Mol Gen Genet (1993) 238:49-58
© Springer-Verlag 1993
Individualization and estimation of relatedness in crocodilians
by DNA fingerprinting with a Bkm-derived probe
J.W. Lang 1, R.K. Aggarwal 2, K.C. Majumdar 2, L. Singh 2
1Department of Biology,University of North Dakota, Grand Forks, USA
2 Centre for Cellular and Molecular Biology, Hyderabad 500007, India
Received: 15 June 1992/Accepted: 10 October 1992
Abstract. Individual-specific DNA fingerprints of
crocodilians were obtained by the use of Bkm-2(8) probe.
Pedigree analyses of Crocodylus palustris, C. porosus and
Caiman crocodilus revealed that the multiple bands (2223 bands with Aludigest) thus obtained were inherited
stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and
reconstruct the DNA profile of a missing parent. Average
band sharing between unrelated crocodiles was found to
be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives.
Similar results obtained in other species/genera, using the
same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.
Key words: Bkm D N A - Genetic/DNA fingerprinting
Crocodilians - Paternity determination - Band sharing
Introduction
DNA fingerprinting utilizing multilocus and single locus
probes is of proven utility for identification of individuals
and for paternity determination (Jeffreys et al. 1991).
Application of DNA fingerprinting to studies of wild
populations has provided the behavioural ecologist with
a powerful tool with which to estimate genetic relatedness among socially interacting individuals (Burke et al.
1991). Among vertebrates, D N A fingerprinting has been
widely applied to studies of fishes, birds and mammals.
These include demonstrations of reduced genetic variation in clonal and colonial species (Turner et al. 1990;
Reeve et al. 1990), genetic parentage in species with
varied mating systems (Westneat 1990; Tegelstrom et al.
1991), and genetic relatedness in species exhibiting social
cooperation in breeding behaviour (Rabenold et al.
Communicated by E. Bautz
Correspondence to: L. Singh
1990; Amos et al. 1991; Gilbert et al. 1991). In contrast,
studies in reptiles have been limited to brief reports on
turtles (Jones et al. 1987; Demas et al. 1990; Demas and
Wachtel 1991) and crocodilians (Demas and Wachtel
1991). In the latter study, which was carried out on five
species of crocodilians, individual-specific variation in
DNA fingerprints was not evident and this was attributed to low levels of heterozygosity thought to be characteristic of the group (Lawson et al. 1989).
The crocodilians are an endangered group and consist
largely of managed wild populations. They exhibit sophisticated communication repertoires, structured social
interactions and mating systems, and extensive parental
care, a feature absent or rare in other reptiles (Lang
1987). Therefore, there is a need to establish direct methods of assessing genetic relatedness for their future management in the wild as well as in captive breeding programmes. In the present investigation, we have used the
approach of DNA fingerprinting using the minisatellite
probe Bkm-2(8), to study genetic relatedness among individual crocodiles. The Bkm sequences were first identified and isolated as W sex-chromosome associated minor satellite D N A from the genomic D N A of the females
of a poisonous Indian snake, Bungarus fasciatus (Singh
et al. 1976, 1980, 1981). These sequences are found in all
eukaryotes and are preferentially concentrated on the
sex-chromosomes of many vertebrate species including
mouse and human (Jones and Singh 1981a, b; Singh et
al. 1981, 1988; Singh and Jones 1982, 1986). The Bkm2(8) probe predominantly consists of GATA repeats
(Epplen et al. 1982; Singh et al. 1984), which are found
scattered all over the genome (Singh and Jones 1982).
This probe shows extensive restriction fragment length
polymorphism (RFLP) in a wide range of higher eukaryotes (Singh and Jones 1986; Jones et al. 1987; Traut,
1987; Singh et al. 1988; Lloyd et al. 1989; Demas et al.
1990) and can be used as an efficient probe for DNA
fingerprinting in humans (Singh 1991).
Here we report that the Bkm-2(8) probe can be used
for generating DNA fingerprints in crocodilians. Using
a representative species (Crocodylus palustris) with in-
50
dividuals from known pedigrees, individual-specific, stably inherited band patterns were obtained which could
be used to assign parentage and to reconstruct the fingerprint of a missing parent. Furthermore, calculations of
band sharing between relatives and non-relatives
provided an estimate of genetic relatedness among individual crocodiles. Our studies of several additional
species (Aggarwal et al. 1992b) suggest that this technique may be applicable to all crocodilians.
Materials and methods
Specimens. Four males and six females of wild-caught
mugger crocodiles C. palustris were sampled from the
Nehru Zoological Park (Hyderabad, India) and ten
males and six females from the Madras Crocodile Bank
(MCB, Madras, India). These were originally caught
from two geographic regions (states of Gujarat and Andhra Pradesh in India) and from four separate localities
in Tamil Nadu (India) respectively. In addition, 31 males
and 71 females of second generation progeny of known
pedigrees, which were produced in captive breeding enclosures (Lang et al. 1989), were also sampled at MCB.
