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The aim of this study was the identification of candidate genomic regions associated with fiber diameter in alpacas. DNA samples were collected from 1011 female Huacaya alpacas from two geographical Andean regions in Peru (Pasco and Puno), and three alpaca farms within each region. The samples were genotyped using an Affymetrix Custom Alpaca genotyping array containing 76,508 SNPs. After the quality controls, 960 samples and 51,742 SNPs were retained. Three association study methodologies were performed. The GWAS based on a linear model allowed us to identify 11 and 35 SNPs (-log 10 ( p -values) > 4) using information on all alpacas and alpacas with extreme values of fiber diameter, respectively. The haplotype and marker analysis method allowed us to identify nine haplotypes with standardized haplotype heritability higher than six standard deviations. The selection signatures based on cross-population extended haplotype homozygosity (XP-EHH) allowed us to identify 180 SNPs with XP-EHH values greater than |3|. Four candidate regions with adjacent SNPs identified via two association methods of analysis are located on VPA6, VPA9, VPA29 and one chromosomally unassigned scaffold. This study represents the first analysis of alpaca whole genome association with fiber diameter, using a recently assembled alpaca SNP microarray.

Small farm producers' sustenance depends on their alpaca herds and the production of fiber. Genetic improvement of fiber characteristics would increase their economic benefits and quality of life. The incorporation of molecular marker technology could overcome current limitations for the implementation of genetic improvement programs. Hence, the aim of this project was the generation of an alpaca single nucleotide polymorphism (SNP) microarray. A sample of 150 Huacaya alpacas from four farms, two each in Puno and Cerro de Pasco were used for SNP discovery by genotyping by sequencing (GBS). Reduced representation libraries, two per animal, were produced after DNA digestion with ApeK1 and double digestion with Pst1-Msp1. Ten alpaca genomes, sequenced at depths between 12× to 30×, and the VicPac3.1 reference genome were used for read alignments. Bioinformatics analysis discovered 76,508 SNPs included in the microarray. Candidate genes SNPs (302) for fiber quality and color are also included. The microarray SNPs cover 90.5% of the genome length with a density of about 39 ± 2.51 SNPs/Mb of DNA at an average interval of 26.45 ± 18.57 kbp. The performance was evaluated by genotyping 30 family trios and comparing them to their pedigrees, as well as comparing microarray to GBS genotypes. Concordance values of 0.93 and 0.94 for ApeK1 and Pst1-Msp1 generated SNPs were observed. Similarly, 290 fiber quality and color candidate gene SNPs were validated. Availability of this microarray will facilitate genome-wide association studies, marker-assisted selection and, in time, genomic selection.

Alpaca is a camelid species of broad economic, biological and biomedical interest, and an essential part of the cultural and historical heritage of Peru. Recently, efforts have been made to improve knowledge of the alpaca genome, and its genetics and cytogenetics, to develop molecular tools for selection and breeding. Here, we report cytogenetic mapping of 35 new markers to 19 alpaca autosomes and the X chromosome. Twenty-eight markers represent alpaca SNPs, of which 17 are located inside or near protein-coding genes, two are in ncRNA genes and nine are intergenic. The remaining seven markers correspond to candidate genes for fiber characteristics ( BMP4, COL1A2, GLI1, SFRP4) , coat color ( TYR ) and development ( CHD7, PAX7 ). The results take the tally of cytogenetically mapped markers in alpaca to 281, covering all 36 autosomes and the sex chromosomes. The new map assignments overall agree with human-camelid conserved synteny data, except for mapping BMP4 to VPA3, suggesting a hitherto unknown homology with HSA14. The findings validate, refine and correct the current alpaca assembly VicPac3.1 by anchoring unassigned sequence scaffolds, and ordering and orienting assigned scaffolds. The study contributes to the improvement in the alpaca reference genome and advances camelid molecular cytogenetics.

