Genetics of coat color and fiber production traits in llamas and alpacas

Abstract

• The genetic mechanisms that regulate economically important fiber traits in South American camelids are not yet fully understood, resulting in low productivity. • In recent years, mutations responsible for some monogenic or oligogenic traits have been identified, enabling molecular testing to assist breeding decisions. • The recently developed 76K SNPs array for the alpaca, will facilitate the identification of genes affecting more complex traits through genome wide association studies. The use of South American camelids by man dates to more than 10,000 years ago, when the first human populations from the region began hunting the wild species, the guanaco (Lama guanicoe), and the vicuña (Vicugna vicugna) for food (Yacobaccio and Vilá, 2013). Archaeological and genetic evidence indicates that the llama (Lama glama) and the alpaca (Vicugna pacos) were domesticated more than 5000 years ago, from L. guanicoe and V. vicugna respectively, mainly in the Peruvian Andes (Kadwell et al., 2001) although independent domestication centers in northern Argentina and Chile could have existed (Wheeler, 2012). Both the llama and the alpaca played a preponderant role in the economy and the culture of the Andean communities, which persists in the present. It is believed that llamas were first selected for meat production and their ability to carry heavy loads, while alpacas were selected for their fiber (Kadwell et al., 2001; Marín et al., 2018). Thus, physical characteristics and their diversity in these species were shaped by domestication. Nowadays, in Peru, Bolivia, and Chile, the alpaca is still primarily raised for fiber production, although its meat is also consumed. In Argentina, where alpaca population is small, llama farming is mainly intended for fiber production. However, in the northwest of that country, as in Bolivia and Peru, llamas are raised for multipurpose use providing other resources such as meat and leather. Additionally, (small populations of) alpacas and llamas can be found throughout the world, where they are bred mostly, but not only, for their fiber. Because of their intelligence and docile nature, domestic South American camelids are also good companion animals and they are used for other purposes too, like golf animal-caddy, tourist attraction or zootherapy (Marcoppido and Vila, 2013). Among all domestic South American camelids uses, fiber production is one of the most important for breeders and for the industry. There are several traits that determine the value of the fiber, being fiber mean diameter the most important, followed by fiber length, fiber uniformity, and color (Mueller et al., 2015; Frank, 2017). Other characteristics such as fleece type and the amount of fiber produced (fleece weight) also impact on the producer´s income. The elucidation of the genetic mechanisms underlying these commercially important fiber traits would help breeders to select and improve the productive characteristics, as well as to conserve and add diversity to the species. In general, traits can be classified into two types: qualitative, such as color and fleece type, and quantitative, like the amount of fiber produced or fineness (diameter). Qualitative traits are under genetic control of one or several genes and are exposed to little or no environmental influence. Conversely, quantitative traits are encoded by many genes, each one contributing a little to the phenotype and their expression is modified by the environment (Mackay, 2003). Advances have been made in the genetic understanding of fiber traits; however, this is a vast field, and a lot remains to be studied. The purpose of this review is to update the reader on the current state of knowledge of fiber genetics in domestic South American camelids and to discuss how genomics and the emergence of modern technologies for sequencing and discovering genetic variants will contribute to the advancement in this field. Llamas and alpacas have more than 22 natural colors ranging from black and brown through gray and fawn to white, including all intermediate shades. Llamas present greater color variation compared to alpacas; tricolor phenotypes may be observed and the presence of white spots is common in llamas. Additionally, this variety of colors and patterns normally occurs in the same herd, unlike alpaca’s herds that tend to be more homogeneous. The difference can be attributed to the selection process during the domestication of each species. The llama, as a multipurpose animal, was selected for greater body size and fiber weight rather than other characters, such as color uniformity or fiber fineness (Mueller et al., 2015). Nowadays, the value of a particular color phenotype depends on the production goal. For example, animals with nonuniform coat are of lesser value for fiber production but they are more in demand as pets or for animal exhibitions. White color has traditionally been preferred by the industry because it can be dyed without bleaching. Because of the pressure in favor of white alpacas, there has been a significant decrease in colored animals, especially in Peru (Hick et al., 2009), with subsequent losses in genetic diversity. Fortunately, in the last years, the demand for natural colors by the textile industry has increased along with the worldwide trend towards consumption of eco-friendly products. In this context, the vast diversity of natural colors offered by domestic South American camelids results very attractive for the current market (Kozłowski and Mackiewicz-Talarczyk, 