Working With Autosomal Genes for Color and Pattern: A Domestic Guppy Breeders best friend and often worst nightmare

Working With Autosomal Genes for Color and Pattern: A Domestic Guppy Breeders best friend and often worst nightmare Alan S. Bias P.O. Box 1508, Lewisburg, WV 24901, USA http://swordtailguppies.com alansbias@aol.com Presented Sept. 5, 2015 to attendees of the 18 th World Guppy Contest held in Tampa, Florida, USA Alan S. Bias Permission granted for nonprofit reproduction or duplication of photos and text with proper credit for learning purposes only. Abstract. The selection for genes in Domestic Guppies has long been heavily based on sex-linked genes. In particular Y- linked for pattern and X-linked for color. Over the last several decades a larger percentage of strains and phenotypes (especially those created by Asian and European breeders) have been produced based on autosomal genes or co-expression of sex-linked and autosomal genes. The attempt of this presentation is to help breeders gain not only an understanding of autosomal genes, but their interactions in combination(s) and with other genes. The development of domestic genes, unlike wild counterparts subject to predation, is only hindered by breeder selection and retention. Results based on 45 years breeding experience of which nearly thirty (30) years have been devoted to strains collectively known as Swordtail Guppies, efforts of other professional breeders, and that of the scientific community are presented. Key Words: Guppy, Domestic Strains, Autosomal Genes, Autosomal Recessive, Autosomal Incompletely Dominant, Wild-type, Breeder, Guppy Color and Pattern, Poecilia reticulata. Introduction Welcome to each and every one of you. I d like to thank you all for taking the time and effort to attend not only the 18th World Guppy Contest, but also this presentation. Your support as Domestic Guppy breeders has ensured the success of this venue and its place in Guppy C history and the memories of those in attendance. Some of you I have known for many years, others I have corresponded with for just as many years, and others are of new acquaintance In today s presentation we will only be dealing with hereditable genes impacting color and pattern. Not those dealing with finnage propagation or others related to fecundity. Discussion Much variation is found in wild guppy colors & patterns as a result of habitat type (upper or lower stream setting), sexual selection, and predation. Yet, only a singular grey body color is found in wild guppies, often referred to as wild-type or grey wild-type. Domestic Guppy A foundation stocks in the early 1900 s all derived from limited importations of such fish. While unexpressed, the majority of all of the diversity in modern phenotypes was contained in these initial imports or subsequent collections that may have contributed additional mutations. A large portion of the past effort of both breeders and researchers from 1920 onward has focused on the use and documentation of sex-linked traits found within genotypes to produce and improve upon domestic phenotypes. While early research linked specific expressions to individual genes, we now find that many are the result of multiple genes or gene interactions. As a pedigree breeder and part-time Page1

researcher I find it best if we refrain from the use of the term gene(s) and instead defer to the use of trait in discussions if you don t have genetic evidence supporting how the gene is inherited. Sex-link genes can be identified, and passed on by either the X or the Y chromosomes, or by both of them. Depending upon just how closely they reside to the SRY; sex determining region of the Y chromosome, they may cross over frequently from X to Y or Y to X, they may cross over rarely, or not at all. In many instances crossover is often overlooked in breeder tanks for a simple reason. That being does the gene express in males only, or both males and females? I d like to take the opportunity to focus on a singular mode of inheritance available within our genetic toolbox ; autosomal genes. Prior to 1930 only a single autosomal gene had been identified for color or pattern in Guppy stocks; Zebrinus (Winge 1927). Autosomal refers to genes located on any chromosome other than a sex chromosome. In theory, autosomal genes should affect the body color of both sexes equally. However, in practice this is not always the case as specific genes may impact color expression limited to males (known as sex-limited genes). Zebrinus is an obvious example of a sex-limited gene that produces a phenotype in only one sex. Females do not express any barred pattern. Autosomal genes can be passed by either sex in several modes of inheritance. 1. Autosomal Incompletely dominant, 2. Autosomal Recessive, 3. Autosomal completely dominant. Initially, basic identification of traits found in Guppy genotype was the product of scientific research and publication. While often lacking in formal documentation, identification of new traits, or at least assemblage of complex phenotypes, has been the product of the breeder community. Where we often fail as breeders is in keeping complete records needed to document our results. This results in much speculation and inaccurate facts when disseminating our results among fellow breeders. Male sex-limited genes require the presence of testosterone for expression. Autosomal genes are passed to male and female offspring, and are expressed in females. The exception would be autosomal sexlimited genes, such as Zebrinus, which also require male hormones for expression. When a gene does not visibly express in females, X-linkage is easily confused with an autosomal dominant gene. Dominance in itself can be established for a particular gene with the first generation of reciprocal crosses. Though this does not distinguish between sex-linkage and an autosomal mode of inheritance. After an initial cross and expression, it requires one or two generations of a reciprocal cross to distinguish between X-link dominant and autosomal dominant. Chromatophores are responsible for projection of color and pattern in Guppies (See below: Figure I Kottler 2013). Generally, five (5) types are acknowledged; black melanophores, orange to yellow xanthophores, red erythrophores, blue/green/silver reflective iridophores and white leucophores. Blue chromatophores (cyanophores) are present in some species and possibly Guppies(?). The autosomal genes we will discuss have impacts on each of these classes. Page2

