K. Byrne

A medium-density genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.USDA-MARC family and data for validating this family. P. Creighton, C. Skidmore, T. Holm, and A. Georgoudis provided some validation data for the BOVMAP families. R. Fries, S. Johnson, S. Solinas Toldo, and A. Mezzelani kindly made some of their FISH assignments available before publication. We wish to thank all those researchers who kindly sent us probes and DNA primers.

Genetic mapping of the black tiger shrimp Penaeus monodon with amplified fragment length polymorphism

Abstract We report construction of an initial genetic linkage map for the black tiger shrimp, Penaeus monodon. Mapping was carried out using polymorphic markers derived from 23 Amplified Fragment Length Polymorphism (AFLP) primer pairs. These were analysed on three reference families of known pedigree. A total of 673 polymorphic AFLP loci that conformed to expected Mendelian segregation ratios were scored in three families, and these were used to construct separate male and female linkage maps for each family. AFLP markers that consisted of a segregating fragment of the same size, amplified with the same primer pair in two or more of the reference families, were considered to be common markers. 116 such common AFLP markers were used to construct a common linkage map across the three families. This linkage map has 20 linkage groups covering a total genetic distance of 1412 cM. Future directions for genetic mapping in P. monodon are discussed in light of these initial data.

The development and application of genetic markers for the Kuruma prawn Penaeus japonicus

Microsatellite and Amplified Fragment Length Polymorphism (AFLP) DNA markers have been characterised for use in establishing pedigrees, linkage mapping and identifying Quantitative Trait Loci (QTL) influencing commercially important traits in P. japonicus. Low efficiency of Microsatellite characterisation from P. japonicus genomic DNA, due to the high frequency and extended length of simple sequence repeats, make these DNA markers unsuitable for linkage mapping studies. Unique sequence flanking repeats necessary for PCR primer design were difficult to obtain due the extended lengths of the repeats. Microsatellites that were characterised displayed between 4-24 alleles and heterozygosities between 47-91% in unrelated animals. No P. japonicus derived microsatellite successfully amplified sequences in P. monodon, P. esculentus or P. stylirostris. AFLPs were developed as an alternative to microsatellites. Over 570 polymorphic loci were defined using different primer combinations. AFLPs are robust with some polymorphisms conserved across families. PCR amplification and sequencing of excised bands allowed development of Sequence Tagged Sites from AFLPs. A primary linkage map based on a three generation pedigree, genotyped at 246 AFLP loci has been constructed. It incorporates 129 markers in 44 linkage groups with an estimated genome coverage of approximately 57%.

Genetic mapping of the kuruma prawn Penaeus japonicus using AFLP markers

Abstract Amplified fragment length polymorphism (AFLP) analysis is a rapid, efficient technique for detecting large numbers of DNA markers for linkage analysis. We have used AFLP markers in a two-way pseudo-testcross strategy to generate genetic maps of a Penaeus japonicus family. A two-stage selective genetic mapping strategy was applied in this study. The initial stage involved the linkage mapping on 46 progeny from two tails (top and bottom 6%) of the size distribution of an intermediate cross family (F1 from HH×LL and F2 from HL×LH) using 54 pairs of AFLP primer combinations. The second stage of linkage mapping involved genotyping an additional 56 progeny (top and bottom 8%) of the same family with the same 54 primer combinations based on the framework map from the first stage. Of 535 polymorphic fragments scored in 46 progeny of the first stage, 355 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. The other 154 markers followed a 3:1 Mendelian segregation ratio. Of 502 bands scored in 56 progeny of the second stage, 359 segregated in a 1:1 ratio and 138 in a 3:1 ratio. When the markers with a 1:1 segregating ratio were combined from both stages (401 in total), 217 markers were ordered into 43 linkage groups (1780 cM) of the paternal map and 125 markers in 31 linkage groups (1026 cM) of the maternal map. The average density of markers was approximately 1 per 10 cM. To investigate the homologies between two parental maps, we included 182 markers segregating 3:1 in the analysis. One homologous linkage group was recognised. The sex marker initially mapped on the maternal parent map was also confirmed in the second-stage mapping with more progeny information. The linkage data developed in these maps will be used to detect loci controlling commercially important traits.

Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus.

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1, 4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.

Tissue-Specific Expressed Sequence Tags from the Black Tiger Shrimp Penaeus monodon

Abstract: Expressed sequence tag data were generated from complementary DNA libraries created from cephalothorax, eyestalk, and pleopod tissue of the black tiger shrimp (Penaeus monodon). Significant database matches were found for 48 of 83 nuclear genes sequenced from the cephalothorax library, 22 of 55 nuclear genes from the eyestalk library, and 6 of 13 nuclear genes from the pleopod library. The putative identities of these genes reflected the expected tissue specificity. For example, genes for digestive enzymes were identified from the cephalothorax library and genes involved in the visual and neuroendocrine system from the eyestalk library. A few sequences matched anonymous EST or genomic sequences, and others contained mini-satellite or microsatellite repeat sequences. The remainder, 31 from the cephalothorax library, 25 from the eyestalk library, and 5 from the pleopod library, were sequences of high nucleotide complexity with no matches in any database searched and thus may represent novel genes.

Gene expression profiling of bovine in vitro adipogenesis using a cDNA microarray

The gene expression profile of bovine bone marrow stromal cells undergoing adipogenesis was established using a custom cDNA microarray. Cells that were treated with adipogenic stimulants and those that were not were collected at each of the six time points, and gene expression differences between the treated and untreated samples within each time point were compared using a microarray. Statistical analyses revealed that 158 genes showed a minimum fold change of 2 in at least one of the five post-differentiation time points. These genes are involved in various cellular pathways and functions, including lipogenesis, glycolysis, cytoskeleton remodelling, extracellular matrix, transcription as well as various signalling pathways such as insulin, calcium and wingless signalling. The experiment also identified 17 differentially expressed (DE) microarray elements with no assigned function. Quantitative real-time PCR was employed to validate eight DE genes, and the PCR data were found to reproduce the microarray data for these eight genes. Subsequent gene ontology annotation was able to provide a global overview of the molecular function of DE genes during adipogenesis. This analysis was able to indicate the importance of different gene categories at various stages of adipogenic conversion, thereby providing further insights into the molecular changes during bovine adipogenesis.