www.nature.com/scientificreports OPEN Skin transcriptome profling of Changthangi goats highlights the relevance of genes involved in Pashmina production Sonika Ahlawat1*, Reena Arora1, Rekha Sharma1, Upasna Sharma1, Mandeep Kaur1, Ashish Kumar1, Karan Veer Singh1, Manoj Kumar Singh2 & Ramesh Kumar Vijh1 Pashmina, the world’s fnest natural fber is derived from secondary hair follicles of Changthangi goats which are domesticated in Ladakh region of Jammu and Kashmir by nomadic pastoralists. Complex epithelial-mesenchymal interactions involving numerous signal molecules and signaling pathways govern hair follicle morphogenesis and mitosis across diferent species. The present study involved transcriptome profling of skin from fber type Changthangi goats and meat type Barbari goats to unravel gene networks and metabolic pathways that might contribute to Pashmina development. In Changthangi goats, 525 genes were expressed at signifcantly higher levels and 54 at signifcantly lower levels with fold change >2 (padj < 0.05). Functional annotation and enrichment analysis identifed signifcantly enriched pathways to be formation of the cornifed envelope, keratinization and developmental biology. Expression of genes for keratins (KRTs) and keratin-associated proteins (KRTAPs) was observed to be much higher in Changthangi goats. A host of transcriptional regulator genes for hair follicle keratin synthesis such as GPRC5D, PADI3, HOXC13, FOXN1, LEF1 and ELF5 showed higher transcript abundance in Pashmina producing goats. Positive regulation of Wnt signaling pathway and negative regulation of Oncostatin M signaling pathway may be speculated to be important contributors to hair follicle development and hair shaft diferentiation in Changthangi goats. Tirty four registered breeds and many non-descript populations represent the diverse caprine genetic resources of India which inhabit four eco-zones viz. Temperate Himalayan, North Western, Southern Peninsular and Eastern region. Tere is great variability in Indian goats with regard to many phenotypic traits such as color, size, reproductive and productive parameters. Additionally, on the basis of utility, they are classifed into milk, meat and fber type breeds (www.nbagr.res.in). An important breed of extremely cold Temperate Himalayan region of India is Changthangi which is a source of world’s most luxurious natural fber, Pashmina/Cashmere. Hence, these goats are also referred to as Pashmina or Cashmere goats. Te ability of this breed to adapt to high altitude hypoxic conditions has enabled it to thrive in difcult terrains of Changthang region of Ladakh and hilly tract of Leh. In contrast, Barbari is a breed of North Western region, which is primarily reared for milk and meat purpose. Tese animals are small sized, short-haired and white coloured with small light brown patches1. Pashmina is considered the world’s fnest natural fber and is obtained from undercoat of goats native to Asia. Indian Pashmina is mainly derived from Changthangi goats which are domesticated in Ladakh region of Jammu and Kashmir by nomadic pastoralists (Changpas) and is a prominent symbol of their cultural heritage. It is also known as Cashmere, Kashmir and pashm in India (http://jksheephusbandrykashmir.nic.in). Te other Cashmere producing countries are China, Mongolia, Iran, Afghanistan, Pakistan and Nepal. Owing to its warmth, fneness, acclaimed aesthetic value and lusture, it has rightly been called “sof gold” or “the king of fbers”. Te premium price it fetches is understandable in light of its greater sofness than even superfne merino of the same diameter2. Cashmere is derived from secondary hair follicles of goats which undergo cyclic variation as a result of complex epithelial-mesenchymal interactions. Tese physiological processes involve interactions of numerous signal mol- ecules and signaling pathways which govern hair follicle morphogenesis and mitosis3. 1ICAR-National Bureau of Animal Genetic Resources, Karnal, India. 2ICAR-Central Institute for Research on Goats, Mathura, India. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:6050 | https://doi.org/10.1038/s41598-020-63023-6 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Percent of Percent Number Number of raw Processed high quality aligned of genes Sample details reads reads data reads expressed Barbari1 42,180,760 40,871,412 96.90 94.33 17,891 Barbari2 44,027,632 42,757,303 97.11 94.41 18,089 Barbari3 43,653,637 42,326,495 96.96 93.24 17,923 Barbari4 46,000,800 44,554,407 96.86 94.22 17,870 Changthangi1 52,572,185 50,994,026 97.00 95.85 18,756 Changthangi2 51,395,618 49,913,541 97.12 94.35 17,721 Changthangi3 55,521,228 53,856,077 97.00 95.14 18,461 Changthangi4 53,981,415 51,928,597 96.20 94.42 19,521 Table 1. Summary of