A preliminary linkage map of the chicken genome

GENOMICS

13,690-697

(19%‘)

A Preliminary

Linkage Map of the Chicken Genome NAT BUMSTEAD AND JAN PALYGA

Institute

for Animal Health, Houghton Received

September

Laboratory, 23, 1991;

Houghton, revised

March

Huntingdon

PE17 ZDA, England

IO, 1992

ation of resistance to Marek’s disease with the major histocompatibility complex, they are not associated with known loci. It is therefore difficult to identify the genes involved or to investigate their individual properties. Analysis of these traits using a linkage map should allow the relationship of these genesto each other and to previously characterized genes to be determined and should make it possible to investigate their effects in controlled experiments. It would also permit marker-assisted selection in a commercial context and perhaps ultimately allow isolation of genes by techniques of chromosomal walking (Soller and Beckmann, 1985). The chicken has a number of advantages for the construction of linkage maps: the genome is small relative to other domesticated animals [approximately 1.2 X 10’ bases (Olofsson and Bernardi, 1983)], and the reproductive potential of chickens is high, allowing large sibships to be derived from single pairs of parents. It is also possible to prepare good quality DNA in large quantities from small volumes of blood since the red blood cells are nucleated. There are, however, a large number of microchromosomes in the chicken karyotype, which consists of 8 pairs of macrochromosomes and 30 pairs of microchromosomes (Yamashina, 1944). In addition, the sex chromosomes consist of a moderately sized 2 chroPress, Inc. mosome paired to the W microchromosome (Shoffner and Krishan, 1965). As in other avian species, the female is the heterogametic sex (ZW), the male being homogaINTRODUCTION metic (ZZ). Although a considerable number of biochemical and Commercial poultry breeding flocks are large, well physiological loci have been identified in chickens in the pedigreed, and closely monitored for both physiological past, and some linkage data have been derived (Somes, and disease-related characteristics, and they provide a 1984), the strains of birds in which variants have been potent source in which to identify genetic traits. Many detected limit their applicability. We have therefore atsuch traits have been identified by classical or biometritempted to develop a linkage map based on restriction cal techniques; however, while it is possible to determine fragment variants, working directly in the lines in which that these properties are genetic in origin, in most cases we are interested. there are no ready means of identifying the underlying For the present map we have chosen two lines of genes. Our particular interest lies in the genes responsible for disease resistance. Differences in susceptibility White Leghorns as parental lines. These lines differ in have been described for most of the diseasesof chickens their susceptibility to a number of diseases,but in partic(Calnek, 1985; Payne, 1985; Wakelin and Rose, 1990; ular, line N is resistant to salmonellosis while line 151 is Bumstead et al., 1991), and in a number of cases these highly susceptible (Bumstead and Barrow, 1987). The appear to be due to a small number of genes of major lines are partially inbred (Bumstead et al., 1987), but effect. However, with the notable exception of the associ- since neither line is fully inbred, restriction fragment

We have used backcross progeny from a cross between two inbred lines of chickens to construct a linkage map of the chicken. The map currently consists of 100 loci, identified using either anonymous cloned fragments of genomic DNA or sequences corresponding to cloned genes. Parent birds were derived from two lines of White Leghorn chickens, which differ in susceptibility to a number of diseases. Restriction fragment length variants were identified by comparison of the DNA of these two parent birds using a panel of seven restriction enzyme digests and the segregation pattern observed in progeny of these two birds. Restriction fragment length variants were detected for approximately 41% of the clones tested, whether these were known genes or random genomic fragments. This high level of variability was also reflected in the presence of variation within the parental lines for some clones. The overall size of the linkage groups and the progressively higher incidence of linkage as further clones were added suggests that the map covers the majority of the genome, although it is unlikely that there are marker loci on all the microchromosomes. The present map will be of use in locating genes affecting disease resistance, but also illustrates the relative ease with which such maps for the chicken can be constructed. c 19~2 Academic

