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Attribution| 4.0 International LicenseDuck (Anas platyrhynchos) linkage mapping by AFLP fingerprinting
Chang-Wen Huang, Yu-Shin Cheng, Roger Rouvier, Kuo-Tai Yang, Chean-Ping Wu, Hsiu-Lin Huang, Mu-Chiou Huang
To cite this version:
Chang-Wen Huang, Yu-Shin Cheng, Roger Rouvier, Kuo-Tai Yang, Chean-Ping Wu, et al.. Duck
(Anas platyrhynchos) linkage mapping by AFLP fingerprinting. Genetics Selection Evolution, BioMed
Central, 2009, 41, online (march), Non paginé. �10.1186/1297-9686-41-28�. �hal-02663467�
Open Access
Research
Duck (Anas platyrhynchos) linkage mapping by AFLP fingerprinting Chang-Wen Huang
1,2, Yu-Shin Cheng
3, Roger Rouvier
4, Kuo-Tai Yang
1,5, Chean-Ping Wu
1,6, Hsiu-Lin Huang
1and Mu-Chiou Huang*
1Address: 1Department of Animal Science, National Chung Hsing University, 250 Kuo-Kung Road, Taichung 402, Taiwan, 2Institute of Cellular and Organism Biology, Academia Sinica, 128 Section 2, Academia Road, Nankang, Taipei 115, Taiwan, 3Livestock Research Institute, Council of Agriculture, Hsin-Hua, Tainan 712, Taiwan, 4Institut National de la Recherche Agronomique, Station d'Amélioration Génétique des Animaux, Centre de Recherches de Toulouse, BP52627, F31326 Castanet-Tolosan Cedex, France, 5Institute of Biomedical Sciences, Academia Sinica, 128 Section 2, Academia Road, Nankang, Taipei 115, Taiwan and 6Department of Animal Science, National Chiayi University, 300 Syuefu Road, Chiayi 600, Taiwan
Email: Chang-Wen Huang - amino0116@yahoo.com.tw; Yu-Shin Cheng - yushin@mail.tlri.gov.tw;
Roger Rouvier - rouvier@germinal.toulouse.inra.fr; Kuo-Tai Yang - ktyang@ibms.sinica.edu.tw; Chean-Ping Wu - wucheanp@yahoo.com.tw;
Hsiu-Lin Huang - hlhuang2001@yahoo.com; Mu-Chiou Huang* - mchuang@mail.nchu.edu.tw
* Corresponding author
Abstract
Amplified fragment length polymorphism (AFLP) with multicolored fluorescent molecular markers was used to analyze duck (Anas platyrhynchos) genomic DNA and to construct the first AFLP genetic linkage map. These markers were developed and genotyped in 766 F2 individuals from six families from a cross between two different selected duck lines, brown Tsaiya and Pekin. Two hundred and ninety-six polymorphic bands (64% of all bands) were detected using 18 pairs of fluorescent TaqI/
EcoRI primer combinations. Each primer set produced a range of 7 to 29 fragments in the reactions, and generated on average 16.4 polymorphic bands. The AFLP linkage map included 260 co- dominant markers distributed in 32 linkage groups. Twenty-one co-dominant markers were not linked with any other marker. Each linkage group contained three to 63 molecular markers and their size ranged between 19.0 cM and 171.9 cM. This AFLP linkage map provides important information for establishing a duck chromosome map, for mapping quantitative trait loci (QTL mapping) and for breeding applications.
Introduction
Amplified fragment length polymorphism (AFLP) is an application of the DNA fingerprinting technique pro- posed by Vos et al. [1], which is a clever combination of two older methods, restriction fragment length polymor- phism (RFLP) [2] and random amplified polymorphic DNA (RAPD) [3-5], generating a large number of genetic markers from any genomic DNA [6]. AFLP markers are inherited in a Mendelian fashion and can be detected as co-dominant markers [7]. Since Ajmone-Marsan et al. [8], several studies have shown that AFLP markers follow
Mendelian inheritance rules and that the technique is highly reproducible, powerful and efficient [9]. Thus AFLP analysis is a useful tool to generate linkage maps [10].
