Late-feathering Chickens Research Articles

Identification of Duplication Genotypes of the Feathering Rate Gene in Chicken by a Multiplex PCR Following Electrophoresis and/or Sanger Sequencing

Sex-linked phenotypes of late feathering (LF) and early feathering (EF) are controlled by a pair of alleles K and k+. Autosexing based on the feathering rate is widely used in poultry production. It is reported that a tandem duplication of 176,324 base pairs linked to the K locus is responsible for LF expression and could be used as a molecular marker to detect LF chicken. So far, there is no genotyping method that can accurately and stably identify the LF homozygote and heterozygote in all chicken breeds. In the present study, a multiplex PCR test was developed to identify EF, LF homozygote, and heterozygote according to electrophoretic bands and the relative height of the peaks by Sanger sequencing. We tested 413 chickens of six native Chinese breeds with this method. The identification was consistent with the sex and phenotype records of the chickens. Band density analysis was performed, and the results supported our genotyping using the new assay. In order to further verify the accuracy of this test in distinguishing homozygote and heterozygote males, 152 LF males were mated with EF females, and the results of the offspring's phenotypes were consistent with our expectations. Our results support tandem duplication as molecular markers of LF, and this new test is applicable to all LF chickens associated with tandem duplication.

RNA sequencing reveals lncRNA-mediated non-mendelian inheritance of feather growth change in chickens

BackgroundLong non-coding RNAs (lncRNAs) play an essential role in biological processes. However, the expression patterns of lncRNAs that regulate the non-Mendelian inheritance feather phenotypes remain unknown.ObjectiveThis study aimed to compare the expression profiles of lncRNAs in the follicles of the late-feathering cocks (LC) and late-feathering hens (LH) that followed genetic rules and the early-feathering hen (EH) and early-feathering cock (EC) that did not conform to the genetic laws.MethodsWe performed RNA sequencing and investigated the differentially expressed lncRNAs (DElncRNAs) between the early- and late-feathering chickens, which function by cis-acting or participate in the competing endogenous RNA (ceRNA) network.ResultsA total of 53 upregulated and 43 downregulated lncRNAs were identified in EC vs. LC, and 58 upregulated and 109 downregulated lncRNAs were identified in EH vs. LH. The target mRNAs regulated by lncRNAs in cis were enriched in the pentose phosphate pathway, TGF-β signaling pathway and Jak-STAT signaling pathway in EC vs. LC and were associated with the TGF-β signaling pathway, Wnt signaling pathway, p53 signaling pathway and Jak-STAT signaling pathway in EH vs. LH. In addition, the lncRNA-mediated ceRNA regulatory pathways of hair follicle formation were mainly enriched in the TGF-β signaling pathway, Wnt signaling pathway, melanogenesis, and calcium signaling pathways. The levels of ENSGALG00000047626 were significantly higher in the late-feathering chickens than in the early-feathering chickens, which regulated the expression of SSTR2 by gga-miR-1649-5p.ConclusionThis study provides a novel molecular mechanism of lncRNA’s response to the feather rate that does not conform to the genetic laws in chickens.

DPRLR causes differences in immune responses between early and late feathering chickens after ALV-J infection

To understand the differences in immune responses between early feathering (EF) and late feathering (LF) chickens after infection with avian leukosis virus, subgroup J (ALV-J), we monitored the levels of prolactin, growth hormone and the immunoglobulins IgG and IgM in the serum of LF and EF chickens for 8 weeks. Moreover, we analysed the expression of immune-related genes in the spleen and the expression of PRLR, SPEF2 and dPRLR in the immune organs and DF-1 cells by qRT–PCR. The results showed that ALV-J infection affected the expression of prolactin, growth hormone, IgG and IgM in the serum. Regardless of whether LF and EF chickens were infected with ALV-J, the serum levels of the two hormones and two immunoglobulins in EF chickens were higher than those in LF chickens (P < 0.05). However, the expression of immune-related genes in the spleen of positive LF chickens was higher than that in the spleen of positive EF chickens. In the four immune organs, PRLR and SPEF2 expression was also higher in LF chickens than in EF chickens. Furthermore, the dPRLR expression of positive LF chickens was higher than that of negative LF chickens. After infection with ALV-J, the expression of PRLR in DF-1 cells significantly increased. In addition, overexpression of PRLR or dPRLR in DF-1 cells promoted replication of ALV-J. These results suggested that the susceptibility of LF chickens to ALV-J might be induced by dPRLR.