Animals of known pedigrees, from a sibling group of
five saltwater crocodiles (C. porosus), bred in captivity by
crossing animals obtained from Tamil Nadu and Orissa
(India), and a family group of eight caimans (Caiman
crocodilus) from an unknown locality in Central America
were also sampled at MCB. Additional samples were
obtained from two siblings of Crocodylus cataphractus at
the Metro Zoo, Miami (Fla., USA), three individuals of
C. moreletii at Zoo Atlanta (Ga., USA), and a single
sample of C. rhombifer at the St. Augustine Alligator
Farm (Fla., USA).
DNA preparation. Blood (0.5 to 60 ml) was drawn from
the caudal vein, brain plexus or from the heart in heparinized, sterile syringes and frozen. The frozen blood cells
were freeze-fractured twice by rapid thawing at 45 ° C in
TES buffer (Singh and Jones 1986). The pellet obtained
after centrifugation at 8000 rpm at 4° C for 10 rain was
homogenized in TES buffer (1:10 v/v), lysed with Sarkosyl NL30 (2-3%), and incubated at 37 ° C for 12-16 h
in the presence of proteinase K (75 gg/ml). DNA was
isolated as described by Aggarwal et al. (1992a) and
dissolved in TE buffer (10 mM TRIS-HC1, 1 mM EDTA,
pH 7.5).
DNAfingerprinting. Genomic DNA (12-15 gg) was digested to completion with AluI or HinfI restriction enzymes according to the conditions specified by the manufacturer (New England Biolabs). Digested samples were
electrophoresed in 30 cm long, 5 mm thick, 0.8% agarose
gels at 60 V for 16-18 h in TPE buffer (15 mM TRISHC1, 18 mM NaH2PO~, 0.5 mM EDTA, pH 7.8).
HindIII and EcoRI double digests of phage )~ DNA
were used as molecular weight markers. Gel-fractionated DNA samples were transferred onto Hybond-N
membrane (Amersham, UK) using a vacuum-blotting
assembly at 30 mm Hg (Olszewska and Jones 1988). The
membrane was baked at 80° C for 2 h under vacuum. The
blots were prehybridized in 7% SDS, 0.5 M sodium
phosphate buffer (pH 7.5) at 60 ° C for 2-3 h, and then
hybridized with 1-2 x 1 0 6 cpm/ml of the 32p-labelled
single-stranded probe (specific activity 0.7-3.0 x 108 cpm/gg) in the same but fresh buffer at 60° C for
14~16 h. The probe used was Bkm-2(8) (Patent no. 1000
DEL 88), a subclone of the clone CS 314 obtained from
Bkm probing of a Drosophila genomic library. The subclone 2(8) contains a 545 bp sequence consisting of 66
copies of the GATA repeats interspersed with variable
numbers of dinucleotide repeats of TA in several locations (Singh et al. 1984). Single-stranded 3Zp-labelled
probe was prepared to a specific activity of 0.7-3.0 x 108
cpm/gg (Hu and Messing 1982), using 32p-dATP (specific activity 3000 Ci/mmol; Jonaki, BARC, India). The
blots were washed once in 3 x SSC (1 x SSC is 0.15 M
NaC1, 0.015 M sodium citrate, pH 7.2) containing 0.1%
SDS at room temperature for 10 rain and thrice in
2xSSC, 0.1% SDS at 60° C for 10 min each. Several
exposures, varying from 1 to 10 days were made of each
hybridized membrane with intensifying screens at - 7 0 °
C, in order to score all the bands of differing intensities.
Analysis of fingerprints. Molecular weight markers and
duplicate samples from the same individual were run on
either side of the gel as controls for mobility distortion.
Samples of known individuals were run in each gel to
facilitate comparison of DNA fingerprints obtained from
different gels. No variation was evident in the band
patterns of the same DNA sample run repeatedly on the
same or on different gels. Autoradiographs were examined visually and also scanned on the Biotrac DNA
fingerprinting system (Foster and Freeman Ltd., UK)
using the Bioworld program to calibrate the fragments
for their molecular weights. The distinct bands in each
genetic profile were scored in the size range of 1.323.0 kb. All bands showing similar molecular weights
(band centres within 0.5 ram) were considered to be
identical for the purpose of pairwise comparisons.
Band sharing was calculated following the methodology used by Burke and Bruford (1987). Mean band
sharing for a particular group of individuals was calculated by averaging the band sharing values for all
possible pairwise combinations. To establish baseline
values for band sharing between non-relatives, independent pairwise comparisons were made (Lynch 1991).
