Yohei Terai’s research while affiliated with The Graduate University for Advanced Studies, SOKENDAI and other places

Like Chinese Silkie, Indonesian Ayam Cemani exhibits fibromelanosis or dermal hyperpigmentation and possesses complex segmental duplications on chromosome 20 that involve the endothelin 3 gene, EDN3. A genomic region, DR1 of 127 kb, together with another region, DR2 of 171 kb, was duplicated by unequal crossing over, accompanied by inversion of one DR2. Quantitative PCR and copy number variation analyses on the Cemani genome sequence confirmed the duplication of EDN3. These genetic arrangements are identical in Cemani and Silkie, indicating a single origin of the genetic cause of Fm. The two DR1s harbor two distinct EDN3 haplotypes in a form of permanent heterozygosity, although they remain allelic in the ancestral Red Jungle Fowl population and some domesticated chicken breeds, with their allelic divergence time being as recent as 0.3 million years ago. In Cemani and Silkie breeds, artificial selection favoring the Fm phenotype has left an unambiguous record for selective sweep that extends in both directions from tandemly duplicated EDN3 loci. This highly homozygous tract is different in length between Cemani and Silkie, reflecting their distinct breeding histories. It is estimated that the Fm phenotype came into existence at least 6600–9100 years ago, prior to domestication of Cemani and Silkie, and that throughout domestication there has been intense artificial selection with strength s > 50% in each breed.

Nucleotide diversity between Cemani and Silkie based on NGS data. Bars with R1–R9 indicate the positions of the nine regions. Green square parentheses indicate the position of EDN3, and a red bar indicates the 71.4-kb region with low divergence between the two breeds. (TIF)

Characteristic morphological traits of several Indonesian chickens and Chinese Silkie. (a) Female Cemani, (b)—(d) female Kedu, (e)—(i) male Kedu, (j) male white Silkie and (k) male black Silkie. (TIF)

Segregating sites in EDN3 haplotypes. (PDF)

Sequences of primers used in this study. (PDF)

Results of the HKA test in each of the nine regions of Cemani (a), Silkie (b), and other domesticated chickens (c). The significant reduction in DNA polymorphism is found in Cemani and Silkie only for DDX27 in region 3, EDN3 in region 4, and TH1L in region 5 are compared. (TIF)

Expected heterozygosity at individual SNP sites in the nine regions in chicken breeds. (a) Domesticated chickens, RJF, and GJF, (b) Ayam Cemani, (c) Silkie chicken. (TIF)

Sequence information for duplication boundaries generated by the A2 and B2 primer sets. The A2 and B2 sequences of Cemani (CM6_A2 and CM6_B2) are identical to those of Silkie (SIB17_A2 and SIB17_B2). The boundary was determined by comparison between A1 (CM31_A1) and A2 (upper panel), and between B1 (CM6_B1) and B2 (lower panel). (TIF)

Reaction mixtures and PCR conditions used in this study. (PDF)

Variable sites in the 71.4-kb region in Cemani, Silkie and Taiwanese L2 as compared to the reference genome. The region ranges from nt 11,183,600 to 11,255,000 and includes part of DR1. Insertions and deletions are excluded. The colored columns indicate the Silkie (green)- or Cemani (red)-specific mutations. (PDF)