parameters of the chicken genome (gallus gallus)

Parameters of the chicken genome (Gallus gallus)J Smith, D W Burt

Summary

As more information on the chicken genome is

gathered, it is becoming increasingly more

important to be able to correlate genetic and

physical maps. Quantitation of the chicken

karyotype is important in establishing para-

meters which define the genome. Here we

report on the physical lengths of the chicken

macrochromosomes and establish the DNA

content of each, thus identifying implicitly

how much of the genome is represented by the

microchromosomal component. For the first

time, genetic and physical data on the chicken

karyotype are presented in relation to one

another.

Keywords: chicken, genetic map, genome size,

physical map

Introduction

The chicken karyotype

The chicken genome consists of eight large

`macrochromosomes' and 30 pairs of cytologi-

cally indistinguishable, `microchromosomes' ±

a karyotype similar to that found in most bird

species (Takagi & Sasaki 1974; TegelstroÈm and

Ryttman 1981; Rodionov 1996). The sex chro-

mosomes are Z and W, with the female being the

heterogametic sex. Tiersch et al. (1989; 1991)

analysed the nuclear content of 45 different

vertebrate species by flow cytometry, followed

by 135 different avian species and concluded

that the chicken had 2.5 pg DNA per diploid

nucleus. This is equivalent to a size of 2.4 ´ 109

bp.

Previous genetic and physical analyses

The five largest macrochromosomes have been

flow sorted (Stubblefield & Oro 1982) as these

are the easiest chromosomes to identify. These

five chromosomes have also been the target for

genetic study. The number of chiasmata formed

in these chromosomes has been analysed (Pol-

lock & Fechheimer 1978; Rodionov et al. 1992a)

and genetic lengths estimated (Rodionov et al.

1992b). In order to estimate the DNA content of

each chromosome as a percentage of the haploid

genome, Bloom et al. (1993) cut out chromo-

some images from photomicrographs and

weighed them, and compared each chromosome

with respect to the others. However, no com-

plete, definitive study has been performed to

determine the physical parameters of the

chicken chromosomes. In this report we present

quantitative physical data on the chicken

karyotype and compare it to the current genetic

data (Burt et al. 1995; Burt et al. 1997; Cheng

et al. 1998).

Materials and methods

Preparation of metaphase cells

Chicken metaphase chromosome spreads were

prepared from 48 h old whole chicken embryos

after treatment with 0.005% colchicine solu-

tion. Cells were swollen by treatment with

hypotonic serum, fixed in 1:3 acetic acid/

methanol and dropped onto ethanol cleaned

slides. Slides were allowed to air dry and were

stored at ±20 °C. Chromosomes were stained by

immersing the slides for 10 s in a 200 ng/ml

propidium iodide solution.

Microscopy and analysis

Chromosome images were captured on a Biorad

MRC-600 laser scanning confocal microscope

fitted with a propidium iodide filter, and linked

to a Viglen 4DX475 PC. These images were then

transferred to a Macintosh Performa 6320

PowerPC, where metaphases were analysed

with NIH-Image version 1.61 software (public

domain; URL = http://rsb.info.nih.gov/nih-

image).

Measurement of DNA content

Metaphase chromosomes were stained with

propidium iodide which does not band DNA

and does not bind preferentially to any parti-

cular region of the chromosomes, thus giving a

uniform fluorescent image (Ponce de Leon et al.

1992). It was therefore assumed that any

Animal Genetics,

1998, 29, 290±294

J SmithD W BurtRoslin Institute (Edin-

burgh), Roslin, Mid-lothian EH25 9PS, UK

ã 1998 International Society for Animal Genetics 290

Correspondence: Dr J Smith.

Accepted 6 May 1998

fluorescence seen was a representation of total

DNA content. The intensity of fluorescence was

measured for each chromosome and compared

to the total amount of fluorescence for that

particular nucleus. Each chromosome in each

diploid was measured twice and eight meta-

phases were analysed for each chromosome,

giving a set of 16 data points for each result.

Chromosome arm lengths

Centromere ratios (length of q-arm/total chromo-

some length) were calculated for each of the eight

macrochromosomes,togetherwiththeZandWsex

chromosomes.Foreachchromosomeanalysed,16

individual chromosomes were measured across

eightdiploidmetaphasespreads.Chromosome6is

acrocentricandonlyhasaverysmallp-arm,andso

directmeasurementsforthischromosomewerenot

possible.