A subset of scored bands that were particularly distinct
and exhibited well defined differences in hybridization intensity were further scored as either heterozygous (less
intense) or homozygous (twofold increase in intensity by
comparison). The differences in band intensities were
inferred mainly by visual inspection but in some cases by
laser densitometry. Only those bands were considered for
this analysis, which repeatedly showed a fixed pattern of
intensity differences in the progeny. Moreover, the differences in intensities of bands due to variation in the
total amount of DNA used in the different lanes being
compared were also taken into consideration. Pedigree
analyses were completed following the methodology used
by Jeffreys et al. (1986). The transmission frequency of
51
each of these bands was calculated as the percentage of
progeny in which the band was present in each family.
Results
D NA fingerprints
For each individual of C. palustris (45 males, 83 females)
tested, an individual-specific pattern was obtained with
the Bkm-2(8) probe. The total number of polymorphic
bands scored in the fingerprints of all the individuals
tested was 41 and 35 in the size range of 1.3-23.0 kb, with
AluI and HinfI restriction enzymes, respectively. No sexspecific band was evident in any of the animals tested,
including the progeny of both sexes.
[ ~
(~P
1
progeny
2
3
4
5
6
7
8
9
10
I
11
In a sample of 14 individuals, originally from six
different localities, the number of bands ranged from 18
to 27 (mean+SD, 23.14-2.81; AIuI digest). The HinfI
digest produced specific but fewer bands (20.7± 2.62)
which were readily distinguishable from the AluI fingerprints. On the other hand, in captive-bred animals the
mean band number in 4 parents with AluI digest was
22.4 ± 2.75 (range, 15-26) and in 45 offspring 22.7 4- 2.44
(range, 16-28).
The inheritance of parental bands in the progeny of
three families (15 individuals per family) was analysed.
The same male fathered these families with three different
females. Without exception, bands in the offspring could
be accounted for as either being inherited from the father
or from the mother. Overall, the offspring shared 30% of
their bands with the father, 33% with the mother, and
37% with both parents. The DNA fingerprint patterns
for two of these families are illustrated in Figs. 1 and 2.
99
kb
23.1 -
9.42-
6.565.15_
4.974.364.273.53-
2.322,03_
1,901.58-
Band heterozygosity
In the above three families, 27 bands (2.5-23.0 kb) were
resolved sufficiently to follow their segregation in the
offspring. Differences in the hybridization intensity at
these loci facilitated discrimination of the number of
copies (single-intensity band, heterozygous; doubleintensity band, homozygous) present per band in each
offspring and their parents (Figs. 1, 2).
The male parent Z (Fig. 1, far left lane) was heterozygous at all 15 loci scored in this analysis. By contrast, all
three females (A, B, C) had varying proportions of
homozygous bands, ranging from 0.37 to 0.44 (Table 1).
These females were homozygous for one band ( ~ 19 kb)
which was inherited by all the 45 offspring tested
(Figs. 1, 2). The absence of double intensity for this band
in female B (Fig. 1 ; 9P lane) was due to the comparatively lower amount of D N A present in this lane. An addiTable 1. Mean transmission frequency (f) of different bands (homozygous vs heterozygous) to the offspring in three families ~ of
Crocodylus palustris
1.33-
Fig. 1. Fingerprints from a pair of mugger crocodiles, Croeodylus
palustris (dr P, far left lane, individual Z; 9 P, far right lane, individual B) and their offspring (lanes 1-6 female progeny; 7-11 male
progeny), obtained with the Bkm-2(8) probe, AluI digest. Segregation analysis of the resolved maternal and paternal bands in 15
progeny (4 others were done but are not shown) revealed no cosegregating bands. Note a major band of ~ 19 kb, present in the
female parent, absent in the male parent, and present in all of the
offspring (homozygous in the female parent; heterozygous in the
progeny). Two bands visible at ~ 6.5 kb are present in the male
parent, absent in the female parent and present in most, but not all
of the offspring (heterozygous in parent and progeny; note lane 6).
Another pair of paternal bands visible at ~ 4.25 kb appear in some
of the progeny, but are absent in the female parent. Other bands are
shared by both parents and are present in variable combinations in
the progeny, e.g. two pairs of major bands of ~ 3 kb
Total number of
bands scored
Male
Female
Z
A
B
15
8
12
3
7
8
3 (37)
5 (42)
7 (44)
15
1.00
5
0.986
7
0.977
9
0.498
0.774
0.513
0.835
0.400
0.783
0.532
0.703
Shared with male
No. ofhomozygous
bands ( + / + )
f (homozygous bands)
No. of heterozygous
bands ( + / - )
f (+/_ ; _/_)b
f(+/--; +/--)°
0 (0)
16
Values in parentheses are % homozygous bands
a The male parent is common to all the three families (number of
offspring is 15 per family)
b Heterozygous bands present in only one parent
° Heterozygous bands present in both the parents
52
. .
i
9P
progeny
. . . .
