ABSTRACT
Phaseolin (α-Phs) is the most abundant protein reserve in seeds of common beans accounting for 40 – 50 % of the total seed protein. Despite having low methionine (0.5 - 0.80 %) content, phaseolin is the primary source of amino acids in common bean seeds. More than 40 genetic variants of phaseolin differing in amino acid composition have been reported. Therefore, phaseolin gene diversity could be used as a strategy to improve the nutritional value of protein in common beans. To date, no information is available on the relationship between natural nucleotide polymorphisms of α-Phs gene and seed protein content in common beans. This study was conducted to determine natural nucleotide polymorphism in α-Phs gene and their association with protein content in dry seeds of common bean. Eleven selected common bean accessions were planted in plastic pots in the greenhouse. Young leaves of 4-week-old plants were used for extraction of genomic DNA, followed by polymerase chain reaction (PCR) amplification using primers specific to different fragments of the phaseolin gene. Amplified PCR products were sequenced, sequences edited and analysed for nucleotide polymorphisms to infer levels of genetic variability, genetic diversity indices and other evolutionary analyses including haplotype diversity, neutrality tests, linkage disequilibrium and recombination events using DNA Sequence Polymorphism (DnaSP) software. Amino acid/codon changes occurring on sequenced α-Phs gene of the common bean accessions were elucidated using Codon Code Aligner software. Dry mature seeds of the selected common bean accessions were harvested and analysed for the total protein content using Lowry protein method. The association of α-Phs gene sequence polymorphisms and protein content was determined. The full-length sequence of α-Phs gene revealed a total of 41 genetic variants which consisted of 24 single nucleotide polymorphisms (SNPs) and 17 indels/parsimony informative sites. Ninety percent of the segregated sites in the coding region of the gene resulted in non- synonymous mutations. The coding region polymorphisms classified the α-Phs gene into 9 distinct haplotypes. The full-length sequence had a nucleotide diversity of π = 0.00271. Some mutated positions of the α-Phs gene were in positive or negative linkage disequilibrium and 6 paired informative sites had a history of recombination. The computed Tajima’s D was significantly less than 0 indicating presence of purifying selection. The association analysis revealed that three non-synonymous indels on the coding region were significantly associated with protein content. The findings from this study indicate that the polymorphisms detected in α- Phs gene can be used for discrimination of the genetic relationships among common bean germplasm. The genetic variants associated with seed protein content in the common bean accessions, could be explored in molecular breeding as well as potential genetic markers in the improvement of protein content in common beans.
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGMENTS iv
TABLE OF CONTENTS vi
LIST OF TABLES x
LIST OF FIGURES xii
LIST OF APPENDICES xiv
LIST OF ABBREVIATIONS AND ACRONYMS xv
ABSTRACT
CHAPTER ONE: INTRODUCTION
1.1 Background to the study 18
1.2 Problem statement 20
1.3 Justification of the study 21
1.4 Objectives 22
1.4.1 General objective 22
1.4.2 Specific objectives 22
1.5 Null hypotheses 22
CHAPTER TWO: LITERATURE REVIEW
2.1 Worldwide domestication, distribution and production of common beans 24
2.2 Production of common bean in Kenya 27
2.3 Nutritional and economic significance of common beans 28
2.1 Major seed storage protein constituents in common beans 29
2.5 Phaseolin protein 30
2.6 The genetic diversity and variation of phaseolin gene 31
2.7 Genome architecture of the phaseolin gene 33
2.8 Genetic improvement of nutritional content of phaseolin in common bean seed 34
2.9 Genetic markers and their use in crop diversity for genetic improvement of crops 35
CHAPTER THREE: MATERIALS AND METHODS
3.1 Plant materials and establishment of plants in the glasshouse 37
3.2 Protein extraction from dry mature seeds of common bean accessions 38
3.3 Determination of seed protein content in common bean accessions 39
3.4 PCR amplification using phaseolin (α-Phs) gene-specific primers 40
3.4.1 Extraction of genomic DNA 40
3.4.2 Quantification and quality assessment of DNA 41
3.4.3 PCR amplification of the extracted DNA 42
3.5 Purification of PCR products 44
3.6 Gel electrophoresis for the PCR products 45
3.7 DNA sequencing 46
3.8 Sequence data and diversity analysis 46
3.8.1 Pre-processing (quality check of the sequences) 46
3.8.2 Assembly of reads into consensus sequence 47
3.8.3 Multiple sequence alignment 47
3.8.4 Single nucleotide polymorphism and allelic diversities 47
3.8.5 Neutrality test analysis 48
3.8.6 Linkage disequilibrium and recombination events 48
3.8.7 Phylogenetic analysis 48
3.8.8 Haplotype and gene diversity analysis 49
