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Am. J. Hum. Genet. 50:107-125, 1991

Y Chromosome Probe p49a Detects Complex Pvull Haplotypes and Many New Taql Haplotypes in Southern African Populations Amanda Spurdle and Trefor Jenkins Medical Research Council Human Ecogenetics Research Unit, Department of Human Genetics, School of Pathology, South African Institute of Medical Research, and University of the Witwatersrand, Johannesburg

Summary Y-specific 49a/TaqI haplotypes were determined for 831 individuals drawn from 21 different southern African populations. A total of 31 new haplotypes were observed, some of which contained new alleles or allelic variants. Duplication, in addition to CpG mutation, is implicated in the generation of certain allelic variants. Cluster analysis of genetic distances between the populations, calculated using the 49a/TaqI haplotype frequencies, revealed a basic split between African and non-African populations. Hybrid groups cluster with the caucasoid groups, indicating that male gene flow has occurred from the latter into the former. Clustering of the negroid and Khoisan groups is not what might have been expected from the known linguistic affinities. It is suggested that the 49a/TaqI haplotype analysis of these populations is not sufficiently sensitive to distinguish between many of the populations. The Y-specific 49a/PvuII polymorphism was studied in 127 individuals from southern African populations, and 17 polymorphic fragments ranging in size from 3.6 kb to >48 kb were identified. A total of 53 PvuII haplotypes were observed, corresponding to only 30 TaqI haplotypes. There appears to be poor correlation between the two polymorphisms. Introduction

Human population genetics has benefited greatly from the use of RFLPs as an analytical tool (Cavalli-Sforza et al. 1986; Wainscoat et al. 1986). Analysis of the frequencies of alleles in different populations permits construction of maps of the genetic distance between these populations (Cavalli-Sforza 1974; Cavalli-Sforza et al. 1986). The potential of human Y-linked RFLPs in human population genetic studies has been discussed (Casanova et al. 1985; Ngo et al. 1986; Breuil et al. 1987; Oakey and Tyler-Smith 1990). The Y chromosome represents the specific paternal contribution to the male genome, and Y-specific RFLPs would thus be useful for studying male gene flow. Southern Africa is a particularly interesting area in Received May 2, 1991; final revision received September 6, 1991.

Address for correspondence and reprints: A. B. Spurdle, Department of Human Genetics, South African Institute for Medical Research, P.O. Box 1038, Johannesburg, 2000 South Africa. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5001-0012$02.00

which to conduct gene flow studies. Its people represent three of the major races of mankind (Nurse et al. 1985), namely, negroid, caucasoid and Khoisan. Present theories on the ancestry of these populations and on the admixture between them are based on archaeological, linguistic, and some genetic data (Nurse et al. 1985). The study of male-specific genetic markers in these populations could confirm, refine, or refute these theories, since it has been the practice in Africa that unilateral flow of genes occurs from only the females of the subservient group to the dominant group (Jenkins 1982), whereas population groups of the same status interchange genes bilaterally. Unfortunately, relatively few Y-linked RFLPs have been discovered, and there appears to be a general paucity of polymorphism on the Y chromosome relative to other chromosomes (Arnemann et al. 1985; Jakubiczka et al. 1989). The ability of Y chromosome probes p49f and p49a to detect numerous Y-specific haplotypes with TaqI (Ngo et al. 1986) has made it a prime candidate for a study of gene flow in southern African populations. These probes are different subclones of cosmid 49 (Bishop et al. 1983), and both 107

