Cheng, Chun Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 164-183). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / General Abbreviations --- p.viii / Abbreviations of Chemicals --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / Table of Contents --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Salt stress in plants --- p.1 / Chapter 1.2 --- Overview of the molecular basis of salt tolerance in plants --- p.2 / Chapter 1.2.1 --- Stress perception --- p.3 / Chapter 1.2.2 --- Signal transduction --- p.3 / Chapter 1.2.2.1 --- Protein phosphatases --- p.4 / Chapter 1.2.2.2 --- The SOS pathway for ion homeostasis --- p.4 / Chapter 1.2.3 --- DNA and RNA helicases in post-transcriptional control --- p.6 / Chapter 1.2.4 --- ROS scavengers --- p.7 / Chapter 1.2.5 --- Proteases and proteinase inhibitors --- p.8 / Chapter 1.2.6 --- Heat shock proteins (Hsps) --- p.9 / Chapter 1.2.7 --- Highlights on DnaJ/Hsp40 --- p.9 / Chapter 1.3 --- Review on functional genomics of salt stress responses in plants --- p.11 / Chapter 1.3.1 --- Genomics on model organisms --- p.12 / Chapter 1.3.2 --- Transcriptomics for identifying salt stress responsive genes --- p.12 / Chapter 1.3.2.1 --- Multiple stress transcriptome analysis --- p.13 / Chapter 1.3.2.2 --- Genome-wide transcriptome analysis on molecular crosstalk --- p.14 / Chapter 1.3.2.3 --- Tissue specific transcriptome analysis --- p.16 / Chapter 1.3.2.4 --- Comparative transcriptome analysis --- p.17 / Chapter 1.3.2.5 --- Transcriptome analysis of soybean --- p.24 / Chapter 1.3.3 --- Proteomics in plant salt stress studies --- p.26 / Chapter 1.3.4 --- Beyond the transcriptome and proteome --- p.27 / Chapter 1.4 --- Significance of using soybean germplasms for identifying salt stress responsive genes --- p.28 / Chapter 1.5 --- Objectives --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Plants, bacterial strains，and vectors" --- p.30 / Chapter 2.1.2 --- Enzymes and major chemicals --- p.33 / Chapter 2.1.3 --- Primers --- p.34 / Chapter 2.1.4 --- Commercial kits --- p.34 / Chapter 2.1.5 --- Equipment and facilities --- p.34 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- cDNA microarray analysis --- p.35 / Chapter 2.2.1.1 --- Construction of cDNA subtraction libraries --- p.35 / Chapter 2.2.1.2 --- Assembly of cDNA microarray --- p.36 / Chapter 2.2.1.3 --- External control RNA synthesis --- p.39 / Chapter 2.2.1.4 --- Probe labelling and hybridization --- p.40 / Chapter 2.2.1.5 --- Hybridization signal collection --- p.41 / Chapter 2.2.1.6 --- Image analysis --- p.41 / Chapter 2.2.1.7 --- Data analysis --- p.42 / Chapter 2.2.1.8 --- Selection of salt responsive genes using fold difference in expression --- p.45 / Chapter 2.2.1.9 --- DNA sequencing --- p.46 / Chapter 2.2.1.10 --- Real-time PCR analysis --- p.47 / Chapter 2.2.2 --- Growth conditions and treatments of plants --- p.48 / Chapter 2.2.2.1 --- Soybean for microarray hybridization and real-time PCR --- p.48 / Chapter 2.2.2.2 --- Soybean for the study of GmDNJ1 expression under ABA treatment --- p.48 / Chapter 2.2.2.3 --- Wild-type and transgenic Arabidopsis for functional analysis --- p.49 / Chapter 2.2.2.4 --- Wild-type and transgenic rice for functional analysis --- p.49 / Chapter 2.2.3 --- "DNA, RNA, and protein extraction" --- p.50 / Chapter 2.2.3.1 --- Plasmid DNA extraction from E. coli cells --- p.50 / Chapter 2.2.3.2 --- RNA extraction from plant tissues --- p.51 / Chapter 2.2.3.3 --- Soluble protein extraction from plant tissues --- p.51 / Chapter 2.2.4 --- Blot analysis --- p.51 / Chapter 2.2.4.1 --- Northern blot analysis --- p.52 / Chapter 2.2.4.2 --- Western blot analysis --- p.53 / Chapter 2.2.5 --- Subcloning of GmDNJ1 into pGEX-4T-1 --- p.53 / Chapter 2.2.5.1 --- "Restriction digestion, DNA purification and ligation" --- p.53 / Chapter 2.2.5.2 --- Transformation of competent Escherichia coli (DH5a and BL21) --- p.54 / Chapter 2.2.6 --- Luciferase refolding assay --- p.54 / Chapter 2.2.6.1 --- Culture of E. coli strain BL21 (DE3) --- p.54 / Chapter 2.2.6.2 --- Cell lysis --- p.55 / Chapter 2.2.6.3 --- Purification of the GST-GmDNJ1 fusion protein --- p.55 / Chapter 2.2.6.4 --- Quantitation of protein --- p.55 / Chapter 2.2.6.5 --- Luciferase refolding assay --- p.56 / Chapter Chapter 3 --- Results --- p.57 / Chapter 3.1 --- Overview of cDNA microarray analysis --- p.57 / Chapter 3.2 --- Identification of salt responsive genes in subtraction libraries concerning two contrasting soybean germplasms --- p.61 / Chapter 3.3 --- Data processing before selection of salt stress responsive genes --- p.75 / Chapter 3.3.1 --- M-A plots --- p.75 / Chapter 3.3.2 --- Boxplots --- p.76 / Chapter 3.3.3 --- Scatterplots --- p.76 / Chapter 3.4 --- Selection of salt responsive genes using fold difference in expression --- p.77 / Chapter 3.4.1 --- Selection of genes with differential expression between tolerant and sensitive germplasms --- p.77 / Chapter 3.4.2 --- Selection of genes with differential expression between cultivated and wild germplasms --- p.89 / Chapter 3.4.3 --- Data validation by real-time PCR analysis --- p.91 / Chapter 3.5 --- Selection of salt responsive genes using statistical tools --- p.95 / Chapter 3.5.1 --- Quantitative trait analysis for salt responsive genes --- p.95 / Chapter 3.5.2 --- Identification of salt stress correlation genes --- p.100 / Chapter 3.5.3 --- Cluster analyses --- p.104 / Chapter 3.5.3.1 --- Clustering genes --- p.104 / Chapter 3.5.3.2 --- Clustering samples --- p.108 / Chapter 3.5.4 --- Data validation by real-time PCR analysis --- p.111 / Chapter 3.6 --- Summary of cDNA microarray analysis --- p.112 / Chapter 3.7 --- Studies on GmDNJ1 --- p.120 / Chapter 3.7.1 --- Sequence analysis of GmDNJ1 --- p.120 / Chapter 3.7.2 --- GmDNJ1 was induced by salt stress and ABA treatment in soybean (Glycine max) --- p.127 / Chapter 3.7.3 --- Expressing GmDNJ1 in transgenic Arabidopsis (Arabidopsis thaliana) enhances the tolerance to salt stress and dehydration stress --- p.129 / Chapter 3.7.4 --- Expressing GmDNJ1 in transgenic rice (Oryza sativa) enhances the tolerance to salt stress and dehydration stress --- p.135 / Chapter 3.7.5 --- The GmDNJ1 protein can replace DnaJ in the in vitro luciferase refolding assay --- p.141 / Chapter Chapter 4 --- Discussion --- p.145 / Chapter 4.1 --- Overview of expression profiling of the 20 soybean germplasms --- p.145 / Chapter 4.2 --- Identification of salt responsive genes from subtraction libraries --- p.146 / Chapter 4.3 --- Normalization of data from microarray experiments --- p.148 / Chapter 4.4 --- The fold difference analysis --- p.149 / Chapter 4.4.1 --- Response to stress --- p.149 / Chapter 4.4.2 --- Gene expression --- p.150 / Chapter 4.4.3 --- Molecular function --- p.150 / Chapter 4.4.4 --- Metabolic activity --- p.151 / Chapter 4.4.5 --- Cellular component --- p.152 / Chapter 4.4.6 --- Genes with 2.5-fold difference in expression between cultivated and wild germplasms --- p.153 / Chapter 4.5 --- Selection of salt responsive genes using statistical tools --- p.153 / Chapter 4.5.1 --- Quantitative trait analysis --- p.153 / Chapter 4.5.2 --- Cluster analyses --- p.154 / Chapter 4.6 --- Studies on GmDNJ1 --- p.157 / Chapter 4.6.1 --- GmDNJ1 is a good candidate for gene studies --- p.157 / Chapter 4.6.2 --- Sequence analysis of GmDNJ1 suggested it to be a DnaJ/Hsp40 homologue in soybean --- p.158 / Chapter 4.6.3 --- GmDNJ1 was induced by salt stress and ABA treatment --- p.158 / Chapter 4.6.4 --- GmDNJ1 has a higher expression in salt tolerant soybean germplasms over sensitive ones --- p.159 / Chapter 4.6.5 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic Arabidopsis --- p.159 / Chapter 4.6.6 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic rice --- p.160 / Chapter 4.6.7 --- Luciferase activity assay showed that GmDNJ 1 functioned as a DnaJ/Hsp40 in vitro --- p.161 / Chapter Chapter 5 --- Conclusion --- p.162 / References --- p.164 / Appendix I - Enzymes and major chemicals --- p.184 / Appendix II - Primers --- p.188 / Appendix III - Major commercial kits --- p.192 / Appendix IV - Major equipment and facilities --- p.193 / "Appendix V - Formulation of buffer, solution, gel, and medium" --- p.194 / Appendix VI - Plots in microarray experiments --- p.198 / Appendix VII - Clones with differential expression (>2.5-fold or >1.8-fold) between germplasms --- p.208 / Appendix VIII - Salt responsive genes revealed by quantitative trait analysis --- p.216 / Appendix IX - Supplementary data in real-time PCR analysis --- p.221 / Appendix X - Supplementary data in functional analyses --- p.233

