Elsevier

Gene

Volume 227, Issue 2, 18 February 1999, Pages 223-230
Gene

Sequence scanning chicken cosmids: a methodology for genome screening

https://doi.org/10.1016/S0378-1119(98)00610-6Get rights and content

Abstract

The chicken genome is relatively poorly studied at the molecular level. The karyotype 2n=78 is divided into three main chromosomal sub-groups: the macrochromosomes (six pairs), the intermediate microchromosomes (four pairs) and the microchromosomes (29 pairs). Whilst the microchromosome group comprise only 25% of the DNA, increasing evidence is proving that this is disproportionate to their gene content. This paper demonstrates the utility of cosmid sequence scanning as a potential method for analysing the chicken genome, providing an economical method for the production of a molecular map. The GC content, gene density and repeat distribution are analysed relative to chromosomal origin. Results indicate that gene density is higher on the microchromosomes. During the scanning process an example of conserved linkage between chicken and human (12q34.2) has been demonstrated.

Introduction

Recent international efforts to create a linkage map of the chicken genome have been focused towards the identification of traits of agricultural and biological interest. Invariably, by their nature, these traits, such as meat and egg production, are polygenic (quantitative trait loci) and require a high marker density. There are currently over 600 loci mapped on the linkage map, distributed over 2600–3000 cM (Cheng, 1996). With a rapidly expanding number of markers being mapped, studies are increasingly moving towards the production of a molecular map and detailed studies of gene structure and synteny (Burt et al., 1995).

As a result, the chicken genome is proving to be of increasing interest to comparative genomics. It has a genome size of 1.2×109 bp (Tiersch and Wachtel, 1991), one third of that of mammals, of which only 17% consists of repeat sequences. This small size is also, in part, due to a reduction in intron sizes (Hughes and Hughes, 1995). Chicken therefore represents a relatively compact and intermediate evolutionary model placed between mammals and more distant vertebrates under study, such as the puffer fish and Amphioxus.

Chicken has a karyotype consisting of 39 pairs of chromosomes (including the ZZ/ZW sex chromosome pair), which are delimited into three groups, determined on the basis of size : six pairs of large, morphologically distinguishable macrochromosomes (Ma), four pairs of intermediate size, intermediate microchromosomes (I.mi) and 29 pairs of very small individually indistinguishable microchromosomes (mi) (Bloom et al., 1993). Initial impressions that the small microchromosomes were probably the more minor genetic components are currently being revised. Whilst they only constitute 25% of the total DNA, they show an increased concentration of CpG islands (McQueen et al., 1996), a feature associated with mammalian coding regions, are very GC rich, have fewer repeats and are therefore potentially more gene dense than the macrochromosomes. So far, the majority of genes sequenced and localised in chicken have been mapped to the larger chromosomes, particularly chromosome 1 (Dominguez-Steglich et al., 1992a, Klein et al., 1996, Palmer and Jones, 1986, Shaw et al., 1991, Tereba et al., 1991). This is due mainly to technical reasons, but has influenced the perception of gene distribution in chicken.

In order to redress this problem, and gain a rapid insight into the molecular content of the different chromosome types, an intermediate method between the levels of genetic mapping and high-resolution sequencing has been used. Sequence scanning is an established technique for sampling insert DNA content in clones with large insert sizes. However, its effectiveness depends upon the gene density of the organism under study and, in particular, it has proved of great utility in analysing more compact genomes such as the Japanese puffer fish (Fugu rubripes) (http://Fugu.hgmp.mrc.ac.uk/). This report demonstrates the utility of the sequence scanning technique to analyse the chicken genome.

Section snippets

Shotgun cloning

The cosmid DNAs [selected at random by McQueen et al. (1998) from a Clontech male Leghorn library] were isolated using the standard alkaline lysis method (Birmboim and Doly, 1979). One microgram of DNA was sonicated to an approximate size of 500 bp, end-filled with T4 DNA polymerase, PEG precipitated and ligated into a phosphatased blunt-ended vector, either SmaI-cut M13 (Amersham) or EcoRV-cut pBluescript (Stratagene).

Sequence generation: M13 clones

After transformation, M13 clone DNAs were isolated using the Qiagen (BioRobot

Gene mapping

Nineteen cosmids were chosen randomly from a chicken genomic library (McQueen et al., 1998). They were sequence scanned with an average of 48 shotgun clones sequenced per cosmid. The average cosmid insert size was relatively small at 32 kb, hence the sequence data generated approximates to 70% single pass coverage of each cosmid. Table 1 gives the general statistics for the sequence scanning of the 19 cosmids.

All clones were searched against the SWISS-PROT, TREMBL and EMBL databases using BLAST

Discussion

The chicken genome is of great interest in comparative genomics. It represents a relatively compact and intermediate evolutionary model, ideally placed between mammals and the more distant vertebrates under study, such as the puffer fish and Amphioxus. Additionally, it appears to have a complex genomic structure, with two or perhaps three distinct chromosomal sub-types, the macrochromosomes, intermediate microchromosomes and microchromosomes. However, current data on the chicken genome are

Acknowledgements

This work was supported by an MRC Project Grant.

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