Aracterization and Genetic Diversity Assessment of African Yam S

Morphological Characterization and Genetic Diversity Assessment of African Yam Bean (Sphenostylis stenocarpa (Hochst. Ex A. Rich.) Harms) Accessions in Ethiopia

Noel Ndlovu (R121740D)

University of Zimbabwe

Faculty of Agriculture

Department of Crop Science

A Research Project Submitted in Partial Fulfillment of the Requirements of the Masters

of Science (MSc.) Degree in Crop Science (Plant Breeding)

Supervisors: Prof. W. A. Gebresselasie, Dr. E. Gasura and Dr. S. Dari

June 2019

DECLARATION

UNIVERSITY OF ZIMBABWE

FACULTY OF AGRICULTURE

The undersigned certify that they have read and recommended to the Department of Crop Science for acceptance, the thesis entitled:

MORPHOLOGICAL CHARACTERIZATION AND GENETIC DIVERSITY ASSESSMENT OF AFRICAN YAM BEAN (SPHENOSTYLIS STENOCARPA (HOCHST. EX A. RICH.) HARMS) ACCESSIONS IN ETHIOPIA

Submitted by: NOEL NDLOVU in partial fulfilments of the requirements for the Master of

Science (MSc) Degree in Crop Science (Plant Breeding)

Approved by

Supervisors:

Prof. W. A. Gebresselasie: Date: 12 August 2019

Dr. E. Gasura…………………………...... Date…………………

Dr. S. Dari………………...... …………………...... Date………………….

Department chairperson:

Dr. E. Gasura…………………...... Date…………………. ABSTRACT

African Yam Bean (Sphenostylis stenocarpa (Hochst. Ex A. Rich.) Harms) (AYB) is an underutilized tropical leguminous plant which produces edible seed grain, leaf spinach and tubers. Africa yam bean possess a greater potential in alleviating food and nutritional insecurities facing Africa in the wake of climate change and its associated effects. However, limited research emphasis has been placed on the crop to establish the extent and magnitude of genetic diversity among the collected AYB accessions. Morphological characterization and genetic diversity analysis study was conducted on 169 AYB accessions sourced from the

International Institute of Tropical Agriculture (IITA), Nigeria. The main objective of the study was to determine the extent of genetic diversity and assess trait inter-relationships among AYB accessions for utilization in future breeding and germplasm conservation. The field experimental trial was conducted during the 2018/19 agricultural season at the Ethiopian

Institute of Agricultural Research (EIAR), Oromia Region, Ethiopia. The study was fitted into a 13 x 13 Alpha lattice design (0.1) with two replications. The morphological characterization experiment was conducted following the method of the IITA Reference

Guide for AYB. Data was collected on quantitative and qualitative traits; and analyzed using

R Studio Version 3.6.1, DARwin Version 6 and IBM SPSS Statistics Version 25 statistical packages. Analysis of Variance (ANOVA) indicated significant variations (p≤0.05) in AYB accessions for some morphological traits evaluated. The significant variations were observed on parameters; seed yield per plant, petiole length, terminal leaf length, days to 50% flowering and number of primary branches. African yam bean accessions with least and highest seed yield per plant recorded means of 22 g and 54 g respectively with accession TSs

357 as the highest yielder. Similarly, 100 seed weight varied widely across the studied accessions and recorded a mean value of 21.36 g. Seed grain yield per plant was highly correlated to terminal leaf length and width; and plant part pigmentation parameters.

Furthermore, 100 Seed Weight was significantly correlated to terminal leaf length (r = 0.441), leaf colour (r = 0.72) and seed yield per plant (r = 0.862). The study further revealed that, there was a negative correlation between seed yield per plant and days to 50% flowering (r =

-1.42). Principal Component Analysis (PCA) was performed to ascertain the main components explaining the total variation observed. The first five components accounted for

78.98% of total variation among accessions. The differential trait loadings on the first two

PCs showed that, the delineation of diversity among accessions emanated from seed yield per plant, 100 seed weight, main stem pigmentation, primary branches pigmentation and petiole pigmentation parameters. Hierarchical clustering analysis based on seed grain parameters grouped the tested accessions into three major clusters (at 100% similarity level) and five sub-clusters (at 50 % level of similarity). The data was further classified into four sections using the Unweighted Neighbour-Joining method according to identified characters loading to the first five principal components. The present research proved the existence of significant genetic diversity and trait association among the studied accessions of AYB. The observed variation and inter-character association should be exploited for AYB genetic advancement and conservation.

Keywords: Africa Yam Bean, Genetic Diversity, Hierarchical Cluster Analysis, Morphological Characterization, Principal Component Analysis, Unweighted Neighbour- Joining Method

iii

ACKNOWLEDGEMENTS

I would like to start by giving special thanks to God Almighty for his everlasting love and favour that has sustained me through this journey. Secondly, I want to extend my profound gratitude to the GENES EU Intra-Africa Mobility Scheme for providing the financial and technical support needed for the swift execution of this project. I would also like to express my deepest appreciation to my research supervisors Prof. W. A. Gebreselassie (Jimma

University, Ethiopia), Dr. E. Gasura and Dr. S. Dari (University of Zimbabwe) for their guidance and technical input throughout the course of the research tenure.

Furthermore, I gratefully acknowledge the University of Zimbabwe and Jimma University for providing me with the necessary skills and tools in executing the various elements of the project. The Ethiopian Institute of Agricultural Research (EIAR) and International Institute of

Tropical Agriculture (IITA) played a pivotal role in ensuring the success of the research and for that I am forever indebted to them.

I would also like to extend my hand of appreciation to the Doyo Community Small Holder

Farmers Group (Oromia Region, Ethiopia) who provided an ideal environment for me to settle and execute my due activities in their region. I would also like to shine light on Norman

Munyengwa, Tedious Choga and Tatenda Mayaya for assisting me in attending to some local administrative procedures when I was out of Zimbabwe. I feel great pleasure to also acknowledge Suzzy Shitta Ndenum and Florence Simbota for their priceless efforts to make this work a success. Lastly, I would like to express my profound gratitude to my family for their unceasing support and belief in me.

DEDICATION

This research study is dedicated to my mother (Mrs. T. Ndlovu), father (Mr. D. Ndlovu) and siblings (Nicole and Nigel). I will always strive to put a smile on your faces.

TABLE OF CONTENTS

DECLARATION...... i

ABSTRACT...... ii

ACKNOWLEDGEMENTS...... iv

DEDICATION...... v

TABLE OF CONTENTS...... vi

LIST OF TABLES...... ix

LIST OF FIGURES...... x

LIST OF APPENDICES...... xi

LIST OF ACRONYMS...... xii

CHAPTER ONE...... 1

1.0 INTRODUCTION...... 1

1.1 Background of the Study...... 1

1.2 Research Problem Statement...... 4

1.3 Justification...... 5

1.4 Objectives...... 6

1.4.1 Main Objective...... 6

1.4.2 Specific Objectives...... 6

1.5 Hypotheses...... 7

CHAPTER TWO...... 8

2.0 LITERATURE REVIEW...... 8

2.1 Africa Yam Bean: Background...... 8

2.1.1 Taxonomy and botanical description of AYB...... 9

2.1.2 Origins, Production and Distribution...... 10

2.1.3 Africa Yam Bean Dietary Diversity...... 14

2.1.4 Nutrient and Anti-nutrient composition of Africa Yam Bean...... 15

2.2 Africa Yam Bean Breeding...... 16

2.3 Characterization and Diversity Analysis of Africa Yam Bean...... 17

2.3.1 Genetic Diversity Assessment of Africa Yam Bean...... 17

2.3.2 Africa Yam Bean Characterization...... 20

CHAPTER THREE...... 22

3.0 RESEARCH METHODOLOGY...... 22

3.1 Plant Experimental Materials...... 22

3.2 Experimental Site...... 22

3.3 Experimental Design...... 22

3.4 Experimental Procedure...... 24

3.5 Data Collection...... 25

3.7 Data Analysis...... 26

CHAPTER FOUR...... 28

4.0 RESULTS...... 28

4.1 Descriptive Statistics of Morphometric Quantitative Characters...... 28

4.2 Descriptive Statistics of Qualitative Characters...... 29

4.3 Analysis of Variance (ANOVA)...... 33

4.4 Trait Correlation Analysis...... 34

4.5 Principal Component Analysis (PCA)...... 36

4.6 Grouping of Accessions based on Morphological Data...... 39

4.6.1 Hierarchical Cluster Analysis...... 39

4.6.2 Unweighted Nearest Neighbour-Joining Analysis...... 40

vii

CHAPTER FIVE...... 41

5.0 DISCUSSION...... 41

5.1 Morphological Trait Variability...... 41

5.2 Morphological Trait Correlations...... 43

5.3 Genetic Diversity Assessment of AYB Accessions...... 45

CHAPTER SIX...... 47

6.0 CONCLUSION AND RECOMMENDATIONS...... 47

6.1 Conclusion...... 47

6.2 Recommendations...... 49

REFERENCES...... 50

APPENDICES...... 60

viii

LIST OF TABLES

Table 2.1: The taxonomic data of Africa Yam Bean...... 9

Table 2.2: Comparison of AYB proximate analysis results from two different research studies...... 16