0888-7543/92 Copyright All rights

$5.00 8 1992 by Academic Press, of reproduction in any form

690 Inc. reserved.

LINKAGE

MAP

OF

THE

length variants (RFLVs) were identified in two individual parent birds and segregation was analyzed in progeny of these two birds. For clarity of interpretation, a backcross rather than an intercross mating was used. Preliminary results indicated that restriction fragment variation is common in chickens and that repetitive sequences were unlikely to affect Southern blots probed at high stringency. Accordingly we have used random cloned genomic fragments as probes, together with clones corresponding to known genes where these were available. As an initial target, we have aimed to map 100 loci to construct an outline map and to identify any problems in this procedure. MATERIALS

AND

CHICKEN

691

GENOME

Barn HI

Eco RI

Xba I

Rsa Taq I I

Msp I

Hae Ill

mfmfmfmfmfmfmf Kb -35.2 -21.2

-

9.0

-

2.5

METHODS

Chickens. Parent birds were derived from the White Leghorn lines N and 151 maintained at the Institute for Animal Health, Houghton Laboratory. These lines were established from eggs provided from the lines held at the Regional Poultry Research Laboratory (East Lansing, MI), in 1982 and 1972, respectively. The genetic purity of the lines is checked by blood group analysis of each generation. Coefficients of inbreeding in these lines have been estimated as 0.8 (line 151) and 0.5 (line N) (Bumstead et al., 1987). Other properties of the lines are described in Bumstead et al. (1991). All birds were maintained in isolation and were free of the major pathogens of poultry. Crosses. An F, hen (No. 2826) was produced by mating a line 151 cockerel to a line N hen, and this bird was mated to a single line 151 cockerel (No. 240) to produce progeny for segregation analysis. Sixtyeight progeny of this mating were included in the analysis; a further 15 progeny of a mating between the same cockerel and a second F, hen (No. 2818) were also analyzed. Preparation ofgenomic DNA. Nuclei were isolated from red blood cells using saponin (Sigma) and DNA prepared by phenol extraction as described in Bumstead et al. (1987). Southern blotting and hybridization. Southern blots were carried out in a conventional manner using either nitrocellulose or nylon membranes (Amersham). Hybridizations were carried out overnight at 42°C in 50% formamide, and blots were washed twice for 30 min at 55°C in 0.1X standard sodium citrate, followed by a further wash for 30 min at 65°C at the same salt concentration (Maniatis et al., 1982). Probes. Random genomic clones were prepared by EcoRI digestion of line N DNA and ligation of fragments into either pBluescript (Stratagene) or pT3T7 (Pharmacia) cloning vectors. In a small number of cases, restriction enzymes other than EcoRI were used to prepare the plasmids. Plasmids containing a number of cloned chicken genes kindly provided by other laboratories were also used; these are listed in Table 1 and were selected solely on the basis of availability. The major histocompatibility complex haplotypes of the birds were determined by agglutination of red blood cells using specific antisera (Bumstead et al., 1987). Two-point linkage estimations were carried Estimation of linkage. out for all combinations of loci (Silver, 1985). The order of linked loci was determined by minimizing the number of recombinations.

RESULTS Restriction Fragment Length Variants In all, 237 DNA clones were screened for variation using panels of the two parental DNAs digested by seven restriction enzymes. Of these, 97 (41%) detected bands present in the F, female parent but not present in the line 151 male. Of 18 cloned genes, 9 detected similar dif-

* FIG. 1. Detection of restriction fragment length variants. DNAs from the line 151 and F, parent birds were digested with the restriction enzymes shown and hybridized to the genomic clone pT5/21. Samples are: m, line 151 male No. 240; f, F, female No. 2826.

