Vol. 56, no. 2: 227-231, 2003
CARYOLOGIA
New methods for comparison of chromosomes
within and between species
JULIE ANNE PLUMMER1, 2, FUCHENG SHAN1, NICHOLAS GALWEY1, 3 and GUIJUN YAN1
1
Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley,
WA, 6009, Australia.
2 Current Address: Oxagen Ltd, 91 Milton Park, Abbingdon, Oxfordshire OX14 4RY, United Kingdom.
Abstract - A new method (called New Relative Length) was developed for comparison of chromosome size when both paired and unpaired chromosomes are
present. All chromosomes were used in the calculation. A method of graphical
display of karyotype data (called a Karyotype Graph), plotting New Relative
Length on the vertical axis and arm ratio on the horizontal axis was used to indicate sampling error, to pair chromosomes and to match similar chromosomes in
related species. Terms for Chromosome Size were also defined namely Small,
Medium or Large depending on New Relative Length as a proportion of total
chromosome length. These terms could also be used to compare chromosomes
within species and between related species.
INTRODUCTION
The standard karyotype descriptions of relative length and arm ratio work well where chromosomes can readily be paired. They were
designed for comparisons within species where
there is little variation between genotypes. However, these measurements are difficult to use
where chromosomes do not pair or for comparisons between related taxa with different karyotypes. Changes in chromosome morphology
reflect evolution and it would therefore be advantageous to have a system whereby karyotypes of
related genotypes and species could be compared. This paper proposes such a system.
Boronia is a genus of the family Rutaceae with
considerable variation in chromosome number
and morphology. Diploid chromosome numbers
vary from 14 to 72 (SMITH-WHITE 1954; STACE et
al. 1993). Intraspecific variation in chromosome
number (SMITH-WHITE 1954; ASTARINI et al.
1999) and morphology is also present, including
* Corresponding author: fax +61 8 9380 1108; e-mail:
[email protected]
different chromosome length and centromere
position and the presence of unpaired chromosomes (YAN et al. 2001). In genera where the
evolutionary relationships are disputed, such as
Boronia (S MITH -W HITE 1954; W ILSON 1971;
WESTON et al. 1984; STACE et al. 1993), a means
to assess differences in chromosome morphology
between taxa would help to elucidate the inheritance of genetic material and the relationships
between species. Therefore Boronia was used to
test the value of the new methods.
MATERIALS AND METHODS
Observations were made on metaphase cells in
which individual chromosomes were clearly distinguishable. A single genotype of Boronia heterophylla
‘Red’ and one of B. molloyae were examined. A minimum of ten mitotic cells were used to determine chromosome number. Photographs of the 5 best individual
cells were enlarged for karyotyping. Chromosomes
were placed in pairs arranged in order of size and
numbered from 1 (largest) to 2n (smallest; 2n =
diploid number). Individual chromosome length, and
long and short arm length were measured.
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PLUMMER, SHAN, GALWEY
Traditional relative length (e.g. LEVAN et al. 1964;
STEBBINS 1971) is calculated for pairs of chromosomes
and the calculations are not designed for unpaired chromosomes. New Relative Length was calculated individually for each chromosomes within a nucleus, and
was given by (chromosome length of the individual
chromosome / total length of all chromosomes) x 100.
Arm ratio (long arm length/short arm length; LEVAN et
al. 1964) was also calculated for each chromosome.
To indicate variation due to sampling errors and to
pair chromosomes within a genotype, arm ratio was
plotted against New Relative Length for four sets of
chromosomes in B. heterophylla ‘Red’. To indicate
similarities between species the mean values for B.
heterophylla ‘Red’ and B. molloyae were presented in
a separate graph, which we called a Karyotype Graph.
For comparisons across genotypes, categories of
Chromosome Size using the New Relative Length
were developed. Total chromosome length was arbitrarily set at 100 so that if all chromosomes were of
equal length, each was 100/2n long, where n = haploid
number. Chromosomes of Small New Relative Length
and YAN
(S) were defined as between 100/(2 x 2n) and 100/(1.5
x 2n), those of Medium New Relative Length (M)
were in the range 100/(1.5 x 2n) to 100/(0.75 x 2n)
with a mid-point of 100/2n, and those of Large New
Relative Length (L) were in the range 100/(0.75 x 2n)
to 100/(0.5 x 2n). This notation can be extended to
define smaller and larger chromosome categories if
required. For example, a category of Very Small can be
created by defining a new boundary using a divisor
twice as large as that used to define the outer boundary of the Small category (i.e. outer limit of Very Small
=100/2 x 2 x 2n). Similarly a category of Very large can
be created by defining a new boundary using a devisor
which is half as big as the outer boundary of the Large
category (i.e. outer limit of Very Large = 100/0.5 x 0.5
x 2n). Further categories can be created by repeating
this pattern. For species with pairs of chromosomes
New Relative Length is half the traditional relative
length. There are equivalent values for Small (100/2n
to 100/1.5n) Medium (100/1.5n to 100/0.75n with a
midpoint of 100/n) and Large (100/0.75n to 100/0.5n)
chromosomes specified above.
a
b
1 µm
c
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
B. heterophylla ‘Red’
b
1 µm
c
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
B. molloyae
Fig. 1 – Karyotypes of Boronia heterophylla ‘Red’ and B. molloyae. a) Chromosomes in nucleus at metaphase of mitosis;
b) karyotype of chromosomes; c) idiogram of chromosomes.
COMPARISON OF CHROMOSOMES WITHIN AND BETWEEN SPECIES
In comparing karyotypes both the length of chromosomes and the position of the centromere are used.
