In search of the genetic background of *Sapphire* Agapornis fischeri.

A more extensive and detailed version of this article has been published in the BVA-International magazine of June 2021

In search of the genetic background of *Sapphire* Agapornis fischeri.
[bl1-bl2]

By Dirk Van den Abeele
Ornitho-Genetics VZW
MUTAVI, Research & Advice Group

In 2017 the first *sapphire* phenotypes appeared in an aviary in South-Africa [1].  The same year I was in South-Africa and I had the chance to see these *sapphire* Agapornis fischeri and to talk with the owner / breeder. He told me that he bought a complete bird collection of a deceased family member.  In this collection there were blue1blue2 birds (those days we called them *turquoise* or *yellow face*), probably all imported from America. Besides the (*turquoise*) Blue1Blue2 types, there was also a different bird, more greenish coloured (probably *Sapphire*Blue1), and he bred the first *sapphire* from green offspring of that bird.

A *sapphire* Agapornis fischeri is a blue bird with an orange-coloured forehead. In the cloaca area and on the wings, we can find a few small green spots.
Main question was: what mutation is this? Some breeders believe it is a normal turquoise, but for us, (and others) there are so many indications that tells us that it is probably not a turquoise.

I quote from my book: “In Agapornis roseicollis this (…turquoise) mutation results in a sixty percent reduction of the coloured psittacine on the wings. On the rest of the body there is a psittacine reduction of about ninety percent. Consequently, this bird has ‘greenish blue’ wings but nearly all of the rest of its body will be blue. I say ‘nearly all’ because if you look at the birds closely you will see that they still have a minimum ‘greenish’ hue on the body. The mask is definitely not completely white, the birds still clearly have a pink hue on the forehead, the rest of the mask is ‘cream’ coloured. This is due to the limited amount of red psittacine created in the mask”[2, p. 305].

This explanation is based on the first descriptions of the turquoise Agapornis roseicollis in aviculture, the different standard rules from the Belgian and Dutch societies in the 1980’s and 1990’s and the turquoise Agapornis roseicollis I had in my collection for many years (those days they called it white face or crème face in Belgium).

I understand the confusion for breeders who are relatively new in the hobby, because now a turquoise Agapornis roseicollis looks almost completely blue, but we have to realise that this phenotype is the result of 45 years of selection on the bluest types. But the original turquoise Agapornis roseicollis still exists, unfortunately no longer in large numbers, but it exists and is clearly different than *sapphire*. BTW if it is a turquoise, believe me, it would have been much easier for us. We have absolutely no reason to deny the existence of a mutation, but we could not ignore the obvious differences in phenotype and important: no one benefits from misinformation.

Here an overview of the most interesting outcomes as mentioned in the first article on this mutation:

a) According to the author: Combination of *sapphire* x *sapphire*. The precise number of young is not known, only that some were *sapphire*.
b) The combination of green/ *sapphire* x *sapphire* resulted in green and *sapphire* young.
c) *Sapphire* x blue resulted in all *SapphireBlue* young. (Those days there was only a picture of these birds and it showed a nearly entirely light blue bird with a light yellow/orange mask).
d) Tests with *SapphireBlue* x *SapphireBlue* resulted in *sapphire*, blue and *SapphireBlue* young.

The info provided by the original breeder suggests a Mendelian inheritance of autosomal recessive alleles. Except the first outcome. We expect in a Mendelian autosomal recessive mutation, that the outcome of autosomal recessive mutant x autosomal recessive mutant (in most of the cases) results in all mutant youngsters.

Later observations provided to us by various breeders:

e) Combination of *sapphire* with blue2, results in blue youngsters with a yellow forehead colour – similar to *sapphire*. (Interesting: Some of these birds are born with a normal white mask colour, but develop after a short of period or a complete ‘normal’ *sapphire* orange or a yellowish forehead. One report mentions that the yellow forehead became white after a few months.)
f) Combinations of *sapphire* and blue1, results in “aqua” youngsters. Greenish birds with an ’orange’ mask.

