Friday, September 6, 2013



Genotypic and Phenotypic Aspects of the Sex-Linked clearbody
By: Inte Onsman, Research coordinator

Research & Advice Group, The Netherlands

The number of cases of albinism in birds that have been studied genetically is rather small. One of the first sex-linked genes known in birds was studied in cinnamon canaries by Durham and Marriyat [5]. They found that affected birds had pink eyes at hatching but these darkened with age. It was presumed that the cinnamon plumage and pink eyes might have been caused by a gene for imperfect albinism.
Sex-linked albinism was first studied in the Budgerigar by Kokemüller in 1935 [8]. The effects of the sex-linked ino allele in the Budgerigar are similar to those seen in birds affected by the non sex-linked a-locus, (the reappearance was reported by Ken Moore in 1990 [11]), causing a "ghost pattern" to show on the wingcover feathers and the back of the head. Microscopical examination of feathers from N.S.L. inos showed elimination of pigment also to be incomplete [13], and therefore this type of albinism is called incomplete albinism.

Imperfect albinism occurring in the Fowl was also found to be caused by a sex-linked recessive gene. Inhibition of the formation of melanin in these birds is also not total, since affected birds also show "ghost barring" in their plumage [20]. The sex-linked gene for imperfect albinism in the Fowl has been found to be multiple allelic, as already was reported by Werret in 1959 [25]. He described a new sex-linked allele of two already described namely silver and gold, they formed a series of three alleles at one locus on the sex-chromosome.

Since the sex-linked clearbody was recognized to be an allele of the sex-linked ino locus [15], we now know that this locus is also multiple allelic in the Budgerigar. The articles published by Ferdinant Wagner were very helpful in this respect. The results of subsequent crosses described by mr. Wagner [23,24], confirmed that the clearbody allele, designated as inocb [15], dominates the ino allele and on the other hand also proved to be recessive to the wildtype allele ino+. In 2006 a new allele named pearly ino was discovered and identified through feather cross-sections and was designated as >inopy. Thus the order of dominance is; ino+ >inocb >inopy>ino whereas the clearbody phenotype is most close to the wildtype with respect to the ino phenotype.
The appearance of sex-linked imperfect albinism in chickens was reported at least five times [20]. In 1990 Silversides and Crawford reported for the sixth time a mutation to imperfect albinism occurring in a line of chickens that had been closed for 15 generations [20]. During their testmatings they opened unhatched eggs and found individuals mosaic for ocular pigmentation who nearly all had died mid-way through incubation. The mosaicism was observed as an area of pigment on an otherwise colourless eye. In their article the suggestion was made that the occurrence of mosaic embryos and a large number of similar mutations in a variety of other avian species such as: Japanese quail, Ring-neck pheasants, Turkeys and Budgerigars, could all be explained if the mutation was the result of integration of a movable genetic element into or near the ino+ locus. In this particular region of the sex-chromosome of the chicken, many mutant genes were shown [21]. However, in the Z-chromosomal region of the Budgerigar where the ino+ locus is located, previous investigations regarding crossover values of four sex-linked mutants called slatecinnamonino and opaline, also suggest close linkage of the first three mentioned [14, 22].

I would like to hypothesize that the appearance of the sex-linked clearbody mutant as a new allele of the ino+ locus in the Budgerigar, could be the result of an incomplete reverse or back mutation as a consequence of the excision of a transposable element from the ino allele. 

Transposable elements
Ever since the first proposal by McClintock [9] that certain genetic instabilities in maize were due to the reversible insertion of transposable "controlling elements", profound advances in understanding these transposable elements have been made. She studied genes affecting pigmentation in maize kernels because pigment defects are easily recognized and quantified. When a transposable controlling element is present in or near a gene, its effect is to block the action of that gene. For example, the presence of a controlling element in or near the sex-linked ino+ gene in the Budgerigar could have lead to inactivation of that gene, resulting in albinos in the blue series and lutinos in the green series. It was also found that an affected pigment gene that had been inactivated by the integration of a controlling element, would "back-mutate" to normal (or more nearly normal) upon departure of the controlling element. That event could also have lead to the sex-linked clearbody phenotype in the Budgerigar and even releases the possibility to hypothesize about the occurrence of the lacewing phenotype.

