that the high level polymorphisms of microsatellite loci were found between duck populations. In accordance with investigations, our results indicated that, in general, the variability in microsatellite DNA loci is much greater than that of traditional genetic markers based on unique-sequence DNA mutations (Kaiser et al., 2000). The differences in allele and genotype frequencies between the selected and control line could be explained by random drift and by effect of the selection. The management of the control line had controlled the increase of inbreeding at a minimum (Cheng et al., 2002) and the random drift variance of the frequencies could be assumed to be low. In order to further investigate the selection effect on the allele and frequency differences, their evolution across the generations of selection should be studied.

Figure 1. The distribution of different genotypes of APH01 microsatellite locus in the different lines of female Brown Tsaiya ducks.

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