108

reveal about 15 Y-specific TaqI bands corresponding to a low-copy-number sequence (Ngo et al. 1986). At least eight of the 15 Y-specific bands have been shown to be present, absent, or variable in length and are considered to be independent loci (Ngo et al. 1986; Guerin et al. 1988; Spurdle et al. 1989; Lucotte et al. 1990b; Torroni et al. 1990). Coinheritance of "alleles" of the A and D series has also been observed (Spurdle et al. 1989; Torroni et al. 1990), while Torroni et al. (1990) have also reported the presence of additional fragments of 5.2 kb and 3.5 kb in Italian caucasoids and of 2.5 kb in an African individual. The TaqI RFLPs were initially thought to result from point mutations within TaqI restriction sites only (Ngo et al. 1986). The possibility of insertion/deletion or rearrangement processes as a mechanism for generating the observed polymorphism was excluded on the premise that variations of hybridization patterns were not observed with other restriction digests (Ngo et al. 1986). However, coexistence of "alleles" at the A and D loci (Spurdle et al. 1989; Torroni et al. 1990) and at the F locus (present study) has led to a reassessment of the nature of the polymorphism detected by 49a, since these coexisting "alleles" are believed to be due to duplication processes (Torroni et al. 1990; present study). It is thus of great interest that p49a and p49f have been shown to detect polymorphism with PvuII (Spurdle et al. 1989), as well as with SstI, Bg11I, HindIII, and PstI (Spurdle and Jenkins 1989). In the present study, the Y-specific 49a TaqI and PvuII haplotypes were determined for a number of different southern African populations. The resulting frequency data were used to assess the affinities of the populations and to elucidate the nature of the historical interactions between them. The relationship between the two different polymorphisms is also discussed. Subjects and Methods Subjects The subjects were unrelated male individuals belonging to various southern African populations. Their appropriate geographical areas of origin are shown in figure 1. Certain individuals were selected for 49a/PvuII analysis, as part of a regional population study, because they possessed 49a/ TaqI Ht7 or Ht8 (for details, see Discussion).

Spurdle and Jenkins The Bantu-speaking negroid group includes different chiefdoms, each speaking a different Bantu language. The Zulu, Xhosa, Ndebele, and Swazi chiefdoms are classified linguistically as the Nguni, whereas the southern Sotho, Pedi (northern Sotho), and Tswana are grouped as the Sotho/Tswana. Likewise, the Herero-speaking group includes the Herero and Himba chiefdoms. The Tsonga sample includes individuals from the Tsonga and Shangaan chiefdoms. The migration of the Bantu-speaking negroids from their postulated area of origin in west-central Africa is believed to have followed at least two routes (Huffman 1982): a general southbound course (the eastern Bantu) and a southwesterly route across central Africa toward the western parts of the continent (the western Bantu). The Herero are the only representatives of the western Bantu group. The Lemba are Venda speakers considered by ethnographers to be of alien origin (Van Warmelo 1974). Many factors distinguish them from the other Bantu speakers (Van Warmelo 1974): many Lemba have a distinctive appearance, i.e., angular features with a prominent hooked nose; the men used to wear a long cotton upper garment (khanzu), as found along the east coast of Africa; among themselves they spoke a language not understood by their hosts in southern Africa; marriage was strictly endogamous; certain foods-e.g., pork, certain other animals, and the flesh of cattle not kosher killed according to their law- are forbidden; circumcision is practiced; and unintelligible prayers are recited and responded to at certain ritual ceremonies. Although the Lemba themselves claim to belong to one of the lost tribes of Israel, certain facts about them suggest that they are descendants of Semitic traders (presumably Arabs) from the east coast. One such fact was the observation that their ritual prayers may represent mangled suras from the Koran (Van Warmelo 1974). The Dama are a Khoisan-speaking negroid group. Although these people have typical negroid features, culturally and linguistically they do not reveal any similarities to the Bantu group (Malan 1980). They speak the same Khoi language as do the Nama, a fact that is in agreement with their historical enslavement by Khoi pastoralists (Jenkins 1982). The Khoisan group is composed of the Khoi (formerly referred to as "Hottentots") and the San (formerly "Bushmen"). These medium-statured people have a yellow skin color, flat triangular face, and steatopygia (Malan 1980), and they speak languages

Y Chromosome Haplotypes in African Populations

Figure I

109

Geographical location of southern African populations

composed of clicks and other guttural sounds (Nurse and Jenkins 1977). The !Kung San group collected at the Omega military camp in northern Namibia originate from southern Angola. The South African caucasoid group includes peoples of western European and Asiatic Indian origin, as well as Ashkenazim Jews from eastern Europe. The Johannesburg (JHB) "colored" group is a population of mixed ancestry, resulting from admixture of European caucasoid, Khoisan, Malay, and Bantu-speaking negroid people. The Richtersveld "colored" group has resulted from admixture between European caucasoid trekboers (farmers), who moved into the northern Cape area during the 18th century, and indigenous Nama women. They have remained relatively isolated over the ensuing years.