Table 3.1: List of collected AYB accessions tested in the study...... 23

Table 3.2: List of quantitative and qualitative parameters utilized in the study...... 25

Table 4.1: Descriptive Statistics of Quantitative Morphological Descriptors...... 28

Table 4.2: Analysis of Variance for Key Morphological Traits...... 33

Table 4.3: Pearson Correlation Analysis...... 35

Table 4.4: Total variance explained by PCA...... 36

LIST OF FIGURES

Figure 2.1: Centre of diversity and origin of Africa Yam Bean...... 11

Figure 2.2: Constraints on the adoption and utilization of Africa Yam Bean...... 13

Figure 2.3: Diversity Hierarchy (Source: Bhandari et al., 2017)...... 18

Figure 3.1: The established AYB Experimental Trial at EIAR Jimma Research Centre,

Ethiopia...... 26

Figure 4.1: Flower Colour Variations: Pinkish White (TSs 8)...... 30

Figure 4.2: Flower Colour Variations: Greyish Ruby (TSs 7)...... 30

Figure 4.3: Pigmentation Variations: Non-Pigmented Primary Branches (TSs 155)...... 31

Figure 4.4: Pigmentation Variations: Pigmented Primary Branches (TSs 330)...... 32

Figure 4.5: Descriptive Statistics Summary Graphs...... 32

Figure 4.6: Scree Plot for the Principal Components...... 37

Figure 4.9: PC1/PC2 Plot (Variable or Factor Map)...... 38

Figure 4.7: Hierarchical Cluster Analysis: Dendrogram using Ward Linkages...... 39

Figure 4.7: Unweighted Neighbour-Joining Cluster...... 40

LIST OF APPENDICES

Appendix 1.1: Descriptive Statistics for the Morphological Trait Data...... 60

Appendix 1.2: Principal Component Analysis Matrix...... 61

Appendix 1.3: Rotated Component Matrix...... 62

Appendix 1.4: Component Transformation Matrix...... 63

Appendix 1.5: Component Score Coefficient Matrix...... 64

Appendix 1.6: Component Plot in Rotated Space...... 65 Appendix 1.7: Predictor Space...... 66

LIST OF ACRONYMS

AFLP Amplified Fragment Length Polymorphism

ANOVA Analysis of Variance

AYB African Yam Bean

EIAR Ethiopian Institute of Agricultural Research

FAO Food and Agricultural Organization of the United Nations

GRIN Germplasm Resource Information Network

IITA International Institute of Tropical Agriculture

LIPS Low Input Production Systems

PCA Principal Component Analysis

PEM Protein Energy Malnutrition

QTL Quantitative Trait Loci

RAPD Random Amplified Polymorphic DNA

SNPs Single Nucleotide Polymorphisms

WAP Weeks After Planting

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CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the study

Food and nutritional insecurities are the major constraints facing Africa in the wake of climate change and its associated effects. The production landscape is currently witnessing low crop yields which has led to pronounced food demand and supply imbalances.

Agricultural production has been presented with a mammoth task to address the existing challenges facing resource constrained communities. The low-income groups in developing countries are at risk of malnutrition and research should provide tailored solutions in addressing this scourge. The current global agricultural research programmes are now shifting focus from staple or major crops towards orphan or under-utilized plant species in a quest of sustainable solutions to food and nutrition insecurities. The drive is further motivated by population increment forecasts, with the African citizenry expected to reach 2,5 billion by year 2050 (Tadele, 2017). The forecasted increase in population dynamics has a negative influence on food security and sustainability.

Drought conditions, poor soil fertility, insect and pest infestations are some of the major constraints derailing progress in the African smallholder crop production systems (Karaya et al., 2009; Tadele, 2017). The trending adverse climatic conditions and other limiting factors call for the adoption of sustainable novel methodologies geared towards scaling-up progress in crop improvement programmes across Africa. The drive can be enhanced by novel germplasm introductions, exploiting orphan or under-utilized crop species, population advancement, and hybrid technology (Adewale et al., 2012; Karaya et al., 2009). The majority of crops conferring a high potential in alleviating food insecurity are neglected, under-utilized and unimproved (Adewale and Kehinde, 2016; Aremu and Ibirinde, 2012).

The diversity pool is deep and rich with possibilities which can be exploited for the betterment of livelihoods in Africa.

African Yam Bean, Sphenostylis stenocarpa (Hochst. Ex A. Rich.) Harms (AYB) cultivation and utilization offers a sustainable remedy to malnutrition especially the protein deficiency associated problems (Ukegbu et al., 2015). Africa yam bean is a leguminous crop that serves a triple purpose by providing edible seed grain (bean), tuber (yam) and leaf (spinach).

Under-utilized legume crop species are an important component of the African food basket, and their continual development through establishment of new traits will certainly have a positive effect in ensuring food security, income generation and nutrition security (Dansiet al., 2012; Ritte et al., 2017). Africa yam bean is documented to have African origins and limited exploited potential (Agbolade et al., 2013; Shitta et al., 2016). It is a tropical crop which has a climbing growing habit reaching a height 3 m, and can be utilized for forming a live fence when grown on high stakes (Mgbeze and Ikhajiagbe, 2010).

Africa yam bean is more popular in the marginal regional niche markets of the world.

Production and utilization of AYB crop has only been documented in Africa (Adewale and

Odoh, 2012) with an average yielding capacity range of 2.5 – 4 Mt/ha (Akinyosoye et al.,

2017). The crop is usually grown in association with cassava, yam, sorghum, okra and maize under intercropping systems. Africa yam bean is drought, acid and low fertility tolerant and has capacity to form nitrogen-fixing nodules under Brady rhizobium bacteria inoculation.

Oagile (2005) asserted that, its ability to fix nitrogen suit well with the resource poor or low input production systems (LIPS) of Africa.

Africa yam bean is a rich source of essential amino acids, manganese, magnesium, potassium, vitamins, phosphorus and carbohydrates (Moyib et al., 2008). Nutrient component analysis on AYB has also shown traces of anti-nutritional elements which to some extent

2 limits its incorporation into dietary preparations in modern-day societies. However, communities in West and Central Africa have developed processing recipes including soaking and dehulling to reduce the level of anti-nutritional constituents to ensure palatability and acceptability for human consumption. Oagile (2005) asserted that, the anti-nutritional substances in AYB such as lectin have insecticidal properties worthy of further research exploitation in entomology experimental trial. Furthermore, the hard seed coat on AYB seeds require more cooking time (4 – 6 hours) and energy. Breeding programmes should therefore seek to reduce the anti-nutrient components and hardiness of the seed coat to ensure maximum accessibility of the crop. Despite the potent capacity of AYB limited research has been tailored to improve the crop and ensure its full utilization. The collected accessions in gene banks should therefore be exposed to genetic diversity characterization and proper identification to set a base for preceding breeding approaches.

The underlying genetic variation is a prerequisite for all crop improvement programmes

(Singh et al., 2011). The success of AYB breeding programme is hinged entirely on the available heritable genetic variability. Genetic diversity is the inherited variation in populations which is formed and maintained by evolution (Moukoumbi et al., 2011). The heritability, genetic advance and selection intensity provides a benchmark for selecting the ideal breeding methodology and tools for any crop improvement scheme (Nwofia et al.,

2013; Tumwegamire, 2011). Genetic diversity assessments within and between populations rely on biochemical, morphological and molecular markers. Moreover, genetic parameter estimates in diversity studies show the total variation and pinpoint characters of economic importance (Abdulkareem et al., 2015; Adewale et al., 2014).

Genetic diversity characterization creates an ideal platform for identifying accessions of greater value in conservation and crop improvement schemes. Morphological characterization encompasses cultivar trait observations to draw a description for comparison with reference

3 cultivars and for future parental selection. Characterization of AYB will provide an overview into the undisclosed allelic variants and earmark the performance of these genotypes under true environmental conditions which is useful in trait improvement schemes. Additionally, genetic polymorphism awareness will play a pivotal role in parental line selection and heterotic grouping of AYB accessions. It is therefore imperative, to place major emphasis on characterizing and determining the genetic diversity extent of AYB so as to promote its development, maintenance and conservation for sustainability, food and nutritional security in Africa.

1.2 Research Problem Statement

The recurrence of drought and prevalence of malnutrition incidences in African small-holder farmer communities have raised the need for exploiting the potential of orphan or underutilized crop species such Africa yam bean in alleviating the existing challenges. Africa yam bean possess a greater potential to provide an inexpensive and sustainable solution to the aforementioned challenges. At the present moment, limited work has been done on the crop to ascertain the extent and magnitude of genetic diversity among the collected accessions.

