SEGREGATION OF TWO ALLELES FOR COLOR OF DOWN IN PARTHENOGENETIC AND NORMAL TURKEY EMBRYOS AND POULTS M. W. OLSENl AND E. G. BUSS2 United States Department of Agriculture and The Pennsylvania State University Manuscript received March 20, 1972 ABSTRACT Data were collected on the down color of 1,386 parthenogenetic embryos and poults from eggs of 306 young virgin and older nonmated bronze turkey hens. Each hen involved was heterozygous (Cc) for alleles which affect feather pigmentation. Six hundred and sixty nine white and 717 colored parthenogenetic embryos and poults were observed, a 1: 1 ratio. Subsequently, 89 of these 306 heterozygous bronze hens were inseminated with semen from BSW (cc) males and down color of embryos and poults from fertilized eggs recorded. the 89 females produced a total of 233 white and 239 colored normal embryos and poults. The 1:l ratio for down color among both parthenogenetic and normal embryos and poults shows that, in both instances, the two alleles at a locus on an autosome segregated equally. Four possible cytological routes which would lead to diploidy were discussed. HE vast majority of cells encountered in incubated, unfertilized turkey eggs Thave been found to be diploid, 2A-ZZ (YAO and OLSEN 1955; SATO and KOSIN 1960). This was likewise true of somatic cells of hatched parthenogens (POOLE 1959). The means by which diploidy occurs has been a matter of much interest. The findings of POOLE et al. (1963) and POOLE (1965) that some parthenogens can be heterozygous at one or more histocompatibility loci led these authors to conclude that restoration of diploidy occurs after meiosis 11, due to the reuniting of the second polar body and the haploid egg nucleus. In 1966, OLSEN presented limited data on the segregation of alleles for down color in parthenogenetic embryos from virgin, heterozygous bronze (Cc) turkey hens. A 1 :1 ratio of white to colored embryos was observed. The present study was conducted to extend the study of segregation for down color of embryos and poults from eggs laid by the heterozygous, Cc, bronze hens. Comparisons were made on embryos produced before and after the hens were mated to Beltsville Small White (BSW) (cc) males. MATERIALS AND METHODS The total experimental population (over a 3-year period) consisted of 461 hens. Two hundred eighty one of these were young virgins and 180 older nonmated females; all hens were hetero- Animal Science Research Division AFS Beltsville Md. 20705. Department of Poultry Science, The Pednsylvania )State University, University Park, Pa. 16802. Genetics 72: 69-75 September, 1972.
~ 70 M. W. OLSEN AND E. G. BUSS zygous for the Cc feather-color alleles. Eggs yielding one or more parthenogenetic embryos in which down color could be established were produced by 306 (210 young and 96 old) of 461 hens. The hens were maintained in individual cages. Daily collections of unfertilized, pedigreed eggs were placed each evening in a forced-draft commercial type incubator operating at 99X"F. Eggs were candled on the 10th day of incubation, at which time all clear eggs and those showing only unorganized growth were removed. Eggs containing live parthenogenetic embryos were returned to the incubator and candled at frequent intervals thereafter. Dead and abnormally developed embryos detected at each candling were removed and examined. Down color was recorded for all dead embryos in which it could be determined and for all parthenogenetic poults hatched. Eggs from mated birds were collected each evening and stored at 55 F. for 1-14 days prior to incubation. Down color of advanced embryos and poults was recorded for each biweekly setting of fertilized eggs. RESULTS Of the 105 young hens involved in tests in 1969, the 72 hens used in 1970, and the 104 in 1971, 55.2%, 73.6% and 95.2%, respectively, produced one or more embryos which survived to an age at which down color could be established. Corresponding numbers and percentages for older bronze hens follow: Fortyseven, 46.8%; 62,58.1%; and 71,53.5% respectively, for years 1969, 1970, and 1971. Numbers and percentages of white and colored parthenogenetic embryos and poults encountered during each of the 3 years are shown in Table 1. Data collected for segregation of down color on parthenogenetic embryos and poults in eggs before and after the hens were mated are presented in Table 2. To determine if the C and c alleles segregated in a ratio 9f 1 :1 during meiosis, the chi-square test was used. These results indicate there was no significant deviation from the expected 1:l ratio of white to colored embryos when all data TABLE 1 Ratio of while io colored parthenogenetic embryos and poulis encouniered in the period 1969-1971 in unfertilized eggs of heierozygous bronze turkey females Parthenogenetic embryos and poults Heterozygous bronze dams White (cc) Colored (CC+Cc) 2 Year Age No. No. Percent No. Percent a b C 1969 Young1 58 91. 44.3 118 55.7 59.03 2.72 56.31 Old2 22 38 55.9 30 44.1 19.29.94 18.35 - -. 80 13247.1 148 52.9.914 1970 Young 53 115 47.3 128 52.7 63.74,695 63.05 Old 36 41 38.7 65 61.3 29.75 5.434* 24.34 - -~ ~ 89 156 193 55.3 3.923* 1971 Young 99 328 50.5 321 49.5 118.40.0755 118.33 Old 38 53 49.1 55 50.9 32.71,037 32.67 - ~ - 137 381 50.2 376 49.7.033 1 yr old. 2 2 yrs and older. * Significant chi square (P =.%). a. Total chi square; b. Pooled chi square; c. Heterogeneity chi square.
