ABSTRACT
Drought is an important abiotic stress in the tropics that highly constrains sorghum production. Sorghum landraces and wild relatives have been known to harbor sources of novel genes but there is hardly any information about their drought tolerance performance during the post flowering period based on the stay green trait. There is need to characterize this stay green expressed drought tolerance and transfer the mapped QTLs into drought susceptible farmer preferred varieties. This research aimed at identifying sorghum genotypes that have the stay green trait through phenotypic and molecular characterization and subsequently, introgress the stay green QTL from mapped donor lines into farmer preferred varieties. This characterization was done phenotypically and also with Diversity array technology (DArT) molecular markers in genotypes grown under well irrigated and induced drought stress conditions. The trials in the field were set in an alpha lattice design of 12*8 replicated three times. The backcross progenies were genotyped using DArT markers. The genotypes and water regimes used had effects on various traits and helped to identify stay green genotypes. Nine genotypes, namely OKABIR, LODOKA, IESV92043 DL, IESV21400 DL, IESV23010 DL, IESV23006 DL, AKUOR-ACHOT, GBK 016109, GBK 048156 outperformed the check varieties, B35 and E36-1 and in their relative chlorophyll content, whereas the genotypes namely, IBUSAR, LODOKA, GBK 047293 AKUOR-ACHOT, OKABIR, F6YQ212, GBK 048917 had more green leaves at maturity than B35 and E36-1 in drought induced conditions. Ten genotypes, namely, AKUOR-ACHOT, LODOKA, GBK 045827, GBK 047293, WAHI IESV23010 DL, IESV23006 DL, IESV92043 DL, GBK 016114, OKABIR that outperformed B35 when ranked using Relative chlorophyll content measurements yielded higher than both B35 and E36-1 which were the check varieties. LODOKA a landrace, recorded the highest chlorophyll content, highest number of green leaves at maturity and a yielded 2.2 tons ha-1. The accessions whose yield was higher than B35 and E36-1 and B35 and also had higher GLAM and RCC values were chosen as novel sources of stay green. The results also indicated the possibility of
finding stay-green alleles from wild genotypes with five wild genotypes, namely, GBK016114, GBK045827, GBK016109, GBK048922, GBK047293 that also clustered separately from B35 and E36-1 in the Neighbor Joining tree. The high significant positive correlation coefficients observed between the relative chlorophyll content and number of green leaves at maturity confirmed that the stay green trait was exhibited as functional stay green. High broad sense heritability estimates of the relative chlorophyll content (0.61) and the number of green leaves at maturity (0.64), indicated the influence of additive gene effects. The narrow sense heritability estimates for the quantity of green leaves at maturity (0.52) and for the relative chlorophyll content (0.45) also indicated the likelihood of a high positive response to selection. This study also identified 20 informative SNP markers that were highly polymorphic and were well distributed across the genome. The F2 genotypes from parental lines, ICSV 111 IN and LODOKA gave high general combining ability (GCA) for relative chlorophyll content and number of green leaves at maturity. Backcrossing for the stay green trait from mapped donor lines into farmer preferred varieties was successful with over 50% of the genotypes having greater than 75% recovery of the genome of the recurrent parent in the first backcross. These genotypes will form a strong basis for selection of superior drought tolerant sorghum varieties and the potential of improving susceptible sorghum genotypes for drought tolerance through marker assisted breeding.
