Study On Molecular Phylogeny Of The Genus Rubus From Southwest China And The Genetic Relationship Between Wild Excellent Germplasm And Introduced Cultivars

The genus Rubus belongs to the family Rosaceae and comprises 750～1000 species in the world,of which 201 species and 98 varieties have been found in China.Considered to be the major center of distribution and diversity of Rubus,Southwest China is exceptionally abundant in Rubus species,varieties and genotypes,from herbaceous plants to woody plants.However,the germplasm resources in this region were little-known and had not to be utilized,for the most important reason was that there was almost no deep study on them.Therefore,it is quite necessary to study the phylogeny and genetic diversity of Rubus distributed in this region,which will be beneficial to further utilization effectively. In this study,more than 30 species collected in Southwest China and 17 cultivars introduced from foreign countries were used as materials.The phylogenetic relationships of wild materials were revealed using sequencing techniques in nrDNA and cpDNA,and the genetic diversity between excellent wild germplasm and introduced cultivars were analyzed using molecular markers.The main results were as follow:1.The internal transcribed spacer(ITS) regions of nuclear ribosomal DNA(including ITS1,5.8S rRNA and ITS2) of 34 species or accessions from Sect.Malachobatus and Sect. ldaeobatus,representing 14 subsections of Rubus were sequenced.The results showed:(1) Length variation for the entire ITS region ranged from 627 to 632 bp.The ITS1 region (254-260bp) was longer than ITS2(207-211 bp),and the GC content of ITS1(55.09%) was slightly lower than ITS2(57.29%).(2) Together with the ITS sequences Rosa and Geum downloaded from GenBank as outgroups,the phylogenetic relationships within and between the 34 Rubus species were explored by employing maximum parsimony analysis using PAUP software.The length of complete ITS region was 639 sites after alignment, with 120 variable sites and 83 parsimony informative sites.The ITS-based tree inferred that Sect.Malachobatus was well-supported monophyletic group,and Sect.Idaeobatus was polyphyletic with five lineages.R.ellipticus and its variety species R.ellipticus var. obcordatus clustered separately.However,R.pinfaensis(Subsect.Pungentes) and R. ellipticus(Subsect.Stimulantes) formed into one group.(3) Ten sequences of other 5 sections of Rubus were downloaded from GenBank as comparison.The results showed that Sect.Malachobatus plus Sect.Dalibardastrum formed a strongly supported monophyletic group.Sect.Idaeobatus and Sect.Cylactis had close relationship.Sect.Chamaemorus had relatively far relationship with other sections,for it located at the basal clade of the ITS tree.The phylogenetic relationship of the materials were discussed according to the ITS data combining with their morphological characteristics and chromosome numbers.2.The trnL-trnF region of 33 species or accessions from Sect.Malachobatus and Sect. Idaeobatus,representing 13 subsections of Rubus were sequenced.The results showed:(1) Length variation for the entire trnL-trnF region ranged from 954 to 998 bp.The mean GC content was 34%.(2) Together with the trnL-trnF sequences Rosa and Geum downloaded from GenBank as outgroups,the phylogenetic relationships within and between the 33 Rubus species were explored by employing maximum parsimony analysis using PAUP software.The length oftrnL-trnF was 1121 sites after alignment,with 191 variable sites and 57 parsimony informative sites.The topological structure of trnL-trnF-based tree was very similar to ITS-based tree.It also supported that Sect.Malachobatus formed a monophyletic group and Sect.Idaeobatus was polyphyletic with five lineages,although some species had different position on the tree.(3) Ten sequences of 4 sections of Rubus were downloaded from GenBank as comparison.The results showed that Sect. Malachobatus plus Sect.Dalibardastrum and some species of Sect.Chamaebatus formed a strongly supported monophyletic group.Sect.Cylactis had parallel relationship with Sect. Malachobatus.(4) We combined ITS and trnL-trnF together to constructed a parsimony tree.It showed that the topological structure of ITS+trnL-trnF-based tree was very similar to the tree based on ITS data or trnL-trnF data separately.The phylogenetic relationships of the materials were discussed according to their morphological characteristics and chromosome numbers.3.Total 29 samples including 17 bramble cultivars introduced abroad and 12 wild excellent Rubus germplasm resources were analyzed by using inter-simple sequence repeat (ISSR).The results showed:(1) By orthogonal design,the satisfactory ISSR reaction system was established,i.e.a total volume of 25μl system,it contained 1×PCR Buffer, 2mmol/L Mg²⁺,1.5U Taq DNA polymerase,0.2μmol/L primer,0.4 mmol/L dNTP and 20ng DNA template.(2) Of the 60 primers screened,10 primers exhibited sufficient polymorphic band patterns.A total of 285 ISSR bands were generated by 10 selected primers,among which 278 bands were polymorphic.The average number of band patterns observed per primer was 28.5 with a range from 21 to 37.The results showed that the genetic relationship between Sect.ldaeobatus and Raspberry cultivars was close,and Sect. Malachobatus had relatively far relationship with other materials.The cluster analysis results were consisted with the morphological classification.