All these outcomes fit into the normal Mendelian autosomal inheritance of alleles controlled by the same gene. Important, it can also explain the breeding outcomes mention under a): if both parents were a combination of *sapphire* and blue2 (*Sapphire*Blue2), we can have indeed normal blue birds in the nest.

g) There was one ambiguity: in 2017 there was a SA breeder who claimed to have bred a *sapphire* young from the combination (what he suspected was) green/blue2 x green/blue2 that developed an orange forehead band after a few weeks. He combined Blue1Blue2 birds with green. With these green ‘splits’, he bred till now already more than ten *sapphire* phenotypes. Most of the blue youngsters are born with a normal mask colour and all they start to develop a yellowish/orange forehead after a few weeks/months.
h) April 2019, we published our findings in an article and suggested the name *sapphire* for this phenotype [1]. As a result of this article, we received e-mails from breeders who claimed they had different breeding outcomes with *sapphire*. As usual, only a few provided clear breeding results.  They had one thing in common: they claimed they have bred, at least one, sapphire young out of the combination of Blue1Blue2 with another blue bird (blue1 or blue2 was not sure for most of them). One of these *sapphire* birds was born with a complete white mask, and developed a yellow/orange forehead after months. One was born with a yellow mask and became almost completely white after months. We asked to do more test mating, but none of them was prepared to do extra test mating with green birds, or they did not have pure green birds, as a result we did not receive any more info of these breeders. So, we could not trust 100% this info, since there was not enough evidence. But we kept this reports in mind.
i) 2019: a Spanish and an Indonesian breeder reported an “aqua” youngster bred out of a ‘yellow face’ (Blue1Blue2) Agapornis fischeri with normal blue. The Spanish breeder, Pere Carbonell, told me and showed me pictures of an “aqua” Agapornis fischeri that he found in a collection of Blue1Blue2 birds that he imported from US in 2013.
j) 2020: A BVA-International judge was approached by a Moroccan breeder who bred a *sapphire* phenotype out of the combination of Blue1Blue2 in combination with a normal blue.
k) 2021: Florian Gouze, a French breeder, reported also that a friend bred a *sapphire* phenotype from Blue1Blue2 x blue.

The ‘aqua’ youngsters mentioned under i), can be explained as a *Sapphire*Blue1 youngster, but these outcomes can never be explained as a normal Mendelian inheritance of autosomal recessive alleles. The fact that Pere Carbonell imported an “aqua” (*Sapphire*Blue1) Agapornis fischeri from US, proves that this *sapphire*Blue1 already existed in the bloodline of American breeders and there is no single relation with an independent South-African bloodline.

The breeding outcomes mentioned under g), h), j) and k) cannot be explained as a normal Mendelian inheritance of autosomal recessive alleles.

To exclude the possibility that normal blue1 or blue2 birds still can develop psittacine in the feathers, we examined feathers of proven blue1 and blue2 Agapornis fischeri and Agapornis personatus with the Raman spectroscopy and none of them had psittacofulvins (psittacine) in the feathers [3]. So the one-million-dollar question remained: what is the genotype behind *sapphire* if it is not an autosomal recessive allele?

Crossing-over
In my opinion, this *sapphire* phenotype, can only be explained as a crossing-over between blue1 and blue2.

The theory that *sapphire* is the result of a crossing-over, can explain the appearance of a *sapphire* phenotype out of the combination of Blue1Blue2 x blue. A Blue1Blue2, has on one chromosome of the pair the blue1 mutated allele, on the other chromosome the blue2 mutated allele. We know that during meiosis crossing-overs can occur. Paired chromosomes align so that similar DNA sequences from the paired chromosomes cross over one another and results in a shuffling of genetic material during meiosis. That way we can combine the blue1 gene with the blue2 gene (or allele) on the same chromosome (bl1_bl2).

If we combine that crossing-over (in that case we indicate it as blue1-blue2) with blue2, we have blue2/blue1-blue2 phenotypes. That also fits into the observation from breeders that states that combinations from *sapphire* with blue2 results in *sapphire* phenotypes – paragraph h), j) and k). The variation on the expression of that gene (colour orange forehead) can also be explained by that theory. Here we can also refer to the cinnamon-ino (crossing-over between cinnamon and SL ino) in Agapornis roseicollis. Here we were able to prove (by breeding results) that there are perfectly looking cinnamon-ino males that are genetically cinnamon-ino/SL ino (Z cin_ino/Z cin+_ino).  As far as we know, there are no proves for the existence of cinnamon-ino/cinnamon birds that have a perfect cinnamon-ino phenotypes.

The outcomes from the pair mentioned under paragraph g), can be explained as green/blue1-blue2 x green/blue2. When he combined Blue1Blue2 bird with a green, in one of the youngsters the crossing-over probably happened. All blue youngsters from that combination are blue1-blue2/blue2 (*sapphire* with a yellowish/orange forehead).

It also explains the outcomes mentioned under paragraph i), here we are probably also dealing with a blue-blue2/blue1 phenotype. – crossing-over happened in the Blue1Blue2 parent during meiosis.

