| Eucommia ulmoides Oliver(2n = 34), the single extant species of the genus Eucommia(Eucommiaceae), is strictly a dioecious perennial tree. It is an economically important plant for both herbal medicine and organic chemical industry. Chemical constituents(e.g., chlorogenic acid and flavonoids) in the bark and leaves possess many pharmacological activities such as anti-hypertension, anti-hyperglycemia, anti-oxidation and anti-mutation. The whole tree except xylem produces trans-polyisoprene which is distinguished as eucommia rubber(EU-rubber). EU-rubber is a hard rubber with thermoplasticity. E. ulmoides is cultivated for EU-rubber to be used as an important raw material in the manufacture of tyres, submarine cables, dental supplies and sealing devices. Conventional breeding of E. ulmoides has mainly focused on the selection of promising plants from existing natural populations. These selected plants were propagated vegetatively and released as clones. Recently, these cultivars were used as parents in crossbreeding. However, classical breeding often takes decades to fully evaluate and release new cultivars. The ability of E. ulmoides breeders to select promising parents for crossing, and to identify progenies with favorable combinations of characters, is hampered by the limited knowledge of the genetic basis of economically important traits. Molecular breeding methods, such as marker-assisted selection(MAS), have the potential to improve the efficiency of E. ulmoides breeding. In this study, molecular marker techniques were used to estimate the genetic diversity and relationships among E. ulmoides cultivars in China and to construct a high-density genetic linkage map. Using the genetic linkage map and phenotypic data measured over five consecutive years, quantitative trait loci(QTL) analysis was conducted for growth-related traits, phenological traits, leaf morphological characters, yield-related traits and secondary metabolite contents. Relationship between progeny growth performance and molecular marker-based genetic distances was evaluated. This provides a basis for understanding the genetic architecture of these economically important traits and for molecular E. ulmoides breeding. The main results are as follows:1. Sequence-related amplified polymorphism(SRAP), amplified fragment length polymorphism(AFLP) and inter-simple sequence repeat(ISSR) markers were used to estimate the genetic diversity and relationships among E. ulmoides cultivars in China. We found a high level of polymorphism among these cultivars. A total of 240, 192 and 150 DNA fragments were detected by 10 SRAP primer combinations, 10 AFLP primer combinations and 10 ISSR primers, among which 89.2, 65.1 and 88.0% of the fragments were polymorphic, respectively. According to the dendrogram, Qinzhong No. 3, Xiaoyexiong, Qinzhong No. 1 and Qinzhong No. 2 were distinctly grouped and showed genetic dissimilarity when compared with the rest of the cultivars. The other 15 cultivars exhibited two clusters, and there was a high similarity among E. ulmoides cultivars in the respective cluster. The results of this study will help in the selection of parents for both genome mapping and crossbreeding purposes. Furthermore, we can conclude that SRAP was a powerful tool for the generation of potential polymorphic markers, and it can be used as a main marker system to construct the high-density genetic linkage map of E. ulmoides.2. A genetic linkage map of E. ulmoides was constructed from a full-sib family using SRAP, AFLP, ISSR and SSR. The population consisted of 152 F1 individuals that resulted from the cross between a wild genotype Xiaoye and a cultivar Qinzhong No.1. In total, 2142 polymorphic markers were scored from 182 primer combinations or primers with an average of 11.8 polymorphic markers per primer combination or primers. Of these, 623(29.0%) markers showed segregation distortion(p ≤ 0.05) and were excluded from mapping. Only 1519 markers conforming to Mendelian segregation ratios were used for the construction of the genetic linkage map using Join Map 4.0. The genetic linkage map consisted of 706 markers distributed over 25 linkage groups(LG) covering 2133 c M. The number of mapped makers per LG ranged from 5 to106. The map size of the LGs ranged from 19.9 to 194.0 c M with a mean of 85.3 c M. The genetic linkage map covered approximately 89% of the estimated E. ulmoides genome with an average of 3.1 c M between adjacent markers. This genetic linkage map provides a tool for QTL analysis of economically important traits and for studies of genome in E. ulmoides.3. Analyzing the phenotypic variation, QTL analysis was applied to identify marker-trait associations for growth-related and phenological traits involving height, basal diameter, date of bud burst, date of leaf expansion, crown diameter, branch number, branching angle, branch length and branch diameter. A total of 97 QTLs were identified for the 9 traits. Phenotypic variance explained by individual QTL ranged from 8.7% to 70.5%. Of these QTLs, 60(61.9%) were significant at genome level, and others had a LOD score greater than or equal to 3. Results obtained from the Kruskal-Wallis nonparametric test revealed that 58(59.8%) flanking markers nearest to the QTL position were significant associated with these traits. Twenty-four QTL clusters that showed significant marker phenotype associations with at least two growth-related or phenological traits were found on LG1, LG3, LG5, LG7, LG9, LG10, LG12, LG14, LG18, LG20, LG21, LG22, LG23 and LG24.4. Analyzing the phenotypic variation, QTL analysis was applied to identify marker-trait associations for 12 leaf traits involving morphology, yield and secondary metabolite. The 12 leaf traits were leaf area, leaf length, leaf width, leaf length-width ratio, petiole length, number of veins, single leaf weight, number of leaves, total dry weight, chlorogenic acid content, rutin content and EU-rubber content. A total of 133 QTLs were identified for the 12 leaf traits. Phenotypic variance explained by individual QTL ranged from 8.7% to 73.7%. Of these QTLs, 62(46.6%) were significant at genome level, and others had a LOD score greater than or equal to 3. Results obtained from the Kruskal-Wallis nonparametric test revealed that 72(54.1%) flanking markers nearest to the QTL position were significant associated with these traits. Twenty-five QTL clusters that showed significant marker phenotype associations with at least two leaf traits were found on LG1, LG2, LG3, LG5, LG7 LG9, LG10, LG14, LG15, LG18, LG20 and LG22. Co-location of these QTLs may be due to pleiotropy or linkage. The instability of QTLs over years may be due to the highly diverging environmental conditions, temporal expression of the genes or maturation in trees. Three QTL regions for chlorogenic acid and rutin content were consistently identified over years. The QTL region on LG1 includes Dca2-2, Dca3-2, Dru3-2, Dru4-1 and Dru5-1. The QTL region on LG7 includes Dca3-3, Dru3-3, Dru4-2 and Dru5-2. The QTL region on LG9 includes Dca2-3, Dca4-1, Dca5-1, Dru2-3 and Dru3-5. On LG1, one QTL region(Deur2-1, Deur3-1, Deur4-1 and Deur5-1) for EU-rubber content was consistently identified over years.5. Progeny performances, variations and combining abilities for growth traits were evaluated in a factorial mating design(5×5) of E. ulmoides. Three marker systems, SRAP, AFLP and ISSR, were used to determine genetic distances between parents. Correlations of genetic distances with progeny performances, within-family coefficients of variation and specific-combining abilities were established for height and basal diameter traits. Significant linear correlations were found between progeny performances of height and genetic distances of parents based on SRAP markers or a combination of all three marker systems. This result represents a potential selection criterion in the breeding program if growth is the requested characteristic. Crosses should then be carried out between parents with a maximal genetic distance. The lack of correlations between parental genetic distances and within-family coefficients of variation or specific-combining abilities suggested that these characteristics were unpredictable. Concerning other economical important traits, investigations should focus on the identification of markers linked to QTLs involved in the expression of the characteristic that could lead to a marker-assisted selection scheme in E. ulmoides breeding programs. |
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