토종 실용닭의 깃털 조만성 형태가 산육능력 및 생존율에 미치는 영향

Sex identification in day-old chicks is essential in the poultry industry. Currently, the feather-sexing using a sex-linked inheritance is a chick sexing method used extensively in the poultry industry. For chicks produced by feather-sexing, all females are early-feathering (EF) chicks and all males are late-feathering (LF) chicks. Therefore, investigating the effects of EF and LF phenotypes on production traits of chickens is critical. The purpose of this study was to analyze and compare the production performance between EF and LF chickens in Korean native commercial chickens. The results showed that the survival rate of the EF chickens was significantly higher than that of the LF chickens, from hatching to 12 weeks of age (<i>P</i>&lt;0.05), with the highest difference observed in females. However, no significant difference was observed in body weight between the EF and LF chickens at almost all ages. In addition, no significant difference was observed between in feed utility, such as average daily feed intake and feed conversion ratio, between the EF and LF groups. In conclusion, although the survival rate of early-feathering chickens was superior to that of late-feathering chickens, no significant difference was observed in growth performance and feed utility among Korean native chickens. The results suggest that production capacity is not influenced by feathering type in the establishment of a breeder structure and production system for feather-sexing Korean native chickens, which is valuable for the practical application of feather-sexing in the poultry industry.

한국토종닭의 깃털 발육성 반성 유전자를 이용한 자가성감별 계통 조성

Although feather-sexing using sex-linked genes related to feather development is a widely used chick sexing method in the poultry industry, the feather-sexing method has yet to be used for Korean native chickens (KNCs). The purpose of this study was to construct a KNC feather-sexing line using early-feathering (EF) and late-feathering (LF) genes for industrial application. Using 557 reddish-brown KNCs as the basal flock, frequencies of the EF (<i>k</i>) and LF (<i>K</i>) genes were estimated to be 0.814 and 0.186, respectively. This indicating that it would be feasible to construct a feather-sexing line using this chicken group, and we accordingly constructed EF paternal and LF maternal lines. On the basis of test-cross for the selection of LF homozygous (<i>KK</i>) males in the maternal line, we confirmed that three of 40 chickens were homozygous males. The survival rate, body weight, days at first egg-laying, hen-day egg production, and egg weight were analyzed to compare the production performance of EF and LF chickens. The results revealed that EF chickens were characterized by a superior survival rate, whereas LF chickens were superior in terms of egg production rate. However, no differences between LF and EF chickens were detected with respect to other production performance parameters. In addition, assessment of the fitness of sexed chicks produced in the established KNC feather-sexing lines revealed that the accuracy of sexing was 98.6%. Collectively, these findings indicate the feasibility of constructing effective KNC feather-sexing lines with potential industrial application.

토종종계에 있어 조우성 개체와 만우성 개체간 생산능력 비교 고찰

Currently, feather-sexing, which is based on differences in feather development at hatching, is a widely used chick sexing method in the poultry industry. For effective chicken feather-sexing, paternal early-feathering (EF) chickens and maternal late-feathering (LF) chickens must be bred. Therefore, it is critical to identify the effect of EF and LF patterns on production traits in chickens. Thus, the purpose of this study is to analyze and compare the production performances between 522 EF and 232 LF chickens in order to establish the Korean native chicken feather-sexing lines. The results showed that the survival rate of the LF group was significantly higher than that of the EF group from hatching to 52 weeks of age (<italic>P</italic>&lt;0.05). Body weight, however, was not significantly different between the two groups at all ages. LF and EF groups did not significantly differ in age at first egg laying. However, the hen-day and hen-housed egg production of the LF group were significantly higher than those of EF group (<italic>P</italic>&lt;0.01). No significant differences were found between the EF and LF groups in all egg quality indicators such as egg weight, eggshell color, albumin height and Haugh unit. Because the breeding target of Korean native commercial chicken is meat-type chicken, feather-sexing strains of Korean native chicken should be established using weighing-based paternal EF lines and laying-based maternal LF lines. Therefore, these results are critical for establishing desirable and effective feather-sexing strains.

