Christian Looft

Combined analysis of data from two granddaughter designs: A simple strategy for QTL confirmation and increasing experimental power in dairy cattle

A joint analysis of five paternal half-sib Holstein families that were part of two different granddaughter designs (ADR- or Inra-design) was carried out for five milk production traits and somatic cell score in order to conduct a QTL confirmation study and to increase the experimental power. Data were exchanged in a coded and standardised form. The combined data set (JOINT-design) consisted of on average 231 sires per grandsire. Genetic maps were calculated for 133 markers distributed over nine chromosomes. QTL analyses were performed separately for each design and each trait. The results revealed QTL for milk production on chromosome 14, for milk yield on chromosome 5, and for fat content on chromosome 19 in both the ADR- and the Inra-design (confirmed within this study). Some QTL could only be mapped in either the ADR- or in the Inra-design (not confirmed within this study). Additional QTL previously undetected in the single designs were mapped in the JOINT-design for fat yield (chromosome 19 and 26), protein yield (chromosome 26), protein content (chromosome 5), and somatic cell score (chromosome 2 and 19) with genomewide significance. This study demonstrated the potential benefits of a combined analysis of data from different granddaughter designs.

Bovine β-defensins: Identification and characterization of novel bovine β-defensin genes and their expression in mammarygland tissue

Abstractβ-Defensin genes code for multifunctional peptides with a broad-range antimicrobial activity. In this project we hypothesized that β-defensin genes may be candidate genes for resistance to mastitis. In this article we describe the identification and genomic characterization of eight bovine β-defensin genes, including six novel defensin genes and two pseudogenes. Expression in the bovine mammary gland of one of the novel genes, DEFB401, has been demonstrated, as well as the expression of LAP, TAP, DEFB1, BNBD3, BNBD9, and BNBD12. For genomic characterization, 20 BACs from two different bovine BAC libraries (RZPD numbers 750 and 754) were isolated by PCR screening with β-defensin consensus primers derived from published sequences. PCR products from BACs generated with consensus primers have been subcloned and sequenced, revealing a total of 16 genes and two pseudogenes. Six novel β-defensin genes share the typical exon–intron structure and are highly homologous to published bovine β-defensin genes. They are named DEFB401–DEFB405 and LAP-like, and two novel pseudogenes are named EBD-P and EBD-P2. Analysis of mammary gland tissue-derived cDNA from nine cows with different clinical findings demonstrated the expression of several β-defensin genes mentioned above. First results indicate that the lactational status of the cow presumably has no influence on gene expression. Competent knowledge of antimicrobial activity of β-defensins from literature, the abundance of β-defensin mRNA in the bovine mammary gland, and the inducibility of some genes give first evidence that β-defensins may play a role in local host defense during udder infections.

A mammary gland EST showing linkage disequilibrium to a milk production QTL on bovine Chromosome 14

As part of a genome scan, ESTs derived from mammary gland tissue of a lactating cow were used as candidate genes for quantitative trait loci (QTL), affecting milk production traits. Resource families were genotyped with 247 microsatellite markers and 4 polymorphic ESTs. It was shown by linkage analysis that one of these ESTs, KIEL_E8, mapped to the centromeric region of bovine Chromosome (Chr) 14. Regression analysis revealed the presence of a QTL, with significant effect on milk production, in this chromosome region, and analysis of variance showed no significant interaction of marker genotype and family. The estimated significant differences between homozygous marker genotypes were 140 kg milk, −5.02 kg fat yield, and 2.58 kg protein yield for the first 100 days of lactation. Thus, there was strong evidence for a complete or nearly complete linkage disequilibrium between KIEL_E8 and the QTL. To identify the biological function of KIEL_E8, we extended the sequence for 869 bp by 5′-RACE. A 560-bp fragment of this shows a 90.9% similarity to a gene encoding a cysteine- and histidine-rich cytoplasmic protein in mouse. Although such a protein may have a regulatory function for lactation and a linkage disequilibrium between the EST marker and the QTL has been observed, it remains to be elucidated whether they are identical or not. Nevertheless, KIEL_E8 will be an efficient marker to perform marker-assisted selection in the Holstein-Friesian population.

A whole genome scan for differences in recombination rates among three Bos taurus breeds

Abstract. Twenty paternal half-sib families of a granddaughter design were genotyped for 265 genetic markers, most of them microsatellites. These were 16 Holstein families, 3 Simmental families, and 1 Brown Swiss family. The number of sires per breed was 872, 170, and 32, respectively. Two-point recombination rates were estimated both jointly for all breeds and each single breed separately. Of 1168 marker intervals, 865 provided estimates for at least two breeds. Differences between breeds were tested by likelihood ratio tests. Four marker intervals, representing three genomic regions on BTA19, BTA24, and BTA27, show a significant impact of the breed at a false discovery rate of 0.23 and indicate a genetic component of observed heterogeneity of recombination. The variability of recombination rates between cattle breeds might not be a common feature of the whole genome, but rather might be restricted to certain chromosomal segments. Thus, attention should be paid to heterogeneities when pooling data of such regions from different breeds.

A high-density linkage map of the RN region in pigs

The porcine RN locus affects muscle glycogen content and meat quality. We previously mapped the RN locus to chromosome 15. This study describes the identification of polymorphisms for four class I and four class II markers located in the RN region. Resource families were genotyped with F-SSCP markers (fluorescent single strand conformation polymorphism) and microsatellite markers. Subsequent multipoint linkage analysis revealed the order FN1-IGFBP5-S1000-S1001-IL8RB-VIL1-RN-Sw936-Sw906. The gene order is identical to the previously reported porcine RH map of the same region. The described map will facilitate positional cloning of the RN gene.