The soybean programme in Zimbabwe is over seventy years old. However, there is lack of information on breeding gains, genetic diversity, heritability, genetic advance, combining ability, gene action and relationships between grain yield and secondary traits available for breeding. Therefore, the aim of the present study was to characterise the genetic diversity of the available germplasm, determine gene action conditioning grain yield and estimate the breeding gains that have been realised since the inception of the breeding programme. Evaluation of 42 soybean genotypes for genetic diversity conducted during 2010/11 and 2011/12 cropping seasons, using phenotypic and molecular characterisation approaches, revealed evidence of wide diversity among the genotypes. The phenotypic traits and SSR markers assigned the soybean genotypes to 8 and 15 clusters respectively. The SSR marker technique was more polymorphic, informative and highly discriminatory. The clustering pattern and relatedness from SSR data was in agreement with the pedigree data while the phenotypic clustering was divorced from pedigree data. Genotypes, G41 and G7; G41 and G1; G41 and G42 were the most divergent; therefore, they could be utilized as source germplasm in cultivar development and commercial cultivars. Investigations on breeding gains involving 42 cultivars (representing a collection of all the varieties that were released in Zimbabwe from 1940 to 2013) showed that improvement in grain yield was slowing down. However, annual genetic gain was estimated to be 47 kg ha-1 year-1 representing an annual gain of 1.67%. Furthermore, grain yield ranged from 2785 to 5020 kg ha-1. Genotypes, G16, G15, G17, G1 and G42 exhibited superior performance in grain yield and other agronomic traits and are therefore, recommended for utilisation in the hybridisation programme. Seed protein concentration decreased by 0.02 year-1 while oil increased by 0.02, 100 seed weight increased by 0.21 g year-1 over time. In addition, number of days to 95% pod maturity and pod shattering increased by 0.35 and 0.38 days year-1 respectively while lodging declined by 0.31%. Results indicated that emphasis should be refocused on grain yield to restore the original linear increase. Assessment of the magnitude of GEI and stability of 42 released cultivars was done over 13 environments and two seasons using additive main effects and multiplicative interaction, cultivar superiority and rank analyses. Results showed that environment and GEI captured larger portion of the total sum of squares, which reveals the influence of the two factors on grain yield, hence, the need for evaluating soybean genotypes in multi-environment trials and over years. Further, the data revealed that GEI was of a crossover type because of differential yield ranking of genotypes. The three stability parameters selected two genotypes, G1 and G15, as the most productive, consistent and stable, thus they could be produced in diverse environments while G2, G4, G5, G7, G16, G40, G17, G18 and G31 were identified as unstable and suitable for specific adaptation. Correlation and path analyses showed that grain yield was positively and significantly correlated with number of branches per plant, number of nodes per plant, shelling percentage, and number of days from 95% pod maturity to first pod shattering, implying that breeding and selection for these traits probably improved grain yield. Number of nodes per plant, plant height and 100 seed weight exhibited highest direct effects on grain yield while, number of nodes per plant and plant height presented the highest indirect effects on grain yield. These results demonstrated that number of nodes per plant and plant height could be recommended as reliable selection traits for developing high yielding genotypes of soybean. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.