Molecular Methods

Probes p49a and p49f were extracted according to standard procedures (Maniatis et al. 1982). The 0.9kb p49a XbaI-BamHI insert and 2.8-kb p49f EcoRI insert (Ngo et al. 1986) were purified by electroelution or phenol-freeze extraction. Human genomic DNA was extracted from packed cells or buffy coats by using the method of Sykes (1983). TaqI restriction-enzyme digests of human genomic DNA (5-10 jig) were separated by electrophoresis on 0.8% agarose gels in 1 x TBE and were run until the bromophenol blue dye front had migrated 20-26 cm from the origin, while PvuII fragments were separated by electrophoresis until the 3.53-kb fragment of lambda digested with HindIII and EcoRI had migrated 17.5 cm from the

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Y Chromosome Haplotypes in African Populations

ill

Table I Frequency of 49a/Taql Alleles in Four Major Population Groups

ALLELIC VARIANT FRAGMENT SIZE(S)a OR ALLELE (kb)

AsC;A3 ............ A4;A3 ............ A4;A3;A2 ........... A3;A2 ............ AS

............

A4..................... A3..................... A2..................... Al..................... AO Bi..................... BO Cl ............ Co Dl;D2 ............ Dl ............ D2 ............ D3 ............

Caucasoid (N = 174)

JHB "Colored" (N = 66)

28.0;18.0'

.019

.000

.000

.000

20.0;18.0 20.0;18.0;17.0 18.0;17.0 28.0d

.006 .006 .038 .019 .045 .474 .346 .000

.002 .000 .005 .000 .007 .123 .147 .695 .020 1.000

.000 .000 .219 .000 .021 .271 .198 .271 .021 .979

.000 .000 .045 .000 .015 .515 .243 .152 .030 .985

.000 .012 .988 .000 .015 .017 .000 .968 .000 .961 .039 .990

.021 .010 .990 .000 .010 .021 .000 .969 .000 .667 .333 1.000

.015 .303 .697 .000 .259 .259 .015 .467 .000 .970 .030 1.000

.006

.010

.000

.000

1.000 .000 .782 .218

1.000 .000 .865 .135

.979 .021

1.000 .000 .833 .167

20.0 18.0 17.0 14.0

.045 .962 .038 .333 .667 .006 .513 .250 .000 .231 .000 .949 .051 .994

10.0 9.0

8.0;7.5 8.0 7.5 6.6

DO

F2;Fle ............ Fl ........ .... FO Gi ............ GO

5.9;5.5

Hi ............

4.5

5.5 5.2

HOf Ii...................... I0

FREQUENCY IN Negroidb Khoisan (N = 407) (N = 96)

3.3

.823 .177

Sizes determined for this study are consistently larger than those reported by Ngo et al. (1986). b Enigmatic Lemba and Khoisan-speaking Dama populations were excluded. c Probably represents same AS fragment as reported by Torroni et al. (1990), but fragment sizes have not yet been compared directly. d Out of range possible with DNA size markers used. ' Variant has been observed only in the Richtersveld population. Fragment F2 may represent 5.2-kb fragment reported by Torroni et al. (1990), but fragment sizes have not yet been compared directly. f Torroni et al. (1990) report absence of H to be associated with absence of bands P and R. The techniques of electrophoretic separation employed in the present study mostly excluded identification of 49a/ TaqI fragments smaller than the 0 fragment. However, in at least one example, HO was shown to be associated with the absence of band P. a

origin. DNA samples from certain individuals of known PvuII haplotype were included as standards on each PvuII gel. DNA was transferred, by the method of Southern (1975), to nylon membranes (Biodyne or Hybond-N). Filters were baked at 801C for 1-2 h.

Hybridization

Baked filters were prehybridized and hybridized according to manufacturer's specifications. The 32p_ dCTP-oligolabelled probe insert (Feinberg and Vo-

Spurdle and Jenkins

112

gelstein 1983) was hybridized to prehybridized blots for 20-48 h, and hybridized filters were washed at low stringency. Biodyne filters were washed twice (15 min each) in 2 x SSC at room temperature and twice (30 min each) in 2 x SSC, 0.1 % SDS at 420 C. Hybond-N filters were washed twice (15 min each) at room temperature, once (30 min) at 420C, and once (10 min) at 65°C in 2 x SSPE, 0.1 % SDS. After 1-6 dexposure at - 70°C, fragments were visualized, by autoradiography with Kodak XAR film backed with Dupont Cronex intensifying screens.

Probes p49f and p49a were initially used consecutively to determine the complete p49a/ TaqI haplotype. Consequently it was discovered that the purified XbaI-BamHI p49a fragment revealed all the Y-specific fragments detected by p49f, with the exception of fragment N (see fig. 2). All five allelic series reported by Ngo et al. (1986) could thus be detected by hybridization with p49a alone. Similarly, the p49a fragment was shown to reveal the same polymorphic PvuII fragments as did p49f and was used alone to detect this polymorphism.