TABLE 2 Numbers of white and colored parthenogenetic embryos and poults encountered among fertilized eggs of heterozygous bronze turkey females prior to mating and from fertilized eggs of the same bronze females after they were mated to Beltsville Small White (cc) males Prior to mating After mating to BSW males ti Embryos and poults Normal embryos and poults +P White Colored White Colored z Age of No. of (cc) (CCfCc) 2 (cc) (CC) Year dams dams No. Percent No. Percent a b C No. Percent No. Percent a C.- b B 1969 Young' 21 Z 31 46.3 36 53.7 141 52.6 127 47.4 Old2 2 6 66.6 3 33.3 0 - - -~ 15 4.6.9 17 53.1 B 2.3 37 48.7 39 51.3 29.182.053 29.130 156 52.0 I44 48.0 26.887.48 26.407 3 1970 Young 25 81 50.6 79 49.4 E 286 55.2 232 44.8 cn Old IO 15 44.1 19 55.9 47 46.5 54 53.5 - -_ -~ 35 96 49.5 98 50.5 39.64.02 39.62 33353.6 2864.6.2 45.04.9 3.568 41.481 c3 1971 Young 21 84 51.9 78 48.1 126 52.1 116 47.9 Old 10 16 40.0 24 60.0 43 46.2 50 53.8 - -~ - -- 31 100 49.5 102 50.5 23.103.02 23.083 169 50.4 166 49.6 18.966.0268 18.939 4 All birds 89 233 49.4 239 50.6 658 52.9 586 47.1 2 s 1 1 yr. 2 2 yrs and older. a. Total chi square; b. Pooled chi square; c. Heterogeneity chi quare,
72 M. W. OLSEN AND E. G. BUSS were pooled for the 3 years. Within years, there was a significant deviation for the old hens used in 1970, but there was no significant difference when the data from the old hens were pooled for the 3 years. Although a few individual hens contributed heavily to the total chi-square values (their progeny did not fit the 1:l ratio of white to colored) for each group, the heterogeneity chi-square test result indicates that the groups were reasonably homogeneous. Data presented in Table 1 are therefore interpreted to confirm the finding of a 1:1 ratio of white to colored parthenogenetic embryos and poults reported previously ( OLSEN 1966). The frequency of white to colored parthenogenetic embryos and poults in unfertilized eggs of individual hens may be of some interest. Records of hens shown in Table 1 were analyzed on an individual basis and the percentage of their embryos and poults having white down calculated. A total of 306 virgin and non-mated, heterozygous (Cc) bronze female were involved. These data revealed that 51 of the 306 hens (16.7%) gave rise to parthenogenetic embryos and poults all of which were colored. Forty-six of the 306 hens (15.0%) gave rise to embryos and poults, all of which had white down. Each of the remaining 209 hens (68.3%) gave rise to both white and colored embryos and poults. As expected, the proportions of white to colored embryos and poults varied with hens. Numbers of hens and percentages of their parthenogenetic embryos and poults with white down follow: Heterozygous (Cc) Bronze Hens (Number) 8 23 35 19 54 41 17 12 Embryos and Poults with white down (Percent) 10.0-19.9 20.0-29.9 30.0-39.9 40.0-49.9 50.0-59.9 60.0-69.9 70.0-79.9 80.0-89.9 Data on down color of embryos and poults from eggs of the 23 hens inseminated in 1969, the 35 females inseminated in 1970, and the 31 hens inseminated in 1971 are shown in Table 2. Data presented show that segregations of white and colored embryos in eggs obtained before and after insemination of the same heterozygous bronze hens fit a 1:1 ratio. Pooled chi-square values shown in Table 2 indicate that alleles determining white or bronze down color segregated independently and randomly during meiosis, both in unfertilized and fertilized eggs. DISCUSSION The data presented appear to indicate that the C and c alleles occurred equally. These data lend support to the suggestion of DARCEY et al. (1971) that the origin of parthenogenesis was likely to be from haploid eggs. However, there are a
PARTHENOGENESIS IN TURKEY 73 number of cytological routes which turkey germ cells might follow in the establishment of diploidy and still give an apparent 1 :1 segregation. BEATTY (1957) lists several such routes as follows: (1) Suppression or re-entry of the first polar body followed by meiosis 11. (2) Suppression or re-entry of the second polar body. (3) A nuclear division at mitosis I in the absence of a corresponding cytoplasmic division. (4) Fusion of two haploid mitotic products. Suppression or re-entry of the first polar body in the case of birds should, theoretically, give rise to some female parthenogens. Data now available make it possible to dismiss route 1 on the basis of sex of turkey parthenogens. All fully developed parthenogenetic embryos examined to date have been males (POOLE and OLSEN 1957). This has likewise been found true of those parthenogens that have survived to maturity (OLSEN and MARSDEN 1954a; OLSEN 1965a). Suppression or re-entry of the second polar body (route 2) is a logical route by which diploidy could be restored. Polar bodies in eggs produced by chickens and turkeys come to lie just beneath the vitelline membrane along the upper surface of the germinal disc and in close proximity to the egg nucleus ( OLSEN 1942), and they are never completely extruded from the ovum. A parthenogen could be heterozygous at a given locus if crossing over occurred between the centromere and that locus, and if the 2nd polar body was not extruded (route 2). But this does not exclude an apparent 1:l ratio because this crossing