Genetic variation at hair length candidate genes in elephants and the extinct woolly mammoth

Background Like humans, the living elephants are unusual among mammals in being sparsely covered with hair. Relative to extant elephants, the extinct woolly mammoth, Mammuthus primigenius, had a dense hair cover and extremely long hair, which likely were adaptations to its subarctic habitat. The fibroblast growth factor 5 (FGF5) gene affects hair length in a diverse set of mammalian species. Mutations in FGF5 lead to recessive long hair phenotypes in mice, dogs, and cats; and the gene has been implicated in hair length variation in rabbits. Thus, FGF5 represents a leading candidate gene for the phenotypic differences in hair length notable between extant elephants and the woolly mammoth. We therefore sequenced the three exons (except for the 3' UTR) and a portion of the promoter of FGF5 from the living elephantid species (Asian, African savanna and African forest elephants) and, using protocols for ancient DNA, from a woolly mammoth. Results Between the extant elephants and the mammoth, two single base substitutions were observed in FGF5, neither of which alters the amino acid sequence. Modeling of the protein structure suggests that the elephantid proteins fold similarly to the human FGF5 protein. Bioinformatics analyses and DNA sequencing of another locus that has been implicated in hair cover in humans, type I hair keratin pseudogene (KRTHAP1), also yielded negative results. Interestingly, KRTHAP1 is a pseudogene in elephantids as in humans (although fully functional in non-human primates). Conclusion The data suggest that the coding sequence of the FGF5 gene is not the critical determinant of hair length differences among elephantids. The results are discussed in the context of hairlessness among mammals and in terms of the potential impact of large body size, subarctic conditions, and an aquatic ancestor on hair cover in the Proboscidea.

into a clean laboratory area. Preventative measures were taken to reduce the introduction of modern DNA to this area by wearing protective gear: a Tyvek™ suit (Kepler), hair net, face mask, an extra pair of Tyvek™ sleeves (Kepler), inner pair of nitrile gloves, outer pair of latex gloves, and safety glasses. In addition, once suited up, each analyst entered an air shower designed to remove any residual surface particulates from the external surface of the Tyvek™ suit before entering the Clean Lab.
The bone and tooth sample were surface sterilized with successive washes of 10% bleach, sterilized water, and 70% ethanol (Fisher). A Dremel™ tool was used with separate bits to cut a small portion of sample. These samples were separately milled into a fine powder using a Retsch mixer mill. Approximately 200 mg of powder was placed into 2.0 mL tubes. The extraction buffer was prepared using a modified protocol [5]: 1.5 mL of 0.5M EDTA (Sigma), 75 µL of 20% N-lauroylsarcosine (Sigma), and 40 µL of Proteinase K (Qiagen). A blank tube was also prepared containing extraction buffer only to act as a negative control. The tubes were placed onto an Eppendorf Thermomixer-R to incubate overnight at 56°C and 1000 rpm.
Next, the sample tubes were centrifuged for 5 minutes at 13,000 rpm and the aqueous extract transferred to a sterile 15 mL tube; 400 µL of phenol (Sigma) and 400 µL of chloroform:isoamyl alcohol (24:1, v/v) (Sigma) were added directly to each 15 mL tube. The tubes were vortexed for 1 minute then centrifuged for 5 minutes at maximum speed. The extract in each tube separated into two layers. The top layer was transferred with a pipette into a sterile 15 mL tube and the above process repeated with this layer.
The bottom layer was discarded. After transferring the top layer to another sterile 15 mL tube for the second time, 800 µL of chloroform:isoamyl alcohol (24:1, v/v) was added.
The tubes were vortexed for 1 minute then centrifuged for 5 minutes at maximum speed.
The top layer was transferred to another sterile 15 mL tube. To each tube was added a 10% volume of 3M sodium acetate (Sigma) and 2.5 times the volume of cold anhydrous ethanol (Commercial Alcohols). These tubes were placed into a -20°C freezer overnight to allow precipitation of product. The next day, the tubes were centrifuged for 5 minutes at maximum speed. The supernatant was discarded. A volume of 1.5 mL of 95% ethanol (Commercial Alcohols) was added to each tube, the tubes vortexed to resuspend the precipitate and centrifuged for 10 minutes at maximum speed. The supernatant was discarded and the precipitate allowed to dry for 1 hour. After this time the precipitate was resuspended in 100 µL TE buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA) and purified additionally with Micro Bio-Spin 30 Chromatography Columns (Biorad).

Indigirka mammoth mtDNA clade
Recent genetic analysis of woolly mammoth populations suggests that there are two distinct mtDNA clades of mammoths with "Clade II" having become extinct much earlier than "Clade I" or the species as a whole. Whether Clade I and Clade II mammoths differ in phenotype is not clear. A 100 bp fragment of the mitochondrial DNA 16S rDNA was sequenced, which indicated that the Indigirka mammoth sample belonged to Clade I, the more common mtDNA clade of woolly mammoth [6].

Jarkov mammoth, exon 2 results
While the complete FGF5 coding sequence was obtained for an Indigirka mammoth, few FGF5 fragments could be retrieved from the Jarkov mammoth. All FGF5 fragments retrieved were identical in sequence between the Indigirka mammoth and the Jarkov mammoth, with the exception of exon 2 amplicons from the Jarkov mammoth, which may have been due to contamination. Exon 2 was amplified in two fragments. The first fragment was identical between all elephants and mammoth samples sequenced and differed from human at two positions. A second exon 2 fragment in the Jarkov mammoth was identical to human but differed from the elephants and the Indigirka mammoth at two positions. It seems likely that the second amplified fragment from the Jarkov mammoth represents contamination rather than variation among mammoth sequences. The difficulty in distinguishing between the alternatives is that exon 2 only differs between elephantids and human at four positions. The controls were negative and the same sequence was obtained from the Jarkov mammoth in all clones of two independent PCR reactions. This does not exclude the possibility that human contamination of the sample itself yielded the result. However, the other amplifications were identical to the Indigirka mammoth suggesting that contamination with human DNA was not present (Additional Figure S2). In addition, primers used to amplify mammoth sequences were designed based on elephant sequences which are quite divergent at the DNA level from human and thus not expected to co-amplify. The Jarkov mammoth failed to yield products for the majority of the PCR reactions successful with the Indigirka mammoth. Thus, it remains unclear whether there is among mammoth diversity in the FGF5 gene. Given the complete sequencing from the Indigirka mammoth, and the limited success for the majority of the PCRs from the Jarkov mammoth, further analysis was restricted to the Indigirka sequence.

Promoter and 5'UTR
Primer names include direction (F-forward, R-reverse) and approximate location on exon; L and R are 5' and 3' to the exon Parentheses indicate estimated Tm for primer, and lower case x indicates design issue as indicated by Primer3: http://frodo.wi.mit.edu/