摘要
冠毛性狀(Crest)是家雞(Gallus gallus domesticus)眾多富有特色的性狀之一，通常可以在波蘭雞及烏骨雞這類品系中發現。該性狀的特徵是頭上會長有一叢冠毛，且有時會伴隨腦疝的產生。過去許多研究致力於找出引起冠毛性狀的因素，而此研究中我們的目標是釐清HOXC8基因與冠毛性狀之間的關聯。為此，我們利用了PCR與Nanopore基因定序的方法分析了不同品系雞HOXC8的一段3端非編碼區域以求找出特定存在於擁有冠毛性狀品系中的突變。然而，序列比對的結果是在該區域並不存在特定發生於冠毛性狀品系中的突變。此外，我們還使用了RCAS反轉錄病毒系統以了解HOXC8基因在雞頭部的異位表現對其造成的影響。接受病毒注射的胚胎在20 ~ 21天的孵育後在破殼前收取樣本，接著我們將樣本進行電腦斷層掃描(Computed Tomography)以得到其頭骨的三維結構影像以對其進行幾何形態測量學分析(Geometric Morphometric Analysis)，並利用主成分分析(Principle Component Analysis)與典型變量分析(Canonical Variate Analysis)表現樣本之間形狀上的異同處。根據我們的發現，與施打帶有GFP的病毒相比，在頭部的HOXC8基因異位表現可能會造成輕微的頭骨型態改變。

關鍵字:冠毛性狀、HOXC8基因、異位表現、RCAS系統、幾何型態測量學分析、Nanopore定序

The Crest phenotype is one of the most intriguing traits in Domestic chickens (Gallus gallus domesticus). This trait is often found in breeds such as Polish chickens and Silkie chickens, characterized by a feather crest and sometimes cerebral hernia. Previous studies have strived to identify the causative factors of this trait, and in this study, we aimed to elucidate the relationship between chicken HOXC8 and the crest phenotype. To this end, we compared the 3’ noncoding region of the HOXC8 gene from multiple chicken breeds using PCR and Nanopore sequencing, focusing on identifying mutations specific to crested breeds. However, the sequence alignment results show no differences specific to crested breeds in the region. Furthermore, we explored the effect of ectopic expression of HOXC8 on the chicken head morphology using the RCAS system. Transgenic embryos were incubated for 20 to 21 days and collected before hatching. Computed tomography (CT) scanning was conducted to document cranial bone 3D structures for geometric morphometric analysis. Variations between samples and groups were evaluated by the principal component analysis and the canonical variate analysis. We found that the ectopic expression of HOXC8 in chicken heads caused mild morphological changes in the skull compared to the GFP-transgenic control group.

Adams, D. C., Rohlf, F. J., & Slice, D. E. (2013). A field comes of age: geometric morphometrics in the 21st century. Hystrix, 24(1), 7.
Arnaout, B., Lantigua, K. E., MacKenzie, E. M., McKinnell, I. W., & Maddin, H. C. (2021). Development of the chicken skull: A complement to the external staging table of Hamburger and Hamilton. The Anatomical Record.
Bartels, T. (2003). Variations in the morphology, distribution, and arrangement of feathers in domesticated birds. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 298(1), 91-108.
Belting, H.-G., Shashikant, C. S., & Ruddle, F. H. (1998). Modification of expression and cis-regulation of Hoxc8 in the evolution of diverged axial morphology. Proceedings of the National Academy of Sciences, 95(5), 2355-2360.
Benchling [Biology Software]. (2020). Retrieved from https://benchling.com.
Bhullar, B.-A. S., Marugán-Lobón, J., Racimo, F., Bever, G. S., Rowe, T. B., Norell, M. A., & Abzhanov, A. (2012). Birds have paedomorphic dinosaur skulls. Nature, 487(7406), 223-226.
Bradshaw, M. S., Shashikant, C. S., Belting, H.-G., Bollekens, J. A., & Ruddle, F. H. (1996). A long-range regulatory element of Hoxc8 identified by using the pClasper vector. Proceedings of the National Academy of Sciences, 93(6), 2426-2430.
Brothwell, D. (1979). Roman evidence of a crested form of domestic fowl, as indicated by a skull showing associated cerebral hernia. Journal of Archaeological Science, 6(3), 291-293.
Danecek, P., Bonfield, J. K., Liddle, J., Marshall, J., Ohan, V., Pollard, M. O., . . . Davies, R. M. (2021). Twelve years of SAMtools and BCFtools. GigaScience, 10(2), giab008.
Davenport, C. B. (1906). Inheritance in poultry: Carnegie institution of Washington.
