Determination of Polymorphisms in Pituitary Genes of the Native Afghani Naked Neck Chicken

Figures

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  Fig. 1.. Determination of the PRL 24 bp indel mutation by agarose electrophoresis. The 154 bp band represents individuals with the In/In genotype (lanes 1, 2, and 5), 154 and 130 bp bands represent individuals with the In/Del genotype (lane 4), and the 130 bp band represents individuals with the Del/Del genotype (lane 3). M shows the molecular weight marker (100 bp ladder).

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  Fig. 2.. Representative profile of the GH T185G mutation by agarose electrophoresis. M shows the molecular weight marker (100 bp ladder).

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  Fig. 3.. The electrophoretic profile of PIT-1 intron 5 digested with TaqI. Lanes 1 and 4 are heterozygous (AB) genotypes, lanes 2 and 3 are homozygous (BB) genotypes, and lanes 5 and 6 are homozygous (AA) genotypes. M shows the molecular weight marker (100 bp ladder).

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  Fig. 4.. RFLP analysis of PIT-1 intron 5 using MspI and confirmed by sequencing. (a) Photograph of the digest patterns of PCR-RFLP for MspI in PIT-1 intron 5. Lanes 1 and 2 represent the expected digestion pattern from a previous report (Rodbari et al. 2011). Lane 8 represents a homozygous genotype (DD) newly identified in the Afghanistan Naked Neck breed. Lanes 3, 4, and 6 are the unclear genotypes. M shows a 100 bp ladder marker. (b) Sequence of the polymorphic site of a novel D allele in which panel (1) shows the MspI recognition site at nucleotide position 280 and panel (2) shows the MspI recognition site at nucleotide position 500. Therefore, the gel shows that novel patterns (320, 280, and 99 bp) were generated.

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  Fig. 5.. RFLP analysis and sequencing result for PIT-1 intron 5 after cloning. (a) Lanes 1, 2 (cloned sample of lane 3 from Fig. 1a), 3, and 4 (cloned sample of lane 6 from Fig. 1a) represent alleles E, B, B, and F, respectively, in PIT-1 intron 5. The 19 bp fragment could not be visualized in this gel. M shows the molecular weight marker (100 bp ladder). (b) Panels (2) (named allele E) and (3) (named allele F) are the Naked Neck cloned sequences (of lane 3 or 4 and lane 6, respectively, from Fig. 1a) and panel (1) is the original sequence released by Gen-Bank.

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  Fig. 6.. Novel restriction sites for the MseI endonuclease in intron 5 of the PIT-1 gene in the Afghani Naked Neck chicken. Arrowhead indicates the SNPs in intron 5 of PIT-1 of the Naked Neck chicken. The upper sequence shows the original sequence reported by GenBank that contains only one MseI restriction site (GenBank accession no. NC_006088.4). The middle and lower sequences exhibit more than one MseI recognition site.

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  Fig. 7.. Schematic representation of MspI recognition sites in PIT-1 intron 5 that are generated by SNPs in the Naked Neck chicken of Afghanistan. Each box shows a different allele (A, B, C, D*, E*, and F*); the solid horizontal line in parentheses represents the 599 bp sequence of PIT-1 intron 5 in which / is the recognition site for MspI created by a point mutation. The double line between the solid circles represents the agarose gel fragment sizes visualized after digestion with MspI in agarose gel. Asterisk (*) indicates a new allele.