Analysis Of Genetic Diversity And Single Nucleotide Polymorphism Of Seven Height Candidate Genes In Jianchang Horse And Anning Pony

Jianchang horse and Anning pony are mainly distributed in the southwest China.They are small in body size and can be used for entertainment,both have important breeding value.In livestock breeding and improvement practice,it is significant to accurately understand the genetic background of the breeds.The main purpose of this study was to analyze the genetic diversity and the single nucleotide polymorphism(SNP)of candidate dwarf genes in Jianchang horse.In this study,genomic DNA was extracted from anticoagulant blood of 40 Jianchang horses(21 males and 19 females)and 4 Anning ponies(2 males and 2 females)raised in Liangshan Prefecture,Sichuan Province.Genetic diversity of cytochrome b(cytb)gene and Y chromosome and single nucleotide polymorphism of seven height candidate genes were analyzed.The main contents and results were as follows:(1)Maternal genetic diversity and evolution of Jianchang horse and Anning pony.The cytb gene of Jianchang horse and Anning pony were amplified by PCR,and the products were directly sequenced.The results showed that 45 variation sites were detected in 44 horses based on the 1140 bp complete sequence alignment of cytb gene,and 20 haplotypes were displayed.Nineteen haplotypes(H1 was the dominant haplotype)were detected in 40 Jianchang horses,and H1,H6,H12 and H20 haplotypes were detected in 4 Anning ponies.The haplotype diversity(Hd)and nucleotide diversity(Pi)of Jianchang horse were 0.894±0.038 and 0.00499±0.00064,respectively,indicating the abundant maternal genetic diversity of Jianchang horse.The phylogenetic tree based on haplotypes showed that Jianchang horses were distributed in 7 branches(A～G),showing multiple maternal origins,and branch A was the dominant.Anning ponies were distributed in branches A,F and G,and its haplotype H20 was distributed in branch A.These results suggested that there was a close relationship of the haplotypes of cytb gene between Jianchang horse and Anning pony.This study found that the genetic distance between Jianchang horse and Chaidamu horse was the minimum(0.0036).These findings suggested that Jianchang horse had abundant maternal genetic diversity and had multiple maternal origins.Jianchang horse and Chaidamu horse had the closest genetic relationship,and the genetic relationship between Jianchang horse and Anning pony was close.(2)Paternal genetic diversity of Jianchang horse and Anning pony.In this study,15 Y chromosome genetic marker loci were amplified by PCR and sequenced in Jianchang horses(n=21)and Anning ponies(n=2),and five of them were further analyzed by fluorescence quantitative PCR for copy number variation(CNV).The results showed that polymorphisms were detected on 11 genetic markers of Y chromosome in Jianchang horses and Anning ponies,and there existed 9 haplotypes(CHT1～CHT9),of which CHT1 was the dominant haplotype.Only one haplotype(CHT1)was detected in Anning ponies(n=2).The Y chromosome haplotype diversity and nucleotide diversity of Jianchang horse were 0.767±0.090 and 0.00038±0.00008,respectively.It was suggested that the Y chromosome of Jianchang horse was rich in haplotype genetic diversity.The phylogenetic tree based on haplotypes showed that Jianchang horses were distributed in two branches,suggesting its two paternal origins.Among the 11 polymorphic loci,5 loci were heterozygous.The qPCR analysis showed that the copy number of these 5 loci were multiple,ranging from 5 to 30,15 to 33,5 to15,2 to 6 and 14 to 35,respectively.The Y chromosome DNA of Jianchang horse has relatively high haplotype genetic diversity but low nucleotide diversity,and might have two paternal origins.The present results had provided new data for the genetic diversity study of Y chromosome in Jianchang horse.(3)Single nucleotide polymorphism of seven height candidate genes in Jianchang horse and Anning pony.Thirteen SNP loci of seven height candidate genes were amplified by PCR,and the genotypes and allele frequencies of each locus were analyzed following by direct sequencing.The results showed that the alleles of g.105547002 of LCORL/NCAPG,g.74798143 and g.75550059 of ZFAT,and g.87287547 of CHRDL1 related to dwarf trait had obvious advantages in Jianchang horse,and the frequencies were 0.8000,0.7125,0.8875 and 0.7368,respectively,suggesting that LCORL/NCAPG,ZFAT and CHRDL1 could be candidate dwarf genes in Jianchang horse.ANKRD1 and HMGA2 might not be associated with the dwarf trait in Jianchang horse.Furthermore,several new SNPs in the amplified sequences nearing g.18101000 of TBX3 gene and g.74798143 of ZFAT were detected in all horse samples,and these SNPs were linked with the loci assayed,and it is suggested that the polymorphisms of the new SNPs were also related to the body size of Jianchang horse.