Studies on expression and function of miRNAs in evolutionarily diverse agricultural species are limited. The alpaca is a domestic mammal specialized in fiber production
 and has more than 22 natural coat colors
[40–44]. In this report, we explored the miRNA transcriptome in alpaca skin using deep sequencing technology, and provided a foundation for future functional studies on the relationship between miRNA expression and development and function of skin including coat color.
Many miRNAs have been identified by traditional Sanger sequencing and microarray methods. However, the high throughout-sequencing method which yields 400,000 reads per run and identifies low abundance tags is a much more comprehensive method for miRNA profiling
. Using this sequencing approach, we found 22 novel, as well as 272 and 267 conserved miRNAs in WA and BA skin libraries
. According to the new miRNA family classifications in Rfam (miRBase 17.0), 272 miRNAs from WA and 267 miRNAs from BA were grouped into 155 families. Most of these identified miRNA families are also conserved in other mammalian species, such as mouse
[23, 46], human
, goat and sheep
. Expression of these conserved alpaca miRNA families differed between WA and BA skin, based on both abundance and representation within miRNA families. For example, miR-146 and miR-8 families both had abundant reads in WA and BA skin libraries, but miR-146b (miR-146 family) and miR-141 (miR-8 family) were of greater abundance in WA than BA. This significant variation maybe attributed to the timing and location of miRNA expression during skin development in adult alpacas. In many miRNA families, there was a predominant member whose reads were significantly higher than other members. For example, the Let-7 miRNA family contains 10 members. The reads for let-7a were more than one million, but let-7i only had five thousand reads. It is possible that the predominant member in each miRNA was responsible for the regulatory role at the developmental stage in which skin samples were collected in the present studies.
We compared the expression of conserved miRNAs between WA and BA skin and identified differentially expressed miRNAs from the two libraries. The expression of miRNAs has an established relationship with cell proliferation and differentiation. As such, differences in miRNA expression between animals possibly reflect alterations in growth and differentiation. Heterochrony, or change in the timing of developmental growth, is known to alter the overall shape of developing tissues, leading to differences in physiology
. Alternatively, there are pronounced phenotypic differences between WA and BA which include fiber diameter, staple length, hair color and other hair characteristics
[48, 49]. Thus, differences in miRNA abundance may also be related to hair color and other fiber characteristics. The functional role of such differentially expressed miRNAs is the focus of future investigation.
In animals, miRNAs target mRNAs with partial sequence complementarity
. One miRNA is inferred to influence expression of hundreds of mRNAs via this flexible recognition. There were many differentially expressed miRNAs identified between WA and BA skin, so the next step was to search for putative specific genes targeted by these miRNAs. The major physiological difference of interest between the samples analyzed was coat color, so we focused on potential target genes regulating pigmentation. Based on KEGG pathway analysis, we found 117 potential miRNA target genes involved in melanogenesis. Many miRNAs targeting these genes have been reported. For examples, miR-137
 and miR-25
 were previously shown to regulate the expression of MITF, which is an important regulator of melanocyte growth, maturation, apoptosis and melanogenesis. In the present study, miR-137 and miR-25 were found in both libraries and their target genes also included MITF. Haflidadóttir et al. showed that miR-148 affects Mitf mRNA expression in melanoma cells through a conserved binding site in the 3’-UTR sequences of mouse and human Mitf
. In our studies, miR-148 showed greater expression in WA versus BA. So, it is possible that miR-148 may contribute to coat color differences between WA and BA.
The present studies also revealed that both miR-193a and miR-193b are differentially expressed in WA and BA skin. Gao et al. reported that Kit expression was subject to post-transcriptional regulation by miR-193a and miR-193b in human acute myeloid leukemia
[53, 54]. In humans, Kit contributes to the regulation of skin pigmentation
 and we have previously reported Kit expression is higher in skin of BA versus WA
. Thus, miR-193a and miR-193b may play a role in coat color determination in alpacas via regulating the expression of Kit.