The established projects on the crop have placed precedence on nutritional and anti- nutritional content of seed components overlooking the value of morphological attributes in future breeding and germplasm conservation schemes. Furthermore, limited strides have been made in establishing the trait association and heterotic clustering or grouping of the studied accessions. Human dependence on plant species should be coupled with counter knowledge development to strike a balance on the food and nutrition sufficiency scale. Over-dependence on major staple crops is also contributing to crop diversity and variability reduction. The prominent genetic resources of AYB are declining in Africa, thus requiring urgent rescue systems to prevent imminent extinction (Centre for Underutilised Crops, 2008). Furthermore,

AYB accession management and planning for genetic resource capacity maximization has

4 not been fully established. Consequently, this has led to a limited source of genetic resources conferring high grain yield and other favourable agronomic traits. Furthermore, the lack of background information on the crop is hindering its sustainable development for the greater good of resource poor farmer communities. Ojuederie et al. (2014) asserted that, the majority of AYB accessions are conserved by small holder farmers and efforts to characterize these landraces will generally lead to subsequent cultivar development. The low uptake and production of AYB by modern middle-aged farmers can be attributed to the lack of improved cultivars on the market conferring high yields and low anti-nutritional properties. The morphological trait variation of AYB accessions has not been fully exploited and documented. Moreover, there is no improved and registered commercial varieties of AYB available for farmer uptake on the seed market. Conclusively, the development and adoption of improved AYB cultivars require the basic information on underlying heterotic patterns and estimated genetic gain values which can only be realized via genetic characterization assessments.

1.3 Justification

The proposed research intended to open a new wave of understanding with precise emphasis being placed on the genomic diversity assessment and characterization of Africa yam bean accessions. The degree of success in a AYB improvement programme depend entirely on the existing genetic diversity among the cultivated accessions and their wild relatives. Identifying the variations among AYB accessions will ultimately offer a valuable source of information on genotypes for improvement and conservation purposes. Germplasm characterization provides essential adjuncts to the conventional approaches (Oagile et al., 2007). Furthermore, characterization for genetic diversity assessment generate useful information for line development and breeding scheme planning and heterotic grouping (Tandzi et al., 2015).

International governing bodies such Food and Agricultural Organization of the United

Nations (FAO) are advocating for the adoption of sustainable, environmentally sensitive and economically feasible production systems (Karaya et al., 2009). The development and identification of climate resilient and high nutrient content crop cultivars confer a high potential in mitigating the resident challenges. Duku (2015) highlighted that, utilization of orphan and under-utilized crop species such as AYB confer a sustainable remedy for buffering environmental, financial and nutritional vulnerabilities. The morphological attributes of AYB are a key component for varietal identification and development. Genetic diversity margins provide an avenue for understanding the expected progress from a selection and aid in determining the ideal selection method for improving a particular character of interest (Mekonnen et al., 2014). To ensure success of future AYB breeding programmes the main thrust of this research is an urgent need. Moreover, the genetic diversity knowledge from this research can be used in screening and selection of desirable traits in separate AYB improvement programmes.

1.4 Objectives

1.4.1 Main Objective

To determine the extent of genetic diversity and trait inter-relationships on the collected

African yam bean accessions for utilization in future breeding and germplasm conservation.

1.4.2 Specific Objectives

a) To characterize the collected Africa yam bean accessions basing on qualitative and

quantitative agro-morphometric characters;

b) To assess the magnitude of genetic diversity among the studied African yam bean

accessions using Principal Component Analysis (PCA) and Cluster Analysis; and

6 c) To determine the extent of association between grain yield and morphological traits of

the African yam bean accessions.

1.5 Hypotheses

The following hypotheses were tested: a) Quantitative and qualitative morphological traits are key in characterizing Africa yam

bean accessions; b) There is an existing significant genetic variation among the collected Africa yam bean

accessions and; c) There is a high association between grain yield and morphological traits which can be

exploited for indirect selection for grain yield.

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 African Yam Bean: Background

African yam bean occupies the first rank among the seven Sphenostylis species basing on the economic value scale (Adewale and Dumet, 2010a; Nnamani et al., 2018). Scientific information on AYB is scanty as compared to other tropical leguminous crops such as soya bean, commercial bean and cowpeas (Adewale and Kehinde, 2016; Popoola et al., 2011).

According to the Centre for Underutilized Crops (2008), AYB is classified under the orphan or underutilized specie category and has received limited research attention over the past decades. AYB is a rich source of micro and macro-nutrients which are essential for human health (Dansi et al., 2012; Ritte et al., 2017). Incorporating AYB in to the modern food systems can aid in scaling up the alleviation of food and nutritional insecurities in resource poor communities of Africa (Adewale and Odoh, 2012; Aremu and Ibirinde, 2012; Centre for

Underutilised Crops, 2008; Duku, 2015; Shitta et al., 2016).

Africa yam bean is cultivated for its edible seeds, leaf spinach and tubers (Evanson and

Atanguma, 2015; Nwosu et al., 2014) and when grown on stakes can also be used as a live fence (Mgbeze and Ikhajiagbe, 2010a). Eneh et al. (2015) asserted that, the tubers resemble sweet and Irish potatoes. It is also grown as an ornamental plant across the globe especially in the developed world (Nwakolo, 1996). Furthermore, AYB has eco-friendly attributes which can be utilized in curbing the negative effects of environmental degradation and climate change (Nnamani et al., 2017). However, despite the known importance of AYB limited research work has been done to improve and conserve the collected accessions (Ohaegbulam et al., 2018). The under-utilization of AYB can be attributed to its low esteem and existing

8 knowledge gap with regard to its nutrient value and composition (Banigo and Kiin-Kabari,

2016; Ohaegbulam et al., 2018).

2.1.1 Taxonomy and botanical description

Aburime (2012) asserted that AYB is classified under the Fabaceae or Leguminosae family and Faboideae sub-family (Table 2.1). The genus Sphenostylis is constituted of a set of leguminous plants with dorsi-ventral cuneate style and flattened stigmatic tip (Adewale and

Odoh, 2012). S. stenocarpa is a diploid with 2n = 22 somantic counts (Popoola et al., 2011).

Table 2.1: The taxonomic data for African Yam Bean

Kingdom Plantae

Sub-kingdom Tracheobionta

Super Division Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Sub-class Rosidae

Order Fabales

Family Fabaceae

Sub-family Papilionoideae

Tribe Phaseoleae

Sub-tribe Phaseolinae

Genus Sphenostylis

Species Sphenostylisstenocarpa (Hochst. Ex A. Rich.)

Harms

Sources: Adewale and Odoh (2012); Adewale and Dumet (2010); Aburime (2012)

Africa yam bean is leguminous crop which produces edible seeds and tubers (Nwosu et al.,

2014). It grows twining vigorous vines of green or pigmented red colour (Adewale and Odoh,

2012; Adewale and Dumet, 2010a). The vines twines in a clockwise direction around the stakes to a height of three or more metres (Adewale and Dumet, 2010a). It also produce compound trifoliate leaves, linear pods and pink or purple flowers (Adewale et al., 2014).

The terminal leaflets can grow up to 140 mm long and 50 mm broad (Adewale and Odoh,

2012). The pods can vary from 12-13 cm in length and can bore 10 - 30 seeds (Evanson and

Atanguma, 2015; Nwakolo, 1996). Evanson and Atanguma (2015) asserted that, the seed colouration varies from whitish to various shades of grey, brown and black with speckling or marbling. African yam bean produce elongated tubers with the same taste as that of Irish potatoes (Moyib et al., 2008).

2.1.2 Origins, production and distribution

Africa yam bean is relatively underutilized and unpopular legume which is grown in the tropical rain-forest and sub-tropical regions of the world (Evanson and Atanguma, 2015).

Archived records have pointed to the Tropical African regions (Fig 2.1) as the main centers of AYB diversity (Adewale and Dumet, 2010a). The Germplasm Reserve Information

Network (GRIN) presented the North east tropical, East tropical, South tropical and West tropical Africa as the centers of AYB diversity (Adewale and Odoh, 2012). Production of

AYB has been widely reported in Northeast, West-central, West and South tropical Africa

(Aburime, 2012; Okeke and Eze, 2007). Adewale and Odoh (2012) asserted that, AYB production is more pronounced in Nigeria. It is largely distributed in the southern parts of

Nigeria (Eneh et al., 2015), which is an important hub of plant genetic resources for African breeding programmes (Nnamani et al., 2017). However, the plant is widely found growing in the wild forestry of Gabon, Congo, Togo, Ivory Coast and Ghana (Adesoye et al., 2012).

Small holder farmers in West Africa grow AYB as a minor crop in intercropping systems and little or no special attention is given to the crop in most instances (Klu et al., 2001).

Moreover, research surveys in Ghana have also shown that, in some localities production of

AYB is placed under the direct supervision of women and children only (Klu et al., 2001).

