Studies On The Genetic Diversity Of Ramie (Boehmeria Nivea L. Gaud) Germplasms And Its Molecular Markers

Ramie (Boehmeria nivea L. Gaud), one of the most important bast fiber crops, has been cultivated and used in China for a long time. With the development of market demand and fiber textile industry, fiber quality of ramie needs to be improved and elite cultivars are urgently required for ramie production. Because of many biological complexities such as natural heterozygote and cross-incompatibility, the analysis of the genetic relationships among inbred line clones is of interest not only for germplasm conservation but also for breeding purposes.Ramie inbred line clones were very important and rare breeding materials. Over 100 ramie inbred line clones had been obtained since 1980 and these would enhance the effectiveness of the breeding program. The study on the genetic diversity of ramie inbred line clones can benefit the conservation, classification, identification, genetic enhancement and effective utilization of the ramie germplasm resources. China, the native home of ramie, possesses many wild species in the genus Boehmeria, including some rare species with stress resistance and good fiber qualities that can be used for biological engineering, genetics and breeding research. The wild species in the genus Boehmeria are the important genetic resources that are not yet utilized by man. Breeding of the new cultivars with high yield, good quality and resistances to stresses and diseases are becoming more and more important and feasible, for the development of breeding techniques.The objectives of this dissertation were:1) to select the ramie materials with perfect agronomic and quality traits and then enrich parental lines for hybridization by field investigation and cluster analysis;2) to examine the correlations between some quality traits and agronomic traits and set up regression equations, by which some quality traits of ramie may be speculated;3) to establish RAPD, ISSR, and SRAP for ramie inbred line clones and do genetic diversity analysis;4) to select some ramie wild materials and determine the genetic diversity and relations between interspecies.Genetic marker technology designed to detect naturally occurring polymorphisms at the DNA level has become an invaluable and revolutionary tool for both applied and basic studies of ramie. In this study, to optimize genetic marker technology for ramie, 7 agronomic traits and 3 quality traits were analyzed by cluster and path analysis in 90 ramie inbred line clones. Forty ramie core inbred line clones were selected. Then, the genetic diversity of these 40 ramie inbred line clones was studied by morphologic identification and molecular marker, cluster and principal component analysis. The ITS (internal transcribed spacer) analysis and genetic diversity analysis were also done for the 45 wild materials collected in 2003-2007. The following results were obtained:1. The 90 ramie inbred line clones were clustered based on 7 agronomic traits and 3 quality traits by SAS System for windows V8.0. The 90 ramie inbred line clones were divided into 5 groups, when Average Distance was 0.5, each group consisted of 1-2 sub-groups. The results suggested that materials with high fiber fineness should be selected from ClusterIV and Cluster V, high yielding materials from Cluster II. The results showed that there were direct or indirect correlative relationships among ramie agronomic and quality traits. In addition, their path relations were established. According to their relations, many regress equations were set up.Eliminating the insignificant factors, regression models were established between agronomic characters and quality characters:y₁=2800.82-1.41x₁-525.18x₂-73.15x₃-3.20x₄+8.56x₅-675.51x₆+2614.28x₇+0.75x₈ y₂= 13.28+0.07x₁-2.28x₂+1.83x₃-0.37x₄-0.13x₅+13.95 x₆-46.78x₇-0.01x₈ y₃=4.57+0.01x₁+0.35x₂-0.01x₃-0.02x₄-0.06x₅-0.47x₆+2.04x₇y1, fiber fineness (m/g); y2, breaking strength (g); y3, breaking elongation ratio (%).x1-x8, plant height (cm); stem diameter (cm); leaf width (cm); leaf length (cm); leafstalklength (cm); leaf sawtooth width (cm); leaf sawtooth depth (cm) and rate of effective tiller(%), respectively.2. Based on preliminary test, 32 sets of 150 RAPD primers and 30 out of 85 ISSR primers, which steadily produced well-defined and scorable amplification products, showed polymorphisms in all 40 genotypes. The genomic DNAs of 40 Inbred line clones were amplified by PCR with 32 polymorphic RAPD primers, which produced 113 bands the rate of polymorphism was 78.76%. The number of polymorphic bands each primer produced was from 4 to 11. The Nei's gene diversity was 0.2317, Shannon's Information index was 0.3568, however the dice coefficient between 0.68-0.88. The presence or absence of each RAPD, SRAP and ISSR single fragment was coded by 1 or 0, respectively and was scored for a binary data matrix, which was also used to