Analyse The Genetic Relationship Of The Native Populus In Sichuan By ISSR And ITS Sequences

Poplar is the general designation of Populus species of Salicaceae and is a kind of deciduous tree. All over the world Poplar contains about100species,which is widely distributed in Europe, Asia and North America,also is one of the most ancient plants. China has rich genetic resources of poplars,Especially in southwest of China,There are much endemic species and native poplar resource,Meanwhile,This area was also considered one of the centers of the poplar variation area.But,In this region the poplar of classification is mainly confined to morphological characteristics.In recent years, all kinds of molecule marker technology were widely known and used. But,There are still many differences and doubt on the classification system and the evolution of relationship in Poplar in this area. In this article, ISSR markers and the rDNA of ITS region sequence(not including5.8S rDNA region sequence)analysis were used to investigate the genetic relationships of22Sichuan native poplar species or hybrid species from the sections of Populus, Tacamahaca and Leucoides.The results are as follows:(1) In the essay, the method named improved CTAB was used to extract the DNA from the native poplar in Sichuan for the experiment analysis.The result of agarose gel electrophoresis testing indicated that the DNA banding patterns were clear and complete, no degradation and protein polution,even with the some of RNA polution would not have an effect on the issr analysis and the ITS region sequence research.(2) Ten out of60ISSR primers were used for amplification. A total of866bands were generated,of which806bands were polymorphic,the percentage of polymorphism sites was93.19%. The most of amplification bands was primer812which was generated134bands;The lowest of amplification bands was813which was generated48bands; the lowest percentage of polymorphism sites was836(59.46%);The average of amplification bands was86.6, And the average of polymorphic bands was80.7(93.62%).(3) In the study, the length of ITS region(exincluding5.8S rDNA region) sequence is429.7bp(ITS1for220.5bp,ITS2for209.5bp)in average;P.tibetica has8bases deletion in ITS1;3species(P cathayana、P. schneideri、P. kangdingensis) and3hybrid species(P. szechuanica×P. cathayana、P. cathayana×P. deltoides、"Erlangshan poplar"×R cathayana)in ITS2have9bases deletion.According to all of the samples in ITS region sequence(exincluding5.8S rDNA region),the base frequencies remains different in different species,which contains A(14.1%-17.1%,14.8%in average), T(15.0%-16.7%,15.7%in average),G(30.3%-33.3%,32.8%in average),C(35.9%-37.4%,36.7%in average).The content of G+C is from66.2%(P.xiangchengensis)to70.4%(P. pseudoglauca and P. trinervis×P. deltoides),with average of68.8%.(4) Compared with the classically morphologic taxonomy,the result of ISSR cluster analysis among sections in the study was consistent,But the analysis of rDNA with ITS sequences can not effectively distinguish the genetic relationship of these species,Probably because of the fewer of Parsim-informative sites of rDNA ITS of poplar.Combined with morphology, analysis of ISSR Molecule Marker Technology and the sequences of rDNA ITS region, the results are as follow:One of the "Erlangshan poplar"and "Zajiao poplar2"have the same parent which is P. pseudoglauca, and it is reasonable that P. pseudoglauca belongs to the section of Tacamahaca;P. schneideri is not the varietas of the P. kangdingensis,But is a natural hybrid with the paternal is P. cathayana and the maternal is P. kangdingensis; P. tibetica is also not a varietas of the P. schneideri, But is a hybrid by P. xiangchengensis and P. kangdingensis;P. gonggaensis should be a species that two or more species in the section of Tacamahaca crossed firstly and formed a new species,then crossed with the species in the section of Leucoides (take P.lasiocarpa for example),Whether P. gonggaensis belongs to the section of Leucoides nor Tacamahaca should be further proofed; Compared with Pxiangchengensis in genetic relationship, has closer relationship with P. pseudoglauca, P. szechuanica, P.yunnanensis and P. kangdingensis, But it is still doubtful that "Kangding Xiangcheng hybrid poplar" is the offspring of P. kangdingensis and P.xiangchengensis or not should be proofed further. Even though, in the morphological characteristics Butuo poplar is very similar to P.lasiocarpa, But it is indicated that it has close paternal relationship with P. adenopada (P. szechuanica take the second place) by the ISSR cluster analysis,Compared with P.lasiocarpa in rDNA clustering analysis, Butuo poplar has close relationship with P. yunnanensis,so Butuo poplar in the relationship of species and family should be proofed further too.