Fig 2.1: Centre of diversity and origin of African yam bean

Extracted from: http://www.zipcodezoo.com/Plants/S/Sphenostylis%5Fstenocarpa/Default.asp

The production of AYB relies entirely upon landrace cultivation and in some instances, collection of wild material (Oagile et al., 2007). African yam bean has a broad ecological suitability spectrum as evidenced by its tolerance to varied climatic and soil conditions

(Adewale and Odoh, 2012). It is usually grown in mixed association with cassava and yam in the Northern parts of Nigeria (Abioye et al., 2015). According to Ajayi (2011), AYB is usually planted 30 – 60 days after the emergence of major crops in mixed cropping systems.

It is usually planted between May and July in Ghana and Nigeria, and exhibits hypogeal germination within a period of 4 – 7 days after planting (Adewale and Odoh, 2012). The planting population requirements ranges from 24 000 – 25 000 plants per ha (Adewale and

Dumet, 2010a). At the present moment, studies have not placed spacing recommendations for

AYB production as a sole crop (Adewale and Odoh, 2012).

Agronomical studies on the crop has exhibited that increasing NPK fertilizer application up to 60 kg/ha enhanced vegetative and yield performance (Adewale and Odoh, 2012). Africa yam bean responds well to nutrient supplement applications from both organic and inorganic sources (Duku, 2015). The crop matures in 170 days (Abioye et al., 2015) and grows a vigorous vine which climbs to a height of approximately 3 m (Aburime, 2012). It also produces flowers with a colour range of purple, pink or green-white (Aburime, 2012). Several studies have recorded an average of 3 Mt/ha for seed yield (Akinyosoye et al., 2017) and tuber yield of 0.5 kg/plant (Adewale and Dumet, 2010a). Dansi et al. (2012) alluded that, a kilogram of AYB seed grain attracts a market price ranging from US$ 0.63 to US$ 0.75 in

Benin.

Africa yam bean is less susceptible to disease and pest infestations as compared to other major tropical legumes (Adewale and Dumet, 2010a). However, Nnamani et al. (2017) asserted that, despite the high depth of advantages of AYB, adoption and full utilization has been constrained by several challenges (presented in Fig 2.2). Cutworm (Agrotis sp.),

12 grasshopper (Zonocerus variegates) and aphids (Aphis craccivora) infestations have exerted major problems in AYB cultivation systems in West Africa (Ogah, 2011). Furthermore,

Phomaspp, Oidiumspp and Aecidumspp are the most prominent pathogens in AYB production transmitting leaf spot, powdery mildew and stem rust diseases (Adewale and

Odoh, 2012). Additionally, production is limited by the long maturation period, high demand for stakes (Adewale and Dumet, 2010a). Nwosu et al. (2014) alluded that, hard seed coat, mould growth in storage and post-harvest defects, long cooking time, photoperiod sensitivity and changes in flavour are some of the constraints limiting the wide adoption of AYB. Seed coat hardness demands more cooking time and consume more heating energy (Adewale and

Dumet, 2010a). The long cooking time requirement of 4-6 hours limit the incorporation of

AYB into daily dietary preparations in modern households (Azeke et al., 2005).

Fig 2.2: Constraints on the adoption and utilization of Africa Yam Bean. Source: Nnamani et al., (2017)

The multi-purpose capacity of AYB render it a sustainable remedy to some of the challenges facing modern-day communities (Ajayi, 2011). Medicinal properties of AYB has been documented in the management of chronic diseases such as hypertension, diabetes and cardiovascular diseases (Abioye et al., 2015; Baiyeri et al., 2018; Obasi et al., 2012). The crop products also contains high levels of anti-nutritional substances which have insecticidal properties (Eneh et al., 2015). Adewale and Dumet (2010) alluded that, the high lectin content of AYB is a biologically potent insecticide for the control of legume field and storage pests. Lectin extracted from AYB has been observed to confer 80% mortality rate of

Callosobruchus macalatus, Maruca vitrata and Clavigrallato mentosicollis insects when applied as a spray in cowpea fields (Adewale and Dumet, 2010a).

2.1.3 Africa Yam Bean Dietary Diversity

Adewale and Kehinde (2016) highlighted that, utilization of neglected crop species can enhance food security, environmental health and income generation. Consumption of the seed, leaf and tuber components of AYB are limited to the cultural and regional preferences of consumers (Ndidi et al., 2014). West Africa communities favour seeds whilst those in the

Eastern and Central African regions prefer tubers in their dietary preparations (Ndidi et al.,

2014). The tuberous roots contain more than twice protein levels to that of sweet potato

(Ipomea batatas) and Irish potato (Solanum tuberosum) (Adesoye et al., 2012).

African yam bean is utilized extensively in several dietary preparations across Africa (Ajayi,

2011). The seeds can be roasted or eaten as porridge in yam preparations (Ndidi et al., 2014).

Africa yam bean is also regarded as a perfect substitute for cowpea and can be consumed with yam, maize and rice (Eneh et al., 2015). It can also be incorporated into cassava to produce African yam bean fufu flour which is a common menu amongst Nigerians (Eneh et

14 al., 2015; Ndidi et al., 2014). Furthermore, AYB and cowpea (Vigna unguiculata) can be processed in different compositions to make moi-moi (Nwosu et al., 2014).

2.1.4 Nutrient and Anti-nutrient composition of Africa Yam Bean

Protein energy malnutrition (PEM) is peaking in African communities and efforts should be put in place to address its associated negative effects (Abioye et al., 2015; Ukegbu et al.,

2015). Ikhajiagbe and Mensah (2012) highlighted that, larger proportions of the rural populace cannot afford animal derived proteins. Africa yam bean offers an inexpensive protein source substitute for the low income and resource constrained communities (Sam,

2018). It has a high content of essential amino acids namely lysine and methionine (Adeyeye et al., 1999; Evanson and Atanguma, 2015). It also has a high crude protein content averaging 20.7 % proximate composition (Aburime, 2012). It is therefore, imperative to establish crop improvement schemes targeting the scaling-up in adoption terms of orphan crop species such AYB (Banigo and Kiin-Kabari, 2016). Adeyeye et al. (2009) and Oshodi et al. (2009) emphasized the importance of AYB in providing a reliable source of carbohydrates, fat, phosphorus and potassium (Table 2.2).

Table 2.2: Comparison of Proximate Analysis results from two different research studies

Nutrient Component Average Composition of the AYB whole seeds

Oshodi et al. (2009) Adeyeye et al. (2009)

Protein 20.50% 20.51%

Fat 8.25% 12.20%

Total carbohydrate 59.72% 50.24%

Total Ash 3.26% 2.60%

Moisture 8.10% 8.36%

Potassium 649.49 mg/100g 625.43 mg/100g

Phosphorus 241.21 mg/100g 206.35 mg/100g

2.2 African Yam Bean Breeding

Africa yam bean improvement depends entirely on identification, maintenance and utilization of the available genetic resources of the crop (Abdulkareem et al., 2015). Research projects should therefore instigate organized AYB management programmes in tandem with genetic enhancement procedures for the crop (Oagile et al., 2007; Umechuruba and Nwachukwu,

1994). Hybridization require the basic information of the breeding system of crop species

(Adewale and Adegbite, 2018; Obatolu et al., 2001; Tumwegamire, 2011). Adewale et al.

(2012) asserted that, breeding for improving AYB depends largely on understanding the pattern of classification and intra-specific variability of the accessions. The vast gene pool of

AYB confer a greater potential for character-based selection and improvement programmes

(Centre for Underutilised Crops, 2008). The differences in diversity studies is an indication of

16 the existence of genetic variations among AYB accessions and should be exploited in improvement schemes (Akinyosoye et al., 2017).

Breeding programmes in the past have not been placing emphasis on the improvement of

AYB genotypes (Oagile et al., 2007). The little breeding research work done on the crop to date has been prioritizing seed grain components at the expense of the leaf and tuber yield

(Zanmenou and Dossou-Yovo, 2017). Tuber formation in AYB is strongly dependent upon the genetic make-up of the accession (Akinyosoye et al., 2017). Adewale and Adegbite

(2018) asserted that, intra-specific hybridization has led to subsequent trait advancement in

AYB. Research should therefore focus on understanding the AYB breeding system which is of prime requisite for tuber improvement (Adewale and Adegbite, 2018).

Flowering characteristics and pollination channels of AYB are not well documented and researched (Adewale and Adegbite, 2018). Adewale and Adegbite (2018) asserted that, AYB exhibit both selfing and out crossing mechanisms. Furthermore, hand pollinations have proved that, the AYB flowers have a wide stigmatic receptivity for pollen from several accessions (Adewale and Adegbite, 2018). The wide adaptability of AYB offer an ecological and breeding advantage over the majority of conventional tropical legumes (Adewale and

Odoh, 2012). Moreover, exploiting the existing genetic variation and wide adaptability provides a platform for identifying major areas to prioritize in conservation and improvement schemes (Mekonnen et al., 2014).

