found to be linked to the rose comb locus. (R) by 16 crossover units. Linkage test matings between AMET*A and (R*R) showed independent segregation, strong-.
Brief Communications
A New Recessive Ametapodia Mutation in the Chicken (Gallus domesticus) J. R. Smyth Jr., G. P. Sreekumar, C. A. Coyle, and J. J. Bitgood An apparently new mutation that is associated with abnormal limb development appeared in a strain of Light Brown Leghorn chickens. Mutants are characterized by the complete absence of the tarsometatarsals, while severely hypoplastic development of the metacarpals is also present. The phenotype of the new mutant (ametapodia-2) closely resembles ametapodia-1, described in 1967, but ametapodia-2 is inherited as an autosomal recessive (AMET*A), while ametapodia-1 was associated with an incompletely dominant gene (MP*A). Only heterozygous ametapodia-1 (MP*N/MP*A) were viable and able to reproduce, while homozygous ametapodia-2 mutants do not normally survive beyond 2–4 days of age. The shankless mutation (SHL*S) also reduces development of the metatarsal and metacarpal bones and has been shown to be associated with a pericentric inversion of chromosome 2. No obvious cytologic abnormality was apparent in ametapodia-2 birds, and offspring of a cross between AMET*A carriers and shankless birds were normal, indicating that the two mutations are not alleles. Ametapodia-1 (MP*A) was found to be linked to the rose comb locus (R) by 16 crossover units. Linkage test matings between AMET*A and (R*R) showed independent segregation, strongly suggesting that the mutation occurred at a relatively distant locus and therefore is probably not allelic to MP*A. A new mutation characterized by the apparent absence of metatarsal and metacarpal bones was first observed in the early spring of 1992 among offspring of a
340
nonpedigreed hatch of Light Brown Leghorns ( LBLs). The LBL line had been a closed population at the University of Massachusetts for 15 years and the skeletal defect had never been observed in this stock previously. The mutant phenotype closely resembled an earlier ametapodia mutation (MP*A) described by Cole (1967), and to a lesser extent the shankless mutation (SHL*S) ( Langhorst and Fechheimer 1985). The major objectives of the present study were to describe the new ametapodia, determine its inheritance, and investigate its possible genetic relationships with the two earlier mutations that also had major effects on the metapodia. Unfortunately sources of MP*A appear to no longer exist; however, shankless is being maintained at the University of Wisconsin poultry unit. Once it was determined that the new Massachusetts mutant was inherited as a monogenic trait, it was assigned the gene symbol AMET*A [following the genetic nomenclature standardization efforts of Crittenden et al. (1996)]. To separate it from the ametapodia (MP) reported by Cole (1967) the AMET*A phenotype will be referred to herein as ametapodia-2 and the MP*A phenotype of Cole as ametapodia-1. Some of the information in this report appeared previously in brief abstracts ( Bitgood et al. 1996; Smyth et al. 1995).
Materials and Methods The first ametapodia-2 mutant chicks appeared among the progeny of a nonpedigreed mating of Light Brown Leghorns ( LBLs) in 1992. The LBL had been maintained as a nonpedigreed, closed population for 15 years and the parents of the above mating were all phenotypically normal. In order to identify carriers, all LBL in the parent population were caged and pedigree matings were made using artificial insemination. Of the 19 LBL females tested, 5 were identified as carriers of the
AMET*A allele, while 1 carrier male was found among 5 that were tested. These six birds were then used in pedigree matings to study the ametapodia-2 phenotype and its mode of inheritance. All chicks of normal phenotype were reared in wire-floored battery brooder pens until 20 weeks of age. At that time breeding populations were selected and housed in conventional single-bird wire cages. Subsequent pedigree matings were made by artificial insemination. Water and commercial poultry rations were available ad libitum. Ametapodia-2 chicks were found to die by 4 days posthatch, even when raised on shredded wood flooring or using chick starter as litter. Since the mutants did not appear to eat or drink, an effort was made to force-feed them a slurry of chick starter administered three times a day using an eyedropper. Possible linkage between the ametapodia2 (AMET) and rose comb (R) loci was also investigated, since ametapodia-1 had been reported previously to be linked by 16 crossover units to the R locus (Cole 1967). Since the MP*A mutation (ametapodia-1) was no longer available, a similar linkage estimate between the AMET and R loci should provide evidence for a close genetic relationship between ametapodias 1 and 2. To conduct this test a heterozygous (R*R/R*S) male from a random breeding gene pool line was crossed with known AMET*A carrier females (R*R/R*S AMET*N/AMET*N male ⫻ R*S/R*S AMET*A/AMET*N females). Since AMET*A homozygotes are not viable, typical backcross linkage tests were not possible. Therefore linkage estimates were made using reciprocal matings of the following types: R*S/R*R AMET*A/AMET*N ⫻ R*S/R*S AMET*A/AMET*N. A 1 rose:1 single comb ratio at the rose comb locus and a 3 normal: 1 ametapodia ratio at the AMET locus would be expected. Expected ratios in the absence of linkage would be 3 rose comb, normal legs:1 rose comb, ametapodia:3 single comb, normal legs:1 single comb, ametapodia. Sta-
Table 1. Results of reciprocal linkage test crosses involving the rose comb (R) and ametapodia (AMET) loci: R*R/R*S AMET*N/AMET*A ⴛ R*S/R*S AMET*N/AMET*A Single comb
Rose comb
a b
Figure 1. Mutant and normal leg bones from newly hatched chicks: S (shankless), N (normal), and A (ametapodia-2). The limb bones from top to bottom are the femur, tibia, tarsometatarsus, and phalanges. The mutant effects on limb development are associated with the tarsometatarsus and phalanges which are reduced and modified in the shankless (S). The tarsometatarsus is absent in the ametapodia-2 (A) phenotype and only digit 1 (rear toe) is present and attached to the distal end of the tibia (A).