Grey Wildtype. There is no notation for a grey gene (See: Table I). The grey color in guppies is primarily due to corolla and dendritic black melanophores. Generally, dendritic melanophores are resident along upper and lower layers of scales. Corolla melanophores are found at lower levels of skin and scattered around the body. Together they produce the reticulated effect Guppies are named for: Poecilia reticulata. Few true mutations have been documented in Guppy research or identified by Domestic Breeders. For the most part we are simply identifying and working with genes. Table I. B not blond G not golden A not albino b blond g golden a albino [Note: If a phenotypical expression is not blond, golden, or albino, it is grey. This list can be expanded to include additional autosomal traits, the same principle applies.] When combining genes into a phenotype, the question then becomes are the various genes alleles of a single gene or are they two or more entirely different genes? If two or more genes are involved, are they located on the same chromosome or on different chromosomes? If they are on the same chromosome, are they close enough to each other that they tend to stay together during meiosis (cell division that produces gametes) or do the separate from each other randomly? When you view results of Grey breeding's that are heterozygous for Albino (Aa), Blond (Bb), and Golden (Gg) the overall intensity of both color pigment and melanophores (See: Table II.) are reduced through epistasis. We are dealing with three pairs of different genes. Epistasis refers to the situation where one gene (or allele of that gene) modifies or inhibits the expression of a different gene, gene pair, or an allele of the second gene (See: Table III). Table II. Melanophore Size & Structure Grey, Blond, Golden, and Cream (left right) (Goodrich et. al 1944). Table III. bb is epistatic to G and A gg is epistatic to B and A aa is epistatic to B, G, bb and gg bb gg is cream Eb bb or Ab bb white Eb gg or Ab gg are silver Two genes are alleles of each other if they are alternate forms of the same gene. In example: the coat color series in cats is full color (C), Burmese (c B ), Siamese (c S ), blue-eyed albino (c b ), and pink-eyed albino (c a ). DNA sequencing has confirmed earlier crossing data indicating each trait (phenotypical expression) is due to different alleles (alternate forms of the same gene). When two pairs of genes are linked, they are located on the same chromosome and are close enough in proximity to each other to avoid random recombination. They recombine at a frequency that is less than 50%. Thus, if the genes A and B are linked, we may find four different possible combinations of A and B on a single chromosome: AB, Ab, ab and ab. Page3

Grey Vienna Emerald DS male & female, photo Alan S. Bias Grey Wild-Type male, photo courtesy of Ramona Osche Grey Wild-Type female, photo Alan S. Bias Autosomal Genes For Color and Pattern Albino Lutino (l ). Recessive. Also known as Wine Red Eye Albino (WREA) or (Type A). Eyes are a dark Burgundy color. When photographed from a certain angles will appear black. Likely tyrosinase positive; enzyme present, with reduced melanin production. Lutino (l) is allelic to Grey (L, not-lutino). Lutino Schimmelpennig Platinum Snakeskin Roundtail, photo courtesy Denise Barbe Albino (a). Recessive. Also known as Real Red Eye Albino (RREA) or (Type B). Inability to produce black melanophores in body and finnage. In effects eliminates all classes of melanophores; dendritic, corolla and punctate. Likely tyrosinase deficient; inability to convert tyrosine into melanin. Albino is Page4

epistatic to both Blond and Golden genes; thus mutant alleles of each may be found in the albino genotype (e.g., aa Bb, aa bb, etc.). Described: Haskins & Haskins 1948 6. Albino Full Red Delta, photo courtesy of Paulo Henrique Keijock Muniz Albino Yellow Vienna LS, photo Alan S. Bias Albino (?? ). Recessive. Also known as Type C - Singapore. Likely tyrosinase positive; enzyme present, with reduced melanin production. Little is known of this allele. It is likely present, but confused with Lutino. [Note: No photo available]. Asian Blau (Ab); also (r2) Europe and (Rr) Asia. Incompletely Dominant. In heterozygous fashion red color pigment is removed, but yellow color pigment and Metal Gold (Mg) is little affected. This produces an iridophore based phenotype. Snakeskin patterns degrade in both heterozygous and homozygous expression. The Purple (Violet) sheen found above the lateral line of both males and females is removed. In homozygous condition certain black melanophores are removed along with red and yellow color pigments. In homozygous condition finnage is also reduced in expression, but genes are still present in genotype for normal finnage. Outcross of homozygous Ab will produce expected finnage in F 1 offspring. [Note: As there are distinct types of red color pigment present in both body and finnage, removal may not be complete in a red Old Fashioned shoulder stripe. A very faint red shoulder stripe is sometimes visible.] Heterozygous Asian Blau Vienna Lowersword, photo Alan S. Bias Homozygous Asian Blau Vienna Lowerswords, photos Alan S. Bias Page5

