S. Ambady

An integrated genetic and physical map of the bovine X chromosome

Genotypic data for 56 microsatellites (ms) generated from maternal full sib families nested within paternal half sib pedigrees were used to construct a linkage map of the bovine X Chromosome (Chr) (BTX) that spans 150 cM (ave. interval 2.7 cM). The linkage map contains 36 previously unlinked ms; seven generated from a BTXp library. Genotypic data from these 36 ms was merged into an existing linkage map to more than double the number of informative BTX markers. A male specific linkage map of the pseudoautosomal region was also constructed from five ms at the distal end of BTXq. Four informative probes physically assigned by fluorescence in situ hybridization defined the extent of coverage, confirmed the position of the pseudoautosomal region on the q-arm, and identified a 4.1-cM marker interval containing the centromere of BTX.

Analysis of the Effect of Endogenous Viral Genes in the Smyth Line Chicken Model for Autoimmune Vitiligo

The Smyth line (SL) chicken, an animal model for autoimmune human vitiligo, is characterized by a spontaneous posthatch pigment loss, determined to be the result of an autoimmune phenomenon. Because endogenous virus (EV) genes have been reported to be associated with a number of autoimmune diseases of human and animal models, we designed this experiment to investigate the role of EV in the SL vitiligo by using the complete sequence of Rous-associated virus-2 as a probe for EV. An F(2) resource population was developed by the matings of SL and parental control (BL) chickens. Linkage disequilibrium between vitiligo and EV was apparent (16.2-kb SacI fragment, P </= 0.05 and a 19-kb HindIII fragment, P </= 0.03). Methylation analyses revealed that the EV and endogenous avian retroviral (EAV) genes were methylated in both the SL and BL sublines of chickens; therefore, methylation does not appear to be responsible for the differences in the expression of vitiligo between SL and BL sublines. Expression of the EV genes correlated with the disease in vitiliginous SL101 birds and also in 5-Azacytidine-induced vitiliginous BL101 parental control chickens. Only one EV locus was detected in the unrelated Light Brown Leghorn control chickens (1q14) by in situ hybridization, whereas 3 EV loci were identified in SL101 and BL101 chickens (1p25, 2q26, and an unidentifiable microchromosome). Our observations indicate that EV genes may play a role in the induction of autoimmune vitiligo in the SL chicken model.

Development and use of a microdissected swine chromosome 6 DNA library

To facilitate the identification of microsatellite genetic markers from a single swine chromosome, chromosome microisolation and microcloning have been used to generate a swine chromosome 6-specific DNA library. Ten copies of swine chromosome 6 were scraped from metaphase spreads, ligated to custom-prepared adaptors, and amplified by PCR. The purity of the amplified product was verified by fluorescent in situ hybridization. The utility of the chromosome painting probe for heterologous painting was demonstrated and confirmed that swine chromosome 6 is syntenic to human chromosomes 1p and 19q. A small insert genomic library of 1.39 x 10(6) clones was generated from the PCR-amplified chromosome 6 genomic DNA and screened for (GT)n microsatellite genetic markers. Nine (GT)n microsatellite markers were developed and genotyped on a Yorkshire x Meishan swine reference family. All nine markers genetically mapped to chromosome 6, confirming the purity of the microisolation method. The method used here should be adaptable to the microdissection of subchromosomal regions of not only the swine genome but also other livestock genomes.

Optimization of RAPD‐PCR conditions in cattle

Abstract Random Amplified Polymorphic DNA polymerase chain reaction (RAPD‐PCR) is a fast and easy way of identifying DNA polymorphisms generated from several regions of the genome. This could expedite the process of identifying informative polymorphic markers that may be linked to important genes controlling economic traits. In cattle, failure to obtain consistent amplification patterns in RAPD‐PCR has been a cause for concern. This has been attributed to the fact that decamer primers that are used in RAPD‐PCR reactions are likely to amplify regions of DNA where the primer‐template base pairing has some degree of mismatch and that these mismatches fail to repeat from reaction to reaction. This paper describes the use of tricine buffer along with changes in reaction components and thermal cycling conditions that has yielded consistent and reproducible RAPD‐PCR amplifications using single primers and double primer combinations on bovine DNA.

Bovine DNA polymorphisms uncovered by RAPD-PCR

Random amplification of polymorphic DNA by the polymerase chain reaction (RAPD-PCR), using arbitrary 10-bp oligonucleotide primers was assessed in 24 young elite bulls selected for progeny testing and, 10 cows. RAPD-PCR amplification patterns were analyzed to identify polymorphic bands among animals within and between groups. Duplicate PCR reactions for each animal were carried out in 1X Tricine buffer, 15/30 ng of primer and 20 ng of genomic DNA for 51 cycles. Amplification products were separated by agarose gel (1.4%) electrophoresis and detected by ethidium bromide staining. Forty primers were screened and 18 of them were selected for further use because of their consistent PCR amplification patterns and large number of amplified bands. An average of 9.66 bands and 10.3 bands per primer was obtained for the elite young bull and cow groups, respectively. Amplified bands ranged from 0.2 Kb to 5 Kb in size. The cow group double (2.1) the average number of polymorphic bands per primer when compared to the young elite bull (1.0) group. Frequencies of dominant polymorphic bands range from 75 to 95% for different primers. Similarity indexes among animals, based on the band sharing method, varied from 0.76 to 1.0. The reduced band polymorphisms found in the elite young bull group is probably due to the intensity of selection applied to this group of animals when compared to the cow group. The trend observed is in agreement with the theoretical expectation of a higher level of homozygosity in the elite young bull group than in the cow group.