tistically significant deviations from this (determined by chi-square test) would suggest genetic linkage. The original heterozygous rose comb male was also heterozygous for polydactyly (PO*N/PO*P), one of the mutations segregating in the gene pool from which the rose comb male was obtained. Polydactyly reappeared among the offspring of the rose comb ⫻ ametapodia-2 linkage testcross.
Results and Discussion Description of Ametapodia-2 The most obvious phenotypic modification of ametapodia-2 chicks is the complete absence of the tarsometatarsus ( Figure 1). The corresponding carpometacarpals are also severely modified, resulting in a much shortened wing. The detailed skeletal modification of the mutant wing is still under study. A single digit at
Male parent
Normal
Ametapodia
Normal
Ametapodia
Pa
R*S/R*R AMET*A/AMET*N R*S/R*S AMET*A/AMET*N Combined
12(12.0)b 31(39.4) 43(51.4)
3(4.0) 21(13.1) 24(17.1)
13(12.0) 37(39.4) 50(51.4)
4(4.0) 16(13.1) 20(17.1)
⬎.95 ⬎.05 ⬎.20
Determined by chi-square analysis based on expected 3:1:3:1 ratio for independent assortment. Observed (expected).
the rear of the foot with two phalanges and a claw was determined by position to be digit 1. This was also verified by a fortuitous observation involving the presence and segregation of the polydactyly (PO*P) mutation in the linkage testcross mating. The single digit on an ametapodia2 chick was observed to be polydactylous. Since only digit 1 expresses the polydactyly phenotype (Somes 1990), this provided additional evidence that the single toe is digit 1. Although ametapodia-2 (AMET*A) homozygosity does not appear to affect embryonic survival, it does act as a delayed lethal gene. Posthatch death of mutants occurred within the first 4 days posthatch, apparently due to inanition. No affected chick survived, even when brooded under heat lamps using chick starter mash as litter and a surplus of chick waterers. Crops were empty at the time of death. In an experiment designed to see if survival could be accomplished by hand-feeding, 11 ametapodia-2 chicks were force-fed three times a day with a slurry of starter mash for the first week posthatch. Five mutant chicks survived for 7 days and these were observed to have commenced to eat and drink on their own. However, four of these died at the ages of 10, 20, 22, and 28 days, while the fifth survived to an age of 5 weeks 3 days. Necropsies of dead chicks failed to reveal any gross anatomic abnormalities; however, some severe metabolic abnormality must be associated with the related lethality in the ametapodia-2 chicks. Inheritance The original appearance of mutant chicks from phenotypically normal parents suggested that the mutation was recessive. When pedigree test matings were made between the five identified carrier females and a single carrier male, 17 of 60 chicks expressed the mutant phenotype. These results fit an expected 3:1 ratio (P ⬎ .30) for a recessive mutation. That the locus is autosomal was based on an approximate 1:1 sex ratio among progeny from the orig-
inal mating and the fact that both males and females were identified as carriers. The results of the linkage analysis between the AMET and the R loci are shown in Table 1. The combined data from the two reciprocal crosses did not deviate significantly from a 3 rose comb, normal legs: 1 rose comb, ametapodia:3 single comb, normal legs:1 single comb, ametapodia ratio (P ⬎ .20). Results from one of the reciprocal crosses did differ in that when the sires were homozygous for single comb and heterozygous for ametapodia, the progeny showed an excess of ametapodia phenotypes. Chi-square analysis of the results from this reciprocal mating was tested for a hypothesized 3:1 ratio resulting in a P value slightly greater than .05. The deviations from expected were due to an excess of the crossover gamete types, assuming linkage, between the R and AMET loci. In contrast, the mating where the males were heterozygous for both R*R and AMET*A closely approximated the expected 3:1:3:1 ratio ( Table 1). The data suggest that MP*A and AMET*A are not allelic, although they could still reside on the same chromosome. In addition, chi-square estimates based on 3:1:3: 1 ratios are not ideally suited for the detection of linkage, but were necessitated by the unavailability of homozygous ametapodia-2 adults. The difference in results between the two reciprocal linkage crosses is not explainable at this time, and warrants a further examination in subsequent studies. The deviations from expected are greatest in the expected crossover class between rose comb and ametapodia-2 classes, which suggests that something other than linkage between the R and AMET loci may be involved. The answer may be as simple as a deviation due to chance. Comparisons of Ametapodia-2 (AMET*A) and Ametapodia-1 (MP*A) with Shankless (SHL*S) Since AMET*A, MP*A, and SHL*S all have a major effect on limb development, particularly the metapodia, it is of interest to
Brief Communications 341