Each chromosome thus has two values represented
by the mean of a series of observations. If chromosomes within a genotype are similar, condensation
errors can sometimes make it difficult to determine
which chromosome is which. Graphical presentation
of the two values simultaneously permits resolution of
some of these ambiguities, and hence fuller identification of pairs of chromosomes. Identification of pairs
is by inspection: a statistical test of the confidence of
this identification is not attempted here.
RESULTS AND DISCUSSION
LEVAN et al. (1964) introduced standardized
definitions of arm ratio when they numerically set
limits for the use of the terms median, submedian,
subterminal and terminal. We have extended this
concept here to include standardized definitions of
New Relative Length and Chromosome Size.
There are 15 chromosomes in Boronia heterophylla ‘Red’ (Fig. 1). Traditional relative length was
therefore difficult to calculate taking account of
the odd chromosome, however New Relative
229
Length indicated the length of all chromosomes
(Fig. 2). The largest chromosome was single with a
New Relative Length of 9.2. All other chromosomes could be grouped into seven pairs in which
mean New Relative Length ranged from 8.0 to 4.8.
Condensation errors resulted in a range of values
for each chromosome. The largest range in New
Relative Length for a chromosome was 5.6-7.8
(Chromosome 11 in Fig. 2) with a mean of 6.4
across the four cells presented. Chromosome 11
was paired with Chromosome 10 (range 6.0-7.1,
mean = 6.4). The smallest range was 6.3-6.7 for
Chromosome 7, with a mean of 6.5, and this was
paired with Chromosome 6 (Fig. 2; range 6.3-7.6,
mean = 6.8).
Arm ratios also varied between cells. The
smallest range in arm ratios was 1.0 - 1.2 for the
single Chromosome 1 (Fig. 2), with a mean of 1.1.
The largest range was 4.8 – 5.5 for Chromosome
12, with a mean of 5.1, and this was paired with
Chromosome 13 (Fig. 2; range 4.7 –5.0, mean =
4.8). Three pairs of chromosomes were subterminal, three were submetacentric and one was
metacentric, according to the classification of
LEVAN et al. (1964). However, the numerical com-
Fig. 2 – Identification of similar chromosomes in Boronia heterophylla ‘Red’ using individual measurements of New Relative
Length and Arm Ratio. For clarity only measurements of four cells are included. Note: paired chromosomes are represented by matching solid and hollow symbols; small and large symbols represent different chromosomes.
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PLUMMER, SHAN, GALWEY
and YAN
Fig. 3 – Small (100/(2 x 2n) to 100/(1.5 x 2n)), Medium (100/(1.5 x 2n) to 100/(0.75 x 2n)) and Large (100/(0.75 x 2n) and 100/
(0.5 x 2n)) category boundaries for New Relative Length for chromosomes of Boronia molloyae (2n=16) and B. heterophylla ‘Red’
(2n=15). 2n = total or diploid chromosome number. Category boundaries for standard relative length are indicated in parentheses.
parisons of arm length given above better indicate
relatedness of chromosomes, especially when
New Relative Length is considered simultaneously (Fig. 2).
Differences in observed chromosome length
and arm ratio were possibly due to differences in
chromosome condensation rates and to arms
bending up or down in the cell preparation which
can only be viewed in two dimensions. Graphical
presentation of the numerical range of New Relative Lengths and arm ratios indicates whether or
not further replication is required.
Fig. 4 – Karyotype Graph of mean values for New Relative Lengths and arm ratios of two species from the genus Boronia,
section Boronia, series Boronia (WILSON 1998; Wilson pers. comm.): B. heterophylla ‘Red’ and B. molloyae.
231
COMPARISON OF CHROMOSOMES WITHIN AND BETWEEN SPECIES
Boronia molloyae has a diploid number of 16
(Fig. 1; SMITH-WHITE 1954) and B. heterophylla
‘Red’ has 2n=15 (Fig. 1; ASTARINI et al. 1999). If
all chromosomes were of average and equal
length within these taxa then the New Relative
Length of all B. molloyae chromosomes would
be 100/2 x 16 = 6.3 and that of B. heterophylla
‘Red’ 100/2 x 15 = 6.7 (Fig. 3). These values represent the center point of Medium length chromosomes. Chromosomes within each species
were classified as Small, Medium or Large based
on New Relative Length (Fig. 3). Comparisons of
chromosomes between species can then refer to
the presence and number of Small, Medium and
Large Chromosomes. The New Relative Length
range for Large chromosomes was 8.9 – 13.3 in B.
heterophylla ‘Red’ and 8.3 – 12.5 in B. molloyae.
Hence B. heterophylla had a single Large chromosome (9.2) and B. molloyae a pair of chromosomes (8.1 and 8.4) just on the Medium/Large
border (Fig. 3, 4). All other chromosomes in both
species were Medium. Thus species of Boronia
contained some similar and some dissimilar chromosomes. Again these could be identified by
simultaneous comparison of graphically presented New Relative Length and arm ratio (Fig. 4).
A relative length is used only within a karyotype to avoid errors generated by different stages
of condensation. Comparison of relative length
between species assumes that the total DNA content is similar within a taxonomic group, such as
a genus (STEBBINS 1971). It is relatively common
for chromosome arms to cross over, with small
rearrangements conserving the DNA compliment.
However, this may not be the case following complicated rearrangements of arms.
Where chromosomes are large enough to
examine and distinguish easily, different species
and even genotypes within species can be given
distinct karyotypes. These methods will provide
a significant extension to the current methods
(STEBBINS 1971; LEVAN et al. 1964) of presenting
such data and comparing chromosomes between
taxa.
Acknowledgements – We thank the Australian
Research Council - Small Grants scheme for financial assistance.
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Received December 11, 2002; accepted February 17, 2003