Origin, Phenotypes, and Plumage Coloration of Golden Pelung Chicken Progenies (G. gallus, Linn.1758)

Pelung chicken has extensively been studied through selective breeding and used by the local poultry sector for ornamental purposes and occasionally as meat-type chicken. However, a well-documented and detailed description of its origins, genealogical backgrounds, unique traits, and diagnostic genotyping of its unique plumage colouration has never been compiled. Therefore, this study aimed to provide a detailed description of Pelung chicken and conduct a diagnostic genotyping of the TYR gene associated with golden plumage colouration accompanied with direct visual observations in Pelung chicken.


Animals and rearing systems
The study was conducted at Berbah, Sleman, DI Yogyakarta, Indonesia. Berbah is located between latitude 7°47'45.1"S and longitude 110°27'55.0"E at the elevation of 489 m above sea level. Animals reared and maintained under the strict regulation of the Animal Welfare Act of Indonesia and all procedures involving the handling of animals were according to the ethics and biosecurity guidelines approved by the Institution of Animal Care and Use Committee, DI Yogyakarta, Indonesia.
Parentals and progenies respectively consisted of the golden Pelung chickens (F 1 Kamper) and inbred golden Pelung chickens (GK). The grandparent stock of Pelung chicken was purchased from a local breeder located in Cianjur, West Java, Indonesia. Local breeders specializing in Pelung chicken breeding have a strict, rigid, and consistent record of the breeding programmes directly under the supervision of HIPPAPI, Indonesia (hippapi.or.id). Under a semi-intensive rearing system, F 1 Kamper hens and roosters were mated in a ratio of two to one, respectively. Both parentals and progenies reared under a semiintensive rearing system with an ad-libitum standard feed diet (PT. Japfa Comfeed, Indonesia) of AD-II and BR-1, respectively. Parentals of each breeding group were fed with ad-libitum AD-II (15% Crude Protein) with the administration of the vaccine and prophylactic medications to ensure the optimal health of chickens. Progenies or DOCs of each breeding group were reared intensively in insulated bamboo pens. DOCs were fed ad-libitum BR-1 (22% Crude Protein, 3,050 Kcal ME/kg). Four-wk-old chickens of each breeding group were then transferred into the larger shed (8m 2 ) under a semi-intensive rearing system with an ad-libitum BR-1 diet for eight weeks.
Direct visual observations, phlebotomy, and DNA isolation Direct visual observations were conducted to characterize the shank colour and plumage colour of parentals and progenies. Progenies and parentals went through the screening phase and were then selected for molecular analysis. A total of thirty selected progenies and ten selected parentals whole blood samples were venipuncture with sterilized syringes (1 mL) via brachial wing vein and stored in EDTA-ready-tube vacutainers (1.5-3 mL). The brachial wing vein is located between the biceps and triceps muscle on the underside of the wing [36]. Whole genomic DNA was isolated through the chelex method [17] from each blood sample. In total, 10 μL of blood sample mixed along with 1 mL of TE buffer and centrifuged at 13,000 rpm for 3 min. The supernatant was removed and 200 μL of 5% chelex solution along with 18 μL of 0.05 M DTT, and 2 μL of 10 mg/mL proteinase K were added to the mixture. The homogenization using vortex followed by incubation at 56 °C for one hour, accompanied by stages of homogenization with 15 min intervals. The sample was further incubated at 100 °C for 8 min and centrifuged at 13,000 rpm for 3 min. The supernatant containing DNA isolates transferred to 1.5 mL microtube DNA was later preserved with the addition of TE buffer (pH 8.0) and stored in the freezer at -20°C for further use.
Primer designs for diagnostic genotyping using TP-PCR Primer designs followed the results reported by [5] with three primer sets based on 1956 bp linear mRNA chicken (G. gallus) TYR gene coding sequences acquired from NCBI GenBank with the sequence accession number D88349. Designed primers as follow; the upstream primer Diag05-cc-up (5′-CCT CTG GCT CTA TTT GAG TAC ACA GT-3′) located in the retroviral sequence, the upstream primer Diag05-nor-up (5′-CAA AAC CAT AAA TAG GAG TGG AAA TAG-3′) located in the normal sequence of intron 4, and the primary downstream Diagnostic05-dw (5′-TTG AGA TAG TGG AGG TCT GAA ATG-3′) located in exon 5 of chicken TYR gene. Primers were produced by Integrated DNA Technologies (IDT, Malaysia) with thirdparty associate Perseroan Terbatas (PT) Genetika Science Indonesia. The expected ampli ed products were 481 bp between Diag05-nor-up and Diagnostic05-dw and 345 bp between Diag05-cc-up and Diagnostic05-dw.

