Genetic Maps: Direct Meiotic Analysis
Genetic Maps: Direct Meiotic Analysis
Maj HulteÂn, University of Warwick, Coventry, UK
Charles Tease, University of Warwick, Coventry, UK
Intermediate article
Cytogenetic analyses provide a direct means of obtaining critical information on the
patterns of meiotic recombination, at the genomic and chromosome-specific levels, in
both male and female germ cells. This information provides insight into the control of
meiotic recombination in human germ cells and allows the construction of accurate
sex-specific genetic maps.
Article contents
Introduction
Chiasmata
MLH1 Foci
Intersex Differences in Recombination
Conclusion
Introduction
Genetic recombination maps illustrate the number and
distribution of crossovers along the length of chromosomes. Originally, it was assumed that the larger the
physical distance between two gene loci, the greater the
probability (and therefore number) of crossovers
between them. By using the rate of recombination,
one could then obtain an estimation of physical
separation. However, it soon became clear that crossovers are not equally distributed along chromosomes:
some chromosomal regions are more favored for
recombination than others (HulteÂn, 1974). This
property has implications for the use of recombination
maps to locate genetic loci of medical importance and
also raises the interesting question of how crossover
numbers and distributions are controlled in human
germ cells.
There are two approaches to analyzing patterns of
meiotic recombination in human germ cells and
therefore of producing recombination maps for the
human genome. The ®rst, which has been used for
constructing most human genetic maps, is an indirect
method that uses genetic linkage studies, that is,
tracing the coinheritance of polymorphic markers
(genes/proteins/DNA) in families. These studies provide the average rate of crossing-over for intervals
bounded by selected polymorphic markers. By combining data from the appropriate intervals, it is then
possible to estimate the rates of recombination for
speci®c chromosomes, and for the genome as a whole.
The second approach makes use of direct analysis
of the cells in which recombination occurs, namely
spermatocytes and oocytes, to identify crossover
numbers and distributions. This is the approach
detailed here; the relative strengths and weaknesses
of these two methods are compared elsewhere (See
Genetic Mapping: Comparison of Direct and Indirect
Approaches).
Two recombination `end points' can be used for
direct assessment of patterns of crossing-over, namely
chiasmata and MLH1 foci. The former approach has
882
been available for many years, whereas the latter is a
recent development. However, as both examine the
same event, that is, meiotic recombination, they should
yield essentially identical results. Similarly, we should
see the same conclusions from direct and indirect
approaches to assessment of meiotic recombination.
These expectations are met for the two direct methods
but some intriguing discrepancies appear between
direct and indirect approaches.
Chiasmata
Chiasmata are the cytologically visible consequences
of crossing-over. They become visible when homologous chromosome pairing lapses at the end of
pachytene of prophase I. Sister chromatid adhesion
proximal and distal to the crossover maintains the
connection between homologous chromosome pairs to
form an X-shaped (chiasma) structure (Figure 1). In
principle, it should be possible to identify chiasmata
from early diplotene (i.e. once intimate chromosome
pairing has completely broken down after pachytene),
but in practice this has not proved to be the case for
human germ cells. At diplotene, bivalents are long,
slender and exhibit many twists; these twists cannot
easily be differentiated from chiasmata (see HulteÂn
et al., 1978). Analysis of chiasmata is therefore usually
performed at later stages (diakinesis and metaphase I)
of meiosis I when chromosomes are more condensed
(Figure 2). Unfortunately, this analytical method is
currently only applicable to spermatocytes. In females,
the diakinesis and metaphase I cell stages take place
just before ovulation, thus limiting the numbers of
oocytes available for investigation. It has also proved
problematical to obtain suf®ciently clear chromosome
preparations from these cells for chiasma analyses.
Consequently, chiasma-based genetic recombination
maps are only available for human males. Cells for
analysis are obtained by testicular biopsy. With the
normal production of several hundred million sperm
NATURE ENCYCLOPEDIA OF THE HUMAN GENOME / &2003 Macmillan Publishers Ltd, Nature Publishing Group / www.ehgonline.net
Genetic Maps: Direct Meiotic Analysis
daily, even small biopsies provide a reasonable sample
size of cells in spontaneous division at the diakinesis/
metaphase I stages (See Meiosis.)
minimum male genetic map length of individual
chromosomes is therefore at least 50 cM (See Genetic
Linkage Mapping; Linkage Analysis.)