2.3 Characterization and Diversity analysis of African Yam Bean

2.3.1 Genetic Diversity Analysis in Africa Yam Bean Genetic resource preservation is a function of proper evaluation and characterization of genetic diversity (Elezi et al., 2013; Manyasa, 2013). According to Phakathi (2015), genetic diversity is the variety of genotypes and alleles within a population. Diversity is made up of

17 four components (Fig 2.3) namely ecological, species, genetic and genomic (Bhandari et al.,

2017). Plant genetic diversity is the most important pre-requisite of any crop improvement programme (Govindaraj et al., 2015; Tuhina-Khatun et al., 2015) since it provides a broader scope for selection (Vandana et al., 2017). Characterization of genetic diversity is important in optimizing the use of genetic resources by researchers, breeders, farmers and local communities (Zannou et al., 2008). It also provides a basis for trait selection and genetic resource conservation (Manyasa, 2013; Nand et al., 2018; Saha et al., 2012). Furthermore, genetic diversity promote the development of farmer-preferred and breeder-preferred traits on a specific crop of interest (Bhandari et al., 2017).

Fig. 2.3: Diversity Hierarchy (Source: Bhandari et al., 2017)

The dependence of commercial staple crop varieties have reduced crop variability and ultimately promoted genetic erosion (Elezi et al., 2013). Wild species, breeding stocks, mutant lines and related species represent the existing genetic diversity and provides a source of desirable alleles for improvement schemes (Bhandari et al., 2017). Wild and related species have been accepted in numerous breeding initiatives as sources genetic variation in

18 staple crop improvements (Riaz et al., 2018). Zanmenou and Dossou-Yovo (2017) asserted that, AYB exhibit greater variability in comparison to other tropical species of its kind. The existing diversity can be captured and stored as plant genetic resources as a DNA library in centralized gene banks (Govindaraj et al., 2015). However, maintenance and characterization of collected accessions is time and financial resource demanding (Hussain et al., 2018;

Kumari et al., 2018).

Genetic diversity studies within accessions assist in screening for individual traits of interest

(Phakathi, 2015; Ringo, 2017). Porbeni et al. (2016) asserted that agro-morphological characters and their trait influence on yield are of greater importance to any crop improvement work. Genetic variation in quantitative traits is controlled by the combined effects of epistasis, quantitative trait loci (QTLs) and the environment (Adewale et al., 2012).

Santos et al. (2012) asserted that, abiotic environmental conditions induce trait variation which may need high genetic variability to ensure adjustment and survival in new environments. The seed component of AYB varies in seed shape, size and coat colour

(Abioye et al., 2015).

The performance of accessions can be subjected to environmental factors thereby enabling trait selection under different agro-ecologies (Akinyosoye et al., 2017; Hussain et al., 2018).

Establishing the inter-trait relationships is pivotal in establishing the hybridization procedures and selection of high performing genotypes (Duku, 2015). Stoilova and Pereira (2013) highlighted that, knowledge on morphological, agronomic and phonological traits of genotypes is important in the development of adaptive and productive cultivars.

Mekonnen et al. (2014) highlighted that, quantitative traits are essential in providing estimates of genetic diversity and several number-based taxonomic techniques are being utilized for classification and measurement of germplasm variability. Multivariate analysis of

19 genotypes sheds light on their genetic identity, potential and characteristics of such genotypes

(Adewale et al., 2012). The resident genetic diversity can be assessed using biometrical components such as variance, range, heritability, standard error and coefficient of variation

(Mekonnen et al., 2014; Syfullah et al., 2018). Ringo (2017) highlighted that, characterization of genetic diversity can also be done using morphological, molecular markers and quality traits in generating information for plant breeding programmes.

2.2.2 Africa Yam Bean Characterization

Genetic diversity assessment can be conducted using morphological, biochemical, molecular and cytological characterization (Bhandari et al., 2017). Phenotypic or morphological characterization is an indispensable tool in ascertaining diversity levels and selecting elite individuals (Efisue, 2016; Moukoumbi et al., 2011). Bhandari et al. (2017) asserted that, morphological characterization utilizes naturally occurring variants of the plant species under study. The variants or morphological indicators of genetic variability helps in determining duplicate accessions within a population (Elezi et al., 2013). Seed, pigmentation and pod are some of the important morphological components of AYB genetic variability (Centre for

Underutilised Crops, 2008; Ikhajiagbe and Kwesi-Mensah, 2012). Aremu and Ibirinde (2012) asserted that, analyzing the association among various plant descriptors assists in ranking traits by the importance levels. Morphological characterization provides a direct, easy and inexpensive evaluation (Bhandari et al., 2017). However, the use of morphological characters is limited by their susceptibility to environmental influences (Bhandari et al., 2017; Mezette et al., 2013; Shitta et al., 2016).

Biochemical marker-based characterization encompasses the separation of isozymes and proteins into specialized banding patterns which can be utilized for identification (Bhandari et al., 2017). The isozymes show products of alleles which can be mapped on chromosomes

(Bhandari et al., 2017). Molecular characterization is another set of methods which have been widely exploited for genetic diversity assessment in modern crop research studies (Abdellatif et al., 2011). Molecular characterization is pivotal in confirming genotypic differences reported from morphologically-based genotype assessments (Centre for Underutilised Crops,

2008). It studies the genotypic variations among the studied genotypes at a DNA or RNA level (Bhandari et al., 2017). Amplified fragment length polymorphisms (AFLPs) and

Random amplified polymorphic DNA (RAPD) have been reportedly used in characterizing

AYB accessions (Moyib et al., 2008). However, the prospects of utilizing single nucleotide polymorphisms (SNPs) and single sequence repeats (SSR) on AYB have not been fully researched (Shitta et al., 2016). Furthermore, cytological characterization involves chromosome length, size and constriction, centromere position, banding characteristics, DNA content and heterochromatic patterns (Bhandari et al., 2017).

The relative contribution of an established trait to the overall yield is accomplished through correlation analysis (Elezi et al., 2013). However, Path Coefficient Analysis is the most ideal methodology that untangles the correlation into direct and indirect effects of the causal factors (Elezi et al., 2013). Path Coefficient Analysis provides an ideal platform for estimating genetic divergence between crop accessions (Akinyosoye et al., 2017).

Furthermore, it partitions the correlation into genetic (realistic) and environmental (inflated) effects (Elezi et al., 2013).

CHAPTER THREE

3.0 RESEARCH METHODOLOGY

3.1 Plant Experimental Materials

Seeds of one hundred and sixty-nine accessions of African Yam Bean (Table 3.1) were sourced from the Genetic Resource Centre of the International Institute of Tropical

Agriculture (IITA), Ibadan, Nigeria.

3.2 Experimental Site

The field experimental trial was conducted at the Ethiopian Institute of Agricultural Research

(EIAR), Jimma Coffee Research Centre, Oromia Region, Ethiopia. The experimental site is situated at a latitude of 7º46' N, longitude of 36º 00'E and an altitude of 1753 m.a.s.l. The agro-ecological zone is characterized by sub-humid tropical to cool mid-highland climatic patterns. The minimum and maximum temperature recordings normally ranges from 9ºC to

28ºC. The Jimma Research Centre receives an average rainfall level of 1561 mm within its characteristic 4-7 months seasonal length. The experimental fields are composed of chromic nitosol, fluvisol and combisol soil types. The field trial was established in the 2018/19

(2010/11 Ethiopian Calendar (E.C)) agricultural season.

3.3 Experimental Design

The experiment was laid out in an Alpha Lattice (0.1) design with two replications. Thirteen incomplete blocks with thirteen plots each completed a replicate (13 x 13). Each replication consisted of 169 unit plots where 169 accessions were randomly allocated.

3.4 Field Layout

The size of an individual experimental or unit plot was 25 m2 (5 m x 5 m). The individual plot was composed of four rows with five plants on each row to make up a plot total of 20 plants. A space allocation of 1 m between plots and 1.5 m between blocks was utilized for the trial.