Direct visual observations of golden Pelung chicken progenies
The selective breeding programmes for golden Pelung chicken are depicted in (Fig. 1). Close inbreeding crossings of the golden Pelung chickens (F 1 Kamper) produced inbred golden Pelung chickens (GK).
Unlike the quantitative nature of polygenic characteristics, qualitative properties are controlled by one or more interrelated genes. Phenotype trait observation of shank colours and plumage colours on inbred progenies indicated a multigenic expression governing the melanogenesis activity displayed as colour variants (Table 1).   Fig. 2 B , the diagnostic genotyping of GK chickens revealed the homozygous dominant (17 samples; C*N/C*N) with single-band fragment 481 bp, the heterozygous (9 samples; C*N/C*C) with dual-band fragment 481 bp and 345 bp, and recessive white phenotype (4 samples; C*C/C*C) with single-band fragment 345 bp. In Fig. 3, the DOCs of GK chickens show early plumage variation, despite future alteration during its lifespan. As described in Fig. 3 A , the wild-type individual possesses normal intron 4 while in Fig. 3 B , the recessive white individual possesses retroviral sequence insertion, with pigmented eyes.

Discussion
Fowls, its origins, and genealogical backgrounds The earliest documentation about Pelung chicken was reported in a study by [21] about the matriarchy ancestor of all domesticated chicken breeds. Based on restriction digest analysis and 400 bases nucleotide sequencing, [21] reported that subspecies of the Thailand RJF (G. g. gallus) as the ancestor of all domestic breeds. Further, based on mtDNA sequences of the D-loop regions of G. gallus, G. g. gallus, G. g. spadiceus, G. g. bankiva, G. lafayettei, G. sonneratii, and G. g. domesticus, [22] solidi ed G. g. gallus as the sole matriarchic origin of all the domestic chicken breeds. Findings suggest that Javanese fowl (G. g. bankiva) contributed to the domestication event and formed a distinct entity in the phylogenetic tree, while G. g. gallus and G. g. spadiceus formed a single monophyletic cluster [21,22]. Four chicken breed lineages (laying-type, game, meat-type, and Bantam), the laying-type of Mediterranean roots, and/or true Bantams were the earliest chicken breeds with the closest similarity with G. gallus [53].
A report of South American chicken breeds also found that G. gallus was the ancestor of Brazilian ghting roosters (G. g. domesticus) [68]. Researchers have always considered that the sport of cock ghting had tremendous in uence not only on the domestication of the chicken but also the fowl dispersal rate and reach throughout the world. The agricultural innovations in East Asia around the early Holocene were the major reason behind chicken domestication and were followed by Neolithic poultry husbandry of chicken along with other species of domesticated animal [65]. The idea that habitat preference and historical, ritual, and leisure activities of ancient man might have resulted in Jungle Fowl being recruited for domestication [12]. In Indonesia, RJF domestication is indirectly affected by human community involvement either by purchasing or hunting in the forest and plantations [77].
The history of chicken domestication is presumed to have occurred more than once throughout Southeast Asia. Some evidence leads to the conclusion that India [33,16,81,65,12] is one of the birthplaces for present days chicken breeds. Four species of genus Gallus inhabit Southeast Asia: RJF (G. g. gallus), La Fayette's Jungle Fowl (G. lafayettei), Grey Jungle Fowl (G. sonnerati), and Green Jungle Fowl (G. varius) [75]. Although domesticated chicken is closely related to wild RJF, evidence suggests that endangered southern India's G. sonnerati [67] also plays an important role in the ancestral tree of domesticated chickens. Through MCMC simulation [75] it was discovered that domesticated chickens divaricate from RJF around 58,000 (±16,000) years ago, with G. varius as their shared common ancestor.
