苎麻种质资源遗传多样性及分子标记研究
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摘要
苎麻(Boehmeria nivea L.Gaud)是我国最重要的韧皮纤维作物之一,在我国有着悠久的栽培和加工历史,加入WTO后,市场需求和麻纺工业的发展,对优良品种的需求越来越迫切。我国苎麻新品种选育虽然取得了较大的成绩,但如要进一步提高显得相当困难,其主要原因是缺乏有效的种质资源。此外,苎麻育种存在遗传背景复杂,缺少纯合的亲本材料,品种选育困难及育种周期长等问题。
高代自交无性繁殖系是苎麻杂交育种极其珍贵的亲本材料,对其进行遗传多样性研究,有利于苎麻种质资源的收集、保存、鉴定和合理利用。我国野生种质资源十分丰富,但是很多尚未开发利用。因此,苎麻野生种和栽培种野生类型种质资源的考察、搜集、保存与鉴定,对于提高我国苎麻育种水平、促进苎麻产品的开发应用、研究苎麻的亲缘关系及起源等具有重要意义。
本研究主要目的在于:(1)进行高代自交无性繁殖系的田间调查和聚类分析,筛选出农艺性状和品质性状均优良的材料,进一步丰富杂交育种亲本资源;(2)明确品质性状和农艺性状的某些内在联系,建立通过田间农艺性状调查来预测部分品质性状的回归方程;(3)建立适合苎麻自交无性繁殖系研究的RAPD、ISSR及SRAP等标记体系;进行苎麻自交无性繁殖系的遗传多样性分析,为亲本选配提供依据。(4)搜集一批苎麻野生种质资源,探索其种内、种间材料的遗传多样性和亲缘关系。
分子标记能直接从DNA水平反映种质之间的遗传差异,是研究植物遗传多样性的有效工具。主要研究结果如下:
1.应用SAS System for Windows V8.0统计分析软件,根据7个农艺性状和3个品质性状指标对供试90份自交无性繁殖系进行聚类。在Average Distance值为0.5时,可以将90份材料分为5个群,每个群又可以分为1-2亚组。研究表明在育种中,要选择纤维细度较优的自交无性繁殖系要在ClusterⅣ和ClusterⅤ两个群组种选择;而要选择产量高的育种材料则在ClusterⅡ群组内选择。通过聚类分析发现了纤维细度极优质的4份自交无性繁殖系材料(ClusterⅤ),纤维细度超过2000m/g的22份材料(ClusterⅣ),农艺性状优良,高产潜力大的13份自交无性繁殖系材料(ClusterⅡ)。
通过田间数据调查分析得知,农艺性状及品质性状之间存在相关关系。建立的农艺性状及品质性状多元一次回归方程如下:y_1=2800.82-1.41x_1-525.18x_2-73.15x_3-3.20x_4+8.56x_5-675.51x_6+2614.28x_7+0.75x_8y_2=13.28+0.07x_1-2.28x_2+1.83x_3-0.37x_4-0.13x_5+13.95x_6-46.78x_7-0.01x_8y_3=4.57+0.01x_1+0.35x_2-0.01x_3-0.02x_4-0.06x_5-0.47x_6+2.04x_7y_1表示纤维细度(m/g);y_2表示断裂强度(g);y_3表示断裂伸长率(%)。x_1-x_8分别表示株高(cm)、茎粗(cm)、功能叶片宽度(cm)、叶片长度(cm)、叶柄长(cm)、叶缘锯齿宽度(cm)、叶缘锯齿深度(cm)和有效分株率(%)。
2.优化了苎麻RAPD分子标记体系;用32个RAPD引物对40份自交无性繁殖系材料的DNA进行PCR扩增,共扩增出113条带,多态性达到78.76%,Nei's平均遗传距离为0.2317,平均Shannon指数为0.3568。Mantel检测得出的聚类结果和相似系数矩阵之间的相关系数为0.635,表明聚类结果很好的体现了种质之间的遗传关系。利用NTSYs 2.0软件进行主成分分析,累计贡献率为24.81%,第一主成分的贡献率为10.47%,结果表明主成分分析和RAPD聚类结果不是很吻合。
3.优化了苎麻ISSR分子标记体系;30条ISSR引物对40份自交无性繁殖系材料的DNA模板进行扩增和电泳检测,总条带数为116条,多态性达到78.45%,Nei's平均遗传距离为0.2405,平均Shannon指数为0.3679,每条引物扩增2-10条带不等。按照材料的地理位置,将40份自交无性繁殖系分为6个居群,多态位点百分率(pp)在23.28%-67.24%之间。Nei's遗传距离在0.0964-0.2285之间,Shannon指数在0.1408-0.3310之间。第一、第二、第三主成分的贡献率分别为72.67%、5.36%和2.11%,累计贡献率为80.15%,结果表明主成分分析与采用ISSR进行聚类的结果十分一致。
4.优化了苎麻SRAP分子标记体系;有72条引物均能扩增出有较高稳定性和重复性、丰富多态性的带型,条带总数为386条,多态性达到72.80%,Nei's平均遗传距离为0.1889,平均Shannon指数为0.2969,每条引物扩增5-11条带不等。根据材料的地理位置,将35份自交无性繁殖系分为6个居群,多态位点百分率(pp)在19.71%-58.81%之间。Nei's遗传距离在0.0794-0.1742之间,Shannon指数在0.1159-0.2699之间。第一、第二和第三主成分的贡献率分别为13.02%、8.47%和6.09%,累计贡献率为27.58%。只有部分聚类结果与采用UPGMA方法聚类得出的结果一致,研究结果表明,RAPD、ISSR和SRAP标记是进行研究苎麻自交无性繁殖系遗传多样性较好的方法。