Table 3.1: List of collected African Yam Bean accessions tested in the study

S/N Accession S/N Accession S/N Accession S/N Accession

1 TSs22 44 TSs311 87 TSs377 130 TSs437

2 TSs209 45 TSs6 88 TSs421 131 TSs333

3 TSs83 46 TSs49 89 TSs309 132 59B

4 TSs82A 47 TSs138B 90 22B 133 TSs26

5 TSs32 48 TSs101 91 TSs42 134 TSs61

6 TSs148 49 TSs82 92 TSs68 135 30B

7 TSS66 50 TSs151B 93 TSs14 136 TSs133

8 TSS437 51 TSs269 94 TSs266 137 TSs166

9 TSs27 52 TSs86 95 TSs445 138 TSs6A

10 TSs293 53 TSs11 96 TSs137 139 TSs430

11 TSs338 54 TSs57 97 TSs39A 140 TSs28

12 TSs3 55 TSs152 98 TSs138 141 TSs89

13 TSs8 56 TSs446 99 TSs439 142 TSs449

14 TSs33 57 TSs22A 100 TSs366 143 TSs358

15 Tss12 58 TSs87B 101 TSs195 144 TSs197

16 TSs23C 59 TSs334 102 TSs422 145 TSs115

17 TSs67 60 TSs357 103 TSs294 146 TSs378

18 TSs155 61 TSs448 104 TSs425 147 3A

19 TSs296 62 TSs44C 105 TSs192 148 TSs331

20 TSs330 63 TSs201 106 TSs150 149 TSs51

21 TSs46 64 TSs34 107 TSs369 150 TSs307

22 Tss363 65 TSs38 108 TSs128 151 TSs337

23 TSs371 66 TSs62 109 TSs56 152 TSs157A

24 TSs9 67 TSs10A 110 TSs-2015-07 153 TSs95

25 TSs81 68 TSs98 111 40A 154 TSs153

26 TSs87 69 TSs417 112 TSs10 155 TSs435

27 TSs117 70 TSs302 113 TSs119A 156 TSs3A

28 TSs30 71 TSs48 114 TSs109 157 TSs15

29 TSs365 72 TSs55 115 TSs4 158 TSs121

30 TSs62B 73 TSs47 116 TSs355 159 TSs5

31 TSs7 74 TSs2 117 TSs5A 160 151B

32 TSs22B 75 TSs7A 118 TSs96 161 TSs60

33 TSs58 76 TSs438 119 TSs29 162 TSs1A

34 TSs112 77 TSs423 120 TSs447 163 TSs16

35 TSs440 78 TSs104B 121 TSs285 164 TSs159A

36 TSs119 79 TSs364 122 TSs56A 165 60B

37 TSs224 80 TSs303 123 TSs354 166 TSs352

38 TSs297 81 TSs431 124 TSs428 167 TSs24

39 TSs91 82 TSs280 125 TSs84 168 TSs63

40 TSs111 83 TSs104 126 TSs313 169 TSs63A

41 TSs424 84 TSs326 127 TSs69

42 TSs1 85 TSs13 128 TSs23

43 TSs443 86 89A 129 TSs301

*Sample Size: 169 Accessions *S/N: Serial Number

3.5 Experimental Procedure

The experimental field was ploughed, leveled and ridged prior to planting. Three seeds were placed per planting station at a spacing of 1 m (Intra-row) x 1 m (Inter-row). The plots were clearly marked with plastics tags prior to seedling emergence. Thinning was done after crop emergence and establishment to ensure one plant allocation per planting station (Fig 3.1). Dry wooden sticks measuring 3 m in length were used to offer support as stakes at 4 weeks after planting (WAP). Insect pest infestations were controlled by the continual 10-day interval sprays of 0.5% Karate (Lambda-Cyhalothrin). Weeds were continuously controlled by the hand-hoeing method at two week intervals. The plants were exposed to natural rainfall and

25 supplementary watering was done during dry spells. Basal dressing was done using

Diammonium Phosphate (DAP) (Composition: 18% N, 46% P2O5, (20%P)) at a rate of 150 kg/ha prior to sowing. Urea (Composition: 46% N) was applied at the fourth week to supplement nutrition at a rate of 100 kg/ha. The field trial was conducted in reference to the methods of Mgbeze and Ikhajiagbe (2010b) and Nwofia et al. (2013).

Fig 3.1. The Established AYB Experimental Trial at EIAR Jimma Research Centre, Ethiopia

3.6 Data Collection

Morphological characterization was conducted using the resource guide of the IITA descriptor list for AYB (Adewale and Dumet, 2010b). A total of 18 morphological descriptors comprising of eleven qualitative (Binary - 2 and Ordinal - 9) and seven quantitative variables as presented by Table 3.2 were used. The qualitative and quantitative

26 descriptors were recorded at an individual plant level using five systematic randomly selected plants per plot. The sample plants were picked from the inner two rows to combat the border effects. The five plants were tagged and consistently scored over all tested morphological traits. Qualitative characters was determined by scoring visual observations in nominal codes.

The Menthuen Colour Chart Book was employed in characterizing leaf and flower colouration parameters.

Table 3.2. List of quantitative and qualitative parameters utilized in the study

Trait Phenotypic Classification Levels Type of Data

Leaf Colour 1. Pale Green (MC 27A3) Qualitative

(Ordinal) 2. Vivid Green (MC27A8)

3. Deep Green (MC27F8)

Flower Colour 1. Pink Rose/Pale Red (MC11A4) Qualitative

(Ordinal) 2. Reddish or Pinkish White (MC12A2)

3. Greyish Ruby or Purple (MC12C3)

Terminal Leaf Shape 1. Ovate, 2. Heart and 3. Lanceolate Qualitative

(Ordinal)

Main Stem Pigmentation 0. Absent and 1. Present Qualitative

(Binary)

Branch Pigmentation 0. Absent and 1. Present Qualitative

(Binary)

Petiole Pigmentation 0. Absent and 1. Present Qualitative

Peduncle Pigmentation 0. Absent and 1. Present Qualitative

(Binary)

Main Stem Pigmentation 1. Slight, 3. Moderate and 5. Extensive Qualitative

Intensity (Ordinal)

Branch Pigmentation 1. Slight, 3. Moderate and 5. Extensive Qualitative

Intensity (Ordinal)

Petiole Pigmentation Intensity 1. Slight, 3. Moderate and 5. Extensive Qualitative

(Ordinal)

Peduncle Pigmentation 1. Slight, 3. Moderate and 5. Extensive Qualitative

Intensity (Ordinal)

Number of Primary Branches Visual counts Quantitative

Petiole Length Measurements in Centimeters (cm) Quantitative

Terminal Leaf Length Measurements in Centimeters (cm) Quantitative

Terminal Leaf Width Measurements in Centimeters (cm) Quantitative

Days of 50% Flowering Visual Counts Quantitative

100 Seed Weight Measurements in Grams (gm) Quantitative

3.7 Data Analysis

Quantitative and qualitative morphological trait data obtained were analyzed using R Studio

Version 3.6.1, DARwin Software Version 6 and IBM SPSS Statistics Version 25 statistical packages. The data was subjected to descriptive and exploratory analysis. The analysis of

28 variance (ANOVA) was performed to ascertain the significance of variation among the studied accessions. A two-tailed Pearson Correlation Analysis was also performed to establish inter-trait relationships and pinpoint traits of importance for future AYB breeding programs. Principal Component Analysis (PCA) was utilized to deduce the total diversity accounted for by the assessed parameters and identify the underlying relationship patterns.

The genotype eigen values in two PC axes were generated to form two-dimensional scattered graph. The accessions were grouped into distinct classes using Hierarchical Clustering and the Unweighted Neighbour-Joining Method with the Euclidian Distance denoting a Similarity

Metric.

CHAPTER FOUR

4.0 RESULTS

4.1 Descriptive Statistics of Morphometric Quantitative Traits

The mean, variance and standard deviation for quantitative morphometric attributes exhibited great variability among the studied 169 AYB accessions (Table 4.1, Fig 4.5 and Appendix

1.1). Variation was observed in number of primary branches per plant which ranged from 2 to 5 with a mean value of 2.99. TSs 201, TSs 448 and TSs338 recorded the lowest number of primary branches (2) whilst the highest value (5) was observed on TSs 363. Petiole length, terminal leaf length and terminal leaf width averaged 5.65 cm, 12.12cm and 4.58 cm respectively. TSs 104 accession recorded the highest petiole length of 7.3 cm whilst TSs 6 surpassed all accessions in terminal leaf length and width with measurements of 16 cm and

5.6 cm respectively.

Table 4.1. Descriptive Statistics of Quantitative Morphological Descriptors

Descriptive Statistics Number of Petiole Terminal Leaf Terminal Leaf Days to 50% 100 Seed Seed Yield Primary Branches Length Length Width Flowering Weight per plant Mean 2.9882 5.6506 12.1186 4.5825 118.7130 21.3580 36.1716

Std. Deviation .52236 .82387 1.31760 1.01049 8.19299 3.28457 6.03256 Variance .273 .679 1.736 1.021 67.125 10.788 36.392 Minimum 2.00 4.00 9.00 3.00 102.00 14.00 22.00 Maximum 5.00 8.00 16.00 13.00 137.00 31.00 54.00

The observed days to 50% flowering ranged from 102 (TSs 333) to 137 (TSs 91 and TSs

303) days with a mean value of 118.7 days. An average of 21.36 g was observed for the 100

Seed Weight measurements across the studied accessions. The highest and lowest yielding

(Seed yield per plant) accessions were recorded to be TSs 357 (with 54 g) and TSs 98 (with

22 g) respectively.