compare the three mutations. Ametapodia-1 and 2 are similar in that they have no tarsometatarsi, but differ in the number of digits present. Ametapodia-1 had phalanges for digits 2, 3, and 4, while ametapodia-2 expresses digit 1 only. Such differences, in themselves, could be attributed to allelic interactions with residual genotypes in the stocks of origin. The shankless phenotype reduces the length of the tarsometatarsus, but does not eliminate it as reported by Langhorst and Fechheimer (1985), according to Bitgood et al. (1996) ( Figure 1). All four digits are present in shankless birds, but these vary from those of normal birds in phalange number and fusions. All three mutations show severely reduced carpometacarpal bones in shortened wings. Another line of evidence that ametapodia-2 is the result of a previously unreported mutation is the difference in modes of inheritance. Ametapodia-1 was found to be due to an incompletely dominant lethal gene (MP*A), the mutant phenotype being due to heterozygosity. The shankless phenotype also segregates like an autosomal recessive mutation. That SH*S and AMET*A are not allelic was shown by a cross between shankless and ametapodia2 heterozygotes that produced 135 normal progeny ( Bitgood et al. 1996). Shankless has been shown to be associated with a relatively large, pericentric inversion of chromosome-2 ( Bitgood et al. 1996; Langhorst and Fechheimer 1985). That it is not allelic to AMET*A is further indicated by the absence in heterozygotes of an observable chromosome rearrangement by cytologic examination at the light microscopic level. There are also survival differences between the two ametapodias and shankless. MP*A was lethal when homozygous, so the mutant chicks were all heterozygous (MP*N/MP*A). These could be reared with special care and soft litter such as straw that helped to cushion their feet (Cole 1967). They matured sexually and could be reproduced by artificial insemination. Shankless individuals, homokaryotypic for a pericentric inversion of chromosome 2, usually die late in incubation or soon after hatching, but a few survive to sexual maturity ( Langhorst and Fechheimer 1985). Those that survive can also be reproduced by artificial insemination. In contrast, ametapodia-2 embryos appear to hatch normally, but most die during the first few days posthatch as discussed pre-
342 The Journal of Heredity 2000:91(4)
viously and none have been raised to sexual maturity. The genetic relationship between the ametapodia-1 and 2 phenotypes may never be determined, since the former is no longer in existence. The present study indicates that the two ametapodias are not due to the same mutation and are probably not alleles. However, the possibility of a translocation of MP*A to another chromosome or to a site further away from the R locus on the same chromosome cannot be eliminated as the source for AMET*A. The final location of AMET*A in the chicken karyotype will probably be determined by molecular marker techniques, which at present have not been as well used for chickens as for mice and humans. It will also be interesting to identify homologous mutations in other species. A good candidate in the mouse is the phenotypically similar hypodactyly mutation (Hummel 1970; Robertson et al. 1996) which is located on mouse chromosome 6. Of interest, it maps very closely to the homeobox cluster, HOXA, which has been identified to be associated with pattern formation in limbs (Innis et al. 1996; Mock et al. 1987). Mortlock et al. (1996) further reported a 50 bp deletion in the first exon of the HOXA-13 gene in mice with the hypodactyly phenotype. Another similar mutation was reported for a human family by Stern et al. (1970), characterized by hand-foot-uterus abnormalities. This has recently been reported to be due to an A-to-G transitional mutation in a highly conserved tryptophan codon in the HOXA13 homeodomain, resulting in an abnormal TGA stop codon (Mortlock and Innis 1997). Further gene characterization and mapping efforts for the AMET locus are under way at present at the University of Wisconsin. Databases will be searched for potential homologous genes such as hypodactyly in the mouse and the HFG syndrome in humans for further mapping purposes. There is considerable interest in the genetics of development, with a particular focus on the genetic control of limb development [for reviews, see Cohn and Tickle (1996), Morgan (1997), and Tickle (1996)]. The ametapodia-2 mutant should be a valuable model for such studies because when homozygous (AMET*A/ AMET*A), the tarsometatarsals do not develop, while the metacarpals show greatly reduced development. The ametapodia-2 phenotype could be a useful addition to such chick limb mutation models as the polydactylous talpid 2, diplopodia, and diplopodia 4 (Rodriguez et al. 1996).
From the Department of Veterinary and Animal Sciences, 260 Animal Sciences Bldg., University of Massachusetts, Amherst, MA 01003 (Smyth and Coyle), Skin Biology Division, Johnson & Johnson, Skillman, New Jersey (Sreekumar), and Dept. 260 Animal Sciences Bldg., University of Wisconsin, Madison, Wisconsin ( Bitgood). Address correspondence to J. Robert Smyth Jr. at the address above. We would like to acknowledge the valuable technical assistance of F. H. Thornton of the University of Massachusetts, Amherst. 䉷 2000 The American Genetic Association
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