Bar (bar). Recessive. Officially the pattern is described as being comprised of 3-5, or more, dark vertical bands. Personally, I view the pattern as being iridophore based stripes with gaps populated by melanophores as a result of the interaction of iridophores and melanophores. If otherwise, the Bar pattern would disintegrate in albino or blond form. Similar in expression to sex-link Tigrinus, which is comprised of strait and forked bands. Described: Phang 1999 13. (Note: See Zebrinus gene. Zebrinus and Bar while similar have noticeable differences in expression. Bar tends to solely express in vertical stripes, while Ze can express forking on top of stripes). Barred Snakeskin Delta, photo courtesy V. Phang (1999) Blond (b). Recessive. Also known as IFGA Gold, Asian Gold, European Blond. This mutation produces a near normal amount of black melanophores of all types; Dendritic, Corolla and Punctate. However, the size of each is greatly reduced and the structure modified as compared to wild-type grey. According to published results Blond is not linked to Golden. However, results were based on limited numbers and interpretation. Results of breeders are often suggestive of partial linkage. This gene should be viewed not as a suppressor of melanophores, but rather one that alters size. Described: Goodrich et al. 1944 4. Blond Vienna Lowerswords, photos Alan S. Bias European Blau (r or r1); also (eb). Recessive. Also known as Dunkel in Asia. European Blau is not epistatic to wild-type grey. It is epistatic to genes for red and yellow; major red and yellow color pigments are removed from the body in homozygous expression. Certain red color pigments may be present in finnage, and to a lesser degree in body. Though reflective qualities are reduced. Ecotopic melanophores may be removed, while basal level melanophores such as found in Half Black (NiII) are only slightly reduced. Snakeskin patterns degrade in homozygous expression. Purple (Violet) sheen found above the lateral line of both males and females is removed. There is minimal impact on finnage reduction. Described: Dzwillo 1959 1. [Note: As there are distinct types of red color pigment present in Page6

both body and finnage, removal is incomplete in a red Old Fashioned shoulder stripe. A reduced red shoulder stripe remains visible.] European Blau Grey Wild-Type, photo courtesy of Tobias Bernsee Glass Belly (gb). Recessive. In homozygous fashion removes / reduces formation of both iridophores and leucophores over the entire body; including peduncle, abdomen, gills and eyes. Producing a clear/ translucent matt finish in areas expressing color pigment and Metal Gold (Mg) or dark black in areas expressing black melanophores, such as the eyes. Glass Belly Pingu, photo courtesy Carl Groenewegen Golden (g). Recessive. Also known as European Gold, IFGA Bronze or Asian Tiger. This gene produces a reduced amount of black melanophores (50% +/-) of all types; dendritic, corolla and punctate. However, the size of dendritic and corolla melanophores are greatly increased and collection of corolla is concentrated into "clumps" or "islands" of melanophores along scale edges. Males and females lack skin melanophores at birth, but develop with maturity. Scale edging will become lighter the higher the inbreeding co-efficient; i.e. long-term Golden x Golden breeding s. According to published results Golden is non-allelic and not linked to Blond. However, results were based on limited numbers and interpretation. Results of breeders are often suggestive of partial linkage. This gene should be viewed as a suppressor of melanophore population numbers. Described: Haskins & Druzba 1938 5 as Fredlini (fr), Goodrich et al. 1944 4 as Golden (g), and adopted by Haskins. [Note: In Europe still often referred to as Fredlini in regard to Golden stocks developed by Swedish Artist / Aquariust Tor Otto Fredlin. This designation did not remain in place as it described a guppy variant B ; Lebistes reticulatus (Peters) var. Fredlini (Schreitm) instead of an allele of grey. Schreitmuller 1932 15, 1933 16, 1934 17.] Page7

