Development and Mapping of Microsatellite Markers Derived From Chicken Chromosome-Specific Libraries S. Ambady,*,1 H. H. Cheng,† and F. A. Ponce de Leo´n*,1,2 *Department of Veterinary & Animal Science, University of Massachusetts Amherst, Massachusetts 01003; and †Avian Disease and Oncology Laboratory, USDA-ARS, Michigan State University, East Lansing, Michigan 48823 ABSTRACT Chromosome-specific painting probes and libraries were developed for chicken Macrochromosomes 1, 2, 3, and 4 by chromosome microisolation and microcloning. Fluorescent in situ hybridization results using the painting probes on normal chicken metaphase chromosomes indicated the purity and specificity of each probe. Chromosome-specific libraries for chicken Macrochromosomes 1, 2, 3, and 4 were prepared in a phage vector. Fifty-two additional unique microsatellite mark-
ers of the (AC)n type were developed from these chromosome-specific libraries. These markers were mapped on the East Lansing reference population to increase the marker density on the four macrochromosomes. Results of the current study suggest that development of markers from chromosome-specific libraries is very useful for constructing high-density linkage maps for chicken macrochromosomes.
(Key words: chicken chromosome, chromosome microdissection, chromosome paint, chromosome-specific library, linkage map) 2002 Poultry Science 81:1644–1646
INTRODUCTION
MATERIALS AND METHODS
Historically, chickens have been one of the most widely used experimental models for studies of genetic inheritance using classical linkage mapping. The most current published linkage map for chicken contains 1,889 markers that span approximately 3,800 cM of the chicken genome (Groenen et al., 2000). The first five pairs of macrochromosomes (autosomes) and the Z-chromosome of chicken represent about 65% of the total genome. Hence, development of chromosome-specific libraries for the macrochromosomes will aid in the development of marker-saturated linkage maps for all those chromosomes. Utility of chromosome-specific libraries for marker saturation on specific chromosomes has been demonstrated in bovines (Ponce de Leo´n et al., 1996; Sonstegard et al., 1997) and in swine (Ambady et al., 1997; Zhao et al., 1999). The present study was undertaken to generate chromosomespecific libraries for Gallus domesticus autosomes (GDA) 1, 2, 3, and 4 for the development of microsatellite markers for linkage mapping of the East Lansing reference population.
Chromosome microisolation and microcloning were performed as described by Ambady et al. (1997). The purity and specificity of the chromosome-specific DNA cocktail were determined by fluorescent in situ hybridization after labeling an aliquot of the DNA probe with biotin (Ambady et al., 1997). Chromosome-specific DNA libraries were prepared in the Lambda ZAP Express vector,3 amplified and stored in 7% dimethyl sulfoxide (DMSO)4 at −70 C. For each library, clones were plated and screened using γ-32P end-labeled adenine, cytosine (AC)12 oligos. Positive clones were sequenced and primers were developed to generate chromosome-specific microsatellite markers. These markers were mapped on the East Lansing reference population (Crittenden et al., 1993) to develop a linkage map as described in Cheng et al. (1995).
2002 Poultry Science Association, Inc. Received for publication May 10, 2001. Accepted for publication May 24, 2002. 1 Current address: 1364 Eckles Ave., 305 Haecker Hall, University of Minnesota, MN 55108. 2 To whom correspondence should be addressed:
[email protected]. 3 Stratagene, La Jolla, CA. 4 Sigma, St. Louis, MO.
RESULTS Chromosome painting probes were hybridized to chicken metaphase chromosomes. Each of the chromosome preparations showed specific hybridization to their respective chromosomes, as detected by the strong fluorescent signal spanning the entire length of the chromosomes (Figure 1). Each chromosome-specific DNA library
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Abbreviation Key: GDA = Gallus domesticus autosomes.