Computer Analysis

Genetic distance measurements and cluster analysis of 49a / TaqI haplotype frequency data were computed using the methods of Harpending and Jenkins (1973) and the computer program Antana written and supplied by Prof. H. C. Harpending, Pennsylvania State University. Results

Screening of southern African populations by p49a has revealed a number of new TaqI allelic variants (table 1). The variants depicted as apparently coinherited A alleles are not believed to be due to partial digestions, since the fragment sizes remain unchanged when digested with increased concentrations of TaqI (data not shown). When DNA from other appropriate family members was available, strict Mendelian inheritance of new allelic variants (A3;A2, GO, and HO) was successfully demonstrated. The frequencies of the alleles/allelic variants in the four major population groups are also shown in table 1. Each new allelic variant results, of necessity, in the creation of a new haplotype. Certain of the new variants are found in more than one haplotype, and new combinations of previously reported alleles have also been revealed. The new haplotypes observed in the present study (including seven haplotypes of disputable nomenclature

Table 2 New Y-specific 49a Haplotypes Reported in Present Study

HAPLOTYPE Taq49aa

A

A3/A2 A3 AO A3 28 ............ A3 29 ............ 30 ............ A3/A2 31 ............ A3/A2 A3 32 ............ 33C ........... A2 Ai 34 ............ 35 ............ A3/A2 A4 36............ 37 ............ A4/A3 38 ............ A3/A2 AO 39 ............ 40 ............ A4/A3 Ai 41'........... AS 42 ............ A4 43 ............ 44 ............ A4/A3 45 ............ A4/A3/A2 46 ............ AS/A3 47 ............ AS/A3 A3 48 ............ AO 49 ............ 50 . ........... A3/A2 A2 51 ............ A3 52 ............ A2 53 ............ A4 54 ............ A2 55 ............ A2 56g ........... A3 57 ............ AO 58 ............ A4 59 ............ A4/A3 60h A4 61 ............ AO 62 ............ 25 26

............

............

...........

B

Bi Bi Bi BO Bi Bi Bi Bi Bi Bi Bi Bi Bi BO Bi Bi Bi Bi Bi B1 Bi Bi Bi BO BO Bi BO Bi BO Bi Bi Bi Bi B1 Bi Bi Bi Bi

=

ALLELE D

F

G

CO

Di

Cl

D2

CO

Di

CO

D2

CO

Dl

Fl Fl FO Fl FO Fl Fl FO FO FO FO Fl Fl Fl FO Fl Fl Fl Fl FO Fl Fl Fl Fl Fl Fl Fl Fl FO FO FO Fl FO FO FO Fl Fl F1/F2

Gl

CO

DO D3 DO DO DO DO DO DO DO DO DO DO D2 DO DO DO DO Dl DO DO D1/D2 DO D2 Dl

C

Ci

CO

CO Cl CO CO CO CO CO

CO CO CO

CO CO CO CO CO CO

CO CO Ci CO Cl

Ci

DO DO DO DO D2 DO D2 D2

CO

Dl

CO CO CO

Ci CO CO Ci

H

I

G1 G1 G1 G1 G1 G1 Gl

Hi

Ii

Hi Hi

I0

Gl G1 G1 G1 G1 G1 G1 G1 G1 GO G1 G1 Gl Gl

Hi Hi Hi Hi Hi Hi

G1 G1 GO GO G1 G1 Gl G1 Gl G1 GO Gl G1 G1 Gi Gi Gi

Hi

Hi Hi Hi

Ii

IO 10

Ii I0

I0 Ii

Ii Ii

I0 Ii Ii

HO I0 Hi

IO

Hi

Ii

Hi

IO

Hi Hi Hi

Ii

I0 Ii

Hi Hi Hi

Ii

Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi Hi

I0 IO

Hi Hi

Ii

Ii Ii

Ii

IO Ii

IO Ii

I0 I0 Ii Ii

I0 Ii

Ii

The Roman numeral system of naming haplotypes (Ngo et al. 1986) has been replaced by the arabic system in the present study, because of the large number of haplotypes recorded. b Ht4O of Torroni et al. (1990). c Ht41 of Torroni et al. (1990). d Ht23 of Torroni et al. (1990). 'Ht38 of Torroni et al. (1990). f Ht34 of Torroni et al. (1990). g Ht27 of Torroni et al. (1990). h Ht37 of Torroni et al. (1990). a