Construction of a high-resolution RH map of the human 2q35 region on TNG panel and comparison with a physical map of the porcine homologous region 15q25

Abstract. This article describes the construction of a high-resolution radiation hybrid map of Hsap 2q35 by using the TNG RH panel generated by irradiation with 50,000 rads. We were able to build a framework map of 1300 cR50,000 including 34 markers ordered with odds higher than 1000:1. The comprehensive map includes 77 loci and describes a region of 3 Mb around the SLC11A1 gene. Because of the very small size of the fragments retained and a reduced retention frequency, it was difficult to build a high-resolution multi-point map of this region by using the TNG RH panel. Nevertheless, this study confirmed the very high potential of this RH panel for constructing a human, high-resolution physical map (2.3 kb/cR50,000). Moreover, human ESTs from Hsap 2q35 were hybridized with porcine BAC contigs to establish a porcine transcript map of the homologous region Sscr 15q25 (greater than 2.5 Mb). We identified 17 new genes in this porcine chromosomal region. We were able to compare the location of 26 genes mapped in both species. The gene order was similar except for two possible minor discrepancies in the Desmin sub-region, suggesting the existence of a porcine micro-region between TNP1 and IL8RB with an unknown origin.

Mapping of 16 ESTs expressed in the bovine mammary gland during lactation.

Abstract. A bovine mammary gland cDNA-library was used to characterize and map genes expressed during lactation. Fifty cDNA clones selected by differential hybridization were sequenced from both ends, and sequences were examined for similarities with database sequences. For 34 of the transcripts, the sequences showed more than 80% similarity to previously characterized genes or expressed sequence tags (ESTs). Twenty cDNAs that could be of interest as candidate genes for milk production are selected for genetic or chromosomal mapping. Twenty-three out of the 39 designed primer pairs representing 16 cDNA clones amplified the expected fragments and were used for subsequent fluorescent single-strand conformation polymorphism analysis (F-SSCP) in the International Bovine Reference Panel families (IBRP). Ten polymorphic loci could be identified and used to genotype the IBRP animals, and nine of them were subsequently genetically mapped on nine chromosomes. In addition, eight loci from the 16 cDNA clones could be mapped by somatic cell hybrids, bringing the total number of mapped genes to 16, one of which was mapped genetically as well as physically. The mapped mammary gland ESTs are potentially useful for cloning economic trait loci by a positional candidate gene cloning approach.

Assignment of the equine S100A7 gene (psoriasin 1) to chromosome 5p12-->p13 by fluorescence in situ hybridization and radiation hybrid mapping

Psoriasin (S100A7) is a member of the S100 gene family and was discovered as a calcium-binding protein with a molecular weight of 11.4 kDa. Psoriasin was first identified as a secreted protein expressed in human skin (keratinocytes) involved in psoriasis (Celis et al., 1990). It was subsequently shown that psoriasin is a potent and selective chemotactic inflammatory protein for CD4(+) T-lymphocytes and neutrophils (Jinquan et al., 1996). Additionally psoriasin was identified in a fraction also containing lysozyme, and it is possible that psoriasin could be a potential antimicrobial peptide. Glaser et al. (2001) found psoriasin to exhibit antibacterial activity, indicating that psoriasin also contributes to the antimicrobial activity in vernix. The mapping of the equine S100A7 gene is the first step for further investigations to understand the biological role of S100A7 in epithelial defense in regard to equine health. Materials and methods

Assignment of two isoforms of the AMP-activated protein kinase γ subunits, PRKAG1 and PRKAG2 to porcine chromosomes 5 and 18, respectively by radiation hybrid panel mapping

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme complex comprising catalytic ·-subunits and regulatory sand A-subunits (Hardie et al., 2003). Each subunit exists as different isoforms encoded by two or three genes (·1, ·2, s1, s2, A1, A2, A3). AMPK has a key role in the regulation of energy metabolism in eukaryotic cells. AMPK is regulated by phosphorylation on the ·-subunit and by AMP allosteric control thought to be mediated by both ·and A-subunits (Adams et al., 2004). A A1 mutation, R70Q, causes a loss of AMP dependence (Hamilton et al., 2001). Mutations in human A2 and pig A3 genes were previously identified to cause an unusual cardiac phenotype and a glycogen storage disease, respectively (Gollob et al., 2002; Milan et al., 2000a). The mapping of the porcine PRKAG1 and PRKAG2 genes is the first step for further investigations helping to understand the regulation of the AMP-activated protein kinase family, an enzyme family with relevance to type 2 diabetes and the metabolic syndrome. Materials and methods

Isolation and assignment1 of the UDP-glucose pyrophosphorylase gene (UGP2) to porcine chromosome 3q21→q22 by FISH and by analysis of somatic cell and radiation hybrid panels

The enzyme UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyses the conversion of glucose-1-phosphate to uridine diphosphate glucose. As a part of the glycogen biosynthesis pathway, the encoding gene UGP2 is of interest as a candidate gene for metabolic dysfunctions of meat producing farm animals. In human UGP2 was assigned to chromosome 2p14→ p13 (Cheng et al., 1997), which is in good agreement with the provisional assignment of the porcine UGP2 gene to chromosome 3 (Milan et al., 1996) and comparative mapping data showing conservation of homology between HSA2 and SSC3q (Jasny and Hines, 1999). The present paper aims at presenting an accurate physical localization of the UGP2 gene in pigs.