Golden Topswords, photos courtesy Denise Barbe Hellblau (r3). Recessive. Red color pigment is entirely removed. Yellow color pigment is almost completely removed, but Metal Gold (Mg) can be present. Unlike Asian Blau (Ab) or European Blau (Eb) snakeskin pattern does not degrade. Any Purple (Violet) sheen found above the lateral line of both males and females remains expressed. As homozygous Hellblau fish mature reflective qualities of iridophores overshadow early formed melanophores, producing an appearance of brightness. Hellblau Snakeskin, photo courtesy of Gernot Kaden Ivory (I); Yoshiki Tsutsui denoted as Violet (v). Incompletely Dominant. Ivory suppresses red color pigment. Resulting in a white appearance on modified red fish and violet on modified blue fish. According to Claus Osche, Ivory first appeared at TFI Farm in Singapore in Red Mosiac (i.e. Blue Variegated), producing a whitish- violet coloration. According to Yuji Yamaguti, Japanese breeders denote Purple Grass genotype as: Ivory (ii) + Asian Blau (Rr). It is possibly Purple Body Mutation (Pb) is also present. While Ivory is the product of a gene, Purple Grass is a trait (phenotype) produced by the action of ii Rr genes. Page8

Ivory Grass, photo courtesy of Shinichi Kobayashi Ivory Half Black, photo courtesy of Shinichi Kobayashi Ivory Galaxy, photo courtesy of Shinichi Kobayashi Ivory Schim Plat. Delta, photo courtesy Akrawat Design Ivory Delta female, photo courtesy Harn Sheng Khor Magenta (M). Incompletely Dominant. Proliferation of red color pigment and iridophores. This gene converts yellow color pigment cells to red, though Metal Gold (Mg) may remain. Also, produces visible concentrations of black melanophores in certain body regions. Reduces fin development. Magenta. Photo courtesy of Krisztián Medveczki Page9

Metal Gold (Mg). Undescribed, likely Incompletely Dominant. Also known as Material Gene in Asian and Metallic in Europe. Yellow-Gold iridophore gene. Comprises most body color found in Yellow strains. Heterozygous expression not visible in females, but visible in homozygous fashion. Heterozygous expression in males often limited to portions of finnage and portions of body. Homozygous expression in males creates an overall metallic sheen. Homozygous Metal Gold (Mg) Vienna Lowerswords, photos by Alan S. Bias Hete. (left) & Homo. (right) Metal Gold (Mg) IFGA Delta, photo courtesy of Bryan Chin Metallicus (Me). Recessive. Also known as Stoerzbach (s) / Störzbach Metal. Blue iridophore gene. Removal of red and yellow color pigments in body, but not finnage. Individuals with a Yellow-Gold cast result from addition of Metal Gold (Mg). Described: Kempkes 2007 7. Homozygous Stoerzbach Grey (left) & Blond Metal Gold (right) Vienna Lowersword, photo Alan S. Bias Midnight Black (mid). Recessive. Also known as Hypermelaninization. A form of non-motile black melanophores, is most commonly associated with Black Moscow strains. Expression of Midnight gene is visible in females. Some breeders report expression in F 1 outcross of midnight females. This is suggestive of Incompletely Dominant mode of inheritance. Page10

Midnight Black Moscow, photo courtesy of Harn Sheng Khor Pink (pk). Recessive. Expression of standalone Pink in itself is easily overlooked in wild-type. Pink is epistatic to the presence of red color cells, resulting in removal in body and a yellow-orange cast in finnage. Homozygous Pink has distinctive impacts on various types of black melanophores; a reduction in co-expression with NiII halfblack, an increase with Moscow Blau Additional Gene (MBAG) or snakeskin genes. In the case of reticulation an increase in concentration of melanophores is visible on scale edging. Does Pink remove melanophores, prevent their development, or convert the cells to a different cell class? Results seem dependent on co-expression with other genes. In pkpk + NiII it appears melanophores are removed or fail to migrate. Yet, in pkpk + MBAG it appears melanophores are converted and proliferate. Pink can remove blue iridophores. This results in opaque body coloration or white appearance in co-expression with Y-link Moscow (Mw) shoulder pattern. The most spectacular phenotypes are the result of homozygous Pink co-expression with other full body or half body melanophore and iridophore genes. Homozygous Pink causes reduction in size and finnage. Each is retained in the genotype and restored with an outcross. Homozygous Pink female, photo courtesy of Yuji Yamaguti Page11