The polyphyletic or hybrid origin of the domestic chicken was proven by the identi cation of yellow skin genes [15,16], which indicated the partly involvement of G. sonneratii.
Introgression and arti cial selection affected the homozygosity, phenotypes, and shared haplotypes between domesticated chickens and Jungle Fowl [75]. The possible involvement of Indian Jungle Fowl or G. g. murghi was also discovered [75]. The Bangladesh Native Chicken is closely related to G. g. murghi, G. g. bankiva, and G. g. gallus thus proves the dispersal of domesticated chickens throughout Southeast Asia and India [33]. India was the original platform for the worldwide dispersal of chicken [16]. On the contrary, [59] identi ed a substantial variation of MHC B-locus of RJF and none of its haplotypes were found in a large sample of commercial and heritage chicken breeds. This phenomenon leading to a conclusion that the RJF population in Vietnam where the study was conducted may not directly the ancestor of domestic chickens or radical arti cial selection that has affected the MHC B-locus in domestic chickens. Based on spatial genetic diversity and population structure, RJF in its natural habitats is widely distributed but tends to form small and isolated populations with strong spatial genetic patterns that occur at both local and regional scales [59]. Jungle Fowl as adaptive species displayed seasonal breeding, well-established social hierarchy, explorative behavior, territoriality, aggression, and short-ranged ight [12].
After centuries of selection and breeding, the variation of colours, shapes, and sizes of chicken has reached around 350 combinations. As a response, an organization was established in 1873 to set the standards of excellence and establishing ways of classi cation of various chicken breeds. The phenotypic variance described in [12] is the result of high phenotypic input from worldwide dispersal and adaptation to a wide range of management and breeding regimes. In the present day, poultry farms are separated into purebred poultry and industrialized poultry. Systematic breeding schemes focused on production traits (i.e., eggs or meat), overcome the negative genetic and phenotypic relationship between reproduction and growth [16]. As the evidence suggests, RJF showed distinct microsatellite alleles distribution and a high level of genetic divergence compared with commercial chicken breeds [81]. Human intervention by comparing microsatellite variations between RJF and commercial chicken breeds [81]. The purebred poultry is mainly driven by hobby and conservation, in which purposively selected and bred fowls for their natural conformity or distinctive unique ornamental traits. On the other hand, industrialized poultry is driven by commercial purposes and science, in which purposively developed and bred meat-type or laying-type breeds. The impact of industrialized poultry is visible in the egg-meat production ratio between commercial breed and purebred RJF [16]. The classi cation of chicken by the U.S. Department of Agriculture is G. gallus, whereas others classify chicken as G. g. domesticus, a subspecies of RJF. The different terms are mainly because taxonomists and ornithologists considered chicken as the domesticated form of wild RJF, its main ancestor. The widely progressive and pivotal importance of domesticated chicken has now reached approximately 60 medium-sized breeds mostly descended from the Javanese wild subspecies of RJF (G. g. bankiva, family Phasianidae, order Galliformes).
Pelung chicken: Indonesian indigenous chicken breed Pelung or locally known as Ayam Kampong is classi ed as a meat-type chicken breed among 34 breeds of Indonesian indigenous chicken. The possibilities of introgression and arti cial selection in the selective breeding programme of Pelung chicken and the commercial breeds have been reported in several concurrent studies. Pelung chicken selection was based on the earliest reports about its potential as the candidate for Indonesian indigenous meat-type chicken breed [32,9] [23] reported its association with growth performance and body composition traits.
Besides its bodyweight performance, another unique trait of Pelung chicken is the ability to sing or produce acoustic melody [70,32,66]. Pelung chicken based on bioacoustics analysis can also be classi ed as ornamental chicken or long crow fowl. Bioacoustics among other economic-related traits have been investigated in different varieties of Indonesia indigenous chicken, for example, Gaga chicken [1] and Kokok Balenggek chicken [71]. The economic value of Pelung chicken is not only its beautiful voice [95] but also as a source of local chicken meat [32]. Appealing ornamental characteristics and relative bodyweight ratios may correlate with the conservation of indigenous chicken germplasm as an attractive factor. In the case of Pelung chicken, the bioacoustics element consists of four phases: initialcrow (tetelur), middle-crow (kukulur-kukudur), end-crow (kukulir), and closing-crow (kook) [32,95]. In Sundanese culture, crow or local term called malewung or melung means rhythmic sounds that could be heard from a long distance and an indication of the end-crow when Pelung extends their neck into a curve shape [3]. Unique morphological traits of Pelung chicken based on comb shape are classi ed into singlecomb with four different variants, including Bajing Turun (squirrels' tail-type comb), Ngabaret (tilted comb), Ngaplek (pendulous comb), and Gobed Nyarande (leaned saw comb) [3]. Based on pigmentation, mostly have yellow eyes, black beak, and black shank, while plumage colour is dominated by black [3].