Results

DNA content

Using fluorescence as a measure of DNA

content, an estimate of the percentage of the

haploid genome as represented by each chro-

mosome is presented in Table 1. Assuming

2.5 pg DNA per diploid cell, the size of the

chicken genome is 2.4 ´ 109 bp, or 1.2 ´ 109 bp

per haploid complement. Based on this, the size

of each chromosome in megabases (mb) is also

given.

Chromosome arm lengths

Centromere ratios for the macrochromosomes

(excluding acrocentric chromosome 6) and

chromosomes Z and W were calculated as

described and are shown in Table 2.

DNA content and chromosome lengths

The measured values for the DNA content of

each chromosome and the measured length of

that particular chromosome were compared for

each of the eight macrochromosomes. Using

chromosome 1 as a standard within each

nucleus, the measured fluorescence intensity

values were compared to the actual measured

lengths (Table 1). Figure 1 shows the measured

values plotted against the physical size of each

chromosome as determined in this study. It has

been established that the association is linear.

Discussion

Physical data

Eight chicken metaphase spreads which

showed the full set of 78 chromosomes upon

propidium iodide staining were analysed in

order to obtain quantitative information on the

Table 1. Physical lengths of chicken chromosomes

Chromosome

Area*

(%)

DNA contenty

� SE (%)

Physical

length (mb)

Measured length � SE

(arbitrary units)

1 20.5 20.8 � 0.8 250 1.14 � 0.07

2 12.8 15.1 � 0.3 181 0.84 � 0.04

3 9.0 11.5 � 0.3 138 0.63 � 0.02

4 7.1 9.1 � 0.3 109 0.48 � 0.02

5 5.8 5.3 � 0.2 64 0.34 � 0.02

6 3.2 3.5 � 0.1 42 0.24 � 0.01

7 3.2 3.4 � 0.2 41 0.22 � 0.01

8 1.9 2.5 � 0.1 30 0.20 � 0.01

Z 7.1 8.4 � 0.4 101 0.49 � 0.03

W 1.9 2.8 � 0.2 34 0.20 � 0.01

*Bloom et al. (1993); ydata from this study.

Table 2. Centromere ratios and arm lengths

Chromosome q/(p + q) � SE

Chromosome

arm

Physical

length (mb)

1 0.613 � 0.011 1p 96.75

1q 153.25

2 0.634 � 0.009 2p 66.06

2q 114.75

3 0.829 � 0.011 3p 23.6

3q 114.4

4 0.747 � 0.010 4p 27.58

4q 81.42

5 0.749 � 0.011 5p 16.06

5q 47.94

6 ± ± 42.00

7 0.667 � 0.012 7p 13.65

7q 27.35

8 0.584 � 0.012 8p 12.48

8q 17.52

Z 0.535 � 0.006 Zp 46.96

Zq 54.03

W 0.576 � 0.007 Wp 14.42

Wq 19.58

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 290±294

291

Parameters of the

chicken genome

chicken karyotype. For each chromosome, the

intensity of fluorescence was measured, as a

representation of DNA content. We have shown

this to be a valid assumption, as chromosome

length is linearly related to DNA content.

According to Bloom et al. (1993), the total

haploid DNA content of the eight macrochro-

mosomes and the Z and W sex chromosomes is

72%. In the results presented here, the macro-

and sex chromosomes (ZW female) are seen to

account for 82% of the haploid genome. The

microchromosomal fraction of DNA has to be

given as a percentage of the diploid genome as it

is not possible to accurately divide up the small

chromosomes into their correct haploid set, as

they are cytologically indistinguishable from

one another. The total intensity of fluorescence

was that of a diploid nucleus and each chromo-

some's fluorescence measured against the total

as such, thus showing the microchromosomes

to represent 23% of the diploid female genome.

Bloom's earlier work, however, indicates a 32%

microchromosomal element in the diploid cell,

a value which is acknowledged as probably

being an over-estimate (Bloom et al. 1993). It

may be that area is not strictly proportional to

DNA content, with coiling of the macrochromo-

somal DNA being a possibility (Rodionov 1996),

thus resulting in an under-estimate of DNA

content on the macrochromosomes. Table 1

compares the physical values obtained in this

study with those of Bloom et al. (1993).

Arm lengths

From direct fractional length measurements of

fluorescent chromosome images, the centromere

ratios for each of the macrochromosomes

(except chromosome 6) and the Z and W

chromosomes have been calculated. This

enabled us to relate these values to the physical

and genetic data and to obtain chromosome arm

length measurements as represented in mb and

the equivalent centiMorgan (cm) distances.

