Phylogenic Relationship Of Houttuynia Thunb. Based On Internal Transcribed Spacer (ITS) Sequences Of Nuclear Ribosomal DNA

Houttuynia cordata Thunb.belongs to genus Houttuynia of Saururaceae.It has the function of antibacterial,anti-virus and diuresis,and could enhance body immunity.Sichuan Province is one of the main producing areas of H.eordata Thunb. H.cordata is the only species in Houttuynia Thunb.according to Florae Reipublicae Populafis Sinicae and Flora Sichuanica.Zhu et al.(2001) classified a new species H. emeiensis Z.Y.Zhu et S.L.Zhang in the genus Houttuynia collected at Emei mountain in Sichuan province.It was called commonly as white Yuxingcao,which was also used as vegetable and HERBA HOUTTYNIAE in Emei and Leshan regions.Houttuynia is widely spread in Sichuan and Chongqing regions of China, including 11 Cytotypes and 2 Chemotypes.There existed plentiful genetic variations among genus Houttuynia gerrnplasm resources.In this paper,were analyzed.The main results were as follows:1.The PCR product length of the entire ITS regions(including 5.8SrDNA) in Houttuynia was found to fluctuate between 670bp and 684bp.The length of ITS1 varied from 231 to 245bp,while that of ITS2 was uniformly 275bp for all accessions. The overall length of the 5.8S subunit of all Houttuynia accessions examined was also uniformly 164bp.The G+C composition of the Houttuynia ITS were similar,with variation from 55.5%to 56.9%for the entire spacer region,from 54.7%to 56.3%for ITS1,from 51.2%to 52.8%for 5.8S,and from 58.9%to 60.4%for ITS2,with an average of 56.4%,55.8%,51.8%,59.8%,respectively.2.Alignment of the entire ITS sequences from 23 accessions were analyzed.It resulted in 684 characters,of which 43 positions(7.02%) were potentially phylogenetically informative sites and 136 positions(19.9%) were variable sites The ITS 1 region had 22 informative sites(3.22%) and 56 variable sites(8.19%),while the ITS2 region had 16 informative sites(2.34%) and 40 variable sites(5.84%).There were 8 informative sites(1.17%) and 16 variable sites(2.33%)3.The lengths of amplified fragments from each intra-individual taxa are similar to each other,with the exception of W01-16,W01-100,and W01-37,which resulted from the variations in ITS1.The overall size of the ITS2 and 5.8S ribosomal RNA gene in W01-16,W01-100 and W01-37 were,respectively,275 and 164bp.Sequence analysis of the ITS1 among different sequence in W01-37 revealed sequence variability,with sizes ranging from 231 to 245bp.W01-37-9 had the shortest ITS1 length among genus Houttuynia,namely 231bp,indicating the presence of a deletion in this accession.4.The average intracytotypic genetic similarity(GS) values within 11 cytotypes(including A,B,C,D,E,F,G,H,I,J and K) were 0.0056,0.0040,0.0096, 0.0077,0.0091,0.0137,0.0092,0.0069,0.0093,0.0058,0.0089 respectively,with the average of 0.0082.The intracytotypic variable sites(42) and the intracytotypic informative sites(15) of cytotype F(chromosome number of 82) were the highest. The intracytotypic variable sites(15) and the intracytotypic informative sites(4) of cytotype B(chromosome number of 54) were the lowest.5.Alignment of the 11 different cytotypes resulted in 684 aligned characters (without gaps).A comparison of 11 different cytotypes revealed that ITS sequence of Houttuynia accessions with the chromosome number less than 72 have less variation than those with the chromosome number more than 72.The average GS between H. emeiensis and H.cordata accessions based on ITS ranged from 0.0051 between cytotype B and A(chromosome number of 36) to 0.0149 between cytotype F and A.6.The neighbor-joining distance method based on Houttuynia ITS sequences analysis with AF203628 as outgroup The accessions in this study were classified as three sole accession groups and two big groups(group A and group B) based on ITS. Group B only consisted of Houttuynia accessions with the chromosome number＞= 72,while group A consisted of almost all Houttuynia accessions,except for w01-99, w01-45 and w01-71,The accessions W01-81-1,W01-81-3 and W01-81-4 in this study were classified as there sole accession groups.This indicated the groups based on ITS GS,to some extent,associated with the chromosome number,although most accessions with the same chromosome number were separated from each other.7.Our result of phylogenetic analysis clearly demonstrated that H.emeiensis accessions could not completely separate from H.cordata accession.It could be observed that there was no significant relationship between the Houttuynia accessions ITS and their geographical distributions.The geographic distribution of the accessions from the two big groups had no definite boundary.