Keywords: Sorghum, drought, stay green, diversity analysis, marker assisted backcrossing
TABLE OF CONTENTS
THE UNIVERSITY OF NAIROBI i
DECLARATION ii
DECLARATION OF ORIGINALITY iii
DEDICATION iv
ACKNOWLEGEMENTS v
TABLE OF CONTENTS vi
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS xi
GENERAL ABSTRACT xii
CHAPTER ONE: INTRODUCTION
1.1 Background information 1
1.2 Constraints in Production 2
1.3 Statement of the Problem 2
1.4 Justification of the study 3
1.5 Objectives 4
1.5.1 Specific objectives 4
1.5.2 Hypothesis 5
CHAPTER TWO: LITERATURE REVIEW
2.1 Sorghum production and utilization 6
2.2 Constraints to sorghum production 6
2.3 Drought response in Sorghum. 7
2.3.1 Drought avoidance 8
2.3.2 Drought escape 8
2.3.3 Drought tolerance 8
2.4 The stay green trait/ non-senescence 10
2.5 Wild relatives of sorghum 10
2.5.1 Morphological characterization 11
2.5.2 Molecular markers 12
2.6 Marker assisted selection 14
CHAPTER THREE; PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF SORGHUM WILD RELATIVES AND LOCAL LANDRACES
3.1 Abstract 16
3.2 Introduction 17
3.3 Materials and Methods 18
3.3.1 Plant material and experimental layout 18
3.3.2 Data collection 21
3.3.3 Drought screening 23
3.4 Statistical analysis 23
3.5 Genotyping, diversity estimation and quality control (QC) panel 24
3.6 RESULTS 25
3.6.1 enotypic variation of traits and heritability among diverse sorghum accessions 25
3.6.2 Comparison of mean values of growth-related parameters of the diverse accessions under well-watered and drought stress conditions and the effect of drought 37
3.6.3 Genetic variation among sorghum accessions 45
3.6.4 Molecular markers for Quality Control (QC) and marker-assisted backcrossing (MABC) 46
3.6.5 New sources of Drought tolerance 47
3.6.6 Genotypic and phenotypic variation and heritability estimate 49
3.6.7 Correlation Analysis 49
3.6.8 Performance of F2 Genotypes for key agronomic traits under drought stress conditions 51
3.7 Narrow Sense Heritability 56
3.8 DISCUSSSION 57
3.9 CONCLUSION 62
CHAPTER FOUR; MARKER ASSISTED BACKCROSSING TO INTROGRESS STAY GREEN FROM MAPPED DONOR LINES INTO FARMER PREFERRED VARIETIES
4.1 Abstract 63
4.2 Introduction 64
4.3 Materials and Methods 65
4.3.1 Experimental Site 65
4.3.2 Plant material 65
4.3.3 Generation of crosses 65
4.3.4 DNA extraction and DArT genotyping 66
4.4 RESULTS 66
4.5 DISCUSSION 69
4.6 CONCLUSION 70
4.7 RECCOMENDATIONS
CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDADTIONS
5.1 GENERAL DISCUSSION 71
5.2 CONCLUSION 72
5.3 RECCOMENDATIONS 72
REFERENCES 75
APPENDICES 84
Appendix I: Allele summary in the genotypes from the cross of KM1 X E36-1 BC1F1 84
Appendix II: Allele summary from the cross of KM1 X B35 BC1F1 85
Appendix III: Allele summary from the cross of GADAM X B35 BC1F1 Alleles Number Proportion Frequency 86
LIST OF TABLES
Table 2. 1: Sorghum production in Kenya 6
Table 3. 1: Sorghum genotypes used in the study 18
Table 3. 2: Agronomic traits measured in the study 22
Table 3. 3: 26
Table 3. 4: ANOVA of mean squares across 37 sorghum genotypes under well-watered conditions 26
Table 3. 5: Combined ANOVA of mean squares under well-watered and drought stress conditions 27
Table 3. 6: Mean comparisons for growth related parameters in the diverse genotypes and effect of drought stress 39
Table 3. 7: Mean comparisons for yield related parameters in the diverse genotypes 43
Table 3. 8. The selected set of 20 most informative SNP markers for the 38 accessions 46
Table 3. 9: Heritability, Phenotypic and genotypic variation estimates of all traits measured under drought stress conditions 49
Table 3. 10: Phenotypic correlations of the traits under drought stress conditions 51
Table 3. 11: Mean performance of F2 genotypes under drought stress conditions 52
Table 3. 12: General combining ability estimates 54
Table 3. 13: specific combing ability effects 55
Table 3. 14: Narrow sense heritability estimates 57