Combining the data thus far presented, enabled

us to calculate the physical size of the arms of

each of the macrochromosomes and the sex

chromosomes (Table 2).

Fig. 1. DNA content in relation to chromosome length (see Table 1 for

values).

Table 3. Genetic lengths of chicken chromosomes

Chromosome

(LG)

No. of

markers

East Lansing

size (cm)

% Expected %

male genome

No. of

markers

Compton

size (cm)

% Expected %

female genome

1 (E01C01) 127 611 16.2 20.5 62 729 18.9 22.0

2 (E06C02) 77 385 10.2 14.7 53 443 11.5 16.0

3 (E02C03) 65 351 9.3 11.2 50 504 13.1 12.2

4 (E05C04) 47 216 5.7 8.8 33 330 8.5 9.6

5 (E07C05) 31 154 4.1 5.2 25 172 4.5 5.6

6 (E11C10)* 33 168 4.5 3.4 12 207 5.4 3.7

7 (E21C07) 20 171 4.5 3.3 20 149 3.9 3.6

8 (E43C12) 21 77 2.0 2.4 5 102 2.6 2.6

Z 29 208 5.5 8.4 ± ± ± ±

W ± ± ± ± ± ± ± ±

Micros 193 1434 38.0 22.1 105 1218 30.6 24.7

Total 643 3774 100 100 365 3855 100 100

LG, linkage group assignments to the eight macrochromosomes.

Genetic data is freely accessible on the Roslin Institute World Wide Web server (URL = http://www.ri.bbsrc.a-

c.uk/genome_mapping.html).

*Pitel et al. (1998).

Genetic data from Burt et al. (1995, 1997) and Cheng et al. (1998).


Comparison of physical and genetic data

After cytological studies of recombination fre-

quency in chicken lampbrush chromosomes,

Rodionov et al. (1992a,b) reported a total

genetic length of 2950±3200 cm for the haploid

genome. However, when genetic linkage infor-

mation from the East Lansing (Crittenden et al.

1993) and Compton (Bumstead & Palyga 1992)

reference crosses is examined, it appears the

total genetic length of the haploid genome is

closer to 3800 cm. However, the microchromo-

somal genetic lengths are probably overesti-

mated, as there is a paucity of markers on these

chromosomes. It is also thought that the micro-

chromosomes have a higher rate of recombina-

tion than their larger counterparts (Rodionov

et al. 1992a; Rodionov 1996), making it difficult

to estimate the true genetic length of these

chromosomes. Table 3 shows the estimated

genetic size (cm) of each macrochromosome

based on current linkage information and the

expected size as determined by the physical

data presented. The genetic data presented to

date is not complete and does not represent

100% genome coverage. However, when the

length of the current genetic linkage map is

compared to the expected sizes based on the

physical measurements in this study, we esti-

mate that over 90% coverage has been obtained

with the East Lansing population (where the

majority of markers have been mapped).

If the physical data presented in this paper

are compared to the genetic linkage informa-

tion, an estimate of the number of mb repre-

sented by 1 cm can be made. When physical

data is compared to East Lansing genetic linkage

data, 1 cm equates to around 440 kb when

averaged over chromosomes 1±8. Using Comp-

ton data, this value is around 350 kb. Figure 2

depicts the correlation of genetic (cm) and

physical (mb) lengths in the eight macrochromo-

somes. On average, 1 cm is equivalent to 396 kb.

It should be noted that chromosomes 6 and 7

show lower values, indicating longer than

expected genetic lengths. Since these two

chromosomes have a marker coverage no less

than any of the other chromosomes, it would

appear that the recombination rate may be

around twofold higher than that of chromo-

somes 1±5.

Conclusion

We have presented a quantitative physical and

genetic analysis of the chicken genome, and set

parameters which will allow integration of the

physical and genetic maps. Having a standard

set of statistical values which define the

karyotype is important for all kinds of further

mapping studies such as scaling radiation

hybrid maps, identifying regions for linkage

analysis and facilitating the use of comparative

mapping.

Acknowledgements

Thanks to Graeme Robertson for excellent

microscopy assistance. This work was sup-

ported by the Biotechnology and Biological

Research Council, UK and by EC grant no.

BIO4-CT95-0287, as part of the ChickMAP

project.

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parameters of the chicken genome (gallus gallus)

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