Molecular Systematics Study On Interspecific Relationship Of Malus Mill.

The genus Malus Mill.is one of the important genera of core Maleae(traditional Maloideae)of the subfamily Amygdaloideae of the Rosaceae family,with about 35 species worldwide and 22 species in China.Species of the genus Malus have economic,ornamental and medicinal value and have continued to receive much attention from researchers in the domestic and overseas over the years,with the wide variety of taxonomic problems affecting their garden applications and breeding.The taxonomic history of the genus Malus spans more than 260 years,with traditional taxonomy,cytology,sporanology,biochemical analysis,chromosomal karyotyping and molecular markers being used in various studies of Malus species,but the phylogenetic relationships among Malus species with complex origins have always been controversial.In this study,we used chloroplast whole genome data sets and high-throughput simplified genome data sets to construct phylogenetic trees of Malus species with high coverage sampling respectively,to deeply resolve the kinship among Malus species,and developed highly variable chloroplast DNA molecular markers and single-copy nuclear DNA molecular markers,which can provide a reference basis for subsequent germplasm mining and species definition of Malus or Malus-related genera,and the origin and identification of begonia species.The main research results are as follows:1.Using chloroplast whole genome sequencing and assembly techniques,40 chloroplast genomes of Malus species and hybrids were successfully assembled,complementing the GenBank database.Using DNA repair and high-throughput sequencing techniques,simplified genome sequences of 39 chloroplast gene fragments and 20 single-copy nuclear genes of Malus species were obtained.2.Combining the GenBank database with the self-test database,a total of 91 chloroplast genome data sets of species of the genus Malus and outgroup were used to construct phylogenetic trees by MP,ML and BI methods,respectively,to classify the major species of the genus Malus into Subgen.Eriolobus G.Z.Qian:Sect.Chloromeles(Dence)Rehd.,Sect.Florentinus(Rehd.)M.H.Cheng ex G.Z.,Sect.Eriolobus(Seringe)Schneid..And Subgen.Malus G.Z.Qian:Sect.Yunnanensis G.Z.Qian,Sect.Docyniopsis Schneid.,Sect.Sorbomalus Zabel ex Schneid.,Sect.Gymnomeles Koehne emend.Langenf.and Sect.Malus.Which agree with Qian Guanze’s new taxonomy system.3.In this study,using the newly developed 10 highly variable chloroplast DNA molecular markers CPF05-1(psaⅠ-ycf4),CPG01-1(psbA-trnK),CPG32-2(ycf1),CPG02-1(rps16),CPG03-1(rps16-trnQ)and CPG06-2(atpH-atpⅠ),CPG08-1(rpoC1),CPG09-1(rpoB-trnC),CPG21-2(petA-psbJ)and CPG32-1(ycf1),were combined to reconstruct the phylogenetic relationships of high-coverage sampled Malus species and to test the results of chloroplast genomics analyses.The association of these 10 highly variable chloroplast DNA molecular markers was also found to be useful for the definition of the Sect.Docyniopsis Schneid.,Sect.Yunnanensis G.Z.Qian,Sect.Chloromeles(Dence)Rehd.,Sect,Florentinus(Rehd.)M.H.Cheng ex G.Z.and Sect.Eriolobus(Seringe)Schneid.,but the delineation of subgenus rank is still recommended by the results of the chloroplast genomics analysis.4.The three newly developed highly variable single-copy nuclear DNA markers(E7514-1,E7669-1 and E7771-1)have a much higher resolution than the chloroplast DNA markers,and when the three highly variable single-copy nuclear DNA markers are analysed together,the subgeneric hierarchy under the genus Malus is in general agreement with the results of the chloroplast-based histological delimitation.It was verified that the Subgen.Malus G.Z.Qian should contain the Sect.Yunnanensis G.Z.Qian,Sect.Sorbomalus Zabel ex Schneid.,Sect.Malus and Sect.Gymnomeles Koehne emend.,and that the Subgen.Eriolobus G.Z.Qian should contain the Sect.Eriolobus(Seringe)Schneid.,Sect Florehtinus(Rehd.)M.H.Cheng ex G.Z.Qian,Sect.Docyniopsis Schneid.,and Sect.Chloromeles(Dence)Rehd..The division of subgeneric groups is in full agreement with the traditional taxonomic classification of Qian Guanze.5.The combination of three highly variable single-copy nuclear DNA markers enabled the identification of four species in the Sect.Yunnanensis G.Z.Qian(M.yunnanensis,M.ombrophila,M.prattii,M.honanensis),Sect.Chloromeles(Dence)Rehd.(M sylvestris,M.sieversii),two species in the Sect.Sorbomalus Zabel(M.kansuensis,M.toringoides),four species in the Sect.Gymnomeles Koehne emend.Langenf.(M.rockii,M.baccata,M.toringo,M hupehensis)and two species in the Sect.Docyniopsis Schneid.(M.doumeri,M.tschonoskii).A total of five groups and 14 species are clearly defined to each other,and it is possible to identify some of the begonia species and to make a preliminary study of their origin.