Identification of variation inFibromelanosisregion on chromosome 20 for determining the purity of Indonesian Cemani chicken

Abstract

Ayam Cemani is a local Indonesian chicken with heavy pigmentation in plumage colour, skin, eyes, and inner body organs. This trait with dermal hyperpigmentation is identical to Fibromelanosis (Fm) mutation in a Silkie chicken. The causal mutation of the Fm trait is due to an inverted duplication and junction of two genomic regions involving the Endothelin3 (EDN3) gene on chromosome 20. There are two duplication boundaries; one is specific to the Fm allele, the other is common for both Fm and fm+ allele. Determining birds that are homozygous or heterozygous at this locus is useful for unifying the Fm trait of Cemani populations. This study develops a method for determining the presence or absence of Fm mutation by PCR amplification using the inverted sequences specific to the Fm allele. Further, it develops the restriction fragment length polymorphism (RFLP) method in regions common to the Fm and wild-type fm+ allele. We aim to establish a simple method for detecting homozygous (Fm/Fm) and heterozygous (Fm/fm+) individuals with Fm mutation and to clarify the degree of fixation of the Fm trait in the Ayam Cemani populations and the association between the phenotype and genotype. The result showed that mostly, the phenotype for Cemani with Fm/ fm+ genotype is reddish black in their comb; meanwhile, the Cemani with (Fm/Fm) genotype showed heavy black pigmentation. Our study concluded that using the PCR-RFLP method. We can discriminate between Fm homozygous and heterozygous birds in the Cemani population. Thus, this briefly genotyping method effectively maintains and protects the pure line of Cemani chicken.

Analysis of Meat Mineral Content in Cemani Chicken with Homozygous (Fm/Fm) and Heterozygous (Fm/fm+) Genotypes

“…The amplification of Fm-specific allele was carried out by multiplex PCR technique combining three primers. Three primers used in this study followed previous study (Dharmayanthi et al 2022) on non-coding genes of Fibromelanosis region: forward primer WT_F : 5'-TTCAGCAGCATTCACTGAAGGC-'3 and reverse primer FMdupl_664_R: 5'-TTCAGCAGCATTCACTGAAGGC-'3 used to amplify 664 bp length of fm + common; forward primer WT_F: 5'-TTCAGCAGCATTCACTGAAGGC-'3 and reverse primer FMspec_588_R: 5'-TGTCCATCTCACATTGC-'3 used to amplify 588 bp length of Fm spesific. DNA samples were mixed in a total volume 15ul consisted of primer WT_F 0.4 μL; primer FMdupl_664_R 0.2 μL; primer FMspec_588_R 0.2 μL; nuclease-free water 5.7 μL; and My Taq TM HS Red Mix 7.5 μL.…”

“…In addition, a recent study discovered that the different genotypes in the Cemani population, i.e. homozygous Fm/Fm and heterozygous Fm/fm + , have differing levels of blackness (Dharmayanthi et al 2017;Dharmayanthi et al 2022). Pigmentation in poultry caused by the amount of melanin and muscle size.…”

“…The pigmentation in the internal tissues of the F1 progeny of BM was significantly lighter than that in WS, indicating that hyperpigmentation is more severe in the Fm/Fm homozygote than in the Fm/fm + heterozygote. A recent study established a polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method that for detecting homozygous (Fm/Fm) and heterozygous (Fm/fm + ) individuals with the Fm mutation in the Cemani population (Dharmayanthi et al 2017;Dharmayanthi et al 2022).…”