Genetic map units
Chiasma-based genetic maps
Genetic map distances are usually expressed in morgans
(or centimorgans, cM). Haldane (1919) originally
de®ned a morgan as that length of a chromatid that
has experienced on average one crossover per meiocyte
(Figure 3). Each chiasma gives rise to two recombinant
and two nonrecombinant chromatids, that is, only half
the products of the chiasma are recombinant. Therefore, each chiasma represents a genetic distance of
0.5 M or 50 cM. In other words, genetic distance in cM
is obtained by multiplying the average chiasma
frequency (of a chromosomal segment, whole chromosome or genome) by 50. Failure of chiasma formation
in spermatocytes from fertile men is very rare and the
The ®rst detailed analysis of chiasma numbers and
distributions in human spermatocytes was produced in
1974. This initial investigation was based on testicular
material from one man (HulteÂn, 1974). Subsequent
studies later con®rmed the generality of the observations made.
The mean number of autosomal chiasmata per cell
in the original study of HulteÂn (1974) was 50.61 (SD
3.87, range 43±60) corresponding to a total genomic
recombination map length of 2530.5 cM (range of
individual spermatocytes 2150±3000 cM). Later studies showed the presence of variation between men of
normal fertility, with a range of means from 46.3 to
56.7 (i.e. 2315±2835 cM; Laurie et al., 1981, 1985).
Statistical analysis also indicated the presence of
signi®cant variation in mean numbers of chiasmata
per cell between individuals.
These estimates of genomic map lengths were exploited to estimate average crossover rates over the whole
genome, often quoted in the literature as around 1 cM
per megabase (Mb), assuming the total physical length
of the human genome to be approximately 3000 Mb.
Through use of appropriate staining methods
(HulteÂn, 1974; HulteÂn et al., 2001), it is possible to
identify each individual chromosome pair in metaphase I spermatocytes. In this way, chiasma-based
genetic map lengths of individual chromosome pairs
(bivalents) can be obtained. Overall, the map lengths
of chromosomes are correlated with physical length,
although smaller chromosomes tend to have a slightly
greater rate of recombination per unit length than
longer chromosomes (HulteÂn, 1974; Laurie and
HulteÂn, 1985; Table 1).
The distribution of chiasmata along the length of
chromosomes is clearly nonrandom with prominent
Figure 1 Cartoon illustrating the generation of homologous pairs of
chromosomes (bivalents) held together by chiasmata following
crossing-over at pachytene. Two chromosome pairs are illustrated:
one acrocentric, the other metacentric. Centromere positions are
indicated by ®lled circles. One homolog of each pair is dark, the
other light. At the end of pachytene, intimate homologous
chromosome pairing (synapsis) lapses and the two homologs are
held together only at the positions of crossing-over (chiasmata). The
acrocentric pair, with one chiasma, forms a rod-shaped structure; the
metacentric pair, with two chiasmata, forms a ring structure.
XY
(a)
(b)
Figure 2 Human metaphase I spermatocyte. (a) Intact cell containing 22 pairs of autosomal bivalents and the XY bivalent.
(b) Individual bivalents have been cut out and the positions of chiasmata are indicated by arrows.
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883
Genetic Maps: Direct Meiotic Analysis
Meiosis I
Gametes
1 chiasma
(or MLHI focus)
=0.5 morgans
0
1
1
0
crossovers
[1 crossover = 1 morgan]
Figure 3 Cartoon illustrating the relationship of a crossover,
as viewed directly (as a chiasma or MLH1 focus) or indirectly
in the products of meiosis (gametes).
Chromosome 15
Table 1 Rates of recombination per unit length in selected
bivalents
Mean
Genetic
Relative rate of
number of length Chromosome recombination
Chromosome chiasmataa (cM)
size (Mb)b
(cM/Mb)
1
3.88
194
263
0.738
2
3.52
176
255
0.69
3
2.82
141
214
0.659
11
12
2.24
2.49
112
124.5
144
143
0.778
0.871
13
1.9
95
114
0.833
20
1.95
97.5
72
1.354
21
1.06
53
50
1.06
22
1.13
56.5
56
1.01
Chromosome 16
From Laurie and HulteÂn (1985).
b
From Morton (1991).