ferences. A typical result is shown in Fig. 1. In a small number of cases, clones detected more than one variant locus, and the total number of loci identified was 100. Clones for which variation was detected are listed in Table 1, which also shows the fragment sizes observed for the enzyme digestion used in screening the segregating progeny. In addition to these clones, a further 6 clones identified bands present in the line 151 but not in the F, parent; in each case the line 151 bird was heterozygous. These clones were not included in the mapping process. The majority of clones showed variation for only one of the seven enzymes tested (65/97 cases), most commonly either TaoI or MspI. Clones showing variation for four or more enzymes were rare (5/97). In all cases, clones detected bands in both parents, and there was therefore little evidence of local deletions or extreme sequence changes. The cloning of random fragments allowed the possibility of two or even more discrete fragments being cloned in the same plasmid. In at least two casesthis appears to have occurred, as plasmids identified more than one segregation pattern. The frequent presence of more than one EcoRI fragment in genomic blots suggests that the inclusion of more than one fragment may have been widespread; however, in most cases it is not possible to tell whether this is due to two linked fragments resulting from incomplete digestion or two disparate fragments incorporated at ligation. In three cases, multiple restriction fragments displaying two distinct segregation patterns were observed for a single plasmid, suggesting that in these cases the latter event had taken place.

692

BUMSTEAD

AND

TABLE Restriction Restriction Probe

Enzyme

d 240 line

Fragments

fragments

151

PALYGA

1

Detected

in Parental

Birds Restriction

(kb) Probe

P 2826 F, (151 x N)

Enzyme

6 240 line

fragments

151

(kb)

P 2826 F, (I.51 x N)

(a) Probes pIg(V + Cl (Reynaud et al., 1983) p5MA (Sudol et al., 1988) LYS (Steiner et al., 1987) pCBAECAT (Ferrari et al.. 19881 pCTR (Ghan et al., 1989) pH2fa (Dalton et al., 19891 pUNl2Iov (Benoist et al., 1980) p6ayes (Sudol et al., 1988) pRAV-1 (Bumstead et al.. 1987)

corresponding

genes

(b) Random

zb9I

4.2, 3.0, 1.0, 0.8, 0.5. 0.1

4.2, .6,” 3.0, 1.0, 0.8, 0.5, 0.1

TqI

8.0, 6.0, 5.3, 4.2, 3.0

8.0,6.0, 5.3, 4.2, 3.0, -2.7

TqI

4.5, 3.3

5.5, 4.5, 3.3

MspI

3.3

@,

HaeIII

0.3

0.8, 0.3

Hue111

2.8, 1.8, 0.6, 0.2

3.6, 2.8, 2.1, 0.6, 0.2

HaeIII

1.5, 1.0

3.J, 1.5, 1.0

MspI

31.5,

EcoRI

Ta91 TuqI A4spI

MspI BamHI XbaI MspI

ImpI TaqI TaqI EcoRI TaqI RsaI BamHI TqI Taql Tu~I MspI Tag1 Xbal TagI MspI MspI MspI TaqI

TaqI Tag1 TaqI

16.6, 11.9, 5.3, 1.8

genomic

3.1, 1.5 6.9, 2.3 4.5 10.5, 3.9, 2.8 15.5 15.5 8.9 6.8 6.6, 4.2 4.7 2.4 7.5 2.0 23.1, 15.5, 5.5 19.4, 6.9 6.9 10.5 4.7 8.0, 5.3 10.0 16.6, 1.2 8.9, 5.7, 1.4 3.7 15.5 8.9, 4.7 15.5 3.5 10.5, 3.2

3.3

16.6, 11.9, u, 1.8

14.4, 7.2, 4.4, 3.4

(b) Random RI1 R2 R33 R34 R54 R55 R2/1 R2/7 R2/14 R3/1 54 s50 S52 Tl T4 T9 T12 T28 T2/11 T2/12 T2/20 T2/22 T2/28 T2/29 T2/38 T2/41 T2/46 T2/47

to cloned

5.3,

14.4, u, 7.2, e, 4.4, 3.4

clones 4.0, 3.1, 1.5 6.9, a, 2.3 8.9, 4.5 10.5,@, 3.9, 2.8 15.5,g.J 15.5, gj 8.9,2 9.0, 6.8 6.6, 4.2, &J 4.7,&4 &I, 2.4 a, 7.5 2.3, 2.0 23.1,-, 15.5, 5.5 23.1, 19.4, 6.9 6.9, $.J 10.5, &9 s.s, 4.7 Q, 8.0, 5.3 12.7, 10.0 16.6,m, 7.2 8.9, 5.7, 1.4, u u, 3.7 15.5, lo.r, 8.9, 3, 4.7, 3lJ 17.9, 15.5 3.5, g3 10.5,3.7, 3.2