Transposable controlling elements in maize have shown the ability to integrate the activity of one gene with that of another or even accomplish a fusion amongst two adjoint genes. The sex-linked clearbody however, could be an imaginable candidate for the evidence that reverse or back mutations are not always able to completely restore the function of the wild-type gene from which they originated. This problem was very well studied in Drosophila melanogaster (the fruitfly) by Rasmuson, Green and Ewertson [17]. They investigated eye pigments in this species, controlled by a sex-linked gene designated as wa. Their results show that original back mutations, indistinguishable from the wild-type, have not been obtained. They concluded that a complete back mutation to wild-type is a rare or even impossible event.

Transposable elements controlling genetic instabilities in mammals have also been studied extensively. It is believed that some mutant states of several mammalian loci, including those of dogs and humans, result from the insertion of transposable elements into or near these loci [26]. Schlager and Dickie [19] collected data from spontaneous mutations and mutation rates involving 1.5 million mice during the period from August 1963, through April 1966, and include data previously reported. They found a large number of reverse mutations at a certain locus, suggesting the presence of a transposable element making this locus multiple allelic. Cattanach and Isaacson [3] described the presence of controlling elements in the mouse X-chromosome and found analogous to the "changes in state" of the controlling elements described in maize. They also mention the possibility that meiotic crossing over may be the mechanism by which some of the changes take place cannot yet be excluded. Nevertheless, wherever the transposable elements arose, they have been found in all species investigated [26].

I believe that the sex-linked clearbody could provide evidence for my hypothesis that its appearance can be explained as a consequence of an incomplete backmutation of the ino allele. Also other phenotypic evidence in support of the hypothesis that transposable elements play a major role in the appearance of some mutants in the Budgerigar, is presented for e.g. the PbPidilas and Sp loci [16], which all affect pigmentation. Even the complex bl locus is a possible candidate for the integration of a transposable element causing the series of yellowface mutants we recognize nowadays [16].

Since Dawn Henderson gave an excellent description of the normal sex-linked clearbody phenotype as well as the cinnamon variety [6], I feel no need to go into this extensively. However, I would like to show that one could breed five phenotypic "normal" sex-linked clearbody cocks and two phenotypic sex-linked cinnamon-clearbody cocks yet having different genotypes. 

Phenotypes -> Genotypes

1. clearbody = Z inocb/ Z inocb 

2. clearbody/ino = Z inocb/ Z ino 

3. clearbody/lacewing = Z inocb_cin+/ Z ino_cin 

4. clearbody/ino_cinnamon Type II = Z inocb_cin/ Z ino_cin+ 

5. clearbody/cinnamon = Z inocb_cin+/ Z inocb_cin 

6. cinnamonclearbody = Z inocb_cin/ Z inocb_cin 

7. cinnamonclearbody/lacewing = Z inocb_cin/ Z ino_cin 

The heterozygous state shown in # 2, 3, 4, 5 and 7, can only be achieved in cocks since hens are always hemizygous for sex-linked genes. The # 1 cock is distinguished from the others by showing a little more body suffusion.
Finally I would like to discuss the mating described by mr. Gordon Davis on page 35 of the august/september issue of the Budgerigar journal [4]. He states that the lacewing does not work well with the clearbody. Mr. Davis raised two ino cocks, two ino hens, two cinnamon hens and one cinnamon-clearbody hen out of a cinnamon-clearbody/ino cock mated to a lacewing hen. (To make this less complicated I left the opaline factor out of the hen involved, this has no influence on the phenotypes of the offspring). Unfortunately I have three major problems with that mating and its progeny. During my analysis it appeared to me that a sex-linked cinnamon-clearbody cock cannot be split ino only, because he must also be homozygous for cin to show cinnamon in the phenotype. Since we determined that only two types of sex-linked cinnamon-clearbody cocks do exist (# 6 and # 7), this cock must have been split for lacewing. However, during microscopical examination of feathers from this particular cock, and comparing them with clearbody feathers obtained from other breeders, it appeared that I could not prove that this bird is actually a cinnamon-clearbody. The offspring of this mating however, shows involvement of the cin factor in the cock for three cinnamon hens hatched. The outcome of the "Davis" mating did not fit in analysis made with several genotypes involving the cin factor as presented in the list above. In order to find an explanation for the peculiar results out of this mating, I even considered the presence of theCl factor in the cock. The cock then would have been a clearbody-clearbody, if you know what I mean. The existence of such birds in the USA was mentioned by the late Sorrita Ramsey in 1988 [18]. His genotype than could have been e.g. Cl/Cl+ Zinocb_cin/ Zino_cin, but if this was the case it would have shown in the cross-sections of the feathers I examined and it did not.
Just to show the genetic differences in offspring out of several complicated matings, I made two analysis. At first the analysis of a sex-linked clearbody/ino_cin Type II cock mated to a lacewing hen and secondly the same mating if the cock had been a SF dominant clearbody/ino_cin Type II. 