The alpaca ( Vicugna pacos ) is an economically important and cultural signature species in Peru. Thus, molecular genomic information about the genes underlying the traits of interest, such as fiber properties and color, is critical for improved breeding and management schemes. Current knowledge about the alpaca genome, particularly the chromosomal location of such genes of interest is limited and lags far behind other livestock species. The main objective of this work was to localize alpaca candidate genes for fiber growth and color using fluorescence in situ hybridization (FISH). We report the mapping of candidate genes for fiber growth COL1A1 , CTNNB1 , DAB2IP , KRT15 , KRTAP13-1 , and TNFSF12 to chromosomes 16, 17, 4, 16, 1, and 16, respectively. Likewise, we report the mapping of candidate genes for fiber color ALX3 , NCOA6 , SOX9 , ZIC1 , and ZIC5 to chromosomes 9, 19, 16, 1, and 14, respectively. In addition, since KRT15 clusters with five other keratin genes ( KRT31 , KRT13 , KRT9 , KRT14 , and KRT16 ) in scaffold 450 (Vic.Pac 2.0.2), the entire gene cluster was assigned to chromosome 16. Similarly, mapping NCOA6 to chromosome 19, anchored scaffold 34 with 8 genes, viz., RALY , EIF2S2 , XPOTP1 , ASIP , AHCY , ITCH , PIGU , and GGT7 to chromosome 19. These results are concordant with known conserved synteny blocks between camelids and humans, cattle and pigs.

Alpacas are one of four South American Camelid species living in the highlands of the Andes. Production of alpaca fiber contributes to the economy of the region and the livelihood of many rural families. Fiber quantity and quality are important and in need of a modern breeding program based on genomic selection to accelerate genetic gain. To achieve this is necessary to discover enough molecular markers, single nucleotide polymorphisms (SNPs) in particular, to provide genome coverage and facilitate genome wide association studies to fiber production characteristics. The aim of this study was to discover alpaca SNPs by genotyping forty alpaca DNA samples using the BovineHD Genotyping Beadchip. Data analysis was performed with GenomeStudio (Illumina) software. Because different filters and thresholds are reported in the literature we investigated the effects of no-call threshold (≥0.05, ≥0.15, and ≥0.25) and call frequency (≥0.9 and =1.0) in identifying positive SNPs. Average GC Scores, calculated as the average of the 10% and 50% GenCall scores for each SNP (≥0.70) and the GenTrain score ≥ 0.25 parameters were applied to all comparisons. SNPs with minor allele frequency (MAF) ≥ 0.05 or ≥ 0.01 were retained. Since detection of SNPs is based on the stable binding of oligonucleotide probes to the target DNA immediately adjacent to the variant nucleotide, all positive SNP flanking sequences showing perfect alignments between the bovine and alpaca genomes for the first 21 or 26 nucleotides flanking the variant nucleotide at either side were selected. Only SNPs localized in one scaffold were assumed unique. Unique SNPs identified in both reference genomes were kept and mapped on the Vicugna_pacos 2.0.2 genome. The effects of the no-call threshold ≥ 0.25, call frequency = 1 and average GC ≥ 0.7 were meaningful and identified 6756 SNPs of which 400 were unique and polymorphic (MAF ≥ 0.01). Assignment to alpaca chromosomes was possible for 292 SNPs. Likewise, 209 SNPs were localized in 202 alpaca gene loci and 29 of these share the same loci with the dromedary. Interestingly, 69 of 400 alpaca SNPs have 100% similarity with dromedary.

Melanocortin 1 receptor ( MC1R ), the agouti signaling protein ( ASIP ), and tyrosinase related protein 1 ( TYRP1 ) are among the major regulators of pigmentation in mammals. Recently, MC1R and ASIP sequence variants were associated with white and black/dark brown coat colors, respectively, in the dromedary. Here we confirmed this association by independent sequencing and mutation discovery of MC1R and ASIP coding regions and by TaqMan genotyping in 188 dromedaries from Saudi Arabia and United States, including 38 black, 53 white, and 97 beige/brown/red animals. We showed that heterozygosity for a missense mutation c.901C > T in MC1R is sufficient for the white coat color suggesting a possible dominant negative effect. Likewise, we confirmed that the majority of black dromedaries were homozygous for a frameshift mutation in ASIP exon 2, except for 4 animals, which were heterozygous. In search for additional mutations underlying the black color, we identified another frameshift mutation in ASIP exon 4 and 6 new variants in MC1R including a significantly associated SNP in 3'UTR. In pursuit of sequence variants that may modify dromedary wild-type color from dark-reddish brown to light beige, we identified 4 SNPs and one insertion in TYRP1 non-coding regions. However, none of these were associated with variations in wild-type colors. Finally, the three genes were cytogenetically mapped in New World (alpaca) and Old World (dromedary and Bactrian camel) camelids. The MC1R was assigned to chr21, ASIP to chr19 and TYRP1 to chr4 in all 3 species confirming extensive conservation of camelid karyotypes. Notably, while the locations of ASIP and TYRP1 were in agreement with human-camelid comparative map, mapping MC1R identified a new evolutionary conserved synteny segment between camelid chromosome 21 and HSA16. The findings contribute to coat color genomics and the development of molecular tests in camelids and toward the chromosome level reference assemblies of camelid genomes.