Figure 4 Comparison of chiasma distributions (in 5% length
intervals) along chromosomes 15 and 16. Chromosome 15 is
acrocentric, chromosome 16 metacentric. Although both
chromosomes are similar in length, they show different distributions.
`hot spots' near the ends of most (Figures 2 and 4).
Nonrandom chiasma distribution is indicative of
preferential crossing-over in particular chromosomal
regions. It is also worth noting that the distribution of
chiasmata, along a given chromosome pair, varies
according to the number of crossovers present and the
position of the centromere (Figure 4). In addition
chiasmata are spaced out as a result of positive
chiasma (crossover) interference (see Figure 2). Interference is a poorly understood phenomenon but its
consequence is that the presence of one crossover
inhibits the formation of a second in its vicinity. This
inhibitory effect stretches over considerable physical
distances. For example, in the study of HulteÂn (1974),
the smallest interchiasma distances were recorded in
chromosomes 9 and 10; even in these instances, the
distances equated to approximately 10 Mb. In other
chromosomes, the smallest distance between chiasmata varied widely up to a maximum of about 44 Mb.
Although this interchromosome variability may to
some extent be due to a sampling effect, it is nevertheless clear that relatively large physical distances
invariably separate successive crossovers along a
chromosome arm. The minimum interchiasma distance appears to be roughly the same along arms of
different chromosomes but is generally longer over the
centromere.
The patterns of chiasma frequency and distribution
along chromosomes found in the human male are
(with few exceptions) very similar to those in other
species that have been investigated in detail (Lichten
and Goldman, 1995). However, it is fair to say that the
mechanism(s) underlying the positional control and
nonrandom distribution of chiasmata is unknown.
Analyses of chiasmata provide a reliable means of
obtaining a `bird's-eye' view of the numbers and
positions of crossovers for the whole genome as well
as for individual chromosomes and chromosomal
a
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Genetic Maps: Direct Meiotic Analysis
segments. However, it must also be remembered that
this overview suffers from limited resolution with
respect to the DNA sequences involved.
MLH1 Foci
A slightly higher resolution of crossover positions may
be obtained by analysis of MLH1 foci in pachytene
stage germ cells (Figure 5). These foci result from aggregations of the DNA mismatch repair protein (MLH1)
on the synaptonemal complex, a meiosisspeci®c, proteinaceous structure present at the axes
of paired homologous chromosomes (Figure 5).
MLH1 is involved in the completion of crossing-over
in yeast, and is assumed to have the same function in
humans. The MLH1 foci therefore mark the positions
of crossing-over between paired homologous chromosomes. The foci can be visualized in human spermatocytes and oocytes by immunostaining with a
monoclonal antibody (Barlow and HulteÂn, 1998;
Lynn et al., 2002; Tease et al., 2002). As this approach
is applicable to both male and female meiosis, it can be
used to gather comparable information on crossover
patterns from both sexes. Pachytene spermatocytes
can be obtained from testicular biopsies from adult
men. However, as pachytene oocytes are only present
in the human ovary during gestation, it is necessary
to obtain fetal ovarian tissue.
MlH1-based genetic maps
The numbers of MLH1 foci in human spermatocytes
have been described in two recent reports (Barlow and
HulteÂn, 1998; Lynn et al., 2002). These found averages
of 50.9 and 49.1 (2545 and 2455 cM respectively)
autosomal foci per cell. As anticipated, on the basis
(a)
that chiasmata and MLH1 foci are manifestations of
the same underlying event of meiotic recombination,
these estimates are very similar to those from
studies of chiasma numbers in human spermatocytes.
Both studies found evidence of considerable intercell
variation in numbers of foci (range 34±66, i.e. 1700±
3300 cM). Lynn et al. (2002) also showed the presence
of signi®cant interindividual variation, similar to that
found for chiasmata, with a range of 46.2±52.8 (2310±
2640 cM) in a sample of 14 men.