The possibility that a crossing-over will occur depends on the distance between both alleles on the chromosome. The larger the distance between both mutations on the chromosome [2, p. 228], the larger the possibility that the chromosome will fracture somewhere in between and a crossing-over between both alleles will occur.

In 2017 scientist discovered that in budgies [Melopsittacus undulatus] the MuPKS gene is responsible for the blue mutation [4]. That gene is 20662 bp long and has 6 exons [5]. So that is a relative ‘short’ gene.  If blue1 and blue2 are alleles of that MuPKS gene, it can explain why *sapphire* phenotype rarely occurs.  It also explains why the combination is really ‘stable’, since there is a limited change on a recombination. I guess that the distance between both alleles is only a few centimorgan. Therefore, this combination can easily be misinterpreted as a normal autosomal recessive mutation, and it would not be the first time. Remember the cinnamon-ino in budgerigars. The appearance of the first cinnamon-ino budgies was already reported in 1946 [6]; Those days, breeders called it “lacewings” because they also believed it is a basic mutation. The possibility that cinnamon-ino was a crossing-over was first discussed by Dr. Taylor and Cyril Warner in 1961 [7]. So, it took 15 years before scientists realised that it was a crossing-over. And believe me, it took other long years before breeders in aviculture realised and accepted that it was not a separate mutation but the result of a crossing-over.  Even now a days, some breeders even deny the fact that cinnamon-ino is a crossing-over. BTW we indicate combinations by crossing-over by naming the basic mutations with a hyphen in between. So in this case: blue1-blue2. See international agreements [8]

Our thoughts:

Phenotype	Genome	Genetic symbol
blue	blue1	bl1
blue	blue2	bl2
*yellowface* *turquoise*	Blue1Blue2	bl1/bl2
*sapphire* phenotype	blue1-blue2	bl1_bl2/bl1_bl2
*aqua* phenotype	blue1-blue2/blue1	bl1_bl2/bl1_bl+
‘light’ *sapphire* phenotype more yellow front colour in the first weeks/months	blue1-blue2/bleu2	bl1_bl2/bl+_bl2

Conclusion:
We now have several indications that the *sapphire* phenotype and the way it inherits could not be explained as a normal autosomal recessive inheritance. It maybe can be explained as a result of a crossing-over between blue1 and blue2. That way it can also explain the different patterns of non-mendelian inheritance presented in some breeding outcomes, provided to us by different breeders.

Now I would like to add some personal thoughts. Let that be clear!! It is certainly not our intention to blame anyone. It is perfectly understandable why breeders believe it is an autosomal recessive mutation. Most of the indications pointed out in this direction and we have to admit, we also followed that theory for a while. But as usual, and as I promised in my article of April 2020, time will tell because further insights provided more knowledge. And this is what happened again.

I only regret that some breeders – for whatever some reason – are unable or unwilling to understand that it sometimes takes a long time before we can figure out these mutations correctly. Unfortunately, others then take the opportunity to turn people and groups against each other, just for their own benefits. These internet and Facebook trolls will unfortunately always be involved in this hobby. What they don’t understand is that they are destroying the hobby for many enthusiasts. Do we want to continue with our hobby like this? Not me, let that be clear.

Dirk

Sources:
[1]          D. Van den Abeele, ‘Blue, blue type2, turquoise, *sapphire*, *teal*,…. and so much more ….’, Agapornis.info, nr. April, 2019.
[2]          D. Van den Abeele, Lovebirds Compendium, 1ste dr. Warffum- The Netherlands: About Pets, 2016.
[3]          D. Van den Abeele, ‘Short communications: blue1 and blue2’, Agapornis.info, nr. 2020/6, p. 184, 2020.
[4]          T. F. Cooke e.a., ‘Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars’, Cell, vol. 171, nr. 2, pp. 427-439.e21, okt. 2017, doi: 10.1016/j.cell.2017.08.016.
[5]          ‘Melopsittacus undulatus isolate bMelUnd1 chromosome 1, bMelUnd1.mat.Z, whole genome shotgun sequence’, mei 2020, Geraadpleegd: feb. 15, 2021. [Online]. Beschikbaar op: http://www.ncbi.nlm.nih.gov/nuccore/NC_047527.1.
[6]          C. H. Rogers, The World of Budgerigars. Nimrod, 1987.
[7]          T. G. Taylor en C. Warner, Genetics for budgerigar breeders. 1961.
[8]          ‘Downloads – Dirk Van den Abeele’. https://www.ogvzw.org/downloads-2/ (geraadpleegd feb. 16, 2021).