T2/53 T2/55 T2/58 T2/59 T2/66 T2/68 T2/69 T2/70 T2/71 I-2/72 T2/73

Hind111 MspI TaqI TuqI TaqI BamHI MspI

T2/75 T2/82 T3/2 T3/4 T3/5 T3/7 T4/2 T4/6 T4/7 T4/12 T4/13 T4/18 T4/19 T4/20 T5/1 T5/3 T5/12 T5/14 T5/21 T5/24 T5/39 T5/41 T5/51 T7NB Tlf40 T12/28

MspI MspI MspI MspI Tag1 MspI MspI

T12/33 T12/40 T12/46 T12/48 T12/53 T12/61 T12/63 T12/65 T12/67 T1”/74 T12/81 T12/88 T12/91 T12/104 T12/106 T12/108 Tl3/2 T13/40 111/26 u3/10

Hue111 TaqI TaqI TaqI MspI TaqI M.spI MspI TaqI BamHI Tag1 EcoRI MspI HaeIII MspI MspI MspI MspI Hue111 MspI

The 0 Fragments

underlined

are those present

in the F, parent

bird

n9I MspI TaqI XbaI

ImpI MspI TaqI XbaI MspI EcoRI MspI MspI

TaqI TayI

MspI XbaI TaqI RsaI TaqI MspI MspI M.spI TaqI

EaB locus

hut not present

was detected

in the line

genomic 11.2, 16.6 2.8 2.7 3.1, 12.7 2.2 4.9, 14.4, 8.5, 11.2,

clones 8.0

2.7, 1.6, 1.2

2.3, 0.8 6.0, 4.4 2.9 7.2, 6.0, 4.5, 4.0 10.5, 4.5 3.4, 1.5 17.9 19.6 9.4, 1.6 13.5 4.0 6.6 12.7 1.6 5.7 2.0 13.5 12.7, 8.9, 5.5 3.7 1.3 10.5 2.7 7.2, 1.8 8.3 1.8 14.4, 6.6 5.3 12.7,5.1, 1.4 11.2, 7.2, 6.0, 4.5, 4.0 2.8, 1.3, 0.7 9.4 2.8 16.9 11.9 6.3 1.4 8.2 4.5 5.5 9.4 3.7 8.5 1.5, 0.5 1.8, 1.4 10.4 5.1 8.5 1.9 4.9 serologically

151 parent.

(continued) 11.2, 8.0, Q 16.6, 5.7 u, 2.8 2.7, z B, 3.1, 2.7, 1.6, 1.2 21.5, 12.7 2.2, u 4.9.4.5. 2.3, 0.8, Q.J 19.4, 14.4, 6.0, 4.4 8.5. 2.9, 2.4 16.6, 11.2, 7.2, 6.0, 4.5, 4.0 10.5, 6.6, 4.5 5.7, 3.4, 1.5 17.9, 11.9 23.1, 19.6 m, 9.4, 1.6 13.5, g 4.0, &j 13.5, 6.6 12.7,8.:, 1.6, u 19.4 5.7, gJ 2.0, u 1Fj,5, 13.5 12.7, Q, 8.9, 5.5 3.7, g.cJ 1.3, 0.8 10.5, 80 s.0, 2.7 7.2, 4.7 1.8 -T 3.0 -t 8.3, Q u, 1.8 15.5, 14.4, 6.6 a, 5.3 12.7, z, 5.1, 1.4 16.6, 11.2, 7.2, 6.0, 4.5, 4.0 3.J, 2.8, 1.3, 0.6 9.4, s.0 2.8, &I 16.9, u 11.9, Q 13.5, 6.3 6.0, 1.4 a, 8.2 4.5, &6 7.4, 5.5 9.4, 5-5 3.7, &2 lo.s_, 8.5 1.5, o.& 0.5 u, 1.8, 1.4 14.7, 10.4 5.1,u 8.5, G 2.3, 1.9 4.9, 4.5

LINKAGE

MAP

OF

THE

FIG. 2. Detection of segregation in 56 backcross progeny. DNAs were digested using the restriction enzyme XbaI and hybridized to pT5/21: m, line 151 male parent No. 240; f, F, female parent No. 2826; l-56,56 progeny of these two birds.