Currently, information on MLH1 foci in human
oocytes is available from four cases, although only one
of these has been studied in any detail (Tease et al.,
2002). Three cases, in which small numbers of cells
were analyzed, gave mean frequencies of foci of 95.0
(4750 cM), 77.3 (3850 cM) 77.3 (3865 cM) and 71.6
(3580 cM). One extensively analyzed case gave a mean
of 70.3 (3515 cM), with a very a large range between
cells of 48±104 (2400±5200 cM).
The numbers and positions of MLH1 foci were also
recorded for chromosome pairs 21, 18, 13 and X in
fetal oocytes. These chromosomes had means respectively of 1.23 (61.5 cM), 2.36 (118 cM), 2.5 (125 cM)
and 3.22 (161 cM). The chromosomes therefore
showed the same general length to crossover frequency
relationship as described above for chiasmata.
The distribution of MLH1 foci along chromosome
arms in oocytes shows the in¯uence of crossover
interference in a similar manner to that described
earlier for chiasma distribution in the human male.
Thus the distribution pattern varies according to the
number of crossovers present (Figure 6). Likewise,
along chromosome arms, foci are rarely closely spaced
and the average interfocus distance varies according to
the number present. In chromosome 13, for example,
where there are two foci, these are separated on
average by 41% of chromosome length, where there
(b)
Figure 5 [Figure is also reproduced in color section.] Pachytene male (a) and female (b) germ cells. In both, synaptonemal complexes are
stained green and MLH1 foci red. In the spermatocyte (a), the centromeres are stained blue. The chromatin cloud that surrounds the
synaptonemal complexes has been omitted to aid clarity. Arrowheads indicate the MLH1 foci.
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Genetic Maps: Direct Meiotic Analysis
1 focus
2 foci
Figure 6 Comparison of the distribution patterns of MLH1 foci (in
5% length intervals) of chromosome 21 with one or two foci in fetal
oocytes.
are three foci by 17%, and where there are four foci by
10%. This latter average still equates to approximately
11 Mb. The minimum interfocus distance recorded was
7% of the chromosome arm, roughly 8 Mb. These
analyses again illustrate the fact that successive crossovers are usually separated by considerable lengths of a
chromosome arm and that even on the rare occasions
when they are closely spaced they are still many Mb
apart. It is also worth noting that the interfocus distance
is generally longer over the centromere than within
chromosome arms.
0 cM
0 cM
(a)
96.5 cM
Chromosome 13
125 cM
0 cM
0 cM
(b)
Chromosome 18
95.5 cM
0 cM
118 cM
0 cM
(c)
Chromosome 21
53.5 cM
61.5 cM
Figure 7 [Figure is also reproduced in color section.] Histograms illustrating the distributions of chiasmata in spermatocytes (blue)
and MLH1 foci in oocytes (pink) on chromosomes 13, 18 and 21. Each chromosome pair is divided into 5% length intervals. These
crossover distribution patterns are also displayed for each chromosome as recombination maps. These maps highlight the different
patterns of crossover numbers and distributions in male and female germ cells and the consequent effect on the recombination map.
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Genetic Maps: Direct Meiotic Analysis
To date, it has not been established in any detail to
what extent crossover interference varies between
different chromosomes or between the two sexes.
The introduction of the MLH1 approach should
make it feasible to directly address these questions.
Intersex Differences in
Recombination
Direct analyses of recombination frequencies have
shown the presence of considerable intraindividual
(between cell) variation and signi®cant interindividual
variation in both human males and females. Comparison of male and female germ cells also demonstrates
clear intersex differences in the patterns of recombination. These differences encompass both the numbers of
crossovers and also their preferred positions along
chromosome arms. The latter is exempli®ed by
comparison of the distribution of MLH1 foci in
spermatocytes and oocytes. In spermatocytes, foci
(crossovers) occur regularly in the chromosomal
segments immediately adjacent to the telomeres. In
the female in contrast, these chromosomal segments
are much less favored for crossing-over and, instead,
foci are generally located in more interstitial chromosomal segments than in the male. A similar effect is
seen when comparing male chiasma distributions with
MLH1 foci in oocytes (Figure 7). In other words, there
is a very distinctive pattern of crossover distribution in
the two sexes. As a consequence, intergenic distances
(expressed in cM) will be different across many
chromosomal segments in oocytes and spermatocytes
(Figure 7).