Linkage maps using AFLP, microsatellite or SNP markers have been established and applied extensively to linkage studies or quantitative trait locus (QTL) mapping in ani- mals such as rats [11], rabbits [12], goats [13], sheep [14], cattle [15], chickens [16-20], turkeys [21], quails [22,23], and fish [24,25]. They have also been much used for genome mapping, studies on disease resistance and drug
Published: 17 March 2009
Genetics Selection Evolution 2009, 41:28 doi:10.1186/1297-9686-41-28
Received: 7 March 2009 Accepted: 17 March 2009 This article is available from: http://www.gsejournal.org/content/41/1/28
© 2009 Huang et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genetics Selection Evolution 2009, 41:28 http://www.gsejournal.org/content/41/1/28
tolerance in economic crops and other experimental plants such as sorghum [26], Arabidopsis thaliana [27], rice [28], corn [29], barley [30] and wheat [31].
Ducks are appreciated for meat and eggs. Research on duck genetics and breeding has been developed only in recent years [32]. For detecting and mapping QTL, the construction of a genetic linkage map is a prerequisite and in duck genetic map data are very limited. Huang et al.
[33] have reported a preliminary genetic linkage map in an inbred Pekin ducks resource population using micros- atellite markers. The advantage of AFLP is that a large number of markers can be generated with a smaller number of primer pairs than required when using micro- satellites. This is especially true when working in a species for which only few microsatellite markers are available. A large number of microsatellite markers may be obtained if enough time and financial support are available. In this study, we have chosen the AFLP technique to develop a duck genetic map. We have used the TaqI/EcoRI restriction enzyme combination and selective PCR primers to gener- ate molecular genetic markers and to establish a duck genetic linkage map from a resource population originat- ing from a cross between two outbred selected lines of lay- ing and meat type ducks. This is a first step to provide vital information to construct chromosome maps and map QTL for future applications in duck breeding.
Methods
Animals and blood collection
All ducks tested in the study originate from the Livestock Research Institute, Council of Agriculture (LRI-COA). In the first generation F0, each of three brown Tsaiya drakes and three Pekin drakes were mated either to two Pekin ducks or to two brown Tsaiya female ducks, respectively.
Six F1 drakes originating from the six F0 sires were mated individually, according to the mating plan, with three (one case) or six (five cases) unrelated F1 dams that were daughters of one F0 drake of the same breed brown Tsaiya or Pekin. F2 birds belonging to six half-sib families were used as the mapping population. The number of birds in the resource population was as follows: six males and 12 females in the F0, six males and 33 females in the F1 and 766 males and females in the F2. A total of 766 F2 animals were genotyped. Blood samples obtained from the vein of the ducks wings were carefully mixed with anticoagulant and kept at 4°C for subsequent DNA extraction.
Genomic DNA extraction
DNA extraction procedures were performed according to the method described by Huang et al. [34]. Eighty μL of each blood sample were mixed thoroughly with 1 mL of TNE buffer solution (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA pH 8.0) in a 1.5 mL centrifuge tube and centrifuged at 1,500 × g (Hermle Model Z233 MK,
Maryland, USA) for 5 min to wash the cells. They were then resuspended in 300 μL 10% NH4Cl, 75 μL proteinase K (10 mg/mL), 25 μL collagenase (3.8 IU/μL), and 200 μL 10% w/v SDS and the mixture was incubated at 42°C for 24 h, with agitation. A series of extractions was performed with a same volume of phenol, phenol/chloroform (con- taining 1/25 v/v isoamyl alcohol), and chloroform, respectively. Centrifugation conditions were 3,000 × g (Model SCT5B, HITACHI) for 10 min, then samples were precipitated with isopropanol. Excess isopropanol was removed using 70% ethanol. The DNA was vacuum-dried (Speed Vac® SC110, Rotor RH 40-11, SAVANT) and resus- pended in double distilled water. The DNA was quantified with an S2000 UV/Vis Diode-Array Spectrophotometer (WAP Co. Ltd., Cambridge, UK) to determine its absorb- ance and to confirm DNA purity and concentration for AFLP analysis.