2020). However, color inheritance in domestic South American camelids is complex and frequently leads to unexpected results for breeders. For example, the mating between two white animals, may produce a black offspring and vice versa, two black parents can have a white descendant. As color prediction is quite difficult based only on the parents’ appearance, it is necessary to better understand how color is inherited and which genes are involved. Based on homology with other species, Frank (2001) described color phenotypes and their segregation in the llama. Using a scheme of test crosses and backcrosses, he concluded that white (absence of pigmentation) is dominantly inherited with incomplete penetrance and that black is recessive with respect to the other pigmentary patterns. Similar conclusions were reached by Valbonesi et al. (2011) when analyzing color segregation in white and pigmented alpacas. However, the molecular bases of color inheritance remained to be proved. It was not until recent years that some of the genes and alleles that govern coat color in alpacas and llamas were identified. Color variation occurs when there are alterations in the process of pigmentation. Briefly, this process comprehends the development, migration, and survival of the pigment-producer cells (melanocytes) as well as the synthesis of pigments (melanogenesis) and its transport to the skin and fibers (Cieslak et al., 2011). During mammalian melanogenesis, only two types of pigments can be produced: eumelanin (black) and pheomelanin (yellow or reddish-brown). Basic coat colors are defined by the relative proportion between these two types of melanin. At molecular level, one enzyme syntheses both pigments, the tyrosinase (TYR), but the eumelanin/pheomelanin ratio is regulated primarily by the interaction of two different proteins with a receptor. This receptor is the melanocortin 1-receptor (MC1R), which is in the cellular membrane of melanocytes and has the ability to signal the cell so that melanogenesis begins. If the protein that bind to MC1R is alpha-melanocyte stimulating hormone (α-MSH), eumelanin will be produced. However, if the agouti signaling protein (ASIP) is present, it will preferably bind to MC1R, switching melanin production to pheomelanin (Figure 1) (Lu et al., 1994). Scheme of melanogenesis simplified within a melanocyte. Both types of melanin are synthetized from the amino acid L-tyrosine in a series of reactions catalyzed by TYR enzyme. Photographs at the right show animals with each kind of pigment. It is thought that ASIP presents four alleles for the alpaca (Munyard, 2011), in the following order of dominance: A = white to fawn Ab = brown with dark trims at = black and tan a = black The existence of dominant black, as occurs in other species, has been discussed in South American camelids, although there is no genetic evidence to support it. Instead, three ASIP mutations have been identified in the alpaca, c.292C>T (allele a1), c.325_381del57 (allele a2), and c.353G>A (allele a3). Black animals carry two copies of one or another of these mutations (here referred as the a allele) (Feeley et al., 2011). The first two mutations are also responsible for the same phenotype in the llama, while the third one is quite infrequent in this species (Daverio et al., 2016; Marín et al., 2018). Considering all these studies, only the a allele has been molecularly identified so far, supporting a recessive inheritance pattern for black, as originally proposed by Frank (2001) and Valbonesi et al. (2011). Black animals can be homozygous or compound heterozygous for two of the mutations, but the effect is the same: due to the mutations, ASIP protein is nonfunctional (unable to bind to MC1R), thus there is no signal for switching from eumelanin to pheomelanin synthesis, and eumelanin continues to be produced. The MC1R gene is highly variable in llamas and alpacas. Among all reported polymorphisms, a combination of them (called “haplotype”), has been associated with the ability to produce or not to produce eumelanin. In the alpaca, animals with at least one allele of MC1R with the combination c.82A/c.901C (allele E) are eumelanic, whereas animals homozygous for the combination c.82G/c.901T (e/e) are unable to produce this pigment and express pheomelanin instead (Feeley and Munyard, 2009). In the llama, there are three alleles of MC1R E, E+ and e. A particular MC1R haplotype (c.259A/c.376A/c.383T), different from that of alpacas, determines the ability to produce eumelanin. One variant of this allele (E) is sufficient to produce a colored coat, which can be black if ASIP is nonfunctional (aa) or reddish brown if ASIP is functional (AA or Aa). In contrast, if the animal is homozygous for the MC1R haplotype c.259G/c.376G/c.383C (ee), its coat will be white, regardless of the alleles present in ASIP (Daverio et al., 2016). So, for instance, a black dam and a black sire (aa), both heterozygous E/e for MC1R, are able to produce white or light fawn offspring (ee aa) when they mate. Moreover, the whole coding sequence of MC1R was studied in guanacos and the ancestral allele (wild allele, E+) was identified (Daverio et al., 2016). White llamas frequently have E+/e or E+/E+. Although not yet molecularly confirmed, we can hypothesize that those animals carry at least one copy of the dominant ASIP allele, A. Table 1 Summarizes ASIP and MC1R allele combinations and the resulting phenotypes for the llama and the alpaca. Brown refers to pheomelanic reddish-brown (See Fig. 1) Brown refers to pheomelanic reddish-brown (See Fig. 1) It can be observed from Table 1 that in some cases