Homo. Pink, photo courtesy of David Liebman Homo. Pink Vienna Swordtails, photo courtesy Björn Lundmark Saddleback (Ht ). Autosomal Dominant. [Note: Some breeder reported results are suggestive of an Autosomal Incompletely Dominant mode of inheritance, while others strictly sex-linked. This suggests that there may be at least two and perhaps three different genes with similar effects.] Also known as Half Tux. A black melanophore band runs horizontally on the upper peduncle quadrant. This is normally found in conjunction with corresponding yellow color pigment on lower peduncle quadrant; this version is considered Y-linked. Another version appears lacking corresponding yellow pigment on lower peduncle quadrant; this version considered X-linked. Saddleback Roundtail, photo courtesy of Tobias Bernsee Zebrinus (Ze). Incompletely Dominant. Officially the pattern is described as being comprised of 2-5, or more, dark vertical bands. Personally, I view the pattern as being iridophore based with gaps being populated by melanophores as a result of the interaction of iridophores and melanophores. If otherwise, I would expect the Zebrinus pattern to disintegrate in blond because their melanophores are much smaller in size. But I would not expect the same with albino, because the melanophore is still present with the same size as in grey, the albino cells fail to synthesize melanin, but they are there. Similar in expression to sex-linked Tigrinus, which is comprised of strait and forked vertical bands. Described: Winge 1927 18. (Note: See Bar gene. Zebrinus and Bar while similar have noticeable differences in expression. Bar tends to solely express in vertical stripes, while Ze can express forking on top of forward stripes). Page12

Grey & Blond Zebrinus Vienna Lowerswords, photos by Alan S. Bias Double Recessive Autosomal Genes Cream. Double Recessive (Blond + Golden). Crème is not due to a single gene, it is a trait (phenotype) produced by the action of two genes. In females chromatophoes are not macroscopically visible. Melanophores are reduced by >80-90%. Corolla & Punctate body cells in Cream remain the same size as in blond. Dendritic cells on scale edges in Cream are smaller than either Golden or Blond. Cream not only greatly reduces the total number of black cells, but also reduces remaining large dendritic cells in size. According to French breeder Denise Barbe, viability is reduced. Described: Goodrich et al. 1944 4. Double Recessive Blond + Golden (bbgg) Coral Red Snakeskin, photo courtesy photo courtesy Denise Barbe Silver. Double Recessive (European Blau + Golden) or (Asian Blau + Golden). Silver is not due to a single gene, it is a trait (phenotype) produced by the action of two genes. Similar in appearance to White. Described by Hans Luckmann as having the appearance of tarnished silver. Body coloration is pale, though some pattern remains subject to limitations of European or Asian Blau s; removal of red and yellow color pigment. Finnage is flatter colored with limitations of European or Asian Blau s. Scales are edged in black with a darker appearance. Named by initial breeder Hans Luckmann who first combined and documented European Blau + Golden. European Blau (EbEb) + Golden (gg); i.e. Silver, photo courtesy of Denise Barbe (left) Half Black (NiII) Asian Blau (AbAb) + Golden (gg); i.e. Silver female, photo courtesy of Tobias Bernsee (right) Page13

European Blau (EbEb) + Golden (gg); i.e. Silver, photos courtesy of Denis Barbe (left) & Stephen Elliot (right) White. Double Recessive (European Blau + Blond). White is not due to a single gene, it is a trait (phenotype) produced by the action of two genes. Melanophore size and numbers are reduced. Body coloration is pale, though some pattern remains subject to limitations of European Blau; removal of red and yellow color pigment. Finnage is flatter colored with limitations of European Blau. Described: Dzwillo 1959 1. White Half Black Reds, photos courtesy of Tobias Bernsee ~~~~~~~~~~~~~~~~~~~~~~~~~~~ When Öjvind Winge published The Location of Eighteen Genes in Lebistes Reticulatus in 1927 (Winge 1927 18 ) he listed a total of 18 ornamental traits. Seventeen (17) deriving from an X or Y sex-linked mode of inheritance, and one as autosomal incompletely dominant; Zebrinus (Ze). The foundation male of this particular trait was obtained in 1918. As Zebrinus was the first identified autosmal trait for color or pattern, he delayed publication for nearly eight years. In his words, sex-linked inheritance with frequent crossing-over between the X and the Y chromosome might easily be confused with ordinary mendelian inheritance, and it was therefore desirable to have an abundance of numerical material. While nearly 90 years have passed since the formal publication of Zebrinus as the first documented autosomal gene for color or pattern, to my knowledge only an additional six autosomal genes have been formally described; Blond (1938/1944), Golden (1944), Albino (1947), European Blau (1959), Bar (1999), Metallicus [Stoerzbach] (2007). Researchers often use the term accessory genes to document unknown grey areas. As breeders we have long been aware of positive & negative autosomal enhancement, or accessory genes through our improvements in color, pattern and finnage through positive selection. Control of gene expression of body and finnage is generally considered to be separate and distinct. But this distinction is not always written in stone. This is easily seen in the distinct effects of European and Asian Blau on finnage coloration (See prior photos). The use of "full body" genes can be used to amplify Page14