5.对2003-2007年采集到的45份荨麻科野生材料进行ITS序列分析,以绿叶种苎麻(B.nivea var.tenacissima)、艾麻(Laportea cuspidata)以及冷水花属3个种的ITS序列作为外类群。所得序列采用Clustal X程序进行对位排列,并经手工校正。利用MEGA3.1软件对序列进行分析。启发式搜索得到了最简约树,其中一棵树树长(tree length)777步,一致性指数(consistency index,CI)为0.6735,保留指数(reserved index,RI)为0.9407。聚类分析表明全部材料可以聚为4类7组,支持采用植物学性状分类的结果,两种不同聚类方法的分析结果基本一致:
苎麻属(Boehmeria Jacq.)21份材料单独聚为一类,又可以分为3个亚组,第一亚组材料为序叶苎麻(B.clidemioides var.diffusa),其自展支持率(bootstrap values)为89%,主要采集自神农架地区;第二亚组材料为水苎麻(B.macrophylla);余下的7份白叶种苎麻(B.nivea L.)聚为一个亚组,主要包括栽培种及其栽培种野生类型。
冷水花属(Pilea Lindl.)的3份材料作为外类群单独聚为一类,自展支持率为100%。采自云南丽江和湖北神农架的3份艾麻属(Laportea Gaudich.)材料单独聚为一类。蝎子草属(Girardinia Gaudich.)6份材料聚为一类。水麻属(Debregeasia Gaudich.)3个种共15份野生材料聚为一大类。
此外,对38份野生材料进行的RAPD和ISSR聚类分析结果基本一致。
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:y_1=2800.82-1.41x_1-525.18x_2-73.15x_3-3.20x_4+8.56x_5-675.51x_6+2614.28x_7+0.75x_8 y_2= 13.28+0.07x_1-2.28x_2+1.83x_3-0.37x_4-0.13x_5+13.95 x_6-46.78x_7-0.01x_8 y_3=4.57+0.01x_1+0.35x_2-0.01x_3-0.02x_4-0.06x_5-0.47x_6+2.04x_7y1, 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.
高代自交无性繁殖系是苎麻杂交育种极其珍贵的亲本材料,对其进行遗传多样性研究,有利于苎麻种质资源的收集、保存、鉴定和合理利用。我国野生种质资源十分丰富,但是很多尚未开发利用。因此,苎麻野生种和栽培种野生类型种质资源的考察、搜集、保存与鉴定,对于提高我国苎麻育种水平、促进苎麻产品的开发应用、研究苎麻的亲缘关系及起源等具有重要意义。
本研究主要目的在于:(1)进行高代自交无性繁殖系的田间调查和聚类分析,筛选出农艺性状和品质性状均优良的材料,进一步丰富杂交育种亲本资源;(2)明确品质性状和农艺性状的某些内在联系,建立通过田间农艺性状调查来预测部分品质性状的回归方程;(3)建立适合苎麻自交无性繁殖系研究的RAPD、ISSR及SRAP等标记体系;进行苎麻自交无性繁殖系的遗传多样性分析,为亲本选配提供依据。(4)搜集一批苎麻野生种质资源,探索其种内、种间材料的遗传多样性和亲缘关系。
分子标记能直接从DNA水平反映种质之间的遗传差异,是研究植物遗传多样性的有效工具。主要研究结果如下:
1.应用SAS System for Windows V8.0统计分析软件,根据7个农艺性状和3个品质性状指标对供试90份自交无性繁殖系进行聚类。在Average Distance值为0.5时,可以将90份材料分为5个群,每个群又可以分为1-2亚组。研究表明在育种中,要选择纤维细度较优的自交无性繁殖系要在ClusterⅣ和ClusterⅤ两个群组种选择;而要选择产量高的育种材料则在ClusterⅡ群组内选择。通过聚类分析发现了纤维细度极优质的4份自交无性繁殖系材料(ClusterⅤ),纤维细度超过2000m/g的22份材料(ClusterⅣ),农艺性状优良,高产潜力大的13份自交无性繁殖系材料(ClusterⅡ)。
通过田间数据调查分析得知,农艺性状及品质性状之间存在相关关系。建立的农艺性状及品质性状多元一次回归方程如下:y_1=2800.82-1.41x_1-525.18x_2-73.15x_3-3.20x_4+8.56x_5-675.51x_6+2614.28x_7+0.75x_8y_2=13.28+0.07x_1-2.28x_2+1.83x_3-0.37x_4-0.13x_5+13.95x_6-46.78x_7-0.01x_8y_3=4.57+0.01x_1+0.35x_2-0.01x_3-0.02x_4-0.06x_5-0.47x_6+2.04x_7y_1表示纤维细度(m/g);y_2表示断裂强度(g);y_3表示断裂伸长率(%)。x_1-x_8分别表示株高(cm)、茎粗(cm)、功能叶片宽度(cm)、叶片长度(cm)、叶柄长(cm)、叶缘锯齿宽度(cm)、叶缘锯齿深度(cm)和有效分株率(%)。