3.8.9 Pairwise individual genetic distances 49
3.9 Protein sequence analyses 49
3.9.1 Structural analysis of α-Phs protein 49
3.9.2 Modelling of α-Phs protein 50
3.10 Correlation analysis for the exonic polymorphisms associated with protein content.50
CHAPTER FOUR: RESULTS
4.1 Seed protein content in selected common bean accessions 51
4.2 Phaseolin gene amplification and sequencing 55
4.3 Phaseolin (α-Phs) gene sequence diversity 57
4.3.1 Phaseolin (α-Phs) gene structure 57
4.3.2 Sequence polymorphism 58
4.4 Predicted amino acid changes 63
4.5 Genetic diversity and differentiation 65
4.6 Pairwise genetic distances 67
4.7 Deviation from a standard neutral model 69
4.8 Mutation as a result of recombination 69
4.9 Phylogenetic tree analysis 70
4.10 Linkage disequilibrium 71
4.11 Estimated gene genealogy using transitive consistency score (TCS) 73
4.12 Physiochemical features (biochemical characteristics) of α-Phs in P. vulgaris 75
4.13 Secondary and tertiary structure (3D modelling) of the phaseolin protein 80
4.14 Relationship between α-Phs gene polymorphism and seed protein content 84
CHAPTER FIVE: DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 Discussion 88
5.1.1 Protein content in mature seeds of selected common bean accessions 88
5.1.2 Nucleotide diversity of phaseolin gene in selected common bean accessions 89
5.1.3 Effect of phaseolin sequence polymorphism on seed protein content 96
5.2 Conclusions 97
5.3 Recommendations 98
REFERENCES 99
APPENDICES 114
LIST OF TABLES
Table 3.1: Accession name seed size and color of common bean landraces used in the study 38
Table 3.2: Forward and reverse primers targeting fragments 1-4 of Phaseolin gene 43
Table 3.3: Forward and reverse primers targeting fragments 5-10 of Phaseolin gene 44
Table 4.1: Significance values at the 95% confidence level in protein content values among the paired common bean accessions 53
Table 4.2: Nucleotide changes in the full length α-Phs gene of P. vulgaris based on the consensus sequences 59
Table 4.3: Mutated loci on all intron regions of α-phaseolin gene for each analyzed common bean accession 60
Table 4.4: Mutation positions on exons of α-phaseolin gene for each of the analyzed common bean accessions 62
Table 4.5: Nucleotide and haplotype diversity in the coding region and conserved motifs of the α- Phs gene of P. vulgaris 66
Table 4.6 Estimates of genetic divergence between sequences 68
Table 4.7: Neutrality test statistical values on the coding region 69
Table 4.8: Significant linkage disequilibrium output on paired informative sites 73
Table 4.9: Relative frequency of predicted amino acids in α-Phs from mature seeds of 11 common bean accessions 76
Table 4.10: Biochemical characteristics/properties of predicted protein by Pritparam 78
Table 4.11: Detailed information on the secondary and tertiary structures of common bean α- Phaseolin 84
Table 4.12: Significant associations between mutation of α-Phs and protein content of common bean accessions 87
LIST OF FIGURES
Figure 2.1: Worldwide cultivation/domestication, production and distribution of common beans. 25
Figure 2.2: Common bean production in the world. 26
Figure 2.3: Two-Dimensional structure of phaseolin (C20H18O4) (Edy Susanto, 2019) 33
Figure 3.1: Seed coat colour of the various accessions used in the study 38
Figure 4.1: Protein concentration in mature seeds of 11 studied common bean accessions using Lowry method. 52
Figure 4.2: A representative gel image of PCR amplification profile for common bean accession KATB1 using 10 primer pairs targeting fragments of phaseolin gene 55
Figure 4.3: Representative chromatogram showing reads generated from sequencing of PCR product of Mbeere2 common bean accession 56
Figure 4.4: Schematic representation of the contigs of the phaseolin (α-Phs) gene 57
Figure 4.5: Gene structure (organization structure) of the Phaseolus vulgaris α-Phs gene for common bean accession KATB1 (representing α-Phs gene structure in all common bean accessions) 58
Figure 4.6: Multiple sequence alignment of the predicted protein/ amino acid sequence (345bp) of the transcript region of α- phaseolin gene from the 11 common bean accessions 64
Figure 4.7: Phylogenetic tree based on the full length α-phaseolin gene sequences inferred by Maximum Likelihood method. 71
Figure 4.8: Plot of R2 (the linkage disequilibrium statistic) versus nucleotide sequence for phaseolin gene among 11 common bean accessions 72
Figure 4.9: Haplotype networks for α-Phs using transitive consistency score (TCS) 75
Figure 4.10: Hydrophobicity values of all the amino acids according to Kyte & Doolittle, (1982). 79
Figure 4.11: Structural characteristics of α-Phs in common bean accessions 81
Figure 4.12: Protein domain prediction from the amino acid sequences predicted from the coding region 82
Figure 4.13: Three dimensional (3D) modelled structure selected common bean accessions 83
Figure 4.14: Output of association analysis 85
LIST OF APPENDICES
APPENDIX 1: DNA extraction reagents preparation 114