expression of another trait, as seen with Asian Blau + Schimmelpennig Platinum. This is formally referred to as epistasis. Asian Blau + Schim. Plat DS, photo Alan S. Bias As a breeder I have observed autosomal genes have direct impact on expression in both heterozygous and homozygous states. Breeders have long been aware of this, though may not have understood the implications. This is most easily seen in incompletely dominant genes such as Asian Blau (See prior photos). Asian Blau produces vastly distinct phenotypes in either homozygous or heterozygous condition. What most breeders often fail to realize is recessives also have an impact on expression in heterozygous condition. This expression is often very subtle, and you must understand not only your strain genetics, but that of each line. The degree of expression of the recessive allele in the heterozygote may vary from strain to strain because of the genetic background of the particular strain. This has occasionally been documented in other species. A good example to cite involves Red or Yellow strains, either solid or halfblack (NiII), with lines maintained in Grey (BB) and Blond (bb). Both red and yellow have documented associations with melanophores; when either color pigment is present, so are melanophores. Each type of color pigment has epistatic repulsion qualities that determine not only not only melanophore locations, but also concentration levels. Normally, melanophores will populate regions absent of color pigment or be forced to extreme edges. It is common practice for breeders to maintain dedicated Grey (BB) and Blond (bb) breeding lines within a strain. Often the Grey line is maintained in homozygous condition. In my breeding program I routinely maintain my Grey lines in heterozygous Grey (Bb) condition. This means a percentage of males are also homozygous for both Grey (BB) or Blond (bb). This allows me to select Blond males for use in outcross to dedicated Blond breeding lines, and avoid heterozygous grey-bodied (Bb) males in F 1 offspring. When lines of grey fish are bred long-term grey x grey in homozygous (BB) condition the intensity of coloration increases, along with the concentration and size of melanophores. In time they become dirty from the excess accumulation of melanophores. In turn breeders routinely outcross them to Blond (bb) counterparts. Resultant F 1 grey bodied offspring, which are heterozygous (Bb) for blond, receive direct benefit both from the Blond gene and outcross. 1. Blond reduces melanophore size in both homozygous and heterozygous fashion. 2. Out crossing reintroduces the dominant allele(s) of the modifier gene(s) and returns the F 1 (or beyond) to the more desired condition. Inbreeding led to homozygosity for modifer genes (QTLs) that caused a dirty appearance. When Blond (bb) fish are bred long-term bb x bb the intensity of coloration decreases, along with the concentration and size of melanophores. In time they become pale from the excess reduction of melanophores. In turn breeders routinely outcross them to heterozygous (Bb) or homozygous (BB) grey counterparts. Resultant F 1 offspring, receive direct benefit from outcross. Again, outcrossing reintroduces the dominant allele(s) of the modifier gene(s) and returns the F 1 (or beyond) to the more Page15

desired condition. Inbreeding led to homozygosity for modifer genes (QTLs) that caused a pale appearance. I have found when grey fish are bred long-term for heterozygous (Bb) condition (Bb x bb or Bb x Bb) there are noticeable differences in the intensity of coloration, along with the concentration and size of melanophores. Homozygous grey-bodied male siblings express more intensity of coloration, the same as found in dedicated Grey lines. Heterozygous grey-bodied males lack the same intensity of coloration as found in homozygous grey-bodied male siblings. As outcross is not a factor, this is highly suggestive of direct influence from the autosomal recessive Blond gene when in heterozygous condition. These recessive genes are not completely recessive, but so close to being recessive that it requires careful examination to detect the heterozygotes. [Note: This observation is not limited to autosomal gene Blond. I see the same correlation in other autosomal recessives in heterozygous condition; Golden (Gg), Stoerzbach (Ss), Albino (Aa), and Pink (PKpk).] To illustrate the effect of heterozygous Blond on Grey wild-type (see below), we show a Grey (Bb) male heterozygous for Blond photographed under three lightings. Notice muted Wingean Orange spots and minimal black ocular spotting in anterior shoulders and finnage. This degree of spotting is commonly found in grey bodied males in my Lowersword strain. While he expresses a fairly high degree of iridescence, overall color pigment expression is minimal and melanophores reduced. [Note: Variegation (Var) is visible in dorsal and caudal, but faintly expressed.] In comparison we have two homozygous Grey (BB) males (see below) with a high degree of iridescence and reflective qualities in color pigment. The male on the left in Purple Body Mutation (Pb), and the male on the right in wild-type orange. Heterozygous Grey male (Bb), photos (left center) Alan S. Bias, photo (right) courtesy of Joe Mason Homozygous Grey (BB) males, photo by Alan S. Bias In the following 2 examples we illustrate the effect of heterozygous Golden (Gg) on Grey (bb) wild-type (see below). No additional Y-linked genes for fragmentary snakeskin have been introduced. The Golden gene was infused into my stocks several years ago through a single homozygous Golden (gg) female. Notice how melanophore collection and locations are altered from the prior examples above. This is the expected norm for heterozygous (Gg) breeding s in my stocks. Thus, is highly suggestive of direct influence from the autosomal recessive Golden gene when in heterozygous condition. Page16