over would be hard to detect if the locus were close to its centromere. Thus when data for all birds shown in Table 1 are considered, it can be calculated that there was an excess of 3.4% colored parthenogenetic embryos. Making the assumption that these should be assigned to a heterozygous category resulting from crossing over, examination of Figure 2 in the paper by NACE et al. (1970) reveals that the gene for bronze plumage color in turkeys is located approximately 2 map units from the centromere. If the locus is, indeed, that close to the centromere, the apparent 1:l ratio observed here would not be incompatible with an explanation involving route 2. By this mechanism, an autosomal diploid equivalent to a fertilized egg would be produced. The unfertilized turkey ovum, therefore, would start mitotic division as a diploid cell. Evidence that cell divisions are not normal and result in the production of some haploid, tetraploid and heteroploid cells has been shown by SATO and KOSIN (1960), DARCEY and Buss (1968) and SARVELLA (1971). This is not surprising, however, since the type of parthenogenetic development encountered in eggs of both chickens and turkeys from its onset, shows every indication of being abnormal. Large yolk-ladened blastomeres become arranged in multiple layers and form compact masses. Necrotic areas and vacuoles are encountered frequently throughout the protoplasmic disc. Many of the cells encountered in the blastoderm of newly laid unfertilized eggs show little affinity for iron hematoxylin stain, indicating that they are either moribund or dead (OLSEN and MARSDEN 195413; HANEY and OLSEN 1958). Cell division occurring in such an unfavorable environment could easily deviate from the normal pattern. The surprising thing is that cells found in such a state of disarray will resume development; and; in rare instances give rise to normal parthenogenetic embryos. Cytological events which make this possible take place within
74 M. W. OLSEN AND E. G. BUSS the incubator, at which time, some of the more viable blastomeres composing these cell masses resume development and eventually form an entirely new, single cell-layered blastoderm from whence develops the future embryo. Construction of a new blastoderm, however, requires time, thus the underlying reason for the characteristic 2-3-day delay regularly observed in the onset of parthenogenetic development (OLSEN 1965b). If nondisjunction occurred at meiosis 11, the sex-chromosome condition of the resulting diploid cell would be either ZZ (male) or YY (female?). In Drosophila, the YY constitution has been found to be lethal (STALKER 1954). The absence of female parthenogens indicates that the YY constitution is likewise lethal in the case of turkeys. Restoration of diploidy at or following mitosis I (routes 3 and 4) should give rise to individuals homozygous for all loci. However, some parthenogens, as already noted, have been shown to be heterozygous at one or more loci controlling histocompatibility (POOLE et al. 1963; POOLE 1965), and at the locus controlling bronze plumage color (OLSEN 1966). These observations were major considerations in leading the aforementioned authors to conclude that cytological route 2 is the one being followed by unfertilized turkey ova in restoration of diploidy. Strong cytological evidence may be cited in support of the contention of POOLE et al. 1963; POOLE 1965; and OLSEN 1966 that diploidy in unfertilized turkey eggs occurs due to suppression of the second polar body. Studies conducted by OLSEN (1942) and OLSEN and FRAPS (1950) with ova of the domestic fowl and by OLSEN and FRAPS (1944) with eggs of domestic turkeys revealed that the initial stages in the formation of the second polar body take place in the ovary just prior to ovulation. In mated birds the ovum, upon entering the infundibulum, is fertilized, usually within 15 minutes. Penetration of the ovum by the sperm provides the necessary stimulus for completion of meiosis I1 (OLSEN 1942; OLSEN and FRAPS 1944). The important question is: What happens when no sperm are present to provide the stimulus required for completion of meiosis II? Available evidence indicates that, in the absence of sperm, chromosomes of the second polar body and those of the egg nucleus probably would not separate. This view is also shared by WATERMAN (1948); RUGH (1964) and BEATTY (1967) among others. Cytological route 2 to diploidy provides an explanation as to why some parthenogens can be heterozygous at certain loci. The data presented in this paper show that alleles at one locus segregate in a 1:l manner. However, it will not be known if all parthenogens arise from eggs that have not extruded the second polar body and are, therefore, 2n, until other studies using other loci more distant from centromeres on autosomes are reported. The author wishes to thank Dr. BERNARD WEINLAND, Biometrical Services, ARS, Beltsville, Maryland, for the statistical analyses. LITERATURE CITED BEATTY, R. A., 1957 Parthenogenesis of polyploidy in mammalian development. Cambridge Univ. Press. -, 1967 Parthenogenesis in vertebrates. Chapter 9. In: Fertilization, Vol. I. Edited by C. B. METZ and A. MONROY. Academic Press, N. Y.
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