Recombinational

Map

Initially, linkage data were derived from segregation data for all 100 loci from a panel of 56 progeny of the two parent birds; a typical segregation is shown in Fig. 2. All data were compared by two-point linkage analysis, and a lod score of greater than 3 (equivalent to a map distance of 25 CM) was taken as evidence of linkage; in no case were mutually incompatible linkage associations found. In these results, all recombinations derive from meioses in the female (heterozygous) parent, and no attempt was made to allow for interference or sex-specific differences in recombination. The order of loci was determined by minimizing the number of double crossovers, and the resulting linkage groupings are shown in Fig. 3, which shows the incidence of different chromosomal types among 56 birds. In most cases double recombinations within a linkage group were rare, and the majority of linkage groups are either parental in type or contain a single recombination. The overall map, with estimated distances between loci, is shown in Fig. 4. This includes data from a further 27 progeny, 12 derived from the original parents and 15 derived from a second F, hen. The total map length included within linkage groups is 585 CM, with an average distance of 8.5 CM between loci (omitting loci having identical segregation patterns). If the correspondence of recombinational length to physical length is similar to that in the mouse (Thomas and Rothstein, 1991), this represents approximately 50% of the genome, not including the regions flanking mapped loci. If the detect-

CHICKEN

693

GENOME

able regions flanking linkage groups are included, the total map size is greater than 2000 CM; this is greater than the expected genome size and suggeststhat at least in some cases loci must be located close to the ends of chromosomes. In calculating map distances, we have made no allowance for interference. This would reduce apparent map distances, but since only a very small proportion of even the longest linkage groups contained more than one crossover (Fig. 3), it seemsunlikely that this would significantly affect the estimated distances. An alternative measure of the extent of the map is the incidence with which additional loci were found to be linked to those already mapped: regression analysis of the proportion found to be linked showed this to be approximately 85% for the later loci mapped. Interestingly, nine clones identified segregation patterns identical to those of other clones. In three cases, these may be due to technical errors leading to the duplication of clones, since fragment sizes for the clones are similar. However, in the remaining six cases the observed fragment sizes are quite distinct. It remains possible that analysis of further progeny may distinguish separate loci, although it appears that these loci are surprisingly closely linked. The total number of recombinational events observed within these linkage groups was 293, an incidence of 5.2 crossovers per meiosis. The presence of crossovers for 18 linkage groups and the absence of identical segregations among the loci that do not at present fall within linkage groups suggest that most, if not all, of the microchromosomes can undergo recombination. Variation within Chicken Lines In addition to the RFLVs detected between line 151 and line N, a number of variants were detected within each of the inbred lines. As described above, six clones identified variants present in the line 151 male parent but not present in the F, female parent. Six further clones showed an inverted segregation pattern relative to linked loci, indicating that the variant allele is located on a line 151chromosome in the F, female. As this could only be observed for loci linked to at least one other locus, it represents an incidence of 8% (6/78). Differences were even more evident between the two F, parent birds (Fig. 5), where in 36% of cases (27/76) the variant either was not present or was present in an altered form in the second F, parent. DISCUSSION

The present map of 100 markers covers a minimum of 585 CM of the chicken genome. The markers identify 18 linkage groups together with a further 22 currently unlinked loci, but this close agreement with the expected chromosomal complement is probably to some extent fortuitous, since it is unlikely that this number of markers would identify a locus on every microchromo-

