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

Abstract:【Objective】Chloroplast gene markers were used to analyze the sequence variation of chloro-plast 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 differenti-ation 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 dif-ferent populations, including gene flow (Nm), population fixation coefficient (Fst), gene differentiation coefficient (Gst) and nucleotide differentiation coefficient (Nst) which were used to evaluate gene ex- change 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 af-ter sequencing, splicing, alignment and merging was 4 120 bp, with 579 polymorphic variation sites and 100 haplotypes, and whose nucleotide diversity (Pi) and haplotype diversity (Hd) were 0.008 52 and 0.879 respectively. Genetic diversity (Hd) of the four chloroplast DNA merging regions in 722 acces- sions 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 par-simony informative sites and 298 insertion-deletion gaps. The region with the lowest nucleotide poly-morphism was 5’trnL-trnF, with only 3 singleton variable sites, 2 parsimony informative sites and 8 in-sertion-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 0.034 09, and that of 5’trnL-trnF was the lowest with 0.000 78. In Tajima’s D test, the four cpDNA regions of 15 spe-cies 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 popu-lation, 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 acces-sion 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 dis-tance, 15 species of Malus were divided into 5 groups, group Ⅰ included Malus toringoides, Malus transitoria, Malus hupehensis and Malus halliana, group Ⅱ included Malus prunifolia, Malus robusta, Malus micromalus, Malus baccata and Malus asiatica, group Ⅲ include Malus domestica subsp. chi-nensis and Malus sieversii, group Ⅳ included Malus yunnanensis, Malus ombrophila and Malus kan-suensis, and group Ⅴ included Malus komarovii. The taxonomic attribution of Malus was obviously re-lated 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-Join-ing network for cpDNA haplotypes of 15 spcecies of Malus Mill. was constructed based on four com-bined 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 ge-netic diversity of Malus Mill. native to China was high. Different species of Malus have different evolu-tionary 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 haplo-types, and experienced population expansion in the process of development and evolution. Some haplo-types of Malus domestica subsp. chinensis, Malus asiatica, Malus prunifolia and Malus robusta arised later than those of Malus sieversii.