Analysis of genotypes using AFLP markers
AFLP analysis was carried out according to the procedures described by Vos et al. [1]. All sequences for the EcoRI and TaqI adapters and primers used in this study are shown in Table 1. Briefly, 400–500 ng of genomic DNA (50 ng/μL) was digested with 0.5 μL EcoRI restriction endonuclease (20 U/μL) with 1 μL of 10× EcoRI buffer (50 mM NaCl, 100 mM Tris-HCl, 10 mM MgCl2, 0.025% Triton X-100, pH 7.5) (New England BioLabs® Inc., Ipswich, MA, USA) in a total volume of 10 μL. The mixture was incubated at 37°C for 4 h and then at 65°C for 10 min. Subsequently, the sample was digested with 0.5 μL TaqI restriction endo- nuclease (20 U/μL) with 1.5 μL of 10× TaqI buffer (100 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 pH 8.4) (New England BioLabs® Inc., Ipswich, MA, USA), then mixed with 0.15 μL of 100× BSA in a total volume of 15 μL and incubated at 65°C for 4 h with a last step at 80°C for 10 min. Adaptor ligation was performed by adding 1 μL of TaqI-adaptor (50 ng/μL), 0.1 μL of EcoRI-adaptor (50 ng/
μL), 1 μL of T4 DNA ligase (1 U/μL) and 5 μL of 5× ligase buffer (250 mM Tris-HCl pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% polyethylene glycol-8000) (Invitro- gen Co., Carlsbad, CA, USA). The mixture was made up to 25 μL with double-distilled water and incubated at 23°C for 12 h. DNA pre-amplification was performed in a GeneAmp® PCR system 2700 thermocycler (Applied Bio- systems, Singapore) with a final volume of 20 μL contain- ing 6 μL of the DNA sample, 1 μL of TaqI+A primer (50 ng/μL), 1 μL of EcoRI+A primer (50 ng/μL), 1.6 μL of 2.5 mM dNTPs, 0.25 μL of DyNAzyme™ DNA polymerase (2 U/μL, F-501L, Finnzymes Oy, Espoo, Finland), and 2 μL of 10× PCR buffer (100 mM Tris-HCl pH 8.8, 15 mM MgCl2, 500 mM KCl, 1% Triton X-100). The following PCR conditions were used: a denaturing step for 5 min at 94°C, 20 cycles at 94°C for 30 s, 56°C for 1 min and 72°C for 1 min and a final extension step at 72°C for 5 min. A second PCR reaction was performed in a final
volume of 5 μL containing 0.5 μL of the pre-amplification PCR products, 1 μL of TaqI+ANN selective primer (50 ng/
μL), 1 μL fluorescent dye-labeled EcoRI+ANN selective primer (50 ng/μL) with either VIC (green), NED (yellow), PET (red) or FAM (blue) 0.3 μL of 2.5 mM dNTPs, 0.25 μL of DyNazyme™ DNA polymerase (2 U/μL), and 0.5 μL of 10× buffer (10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl). Conditions for the selective amplification PCR are shown in Table 2.
Equal volumes of each of the four PCR products with dif- ferent color fluorescent markers (either VIC, NED, PET or FAM) were combined, diluted and mixed with double- distilled water and mixed. Then, 1 μL of the diluted PCR product mixture was added to 0.2 μL of GeneScan-500 LIZ internal lane size standard (Applied Biosystems, Foster City, CA, USA) and 10.8 μL of deionized formamide, denatured for 3 min at 94°C and immediately after placed
on ice for 5 min. Capillary electrophoresis was performed on an ABI PRISM® 3100 Avant Genetic Analyzer using the GS STR POP-6 F module column (Applied Biosystems, Foster City, CA, USA). Fluorescent peak signals for each primer combination were collected with the ABI PRISM® 3100 Genetic Analyzer Data Collection 1.1 (Applied Bio- systems, Foster City, CA, USA). The resulting genotyping data were scanned and analyzed with the software ABI PRISM™ GeneScan 3.7 and Genotyper 3.7 software pack- age (Applied Biosystems, Foster City, CA, USA), which displayed the AFLP fingerprints and quantified the poly- morphic peaks. AFLP markers were named according to the serial number based on the extension sequence of TaqI and EcoRI primer combination (Table 3) and to the size of the fragment in base pairs. Polymorphic markers from duck individuals belonging to the same family were scored according to the different heights and distributions of peak signals using the Genotyper software.