694

BUMSTEAD

1ff3Of

3122

17

f4fI

12

AND PALYGA

If

ff

am

~2/53 Tf2W7

nfl ummumc3mnm~m0 RI5

1822310

10

00

Of

f

2

5 772149

R/82=

FIG. column column. identical

728

CID q IWq Mq lmf2,56 CID 1616

DO 5 7

DO B4

mu 20

3. Summary of results of backcross analysis. Fifty-six progeny were typed for all depicts a particular pattern of recombination among the loci within the linked group. The number of crossovers observed between pairs of loci is shown on the right of segregation patterns are shown as =. Open boxes indicate the line 151 allele; solid

some. Similarly the longest linkage group detected here is about 164 CM in length (including the detectable region flanking the terminal loci); this compares with a linkage group of 220 CM described by Somes (1984) using phenotypic markers. This may partly reflect compression of map differences in the heterogametic map described here, but it is also very possible that the addition of further loci will extend these linkage groups. While the data do not represent a complete map, they cover a substantial part of the genome. This is indicated

the 100 loci shown. For each linkage group, each The incidence of each pattern is shown below the each linkage group. Different probes identifying boxes indicate the line N allele.

by the total map distance, but particularly the increasing incidence of linkage of new loci to existing loci, which suggests that 85% of the genome falls within detectable linkage of the existing markers. Without considerable physical data from in situ hybridization, it is difficult to show conclusively that the marker loci are well distributed over the genome; however, the size of the overall map and the length of individual linkage groups suggest that the loci are widely spread and that clones of known genes are also spread throughout the map.

LINKAGE

MAP

OF

THE

CHICKEN

GENOME

595

18.

2778

0

17

FIG.

3-Continued

The map is derived entirely from recombinations in the female parent, and therefore map distances are those for the heterogametic sex. It will be interesting to seethe corresponding distances in the male homogametic bird, since this situation is reversed relative to that of mammals. In this cross there was no opportunity for recombination between 2 chromosomes. Probes derived from a Z chromosome would not have provided variants detectable between the parent birds and would not have been processed further. A probe specific for the Z chromosome, kindly provided by Dr. E. Smith (Avian Disease and Oncology Laboratory, East Lansing, MI), did detect variation between pure line N birds and line 151 birds,

but as expected showed only the line 151 pattern in the parent F, hen used here, since this bird would carry a Z chromosome derived from line 151. Perhaps the most striking aspect of these results is the high level of variation’ found for simple biallelic marker loci both between and within the two parent lines. This has allowed the mapping process to be very straightforward, since a high proportion of clones yielded variants and restriction fragment patterns were generally simple to interpret. Limited results for other inbred lines and outbred commercial chickens suggest that this level of variability may be general in chickens (unpublished results). A number of probes identifying variable number

696

BUMSTEAD

AND

PALYGA 5

4

6

Tf2lUl

I

T4/12 103 214

7vff

19.3

7x3 4.9

T?W67 73s

4.6 6.6

737

17.9 ?2m cm?= 726 I

I-

3.6

13

15

14

T4 19.6

14.7

19.1

72

-1

6.6

z22

1-6

w

TlW48 7

7wm I

16

pCdWCsT

6 pRpiVb

R..ul

.

.

3.6

s4 .

me

25.0

7wmb

mm.

T4/1#

TTWSJ -I

7wa.

l

TtZLQl 4

FIG. 4. Linkage map of the chicken genome. The estimated distances (in centimorgans) the linkage groups. Estimates of recombination were based on between 56 and 83 progeny, unlinked loci are indicated as 0.

tandem repeat (VNTR) sequences have been described in chickens (Buitkamp et al., 1991), but the use of these more variable probes may not be necessary; however, more data is required on this point. Such a high level of polymorphism may cause problems in analysis of highly outbred commercial chickens, especially in crosses using multiple parents. The reason for this variability may lie in the widespread gene pool used to initiate selection of the lines (Cole, 1968; Stone, 1975) or the large number of recessive lethal and sublethal genes that hinder inbreeding in chickens, although more fundamental properties may be involved. Interestingly, in one instance a mutation was observed among the progeny: a change in size m

1

2

FIG. 5. Restriction fragment length line 151 male parent No. 240; f, F, female 2818.

3

4

6

6

7

6

9 loll

between depending

72/m.

74/B

.

urns

l

riw74

l.wfO

.