1
2
3
4
.
5
.
.
.
.
6
.
I- . . . . . . .
.
7
d'P
8
9
progeny
10
kb
23.1 -
9.426.565.15_
4.974.36_
4.273.53-
2.32tional band ( > 2.3 kb), which was homozygous in two of
the females from different localities (females B and C, ~P
tracks in Figs. 1, 2), was absent in the male.
The mean transmission frequencies of the homozygous versus heterozygous bands were consistent with the
assumption that these bands were inherited in a Mendelian pattern following a binomial distribution (Jeffreys
et al. 1986). Bands transmitted to virtually all the offspring (mean transmission frequency, 0.977 to 1.00) were
also judged to be homozygous on the basis of hybridization intensity. Heterozygous bands that were shared in
both parents were transmitted to the offspring with a
mean transmission frequency of 0.703-0.835, and those
absent in one parent were transmitted with a frequency
of 0.400-0.532 (Table 1). In our analysis, no bands were
detected that were either allelic or cosegregating pairs.
The mutation rate (mutation/band per generation),
based on the difference between observed and expected
transmission rates of bands judged to be homozygous,
was estimated to be 1.3 x 10 -3 (Kuhnlein et al. 1990).
Band sharin 9
As a baseline for comparisons among known relatives,
band sharing was calculated between pairs of crocodiles
from different localities in India. We have assumed that
these individuals were unrelated. Among these unrelated
individuals, the mean band sharing coefficient, calculated
on the basis of all possible pairwise combinations, was
0.36. In contrast, wild-caught individuals from the same
localities exhibited relatively high band sharing (mean
coefficient, 0.59), a value closely approximating the mean
observed value for half siblings (Table 2).
Mean allele frequency was calculated using x = 2q - q2
(Jeffreys et al. 1985); q equalled 0.213 for the mean band
sharing value (x) of 0.38 (Table 2). If shared bands
represent indentical alleles and all alleles have equal
frequencies, the expected band sharing between first
11
12
......... 1
13
14
15
?P
Fig. 2. Fingerprints from a pair of mugger crocodiles (d P, two centre lanes, individual Z; c?p, outer lanes, individual
C) and their offspring (lanes 1-7 female
progeny; 8-15 male progeny), obtained
with the Bkm-2(8) probe, AluI digest.
Segregation analysis of bands in this
family, like the family in Fig. 1, showed:
no cosegregating bands; a major band of
19 kb homozygous in female parent
and heterozygous in all the offspring; a
pair of heterozygous paternal bands of
6.5 kb, present in some of the
progeny; and most of the other bands
shared by both parents and present in
variable combinations in the progeny,
e.g. a series of bands around 3 kb in
size. Male parent in this family is the
same as in Fig. 1; note identical band
patterns
order relatives (parents-offspring; siblings) with q = 0.213
would be 0.67 (Jeffreys et al. 1985). By comparison, the
observed mean band sharing (based on all pairwise combinations) for parents-offspring and for full siblings in
these three families was 0.68 and 0.70, respectively
(Table 2). This close agreement between the expected and
the observed band sharing probabilities substantiates
the assumption of independence for the bands produced
with the enzyme/probe combination used in this study
(Burke et al. 1991).
The band sharing was higher among relatives than
among non-relatives. It was the highest between parents
and offspring and between full sibs with, as expected,
nearly comparable mean values (see above; Table 2).
Within each family, the band sharing coefficient between
mother-offspring was higher, relative to the fatheroffspring value (combined mean 0.70 and 0.66, respectively; Table 2). There was, however, considerable variation in the values for mother-offspring among the three
families (mean 0.63, 0.73 and 0.75 for the females A, B
and C, respectively). Likewise, the father-offspring values
also varied among families (mean 0.63, 0.71 and 0.64 for
families from the females A, B and C, respectively).
The parent-offspring values reflected the relative differences in band sharing among the parents. Band sharing between the parents of each family (fathered by the
same male) was consistent with the assumption that the
male and female parents were not closely related. On the
other hand, two of the females, A and C, were from the
same locality, and possibly were sibs. For them the band
sharing coefficient was 0.58. The relatively high proportion ofhomozygous bands in the females A, B and C, and
the fact that some of these bands were shared by all of
these, were reflected in the band sharing values among
the progeny in these families (Table 2; also see above).
The D N A fingerprints from these three groups of sibs
were compared within and between the families. Mean
band sharing among the full sibs was higher than among
the half sibs (combined mean 0.70 and 0.61, respectively,
53
Table 2. Mean band-sharing coefficient (x)
among crocodilians
Comparisons a
Croeodylus palustris
Wild population
Non-relatives:
Different localities (6)
Different localities (6)
Individuals :
Same localities (5)
Number
Pairwise
Mean_+ SD
of individuals comparisons b
Average
relatedness
14
12
77
6c
0.36+0.08
0.37 -+0.07
-
13
14
0.59+_0.16
?