Table 1: Sequences of adapters and primers used in the AFLP detection
Name Sequence
Adapter EcoRI
Eco Top Strand 5-CTCGTAGACTGCGTACC
Eco Bottom Strand 5-AATTGGTACGCAGTCTAC
Adapter TaqI
Taq Top Strand 5-GACGATGAGTCCTGAC
Taq Bottom Strand 5-CGGTCAGGACTCAT
Primer EcoRI
EcoR+A 5-GAC TGC GTA CCG TAC CA
E1 VIC-EcoR+AAA 5-GAC TGC GTA CCG TAC CAA A E2 NED-EcoR+AAC 5-GAC TGC GTA CCG TAC CAA C E3 PET-EcoR+AAG 5-GAC TGC GTA CCG TAC CAA G E4 FAM-EcoR+ACA 5-GAC TGC GTA CCG TAC CAC A
E5 VIC-EcoR+AC 5-GAC TGC GTA CCG TAC CAC
E6 FAM-EcoR+AG 5-GAC TGC GTA CCG TAC CAG
Primer TaqI
Taq+A 5-GAT GAG TCC TGA CCG AA
T1 Taq+AAC 5-GAT GAG TCC TGA CCG AAA C
T2 Taq+AAG 5-GAT GAG TCC TGA CCG AAA G
T3 Taq+AAT 5-GAT GAG TCC TGA CCG AAA T
T4 Taq+ACA 5-GAT GAG TCC TGA CCG AAC A
T5 Taq+AC 5-GAT GAG TCC TGA CCG AAC
T6 Taq+AG 5-GAT GAG TCC TGA CCG AAG
Table 2: Conditions of selective amplification PCR
Hold Cycle Number of cycles
94°C, 5 min 94°C, 30 s 66°C, 30 s 72°C, 1 min 2
- 94°C, 30 s 64°C, 30 s 72°C, 1 min 2
- 94°C, 30 s 62°C, 30 s 72°C, 1 min 2
- 94°C, 30 s 60°C, 30 s 72°C, 1 min 2
- 94°C, 30 s 58°C, 30 s 72°C, 1 min 2
- 94°C, 30 s 56°C, 30 s 72°C, 1 min 25
- - - 72°C, 5 min 1
4°C, forever - - - 1
Genetics Selection Evolution 2009, 41:28 http://www.gsejournal.org/content/41/1/28
Construction of linkage maps
Each polymorphic marker was analyzed by Chi-square tests. Markers heterozygous in both F1 parents and signif- icantly (P = 0.05) fitting a 1:2:1 ratio (Mendelian inherit- ance) with the ratio of the numbers of individual genotypes A, H and B, were counted. Linkage analysis was performed by CarteBlanche software (Keygene, Wagenin- gen, Netherlands) following the instructions of the man- ufacturer. Briefly, each F2 genotype data from every family was imported. Linkage groups were constructed by the 'linkage phase establishment' function, calculating the recombination frequency (θ) between pairs of markers and the decimal logarithm of the odds ratio score (LOD score). Significant linkage was defined by a LOD score ≥ 3.0. Map distances were calculated according to the Kosambi mapping function. The linkage maps were drawn by MapChart 2.2 [35] and denominated in accord- ance to the calculated length orders of linkage groups.