calculate Dice coefficient. Cluster analysis and dendrogram construction were performed with the SHAN program in NTSYs 2.0 software. The FIND module was used to identify all trees that could result from different choices of tied similarity or dissimilarity values. At the level of 0.74 dice coefficient, 40 ramie inbred line clones can be divided into 4 groups. The Mantel examination between cluster analysis and coefficients matrix showed good fit. The results of principal components analysis showed accumulative variance contribution of the first three principal components was 24.81%. The variance contribution of the first principal component was 10.47%. The results of hierarchical cluster analysis were not in accordance with principal components analysis.3. The genomic DNAs of 40 inbred line clones were amplified by PCR with 30 polymorphic ISSR primers, which produced 116 bands from 200bp to 2000bp bands were polymorphism. While the polymorphic of ISSR was 78.45% (91 bands out of 116), ranging from 2 (ISSR6) and 10 (ISSR22), with an average of 3.87. Pairwise comparison was made between all the inbred line clones included in this study. The Nei's gene diversity was 0.2405, Shannon's Information index was 0.3679. Based on the appearance of the markers, the genetic relationships were analyzed using UPGMA and the genetic Dice coefficients were calculated. According to the region of origin of ramie inbred line clones (these genotypes included 8 ramie inbred line clones from Brazil and 32 from 5 provinces of China) 6 groups produced. The Nei's gene diversity was between 0.0964 and 0.2285, Shannon's Information index was from 0.1408 to 0.3310. The Mantel examination between cluster analysis and coefficients matrix showed good fit. The results of principal components analysis showed accumulative variance contribution of the first three principal components was 80.15%. The variance contribution of the first principal component was 72.67%. The results of hierarchical cluster analysis were commendably in accordance with principal components analysis.4. The genomic DNAs of 35 inbred line clones were amplified by PCR with 72 polymorphic ISSR primers, which produced 386 bands were polymorphism, while the polymorphic of ISSR was 72.80%. The Nei's gene diversity of six group was between 0.0794 and 0.1742, Shannon's Information index was from 0.1159 to 0.2699. The Mantel examination between cluster analysis and coefficients matrix showed good fit. The results of principal components analysis showed accumulative variance contribution of the first three principal components was 27.58%. The variance contribution of the first principal component was 13.02%. The results of hierarchical cluster analysis were not in accordance with principal components analysis.SRAP and ISSR marker may have various applications for genetic studies and practical breeding programs in ramie. Among these ramie inbred line clones, we obtained good amplification and easily found polymorphism. So, SRAP and ISSR marker were suitable for the molecular characterization and the investigation of phylogenic relationships in ramie. Furthermore, these researches proved that the use of SRAP and ISSR approach were more efficient to examine the genetic diversity in ramie.5. Complete internal transcribed spacer (ITS) sequence and flanking regions from 48 Urticaceae species were PCR-amplified, cloned, sequenced, and their variability compared. Only the ITS1 and ITS2 regions were included in the analysis since sequence data for the 5.8S subunit were not sufficiently variable to warrant additional sequencing. Resulting DNA sequences of the entire ITS region of all samples were multiply aligned using the Clustal X program and MAGA 3.1 program, and these alignments were used for further analysis.The partial ITS sequence (542bp to 643bp) of 45 Urticaceae species and four outgroups were performed and the obtained sequences used for phylogenetic analysis. The phylogeny derived from ITS sequences estimated using N-J methods indicated that recovered four well-defined clades and monophyly of the genus Urticaceae. ITS (including ITS1 and ITS2) phylogenetic tree using the maximum parsimony method. The tree has 777 steps with CI=0.6735 and RI=0.9407. The phylogenetic relationships derived from sequences of ITS fragments corroborate the taxonomic classification of Urticaceae based on morphological characters.Group I was formed by all the 21 accessions of Boehmeria Jacq., B. clidemioides var. diffusa, B. macrophylla and B. nivea included. Group II consisted of 3 accessions of Pilea Lindl., which was used as outgroup species. Three accessions of Laportea Gaudich. collected from Lijiang, Yunnan and Shennongjia, Hubei clustered in group III. Six Girardinia Gaudich. materials and 15 Debregeasia Gaudich. materials were clustered in group IV and V respectively.38 wild materials of Boehmeria Jacq. based on RAPD analysis were in accordance with ISSR analysis.