Changes in prolactin receptor homodimer availability may cause late feathering in chickens

Chicken early (EF) and late feathering (LF) are sex-linked phenotypes conferred by wild-type k+ and dominant K alleles on chromosome Z, respectively. Besides prolactin (PRL) receptor (PRLR) and sperm flagellar 2 (SPEF2) genes, the K allele contains a fusion gene in which partially duplicated PRLR (dPRLR) and SPEF2 (dSPEF2) genes are linked in a tail-to-tail manner. The causative dPRLR gene encodes a C-terminal truncated receptor. LF chickens have short or no primaries at hatching; however, their feather growth rate is higher than that of EF chickens. This study aimed to elucidate the molecular basis of the K allele’s biphasic effect on feather development. By 3′RACE and RT-PCR analyses, we demonstrated that dSPEF2 gene transcription occurred beyond all coding exons of the dPRLR gene on the opposite strand and that dPRLR mRNA was less abundant than PRLR mRNA. In addition, a 5′UTR splice variant (SPV) of PRL receptor mRNAs was increased in LF chickens. In vitro expression analysis of 5′UTR linked to the luciferase reporter gene revealed higher translation efficiency of SPV. RT-qPCR showed that the dPRLR mRNA level was higher in embryos; conversely, SPV was higher in hatched chickens, as was dSPEF2 mRNA. These findings suggest that the K allele inhibits feather development at the fetal stage by expressing dPRLR to attenuate PRLR function and promotes feather growth after hatching by increasing PRLR through dSPEF2 mRNA expression. Increased SPV may cause greater feather growth than that in EF chickens by increasing the availability of PRLR homodimers and enhancing PRL signaling.

Analysis of a genetic factors contributing to feathering phenotype in chickens.

In the current study, we sought to determine whether or not the endogenous retroviral ev21 influences feathering type of chickens, and if one mutation locus in the unoccupied repeat (UR) region can be used to predict the corresponding feathering type and genotype. The distribution of ev21 as well as the mutation locus in UR and occupied site (OR) regions was detected in HY-line gray progenitor (HYGP) 4 lines, HY-line brown (HYB) and Taihang chickens (TH). Furthermore, a detection method for the genotype resulting in late feathering (LF) phenotype was developed by double PCR using C line of HYGP, C line of Dawu progenitor, commercial line of HY-line gray (HYG) males, LF males of TH and Bashang long-tail chickens (BS). Results indicated that a product of 7590bp from the long fragment amplification was observed to be a partial segment of ev21, and was linked with the LF phenotype in HYGP but not in HYB and TH chickens. A total of 2 of 35 males and 10 of 29 females of TH LF chickens were found to be ev21 negative. HaeIII RFLP mutations of 1450bp of UR, 1440bp of OR, and 538bp in the UR and OR common region were analyzed, and genotypic features at the locus correlated with the feathering type phenotype in HYGP, but exhibited no significant effects in HYB and TH chickens. The cut-off of relative intensity of 857 and 1305bp from the double PCR for distinction between homozygous and heterozygous LF males was 1.37. In conclusion, ev21 and the HaeIII RFLP patterns within the locus in UR cannot be used for prediction of feathering type phenotypes in Chinese heritage chickens. However, the partial duplication of PRLR and SPEF2 were able to predict the LF phenotype. Therefore, the double PCR detecting products of 857 and 1305bp described herein could be used for the accurate identification of genotypes influencing feathering type.

Characterization of microRNA and mRNA expression profiles in skin tissue between early-feathering and late-feathering chickens