Results
Polymorphisms of fluorescent markers
The number and the size range of the detected AFLP poly- morphisms are shown in Table 3. Two hundred and ninety-six polymorphic markers (64% of all peaks) were produced. Each primer pair produced between seven and 29 polymorphic markers (16.4 markers on average). This indicated that multicolor fluorescence detection with AFLP markers is a high throughput, timesaving and easily analyzed DNA fingerprinting technique. It can be applied
to investigate genetic linkage and polymorphism in a pop- ulation.
Linkage mapping
Histograms, created by ABI PRISM™ Genotyper 3.7 of sig- nal heights from an AFLP marker, are shown in Figure 1 and can be classified into three genotypes: homozygous present (A), heterozygous (H) and homozygous absent (B). Genotype data that were missed or could not be scored are indicated as genotype (U). After polymorphism analysis and χ2 tests, 281 AFLP markers obtained from the genomic DNA of six duck families could be used for link- age analysis. Phases of all the linkage group markers were established by the 'linkage phase establishment' function in the CarteBlanche software (Keygene, Wageningen, Netherlands). Calculating recombination frequencies (θ), LOD scores and map distances for markers in each linkage group provided an optimum order of markers. Then, link- age maps were constructed using MapChart 2.2 [35] and they were denominated according to the calculated length orders of the linkage groups. Figure 2 shows the linkage group maps comprising 260 markers placed in 32 linkage groups. Twenty-one markers were not linked with any other marker. The number of markers in each linkage group ranged between three and 63 with 11 major groups containing 7 to 63 markers and 21 minor groups contain- ing three to four markers. One hundred and fifty-seven of the mapped markers (60%) originated from seven linkage groups containing 10 to 63 markers. The lengths of the
Table 3: Number of detected polymorphisms per primer pair 3' end extensions of EcoRI and TaqI primers are shown; EcoRI primers are fluorescently labeled
Primer extensions1 Nb of peaks Polymorphic markers Mapped marker
TaqI EcoRI, labeled No. % No. % Size range of peaks (bp)
AAC AAA, VIC 33 20 61 18 90 61–399
AAC AAC, NED 28 19 68 13 68 60–260
AAC AAG, PET 15 9 60 8 89 84–282
AAC ACA, FAM 34 24 71 21 88 91–467
AAG AAA, VIC 36 22 61 18 82 41–261
AAG AAC, NED 14 9 64 8 89 61–205
AAG AAG, PET 17 11 65 11 100 45–195
AAG ACA, FAM 21 13 62 13 100 46–349
AAT AAA, VIC 41 25 61 21 84 44–325
AAT AAC, NED 12 8 67 7 88 52–216
AAT AAG, PET 16 9 56 7 78 108–282
AAT ACA, FAM 29 18 62 17 94 91–-239
ACA AAA, VIC 27 20 74 19 95 39–354
ACA AAC, NED 23 14 61 12 86 39–284
ACA AAG, PET 19 13 68 10 77 41–233
ACA ACA, FAM 13 7 54 7 100 81–283
AC AC, VIC 42 26 62 23 88 46–349
AG AG, FAM 45 29 64 27 93 56–382
465 296 64 260 88
1 Sequence of the two or three selective nucleotides at the 3' end of the AFLP primer
linkage groups varied between 19.9 and 171.9 cM. The total length of the map was 1,766 cM, with an average interval distance of 7.75 cM between two consecutive markers, the spacing between adjacent markers ranging from 0.0 cM to 33.3 cM. The results of the marker density analysis showed that the linkage group LG-1 had the high- est density with 63 markers for 171 cM, whilst the LG-11 linkage group had the lowest density with three markers for 61.4 cM.