Y Chromosome Haplotypes in African Populations

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113

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Population Figure 3

Cluster analysis generated using 49a/ TaqI haplotype genetic distance data of southern African populations

reported by Torroni et al. 1990) are shown in table 2. The hybridization patterns for most of the new haplotypes are displayed in figure 2. Haplotype frequencies were calculated for each of the 21 populations (Appendix). Cluster analysis of the populations, as determined from 49a/ TaqI haplotype frequencies, is shown in figure 3. Previous research revealed that p49f detects a complex PvuII polymorphism, involving at least 10 polymorphic fragments (Spurdle et al. 1989), all of which are inherited in a Mendelian manner. Improved resolution of the larger polymorphic bands has shown 17 polymorphic fragments to be present in the 127 individuals screened (table 3 and fig. 4). The polymorphic fragments present in a single individual can be said to represent a PvuII haplotype of that individual. A total of 53 such haplotypes have been observed in the present study (table 4), indicating extreme diversity of this

polymorphism. In different haplotypes, differences in intensity can be observed for the same fragment. As seen in figure 4, band D is clearly more intense in HtlS than in Htl2, while band E is less intense in Htl6 than in Htl 1. This suggests that some fragments may exist as a variable number of copies. However, the intensity observed for a given haplotype appears to be consistent. PvuII fragment and haplotype frequencies in the caucasoid, negroid, and Khoisan populations are displayed in tables 3 and 5, respectively. The relationship between the 49a/ TaqI and 49aIPvuII polymorphisms is summarized in table 6. Discussion Origin of 49a/Taql Polymorphism Probes 49f and 49a are said to detect a family of moderately repeated sequences (Ngo et al. 1986).

Spurdle and Jenkins

114 Table 3 Frequencies of Polymorphic Pvull Fragments Detected by Y Chromosome Probe 49a

FREQUENCYb FRAGMENTa A+ .. .......... A .......... B .......... B2 .......... C .......... D .......... D2 .......... E .......... E2 .......... F .......... F2 .......... G .......... G2 .......... H ..........

J

..........

L2 M

..........

..........

IN

SIZE (kb)

Khoisan (N = 35[36])

Caucasoids (N = 30[40])

Negroids (N = 34[51])

>>>48c >>48 >48 24.0 20.2 18.5 16.0 15.0 13.5 12.8 11.0 10.5 8.8 8.3 6.0 4.8 3.6

.00(.00) .31(.33) .60(.58)

.17(.15) .51(.38) .41(.45) .55(.68)

.00(.00) .12(.14) .85(.90)

.34(.36) .51(.50) .63(.64)

.10(.10) .79(.85)

.03(.29) .74(.55) .91(.84)

.06(.06) .54(.56) .00(.00) .06(.06) .91(.89)

.07(.13) .45(.58) .14(.10) .51(.43) .66(.63)

.00(.02) .85(.76) .00(.00) .09(.08) .97(.94)

.69(.67)

.28(.30)

.18(.22)

.00(.00)

.03(.03) .07(.05)

.03(.06) .91(.92) .00(.00) .71(.72)

.93(.95) .03(.03) .97(.95)

.00(.00)

.03(.02) .97(.98) .00(.00) .97(.98)

Constant fragments 1 (7.7 kb), K (5.6 kb), and L (4.7 kb) (not shown) were encountered in all three populations at a frequency of 1.00. Fragment L is an autosomal fragment detected in females. b Calculated for random individuals. Certain individuals were selected for 49a/PvuII analysis, as part of a regional population study, because they possessed 49a/ Taql Ht7 or Ht8 (see text for details). Data in square brackets include individuals with TaqI Ht7 or Ht8. c Fragment migrates more slowly than undigested lambda. a

