Studies On The Genetic Relationship Of Some Species Of Malus Based On Genomic In Situ Hybridization(GISH)

As a difficult genus to classify,there exists the phenomena of interspecific hybridization and apomixis commonlyin in the plant of Malus Mill.?Rosaceae subfam Maloidean?,and M.hupehensis?Pamp.?Rehd.is regard as a typical heterologous triploid species,it's hybrid origin and classification is very important for the taxonomic study of Malus.Cytology,palynology,biochemical analysis and molecular marker techniques have all been applied in Malus,but still can not solve the problems.Therefore,it is very significative to find a direct and effective experimental method to verify the kidnip distance of various relationships,and to screen out the possible parents of M.hupehensis?Pamp.?Rehd.Wang Yan has basically determined that M.baccata?L.?Borkh is a parent of M.hupehensis?Pamp.?Rehd.var.mengshanensis G.Z.Qian and W.H.Shao by GISH.In this study,the M.hupehensis?Pamp.?Rehd.and their related species were studied by means of GISH combined with karyotype analysis,exploration the relationship among them,and obtained the evidence of hybrids origin of Malus.1.The optimal experimental conditions for the seeds germination of M.hupehensis?Pamp.?Rehd.var.mengshanensis G.Z.Qian and W.H.Shao and M.baccata?L.?Borkh were explored.deal with at 4?for 21 days and transferred to 28?,the germination rate were up to 86%and 70%respectively.GA3 with 50⁸00 mg·L^-11 concentration gradient and NAA with 10^-101 mol·L^-1concentration gradient had no obvious effect on breaking dormancy of seeds.By optimizing germination conditions of seeds,a large number of experimental materials were obtained.2.By optimized the chromosome production technique of Malus,the metaphase chromosome with better morphology and dispersibility was obtained after pretreatment the tips at 4?ice-water for 24 h or 2 mmol·L^-11 8-hydroxytryptamine for 4 h,enzymolysis the tips with 2.5%cellulase and Pectinase mixture for 90 min at 37?,Metaphase division phases of 15 taxa of the genus Malus were observed.Among them,M.sikkimensis?Wenz.?Koehne is tetraploid,M.hupehensis?Pamp.?Rehd.var.mengshanensis G.Z.Qian and W.H.Shao,M.hupehensis Rehd.var.taiensis G.Z.Qian,M.toringo De Vriese and M.toringoides?Rehd.?Hughes?nom.cons?are triploid,and the rest are diploid.The chromosome numbers of M.hupehensis Rehd.var.taiensis G.Z.Qian,Tianmu crabapple Riwa crabapple,M.purpurea and M.‘Donghongguo'were published for the first time.The karyotypes of the 15 taxa of the genus Malus are mostly belong to metacentric chromosome?m?and submetacentric chromosome?sm?,which belong to the more primitive type.The karyotype of M.ombrophila Hand.-Mazz.is 1A,which is the most primitive,and the karyotype of M.toringo De Vriese is 3B,which is the most evolutionary one.3.When total DNA of M.hupehensis?Pamp.?Rehd.var.mengshanensis G.Z.Qian et W.H.Shao as probe,hybrided with templates of Tianmu crabapple,M.hupehensis Rehd.var.taiensis G.Z.Qian,M.rockii Rehder,Malus toringo De Vriese,M.toringoides?Rehd.?Hughes?nom.cons?,M.sikkimensis?Wenz.?Koehne,Riwa crabapple,the result of GISH were that the number of homologous chromosomes was 16,18,8,20,20,30,14,the homologous chromosome ratio was 47.06%,35.29%,23.53%,39.22%,39.22%,44.12%,41.18%,respectively.When DNA probe of Tianmu crabapple hybrided with chromosome templates from other 7 simples,the result of GISH were that the number of homologous chromosomes was 24,22,14,22,24,26,12,the homologous chromosome ratio was47.06%,43.14%,41.18%,43.14%,47.06%,38.24%,35.29%,respectively.When DNA probe of M.hupehensis Rehd.var.taiensis G.Z.Qian hybrided with chromosome templates from other 7 simples the result were that the number of homologous chromosomes was 12,14,12,18,12,20,10,the homologous chromosome ratio was23.53%,41.18%,35.29%,35.29%,23.53%,29.41%,29.41%,respectively.M.toringoides?Rehd.?Hughes and M.sikkimensis?Wenz.?Koehne have high homology with M.hupehensis?Pamp.?Rehd.var.mengshanensis G.Z.Qian and W.H.Shao,this suggested that there is a close relationship between M.baccata?L.?Borkh.and M.sikkimensis?Wenz.?Koehne,M.toringoides?Rehd.?Hughes.And as varieties of M.hupehensis?Pamp.?Rehd.,although their morphological character are similar,the chromosome homology of M.hupehensis Rehd.var.taiensis G.Z.Qian and M.hupehensis?Pamp.?Rehd.var.mengshanensis G.Z.Qian and W.H.Shao seems not high.Tianmu crabapple,identified as M.hupehensis?Pamp.?Rehd.formerly,was found to be diploid,thus it was speculated that it is not the M.hupehensis?Pamp.?Rehd.,but an independent species,with name Malus tianmuensis suggested.While the hybrid ratio of Tianmu crabapple with M.hupehensis?Pamp.?Rehd.var.mengshanensis G.Z.Qian et W.H.Shao and M.hupehensis Rehd.var.taiensis G.Z.Qian were comparatively high,and it is similar to M.hupehensis Rehd.var.taiensis G.Z.Qian in shape,so can be speculated that there is a close relationship among them.In like wise,M.rockii Rehder was supported to be an independent species.There was a great difference between Riwa crabapple and M.sikkimensis?Wenz.?Koehne,which supported Riwa crabapple to be a new species.It was suggested that M.hupehensis?Pamp.?Rehd.might be multi-points origined.