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

In this study, we show how Y-specific interspersed multilocus microsatellites, which are loci that yield several amplified bands differing in size from the same male individual and PCR reaction, are a powerful source of information for tracing the history of cattle. Our results confirm the existence of three main groups of sires, which are separated by evolutionary time and clearly predate domestication. These three groups are consistent with the haplogroups previously identified by Götherström et al. (2005) using five Y-specific segregating sites: Y1 and Y2 in taurine (Bos taurus) cattle and Y3 in zebu (Bos indicus) cattle. The zebu cattle cluster clearly originates from a domestication process that was geographically and temporally separated from that of taurine clusters. Our analyses further suggest that: (i) introgression of wild sire genetic material into domesticated herds may have a significant role in the formation of modern cattle, including the formation of the Y1 haplogroup; (ii) a putative domestication event in Africa probably included local Y2-like wild sires; (iii) the West African zebu cattle Y-chromosome may have partially originated from an ancient introgression of humped cattle into Africa; and (iv) the high genetic similarity among Asian zebu sires is consistent with a single domestication process.

Five cattle Y-specific microsatellites, totalling six loci, were selected from a set of 44 markers and genotyped on 608 Bos taurus males belonging to 45 cattle populations from Europe and Africa. A total of 38 haplotypes were identified. Haplogroups (Y1 and Y2) previously defined using single nucleotide polymorphisms did not share haplotypes. Nine of the 27 Y2-haplotypes were only present in African cattle. Network and correspondence analyses showed that this African-specific subfamily clustered separately from the main Y2-subfamily and the Y1 haplotypes. Within-breed genetic variability was generally low, with most breeds (78%) showing haplotypes belonging to a single haplogroup. AMOVA analysis showed that partitioning of genetic variation among breeds can be mainly explained by their geographical and haplogroup assignment. Between-breed genetic variability summarized via Principal Component Analysis allowed the identification of three principal components explaining 94.2% of the available information. Projection of principal components on geographical maps illustrated that cattle populations located in mainland Europe, the three European Peninsulas and Mediterranean Africa presented similar genetic variation, whereas those breeds from Atlantic Europe and British Islands (mainly carrying Y1 haplotypes) and those from Sub-Saharan Africa (belonging to Y2-haplogroup) showed genetic variation of a different origin. Our study confirmed the existence of two large Y-chromosome lineages (Y1 and Y2) in taurine cattle. However, Y-specific microsatellites increased analytical resolution and allowed at least two different Y2-haplotypic subfamilies to be distinguished, one of them restricted to the African continent.

The Deleted in AZoospermia Like (DAZL) gene is a member of the DAZ family and encodes an RNA-binding protein that is expressed in prenatal and postnatal germ cells of males and females. In the human, there are five highly-related members in the DAZ family, four (DAZ1-4) on the Y chromosome and one (DAZL) on an autosome (HSA3). Mutations in these genes have been linked to severe spermatogenic failure and infertility in men. In the present study, we have cloned and characterized the bovine DAZL (bDAZL) gene. The full-length bDAZL cDNA is predicted to encode a protein of 295 amino acids with an RNA recognition motif. The deduced protein sequence of bDAZL is 96 and 97% similar to human and mouse DAZL, respectively. Fluorescence in situ hybridization (FISH) maps bDAZL to the distal region on BTA1q. The bDAZL gene consists of 11 exons and 10 introns. A bDAZL pseudogene was identified on BTA16. Expression analysis of bDAZL in 13 different tissues by RT-PCR shows that two transcripts, variant 1 (2,996 bp) and variant 2 (1,373 bp), of the bDAZL gene are detected only in testis mRNA. The variants probably result from alternative RNA splicing as variant 1 contains an additional 1,623-bp insertion in the 3' UTR. Our results lay the groundwork for possible single nucleotide polymorphism (SNP) and functional studies of the DAZL gene in cattle., (Copyright 2007 S. Karger AG, Basel.)