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RESEARCH NOTE TABLE 1. Primers developed from chromosome-specific libraries for Gallus domesticus autosomes (GDA) 1, 2, 3 and 4 Primer name
Allele size (bp) Repeat Type
UMA1003 (GT)16 UMA1015 (GT)19 UMA1017 (GT)13 UMA1019 (AC)13 UMA1031 (CA)16 UMA1034 (AC)14 UMA1048 (CT)6 (CA)20 UMA1051 (GT)14 UMA1070 T25 (TG)13 UMA1071 (GT)19 UMA1074 (CA)14 UMA1100 (TG)21 UMA1104 T22 (GT)25 UMA1107 (AC)13 UMA1117 (TG)15 UMA1124 (GT)15 UMA1125 (CA)21 UMA1126 (CA)26 UMA1136 (AC)14 UMA1220 (CA)18 UMA2021 (AC)25 UMA2025 (GT)10 GCAT (GT)3 UMA2032 (TT)3 TC (TT)4 TC( TT)5 UMA2037 (GT)11 (GA)3 UMA2052 (TG)22 UMA2053 (TG)12 UMA2080 (TG)22 UMA2105 (TG)12 (AG)2 UMA2123 (AT)5 (GT)10 UMA2183 (TG)13 UMA2187 (CT)2 (GT)11 UMA2195 (TA)6 (TG)11 (CG)3 UMA2283 (AC)14 (AA)6 UMA3001 (TG)12 UMA3006 (CA)21 UMA3028 (CA)16 UMA3029 (GT)12 UMA3036 (CA)15 UMA4002 (AC)17 (AT)6 UMA4005 (TG)11 UMA4020 (AC)17 (A)15 UMA4021 (TG)16 (AG)11 UMA4024 (GT)17 UMA4025 (AC)15 UMA4027 (CA)12 UMA4033 (TG)17 UMA4034 (TG)17 UMA4036 (CA)14 CG( CA)8 UMA4038 (CA)20 UMA4046 (TG)15 UMA4056 (GT)14 UMA4059 (T)6 (GT)13 1 0
T
A
1
Forward primer
Reverse primer
TCCTGGCATGCGGATTGGTT TTTCCTCGAGGCTGTAAACA TTGCAGAAAACCAAAGATTT TTGTTAATGCCAAAGTGGAA CACCCAAATTTACTCAAAGA TGAAGGAAGCTGTCATAATA ACACTGGCAGGCGTGCTTAG GCTTGAGGACAGGGGTCAGG TCTTTCCCTGCTACTCTGGA GGAAACAGCTATGACCTTGA AAATAAAGGTGTTGGCAGTT CCCTGAAGTTGTGCTACCAC ACCCTGAACCAGTCATCACA CAGGACAATACTATGGAACA TTTTTAAATATCACGGGTAA TCTTCTTTTCCCTTCACAGG AAGCTTTTAAACCAATCTGA TCCTGCATGTGCCCTTTGTA ATGGCTGAAAGTGGTCATCC CCTTGAAAATACCTTCTTGG TGCATTTTACAGAAAGGAAA TGCTAATTAAACTGGCATTT GGCAGCCAATCTGTCTTATT TTGATTTTGGTCAGTGCTTT CCTGCGGTTTTCCTTTACAC GGGCAGACATAAGCACAGTT AGGGGCCTCACTATTTTCCT GTAAACGGGGGTGGATTCAG TTAGAATGCACTGGACACAG TGTTCTTTTGAGGGATGATT TTCTTTAAACTTTCCTTTCC TTTGTGAATGTCCTGTAACA CCAGCATGTGATTCCCAAGT AGTGTTTCCAGGGGCAAGGA AAAACGTTTCTTTCCAGTTG TGTAGACCTTCCAGGCTTTC AGCTCCTTGTACCCCTTTGC CACCATTTGCCTCACCAACT TGAAAAGTAAACAGGGAAAA GACTGCAACAGCATTTGAGT TCAGATGCACAGTGAGTC CTGCCACCATTAGCTAGAAGT GATCCATGTAAAGGCTAAAC ATGTATGGCTCAGGAGAAAT GATTTGGTGCCTACTTGAATG AATCTACAGTGGCCAGGAATA GAGGTGCACAGCAAGCTTTAT GTTCACTTGCCATCTGGTTCT ATGCAAGGCTAGGTGGTAGTT ACACGTCCACTCACAATTCAG GGCTTGCATATCGATTCACTG TGTCTGGCAAGTGCATACTCA GAGCATTCTGCATTATGGAGT TTTGTTCTTACAGCTGAGTGG GATATGAGGTGCACAGCAAGC AAGCACTCTGAGCCCTAGAAG GTGGTTCCTCACTATGGCAAA CCACCCAAGCAACTTCATTAC ATCCTTGAGGTCCCATCCA GGGAGGCAAACAACTATGAG TGATGATGGGTTGAATTGAG GCTTAATGGGATACCATGAA TACGTAGTGTGCAGGTAT AATCTGGAAAGCACAAAC CTGTGTTTCAGTTTGTACCCA GAGGAGGAATCTTACAAATGC AGCAATCCGACTCCTGAGCA TTCATTCTTTTTGATGGTTG AGCCCAAATGCCCATCAATC