4.2 Descriptive Statistics of Qualitative Characters

The tested accessions showed great similarity in plant pigmentation parameters (main stem, primary branches, petiole and peduncle pigmentation). The peduncles of all the accessions was not pigmented. The leaf colour phenotypic levels of classification showed that deep green (Menthuen colour code: 27F8) was common in 99.4% of the observations or among the

168 accessions including TSs 22, TSs 209, TSs 338 and TSs 371 (Appendix 1.1). Pale green

(27A3) was not observed in any of the accessions whereas vivid green (27A8) was observed on TSs32 accession trial plots only.

Flower colouration showed a significant variation with pink rose (11A4) commanding 6.5% or 11 accessions (including TSs 363, TSs 338, TSs 363 and TSs 371). Reddish or pinkish white (12A2) was common in 89.1% of the observations or 150 accessions including TSs 8,

TSs 22, TSs 67 and TSs 155 (Fig. 4.1). Greyish ruby or purple flower colour (12C3) was observed in 4.4% of the accessions including TSs 7, TSs 27, TSs 47 and TSs 137 (Fig. 4.2).

Leaf Shape was mainly dominated by the ovate morphological class which was exhibited on

98.2% of the accessions (such as TSs 23C, TSs 82 A, TSs 148 and Tss 293). The heart leaf shape class was not recorded in any of the accessions tested. Lanceolate leaf architecture class was observed in 5 accessions (1.7%) namely TSs 6A, TSs 23C, TSs 98, TSs 109 and

TSs 330.

Fig.4.1 Flower Colour Variations: Pinkish White (TSs 8)

Fig.4.2. Flower Colour Variations: Greyish Ruby (TSs 7)

The larger proportion (76.9 %) of the tested accessions do not have pigmented main stem, primary branches and petiole. The non-pigmented classification comprised of 130 accessions including TSs 22, TSs 32, TSs 33, TSs 67, TSs 82A and TSs 148. Pigmentation was however observed in 36 accessions (23.1%) such as TSs 66, TSs 83, TSs 187 and TSs 338. Main stem pigmentation was common in TSs 66, TSs 209, TSs 297 and TSs 437. Accessions such as

TSs 30, TSs 81, TSs 87 and TSs 330 exhibited pigmentation trait on primary branches (Fig.

4.4). Furthermore, petiole pigmentation were observed in several accessions including TSs

30, TSs 87, TSs 293 and TSs 338.

Fig.4.3. Pigmentation Variations: Non-Pigmented Primary Branches (TSs 155)

Fig.4.4. Pigmentation Variations: Pigmented Primary Branches (TSs 330)

Fig 4.5: Descriptive Statistics Summary Graphs

4.3 Analysis of Variance (ANOVA) for Quantitative Traits

Analysis of Variance: Seed Yield Per Plant Df Sum Sq Mean Sq F value Pr(>F) Accession 168 3617.6 21.533 1.3247 0.04139 * Residuals 144 2340.8 16.255

Analysis of Variance: Number of Primary Branches Df Sum Sq Mean Sq F value Pr(>F) Accession 168 27.3464 0.162776 2.8345 1.967e-10 ** Residuals 144 8.2696 0.057428

Analysis of Variance: Petiole Length Df Sum Sq Mean Sq F value Pr(>F) Accession 168 34.878 0.20761 1.5897 0.002198 * Residuals 144 18.805 0.13059

Analysis of Variance: Terminal Leaf Length Df Sum Sq Mean Sq F value Pr(>F) Accession 168 114.94 0.68416 1.5908 0.002169 * Residuals 144 61.93 0.43007

Analysis of Variance: Terminal Leaf Width Df Sum Sq Mean Sq F value Pr(>F) Accession 168 63.418 0.37749 1.286 0.06029 . Residuals 144 42.270 0.29354

Analysis of Variance: Days to 50% Flowering Df Sum Sq Mean Sq F value Pr(>F) Accession 168 6646.4 39.562 2.2364 5.144e-07 ** Residuals 144 2547.3 17.690

Analysis of Variance Table: 100 Seed Weight Df Sum Sq Mean Sq F value Pr(>F) Accession 168 1005.35 5.9842 1.2402 0.09194 . Residuals 144 694.86 4.8254 *P≤0.05 - Significant **P≤0.01 - Highly Significant

The ANOVA was performed on quantitative parameters to determine their level significance across the tested accessions. The mean squares and significance values from ANOVA for characters observed are presented in Table 4.2. The analysis showed that the 169 AYB accessions showed wide diversity for some of the observed parameters and the differences were significant (P≤0.05) and highly significant at P≤0.01. Number of primary branches and days to 50% flowering showed significance (P≤0.05) whilst seed yield per plant, petiole

35 length and terminal leaf length showed high significance. However, terminal leaf width and 100 seed weight parameters were not significant across all the studied accessions.

4.4 Trait Correlation Analysis

The Pearson Correlation analysis was done to identify morphological descriptors which showed significant relationships among the tested accessions. The output of the data (Table 4.3) shows that, there was a negative correlation between seed grain yield and Days to 50% Flowering (r = -1.42). Pigmentation parameters (main stem, primary branches and petiole pigmentation and intensity levels) were significantly correlated at all levels (r = 1). Leaf Colour and 100 Seed Weight also showed a positive correlation (r = 0.72). Seed grain yield per plant was highly correlated to terminal leaf length and width; and all pigmentation parameters. Furthermore, 100 Seed Weight was significantly correlated to terminal leaf length (r = 0.441) and seed yield per plant (r = 0.862).

Table 4.4: Pearson Correlation Analysis

LC FC LS MSP BP PP MSPI BPI PPI NPB PL TLL TLW D50F S100W SY P

LC 1 . .

FC -.003 1

LS .007 .008 1

MSP .030 -.029 -.067 1

BP .030 -.029 -.067 1.000** 1

PP .030 -.029 -.067 1.000** 1.000** 1

MSPI .029 -.033 -.065 .963** .963** .963** 1

BPI .025 -.011 -.056 .835** .835** .835** .829** 1

PPI .029 -.027 -.065 .970** .970** .970** .958** .843** 1

NPB -.001 .050 .050 -.041 -.041 -.041 -.034 -.100 -.039 1

PL -.023 -.038 .141** -.058 -.058 -.058 -.042 -.094 -.066 .039 1

TLL .046 -.096 .063 .069 .069 .069 .096 .087 .064 -.007 .271** 1

TLW .031 -.079 -.022 .094 .094 .094 .124* .087 .081 -.009 .309** .443** 1

D50F -.035 -.005 -.038 -.094 -.094 -.094 -.098 -.125* -.092 .078 -.169** -.257** -.180** 1

S100W .072 -.149** -.021 .148** .148** .148** .150** .105 .126* -.003 .045 .441** .246** -.111* 1

SYP .011 -.140* .045 .106 .106 .106 .121* .062 .091 -.066 .080 .471** .273** -.140** .862** 1

**. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed). LC = Leaf Colour, FC = Flower Colour, LS = Leaf Shape, MSP = Main Stem Pigmentation, BP = Branch Pigmentation, PP = Petiole Pigmentation, MSPI= Main Stem Pigmentation Intensity, BPI = Branch Pigmentation Intensity, PPI = Petiole Pigmentation Intensity, NPB = Number of Primary Branches, PL = Petiole Length, TLL = Terminal Leaf Length, TLW = Terminal Leaf Width, D50F = Days to 50% Flowering, S100W = 100 Seed Weight and SYP = Seed Yield Per Plant.

4.5 Principal Component Analysis (PCA)

Table 4.4. Total Variance Explained by PCA

Total Variance Explained Component Initial Eigen values Extraction Sums of Squared Loadings Total % of Cumulative Total % of Cumulative Variance % Variance % 1 5.762 37.60 37.60 5.762 37.60 37.60 2 2.531 18.3 55.9 2.531 18.3 55.9 3 1.309 6.182 62.082 1.309 6.182 62.082 4 1.063 5.646 67.728 1.063 5.646 67.728 5 1.012 5.255 72.983 1.012 5.255 72.983 6 .974 6.086 79.068 7 .961 6.007 85.075 8 .802 5.011 90.086 9 .633 3.957 94.044 10 .493 3.080 97.123 11 .238 1.489 98.612 12 .129 .806 99.418 13 .055 .341 99.760 14 .038 .240 100.000 15 4.836E-017 3.022E-016 100.000 16 -2.198E-018 -1.374E-017 100.000

The principal component analysis output (Table 4.3) was done to establish the major parameters explaining the recorded variations among the 169 AYB accessions. The analysis pin-pointed five principal components which accounted for 72.983 % of the existing variation among the studied accessions. PC1, PC2, PC3, PC4 and PC5 contributed 37.6 %, 18.3 %,

6.182 %, 5.646 % and 5.255 % variance respectively. Furthermore, PC1, PC2, PC3, PC4 and

PC5 had eigen values of 5.762, 2.531, 1.309, 1.063 and 1.012 respectively. The Rotation

Method of Varimax with Kaiser Normalization (Appendix 1.3) was utilized to identify the parameters loading to each principal component. Main stem, petiole and primary branches pigmentation parameters loaded to the variation explained by PC1. PC2 was associated with

100 seed weight and seed yield per plant parameters. Petiole length and terminal leaf width

38 contributed positively to PC3 whilst number of branches and leaf shape loaded to PC4.

