A Study On The Genetic Diversity And Evolution Of Malus Toringoides (Rehd.) Hughes

Malus toringoides belongs to Series kansuenses Rehd.of the genus Malus Miller of the family Rosaceae.M.toringoides is a facultative apomictic species.It shows high diversities in morphology,germplasm characteristics,and ecological habitats.Used as rootstock,M.toringoides showed high resistance to various environmental stresses such as tolerances to salt,drought,heat,cold,water-logging,and resistance to valsa mail Miyabe et Yamada and Xylaria mali Fromme,and had important effects on the grafted trees including dwarfing,early-fruiting,fruit production and quality. Therefore,M.toringoides was considered as a very important apple germplasm resource in China.In the present study,the genetic diversity and population genetic structure of M.toringoides were analyzed using morphological characters,nuclear ribosomal DNA(nrDNA) internal transcribed spacer(ITS) and chloroplast DNA (cpDNA) sequence.A total of 90 accessions collected from three natural populations (Maerkang,Xiaaba and Kehe of Aba Zang Autonomous Region,Sichuan Province, China) of M.toringoides were used as plant materials.Based on the sequence variations of nuclear ribosomal DNA(nrDNA) internal transcribed spacer(ITS),and the intergenic spacer regions of chloroplast genome,trnG-trnS and rps16-trnQ,the phylogenetic relationship,between M.toringoides and its putative parents,M. transitoria and M.kansuensis were analyzed.In addition,the molecular evolutionary pattern of the ITS sequences of M.toringoides were analyzed,and the maternal evolutionary relationship of M.toringoides to its parents were discussed.The main results obtained are summarized as follows:1.An analysis of the phenotypic diversity including 12 morphological characters (leaf length,leaf width,leaf length/leaf width ratio,dent distance,dent base distance, leaf width/dent distance ratio,leaf length/dent base distance ratio,fruit longitudinal diameter,fruit transversal diameter,fruit longitudinal diameter/fruit transversal diameter ratio,number of flowers per inflorescence,numberof styles per flower) of M. toringoides were carried out using 90 accessions collected from three natural populations.The results showed that M.toringoides was rich in morphological variations and different characters showed a different degree of variations,among which the leaves had the highest coefficient of variation(CV=22.83%).Our data also revealed that rich phenotypic variation existed both within and between populations. The results indicated that extensive variation of phenotypic traits existed within populations and among populations of M.toringoides.The phenotypic differentiation coefficient Vst was 18.92%.The high variation of phenotypic traits in M.toringoides occurred within populations(71.08%).The Shannon's diversity index was 0.335 among populations and 1.699 within population.The genetic variation of phenotypic traits within population was the main sources of genetic diversity in M.toringoides.2.Using M.angustifolia Michx.,M.ioensis Britt.,and M.doumeri Chev.as outgroups,the species-specific nucleotides of M.kansuensis and M.transitoria and the phylogenetic relationship between M.toringoides and its putative parents were analyzed using the sequences obtained in this study and those deposited in GenBank.The recombination detection of M.toringoides sequences was also carried out using RDP3beta27 computer program.Three different types of ITS sequences were found in M.toringoides:(1) type one sequences were identical to those of M.kansuensis.(2) type two sequences were identical to those of M.transitoria,and(3) type three sequences were the mosaic sequences between M.kansuensis and M.transitoria.On the gene tree,three different types of ITS sequences clustered separately with M.kansuensis (CladeA) and M.transitoria(CladeB) with strongly statistical supports.All type three ITS sequences from M.toringoides formed a subbranch within the CladeB with a bootstrap value of 94%.Recombination detection suggested that all mosaic ITS sequences were the evolutionary products of a recombination event between ITS sequences of M.kansuensis and M.transitoria.These results provided new evidence for the hybrid origin hypothesis of M.toringoides on the basis of our previous work.3.The M.transitoria specific ITS copy was detected within KH population and XB populations of M.toringoides.The M.kansuensi specific ITS copy was not detected within the two populations of M.toringoides.In addition,the mosaic sequences which showed a continuous variation pattern between M.kansuensis and M.transitoria were detected within three natural populations of M.toringoides.These results suggested that the concerted evolution within M.toringoides was unidirectional toward M.transitoria4.The average number of nucleotide differences(κ=17.597) and nucleotide diversity of M.toringoides were p=0.03359±0.00201,θ_w = 0.03874±0.00943 respectively,which were s higher than those of M.kansuensis(p=0.01893±0.00128,θ_w =0.02174±0.00917) and M.transitoria(p=0.01518±0.00203,θ_w=0.01710±0.00773). These results suggested an abundant genetic diversity in M.toringoides.The different levels of gene flow were also detected between M.kansuensis and each of M. toringoides population.The value of gene flow between M.kansuensis and MK population(Nm = 0.2662) was higher than those between M.kansuensis and KH population(Nm = 0.1824),and between M.kansuensis and XB population of M. toringoides(Nm = 0.1801).Higher gene flow between M.transitoria and each population of M.toringoides were detected(Nm＞1).Low level of inter-population genetic differentiation(Gst and Fst varied from 0.00227 to 0.00988 and 0.01896 to 0.05329,respectively ) and high gene flow(Nm ranged from 3.7252 to 15.7688＞3) were detected among the three natural populations of M.toringoides.Moreover,an analysis of molecular variance(AMOVA) of the populations of M.toringoides showed that the genetic variation mainly occurred within populations(97.41%).5.An analysis of the sequence variation between M.toringoides and its putative parents based on trnG-trnS and rps16-trnQ revealed that the cpDNA sequences of M. toringoides were similar to M.transitoria sequence.Using Prunus hortulana as outgroup,phylogenetic relationship between M.toringoides and its putative parents were analyzed based on TrnS-TrnG and TrnQ-rps16 sequences.A combined analysis of TrnS-TrnG and TrnQ-rps16 sequences was also carried out.The results obtained strongly supported that there were close maternal relationships between M.toringoides and M.transitoria.Higher genetic distance between M.kansuensis and M.toringoides than that between M.transitoria and M.toringoides were detected.There was limited variation between M.toringoides populations.An analysis of molecular variance (AMOVA) of the populations of M.toringoides based on cpDNA data showed that the genetic variation mainly occurred within populations(89.81%).