Discussion
One purpose of the resource population produced in this work was to generate individuals with a maximum of het- erozygous markers in its F1 generation. This resource pop- ulation originated from a cross between two genetically
different lines: a laying brown Tsaiya line selected for long duration of fertility [36,37] and a Pekin duck line selected as grand parent to produce mule ducks for roasting. Six F1 drakes from the six F0 sires were each mated with three (one case) or six (five cases) unrelated F1 dams, which were daughters of one F0 drake of the same breed brown Tsaiya or Pekin. Using AFLP markers to screen genotypes on every F2 individual from each family, we found that 281 markers (60% of all bands) conformed to Mendelian segregation. These genotype results demonstrate that ped- igree information from integrated family generations is important for scoring AFLP marker genotypes. In this duck population, we observed very little segregation dis- tortion and genotyping errors. These results show also that AFLPs can be scored as bi-allelic co-dominant mark- Histogram created by ABI PRISM™ Genotyper 3.7 of signal heights from an AFLP marker in 179 F2 ducks from a single half-sib family
Figure 1
Histogram created by ABI PRISM™ Genotyper 3.7 of signal heights from an AFLP marker in 179 F2 ducks from a single half-sib family. Three categories are manually defined, displaying signals characterized as genotype (B) when the marker is homozygous absent, genotype (H) when the marker is heterozygous, and genotype (A) when the marker is homozygous present. Signals outside the categories are characterized as genotype (U).
Genetics Selection Evolution 2009, 41:28 http://www.gsejournal.org/content/41/1/28
ers in ducks, increasing the information content when compared to bi-allelic dominant markers and facilitating linkage and QTL analyses.
Using primer combinations labeled with multicolor fluo- rescent dyes and a fragment scanning system from ABI PRISM® 3100 Avant Genetic Analyzer, it will be possible to greatly increase the quantity and density of markers in a linkage group to build more detailed and better integrated genetic linkage maps. Due to the GC rich and gene-dense nature of bird microchromosomes [38,23], double diges- tion with EcoRI and TaqI restriction enzymes was per- formed. The sequences of adapters and primers (Table 1) and the conditions of selective amplification PCR (Table 2) were designed and adapted according to the method described by Herbergs et al. [19]. The average number of polymorphic fragments generated by each primer pair was
8.5 [19], 10.5 [20] in chickens and 18 in quails [23]. Our results indicate that in duck the average number of frag- ments is 16.4. This discrepancy may be due to species dif- ferences and to differences in the selection of primer combinations. The present results demonstrate that AFLP can produce a large amount of polymorphic markers in duck genomic DNA (Table 3). Therefore, AFLP markers are useful for linkage analysis in ducks.
For a given number of informative meiosis, the higher the LOD score, the closer the distance between two markers, which means there is a high probability that the two markers are located in the same linkage group. The map is relatively dense with an average interval distance between adjacent markers of 7.75 cM. The large number of chro- mosomes (2n = 80) and especially the presence of micro- chromosomes [39], make it difficult to build an AFLP genetic linkage map of the ducks
Figure 2
AFLP genetic linkage map of the ducks. Two hundred and sixty of the markers were assigned to 32 linkage groups in six families by CarteBlanche linkage software. Map distances (centimorgan, cM) were indicated to the left of the maps and calcu- lated using the Kosambi mapping function. The names of the markers are indicated to the right of the maps.
