Study On Phylogenetic Relationships Of Cypripedium In West Of Sichuan Based On ITS And TrnH-psbA

Cypripedium is a perennial herb belonging to the subfam Cypripedioideae of orchidacea.They are very important wildlife and endangered plants with great ornamental and medicinal value.China is the center of diversity of the world’s Cypripedium.The genus of Cypripedium is divided into12groups and2subgroups according to the main morphological differences.However, because of the form shape is susceptible to environmental factors, the intergeneric and interspecific classification within the subfam. Cypripedioideae are Controversial somewhere.so it is urgent to supplement it with the classification research results from other areas.In this study, We selected part of the representative varieties of subfam. Cypripe-dioideae, which growth in southwest mountains of Sichuan Province,and taked ITS of nrDNA and trnH-psbA of cpDNA nucleotide sequence as the object of the research,trying to provide molecular biology basises of phylogenetic studies within subfam. Cypripedioideae and cypripedium.The main test results are as follows:1.It is easy to access the PCR amplification products of ITS and results in the genus of Cypripedium.The length of Cypripedium’s ITS sequence is about678bp with low interspecific length differences and no pseudogene.The length of Cypripedium’s trnH-psbA is about1014bp and it is varied Obviously between different species.In addition, there is a rps19gene with the length of279bp in trnH-psbA sequence.2.The intergeneric phylogenetic studies based on ITS sequence shows that:subfam. Cypripedioideae is divided into5groups Obviously.Mexipedilu, Phragmipedium and Paphiopedium belong to conduplicate-leaved group have Closer relationship. cypripedium and selenipedium with plicate-leaves have independent evolutionary status in the subfamily.3.The Interspecific phylogenetic studies based on ITS sequences shows that: Cypripedium irapeanum has an independent evolutionary statu in the Cypripedium group.It is not classified to one group with subtropicum and wardii.In addition, ITS sequences shows that Paphiopedilum.malipoense is independent outside the genus Cypripedium4.Combination of ITS and trnH-psbA sequence information shows that among Cypripedium species in southwest Sichuan C.henryi C tibeticum C.yunnanense C.franchetii and C.fasciolatum have relative close relationship.C.flavum C.guttatum and C.plectrochilum have more independent evolutionary status.All of these results are similar with morphological findings of Cribb (1997,1998). However interspecific phylogenetic relationship within section Cypripedium is Controversial. ITS and trnH-psbA support C.fasciolatum of Subsection Cypripedium closely related to cypripediums of Subsection Macrantha.we suggest that it is better to reconsider the group status of C.fasciolatum.Of course, the involved groups is not comprehensive enough, and may affect the support for interspecific phylogenetic relationship.5.ITS and trnH-psbA DNA barcoding has a strong interspecific identify in the genus Cypripedium.There may be some limitations to deal with the genetic relationship of closely related species because of the large number of insertions/deletions winth trnH-psbA.but it has important evolutionary evolutionary significance among the genus of Cypripedium.