read statistics of 8 libraries from Barbari and Changthangi breeds. Transcriptomic profling using the Illumina high-throughput sequencing platform has opened new vistas for unraveling of global gene expression and annotation of whole transcriptomes underpinning phenotypic and physiological variability. Such advances in genome research have facilitated an improved understanding of sys- temic gene expression and ensuing regulatory mechanisms in several species4. Over the last fve years, Cashmere goats have become focus of intense study and there have been attempts to identify molecular mechanisms gov- erning hair follicle morphogenesis5, hair follicle cycling under natural and shortened photoperiod conditions4, and also, delineating the gene networks controlling the coat color in these goats6. However, to the best our knowl- edge, there are no reports on comparative transcriptome profling of skin of goats inhabiting contrasting climatic regions and difering in terms of utility, particularly from the Indian subcontinent. Terefore, the present study was planned to elucidate complete repertoire of transcripts expressed in the skin of fber type Changthangi goats from cold desert region of India and compare it with meat type Barbari goats from hot arid regions of the coun- try. Novel information from the caprine skin transcriptomes can contribute to elucidation of genetic networks determining adaptation to divergent agro-ecological zones and diferences in the quality of the fber produced by the two breeds under study. Results In order to quantify the gene expression patterns of goat skin samples, cDNA libraries were constructed from 4 animals each of Changthangi and Barbari breeds and these libraries were subjected to deep sequencing using Illumina HiSeq platform. Summary of RNA Seq data. Te number of raw reads and processed reads varied from 42.1 to 55.5 mil- lion and 40.8 to 53.8 million, respectively for diferent samples. Mapping with the Capra hircus reference assem- bly ARS1 yielded 93.24 to 95.85% aligned reads, suggesting good quality of RNA-seq data for further analysis. Similarity in the percentage of mapped reads eliminated any sequencing biases in the dataset generated. Detailed results are presented in Table 1. Functional enrichment analysis. Based on annotation of the Bos taurus genome, the top 30 genes with highest expression in both breeds were linked to biological functions such as cellular macromolecule metabolic process, cellular biosynthetic process, skin development, intermediate flament cytoskeleton organization, hair follicle morphogenesis and epidermis morphogenesis. A total of 1147 genes were expressed at signifcantly higher levels and 948 at signifcantly lower levels in Changthangi goats (padj < 0.05) as compared to Barbari goats. Gene Ontology enrichment analysis was performed for the diferentially expressed genes (DEGs). Top 5 enriched func- tional categories included signal, coiled coil, intermediate flament, cytoskeleton, keratin and diferentiation. Classifcation of the DEGs into biological process (BP), cellular components (CC) and molecular function (MF) is detailed in Fig. 1. On the basis of KEGG enrichment analysis, the DEGs were observed to be involved in several pathways which include protein processing in endoplasmic reticulum, transcriptional misregulation in cancer, ribosome biogene- sis in eukaryotes, adherens junction, FoxO signaling pathway, AMPK signaling pathway and extracellular matrix receptor interaction. Genes related to adherens junction were ACTB, PTPRJ, CREBBP, CSNK2B, LEF1, SMAD3, CTNND1, ACTN1, NECTIN4, TCF7L1, VCL, IGF1R, SORBS1, FYN and SSX2IP. Genes representing extra- cellular matrix receptor interaction pathway included ITGA1, HSPG2, VWF, ITGA9, CD47, LAMC3, LAMA5, ITGB7, COMP, ITGB6, SV2B, THBS1, COL11A1, FN1 and SPP1. Amongst the DEGs, 524 genes with higher expression and 53 genes with lower expression with a fold change >2, were considered for further analysis. For the highly expressed genes, a total of 14 annotation clusters were identifed (enrichment score of >0.5 and padj < 0.05). Te most enriched clusters in decreasing order of enrich- ment score were structural molecule activity, cornifed envelope, diferentiation, serine-type endopeptidase inhibitor activity and cell adhesion. Te most prominent clusters for the genes with lower expression were trans- membrane helix, immunoglobulin subtype, signal peptide and calcium ion binding. Signifcant Gene Ontology terms for the genes with higher expression in Changthangi goats included cell diferentiation, multicellular organism development, keratinocyte diferentiation, hair follicle morphogenesis and establishment of skin bar- rier. Most of these genes were components of nucleus, cytoplasm, extracellular