LG-5
T4E1-206 0.0
T1E4-150 12.5
T1E2-238 T4E1-079 15.8
T4E1-064 24.0
T2E3-195 28.1
T1E3-117 36.5
T1E1-061 45.9
T3E2-117 48.2
T6E6-254 51.5
T2E1-078 59.4
T2E3-141 66.8
T3E4-195 71.7
LG-6
T2E4-080 0.0
T2E2-205 8.9
T4E4-107 25.0
T4E4-230 31.0
T6E6-119 39.6
T3E4-209 48.8
T3E3-145 56.4
T1E1-255 64.4
T2E1-174 67.8
T5E5-123 71.3
LG-7
T4E1-141 0.0
T1E1-201 27.6
T2E1-152 52.3
T6E6-247 68.1
T6E6-264
LG-8T3E4-154
0.0
T2E3-062 1.6
T4E2-204 6.5
T1E4-154 13.3
T2E3-045 13.4
T3E3-113 17.0
T1E2-207 26.1
T4E2-114 36.7
T1E3-176 40.2
T3E1-202 49.9
T3E3-108 54.1
T3E1-132 58.7
T3E3-282 67.1
T4E2-284 67.4
T4E2-060 67.9
LG-1
T1E1-147 T4E3-064 0.0
T4E3-091
8.0 T1E4-270 T2E3-048
10.7
T1E4-265 T3E1-166 T3E4-181 18.2
T1E1-115 26.2
T4E2-046 26.3
T2E2-072 T4E1-238 27.5
T1E4-115 T2E2-075 T1E2-122 36.1
T1E1-114 44.2
T2E1-050 T3E1-061 T4E1-185 52.9
T1E1-271 T2E2-159 61.0
T2E1-128 T3E2-116 68.2
T1E4-180 T2E1-041 T2E4-046 76.3
T2E1-055 T5E5-209 78.7
T4E1-354 79.7
T5E5-076 81.4
T5E5-110 83.1
T4E1-324 84.1
T4E3-041 84.7
T3E1-112 T1E4-195 91.7
T1E1-230 T1E4-197 T2E4-189 100.6
T2E4-194 107.3
T2E4-125 112.2
T2E3-113 T3E1-044 119.2
T4E1-165 120.3
T3E1-163 122.1
T2E3-115 T2E3-053 T3E4-091 126.3
T5E5-171 132.9
T5E5-126 137.7
T3E1-215 T5E5-162 143.4
T3E1-247 T3E2-052 151.0
T1E2-216 T1E4-178 159.2
T1E4-161 159.3
T4E2-245 T4E1-089 163.4
T1E4-127 T1E4-209 166.2
T3E3-117 T1E2-111 169.9
T3E1-088 171.9
LG-2
T1E2-260 T5E5-185 0.0
T3E4-092
11.2 T4E1-292 T3E2-216
14.3
T6E6-276 19.6
T3E3-236 24.5
T1E3-107 25.4
T2E3-168 T3E4-116 30.9
T4E2-158 T4E3-137 34.0
T2E4-171 43.2
T1E3-212 44.5
T3E1-127 50.3
T2E1-065 53.6
T1E1-125 T2E4-058 58.6
T3E4-226 64.4
T5E5-065 68.6
T2E2-061 71.7
T4E1-113 71.9
T3E4-127 76.6
T1E4-132 81.0
LG-3
T3E4-150 0.0
T2E4-261 3.4
T2E4-118 9.1
T1E2-084 T2E4-349 16.4
T2E1-141 22.7
T3E1-258 27.2
T1E1-096 31.3
T4E1-039 33.3
T3E4-180 40.4
T1E2-069 40.5
T3E1-162 44.5
T1E1-144 49.3
T5E5-050 53.6
T3E1-156 59.0
T2E1-173 T4E1-045 64.5
T2E1-257 69.1
T1E1-110 T3E2-100 78.5
LG-4
T4E1-107 0.0
T1E2-060 T2E1-261 17.0
T1E1-399 28.6
T4E2-039 41.4
T2E1-206 50.0
T1E4-288 64.1
T2E1-134 68.0
T4E2-124 75.3
LG-9
T2E4-278 0.0
T2E4-066 6.7
T6E6-153 18.8
T4E3-216 21.2
T1E1-243 34.9
T1E3-084 40.3
T2E1-096 56.6
T4E2-055 57.4
T1E4-219 66.3
LG-10
T2E1-061 0.0
T2E3-092 7.0
T2E4-256 9.7
T1E3-183 18.6
T6E6-199 28.6
T1E4-166 38.7
T1E1-087 45.9
T5E5-058 50.2
T3E1-100 53.3
T4E1-116 59.8
T4E1-051 66.1
LG-11
T1E1-138 0.0
T3E1-113 30.4
T3E3-152 61.4
LG-12
T4E1-061 0.0
T1E4-225 7.8
T1E4-467 T3E1-122 16.3
T5E5-193 19.7
T2E3-100 34.2
T5E5-230 47.0
T5E5-221 53.7
T5E5-218 60.4
LG-13
T1E1-215 0.0
T4E3-197 6.2
T3E1-189 17.8
T3E4-187 31.5
T6E6-056 38.6
T6E6-142 51.4
T3E2-201 60.1
LG-14
T6E6-205 0.0
T6E6-107 25.6
T5E5-178 52.5
LG-15
T3E4-175 0.0
T5E5-063 18.7
T6E6-287 42.4
T3E1-325 51.5
LG-16
T1E1-298 0.0
T5E5-349 33.1
T5E5-292 41.9
T5E5-264 49.4
LG-17
T5E5-212 0.0
T3E2-187 26.6
T6E6-168 37.2
T2E1-156 47.9
LG-18
T4E3-233 0.0
T4E3-152 25.2
T2E2-150 35.8
T2E2-103 45.2
LG-19
T1E4-116 0.0
T2E2-173 33.3
T6E6-228 44.0
LG-20
T3E1-207 0.0
T6E6-093 32.9
