建昌马mtDNA D-loop区遗传多样性及系统进化分析

试验旨在以线粒体DNA(mitochondrial DNA,mtDNA)为切入点,研究建昌马的母系遗传多样性与系统进化。从建昌马(n=39)血液中提取基因组DNA,用PCR方法扩增mtDNA D-loop区并直接测序,分析其高变区247 bp序列信息,统计mtDNA D-loop区的单倍型及变异位点,计算单倍型多样性(haplotype diversity,Hd)、核苷酸多样性(nucleotide diversity,Pi)和平均核苷酸变异数(average number of nucleotide differences,K)。构建包括建昌马在内的19个品种马的NJ系统进化树,计算各品种间的遗传距离。结果显示,试验获得了清晰的PCR扩增产物,并通过直接测序方法获得了约1200 bp的序列。39匹建昌马mtDNA D-loop区247 bp序列(其中1个样品缺失1 bp)的AT碱基含量为61.45%,属AT碱基对富集区,检测到33个多态性位点,共显示26种单倍型,其中4种为共享单倍型,且Hap7和Hap1为优势单倍型,单倍型多样性为0.947,核苷酸多样性为0.02399,平均核苷酸变异数为5.901,显示丰富的母系遗传多样性;NJ系统进化树显示,建昌马分布在A、C、D、E、F、G共6个支系中,约50%的样品分布在A支系,显示出复杂的母系起源;建昌马与关中马的遗传距离最小(0.021),其次是三河马、文山马、韩国车巨马(0.024),与韩国济州岛马遗传距离最大(0.032)。本研究结果表明,建昌马的mtDNA D-loop高变区遗传多样性丰富,具有多个母系起源,且A支系占有明显优势,与关中马、文山马可能有共同的母系起源。

Genetic Diversity and Evolutionary Analysis of mtDNA D-loop Region in Jianchang Horse

In order to study the maternal genetic diversity and phylogenetic evolution of Jianchang horse using mitochondrial DNA (mtDNA),the genomic DNA was extracted from the blood of Jianchang horses (n=39),and the genetic diversity of mtDNA D-loop was assayed by PCR and direct sequencing.A 247 bp mtDNA D-loop hypervariable sequence was analyzed,the haplotypes and mutation sites of this region were assayed,and the haplotype diversity (Hd),nucleotide diversity (Pi) and average number of nucleotide differences (K) were calculated.The Neighbour-Joining (NJ) phylogenetic tree of 19 horse breeds including Jianchang horse was constructed,and the genetic distances among the breeds were calculated.The experiment obtained the PCR products with good resolution,and about 1 200 bp sequences were obtained by direct sequencing.The AT base content of the 247 bp mtDNA D-loop sequences from 39 samples (one of which lacked 1 bp) was 61.45%,with a characteristic of AT base pair enrichment region.26 unique haplotypes with 33 polymorphic sites were detected,and 4 of which were shared haplotypes,Hap7 and Hap1 were the dominant haplotypes.The Hd, Pi and K were 0.947,0.02399 and 5.901,respectively,indicating abundant maternal genetic diversity;The NJ phylogenetic tree demonstrated the presence of 6 major lineages (A,C,D,E,F and G),and about 50% were distributed in the A branch,indicating the complicated maternal origins of Jianchang horse.The genetic distance were the minimum (0.021) between Jianchang and Guanzhong horses,followed by Sanhe,Wenshan and Cheju horses (0.024),and the genetic distance with Jeju native horse was the maximum (0.032).In conclusion,the present study demonstrated that the mtDNA D-loop hypervariable region was rich in genetic diversity in Jianchang horse,showing multiple maternal origins,and the A branch had obvious advantages,which suggested that Jianchang,Guanzhong and Wenshan horses might have a common maternal origin.