Heterozygous Golden (Gg) males, photo by Alan S. Bias Summary & Conclusion When taken seriously, Domestic Guppy breeding may not be Rocket Science, but it is a science within itself as is any form of pedigree stock breeding. Longtime successful stock breeders breed by eye; through the power of observation to achieve if not understand results. We are visual thinkers to varying degrees. Successful breeders have an inherent and often above average ability to observe the natural world and create domestic results (Arnheim 1969 19, Grasseni 2004 2, 2005 3 ). Yet few take the time to learn the applied genetics needed understand both past, present and future results of planned breeding s. The Poecilia reticulata genotype is contained in a diploid chromosomal set of 46 chromosomes found in pairs, 44 of which are autosomal and 2 sex determining. Nearly all of the genes for color and pattern are identified as sex-linked. In a loose sense, assuming equal chromosome sizes, this equates to color and pattern genes being located on 4% of the total genome, leaving for us as breeders 96% of the total genome to search for unknown autosomal modifiers of color and pattern to hide. No new color is being added to our genetic toolbox as breeders. Genotype has been fixed since the inception of Domestic Guppy Breeding by the five identified types of chromatophores that determine color and pattern. Or has it? Much new color and pattern has been added to phenotypical expressions in recent decades. Some of which results from the identification of autosomal modifiers found in existing stocks. However, the greatest potential source of new color genes and gene combinations is wild populations, as evidenced by the incredible color traits being developed from infusion of variant populations. The implications should be readily visible to any long-term breeder. We still have a vast unknown in the Guppy genome to explore in our search for additional autosomal genes. Modifiers which can be used to further create new and diverse autosomal combinations with existing sex-linked genes. Regardless of your interests as a breeder; production to set show standards, creation, exploration or an overlapping interest of all. Don t be so quick to cull that anomaly that shows up. Keep some notes or take a digital photograph to record. More importantly, pass it on to a breeder who may have the interest to study and develop it. Just because a class does not exist to exhibit results today, does not mean it will not exist in the future. Just because a new expression does not meet a standard of today, does not mean it cannot be improved upon in the future. I sincerely thank you all for taking the time to attend this presentation today Page17

DEFINITIONS Alleles: Alternate forms of the same gene. Domestication: To bring or keep (wild animals or plants) under control or cultivation. http://dictionary.reference.com Dominance: One gene contributes more, or less, to the final phenotype. In complete dominance one allele completely hides the effect of the other allele; while in the case of incomplete dominance, the phenotype is part way between the phenotype of the two homozygous forms of the allele. Heterozygosity: If two different alleles for a specific gene are present. Homozygosity: When an allele inherited from a sire is the same as the one inherited from the dam. Inbreeding Depression: A reduction in fitness due to the mating of closely related and often genetically similar individuals; creating a loss of robustness due to expression of deleterious recessive alleles. This may result in an inability to respond to selection from lack of residual heterozygosity. Inbreeding: The mating of highly related individuals such as father to daughter or brother to sister. An inbred individual or population often shows increased homozygosity. Progeny may exhibit a reduction in desired genes or loss of fitness. Linebreeding: Planned mating to maintain a high degree of relation to a common ancestor or ancestral group of high regard. The intent is to avoid a reduction in desired genes or loss of fitness. A linebred individual has at least one common ancestor on the sire and dam side within 3-5 generations. Meiosis: A type of cell division which reduces chromosome numbers by half. Two successive nuclear divisions occur, Meiosis I (Reduction - ploidy level from 2n to n) and Meiosis II (Division - divides the remaining set of chromosomes). Meiosis produces 4 haploid cells, while Mitosis produces 2 diploid cells. http://www2.estrellamountain.edu/faculty/farabee/biobk/biobookmeiosis.html#meiosis Mutation: A mutation is a permanent change in the DNA sequence of a gene. Mutations in a gene's DNA sequence can alter the amino acid sequence of the protein encoded by the gene. http://learn.genetics.utah.edu/archive/mutations Nonadditive Gene Interaction: Non-additive gene interactions include the effects of dominance of one allele over another and epistatic effects of one gene (or gene pair) on another, non-allelic gene (or gene pair). Outcrossing: The mating of unrelated or distantly related individuals. This results in increased heterozygosity. QTL: Quantitative Trait Loci Section of DNA (the locus) correlating to variation in a phenotype. Statistical analysis is then used identify linkages with genes controlling phenotype. Acknowledgments Richard Squire, Ph. D. (Genetics), Retired Full Professor of Biology, University of Puerto Rico, Mayaguez Campus. For his suggestions, technical edits and proof reading of this publication. Carl Groenewegen, Guppy Gene Collector & Breeder, Liberty Twnshp., Ohio, USA For his insights as a breeder, edits and creation of a supporting PowerPoint presentation. Claus Osche, Guppy Breeder & Gene Researcher, Altenburg, Thuringia, Germany For his insights as a breeder. Yuji Yamaguti, Guppy Breeder & Gene Researcher, São Paulo, Brazil For his insights as a breeder. Notes A: The term Domestic Guppy in this article shall collectively refer to any and all captive bred strains, regardless of genetic inputs from variant populations of P. reticulata, P. reticulata (wingei), P. reticulata (obscura). B: The terms Variant Populations or Variant in this article shall collectively refer to any and all described species of Guppy; P. reticulata, P. reticulata (wingei), P. reticulata (obscura), P. reticulata (guppii). C: The term Guppy in this article shall collectively refer to any and all captive bred or wild-type populations of P. reticulata, P. reticulata (wingei), P. reticulata (obscura). Page18