De Coster, W., D’Hert, S., Schultz, D. T., Cruts, M., & Van Broeckhoven, C. (2018). NanoPack: visualizing and processing long-read sequencing data. Bioinformatics, 34(15), 2666-2669. doi:10.1093/bioinformatics/bty149
Deamer, D., Akeson, M., & Branton, D. (2016). Three decades of nanopore sequencing. Nature biotechnology, 34(5), 518-524.
Deng, Y., Hu, S., Luo, C., Ouyang, Q., Li, L., Ma, J., . . . Hu, J. (2021). Integrative analysis of histomorphology, transcriptome and whole genome resequencing identified DIO2 gene as a crucial gene for the protuberant knob located on forehead in geese. BMC Genomics, 22(1), 1-16.
Di Tommaso, P., Moretti, S., Xenarios, I., Orobitg, M., Montanyola, A., Chang, J.-M., . . . Notredame, C. (2011). T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic acids research, 39(suppl_2), W13-W17.
Duboule, D. (1998). Vertebrate hox gene regulation: clustering and/or colinearity? Current opinion in genetics & development, 8(5), 514-518.
Fan, W.-L., Ng, C. S., Chen, C.-F., Lu, M.-Y. J., Chen, Y.-H., Liu, C.-J., . . . Mao, C.-T. (2013). Genome-wide patterns of genetic variation in two domestic chickens. Genome biology and evolution, 5(7), 1376-1392.
Farré, D., Roset, R., Huerta, M., Adsuara, J. E., Roselló, L., Albà, M. M., & Messeguer, X. (2003). Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic acids research, 31(13), 3651-3653.
Fisher, R. A. (1935). VI—Dominance in poultry. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 225(523), 197-226.
GelAnalyzer 19.1 (www.gelanalyzer.com) by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc
Goodman, F., & Scambler, P. (2001). Human HOX gene mutations. Clinical genetics, 59(1), 1-11.
Halder, G., Callaerts, P., & Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science, 267(5205), 1788-1792.
Hamburger, V., & Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. Journal of morphology, 88(1), 49-92.
Hammer, Ø., Harper, D., & Ryan, P. (2001). PAST-palaeontological statistics, ver. 1.89. Palaeontol. electron, 4(1), 1-9.
Hata, A., Nunome, M., Suwanasopee, T., Duengkae, P., Chaiwatana, S., Chamchumroon, W., . . . Srikulnath, K. (2021). Origin and evolutionary history of domestic chickens inferred from a large population study of Thai red junglefowl and indigenous chickens. Sci Rep, 11(1), 2035. doi:10.1038/s41598-021-81589-7
Herr, W., Sturm, R., Clerc, R., Corcoran, L., Baltimore, D., Sharp, P., . . . Ruvkun, G. (1988). The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products. Genes Dev, 2(12A), 1513-1516.
Herzlinger, G., Goren-Inbar, N., & Grosman, L. (2017). A new method for 3D geometric morphometric shape analysis: The case study of handaxe knapping skill. Journal of Archaeological Science: Reports, 14, 163-173.
Himly, M., Foster, D. N., Bottoli, I., Iacovoni, J. S., & Vogt, P. K. (1998). The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology, 248(2), 295-304.
Hoegg, S., & Meyer, A. (2005). Hox clusters as models for vertebrate genome evolution. Trends in genetics, 21(8), 421-424.
Joller, S., Ammann, P., Flury, C., & Drögemüller, C. (2018). Evaluation of HOXC 8 in crested Swiss chicken. Animal genetics, 49(4), 334-336.
Juan, A. H., Lei, H., Bhargava, P., Lebrun, M., & Ruddle, F. H. (2006). Multiple roles of hoxc8 in skeletal development. Annals of the New York Academy of Sciences, 1068(1), 87-94.
Kanginakudru, S., Metta, M., Jakati, R., & Nagaraju, J. (2008). Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken. BMC evolutionary biology, 8(1), 174.
Kel, A. E., Gossling, E., Reuter, I., Cheremushkin, E., Kel-Margoulis, O. V., & Wingender, E. (2003). MATCHTM: a tool for searching transcription factor binding sites in DNA sequences. Nucleic acids research, 31(13), 3576-3579.
Kent, W. J. (2002). BLAT--the BLAST-like alignment tool. Genome Res, 12(4), 656-664. doi:10.1101/gr.229202
Klingenberg, C. P. (2011). MorphoJ: an integrated software package for geometric morphometrics. Molecular ecology resources, 11(2), 353-357.
Klingenberg, C. P., & Monteiro, L. R. (2005). Distances and directions in multidimensional shape spaces: implications for morphometric applications. Systematic Biology, 54(4), 678-688.
Lemons, D., & McGinnis, W. (2006). Genomic evolution of Hox gene clusters. Science, 313(5795), 1918-1922.