Familial analysis
Parents:
Overall
Mother-father
Mother-mother
Parent-offspring:
Overall
Father-offspring
Mother-offspring
4
4
3
6
3
3
0.45 _+0.09
0.38 _+0.07
0.52_+ 0.06
-
49
46
48
90
45
45
0.68 _+0.10
0.66 ± 0.08
0.70 ± 0.07
0.5
0.5
0.5
Full sibs (overall)
Half sibs (overall) "
45
45
315
630
0.70_+0.10
0.61_+0.11
0.5
0.25
C. porosus family
Full sibs (overall)
Sibs-unrelated individual
5
6
10
5
0.60_+0.14
0.40 _+0.07
0.5
?
Caiman crocodilus family
Full sibs (overall)
Mother-offspring
Mother-other female
Sibs-other female
Sibs-male individual
5
6
2
6
6
10
5
1
5
5
0.60_+0.12
0.65 _+0.08
0.65
0.53 _+0.09
0.41 _+0.08
0.5
0.5
?
?
a Figures in parentheses indicate number of localities
b Includes all possible combinations
c Independent comparisons only between putative non-relatives from different localities
(Lynch 1991)
Table 2). However, extensive overlap in these distributions precludes a determination of relatedness between
sibs versus halfsibs, based only on the band sharing value
of the two individuals. Furthermore, the band sharing
values between families reflected the relatedness of the
four parents. Thus in each of the three families tested, the
mean band sharing coefficients for sibs and half-sibs
differed numerically (0.66 and 0.61; 0.73 and 0.60; 0.71
and 0.61 in the families from the females A, B and C,
respectively), and were consistently higher for the sibs in
comparison to their halfsibs.
Fingerprint reconstruction
F o r two additional crocodile families, D N A samples
were available for 12 and 11 offspring from two female
parents (females B and C, which were the same female
parents as for the families shown in Figs. 1, 2), but the
male parent (Y) had died 4 years earlier. The band pattern of the missing male parent was reconstructed
separately f r o m the fingerprints of each family (female
parent and representative progeny). For this reconstruction, discernible bands within the range of 2 . 0 ~ 3 . 0 kb
were used. In each family (Fig. 3), the unique bands were
identified that were not assignable to the female parent,
but were evident in the progeny. These consisted of 13
unique bands in family 1 (female B) and 17 bands in
family 2 (female C). Within each family, the bands that
were shared by both the parents were identified on the
basis of their hybridization intensity and the segregation
in the offspring. The n u m b e r of such shared bands was
11 in family 1 (female B) and 7 in family 2 (female C).
Thus the total number of bands attributable to the male
within each family was 24 (13 unique and 11 shared with
female B; 17 unique and 7 shared with female C) which
on comparison, were found to be identical (see legend to
Fig. 3). Using the reconstructed fingerprint of the missing
male Y, the band sharing coefficients were also computed
for the above families which were consistent with the
results obtained in the three families described earlier
(Table 2).
The validity of this method was confirmed by reconstructing the fingerprint pattern of the male (Z) of the
three families described earlier, from the D N A fingerprints of the progeny and the female parents alone (using
bands in the size range of 2.5-23.0 kb; Figs. 1, 2). A total
of 15 bands were reconstructed which corresponded in
number and size (molecular weight) to the actual number
of bands scored for the male Z (Table 1).
54
F. . . . . .
~P
kb
I
2
PROGENY . . . . . . . .
3
4
5
6
7
1
8
F. . . . .
SP
ZP
SP
1
PROGENY . . . . . . .
2
3
4
5
6
23.10
9.42
6.56
5.15
4-97
4.36
4.27
3.53
2.32
2.03
1.90
1.58
FAMILY
I
2
3
4
7
8 SP
Fig. 3. Fingerprint reconstruction for
missing male parent Y (~ P, line
drawing in centre) based on Bkm-2(8)
probe-generated AluI fingerprints, of
two mugger families from females C
(far left lane) and B (far right lane).
On the basis of segregation analysis
of the families, 11 bands were unique
to the male parent (unmarked bands
in the line diagram), and 5 bands
(marked by dots) were shared by all
parents. In addition, 6 bands (marked
with arrowheads' at left side) were detected as unique paternal bands in
family 1, but as shared (with female
parent B) in family 2. Likewise, 2
more bands (marked with arrowheads
at right-hand side) appeared as unique paternal bands in family 2, but
as shared among parents in family 1.