.

pairs of loci are indicated to the right of on the pairs of loci involved. Currently

from 17.9 to 5.2 kb in fragment size for a TuqI digest probed with pT3/5. We were able to identify variants in this cross for approximately half of the cloned genes used as probes. To map further cloned genes, it would be possible to use additional enzyme digests or to make use of a different cross; however, Dr. Crittenden (personal communication) is currently carrying out an interspecific mapping cross between Jungle Fowl and chicken, and this should provide a more powerful means of mapping cloned genes in the future. We have not attempted to allocate linkage groups to chromosomes, since our principal objective is genetic

1213141516171819

2021

222324252627

variation between F, parent birds. DNAs were digested parent No. 2826; f, F, female parent No. 2818; l-12,12

f f’

using TaqI and were hybridized progeny of No. 2826; 13-27,15

to pT2/38: m, progeny of No.

LINKAGE

MAP

OF

THE

analysis of observed traits; however, an increasing number of loci have been identified to chromosomes (Somes, 1984; Dominguez-Steglich et al., 1990), and the progressive addition of these loci to the map will provide information at least for the larger chromosomes. Chicken microchromosomes are in most cases indistinguishable cytologically, and here perhaps only a genetic designation of chromosomes is practicable. We are also attempting to construct a set of somatic hybrid cell lines for the chicken chromosomes. This is intended to provide a means of generating chromosome-specific clones; however, it will also allow the rapid allocation of existing clones to chromosomes. In chickens it is difficult to observe chiasmata cytologically in the smaller chromosomes. It is therefore not possible to compare the numbers of visible crossovers with those detected genetically. Recombination is known to occur within at least some of the microchromosomes. Bloom et al. (1987) have shown that the chicken major histocompatibility complex (B) is located on a microchromosome, and recombinants among loci in this complex have been described (Pink et al., 1977). Our results indicate that other microchromosomes also regularly recombine, but we are some way from identifying the minimum of two loci on each chromosome required to demonstrate that this is the case for all the microchromosomes. The map described is intended primarily as a resource map for locating the genes responsible for resistance to Salmonella infection. We intendto determine the susceptibility of the genotyped birds described above by progeny testing; this should provide an initial location of the genes responsible, which will be refined in further backcrosses of selected lineages. The gradual development of a more complete linkage map of the chicken genome should also allow remarkable progress in the analysis of other more complex traits. ACKNOWLEDGMENTS We thank other investigators who kindly this work and S. Sanderson, M. F. Quinn, Manning for excellent technical assistance.

provided DNA probes for N. Salmon, and A. C. C.

REFERENCES Benoist, C., O’Hare, K., Breathnach, R., and Chambon, P. (1980). The ovalbumin gene-sequence of putative control regions. Nucleic Acids Res. 8: W-142. Bloom, S. E., Briles, W. E., Briles, R. W., Delany, M. E., and Dietert, R. R. (1987). Chromosomal localisation of the major histocompatibility (B) complex (MHC) and its expression in chickens aneuploid for the major histocompatibility complex/ribosomal deoxyribonucleic acid microchromosome. PO& Sci. 66: 782-789. Buitkamp, printing

J., Ammer, in domestic

H., and Geldermann, animals. Ekctrophoresis

Bumstead, N., and Barrow, Salmonella typhimurium 29: 521-530. Bumstead, N., Greenwood,

H. (1991). DNA 12: 169-174.

finger-

P. A. (1987). Genetics of resistance in newly hatched chicks. Br. Poult. N. M.,

and Messer,

L. I. (1987).

to Sci.

Use of eu

CHICKEN

697

GENOME

loci as a measure 717-726.

of inbreeding

in domestic

fowls. Br. Pot&.