2.优化了苎麻RAPD分子标记体系;用32个RAPD引物对40份自交无性繁殖系材料的DNA进行PCR扩增,共扩增出113条带,多态性达到78.76%,Nei's平均遗传距离为0.2317,平均Shannon指数为0.3568。Mantel检测得出的聚类结果和相似系数矩阵之间的相关系数为0.635,表明聚类结果很好的体现了种质之间的遗传关系。利用NTSYs 2.0软件进行主成分分析,累计贡献率为24.81%,第一主成分的贡献率为10.47%,结果表明主成分分析和RAPD聚类结果不是很吻合。
3.优化了苎麻ISSR分子标记体系;30条ISSR引物对40份自交无性繁殖系材料的DNA模板进行扩增和电泳检测,总条带数为116条,多态性达到78.45%,Nei's平均遗传距离为0.2405,平均Shannon指数为0.3679,每条引物扩增2-10条带不等。按照材料的地理位置,将40份自交无性繁殖系分为6个居群,多态位点百分率(pp)在23.28%-67.24%之间。Nei's遗传距离在0.0964-0.2285之间,Shannon指数在0.1408-0.3310之间。第一、第二、第三主成分的贡献率分别为72.67%、5.36%和2.11%,累计贡献率为80.15%,结果表明主成分分析与采用ISSR进行聚类的结果十分一致。
4.优化了苎麻SRAP分子标记体系;有72条引物均能扩增出有较高稳定性和重复性、丰富多态性的带型,条带总数为386条,多态性达到72.80%,Nei's平均遗传距离为0.1889,平均Shannon指数为0.2969,每条引物扩增5-11条带不等。根据材料的地理位置,将35份自交无性繁殖系分为6个居群,多态位点百分率(pp)在19.71%-58.81%之间。Nei's遗传距离在0.0794-0.1742之间,Shannon指数在0.1159-0.2699之间。第一、第二和第三主成分的贡献率分别为13.02%、8.47%和6.09%,累计贡献率为27.58%。只有部分聚类结果与采用UPGMA方法聚类得出的结果一致,研究结果表明,RAPD、ISSR和SRAP标记是进行研究苎麻自交无性繁殖系遗传多样性较好的方法。
5.对2003-2007年采集到的45份荨麻科野生材料进行ITS序列分析,以绿叶种苎麻(B.nivea var.tenacissima)、艾麻(Laportea cuspidata)以及冷水花属3个种的ITS序列作为外类群。所得序列采用Clustal X程序进行对位排列,并经手工校正。利用MEGA3.1软件对序列进行分析。启发式搜索得到了最简约树,其中一棵树树长(tree length)777步,一致性指数(consistency index,CI)为0.6735,保留指数(reserved index,RI)为0.9407。聚类分析表明全部材料可以聚为4类7组,支持采用植物学性状分类的结果,两种不同聚类方法的分析结果基本一致:
苎麻属(Boehmeria Jacq.)21份材料单独聚为一类,又可以分为3个亚组,第一亚组材料为序叶苎麻(B.clidemioides var.diffusa),其自展支持率(bootstrap values)为89%,主要采集自神农架地区;第二亚组材料为水苎麻(B.macrophylla);余下的7份白叶种苎麻(B.nivea L.)聚为一个亚组,主要包括栽培种及其栽培种野生类型。
冷水花属(Pilea Lindl.)的3份材料作为外类群单独聚为一类,自展支持率为100%。采自云南丽江和湖北神农架的3份艾麻属(Laportea Gaudich.)材料单独聚为一类。蝎子草属(Girardinia Gaudich.)6份材料聚为一类。水麻属(Debregeasia Gaudich.)3个种共15份野生材料聚为一大类。
此外,对38份野生材料进行的RAPD和ISSR聚类分析结果基本一致。
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:y_1=2800.82-1.41x_1-525.18x_2-73.15x_3-3.20x_4+8.56x_5-675.51x_6+2614.28x_7+0.75x_8 y_2= 13.28+0.07x_1-2.28x_2+1.83x_3-0.37x_4-0.13x_5+13.95 x_6-46.78x_7-0.01x_8 y_3=4.57+0.01x_1+0.35x_2-0.01x_3-0.02x_4-0.06x_5-0.47x_6+2.04x_7y1, 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.
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