T5E5-046 42.9
LG-21
T2E1-124 0.0
T3E4-138 33.0
T3E4-132 42.5
LG-22
T1E4-383 0.0
T6E6-239 33.2
T4E3-202 41.9
LG-23
T4E2-176 0.0
T3E4-107 32.9
T6E6-130 41.9
LG-24
T4E2-129 0.0
T6E6-083 31.5
T6E6-317 41.7
LG-25
T3E4-239 0.0
T6E6-296 30.9
T1E2-136 41.5
LG-26
T6E6-133 0.0
T5E5-271 31.3
T6E6-061 41.2
LG-27
T4E4-156 0.0
T6E6-076 30.8
T1E2-182 40.4
LG-28
T4E1-123 0.0
T6E6-174 30.8
T3E1-210 40.3
T4E4-201 0.0
T6E6-341 29.6
T4E4-283 39.8
LG-29
T4E3-114 0.0
T6E6-222 10.6
T1E2-081 21.0
T1E3-282 21.5
LG-30
T4E4-122 0.0
T6E6-382 10.9
T1E4-091 20.3
LG-31
T1E3-192 0.0
T4E4-081 10.6
T1E2-222 19.9
LG-32
exhaustive map and thus the number of linkage groups is smaller than the number of chromosome pairs. However, AFLP markers are expected to provide a better coverage of microchromosomes than microsatellite markers [38,23].
Currently, the use of AFLP marker analysis to establish a genetic linkage map is mainly restricted to plant studies [6]. A recent study applied the microsatellite technique to establish a preliminary genetic linkage map in an inbred Pekin duck resource population [33]. When comparing the results with our current study (Figure 2), AFLP markers produced a higher number of linkage groups (32 vs 19) and an increased marker density (average interval distance 7.75 cM vs 15.04 cM). This difference is mainly caused by the use of different molecular markers, resource popula- tions and analysis methods. However, the microsatellite map made it possible to construct in parallel a cytogenetic map, which is not possible with AFLP markers. Thus, AFLP and microsatellite markers each have their advan- tages and drawbacks. To date, no large and integrated duck map is available for analysis and comparison. The successful establishment of a duck linkage map using AFLP genetic markers (Figure 2) in this study provides important information to integrate the published micros- atellite markers, to set up a duck chromosome map, to map QTL and to develop future breeding applications.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
C-WH carried out the AFLP detection, performed the con- struction of the map, and wrote the first draft of the man- uscript. Y-SC participated in the design and supervising the study, provided the duck samples, pedigree and per- formance information. RR participated in the design and supervising of the study, directed the data analysis, and helped to improve the manuscript. K-TY and C-PW partic- ipated in the collection of samples, prepared the genomic DNA and helped to the AFLP detection. H-LH participated in the collection of samples, prepared the genomic DNA, and helped to interpret the data and draft the manuscript.
M-CH conceived, designed and supervised the study, suc- ceeded in finding funding and coordination, and final- ized the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This study was funded by a grant awarded by the National Science Council, Executive Yuan, Taiwan (Grant No. NSC92-2313-B005-106).
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