Sci. 28:

Bumstead, N., Millard. B. J., Barrow, P., and Cook, J. K. A. (1991). The genetic basis of disease resistance in chickens. In “Breeding for Disease Resistance in Farm Animals” (J. B. Owen and R. F. E. Axford, Eds.), pp. 10-23, C.A.B. International, Wallingford. Calnek, B. W. (1985). Genetic resistance. In “Marek’s Disease” (L. N. Payne, Ed.), pp. 293-328, Nijhoff, Boston. Chan, L. N. L., Grammatikakis, N., Banks, J. M., and Gerhardt, E. M. (1989). Chicken transferrin receptor gene: Conservation of 3’ noncoding sequences and expression in erythroid cells. Nucleic Acids Res. 17: 3763-3771. Cole, R. K. (1968). Studies on genetic resistance to Marek’s disease. Avian Dis. 12: 9-28. Dalton, S., Robins, A. ,J., Harvey, R. P., and Wells, J. R. E. (1989). Transcription from the intron-containing chicken histone H2A.f gene is not S-phase regulated. Nucleic Acids Res. 17: 1745-1756. Dominguez-Steglich, Schmid, M. (1990). microchromosome

M., Meng, G., Bettecken, T., Muller, C. R., and The dystrophin gene is autosomally located on a in the chicken. Genomics 8: 536-540.

Ferrari, S., Drusiani, E., Battini, R., andFregni, M. (1988). Nucleotide sequence of the promoter region of the gene encoding chicken calbindin D28K. Nucleic Acids Res. 16: 353. Maniatis, T.. Fritsch, E. F., and Sambrook, J. (1982). In “Molecular Cloning: A Laboratory Manual,” pp. 382-389, Cold Spring Harbor Laboratory, Cold Spring Harbour, NY. Olofsson, B., and Bernardi, G. (1983). Organisation quences in the chicken genome. Eur. J. Biochen.

of nucleotide 130: 241-245.

se-

Payne, L. N. (1985). Genetics of cell receptors for avian retroviruses. In “Poultry Genetics and Breeding” (W. G. Hill, J. M. Manson, and D. Hewitt, Eds.), pp. I-16, British Poultry Science Ltd., Longman Group, Harlow, Essex. Pink, J. R. L., Droege, W., Hala, K., Miggiano, V. C., and Ziegler, A. (1977). A three locus model for the chicken major histocompatibility complex. Immunogenetics 5: 203-216. Reynaud, C. A., Dahan, A., and Weill, J. C. (1983). Complete sequence of a chicken X light chain immunoglobulin derived from the nucleotide sequence of its mRNA. Proc. Natl. Acad. Sci. USA 80: 40994103. Shoffner, R. N., and Krishan, A. (1965). The karyotype of Gallus domesticus with evidence for a W chromosome. Genetics 52: 474. Silver, J. (1985). Confidence limits for estimates of gene linkage based on analysis of recombinant inbred strains. J. Hered. 76: 436-440. Soller, M., and Beckmann, J. S. (1986). Restriction fragment length polymorphisms in poultry breeding. PO&. Sci. 65: 1474-1488. Somes, R. G. (1984). “International Registry of Poultry Genetic Stocks,” Bulletin 469, Storrs Agricultural College, Storrs, CT. Steiner, C., Muller, M., Baniahmad, A., and Renkawitz, R. (1987). Lyzozyme activity in chicken macrophages is controlled by positive and negative regulatory elements. Nucleic Acids Res. 15: 41634178. Stone, H. A. (1975). Use of highly Dept. of Agriculture, Agricultural letin No. 1514, Washington, NY.

inbred chickens in research. Research Service Technical

US Bul-

Sudol, M., Kieswetter, C., Zhoa, Y. H., Dorai, T., Wang, L. H., and Hanafusa, H. (1988). Nucleotide sequence of a cDNA for the chick yes proto-oncogene: Comparison with the viral yes gene. Nucleic Acids Res. 16: 9876. Thomas, B. J., and Rothstein, R. (1991). Sex, maps and imprinting. Cell 64: l-3. Wakelin, D., and Rose, M. E. (1990). Immunity to coccidiosis. In “Coccidiosis in Man and Animals” (P. L. Long, Ed.), pp 281-306, CRC Press, Boca Raton, FL. Yamashina, M. Y. (1944). Karyotype studies in birds. I. Comparative morphology of chromosomes in seventeen races of domestic fowl. Cytologia 13: 270-296.