Pelung chicken purebred poultry on local farms with medium-sized housing under a semi-intensive rearing system has high potential commercial bene ts. The average yearly revenue from thirty Pelung chicken farmers around Cianjur, West Java reaching 782.71 million rupiahs/yr [69]. Pelung chicken purebred poultry is highly concentrated in Warungkondang, Cianjur, West Java, Indonesia. Cianjur is located in longitude 106° 42' -107° 25' E and latitude 6° 21' -7° 25' S with an elevation range from 7 -2,962 meters above sea level [3] (Fig. 4). The average temperature in Cianjur is 24.4 °C with an annual rainfall of around 2,610 mm [3]. Cianjur, West Java is known as the natural habitat of Pelung chicken where it is rst documented. The earliest historical accounts regarding the possible origin of Pelung chicken point out several villages in Warungkondang, Cianjur, West Java, including Jambu Dipa village, Bumi Kasih village, Songgom village, and Tegal Lega village [32]. Based on local folklore around 1850 Pelung chicken has been nurtured as an animal with its distinct vocal character and strong appearance/body posture. In 1978, the local government set up a breeding center for Pelung chicken as part of preserving local chicken germplasm and cultural heritage. The rich and deeply rooted cultural philosophy behind the Pelung chicken based on sociocultural, historical events, and old literature sources are unquestionable [66]. From the economic perspective, a sustainable breeding programme, continuous funding support, and local feed supply can signi cantly improve the Pelung chicken poultry business [19]. However, these strategies have proven to be conventional and are not su cient to cope with evergrowing demands for rapid and reliable solutions. Hence, the necessity for a creative and integrative approach must be taken based on multidisciplinary scienti c innovation including molecular genetics, genetic engineering, and bioinformatics to assist the breeding programme of Pelung chicken. Inbreeding crossings were performed to increase the homozygosity of progenies, however, the risk of heterosis was also high. The inbreeding crossings produced inbred golden Pelung chickens with a wide range of phenotypic variance, including plumage colour and shank colour. Based on plumage colour and shank colour, the heterozygous state of the inbred progenies GK (Fig. 1) can still be observed as early as DOCs (Fig. 3).
The selective breeding programme has achieved numerous variances in colouration among and within individual progenies, compared to their wild ancestors as the result of human intervention to gain speci c preference or novelty [16]. The phenotypic variance can be reduced through selecting with MAS. Diagnostic genotyping of retroviral sequence insertion in the intron 4 of G. gallus TYR gene is the preliminary stage in developing a reliable and robust MAS. solely relied on phenotype preferences through visual observation, including posture, plumage similarity, bodyweight performance, and egg productivity, without any solid footprint of molecular genetics observations. However, these results indicate that golden Pelung has a unique trait quality compared with the purebred Pelung chickens. As stated in [3] a decrease in phenotypic variance speci cally the plumage colour and the absence of unique colour or pattern were observed in purebred Pelung chickens. Thus, the selective breeding programme succeeded in altering the phenotypic and genotypic variability of purebred Pelung chickens.
Despite the probability of selection errors, other highly considered factors are genetic variability and external factors, including the possibility of mutation or nutrition. The complex interplay between genetic variability and environmental factors [14] are two probable cause for the high degree of variations occurred in GK chickens. In the grandparent stock of Pelung chicken, considerable phenotypic variance appeared, and for generations, local breeders have selected the plumage colour based on their preferences. Local breeders of Pelung chicken in Cianjur, have slightly different preferences when selecting chicken especially hens, based on plumage colour, some preferred black and yellow, while others brown [3]. Genetic inheritance derived from a different state of the allelic structure in the parental generation, especially genes related to qualitative traits, together with environmental pressure (i.e., nutrition, temperature, and lighting) is known to cause phenotype alterations in the inbred progenies.