APPENDIX 2 : Representation of multiple sequence alignment for all consensus sequences 115
APPENDIX 3: Mutations registered across common bean accessions 104
APPENDIX 4: Linkage disequilibrium output on the entire gene 106
APPENDIX 5: Amino acid composition on all accessions 126
LIST OF ABBREVIATIONS AND ACRONYMS
% Percent
°C Degree Celsius
µL Microliter
AFLP Amplified Fragment Length Polymorphism
AM Arbuscular Mycorrhiza
BC Before Christ
BSA Bovine Serum Albumin
cDNA Complementary DNA
CEBIB Center for Biotechnology and Bioinformatics
DNA Deoxyribonucleic Acid
DnaSP DNA Sequence Polymorphism
dNTPs dwb Deoxyribonucleotide Triphosphates
Dry weight basis
EDTA Ethylenediaminetetraacetic acid
HCl Hydrochloric acid
KDa Kilodalton
M Molar
MCL Maximum Composite Likelihood
MEGA Molecular Evolutionary Genetic Analysis
mg Milligram
mL Milliliter
MLM Mixed Linear Model
MSA Multiple Sequence Alignment
MUSCLE NJ Multiple Sequence Comparison by Log- Expectation\
Neighbor joining
NCBI National Center for Biotechnology
PCR Polymerase Chain Reaction
pH Potential of Hydrogen
RFLP Restriction Fragment Length Polymorphism
Rpm Revolutions Per Minute
SDS Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SNP Single Nucleotide Polymorphisms
SSRs Simple Sequence Repeats
T CS Transitive Consistency Score
TASSEL Trait Analysis by Association
TBE UPGMA Tris Boric Ethylenediaminetetraacetic acid
Unweighted Pair Group Method with Arithmetic Mean
UV Ultra violet
V Voltage
V/v Volume by Volume
W/v Weight by Volume
WHO World Health Organization
CHAPTER ONE
INTRODUCTION
1.1 Background to the study
Protein dietary malnutrition is the most dangerous form of malnutrition affecting many people worldwide, mostly children, due to insufficient protein in the diet (Schönfeldt & Hall, 2012). Plant foods that are protein-rich such as common beans have the potential to provide solutions for malnutrition more so in low income countries in the world where there is low intake of animal protein. Common bean (Phaseolus vulgaris L.) is an important leguminous crop used by humans for direct nutritional purpose and serves as an important source of dietary protein to more than one billion people globally (Lioi et al., 2019). It serves as a major source of vegetable protein (Bitocchi et al., 2011). In addition, its seeds contain significant amounts of other valuable nutrients including vitamins, energy, fiber, minerals and low content of fat (Celmeli & Sari, 2018). Common beans are also known to have substantial health promoting properties such as reducing the risk of coronary heart disease, renal and diabetes type- II diseases, protecting against many cancer types as well as controlling overweight and obesity (Mullins & Arjmandi, 2021).
The main protein components of common beans include globulins (54 – 79 %) and albumins (12 – 30 %). However, common bean is known to have a poor balance of essential amino acids relative to human nutritional requirement. The nutritional quality of common beans is therefore considered low as a protein source due to the presence of sub-optimal amounts of Sulphur amino acids i.e. methionine, cysteine and S-homocysteine. Furthermore, common bean proteins are poorly digested even after cooking (low protein digestibility) because of the components of its protein fractions, presence of anti-nutritional compounds such as phytic acid, proteinase inhibitors as well as the presence of oligosaccharides, that are water soluble, hence causing accumulation of gas in the alimentary canal, resulting in eructation (Montoya et al., 2013).
Phaseolin (encoded by α-Phs gene) is the most abundant protein reserve in beans, constituting 40 – 50 % of the total protein content and hence a major source of amino acids in the common bean seeds (Montoya et al., 2010). Phaseolin protein is an important genetic marker specifically in the understanding of genetic- based diversity/variability of different landraces of common bean (Fuente et al., 2012). Previous studies have utilized the molecular diversity of the α-Phs locus to distinguish genetic variation/evolutionary relationships across the species and it has not been considered as a parameter to improve the protein nutritional value in the common beans using genetic approaches (Qureshi et al., 2019). More than 40 genetic variants/forms of phaseolin differing in amino acid composition have been reported both in the wild and domesticated beans based on their composition of polypeptides and the most common ones include: Tendergreen (T), Sanilac (S) and Contender (C) (Gepts et al., 1986). The genetic variability of phaseolin gene may hence be used as an opportunity to enhance protein nutritional value in common bean seeds (Yildiz et al., 2017). Modern genetic approaches for the improvement of nutritional quality of proteins in common bean seeds, require the knowledge of nucleotide diversity to understand the general composition, structure and organization of its genetic based diversity in the different landraces.