Since 49a and 49f also detect complementary sequences mapped to chromosome 3 (Cohen-Hagenhauer et al. 1987), it has been suggested that the Y sequences represent a pseudogene and have resulted from duplication or retrotranscription (Leroy et al. 1987). Lucotte et al. (1990a) conclude, from comparative hybridization studies in primates, that the autosomal sequence was retrotranscribed onto the Y chromosome during primate evolution. Only one and two autosomal TaqI fragments of apparent uniform intensity are detected by 49a and 49f, respectively, as opposed to a number of varying Y-specific fragments. This implies that the copy-number increase detected on the Y occurred only after retrotranscription, probably by gene amplification (Maeda and Smithies 1986). It is also not unforeseeable that, during the course of human evolution, divergence by duplication or deletion may have taken place because of polymerase slippage (Wolff et al. 1988). The generation of the TaqI RFLPs detected at each locus has been proposed to be due to point mutations within TaqI sites (Ngo et al. 1986), arising in or lead-

ing to fragments that could comigrate with other major nonpolymorphic bands, such as E, N, and 0, and thus remain undetected. This behavior was illustrated by the apparent comigration of a putative DO fragment with B (Ngo et al. 1986), but haplotype data presented by Torroni et al. (1990) and in table 2 indicate that this comigration is at the very least inconsistent, since Ht3O0 and Ht33 of Torroni et al. (1990) and Ht28 and Ht3 8 of the present study each include both allele BO and allele DO, suggesting that DO may comigrate with a smaller nonpolymorphic band. The coexistence of "alleles" at several loci (Spurdle et al. 1989; Torroni et al. 1990; present study) has led to a reassessment of the nature of the polymorphism detected by 49a. The presence of three A bands in one individual (table 1 and fig. 2) suggests that duplication processes, followed by differential CpG mutation of the duplicated loci, may be responsible for the observed coexistence. This explanation for coexisting alleles is also that favored by Torroni et al. (1990). Each combination would thus represent one allele in itself. Densitometric studies could be used to quantify

Table 4 Nvull Haplotypes Detected in Southern African Populations PvulI FRAGMENTa

HAPLOTYPEA I 1 .......... 2 .......... 3 .......... 4 .......... 5........A 6 .......... 7 .......... 8 .......... 9 .......... 10.......A 11 .......A 12 ......... 13 ......... 14 ......... 15 ......... 16 ......... 17 ......... 18 ......... 19 ......... 20 ......... 21 ......... 22 ......... 23 ......... 24 ......... 25 ......... 26 ......... 27 ......... 28 ......... 29 ......... 30 ......... 31 ......... 32 ......... 33 ......... 34 ......... 35 ......... 36 ......... 37 ......... 38 ......... 39 ......... 40 ......... 41 ......... 42 ......... 43 ......... 44 ......... 45 .......A 46 ......... 47 ......... 48 ......... 49 ......... 50 ......... 51 ......... 52 ......... 53 ......... a

A

B

B2

C

D

D2

B A

A A

B

A

E

E2

B B2 B2 B2 B2 B2 B2 B2 B2

F F

B

D D

F

F2

F2 F2

E

E E E

D D

G2

F2 F2 F2 F2

C

C

C B B B A

A

B B B B

B2 B2 B2 B2

C C C

A A. A

B2

C B2

C C

B B2

A B B B

B2

C C C C C

B B B

C C B2

B B B B B B B B

E B E E

D D

F

D D

B2 B2 B2 B2 B2 B2 B2 B2 B2

F2 F2 F2 F2 F2

E2 E2

B

F2 F2 F2 F2

F2 F2 F2 F2 F2 F2 F2 F2

D. B2

A

F F F F

D

B B B

E

D D D. D D

C

D D D. D D D. D D. D

E D2

E

E

F B B B E

D D D D D D D D D

E2

F

E E B B

F2 F2 F2

B B

D2 D2

B E

F F

F2 D2

An ellipsis (. . .) indicates absence of bands.

B

F

G G C. C. C. C.

G C. C. C. C. C.

J

J J J J J I J J

G C

J J I J J

C

C2 F2 F2

B

D D

F2 F2 F2 F2 F2 F2

H

I J J J J i J i i J i J I I I J J J J J J

C

F

D2

G

J J J J J J J J J J J J J J J J

L2

M M M M M M M M

M.M

L2

M M M M M M M M M M M M

M

M. M. M M. M M M. M M M. M. M. M M M. M M M M M M

Spurdle and Jenkins

116 0

39

12 15

11

12

16 17

As.

BA

B2. A. B. B2E

EE2

C.

F2

D2-D

G2* He

I.

J.

Ken LB!

L2-

49a/PvuII hybridization pattern. The positions of the PvuII fragments Figure 4 Haplotype numbers are shown at the top of each lane.

the number of A loci present in a given A band and to thus distinguish between single- and multiple-copy alleles. Unfortunately, such studies have proved unreliable because of the large range of hybridization intensities present in any given 49a / TaqI haplotype, as well as because of the large area (>23 kb-

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