different phenotypes share MC1R and ASIP genotypes. This suggests other unidentified alleles (probably of ASIP) are involved. Besides variation within genes coding regions, their expression plays a role in modifying hair pigmentation in camelids. ASIP has a complex structure with noncoding exons which are alternatively transcribed into different mRNAs isoforms. Differential expression of those transcripts in distinct parts of the body results in the diverse pigmentation patterns observed in mammals. Although the expression of ASIP in relation to coat color patterns has not been analyzed in domestic South American camelids, it was studied for the solid phenotypes. ASIP expression resulted significantly higher in white llamas and alpacas compared to black ones. A chimeric transcript of ASIP containing part of the NCOA6 gene was found in the skin of white animals while it was not present in black animals, accounting for the ASIP expression differences observed between these two phenotypes. NCOA6-ASIP transcript was also highly expressed in brown llamas, at similar levels to those observed in white animals (Chandramohan et al., 2013; Anello et al., 2022). Other genes, such as KIT and MITF, which are involved in the development, migration and survival of melanocytes are obvious candidates for white color. KIT and MITF genes have been sequenced in white llamas but no mutations associated with this phenotype were identified (Anello et al., 2019a). Nonetheless, analysis of gene expression profiles in different studies showed that KIT and MITF are downregulated in white llamas, as well as other important genes of melanogenesis such as TYR and SLC7A11 (Anello et al. 2019b, 2019c). The expression of pigment-related genes has also been associated with diluted phenotypes in alpacas and llamas (Figure 2A and B). Eight genes (RAB38, SLC24A5, TYRP1, SILV, MATP, KRT4, OCA2, and TYR) were observed to be expressed in a common pattern in alpacas: high in black, moderate in bay, and low in white (Munyard, 2011). Similar results were observed for TYR and SLC7A11 in llamas (Anello et al. 2019b, 2019c) where white, diluted and nondiluted pheomelanic animals where compared. It was observed that the expression levels were the highest for reddish brown animals and the for the white while fawn llamas intermediate gene these studies no the how diluted phenotypes are they show that gene expression is with color Photographs of llamas and alpacas color phenotypes. A. llama. fawn llama llama. llama. gray llama. gray alpaca. white alpaca. alpaca. South American camelids also present a of phenotypes (Figure and including and by phenotypes like the the and 2001; Munyard, 2011). There are spots and phenotypes are which its is quite and to present a recessive This was by segregation studies by Frank et al., in llamas, observed that white is dominant This that patterns are unable to be in animals coat color is white (Munyard, 2011), the identification of the Moreover, it is not if the different phenotypes are of genes, or different alleles of the same KIT has been proposed a gene for this it is responsible for similar white phenotypes in other species (Munyard, 2011). However, no molecular studies have this in South American camelids and the responsible for remains phenotype for breeders is alpacas present gray body with especially the and from gray llamas which present a body color and a (Figure and The of the gray phenotype in alpacas has recently been identified in of the KIT gene et al., alpacas are heterozygous for the as are alpacas, supporting the by breeders that the phenotype is by a combination of the allele gray and a white allele identified et al., the other no homozygous have been found for the gray the that this et al., another is a common in domestic South American camelids that present the which is white hair coat and solid as to white animals which have pigmented (Figure and is in South American but et al., reported that more than of animals are to be based on in domestic South American camelids from This may be as by a by et al. the of llamas and alpacas and found that of animals were and have been associated to the are in with the which could be in the KIT gene or its region et al., South American camelids produce diverse types of but not all of them present the to the The traits that determine fiber are mean fiber type of fiber length, and uniformity of diameter (Mueller et al., 2015; Frank, 2017). fiber is worldwide for its fineness and fiber not as although different studies showed that most of llamas have fiber to those of the alpaca fiber to the et al., 2016; et al., 2009). However, there is a low of and industry llama fiber to The structure of the fleece of domestic camelids is by the combination of fiber length, and the presence and type of of these determine different types of In this a fleece is by two types of fibers and and fibers and the other animals have fibers with a of and not present 2001) (Figure of fleece and modified from Frank, fleece presents and is the one with the most fibers are not or while has the In general, the and the fiber the higher its the alpaca which is the llama has been traditionally a species. However, studies in llama herds found that there is a of with of and et al., to fleece characteristics, llamas are classified with very fleece and no fibers in the and and with fleece the body (Figure and et al., to alpacas, two different types of are with fiber and with fleece of fibers (Figure and et al., 2009). of llamas and alpacas: llama llama alpaca alpaca of In the fiber is a highly mainly of the the and the et al., (Figure