Conclusion
Direct cytogenetic analysis offers a rapid and reliable
means of assessing meiotic recombination in human
germ cells. A particular advantage of the direct
approach is its ability to provide an overview of
recombination across the whole genome, while at the
same time being able to determine chromosomespeci®c patterns of crossing-over.
Acknowledgement
Work supported by the Wellcome Trust (grant no
0611202/Z/00/Z).
See also
Genetic Maps: Integration
Genetic Mapping: Comparison of Direct and Indirect
Approaches
Meiosis
References
Barlow AL and HulteÂn MA (1998) Crossing over analysis at
pachytene in man. European Journal of Human Genetics 6:
350±358.
Haldane JBS (1919) The combination of linkage values and the
calculation of distance between the loci of linked characters.
Journal of Genetics 8: 299±309.
HulteÂn M (1974) Chiasma distribution at diakinesis in the normal
human male. Hereditas 76: 55±78.
HulteÂn M, Barlow AL and Tease C (2001) Meiotic studies in
humans. In: Rooney DE (ed.) Human Cytogenetics: Constitutional
Analysis, pp. 211±236 Oxford, New York, NY: Oxford University Press.
HulteÂn MA, Luciani JM, Morrazinni ML and Kirton V (1978) The
use and limitations of chiasma scoring with reference to human
genetic mapping. Cytogenetics and Cell Genetics 22: 37±58.
Laurie DA and HulteÂn M (1985) Further chiasma studies on bivalent
chiasma frequency in human males with normal karyotypes.
Annals of Human Genetics 49: 189±201.
Laurie DA, HulteÂn M and Jones GH (1981) Chiasma frequency and
distribution in a sample of human males: chromosomes 1, 2, and
9. Cytogenetics and Cell Genetics 31: 153±166.
Laurie DA, Palmer RW and HulteÂn MA (1985) Chiasma derived
genetic lengths and recombination fractions: a 46,XY,t(9;10)
(p22;q24) reciprocal translocation. Annals of Human Genetics 49:
135±146.
Lichten M and Goldman AS (1995) Meiotic recombination hotspots. Annual Review of Genetics 29: 423±444.
Lynn A, Koehler KE, Judis L, et al. (2002) Covariation of
synaptonemal complex length and mammalian meiotic exchange
rates. Science 296: 2222±2225.
Morton NE (1991) Parameters of the human geneome. Proceedings
of the National Academy of Sciences of the United States of America
88: 7474±7476.
Tease C, Hartshorne GM and HulteÂn MA (2002) Patterns of meiotic
recombination in human fetal oocytes. American Journal of
Human Genetics 70: 1469±1479.
Further Reading
Barlow AL, Benson FE, West SC and HulteÂn MA (1997)
Distribution of the Rad51 recombinase in human and mouse
spermatocytes. EMBO Journal 16: 5207±5215.
Hassold TA, Sherman S and Hunt P (2000) Counting cross-overs:
characterizing meiotic recombination in mammals. Human
Molecular Genetics 9: 2409±2419.
HulteÂn MA and Lawrie NM (1993) Chiasma density and interference maps of the normal and translocated chromosome 9 in
the human male. First International Workshop on Chromosome
9. Annals of Human Genetics 56: 194±195.
HulteÂn MA, Lawrie NM and Laurie D (1990) Chiasma-based
genetic maps of chromosome 21. American Journal of Medical
Genetics 7: 148±154.
HulteÂn MA, Palmer RW and Laurie DA (1982) Chiasma derived
genetic maps and recombination fractions: chromosome 1.
Annals of Human Genetics 46: 167±175.
Jones GH (1987) In: Moens PB (ed.) Chiasmata in Meiosis, pp. 213±
244. Cambridge, UK: Cambridge University Press.
Laurie DA and HulteÂn MA (1985) Further studies on chiasma
distribution and interference in the human male. Annals of Human
Genetics 49: 203±214.
Laurie DA, Palmer RW and HulteÂn MA (1982) Chiasma derived
genetic lengths and recombination fractions: Chromosomes 2
and 9. Annals of Human Genetics 46: 233±244.
Saadallah N and HulteÂn MA (1983) Chiasma distribution, genetic
lengths and recombination fractions: A comparison between
chromosome 15 and 16. Journal of Medical Genetics 20: 362.
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