## 4 Discussion
Therefore, the development of a simple and efficient molecular detection technology to screen male and female donkeys with multiple thoracolumbar numbers is of great significance to accelerate the breeding of Multi-TLN Dezhou donkeys. A series of studies have been performed to identify genes regulating vertebrae number variation in livestock. By direct sequencing and association analysis, we found that g.18093100G > T, g.18094587G > T, g.18106043G > T, g.18108764G > T, g.18110615T > G, g.18112000C > T and g.18114954T > G were significantly correlated with body size traits and thoracolumbar vertebrae number in our experimental sample population. It is particularly noteworthy that g.19093100G > T is significantly associated with BH, SW, and SLL. Individuals with TT genotype had significantly lower BH and SLL than wild type and heterozygote (\(p\) < 0.05), TL, TLL, and CW had the same decreasing trend, but did not reach the significant level. For g.18094587C > G and g.18106043A > C, individuals with mutant genotype had significantly smaller CHL than other genotypes (\(p\) < 0.05). For g.18108764A > G, individuals with heterozygote had the highest CHC, mutant genotype individuals had significantly smaller CHC, but wild genotype individuals did not differ significantly to others. For g.18112000T > G, individuals with GG and GT genotype had significantly higher CW value than wild type genotype (\(p\) < 0.05). TN, LN, and TLN did not have such a tendency, but BL, STL, SLL and STLL had the same trend (did not reach a significant level). Therefore, the function of this locus may be to increase carcass length by affecting the length of one vertebra, and then increase carcass weight by increasing carcass length. For g.18114954C > T, individuals with CT genotype had significantly bigger LN and TLN than wild type (\(p\) < 0.05), but had no significant difference with mutant type. For CW, heterozygous individuals have the highest value, followed by wild-type and mutant homozygous individuals. Statistically, the CW value of mutant homozygous individuals was significantly lower than that of others. It can be seen that different SNVs in the same gene may have contradicting functions in one trait. Moreover, several SNVs have been found associated with CHC, which indicates that these loci in _NR6A1_ may have had some functions in the development of ribs. We constructed 16 haplotypes (n \(\geq\) 5) by seven SNV loci as Haplotype composed of SNVs could provide more accurate information than single loci association analysis for phenotypic traits [14]. We found that individuals with Hap2 Hap7 had significantly higher TL, STL and STLL than Hap3 Hap3 individuals (\(p\) < 0.05), while individuals with Hap3 Hap3 haplotype had bigger LN and LL than Hap2 Hap7 samples (did not reach a significant level). Therefore, we speculate that Hap2 Hap7 is the superior haplotype for TL, while Hap3 Hap3 is the superior haplotype for the development of lumbar vertebrae. As a first-class candidate gene, _NR6A1_ has been studied in several animals and association with the vertebrae number has been proved. [PERSON] et al. detected a 13 bp Indel in the intron 1 of the Dezhou donkey _NR6A1_ gene, and found it was significantly associated with growth traits [35]. [PERSON] et al. [12] found that there was a significant association between SNV (rs414302710: A > C) in the exon-8 of _NR6A1_ gene with the number of lumber vertebrae in sheep (\(p\) < 0.01). [PERSON] et al. [18; 19] found that a missense substitution (c.575T > C, p.Pro192 Leu) in the _NR6A1_ gene was the causative mutation of a quantitative trait locus affecting the number of vertebrae in pigs. Unlike the Asian alleles, all European alleles in that study had the effect of increasing the number of vertebrae by 0.44 to 0.49 and acted additively without dominance. Owing to the lack of pedigree information, it is difficult to calculate the effect of these SNPs on the number of vertebrae between parental generation and offspring.