The diversity of phaseolin glycoprotein has been studied in many genotypes of common beans using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Fuente et al., 2012). Gel electrophoretic band patterns of phaseolin have revealed the diversity/variation of common beans as being organized into distinct eco geographic pools of genes mainly Mesoamerican and Andean (Bitocchi et al., 2011). In the present study, nucleotide polymorphisms of α-Phs gene was analysed among 11 common bean accessions of agronomic importance in Kenya and association of polymorphisms with seed protein content evaluated.
1.2 Problem statement
Common bean (Phaseolus vulgaris L.) is a main food legume used directly by humans as a protein source mainly because it contains high amount of protein. However, the protein quality in the different common bean germplasm is variable. For example, some common bean accessions have sub-optimal amounts of sulphur-containing essential amino acids mainly cysteine, methionine and S-homocysteine. These amino acids are important to humans ; methionine is used in initiating the synthesis of amino acids in proteins while cysteine, plays a significant role in protein-folding pathways and structure (Brosnan & Brosnan, 2006) and is a component of antioxidants such as glutathione. Phaseolin, its main seed storage protein contains only 0.5 - 0.80 % methionine content, which is below the human nutritional need (Aylor et al., 2008; Celmeli & Sari, 2018). The suggested nutritional requirements for methionine-cysteine in the human diet are between 2.5 and 2.6 % which is equal to between 26 and 25 mg methionine-cysteine gram per protein (McLarney et al., 1996; Millward, 2015).Thus depending on common bean diet entirely, especially from common bean germplasm with low protein can result in malnutrition. Previous studies have utilized variability of the α-Phs locus to distinguish genetic variation/evolutionary relationships in common bean germplasm, while the improvement on the quality of protein, has been neglected (Qureshi et al., 2019). Polymorphisms present in the α-Phs locus gene in common beans have been scarcely studied, even though its variants can be used as potential genetic markers for improving the nutritional quality in common bean germplasm. Phaseolin as a dietary protein is also faced with the problem of being poorly digested in its original form; due to its inability to be degraded by gastrointestinal tract enzymes of various monogastric animals. Common beans protein quality is further compromised by anti-nutritional compounds such as phytic acid, proteinase inhibitors and oligosaccharides that are water soluble and can cause accumulation of gas in the alimentary canal, resulting in eructation (Montoya et al., 2010).
1.3 Justification of the study
Common beans are considered to be the main legume /grain food in many low income and developing countries especially in sub-Saharan Africa and Latin America (Bitocchi et al., 2011). In order to improve the quality of seed protein in beans for nutritional enhancement; an understanding of the nucleotide diversity of α-Phs locus is essential. Seed protein content and quality varies in different common bean germplasm, with different landraces exhibiting varied protein fractions and amino acid composition (Bernal et al., 2014). Phaseolin as a main protein reserve in the beans is known to be genetically diverse. The variability /genetic-based diversity of phaseolin can be considered as an important strategy to target enhancement of protein in common bean using marker-assisted breeding or genetic engineering approaches (Montoya et al., 2010). Modern genetic improvement methods of protein quantity and quality require the knowledge of nucleotide diversity of genes encoding for protein in different common bean germplasm.
1.4 Objectives
1.4.1 General objective
To determine the nucleotide diversity of common bean phaseolin (α-Phs) gene and association with seed protein content in selected germplasm.
1.4.2 Specific objectives
The specific objectives of the study were:
(i) To determine protein content in mature seeds of selected common bean accessions of agronomic importance in Kenya
(ii) To analyse the nucleotide polymorphisms of phaseolin (α-Phs) locus in selected common bean accessions of agronomic importance in Kenya.
(iii) To evaluate the association between the nucleotide polymorphisms of common bean α- Phs gene and mature seed protein content.
1.5 Null hypotheses
(i) There is no variability in seed protein content of selected common bean accessions of agronomic importance in Kenya
(ii) There is no nucleotide polymorphism of phaseolin (α-Phs) locus in common
bean accessions of agronomic importance in Kenya.
(iii) There is no relationship between nucleotide polymorphisms of common bean α-Phs gene and seed protein content.
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