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Genes, development and cancer, 205-217.
Li, H. (2013). Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiv:1303.3997.
Li, J., Lee, M.-O., Davis, B. W., Wu, P., Hsieh-Li, S.-M., Chuong, C.-M., & Andersson, L. (2021). The crest phenotype in domestic chicken is caused by a 197 bp duplication in the intron of HOXC10. G3 Genes| Genomes| Genetics.
Mallo, M., & Alonso, C. R. (2013). The regulation of Hox gene expression during animal development. Development, 140(19), 3951-3963.
Marugán-Lobón, J., & Buscalioni, Á. D. (2004). Geometric morphometrics in macroevolution: morphological diversity of the skull in modern avian forms in contrast to some theropod dinosaurs Morphometrics (pp. 157-173): Springer.
Marugán-Lobón, J., Blanco-Miranda, D., Chamero Macho, B., & Martín-Abad, H. (2013). On the importance of examining the relationship between shape data and biologically meaningful variables. An example studying allometry with geometric morphometrics.
Messeguer, X., Escudero, R., Farré, D., Nuñez, O., Martı́nez, J., & Albà, M. M. (2002). PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics, 18(2), 333-334.
Montavon, T., & Soshnikova, N. (2014). Hox gene regulation and timing in embryogenesis. Paper presented at the Seminars in cell & developmental biology.
Navailles, S., Zimnisky, R., & Schmauss, C. (2010). Expression of glucocorticoid receptor and early growth response gene 1 during postnatal development of two inbred strains of mice exposed to early life stress. Developmental neuroscience, 32(2), 139-148.
Ng, C. S., & Li, W. H. (2018). Genetic and Molecular Basis of Feather Diversity in Birds. Genome Biol Evol, 10(10), 2572-2586. doi:10.1093/gbe/evy180
Ng, C. S., Wu, P., Foley, J., Foley, A., McDonald, M.-L., Juan, W.-T., . . . Chen, C.-F. (2012). The chicken frizzle feather is due to an α-keratin (KRT75) mutation that causes a defective rachis. PLoS Genet, 8(7), e1002748.
Rhoads, A., & Au, K. F. (2015). PacBio sequencing and its applications. Genomics, proteomics & bioinformatics, 13(5), 278-289.
Rohlf, F. J. (2015). The tps series of software. Hystrix, 26(1).
Santini, S., Boore, J. L., & Meyer, A. (2003). Evolutionary conservation of regulatory elements in vertebrate Hox gene clusters. Genome research, 13(6a), 1111-1122.
Schadt, E. E., Turner, S., & Kasarskis, A. (2010). A window into third-generation sequencing. Human molecular genetics, 19(R2), R227-R240.
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., . . . Rueden, C. (2012). 714 Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. 715 Fiji: an open-source platform for biological-image analysis. Nat Methods, 9, 676-682.
Su, J., Guan, K., Wang, J., & Yang, Y. (2015). Significance of hind wing morphology in distinguishing genera and species of cantharid beetles with a geometric morphometric analysis. ZooKeys(502), 11.
Tao, Y., Zhou, X., Zheng, X., Li, S., & Mou, C. (2020). Deciphering the Forebrain Disorder in a Chicken Model of Cerebral Hernia. Genes, 11(9), 1008.
Wang, T., Zhang, Y., Guo, Y., Zhang, X., Yang, H., Tian, X., . . . Luo, K. (2021). RNA-sequence reveals differentially expressed genes affecting the crested trait of Wumeng crested chicken. Poultry Science, 101357.
Wang, W., Dong, B., Ittmann, M. M., & Yang, F. (2016). A versatile gene delivery system for efficient and tumor specific gene manipulation in vivo. Discoveries, 4(2).
Wang, Y., Gao, Y., Imsland, F., Gu, X., Feng, C., Liu, R., . . . Li, Q. (2012). The crest phenotype in chicken is associated with ectopic expression of HOXC8 in cranial skin. PLoS One, 7(4), e34012.
Yoshimura, K., Kinoshita, K., Mizutani, M., Matsuda, Y., & Saito, N. (2012). Inheritance and developmental pattern of cerebral hernia in the crested Polish chicken. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 318(8), 613-620.
Yravedra, J., Aramendi, J., Maté-González, M. Á., Austin Courtenay, L., & González-Aguilera, D. (2018). Differentiating percussion pits and carnivore tooth pits using 3D reconstructions and geometric morphometrics. PLoS One, 13(3), e0194324.
Yuan, X., Zheng, S., Zhang, Y., Guo, Q., Wang, S., Bi, Y., . . . Pan, R. (2019). Embryonic morphology observation and hoxc8 gene expression in crest cushions of chinese crested duck. Gene, 688, 98-106.