CGTGCTCAGGTGCTCAGGAC GTGACCAGTACCCAAGACAA TGCAGGAAAGAGGAAATCTC TTCTGGAGGCAAATCTGCTG GGGTTATCTCCTTTCTGTCC AATTCAAATTTCAGAAGCAA TGTTATAAAATGGTTACAGA TCTATTGCTTTGGGTTATTC TGAGCGACTGAACTGAACAC CGTGGAGGGAGGGCTCAGTG GCGGGAGGGCACCGAGAATG TAACCACAACAGCGAACAAC TTAGGAAAGACTGCCAGAGG GTGGAGCAGTTTACTTCCTG TTTGTATCTTGCCCCTTTCC TGAAAGCAGTGAAGGCAAAC GGTGGGAACTGGAAGAAGAG GGGCATGATAGAACTTTACC CATGCCAAATTGAGAAGAAA GCACAAGAACAGTGCCACAC CTCCCTGAAACAACATCAAG ATGTTGGGAAAAAGGACTTC TTCTGCAAATGTAAATGCTC GGTGATTTGGGGAGAATGAG AGGGAGGAGGGGCTTTACTC GACCACAGCAAAGGGCTTAC TGCAACAAATTCCAGAGTTT CATTTGCAAGTGCCATACAG GCCCTGGTAAACTGGTGTCC GCCCTATTGCTCCCATTGCT TGCAACTCATAAGCAGAAAA GGGTAGTTGGGATTTTTGAC ATGGATCAGCTCTTTCTAAC ACAGGAGATCAAGGTTTTTG CTGTGCACACATCCAATACA
T0A
WL
JF
Gen Bank no.
52 48 47 56 53 52 49 47 48 52 49 50 50 53 50 47 52 50 54 48 52 52 55 47 52 52 50 55 55 56 52 52 56 48 55 49 51 44 47 59 49 50 51 49 49 49 52 50 51 48 47 49
169 147 129 145 NP 153 NP 211 184 NP 141 189, 205 NP 131 133 180 167 181 222 109 144 106 NP NP 115 NP 125 NP NP NP 100 106 ND NP NP 150 NP NP NP 166 162 NP 215 158 142 NP 106 170 202 163 125 143
169, 171 136 129 139, 151 NP 157 NP 213 167 NP 148 205 NP 125, 127 133 180 146 194 231 103 159 100 NP NP 104 NP 150 NP NP NP 106 69, 73 ND NP NP 146 NP NP NP 168 163 NP 155, 163 152 140 NP 89 170 193 163 123 143
G29056 G29057 G29058 G29059 G29060 G29061 G29062 G29063 G29064 G29065 G29075 G29066 G29067 G29068 G29069 G29074 G29070 G29071 G29072 G29073 G64890 G64862 G64863 G64864 G64865 G64866 G64867 G64891 G64868 G64869 G64870 G64871 G64872 G64873 G64874 G64875 G64876 G64877 G64878 G64879 G64880 G64881 G64882 G64883 G64884 G64885 G64886 G64887 G64888 G64889 G64892 G64893
= Annealing temperature; WL = White leghorn; JF Jungle fowl; ND = not determined; NP = not polymorphic.
represented at least threefold chromosome coverage. After DNA library screening, selected clones were sequenced and primers were designed to develop unique microsatellite markers. This work resulted in the development of 20, 13, 5, and 14 primer sets for chromosomes 1, 2, 3, and 4, respectively. Of these, 11, 6, 1, and 5 markers, for GDA 1, 2, 3, and 4, respectively, were mapped to the respective chromosomes of the East Lansing mapping population. However, one marker from the GDA 4 library (UMA 4.005) mapped to GDA 1. The linkage map positions of the markers developed from our chromosomespecific libraries can be viewed on the World Wide Web
site (http://poultry.mph.msu.edu/resources/Conmap/ Chromosomesmain.htm). The primer sequences for all markers and polymorphism information for the mapped markers are presented in Table 1.