Mating # 1

Formula COCK : Z inocb_cin/ Z ino_cin+ (clearbody/ino_cin Type II cock)
Formula HEN : Zino_cin/ W (lacewing hen) 

Number of all possible combinations cross-overs included : 8
Number of different genotypes : 8 

1) 24.25% Z inocb_cin/ Z ino_cin (cinnamonclearbody/lacewing cocks) 

2) 0.75% Z inocb_cin+/ Z ino_cin (clearbody/lacewing cocks) 

3) 24.25% Z ino_cin+/ Z ino_cin (ino/lacewing cocks) 

4) 0.75% Z ino_cin/ Z ino_cin (lacewing cocks) 

5) 24.25% Z inocb_cin/ W (cinnamonclearbody hens) 

6) 0.75% Z ino_cin+/ W (clearbody hens) 

7) 24.25% Z inocb_cin+/ W (ino hens) 

8) 0.75% Z ino_cin/ W (lacewing hens) 

Mating # 2

Formula COCK : Cl/Cl+ Z ino+_cin/ Z ino_cin+ (SF clearbody/cin_ino Type II cock)
Formula HEN : Cl+/Cl+ Z ino_cin/ W (lacewing hen) 

Number of all possible combinations crossovers excluded: 16
Number of different genotypes : 8 

1) 12.5% Cl/Cl+ Z ino_cin+/ Z ino_cin (SF clearbody-ino/lacewing cocks)
2) 12.5% Cl+/Cl+ Z ino_cin+/ Z ino_cin (ino/lacewing cocks) 

3) 12.5% Cl/Cl+ Z ino+_cin/ Z ino_cin (cinnamon-clearbody/lacewing cocks)
4) 12.5% Cl+/Cl+ Z ino+_cin/ Z ino_cin (cinnamon/lacewing cocks) 

5) 12.5% Cl/Cl+ Z ino_cin+/ W (SF clearbody-ino hens) 

6) 12.5% Cl+/Cl+ Z ino_cin+/ W (ino hens) 

7) 12.5% Cl/Cl+ Z ino+_cin/ W (SF clearbody-cinnamon hens) 

8) 12.5% Cl+/Cl+ Z ino+_cin/ W (cinnamon hens) 

The results of the "Davis" mating only fit in the second mating where I left the crossovers out to keep it less complicated. All clearbodies in this mating however are dominant clearbodies with about the same phenotype as the sex-linked clearbodies. The only difference is the color of the flightfeathers which are black in a dominant clearbody and silver in a sex-linked clearbody. The author even considered the brownwing gene causing the cinnamon phenotypes. Both parents must have been split brownwing than to show in 25% of their progeny, but since we have no prove of its reappearance I will not go into this possibility for now. Crossbreeding with both Australian greys as well as opalines is recommanded for improving body color in sex-linked clearbody cocks. The spangle gene (Sp) does not show very well in sex-linked clearbodies. The typical spangle wingmarkings will be diminished by the clearbody gene [10]. 

Due to the lack of communication between European and American breeders as well as Budgerigar societies, it took almost fourty years for us to discover this important and attractive mutation.

I am very much indepted to the following persons: Dawn Henderson for her interesting article about the clearbodies and giving me the opportunity to publish, Ferdinant Wagner for his information and feathers, Gordon Davis for giving me the inspiration to write this article, sending me feathers and his wise decision to allow a second round to produce more offspring, Ken Moore for sending me feathers from NSL inos, Mary Morphet for sending pictures and feathers, Jim Bratt for sending information and feathers, Jeff Attwood (England) for sending feathers and Didier Mervilde for sending feathers.