Table 4. 1: Background screening of the backcross progenies 68
LIST OF FIGURES
Figure 1. 1: (Source: FAOSTAT 2018) 2
Figure 3. 1: A dendrogram illustrating two major clusters of the 38 genotypes analyzed. 45
Figure 3. 2: A dendrogram drawn using the 20 selected informative markers. Two clusters previously identified with 803 SNP markers (Figure 3.1) were still revealed 47
Figure 3. 3: Performance of 37 out of the 44 sorghum genotypes that did not senesce under water stress conditions in comparison with known stay-green sources, E36-1 and B35 as measured using RCC (A), GLAM (B) and Yield (C). 48
Figure 4. 1: A snapshot of some informative SNPS in the backcross progenies of Kari Mtama1 X B35 BC1F1 67
Figure 4. 2: A snapshot of some informative SNPS in the backcross progenies of Kari Mtama1 X E36-1 BC1F1 68
Figure 4. 3: A snapshot of some informative SNPS in the backcross progenies of Gadam X B35 BC1F1 68
LIST OF ABBREVIATIONS
AFLP Amplified fragment length polymorphism
ANOVA Analysis of variance
BC1F1 Backcross one F1
CAPS Cleaved amplification polymorphic sequences
CWR Crop wild relatives
DArT Diversity Array Technology
DFL Days to flowering
GBK Gene Bank of Kenya
GCV Genotypic coefficient of variation
GLAM Number of green leaves at maturity
HSW Hundred seed weight
ISSR Inter simple sequence repeats
FLA Flag leaf area
MABC Marker Assisted Backcrossing
MAS Marker Assisted Selection
PCR Polymerase chain reaction
PCV Phenotypic coefficient of variation
PHT Plant height
PWT Panicle weight
QTL Quantitative Trait Loci
RAPD Random amplified polymorphic DNA
RCC Relative chlorophyll content
RFLP Restriction fragment length polymorphism
RP Recurrent parent
SCAR Sequence characterized amplification region
SNP Single nucleotide polymorphism
SPAD Soil plant analysis development
SSR Simple sequence repeats markers
STG Stay Green
YLD Grain Yield
CHAPTER ONE
INTRODUCTION
1.1 Background information
Sorghum (sorghum bicolor L. Moench) is a grass that uses the C4 pathway (Kresovich et al., 2005). Sorghum is part of the Poaceae family and the Andropogonea tribe. Sorghum is diploid (2n=2x=20), belongs to the genus Sorghum together with the two perennial species Sorghum halepense (2n=4x=40) and Sorghum propinquum (2n=2x=20). Sorghum is highly diverse and it is made of five botanical races (Durra, Bicolor, Caudatum, Kafir and Guinea) characterized according to the different inflorescence types (Harlan and Dewet, 1922). In order of importance of cereal crops worldwide, sorghum is the fifth (FAO, 2005) and ranks second in the semi-arid tropics. Sorghum is important for food security (Kidanemaryam et al., 2018) for many people in Asian and sub-Saharan African countries (Mindaye et al., 2016). Sorghum provides proteins, vitamins and minerals (Kumar et al., 2011), this could be due to its wide adaptability in comparison to other field crops like wheat and maize (Ali et al., 2009).
Over the last seven years, global sorghum production has fluctuated between 57 and 66 MMT. The USA currently leads with an annual output of around 9 million metric tons, followed by Nigeria (6.9 MMT), Ethiopia (5.0 MMT), Mexico (5.0 MMT), India (4.5 MMT), and China (3.6 MMT) although by area, more than 90% of the world's sorghum is from the developing countries, in Africa and Asia (USDA 2019). The top sorghum producing countries in Africa
are Nigeria (6.9 MMT), Ethiopia (5.2 MMT), Sudan (4.0 MMT), Niger (1.9 MMT) and Burkina Faso (1.8 MMT), Kenya produces 0.15 MMT (USDA 2019).
1.2 Constraints in Production
Sorghum production is limited by biotic and abiotic constraints, numerous pests and diseases, water deficit and low soil fertility (Orr et al., 2020). Together these may significantly reduce yields. To address most of these constraints, genetic enhancement through exploiting host plant resistance is the best approach used that forms a basis for integrated control programs (Olembo, 2010).
