苹果属15个种的叶绿体DNA变异与遗传分化

【目的】利用叶绿体基因(cpDNA)标记,对近十年间在我国苹果属植物的主要密集分布区收集的15种苹果属植物的叶绿体DNA进行序列变异分析,开展其遗传分化和遗传结构研究。【方法】利用4对叶绿体DNA引物扩增722份种质的4个非编码区trn H-psb A、trn S-trn G spacer+intron、trn T-5’trn L和5’trn L-trn F,对每个基因间区正反向测序获得的序列进行人工校对后,使用MEGA 7.0进行序列拼接和比对,使用Dna SP ver5.10.01计算叶绿体DNA的遗传多样性参数,利用Arlequin v3.5分析标准分子变异(AMOVA),运用Net Work ver4.6.1.2构建种内居群间的叶绿体DNA单倍型邻接网络关联图。【结果】4个cp DNA区域经测序、拼接、比对和合并之后的片段长度为4 120 bp,共有579个多态性变异位点,单倍型为100个,核苷酸多样性(Pi)和单倍型多样性(H_d)分别为0.008 52和0.879。Tajima’s D检验中,15种苹果属植物的4个cp DNA区域合并后遵循中性模型。AMOVA分析表明,遗传变异主要存在于种群内,但种群间遗传变异也占较大比重。【结论】中国原产苹果属植物在叶绿体DNA水平具有较高的遗传多样性,苹果属植物不同种具有不同的演化路线,各个种间以及种内起源演化关系错综复杂。山荆子和新疆野苹果的部分种质占据较为古老的单倍型,并在发展演化过程中经历过种群扩张。苹果属栽培种中国苹果、花红、楸子和八棱海棠的部分单倍型晚于部分新疆野苹果的单倍型。

Genetic divergence and population structure of chloroplast DNA in fifteen species of Malus Mill.

【Objective】Chloroplast gene markers were used to analyze the sequence variation of chloroplast genes of 722 accessions of 15 species of Malus Mill.collected from the main densely distributed areas of Malus Mill.in China in the past decade,and to carry out the research on their genetic differentiation and genetic structure.【Methods】Four non-coding region,trnH-psbA,trnS-trnG spacer+intron,trnT-5’trnL and 5’trnL-trnF of 722 germplasms were amplified by the four primers.After manually proofreading sequences obtained through forward and backward sequencing,MEGA 7.0 was used for sequence splicing and alignment,and the Neighbour-Joining phylogenetic tree was constructed among different populations of Malus Mill.based on genetic distance.DnaSP ver5.10.01 was used to calculate the genetic diversity parameters of the chloroplast DNA,gene flow and gene differentiation among different populations,including gene flow(Nm),population fixation coefficient(Fst),gene differentiation coefficient(Gst)and nucleotide differentiation coefficient(Nst)which were used to evaluate gene exchange among different populations.Arlequin v3.5 was used to analyze standard molecular variation(AMOVA),and NetWork 4.6.1.2 was used to construct Median-Joining network for cpDNA haplotypes among intraspecific populations of Malus Mill.【Results】The length of four chloroplast gene regions after sequencing,splicing,alignment and merging was 4120 bp,with 579 polymorphic variation sites and 100 haplotypes,and whose nucleotide diversity(Pi)and haplotype diversity(Hd)were 0.00852 and 0.879 respectively.Genetic diversity(Hd)of the four chloroplast DNA merging regions in 722 accessions of Malus Mill.was significantly higher than the average of 170 species(0.670-0.826).The region with the highest nucleotide polymorphism was trnT-5’trnL,including 3 singleton variable sites,20 parsimony informative sites and 298 insertion-deletion gaps.The region with the lowest nucleotide polymorphism was 5’trnL-trnF,with only 3 singleton variable sites,2 parsimony informative sites and 8 insertion-deletion gaps.There were 579 heterozygous sites in the 4 cpDNA regions of 722 accessions of Malus Mill.native to China,among which 321 were in region trnT-5’trnL,which was the region with the most mutation sites.The nucleotide diversity(Pi)of trnH-psbA was the highest with 0.03409,and that of 5’trnL-trnF was the lowest with 0.00078.In Tajima’s D test,the four cpDNA regions of 15 species of Malus followed the neutral model.There was obvious population expansion in the evolutionary process of Malus Mill.,and its genetic evolution was mainly based on mutation or random drift within populations.The number of haplotypes in the region trnH-psbA was 48 at most,and that in the region 5’trnL-trnF was 10 at least.The haplotype diversity(Hd)of trnH-psbA was the highest with 0.808,and the haplotype diversity(Hd)of 5’trnL-trnF was the lowest,which was 0.577.AMOVA analysis showed that the genetic variation mainly existed in the population,but the genetic variation among populations also accounted for a large proportion.44.63%of population genetic variation come from the inter population,and 55.37%from the intra population.Although the population genetic variation was mainly from the intra population,but the population genetic variation from the inter population also accounted for a relatively heavy proportion.The genetic differentiation coefficients of the other 11 populations,except Malus kansuensis,Malus yunnanensis,Malus ombrophila,Malus komarovii with only one accession respectively,were from 0.014 to 0.840.Compared with the wild species of Malus in China,gene exchange between cultivated species was more frequent.Malus baccata and Malus halliana played a role in the evolution of cultivated species.Based on the NJ cluster analysis of population genetic distance,15 species of Malus were divided into 5 groups,groupⅠincluded Malus toringoides,Malus transitoria,Malus hupehensis and Malus halliana,groupⅡincluded Malus prunifolia,Malus robusta,Malus micromalus,Malus baccata and Malus asiatica,groupⅢinclude Malus domestica subsp.chinensis and Malus sieversii,groupⅣincluded Malus yunnanensis,Malus ombrophila and Malus kansuensis,and groupⅤincluded Malus komarovii.The taxonomic attribution of Malus was obviously related to its species relationship and geographical distribution.A total of 100 haplotypes were found in the combined chloroplast gene segments of the 4 regions.Malus sieversii had the most haplotypes,242 accessions of Malus sieversii had 52 haplotypes,and 72 of which were concentrated on haplotype H15,and 76 of which were in haplotype H42,and the rest scattered in the other 50 haplotypes.Median-Joining network for cpDNA haplotypes of 15 spcecies of Malus Mill.was constructed based on four combined cpDNA intergenic regions.The tarso of haplotype network consisted of 23 intermediate vector sites and 20 haplotypes,and 23 intermediate vector sites were missing haplotypes.【Conclusion】The genetic diversity of Malus Mill.native to China was high.Different species of Malus have different evolutionary routes,and the relationship of the origin and the evolution among species and within species was complex.Some of the germplasms of Malus baccata and Malus sieversii occupied the older haplotypes,and experienced population expansion in the process of development and evolution.Some haplotypes of Malus domestica subsp.chinensis,Malus asiatica,Malus prunifolia and Malus robusta arised later than those of Malus sieversii.