Thus in total, the reconstructed band
pattern of the missing male consists
of 24 bands (11 unique to the male in
both the families, 5 shared by all
parents and 8 unique/shared in the
two families)
FAMILY II
I
r--Progeny--1
GM ~P
kb
7
r--Progeny ---i
SP GM
dP
I
2
3
4
9P
dP
25.10
9.42
6.56
5.15
4.97
4.36
4.27
3.53
2.32
2.03
1.90
1.58.
1.33
I
FAMILY I
II
FAMILY 11
Paternity determination
The families discussed above were sired by lone males
(Z and Y) living in separate breeding enclosures with
multiple females. In an adjacent pen there were 10 males
housed with 18 females. One of the males (X) was clearly
I
Fig. 4. Paternity determination in two crocodile families based on DNA fingerprinting with Bkm-2(8)
probe, after AluI digestion. Left panel shows family 1
and right panel family 2 (lane identification: GM,
grandmother; d P, putative male parent X; 1-4,
progeny; ~ P, female parent; single far right lane (3' P,
shown separately, is putative male parent W). The fingerprints of progeny 3 and 4 in family 1 and of all the
progeny in family 2 contain a total of 12 unique bands
(indicated by arrowheads) which are absent in the
female parents and male X, but present in male W. By
contrast, the fingerprints of progeny 1 and 2 in family
1 lack these bands, but show bands (at 4.5 and 6.0 kb)
which are evident in the putative male parent X (indicated by dots)
dominant in mating behaviour, whereas others were subordinate in social rank and were rarely observed to mate.
The parentage of the two sib groups from the latter
breeding enclosure was tested by fingerprinting the
progeny along with the putative father (dominant male,
X) and the known mother.
55
In one family, the putative father was judged to be the
genetic parent of offspring 1 and 2 (Fig. 4; family 1, lanes
1 and 2) on the basis of bands unique to male X (Fig. 4;
left panel, lane c? P). However, progeny 3 and 4 (Fig. 4;
family 1, lanes 3 and 4) exhibited other bands which were
not assignable to male X, the putative parent. Similarly
all the four offspring in family 2 (Fig. 4) showed many
bands not attributable to the putative father X. These
paternal bands (not accounted for by the female parents;
shown by small arrowheads in Fig. 4) in offspring 3 and
4 in family 1 and in offspring 1-4 in family 2 were found
to be identical, indicating that the unidentified male
parent in both the families was the same. Subsequent
examination of the D N A fingerprints of 10 candidate
males from this breeding enclosure, clearly identified one
of the subordinate males W (Fig. 4; right hand lane d' P)
as the father of these offspring. The mean number of
paternal bands per offspring inherited from W was
6 . 3 i 1.8 (range 4 9). A total of 12 such bands unique
to W were identified from the fingerprints of 6 progeny individuals (Fig. 4; lanes 3-4 of family 1; 1-4 of
family 2).
Thus in family 1, offspring 1 and 2 were fathered by
X and offspring 3 and 4 by W. In this family the four
progeny individuals were, in fact, produced from two
clutches of eggs laid in different years by the same female
(offspring 1 and 2 from one clutch, and offspring 3 and
4 from another clutch 1 year later). These individuals
were, therefore, not clutch-mates; thus, this is not an
instance of multiple paternity within a single clutch of
eggs.
Fingerprinting other croeodilians
In a separate study (Aggarwal et al. 1992b), samples from
18 of the 23 extant species of crocodilians were used to
generate D N A fingerprints, specific at the individual,
species, and generic levels, using Bkm-derived probe 2(8).
These included species in the genera Alligator, Caiman,
Paleosuchus, Gavialis, Tomistoma, Osteolaemus, and
Crocodylus. In the three Crocodylus species tested in the
present study, band patterns differed significantly from
each other between the species but the overall profiles
were similar to those of C. palustris (Fig. 5). In the
saltwater crocodile, C. porosus, five sibs and one unrelated animal were tested with the Bkm-2(8) AluI digest.
The resultant band patterns were individual-specific and
revealed a number of shared bands among relatives. The
mean number of bands per individual was 18.2:k2.93
(range 14-23) of 1.3 23.0 kb in size. Mean band sharing
was 0.60 :k 0.07 among sibs and 0.40 + 0.07 between the
single unrelated animal and the sibs (fingerprints not
shown).
For the spectacled caiman, Caiman crocodilus, samples were available for five sibs, the female parent, the
putative male parent, and another adult female from the
same breeding group in which the sibs were produced. As
in C. porosus, the resultant band patterns were unique
for each individual and higher band sharing was evident
in related animals (fingerprints not shown). The number of bands in HinfI profiles per individual aver-
Cr.m.
kb
23.10
Cr.r, Cr.c.
I
i r"']
1
2
3
4
I - - 1
5
Cr.p.