Although remain untouched, the phenomenon of epigenetic changes in wild populations is suspected to underlie certain colour patterns in domestic or laboratory animals [72]. Spatial and temporal modularization of gene expression via transcription factors or epigenetic changes is also expected to be of great importance to differently use the same genetic machinery at distinct body parts [72]. Nonetheless, the domestication has achieved phenotypic changes in present-day chickens, including external and internal morphology, physiology, development, and behavior [16].
Avian species exhibits a diversity of plumage colour among different species and uniformity of plumage colour within the same species. Plumage colour and patterns correlate with the adaptive ability and environmental awareness assist by a well-developed visual perception among avian species. Plumage colour and patterns in some ways act as the species-recognition mechanism [20], the ability in which birds belong to a certain species can differentiate between birds from similar species or outgroup birds. Sensitivity towards the environment dynamics or changes in uence the survivability rate, for example in mating rituals and behavior, and provides means of intra-or interspeci c communication. The pigmentation serves as camou age, mimicry, intraspeci c communication, protection against ultraviolet radiation, and mate attraction [16, 58]. Environmental awareness is displayed from an intraspeci c variation which highly correlates with chromatic variability and plumage colour conspicuousness [11]. Thus, the diversity of plumage colour, in this case, can affect the behavior, physical condition, and performance of chicken breeds. During the rearing under a semi-intensive system, GK chickens belong to group A were observed to have aggressive and dominant behavior than the other group (i.e., group B; C; D). The GK chicken belongs to group D were observed to be non-aggressive or more submissive and simply lack the ability of aggression due to smaller body size and agility. Thus, gradually group D started to show a decrease in health state as the result of unproportionate feed intake. As the result, during the observation, GK chickens with the same age group were divided into four different solitary pens corresponds to each phenotype group to avoid cannibalism or aggression. Overall welfares of recessive white chicken (group D, Fig. 1) showed lower health conditions than wild-type chicken, indicated by slower growth performance and smaller posture. At the observation during day old period, the DOCs of group D had indicated a similar condition. Despite these observation results, group D chickens were still able to reproduce and grow normally, furthermore, recessive white allele appeared to have not directly correlated with a physical condition or physiological state. Several studies only indicated a tendency for a phenotypic variance to correlate directly with the physiological state, physical condition, and genetic quality of chickens attributed to certain plumage colour, patterns, and shank colour [16,40]. The possibility of correlation between aspects of a multiple plumage ornamentation system may re ect together some aspects of individual quality, thereby functioning as a composite signal [40]. In the case of golden Pelung chickens, the plumage colour, crowing duration, volume, and rhythm have been used by breeders to select potential hens and roosters [3].
Multiple allelic interactions appeared in GK chickens ( The E locus is located on GGA11 [34] and in GK chickens the effect was not only seen in shank skin colour but also plumage colour. The Extension (E) locus is classi ed as the primary pigmentation of plumage and consists of two alleles, one being autosomal dominant E is responsible for the extension of melanic pigmentation for plumage and shank skin colours, while the autosomal recessive e is responsible for the non-extension of the black colour [38, 39,54,80,26,34,76,47]. The polyallelic Extension locus E determines the basic or zonal distribution of black eumelanin across the body of a chicken and depends on the MC1R gene [10], sex, and other interacting loci [34,76,47]. The E locus expression is sex-dependent and consists of eight alleles, including E-extended black; E R -birchen; e Whdominant wheaten; e + -wild-type; e b -brown; e bc -buttercup; e s -speckled; and e y -recessive wheat [27,76,47].