Consulted and cited literature:
[1] Bingham P.M., Judd B.H., (1981) A Copy of the Copia Transposable Element is very Tightly Linked to the Wa Allele at the White Locus of Drosophila Melanogaster Cell Vol.25: p.p.705-711 [2] Calos M.P., Miller J.H., (1980) Transposable Elements Cell Vol.20: p.p.579-595 [3] Cattanach B.M., Isaacson J.H., (1967) Controlling Elements in the Mouse X-Chromosome Genetics Vol.57: p.p.331-346 [4] Davis G., (1992) Letters to the Editor Budgerigar Journal (Aug/Sept Iss.: p.p.35) [5] Durham F.M., (1927) Sex-Linkage and Other Genetical Phenomena in Canaries Journal of Genetics Vol.17 no.1: p.p.19-33 [6] Henderson D., (1992) The Clearbodies, Our American Mutations The Budgerigar Journal no.4: p.p.13-15 [7] Ingels S.C.,Bennetzen J.L.,Hulbert S.H, (1992) Mutator Transposable Elements that Occur in Clusters in the Maize Genome Journal of Heredity Vol.83: p.p. 114-118 [8] Kokemüller K., (1935) Geschlechtsgebundene Vererbung bei der Totalalbinotischen Aberration des Melopsittacus undulatus [Shaw] Zeitschr.Ind.Abst.Vererb.Vol.21: p.p.299-302 [9] McClintock B., (1951) Chromosome Organization and Genic Expression Cold Spr.Harb.Symp.Quant.Biol.16: p.p.13-47 [10]Mervilde D., (1992) Personal communication [11]Moore K., (1990) The Reappearance of the Non Sex-Linked Red Eye Budgerigar World issue 89, no.1: p.p.18-19 [12]Nevers P., Saedler H., (1977) Transposable Genetic Elements as Agents of Gene Instability and Chromosomal Rearrangements Nature Vol.268: p.p.109-115 [13]Onsman I., (1991) Unpublished results [14]Onsman I., (1992) Crossing-over in the Sex-Chromosome of the Male Budgerigar (In press) [15]Onsman I., (1992) Brief Communications The Budgerigar Journal no.6: p.p.19 [16]Onsman I., (1992) Rules for a Standard Genetic Nomenclature for Budgerigars The Budgerigar Journal no.6: p.p.19-21 [17]Rasmuson B., Green M.M., Ewertson G., (1961) Qualitative and Quantitative Analysis of Eye Pigments and Pteridines in Back- Mutations of the Mutant Wa in Drosophila Melanogaster Hereditas Vol.46: p.p.635-650 [18]Ramsey S., (1988) Facts About the American Clearbodies Budgerigar World Vol.69 no.5: p.p.15/17 [19]Schlager G., Dickie M.M., (1967) Spontaneous Mutations and Mutation Rates in the House Mouse Genetics Vol.57: p.p.319-330 [20]Silversides F.G., Crawford R.D., (1990) Genetic Aspects of a New Mutation (Sal-s) to Sex-Linked Imperfect Albinism in Chickens [21]Somes R.G. Jr. (1984) International Registry of Poultry Genetic Stocks University of Connecticut, Storrs [22]Taylor T.G., Warner C., (1986) Genetics for Budgerigar Breeders (second edition) Publ. The Budgerigar Society England [23]Wagner F., (1988) What is the Clearbody Budgerigar? Budgerigar World issue 71: p.p.15/20 [24]Wagner F., (1988) The Texas Clearbody and the Laced Clear Budgerigar World Issue 72: p.p.26-27 [25]Werret W.F., Candy A.J., King J.O.L., (1959) Semi-Albino: A Third Sex-Linked Allelomorph of Silver and Gold in the Fowl Nature Vol.184: p.p.480-482 [26]Whitney B., Lamoureux M.L., (1982) Transposable Elements Controlling Genetic Instabilities in Mammals Journal of Heredity Vol.73: p.p.12-18 ©Inte OnsmanMUTAVI Research & Advice Group

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