I
6
I
7
8
9,42
6.56
5.15
4.97
4.36
4.27
3.53
2.32
2.03
1.90
1.58
1.33
Fig. 5. Representative fingerprints of four species of Crocodylus,
obtained with Bkm-2(8) probe after AluI digestion. Individualspecific bands are evident in all the fingerprints, primarily in
the 2.0-20.0 kb size range, except in the case of C. rhombifer
(Cr.r., lane 4) for which only one sample was tested. Among individuals (putative siblings) of C. moreIetti (Cr.m., lanes 1-3) and
of C. cataphractus (Cr.c., lanes 5-6), the banding patterns are
similar but not identical. Individuals of C. palustris (Cr.p., lanes
7-8) are from different geographic regions. In these species, band
patterns in the size range of 1.3 to 3.0 kb are distinctive, and appear
to be species-specific
aged 24.9:t:3.44 (range 19-30) of 1.3-23.0 kb in size.
Mean band sharing was 0.60 i 0.10 among the sibs, and
0.65 + 0.07 between the offspring and the female parent.
It was evident from the fingerprint patterns that many
bands in the sibs were shared among siblings and with the
mother. In contrast, few bands were shared between
siblings and the putative father. Mean band sharing
between this male and the sibs was 0.41 :~ 0.07.
The lack of correspondence between band patterns of
the putative father and offspring, and the relatively low
mean band sharing value, indicate that this male was not
the parent. Unfortunately, other candidate fathers living
in the same breeding enclosure were not tested. Further
analysis of these Caiman suggested that the two females
(mother and the other adult female) were closely related
(band sharing coefficient, 0.65). As expected, the mean
band sharing between the sibs and this female was higher
(0.53 i 0.07) than between the non-parent male and the
sibs.
56
Discussion
Utilizing the Bkm-2(8) probe, individual-specific DNA
fingerprints have been produced in 18 species belonging
to seven of the eight extant genera of the order Crocodilia
(Caiman, Crocodylus, Alligator, Gavialis, Osteolaemus,
Paleosuchus, and Tomistoma; this study and Aggarwal
et al. 1992b). Our results, based on a large data set within
the species and also across genera, contradicts a recent
report in which identification of individual crocodilians
was not possible from Bkm-generated band patterns,
although species-specific differences were noted (Demas
and Wachtel 1991). This discrepancy may be due to the
different combination of restriction enzymes used by
these authors. They suggested that the absence of individual-specific fingerprints may be due to the high
levels of homozygosity noted previously in crocodilian
populations (Lawson et al. 1989). On the other hand, we
have demonstrated band heterozygosity in the size range
of 1.3 to 23.0 kb in the DNA fingerprints, and this is
comparable to those observed in other species with commonly used probes (Burke et al. 1991 ; Jeffreys et al. 1991 ;
Singh 1991).
A captive breeding population was studied of known
pedigree mugger crocodiles, originating from wild animals collected at various localities in India. For each
animal tested, the DNA fingerprint was unique and reproducible. In a sample of non-relatives, on an average
23 bands (n) per individual were resolved and the mean
band sharing coefficient was 0.37. Thus the probability
of an exact match, calculated according to Jeffreys et al.
(1985), between any two mugger crocodiles is x", or
0.3723. The assumption of linkage equilibrium implicit in
this approach (Burke et al. 1991) was tested by comparing the DNA profiles of three large families of crocodiles
of known pedigree. All of the parental bands were transmitted to representative progeny in varying combinations. Within each family, frequencies of individual
bands were predictable and consistent with the assumption of band independence. This was also confirmed by
segregation analyses on the basis of discernible differences in hybridization intensity of specific bands. Thus
for the crocodile, we have confirmed by a detailed pedigree analysis that DNA fingerprints generated with the
enzymes HinfI or AluI and Bkm-2(8) probe combinations
are especially useful for individual identification and
paternity determination.
Our study failed to reveal any sex-specific fingerprint
pattern or sex-specific inheritance of any discernible
band in any of the crocodilian species. This suggests that
the Bkm-related sequences in these species are probably
not preferentially concentrated in a specific sex-determining region of a chromosome as in the mouse (Singh and
Jones 1982), or distributed along a specialized sex chromosome as in snakes (Singh et al. 1980). This is in agreement with the cytogenetic finding of the universal absence of heteromorphic sex chromosomes among crocodilians (Cohen and Gans 1970; Singh and Ray-Chaudhuri 1973; King et al. 1986). This however, is in sharp
contrast with the observation of Demas et al. (1990) that
Bkm-2(8) probe gives sex-specific fingerprint patterns in
sea turtles, Chelonia mydas and Lepidochelys kempi,
which like crocodilians also have temperature-dependent
sex determination (Morreale et al. 1982; Shaver et al.
