Florencia Di Rocco

The South American fauna has endured challenges such as illegal hunting, cattle expansion, climate change, and landscape fragmentation. Despite ongoing conservation efforts that aim to address these issues, genetic diversity remains overlooked. We genotyped 126 Lama guanicoe individuals from an area of 2 million km² in Patagonia and Cuyo using the 60K SNPs Axiom-Camelids, and we assessed population viability through genetic diversity, inbreeding, and population size. We then explored population connectivities through network analyses, genetic differentiation, and Bayesian approaches. Our findings reveal six genetic clusters: Cuyo, Somuncura, North Patagonia, Central Patagonia, South Patagonia, and Tierra del Fuego Island (TDFI). Cuyo and TDFI guanacos differed from others due to isolation-by-distance and geographical barriers, respectively. Cuyo exhibited the highest degree of allele conservation, but TDFI faced heightened vulnerability due to isolation and increased homozygosity. Sharp bottlenecks in Cuyo and North Patagonia coincided with the expansion of sheep farming, while bottlenecks in the other populations occurred during the Little Ice Age. North Patagonia acted as a probable contact zone, facilitating mixing of the Cuyo, Somuncura, and Central Patagonia populations. The Central and South Patagonian populations presented the highest gene flow. Thus, genomic advances help to define distinct conservation units (genetic stocks) to guide management practices and decisions.

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).

Tyrosinase, encoded by TYR gene, is an enzyme that plays a major role in mammalian pigmentation. It catalyzes the oxidation of L-dihydroxy-phenylalanine (DOPA) to DOPA quinone, a precursor of both types of melanin: eumelanin and pheomelanin. TYR is commonly known as the albino locus since mutations in this gene result in albinism in several species. However, many other TYR mutations have been found to cause diluted phenotypes, like the Himalayan or chinchilla phenotypes in mice. The llama (Lama glama) presents a wide variety of coat colors ranging from non-diluted phenotypes (eumelanic and pheomelanic), through different degrees of dilution, to white. To investigate the possible contribution of TYR gene to coat color variation in llamas, we sequenced TYR exons and their flanking regions and genotyped animals with diluted, non-diluted, and white coat, including three blue-eyed white individuals. Moreover, we analyzed mRNA expression levels in skin biopsies by qPCR. TYR coding region presented nine SNPs, of which three were non-synonymous, c.428A > G, c.859G > T, and c.1490G > T. We also identified seven polymorphisms in non-coding regions, including two microsatellites, an homopolymeric repeat, and five SNPs: one in the promoter region (c.1-26C > T), two in the 3′-UTR, and two flanking the exons. Although no complete association was found between coat color and SNPs, c.1-26C > T was partially associated to diluted phenotypes. Additionally, the frequency of the G allele from c.428A > G was significantly higher in white compared to non-diluted. Results from qPCR showed that expression levels of TYR in white llamas were significantly lower (p < 0.05) than those in diluted and non-diluted phenotypes. Screening for variation in regulatory regions of TYR did not reveal polymorphisms that explain such differences. However, data from this study showed that TYR expression levels play a role in llama pigmentation.

The llama (Lama glama) is a fiber‐producing species that presents a wide range of coat colors, among which white is one of the most important for the textile industry. However, there is little information about the molecular mechanisms that control the white phenotype in this species. In domestic mammals, a white coat is usually produced by mutations in the KIT proto‐oncogene receptor tyrosine kinase (KIT) and microphthalmia‐associated transcription factor (MITF) genes. In this work we have sequenced and described the coding regions of KIT and MITF‐M, the melanocyte‐specific isoform, and the two transcriptional variants MITF‐M(−) and MITF‐M(+). Moreover, we studied the expression of these genes in the skin of white and colored llamas. Although no variants were revealed to be associated with white coat color, significant differences between phenotypes were observed in the expression levels of KIT and MITF‐M. Interestingly, white llamas expressed less MITF‐M(+) than did colored ones, which is consistent with a consequent reduction in the synthesis of melanin. Even though our results indicate that downregulation of KIT and MITF‐M expression is involved in white phenotype production in llamas, the causative gene of white coat color remains unknown.

Summary: Llama, the most numerous domestic camelid in Argentina, has good fiber‐production ability. Although a few genes related to other productive traits have been characterized, the molecular genetic basis of fiber growth control in camelids is still poorly understood. Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that controls hair growth in humans and other mammals. Mutations in the FGF5 gene have been associated with long‐hair phenotypes in several species. Here, we sequenced the llama FGF5 gene, which consists of three exons encoding 813 bp. cDNA analysis from hair follicles revealed the expression of two FGF5 alternative spliced transcripts, in one of which exon 2 is absent. DNA variation analysis showed four polymorphisms in the coding region: a synonymous SNP (c.210A>G), a single base deletion (c.348delA), a 12‐bp insertion (c.351_352insCATATAACATAG) and a non‐sense mutation (c.499C>T). The deletion was always found together with the insertion forming a haplotype and producing a putative truncated protein of 123 amino acids. The c.499C>T mutation also leads to a premature stop codon at position 168. In both cases, critical functional domains of FGF5, including one heparin binding site, are lost. All animals analyzed were homozygous for one of the deleterious mutations or compound heterozygous for both (i.e. c.348delA, c.351_352insCATATAACATAG/c.499T). Sequencing of guanaco samples showed that the FGF5 gene encodes a full‐length 270‐amino acid protein. These results suggest that FGF5 is likely functional in short‐haired wild species and non‐functional in the domestic fiber‐producing species, the llama.