BackgroundEarly feathering and late feathering in chickens are sex-linked phenotypes, which have commercial application in the poultry industry for sexing chicks at hatch and have important impacts on performance traits. However, the genetic mechanism controlling feather development and feathering patterns is unclear. Here, miRNA and mRNA expression profiles in chicken wing skin tissues were analysed through high-throughput transcriptomic sequencing, aiming to understand the biological process of follicle development and the formation of different feathering phenotypes.ResultsCompared to the N1 group with no primary feathers extending out, 2893 genes and 31 miRNAs displayed significantly different expression in the F1 group with primary feathers longer than primary-covert feathers, and 1802 genes and 11 miRNAs in the L2 group displayed primary feathers shorter than primary-covert feathers. Only 201 altered genes and 3 altered miRNAs were identified between the N1 and L2 groups (fold change > 2, q value < 0.01). Both sequencing and qPCR tests revealed that PRLR was significantly decreased in the F1 and L2 groups compared to the N1 group, whereas SPEF2 was significantly decreased in the F1 group compared to the N1 or L2 group. Functional analysis revealed that the altered genes or targets of altered miRNAs were involved in multiple biological processes and pathways related to feather growth and development, such as the Wnt signalling pathway, the TGF-beta signalling pathway, the MAPK signalling pathway, epithelial cell differentiation, and limb development. Integrated analysis of miRNA and mRNA showed that 14 pairs of miRNA-mRNA negatively interacted in the process of feather formation.ConclusionsTranscriptomic sequencing of wing skin tissues revealed large changes in F1 vs. N1 and L2 vs. N1, but few changes in F1 vs. L2 for both miRNA and mRNA expression. PRLR might only contribute to follicle development, while SPEF2 was highly related to the growth rate of primary feathers or primary-covert feathers and could be responsible for early and late feather formation. Interactions between miR-1574-5p/NR2F, miR-365-5p/JAK3 and miR-365-5p/CDK6 played important roles in hair or feather formation. In all, our results provide novel evidence to understand the molecular regulation of follicle development and feathering phenotype.

한국재래닭 깃털 성감별 계통에 있어 조우성과 만우성이 개체의 생산능력에 미치는 영향

The vent sexing and the auto-sexing by using sex-linked traits are general sexing methods of day-old chicks. Currently, the feather sexing which is based on the differences in the feather characteristics at hatching is the representative sexing method of chicken, because the late-feathering is sex-linked trait. The feather sexing can be used if the breed has dominant feathering gene (K) in maternal and recessive gene (k + ) in paternal. Therefore it is necessary to identify the associa- tion of feathering genes and quantitative traits in chickens. In this study, we investigated the influence of the rate of feathering on productive traits in Korean Native Chicken. In results, there was no significant difference between early-feathering chickens and late-feathering chickens in reproductive performance such as fertility and hatchability. Livability, body weights, egg production, egg weight and egg quality also did not significantly differ between early- and late-feathering chickens. Age at first egg was the only trait of those tested in which significant difference was observed. The early-feathering chickens laid eggs 3 days earlier than late-feathering chicken. As a result, there is no influence of feathering phenotypes on productive performance in Korean Native Chickens. Consequentially, establishing the feather sexing strain is available using the Korean Native Chicken breed without considering of the effect of feathering genes on productive traits.

Differences of Z chromosome and genomic expression between early- and late-feathering chickens

One duplicated segment on chicken Z chromosome is a causal mutation to the late-feathering phenotype. However, understanding biological process of the late-feathering formation is also of interest to chicken breeding and feather development theory. One hundred and thirty-seven valid single nucleotide polymorphisms (SNPs) from an SNP database were used to perform an association study of the Z chromosome in Xinghua chickens. Two SNPs, which were respectively on 9607480 bp and 10607757 bp, were significantly associated with feathering phenotypes. This result indicated the causal mutation of the late-feathering formation in Xinghua chickens was consistent with the previous report which showed the late-feathering locus ranged 9966364–10142688 bp on Z chromosome. Microarray expressions were implemented for six 1-day-old female Xinghua chicks. Compared to the early-feathering chicks, there were 249 and 83 upregulated and downregulated known genes in the late-feathering chicks. Forty-one genes were expressed in late-feathering chicks, but not in early-feathering ones. At least 14 significantly differentially expressed genes were directly related to keratin. In the region of the sex-linked feathering gene, only prolactin receptor (PRLR) gene was a significantly differentially expressed gene. Expression of PRLR in late-feathering chicks was 1.78-fold as that in early-feathering chicks. Late-feathering Wenchang chicks also had higher expression level of PRLR than early-feathering ones. This study suggested that increasing PRLR expression that resulted from the special variant on chicken Z chromosome caused the late-feathering phenotype.