Drought is a very significant cause of crop yield losses (Boyer and Westgate, 2004), drought prone areas are apparently where most of the resource poor farmers are found. In these areas with moisture and temperature stress, sorghum and millets are important crops due to their ability to cope (Atokple, 2003). Sorghum is better adapted to drought prone areas, extensive studies on drought tolerance in sorghum have been done (Blum, 1979; Doggett, 1988), therefore making it a model crop used in studies for various mechanisms of drought tolerance.
1.3 Statement of the Problem
Drought is a complex natural hazard which affects all climates and results in socio-economic impacts, the extent of which varies depending on several factors and conditions. Agriculture is the first and most drought affected sector.
According to (FAO, 2020), a direct impact of drought is the reduced water levels which cause reduced crop productivity. A reduction in crop productivity usually impacts the livelihoods of local populations resulting in less income for farmers, hunger and mass starvation, increased food prices, unemployment, and migration. Responding to drought after the impacts have taken their toll is commonly referred to as crisis management. It is known to be untimely, poorly coordinated and ineffective (FAO, 2020).
Drought in the tropics has significantly limited the sorghum yield potential (Kidanemaryam et al., 2018). Changes in climate are definitely going to accelerate the occurrence and intensity of episodes of drought in many African countries. For example, by 2050, limited water availability is expected to affect about a large proportion of the population which will lead to severe food insecurity (UNESCO, 2017). Drought is unpredictable, it can happen during any crop stage, the stage of anthesis and grain filling are the most critical stages in sorghum, drought occurring at this point is able to cause severe yield losses. For sorghum, drought tolerance is quantified by the plants ability to avoid senescing prematurely often called stay- green which most sorghum genotypes currently lack.
1.4 Justification of the study
Sorghum genotypes grown in semi-arid areas either have to deploy drought escape or inherent drought tolerance to maintain their yields in drought (Ngugi et al., 2013). The drought escape mechanism helps in managing drought stress however in most cases it is accompanied by yield penalties. An important drought tolerance trait that ensures non senescence of the leaves and consistent yields even in drought conditions is the stay green. Genotypes having this trait express it during post flowering drought tolerance period by maintaining a larger leaf area which is photosynthetically active. In comparison to the non-stay green genotypes, stay green genotypes are able to continue grain filling normally under drought stress conditions.
The contribution of this trait is reported for a number of crops, its utilization has led to increased grain yields and established tolerance to drought and heat. The challenge to produce sorghum under water scarcity conditions requires integrated actions and strategies to remodel the crop genetic background and the cropping systems. Landraces and wild relatives of crops have been established to be reservoirs of useful genes for crop breeding including drought tolerance. Utilization of landraces from dry habitats has been used successfully in breeding maize for water-limited environments, and wild species that are relatives of cultivated crops have been on the agenda as possible donors for drought tolerance (Xu et al., 2009). It is important to limit depending on few stay green sources, this is the current case in many breeding programs currently. There is therefore need to gather, characterize and identify genotypes possessing the stay green trait among the wild relatives and local landraces in order to alleviate negative effects of drought stress on grain yield. These stay green alleles can be introduced into sorghum genotypes grown in drought prone agro ecologies of Eastern Africa to harness adaptation to drought. Having sorghum genotypes that have the stay green trait will improve sorghum yield under moisture stress conditions, this will limit the adverse effects of drought on food security.
1.5 Objectives
The main objective of the research was to contribute to improved sorghum production in drought prone areas in Kenya through identification of new drought tolerant genotypes among wild relatives and local landraces.
1.5.1 Specific objectives
1. To identify new stay green genotypes in sorghum wild relatives and local landraces
2. To introgress the stay-green alleles from two mapped donor sources into two drought susceptible farmer preferred varieties through marker assisted backcrossing.
1.5.2 Hypothesis
1. There is no genetic variation for drought tolerance among the sorghum wild relatives and local landraces.
2. Marker assisted backcrossing is not effective in the introgression of drought tolerance alleles from mapped donor sources into recipient farmer preferred varieties.
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