In GK chickens, genes-modi ers were observed including dark brown (Db) and golden (s). The autosomal Db locus is located on GGA1 [26, 25,47], and its mutation in chickens reduces the expression of black eumelanin and increases the expression of pheomelanin, in certain parts of the plumage. The Db phenotype is highly associated with SOX10 which regulates a shift towards pheomelanin, as displayed in altered nature the pigmentation, not in the absence or presence of pigmentation [47]. consists of a total of three alleles, including S and sex-related imperfect albinism (s Al ) [26,47]. In its mode of inheritance, the S locus is quite complex due to the strong in uence of gene-modi ers. The SLC45A2 gene on chrZ of Al locus regulates the sorting of vesicles in melanocytes and involves speci c inhibition of red pheomelanin in silver chickens [49,47]. Secondary pigmentation of plumage in GK chickens caused by the expression of the motley colour of the plumage by the autosomal mottle (mo) locus associated with the EDNRB2 gene located on GGA4 [37]. The mo locus consists of two alleles, the recessive white (mo w ) and the mottled (mo). The mottled (mo/mo) chicken from some studies appeared to express six different phenotypes based on a different combination of the gene mo with other colour genes, for example, the rare and endangered chickens expressing black-and-white Australorp, Mille eur breed chickens, and Pushkin breed [47].
The chicken TYR gene cDNA clones suggested 73% amino acids sequence similarity with human tyrosinases [52], and a six-nucleotide deletion (−∆GACTGG) at a Cu-binding site of tyrosinase cDNA sequence is speculated to be responsible for albinism in chickens [83]. The insertion of a complete avian retroviral sequence in intron 4 of the TYR gene causes aberrant transcripts lacking exon 5 and is the causal mutation for the recessive white mutation in chickens [5]. Melanin biosynthesis and types of melanin including eumelanin and pheomelanin [24] that involves in plumage, skin, and coat colouration in avian and mammalian species depend on the activity and mutation of the TYR gene [74,92,58,47].
One of the key genes that correspond to plumage colouration in chicken is the TYR gene [47], thus its potential to be implemented as the MAS is highly promising.
Diagnostic genotyping of retroviral sequence insertion in intron 4 G. gallus TYR gene found a similar result described in [5,44]. In GK chickens, the presence of different phenotype groups, particularly group D (white plumage) indicated different expressions of the TYR gene. The heterozygous state of parentals F 1 Kamper based on diagnostic genotyping con rmed the possibility of mutational inheritance into the inbred progenies. The C locus is autosomal multiallelic locus located on GGA1 and associated with TYR gene, consists of four alleles, including dominant full pigmentation wild-type C*N or C + , recessive white C*C or c due to retroviral sequence insertion, autosomal albino C*A or c a due to deletion of six nucleotides (−∆GACTGG), and the red-eye white C*RE or c re [83,5,74,47]. The day-old chicks may exhibit a lightly pigmented down hatch in homozygous carriers of the C*C mutation [5]. This explained the characters exhibited by DOCs of GK chicken (Fig. 3). The lack of pigment formation in recessive white GK chickens indicated insertion mutation of the TYR gene. In chicken breeds, a mutation in any gene in the pigment synthesis pathway can disrupt pigment formation [58].
In GK chickens, the retroviral sequence insertion mutation of the TYR gene caused recessive white progenies with pigmented eyes. The retroviral insertion causes aberrant mRNA by modifying the splicing procedure, thus affects the transcription pattern [5]. Exon 5 (Fig. 3) involves the proper positioning of the tyrosinase enzyme in the melanosomes, thus any defect may cause signi cant consequences in the biosynthesis of melanosomes. As described in [5], the recessive white mutation could affect the translation of the membrane-spanning domain due to the lack of the exon 5, which in turn disturb the melanogenesis despite the transcript being absent of any stop codon before the polyadenylation. The pigmented eyes in the recessive white chicken (Fig. 3 A-B ) can be the result of different precursor cells and pigment transfer, thus indicated the tyrosinase activity.