Sequence Characterization of K-gene Linked Region in Various Chicken Breeds

The avian endogenous virus gene (ev21) and dominant sex-linked late feathering (LF) gene (K) are closely associated on the Z chromosome of LF chickens. The K gene is linked to two large repeats: ev21-unoccupied repeat region (URa) and ev21-occupied repeat region (OR). On the other hand, the recessive allele (early feathering, EF: k+) is linked to ev21-unoccupied region (URb). These three regions show high similarity but due to minor sequence differences, restriction enzyme treatment distinguishes URa and URb in White Leghorn. However, the applicability of the PCR-RFLP technique or the association between ev21 and K gene are not well investigated in the other breeds or strains. In this study, ev21 loci were detected in White Leghorn, Nagoya, Silky, Geline but not Gifujidori breeds of chickens, and the URa and URb from the various breeds were sequenced. Four types of sequence (URa-1 and URa-2 from LF or ev21 positive, URb-1 and URb-2 from EF or ev21 negative) were identified. At position 1072-1075, URa-2, URb-1 and URb-2 had “GGCC” whereas URa-1 had “AGCC”. The transition at 1072 clearly indicates that the PCR-RFLP technique used for White Leghorn is not applicable to Nagoya. Interestingly, both URa-1 and URa-2 were obtained from the ev21 positive sample of Geline. Presence of different sequence of URa in the Geline may explain that fixation of one of sequences to White Leghorn or Nagoya had occurred during breed establishment.

Role of contact and genetic transmission of endogenous virus-21 in the susceptibility of chickens to avian leukosis virus infection and tumors

The role of contact and genetic transmission of endogenous virus-21 (EV21) on response of chickens to avian leukosis virus (ALV) infection and tumors was studied. F1 progeny of a cross between RPRL late-feathering (LF) line EV21+ males and RPRL early feathering (EF) line 15B1 females harboring or lacking EV21 were used. The EF chicks lacking EV21 were inoculated with a field strain of subgroup A ALV at hatch and contact exposed to LF, EV21+ hatchmates for various time intervals. In a second experiment, EV21 contact-exposed and unexposed EF chicks as well as LF, EV21+ hatchmates were inoculated with ALV at various ages. Chickens were tested for ALV-induced viremia and antibody and were observed for tumors until 24 wk of age. Antibody to EV21 in EF chickens contact-exposed to LF, EV21+ hatchmates varied from 10 to 65%, and was detected by 10 wk of age. By 24 wk of age, ALV-induced viremia and tumors in EF chickens varied from 5 to 30%, and from 15 to 32%, respectively, regardless of exposure to EV21. The incidence of ALV-induced tumors was significantly higher in LF chickens genetically infected with EV21 than in EV21 contact-exposed or unexposed EF chickens, but only in chickens inoculated with ALV at hatch. The data suggest that contact infection with EV21 has no influence on ALV infection and tumors. The data also suggest that genetic transmission of EV21 may increase susceptibility of chickens to ALV infection and tumors following infection with ALV at hatch, but not at 4 wk of age or older.

Growth and Carcass Characteristics of Early- and Late-Feathering Broilers Reared Under Different Feeding Regimens

Growth, immunological characteristics, and carcass traits were measured in early- and late-feathering chickens maintained under three feeding regimens. The regimens were 1) AL, ad libitum, 2) RL, alternate day feed restriction from 6 to 27 days of age and then released to ad libitum, 3) SOD, alternate day feeding restriction from 6 days of age to the end of the experiment. Sexual dimorphism was observed for accelerated growth with convergence in BW of RL with AL chickens occurring at a younger age in males than females. Absolute BW, feather weight, and gastrointestinal tract (GIT) weights were lower for SOD, after 45 days of age, than for AL and RL chickens. Relative to BW, however, GIT and liver weights, at 45 days of age but not 64 days of age, were heavier for SOD than for AL and RL chickens. Percentage of carcass lipid was lower for SOD than for AL and RL chickens. Generally, carcass traits and components of the GIT were similar for Regimens AL and RL at 45 and 64 days of age. Exceptions were lower percentages for carcass lipid and liver lipid for RL than AL chickens at 64 days of age. There was a significant feeding regimen by age interaction for heterophil to lymphocyte ratios caused by a greater increase in ratios with age for AL than for RL and SOD chickens. At 37 days of age, AL chickens had lower antibody titers to SRBC than SOD or RL chickens that did not differ. The AL chickens had reduced bursa of Fabricius and thymus weights relative to BW, indicative of immunological involution, along with reduced livability, compared with RL and SOD chickens, which did not differ in livability. Early- and late-feathering chickens were similar for most of the traits measured. One exception was higher antibody titers to SRBC in early- than late-feathering chickens.