References 1. Dwzillo (1959); Genetische Untersuchungen an domestizierten Stammen von Lebistes reticulatus (Peters). Mitt. Haburg. Zool. Mus. Inst., pgs 143-186. (European Blau & Silver) 2. Grasseni, C (2004); Skilled vision. An apprenticeship in breeding Aesthetics, Social Anthropology (2004), 12, 1, pgs. 41-55. 3. Grasseni, C (2005); Designer Cow: The Practice of Cattle Breeding Between Skill and Standardization, Society & Animals, 13:1. 4. Goodrich (1944); The cellular expression and genetics of two new genes in Lebistes Reticulatus. Genetics 29: 584, Nov. 1944. (Blond & Golden). 5. Haskins & Druzba (1938); Note on Anomalous Inheritance of Sex-Linked Color Factors in the Guppyi. The American Naturalist, Vol. 72, No. 743 (Nov. - Dec., 1938), pp. 571-574. 6. Haskins & Haskins (1948); Albinism, A semi-lethal Autosomal Mutation in Lebistes Reticulatus. Heredity 2:251-262. (Albino). 7. Kempkes (2007); New colour genes in the guppy, Poecilia reticulate (Peters, 1850). Bulletin of Fish Biology, Vol. 9, Nos. 1-2, pgs. 93-97. (Metallicus / Stoerzbach). 8. King, R (1975); Handbook of Genetics: Volume 4 Vertebrates of Genetic Interest. 9. Klee (1964); Non Sex-Linked Factors In The Body Coloration Of The Guppy. IFGA Extracts, Pg 136, Vol I. 10. Khoo (1999); Linkage Analysis and Mapping of Three Sex-Linked Color Pattern Genes in the Guppy, Poecilia reticulate. Zoological Science 16: 893-903 (1999). 11. Kottler (2013); Pigment Pattern Formation in the Guppy, Poecilia reticulate, Involves the Kta and Csf1ra Receptor Tyrosine Kinases. Genetics Society of America, doi: 10.1534/genetics.113.15 1738. 12. Kottler (2014); Multiple Pigment Cell Types Contribute to the Black, Blue, and Orange Ornaments of Male Guppies (Poecilia reticulata), PLoS ONE 9(1): e85647. doi:10.1371/journal.pone.0085647 13. Phang (1999); Interaction between the Autosomal Recessive bar Gene and the Y-linked Snakeskin Body (Ssb) Pattern Gene in the Guppy, Poecilia Reticulata, Zoological Science 16: 905-908. (Bar). 14. Schröder (1983); The guppy (Poecilia reticulate PETERS) as a model for evolutionary studies in genetics, behavior, and ecology. Ber. Nat.-med.Verein Innsbruck, Band 70, pgs. 249-279. 15. Schreitmüller, V. (1932); Lebistes reticulatus (Peters) var. Fredlini (Schreitm.). Akvariet, 6: 118 119. (Fredlini). 16. Schreitmüller, W. (1933); Neuimporte und anderes. Hyphessobrycon bifasciatus Ellis, Cichlosoma cutteri Fowl., Colossomaspecies, Hyphessobrycon species I und II und Lebistes reticulatus (Pet.) var. fredlini (Schreitm.). Wochenschrift für Aquarien- und Terrarienkunde, 30: 145 149. 17. Schreitmuller, W. (1934); Der Goldguppy und ein Totalalbino von Xiphophorus hellerii Heckel. Wochenschrift für Aquarien- und Terrarienkunde, 31: 242 243. 18. Winge (1927); The Location of Eighteen Genes In Lebistes Reticulatus. Journal of Gen. XVIII. (Zebrinus). 19. Arnheim, R. (1969), Visual thinking. Berkeley; Univ of CA Press. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~ Page19