The of the fiber and it of intermediate in a of proteins and the support for the fiber and its such as and and 1994). The of and that the fiber suggests that their relative and are of fiber characteristics Scheme of the of the fiber, in is a process with followed by This is under and of three defined and produce and fiber cells and is in a and the hair a of relative in and of to at the and it is believed that may be the that control the expression of fiber The proteins that the fiber are encoded by genes and proteins which are expressed in a highly regulated during hair et al. 2009). A genetic selection have been in domestic camelids to improve fleece characteristics, them the the et al., and the genetic and by genetics mechanisms fiber traits in llamas and alpacas are not fully It is through segregation studies that phenotype is dominant with a penetrance effect that some to show intermediate fiber Instead, is by genetic et al., The and inheritance in alpacas is described by a gene with two or alternatively by a two being dominant et al., The presence of genes affecting quantitative fiber traits such as fiber of fiber variation and in both and alpacas has been proposed by et al., based on segregation Thus, molecular identification of the genes involved in the traits would facilitate genetic There are many genes that hair them the hair and hair of the gene results in two a a et al., transcript the a that is by the during the et al., mutations in gene have been associated with phenotypes in some species, including camelids. In alpaca, fiber in different of the body is by differences in the expression of et al., 2011). Moreover, et al., that has higher expression in the skin of the alpaca compared to the The same was observed for protein the other a that a was identified in the of alpacas et al., 2018). However, analysis not effect on that a This gene was also studied in llamas for which the two transcript variants were identified. Besides four in the coding region were a a a and the same found in alpacas The haplotype for a llamas analyzed were homozygous for one of the mutations or compound heterozygous for in the presents no mutations were So, we proposed that not be functional in hair llamas while guanacos have a functional protein (Daverio et al., 2017). The fiber diameter or the fineness the most important for the textile industry. It is by the presence of the the diameter when the or is present and in its and 2020). At the same fiber uniformity is to the fiber diameter et al., The and genetic of these characteristics were analyzed for both llamas and alpacas and it was found that fiber diameter was moderate to low and there was a between variation and fiber diameter et al., et al., in and genes has been associated with fiber diameter and fleece weight in and et al., et al., 2011). studied these genes in llamas and found they were highly of the produce amino acid thus they the fiber characteristics, but it remains to be (Daverio et al., In alpacas, et al., several genes to fiber and all for found a of more genes and in which this a good to sequence variants associated with hair and In another were four of which to genes for fiber traits et al., 2020). if these studies significant it still remains to be how these genes fiber In the last significant has been made in the genes that control coat color in South American camelids. The of mutations of phenotypes for such as the gray in alpacas, has the of a test by et al., This is the first test for color genes in camelids on the but it is that will very testing presents diverse from the of a phenotype to color allele diversity. For example, to a black alpaca a of the three different alleles is thus mating could be between black animals with different to conserve the color allele Additionally, genetic testing can be to some associated to coat like a test on and alpaca breeders could animals gray alleles and have also been made in understanding the inheritance mechanisms of other fiber traits in llamas and alpacas, ranging from gene to more complex on the Moreover, significant genetic has been found in genes protein of the fiber, that genetic would be when the variants associated to traits are the there is still a of studies traits to genes in South American camelids. In this the of the in a in the of genes for fiber the identification of of SNPs and the of a The of this array in genome wide association studies in both the alpaca and the llama, will contribute to the of genes and to pigmentation and will to towards the identification of genetic variation more complex like the that fiber in from the of in is the of the at the on llama coat color genetics as well as wild genetics and and human genetics are other has from the of is of the of of at of of of are to molecular genetic and animal production and has been on the and of genes, in the identification and association of with of productive in domestic camelids, mainly in traits associated with fiber also has been involved in with coat color in llamas and vicuña Anello is a at the has a in and a in of During studied genes to pigmentation in the llama. continues in to the genetics and genomics of South American camelids. Among in and in Color (Anello et al., and expression analysis of KIT and genes in llamas and their relation to white coat (Anello et al., ASIP gene in the llama (Lama expression and relation with color (Anello et al., 2022). would like to Frank, and by providing with of llama fleece and alpacas color phenotypes. is a from the of of the of of The that they have no of

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