Previous genetic mapping identified three linkage groups (M1, M18 and M26) in the turkey corresponding to chicken chromosome 1 (GGA1). This is inconsistent with previously described chromosomal differences between these species. FISH analysis of BAC clones corresponding to microsatellite markers from each of the three turkey linkage groups, assigned all three linkage groups to a single chromosome (MGA1)., (Copyright 2006 S. Karger AG, Basel.)

Characterization of the Gallus gallus alpha-amylase gene family revealed that the chicken genome contains two distinct amy loci. One of the two loci is expressed in the chicken pancreas while cDNA clones for the second locus were detected in a library constructed from liver mRNA. Fluorescent in situ hybridization to chromosome spreads showed that the two loci are both located on chromosome 8 within the chicken genome. Moreover, each locus contains both an intact, expressed gene copy as well as a pseudogene. The expressed gene and the pseudogene are arranged in a divergent configuration in the pancreatic amy locus, while in the hepatic locus the intact gene and the pseudogene are arranged in tandem. The data suggest a complex pattern of evolution for the chicken amylase gene family which includes multiple gene duplication events, insertion/deletion events, as well as changes in spatial expression patterns.

Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.

Thirty-eight bovine Y chromosome (BTAY) microsatellites (MS) were assessed for polymorphisms in DNA samples obtained from 17 unrelated bulls. Thirty-three of these microsatellites are new and were used for the construction of a first generation radiation hybrid map for BTAY (Liu et al., 2002). Five MS had been previously reported and were used as positive controls. Fourteen out of 38 MS were found to be polymorphic; the remaining 24 were uninformative among the animals tested. The number of hemizygous loci per MS within individual ranged from two to over 20. Seven MS presented smear- or ladder-like bands, a unique feature for Y chromosome multi-copy hemizygous MS loci. The locus length variance, within individual, ranged from 2 to 42 bp corresponding to the MS with the minimum and maximum number of loci observed, respectively. Within the 14 polymorphic MS, the five pseudoautosomal MS, on average, were more polymorphic (35.3%) than the nine Y-specific MS (19.6%). Haplotypes resulting from combinations of these polymorphic loci will provide a powerful tool for future studies on the origin of domestic cattle and the evolution of bovid species., (Copyright 2003 S. Karger AG, Basel)

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 markers 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.

Chromosome-specific libraries provide a means to isolate genetic markers from specific chromosomal regions. A small-insert BTA6 library, constructed by microdissection, was screened for dinucleotide repeats (CA)15 and (GA)15. A total of 47 new microsatellite loci were developed and tested for polymorphism and informativeness using the MARC bovine mapping family.

Screening a bovine Y Chromosome-specific DNA library resulted in 34 new microsatellites, six of which mapped to the pseudoautosomal region (PAR), and 28 localized to the Y-specific region. These microsatellites, together with 23 markers previously mapped to the bovine Y Chr, were scored on a 7000-rad cattle-hamster radiation hybrid (RH) panel. Retention frequency of individual markers ranged from 18.5% to 76.5% with an average of 48.4%. Markers with high retention frequency (>55%) were found to exist in multiple copies on the Y Chr. Thirteen markers were placed on the PAR RH map with the AmelY gene proximal to the pseudoautosomal boundary and 46 markers, including Sry and Tspy gene, on the Y-specific region of the RH map. The microsatellites developed and mapped in this work will be useful for comparative mapping of cattle, sheep, and goat, studying the origin, evolution, and migration of bovidae species and provide an initial platform to develop a high-resolution map of the Y Chr and positional cloning of Y-specific genes.

Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome., (Copyright 2002 S. Karger AG, Basel)

A chromosome-specific library was developed for Bos taurus autosome 11 by chromosome microdissection and microcloning using a bovine primary fibroblast culture, obtained from a t(X;23) heifer, that spontaneously developed a translocation chromosome involving bovine chromosome 11. The library was screened using (AC)12 oligos, positive clones selected, sequenced and primers developed to generate bovine chromosome 11-specific microsatellite markers. This study suggests that chromosome-specific libraries have great potential for development of microsatellite markers for the construction of marker-saturated linkage maps for each chromosome.