DISCUSSION Marker density on specific chromosomes can be increased by developing microsatellite markers from chromosome-specific libraries as demonstrated in cattle gene mapping (Ponce de Leo´n et al., 1996; Sonstegard et al., 1997) and in swine (Ambady et al., 1997; Zhao et al., 1999).
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indication of a relatively small degree of contamination of the GDA 4 library from GDA 1 that might have occurred during chromosome microisolation. However, the chromosome-specific DNA is an excellent microsatellite marker resource to develop high-density chromosome maps. The painting probes can be used to study gross chromosomal morphology in other species of domesticated and wild birds and also to look into the chromosomal repartitioning that has occurred through evolution across different avian species. The markers developed for the chicken can also be of great value for gene mapping studies in other commercially important birds such as turkey and ostriches.
ACKNOWLEDGMENTS This project was supported by grants from Avian Farms International, Inc., to F. Abel Ponce de Leo´n.
REFERENCES
FIGURE 1. Fluorescent in situ hybridization (FISH) using Gallus domesticus autosomes (GDA) 1, 2, 3, and 4 painting probes on chicken metaphase chromosomes. Panels on the left (A, C, E, and G) were stained with propidium iodide, G-banded partial metaphase spreads. Panels on the right (B, D, F, and H) correspond to A, C, E and G and show FISH signals for GDA 1, 2, 3, and 4, respectively. Arrows on the left panel indicate the chromosome pair corresponding to fluorescent signals in the right panel.
In the present study, 52 marker sets were developed for four macrochromosomes from chromosome-specific libraries. Of these, 24 (46%) were polymorphic on the East Lansing mapping population and were used to enhance marker saturation of the respective chromosomes. One of the markers developed from the GDA 4 library (UMA 4.005) mapped to chromosome 1. This result may be an
Ambady, S., J. R. Mendiola, C. F. Louis, M. A. Janzen, L. B. Schook, L. Buoen, J. K. Lunney, D. R. Grimm, and F. A. Ponce de Leo´n. 1997. Development and use of a microdissected swine chromosome 6 library. Cytogenet. Cell Genet. 76:27– 33. Cheng, H. H., I. Levin, R. L. Vallejo, H. Khatib, J. B. Dodgson, L. B. Crittenden, and J. Hillel. 1995. Development of a genetic map of the chicken with markers of high utility. Poult. Sci. 74:1855–1874. Crittenden, L. B., L. Provencher, I. Levin, H. Abplanalp, R. W. Briles, W. E. Briles, and J. B. Dodgson. 1993. Characterization of a red jungle fowl by white leghorn back cross reference population for molecular mapping of the chicken genome. Poult. Sci. 72:334–348. Groenen, M. A. M., H. H. Cheng, N. Bumstead, B. F. Benkel, W. E. Briles, T. Burke, D. W. Burt, L. B. Crittenden, J. Dodgson, J. Hillel, S. Lamont, F. A. Ponce de Leo´n, M. Soller, H. Takahashi, and A. Vignal. 2000. A consensus linkage map of the chicken genome. Genome Res. 10:137–147. Ponce de Leo´n, F. A., S. Ambady, G. A. Hawkins, S. M. Kappes, M. D. Bishop, J. M. Robl, and C. W. Beattie. 1996. Development of a bovine X chromosome linkage group and painting probes to assess cattle, sheep and goat X-chromosome segment homologies. Proc. Natl. Acad. Sci. USA. 93:3450–3454. Sonstegard, T. S., N. L. Lopez, S. M. Kappes, R. T. Stone, S. Ambady, F. A. Ponce de Leo´n, and C. W. Beattie. 1997. An integrated genetic and physical map of the bovine X chromosome. Mammal. Genome 8:16–20. Zhao, F., S. Ambady, F. A. Ponce de Leo´n, M. Miller, J. K. Lunney, D. R. Grimm, L. B. Schook, and C. F. Louis. 1999. Microsatellite markers from a microdissected swine chromosome 6 genomic library. Anim Genet. 30:251–255.