In Fig. 2 A , the diagnostic genotyping of F 1 Kamper shows the C*N/C*N and C*N/C*C alleles, translates into wild type with dominant full pigmentation and carrier for recessive white. Both the recessive white and albino could produce tyrosinase-like molecules that are inactive or silenced due to functional, antigenic, and electrophoretic change [83,5]. In GK chickens, the carriers produced inbred individuals with homozygous recessive white C*C/C*C and more carrier individuals with heterozygous wild type C*N/C*C. However, the number of homozygous wild-type C*N/C*N was larger than the rest. Age is also a valuable consideration regarding the ideal selection period for the expression of the TYR gene. In the observation of plumage colour and shank colour between DOCs (Fig. 3) and the eight-wk-old ( Fig. 1) GK chickens, the appearance might gradually transform, as it corresponded with the expression level of tyrosinase. The expression levels of TYR declined dramatically according to age, and expression at hatch was the highest, while the expression of MC1R gene was the highest during 28-d of age than the younger and older ages, also the expression of TYR in chickens carrying E/E and E/e alleles on MC1R loci were higher than those carrying e/e alleles from hatch to 28-d of age [43]. The obtained ndings in GK chickens con rmed that the retroviral sequence insertion in the C locus is stable and heritable in a Mendelian way, both progenies and parentals genotypes were consistent.
The molecular genetics observation con rmed the reliability of genetic-based selective parameters to screen the individuals for a future breeding programme. The problem with recessive white and carrier individuals is due to the fact it may cause inconsistency with the aim of producing golden Pelung chicken through a selective breeding programme. The effect can be traced back to the biosynthesis of pigments regulated by the C locus as the structural tyrosinase gene locus. The TYR gene together with the MC1R gene has been reported in numerous studies as the major genes involved in the plumage pigmentation of chickens [43,42,27,91,29,24,90].  47,89]. The process continues with the transcription of the TYR gene and the TYRP1 and TYRP2/DCT. The tyrosinase catalyzes the conversion of L-tyrosine or L-dopa to dopaquinone, the precursor of eumelanin and pheomelanin, which in eumelanogenesis the TYRP1 and TYRP2/DCT involves in catalyzing the dopaquinone to produce brown/black eumelanin. In pheomelanogenesis, the interaction between ASIP and MC1R reduces the cAMP levels and induces pheomelanin production using only cysteine and dopaquinone. Higher MC1R activity usually results in darker pigmentation [47]. Eumelanosomes can be affected by the I locus associated with the PMEL17 gene on GGA33 [5,47]. Besides TYR and MITF genes, [93] reported other genes, including four homeobox genes, two GSH, and TGF-β. The eumelanin pigment deposition in the chicken plumage involves the migration of melanoblast from the neural crest to the epidermis and plumage follicles, where the synthesis is gene-controlled by NUAK1 and SHH [90].
Most interspecies diversity is caused by regulatory changes affecting gene expression involved in pigment synthesis and pigmentation patterns, which vary more signi cantly between species than in the ability to produce pigments.
The future selective breeding programme The future is here as it has been proven by numerous pioneering studies in the chicken selective breeding programme around the world with a various approach such as phenomics, sequencing analysis [78, 35], transcriptome analysis [57,94], QTL mapping of complex traits [14,30,20], and genome-wide association study [62,2,30,50]. This study demonstrated the possibility of using a genetic marker in the selective breeding programme of golden Pelung chicken with the addition of conventional approaches like morphometrical analysis and phenotypes observation. The inbreeding crossings of F 1 Kamper were conducted to increase homozygosity, therefore, produce progenies with uniform phenotypic variations.
However, misconduct and errors still present, thus with genetic-based observation, it is expected to be realized in a future breeding programme. This research provides a genetic marker speci ed for MAS of plumage colour in the GK progenies.

Conclusions
Direct visual observations of GK resulted in a dominant white shank governs by two autosomal loci and one sex-linked locus. Plumage colours were divided into four variants: black-brown barred, brown-golden barred, brown, and white. Each plumage colour group governs either by both the autosomal loci and recessive sex-linked locus or only autosomal locus. The diagnostic genotyping detected the presence of intron 4 retroviral sequence insertional mutation of tyrosinase (TYR) gene in both F 1 Kamper and GK. Fulllength retroviral insertional mutation of the G. gallus TYR gene is associated with the appearance of recessive white (C*C/C*C) chickens, with pigmented eyes.
In this study, we also concluded that golden Pelung chicken was originated from inbreeding crossings between    The primers and recognition site of intron 4 retroviral sequence insertional mutation G. gallus TYR gene (NCBI GenBank with the sequence accession number D88349) based on [5]. The TP-PCR-ampli ed region corresponding to the retroviral sequence on intron 4 is indicated with the red dotted line. The DOCs of GK chickens A) wild-type and B) recessive white with pigmented eyes.