A number of non-virally and non-chemically immortalized chicken embryo fibroblast (CEF) cells have been established recently in continuous cell culture. All immortal CEF cells tested showed common genetic alterations in the expression patterns of p53 and E2F-1 mRNA and protein which were down- and up-regulated, respectively. The biological effects of differentially regulated p53 and E2F-1 were determined by reporter gene transcriptional activity assays, DNA binding assays, and Northern blot analysis of the expression patterns of down-stream genes. In addition, expression of most of the cyclin genes was up-regulated in immortal CEF cells, which may be associated with the rapid cell division rates and serum-independent growth patterns seen in immortal CEF cells. The telomeric lengths and chromosome integrity were maintained in all immortal CEF cell lines without detectable telomerase activity. Although the functional inactivations of the p53 and Rb regulatory pathways are known to be common events for cellular immortalization, the genetic changes leading to alteration of p53 and E2F-1 function through transcriptional and post-transcriptional regulation seem to be unique in immortal CEF cells.

Testicular activity and semen characteristics of bulls carrying an X-autosome translocation t(Xp +;23q-) revealed all stages of spermatogenesis although their semen consisted of few and, exclusively, of malformed spermatozoa. Chromosome painting on metaphase spreads of their mother and synaptonemal complex analysis on these and normal bulls were carried out to test whether the location and meiotic pairing behaviour of the rearranged segments could have contributed to the sperm head malformation and oligospermia in our X-autosome translocation (X-AT) carrier bulls. Spermatocytes of X-AT carriers displayed the rearranged chromosomes in a univalent-trivalent association, with 23q- always remaining as a univalent and Xp + in synapsis with normal chromosome 23 and the Y chromosome. Chromosome painting studies to test whether the total absence of meiocytes showing a quadrivalent is due to the non-reciprocal nature of this translocation, identified Xp sequence homology with the distal end of 23q- confirming its relocation to the terminal segment of 23q-. Our synaptonemal complex analyses also confirmed that the bovine pseudo-autosomal region (PAR) is at the distal ends of Xq and Yp and further revealed that over 85% of spermatocytes of X-AT carriers (and up to 13% of spermatocytes of normal bulls) sustain a Y-axis break adjacent to the PAR. Although the exact cause of a Y-axis break in bovine spermatocytes is not known at present, we believe that the break and possible loss of Yq in such high proportions of spermatocytes of X-AT carriers could have contributed to the sperm head malformation and oligospermia in our X-AT carrier bulls., (Copyright 2001 Wiley-Liss, Inc.)

To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR-amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinois Yorkshire x Meishan swine reference population. These 22 markers were all assigned within a 50.7-CM region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.

Conserved genome homologies between the chicken and partridge have been demonstrated for chromosomes 1 and Z in previous studies. Morphological differences between the chicken and partridge for chromosome 4 have also been identified. The chicken chromosome 4 is submetacentric while the partridge chromosome 4 is acrocentric. We now report that in spite of this morphological difference, both species share extensive homology for chromosome 4 as determined by fluorescent in situ hybridization (FISH). Since only two chromosomes of the partridge karyotype showed FISH signals, our observation suggests that a chromosome rearrangement (peri- or paracentric inversion) has occurred in the partridge chromosome 4.

A novel insertion 78,ZZ or 78,ZW, ins(3;1)(q25q27;undetermined) was revealed in chicken by double color fluorescent in situ hybridization (FISH). A fragment of chromosome 1 spanning either from q13-14 to q34-35, or from q14-21 to q36-41 bands, had been translocated to chromosome 3 at a site located between q25 to q27 bands. This has resulted in the generation of an interstitial deletion in chromosome 1 and an insertional translocation in chromosome 3. Chickens with this balanced insertional translocation are asymptomatic carriers and their fertility is not affected, but embryo mortality increases. Greater than 50% occurrence of unbalanced gametes are observed. However, progeny sex ratio is not affected.

We have developed a method, using nuclear transplantation, to produce transgenic embryonic stem (ES)-like cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, differentiated into derivatives from the three embryonic germ layers, ectoderm, mesoderm, and endoderm, in 5-month-old animals. Six out of seven (86%) calves born were found to be chimeric for at least one tissue. These experiments demonstrate that somatic cells can be genetically modified and then de-differentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy.

