Abstract: 【Objective】 To investigate chicken gga-mir-1658 precursor area gene genetic variations/haplotypes and its distribution among all varieties. To analyze its effect on microRNA secondary stem-loop structure and target gene selection. The purpose was to screen the mutation sites as having potential biological function, which might lay the foundation to further reveal its influence on gga-mir-1658 gene expression regulation and phenotypic effects. 【Method】 A pair of specific primers were designed according to the chicken gga-mir-1658 genome sequence (GenBank accession number: NR_035151.1). The polymorphism detection was performed on gga-mir-1658 gene precursor area of 220 individuals including three chicken breeds: Taihang chicken (95), Beijing Fatty Chicken (83) and Leghorn chicken (42) by the PCR products direct sequencing. The comparison analysis and secondary structural simulation of gga-mir-1658 genome sequences were performed using DNAman, MEGA and mfold software. The matching linkage disequilibrium analysis and haplotype analysis were performed by SHEsis and Haploview software. The predictive parsing on the free energy changes of gga-mir-1658 target genes and their complexes was performed by miRanda software. 【Result】There were 6 mutation sites in pre-gga-mir-1658 gene. Among them, secondary allele frequency ≥5% included g.28 C＞G, g.31 C＞T, g.70G＞A and g.71G＞-. Four variable sites were positioned in the seed zone of gga-mir-1658 gene mature body. The genetic variation analysis showed that g.70G＞A site manifested low polymorphism (PIC＜0.25), and the other three sites manifested moderate polymorphism (0.25＜PIC＜0.50). The compatibility test showed that the other variation sites were in a Hardy-Weinberg equilibrium state in all breeds except for Leghorn chicken g.31 C＞T, g.71G＞-, Taihang chicken g.71G＞- and Beijing Fatty Chicken g.70G＞A site (P＞0.05). Linkage disequilibrium and haplotype analysis showed that there was a weak linkage equilibrium between mutated sites; 11 haplotypes were detected among the three breeds. Of which, H1 (C C G -) and H11 (G T G G) were the dominant haplotypes. The frequencies were greater than 25%. Bioinformatics analysis showed that the mutation in the seed zone could influence the space configuration and free energy of gga-mir-1658 gene precursor secondary structure. Of which, H6 haplotype mutants was highest (41.00 kcal·mol^-1). H2 and H5 haplotype mutants were lowest (35.70 kcal·mol^-1); The predominant haplotype H1 and H11 mutants were -36.10 kcal·mol^-1 and 40.04 kcal·mol^-1. Different haplotype mature seed zone sequences of gga-mir-1658 gene were different. The gga-mir-1658-5p included two kinds of seed sequences: “AUACCAU” and “AUACCAC”. The gga-mir-1658-3p included four seed zone sequences: “AACUCUG”, “AGCUGUG”, “AACUGUG” and “AGCUCUG”. The bioinformatics analysis of gga-mir-1658 predictable target gene showed that seed zone could affect the selection of gga-mir-1658 mature body on the target gene. They were mainly enriched in gene expression regulation, cell apoptosis, immune system development and B cell activation, other basic biological processes. 【Conclusion】 (1) There were four mutation sites having potential biological function and phenotypic effects in gga-mir-1658 gene seed zone. They could constitute 11 haplotypes. Of which, H1 (C C G -) and H11 (G T G G) were the dominant haplotypes in Beijing Fatty Chicken, Taihang chicken and Leghorn chicken. (2) The seed zone mutation could affect the stability of gga-mir-1658 gene precursor secondary structure and the selection of target genes, which might be the important functional site having potential phenotype effect.

Key words: chicken, gga-mir-1658, RNA secondary structure, genetic variation