Phylogenomic Study Of Malus In China

The genus Malus is a kind of flowering plants in the subfamily of Rosaceae,which are mainly distributed in the northern temperate zone and have important economic and ornamental value.Due to the blurring of morphological characteristics and frequent hybridization and introgression among species during the evolution of the genus Malus,the phylogenetic relationship of Malus has not been completely resolved.There are 21 species of the genus Malus in China.The cultivated varieties can be divided into ornamental and edible aspects after domestication and cultivation.Chloroplast genome sequence is conserved and inherited by the female line.The simplified genome was inherited by parents,and a large number of tag sequences were obtained to represent the genetic information of the whole genome.These characteristics are of great significance in the study of phylogenetic relationships in plants.In order to clarify the phylogenetic relationship among the species and origin of cultivar from Malus,we reconstructed the phylogenetic relationship of Malus based on the plastid genome and RAD-seq data,and further inferred the history of biogeography of Malus,resolve the issue of the ownership of disputed species,speculate whether Asian species have participated in the domestication of imported ornamental crabapples,the origin of cultivated apples(Malus×domestica),and confirm whether the genus Malus has experienced adaptive evolution at the molecular level in the process of continuous evolution.In this study,In terms of the chloroplast genome,we conducted DNA extraction,high-throughput sequencing,assembly,annotation,and construction of physical maps of 13 species and 13 cultivated varieties of the genus Malus,and compared and analyzed them with the 18 chloroplast genome sequences released by Gen Bank.In terms of simplification of the genome,we performed DNA extraction and simplified genomic sequencing(RAD-seq)of 35 Malus plants.For the above two data sets,we use the maximum likelihood method(ML)and Bayesian inference(BI)methods to reconstruct the phylogenetic relationship of the genus Malus.Then,based on BEAST,RASP and Paml software,respectively,the differentiation time of Malus was estimated,the ancestral distribution area was reconstructed to infer the evolution history of Malus,and the positive selection genes were screened.The main results obtained are as follows:(1)The chloroplast genomes of 26 Malus genus species were sequenced and analyzed.The length of the assembled chloroplast genomes was 159,908-160,230 bp.Like most angiosperms and published Malus chloroplast genomes,the chloroplast genomes of all samples have a typical tetrad structure,including a large single-copy region(LSC: 87,964–88,349 bp)and a small single-copy region(SSC):19,176–19,214 bp),two inverted repeat regions(IR: 26,348–26,370 bp).The GC content of the genome of this genus is very similar to 36.50–36.58%.All genomes encode the same 113 unique genes,including 79 protein-coding genes,30 t RNA genes,and 4 r RNA genes.The rps12 gene has a trans-splicing structure,and its5’exon at the end is located in the LSC region,and the 3’exon is located in the IR region.Among all the genes,15 genes contain one intron,and 2 genes contain two introns.(2)Comparative genomic analysis of species of the Malus showed a relatively conserved genome structure with no gene rearrangement events.The M.sieversii chloroplast genome was used as the reference genome for visual analysis,and it was found that the sequence variation of IR region was lower than that of LSC and SSC,and the high variation regions were mostly located in non-coding regions.In addition,we separately identified the coding and non-coding regions.The region was extracted and successfully screened by nucleotide polymorphism analysis to screen 3 highly variable regions(Pi>0.01): rps12,ndh C-trn V,trn R-atp A.In addition,a total of 788 SNP sites and a large number of simple repetitive sequences(SSR)were detected from 21 domestic species of Malus.In addition,the adaptive evolution analysis of the genus Malus revealed that the rpo B gene contains a positive selection site.(3)Use the whole chloroplast genome sequence,coding region sequence and non-coding region sequence data set for phylogenetic analysis to reconstruct the phylogenetic relationship of Malus.The topological structure obtained is basically the same,indicating that the phylogenetic relationship is relatively reliable,but the former has a relatively high support Higher.The results support that the genus Malus is polyphyletic,mainly divided into two major branches,the Asian branch and the European and American branches,which is not completely consistent with traditional taxonomy.The classification issues between genera and subordinates need to be further revised.The ornamental crabapples imported from abroad and the American specises Malus.angustifolia and Malus.fusca are clustered with Asian branches.The Malus×domestica are more closely related to the European wild apples than M.sieversii.Malus doumeri and European and American branches are located at the base of the phylogenetic tree of the genus Malus,which may be related to the primitive evolution of these species.(4)Based on the fossil calibration node,we used BEAST software to estimate the differentiation time of the genus Malus.The results of the study show that the origin time of the genus Malus is about 40.15 Ma,and the divergence time of the Asian branch is 33.14 Ma.The divergence time of the European and American branches is 25.0 Ma.Based on the phylogenetic tree and species geographic distribution information,the results of biogeographic analysis infer that the common ancestor of the Malus genus is mainly distributed in Asia.After that,it moved from East Asia to North America via the Bering Land Bridge and then to Europe and the Mediterranean.(5)Based on the simplified genome data(RAD-seq)of 35 plants of the genus Malus,we used different methods to construct phylogenetic trees(ML and BI),the topological structure of which is exactly the same,the genus Malus is divided into 4branches,and support rate is higher.Based on the analysis of population genetic structure,gene pool mixing has been detected in some specises of the genus Malus,laying a foundation for further revealing the complicated network evolution pattern of Malus.