An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.

Chromosomal homologies among the X chromosomes of species representative of eight bovid subfamilies and most of the recognized tribes were established using a combination of FISH and conventional G- and C-banding. Our analyses allowed for the delimitation of three X chromosome types represented, respectively, by cattle (Bovinae, tribe Bovini), the tragelaphines (Bovinae, tribe Tragelaphini), and a large assemblage comprising all the remaining subfamilies and their tribes (the Cephalophinae, Hippotraginae, Alcelaphinae, Antilopinae, Aepycerotinae, Peleinae, and Caprinae). The use of the bacterial artificial chromosome probe BAC 101 (which maps to Xp12 in cattle) and an Xp painting probe comprising sequences specific for the short arm of cattle Xp (Xp24-->p12) allowed us to orient this region, which has moved as a conserved euchromatic block during the evolution of the bovid X chromosome. We show that the differences between the three chromosomal types are attributable to a transposition, two inversions, and heterochromatic additions/deletions. A paucity of comparative mapping data precludes the assignment of the sequences contained in cattle Xp to either the presumed conserved (XCR) or the recently added (XAR) region of the eutherian X chromosome, and the reasons for the retention of these sequences as an evolutionarily conserved unit in the intrachromosomal restructuring of the bovid X across lineages remain enigmatic.

We have developed a chicken (Gallus domesticus) Z-chromosome-specific DNA library in a phage vector by means of chromosome microisolation and microcloning. The chromosomal origin, specificity, and purity was evaluated by fluorescent in situ hybridization (FISH) on chicken metaphases. Heterologous chromosome painting using this Z-chromosome-specific probe on turkey (Meleagris gallopavo) metaphases identified its homologous Z-chromosome, under the same stringent conditions as that used in the chicken, indicating a high degree of Z-chromosome sequence homology among these two species. This chicken Z-chromosome library will facilitate the development of Z-chromosome-specific DNA markers that will be useful for genetic mapping in the domestic chicken and related avian species. The Z-chromosome-specific DNA probe will also be useful for studies pertaining to the sex chromosome evolution in avian species.

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.

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.

A rapid method for detecting nucleolus organizer regions (NORs) in fish chromosomes based on thermal denaturation and staining with propidium iodide is described. Under epifluorescence, the NORs of 15 fish species from six families could be detected. This protocol differentiates constitutive heterochromatin in mammalian and avian chromosomes, and in some cases, heterochromatic blocks in fish chromosomes. The staining of NORs of fish chromosomes with propidium iodide following denaturation with formalin is likely the result of differential denaturation of the rDNA due to the thermal characteristics of AT- and GC-rich domains of the rDNA cistron. This technique provides a new useful marker for descriptive fish cytogenetic studies.

The chicken natural resistance-associated macrophage protein 1 (NRAMP1) gene has been mapped by linkage analysis by use of a reference panel to develop the chicken molecular genetic linkage map and by fluorescence in situ hybridization. The chicken homolog of the murine Nramp 1 gene was mapped to a linkage group located on Chromosome (Chr) 7q13, which includes three genes (CD28, NDUSF1, and EF1B) that have previously been mapped either to mouse Chr 1 or to human Chr 2q. Physical mapping by pulsed-field gel electrophoresis revealed that NRAMP1 is tightly linked to the villin gene and that the genomic organization (gene order and presence of CpG islands) of the chromosomal region carrying NRAMP1 is well conserved between the chicken and mammalian genomes. The regions on mouse Chr 1, human Chr 2q, and chicken Chr 7q that encompass NRAMP1 represent large conserved chromosomal segments between the mammalian and avian genomes. The chromosome mapping of the chicken NRAMP1 gene is a first step in determining its possible role in differential susceptibility to salmonellosis in this species.

Peripheral blood cultures were made on 20 Dorset sheep including 16 ewes and 4 rams. Photomicrographs of 100 cells were examined for trypsin-induced G-bands, and the length of each chromosome was measured. The banding patterns of each of the 27 pairs of chromosomes were identified and described, and the results observed in this study were compared with those previously reported. Measurements of all chromosomes in which both members of a homologous pair were identifiable were used to calculate mean relative length. Karyotypes, in which chromosomes were arranged to mean relative length, were prepared and an idiogram illustrating major bands was constructed.