1988). It is possible that in crocodilians, stretches of Bkm
repeats associated with the sex-determining region are
too small to be detected as a sex-specific band; or such
sex-specific fragment(s), if generated, may be small and
hidden in the smears in the low molecular weight region
of the fingerprints. It is also possible that the sex-determining region may not be polymorphic for AluI or HinfI
used in the present study but may reveal sex-specific
restriction polymorphism with other restriction enzymes.
The large number of bands unique to each parent and
the differing band intensities at specific sites facilitated
the reconstruction of fingerprints and the assignment of
parentage. Using samples from the maternal parent and
a number of offspring, it was possible to reconstruct the
paternal band pattern and, importantly, to identify
which bands were unique and which were shared in each
parent. In addition, paternity testing of progeny
produced in a multi-male breeding enclosure revealed
which progeny were sired by a particular male. In all
these analyses, approximately one-third of the bands in
the progeny were shared only with the mother, another
third shared with only the father, and the remaining third
shared with both the parents. The expected number of
paternal bands was 8.0. Based on a mean allele frequency
(q) of 0.213, the observed paternal bands in the three
families initially analysed was 6.7. The probability that
an unrelated male has all the paternal fragments by
chance (Jeffreys et al. 1985) is x", or approximately
0.378=3.5x 10 -4, giving an exclusion probability of
0.99965. Maternal exclusion probability also is equally
robust.
The unique, individual-specific feature of DNA fingerprints has fostered the prospect that this technique
may be extended to estimate genetic relationships beyond
first-order relatives (Lynch 1991). The most direct approach is to establish baseline data on the empirical
relationship between band sharing and relatedness for
particular populations and/or species of interest. To
serve as a reference point for comparisons of band sharing among closely related crocodiles, we determined the
mean band sharing coefficient between non-relatives.
The observed band sharing between known pedigree
crocodiles varied predictably with relatedness. A clear
rank order was apparent in the sample population studied. Between first-order kin, band sharing was highest;
between non-relatives, band sharing was lowest. Overlap
in the distribution of band sharing values between firstand second- order kin, e.g. sibs and halfsibs, blurred the
distinction in these categories. As a result, an estimate of
relatedness between any two crocodiles on the basis of
band sharing alone was possible only at the level of
relative versus non-relative. Nevertheless, band sharing
provides a useful first approximation of relatedness, particularly in assessing the status of known groupings of
crocodiles, e.g. when comparing clutches suspected to be
sired by the same male. Further refinement of this approach will be dependent on developing an empirically
determined calibration curve of kinship versus band
57
sharing. Additional values f r o m third-order kin and f r o m
non-relatives will be needed to determine whether the
calibration curve is linear or curvilinear. Examples o f
b o t h types o f curves have been described recently in birds
(Jones et al. 1991) and m a m m a l s , even between populations o f the same species (Gilbert et al. 1991).
Fingerprint analyses o f two additional species, saltwater crocodiles and caiman, indicated that the metho d o l o g y outlined here for the m u g g e r crocodile m a y be
usefully extended to other species o f crocodilians. Firstorder kin were clearly identified on the basis o f b a n d i n g
patterns. The sibling and the parent-offspring groups
exhibited levels o f b a n d sharing c o m p a r a b l e to those
observed in the m u g g e r crocodile families (Table 2).
Genetic analysis using D N A fingerprinting has the
potential to reveal the breeding structure o f wild p o p u l a tions as well as elucidate the genetic relationships underlying specific social interactions, e.g. parental care o f
hatchlings, This technique is equally applicable to captive breeding p o p u l a t i o n s o f rare a n d / o r endangered
species o f reptiles wherein there is a need to verify individual identity, establish paternity, and to determine
pedigree. Finally, preliminary fingerprinting o f two additional species suggests that these techniques will be applicable n o t only to the crocodilians included in this
study, but also to other m e m b e r s o f the order Crocodilia
and to other reptiles such as turtles, lizards, and snakes.
F u r t h e r studies (unpublished) revealed that Bkm-derived
p r o b e 2(8) can be used as a universal p r o b e for D N A
fingerprinting in eukaryotes.
Acknowledgements. We thank S. Bhaskar for her excellent technical
assistance and P.M. Bhargava for going through the manuscript,
both of CCMB, Hyderabad, India. We are grateful to M. Wise at
St. Augustine Alligator Farm, Florida; B. Ziegler at Miami Metro
Zoo, Florida~ H. Hunt at Zoo Atlanta, Georgia (all in USA); and
P. Kumar at Nehru Zoological park, Hyderabad, India, for providing crocodilian samples. We are also grateful to R. Whitaker and
H. Andrews for encouragement, field assistance, and for providing
access to specimens and records at the Madras Crocodile Bank.
Support for J.W.L. was provided by Smithsonian Institution, National Science Foundation, and National Geographic Society of
USA.
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