A heterozygous deletion and inversion at the NHEJ1‑IHH locus associated with shank length in Yunlong short-leg chicken

Abstract Background The Yunlong Short-Leg chicken is a local dual-purpose breed in the Dali Bai Autonomous Prefecture of Yunnan Province, China, characterized phenotypically by notably shortened shank and a compact body stature. Nevertheless, the genetic mechanisms responsible for its short-legged phenotype have yet to be elucidated. Results In this study, Yunlong Short-Leg chickens with different shank lengths—Long-legged and Short-legged phenotypes—were selected as experimental subjects ( n = 60, 30 males and 30 females). We performed genome-wide analyses of single nucleotide polymorphisms (SNPs) and insertion/deletion variants (InDels), followed by genome-wide association studies (GWAS) correlating these variants with body weight and morphometric traits. Population genetic analysis indicated no significant differences in genetic background between the Long‑legged and Short‑legged groups, confirming they belong to the same population. GWAS identified shank length as the only trait significantly associated with eight genes on chromosome 7, including IHH , NHEJ1 , and MNR2 . A comparison of variants on chromosome 7 revealed that the Long‑legged samples exhibited no structural changes, whereas Short-legged samples were categorized as creeper phenotype, exhibiting two non-contiguous deletions and an inversion on chromosome 7: a 13,480 bp deletion (positions 22,287,390–22,300,869 bp) and an 11,559 bp deletion (positions 22,301,448–22,313,006 bp), totaling 25,039 bp, as well as a 578 bp inversion (positions 22,300,870–22,301,447 bp). Lethal samples carried a homozygous deletion and the same inversion in this region on chromosome 7. Multiplex PCR with different primers was performed to genotype shank-length phenotypes based on structural genomic variants. Finally, qRT‑PCR analysis confirmed that the relative mRNA expression levels of IHH and NHEJ1 correlated with corresponding locus variation and shank-length phenotypes in Yunlong Short-Leg chicken. Conclusions These findings provide insight into the genetic basis of the short‑leg phenotype in Yunlong Short-Leg chicken and offer theoretical support for genetic resource conservation and future breeding programs.