Conclusively, the contribution of leaf colour was shown to have a positive bearing on PC5.

Fig 4.6. Scree Plot for the Principal Components

The scree plot output was generated to display the number of components against their corresponding eigen values. The scree plot presented by Fig 4.6, shows that five principal components contributed the major share of the existing variation as indicated by the give eigen values. The first five components have an eigen value greater than 1 and explaining

72.893% of the recorded variability. The scree plot line is almost flat from the fifth component implying that the successive components accounted for small amounts of total variance.

Fig 4.7: PC1/PC2 Plot (Variable or Factor Map)

The plot (Fig 4.7) of the first two principal components (PC1 and PC2) was generated to give a direct visualization of correlated variables from the study. Variables that grouped together were considered to be highly correlated. The factor map shows that 100 seed weight and seed yield per plant were highly correlated. Furthermore, terminal leaf length, terminal leaf width and petiole length also showed some degree of correlation among the studied accessions.

4.6 Grouping of Accessions based on Morphological Data

Cluster analysis was performed using the Hierarchical Method (R Studio Software) on seed grain yield components and the Unweighted Neighbour-Joining Method (DARwin 6 Software) based on the morphological characters associated with the first five principal components.

4.6.1 Hierarchical Cluster Analysis

Fig. 4.8 Hierarchical Cluster Analysis: Dendrogram using Ward Linkages

The hierarchical clustering using Ward's Squared Euclidian Method output (Fig 4.8) was generated to classify the accessions basing on seed yield per plant and 100 seed weight parameters. The procedure classified the accessions into three distinct clusters at a similarity level of 100% and five sub-clusters at 50% level of similarity. Cluster I (occupying the first left-position on Fig 4.8) represented the low yielding genotypes and comprised of accessions such as TSs 9, TSs 12 and TSs 98. Accessions including TSs 363, TSs 155 and TSs 82A T

41 made up Cluster II. The third cluster constituted of accessions such as TSs 22, TSs 62 B and

TSs 81. TSs 330, TSs 7 and TSs 27 accessions were among the members of Cluster IV.

Lastly, Cluster V depicted the highest yielding group housing accessions such as TSs 23C,

TSs 338, TSs-2015-07 and TSs 357. A significant variability was observed among the accessions within major clusters which were further divided into sub-clusters upon truncation between similarity level rescaled percentages.

4.6.2 Unweighted Nearest Neighbour-Joining Analysis

Fig 4.9 Unweighted Neighbour-Joining Cluster

The Unweighted Neighbour-Joining Cluster Analysis based on quantitative morphological traits grouped the tested AYB accessions into four major clusters as presented by Fig 4.9. The

42 analysis yielded a dissimilarity minimum and maximum of 0.4690 and 55.2924 respectively. The first cluster constituted of accessions such as TSs 47, TSs 3, TSs 297 and TSs 355 whilst TSs 307, TSs 87B, TSs 24 and TSs 313 accessions were grouped in to the second cluster. TSs 23C, 40A, TSs 159A and TSs 46 accessions were among the selected elements of cluster three. The fourth cluster housed accessions such as TSs 293, TSs 10A, TSs 87 and TSs 14. Furthermore, TSs 32 presented in red on Fig 4.9 appeared at an outlier position and distant from other accessions.

CHAPTER FIVE

5.0 DISCUSSION

5.1 Morphological Trait Variability

Morphological characterization is a prerequisite of germplasm identification, improvement and conservation. Qualitative and quantitative morphological descriptors were examined across the 169 AYB accessions following the IITA reference guide and additional consultations to the method of Adewale and Kehinde (2016), Nwofia et al. (2013) and

Popoola et al. (2011). Manyasa (2013) asserted that, the most important traits are the ones showing variability among the tested accessions. Number of primary branches and days to

50% flowering showed significant variation (P≤0.05) across the studied accessions. Seed yield per plant, petiole length and terminal leaf length showed high significance (P≤0.01) across the AYB accessions. Adewale and Kehinde (2016) who also recorded significant variations in morphological attributes among the collected AYB accessions. However, terminal leaf width and 100 seed weight parameters were not significant across the tested treatments. The recorded morphological dissimilarities denotes the resident potential of AYB accessions for improvement.

The mean values for number of primary branches, petiole length, terminal leaf length, terminal leaf width, days to 50% flowering, 100 seed weight and seed yield per plant were

2.98, 5.65 cm, 12.12 cm, 4.58 cm, 118.7 days, 21.36 g and 36.17 g. The seed yield per plant mean value was in discord with the results from Akinyosoye et al. (2017) who recorded a mean value of 46.32g. The seed yield per plant measurements from the study ranged from a minimum of 22g to a maximum of 54 g. Accession TSs 357 recorded the highest seed yield per plant measurement. The terminal leaf length and width measurements were in accord with

Adewale and Odoh (2012) recordings of 140 mm and 50 mm respectively. The ovate leaf

44 morphology was common in 98.2% of the studied accession. Furthermore, 99.4% and 89.1% of the studied accessions were classified under the deep green leaf and pinkish-white flower colouration classification bracket respectively. Therefore, leaf shape, leaf colour and flower colour parameters confer limited potential in discriminating the studied accessions of African yam bean. However, the flower colour ranges observed in the study were in agreement with remarks made by Aburime (2012).

On the other hand, the majority of quantitative parameters showed a great depth of variability across the accessions. Nwofia et al. (2013) also observed similar results of higher variability among AYB accessions on yield components. The aforementioned parameters contributed to the greatest variation proportion observed among the genotypes. The recorded observation was also in agreement with the research findings of Popoola et al. (2011) who emphasized the importance of quantitative traits in accession discrimination. Morphological dissimilarities of the these parameters is proof of the existence of genetic variation among the

169 AYB accessions.

The high yielding accessions such as TSs 23C, TSs-2015-07 and TSs 357 can be utilized in breeding for high gain yield programmes. Additionally, days to 50% flowering can also be used as a maturity index in selecting for early maturing parental lines. The days to 50% flowering ranged from 102 to 137 days across the accessions. The observation is in discord with that of Akinyosoye et al. (2017) who recorded a minimum and maximum of 134 and

157 days to 50 % flowering respectively. The divergence can be attributed to the environmental influences exerting differential pressure on the tested plant material.

Accessions such as TSs22, TSs 333 and TSs 11 had the minimal number of days to flowering can be classified as the early maturing genotypes and can be used in drought tolerance breeding schemes. Moreover, leaf length and width parameters can also be used in selecting for grain yield since leaf size determines the photosynthate sink magnitude (Abdulkareem et

45 al., 2015). TSs 6 exhibited high values for terminal leaf length and width and can perfectly be incorporated into grain yield advancement schemes.

Peduncle pigmentation was not observed in all of the tested accessions. It is therefore of no value in discriminating or characterizing the accessions of AYB. The observations made on the experimental unit also showed patches of leaf colourations on leaf surfaces of several accessions which can be attributed to localized mutations as noted also by Abdulkareem et al.

(2015). The phenomenon can also be a result of environmental (biotic and abiotic agents) influences. The general outcome of the morphological characterization showed that the studied accessions varied significantly on the observed characters. In conclusion, the recorded significant variation across the studied accessions in this research is implicative of differences among the accessions and the presence of genetic divergence in S. stenocarpa.

The existence of high population variability can be utilized for heterosis schemes to produce superior hybrids.

5.2 Morphological Trait Correlations

Correlation studies assist in determining the existing relationships in the expression of important traits. Positive and significant correlation between two parameters imply that those parameters can be simultaneously improved under selection. The combined Pearson's correlation analysis on the present study proved that, seed yield components (Seed yield per plant and 100 seed weight) had a positive significant (p<0.05) correlation across the assessed accessions (r = 0.862). Nwofia et al. (2013) recorded a similar positive correlation between seed yield per plant and 100 seed weight (r = 0.627) in their research. Yield is a product of numerous traits operating interdependently (Desissa, 2017; Khan et al., 2016). The relationship between grain yield and associated morphological traits is therefore of paramount importance in seed grain yield breeding (Cervantes et al., 2016).

Plant breeding schemes set major objectives on identifying characters with a positive significant association with the traits of interest (Syfullah et al., 2018). Identifying the parameters correlated to grain yield can hasten the breeding process and ensure successful selection (Kumari et al., 2018; Saha et al., 2012). Leaf colour, leaf shape, pigmentation parameters and terminal leaf length showed a positive significant correlation with grain yield parameters. However, number of primary branches, days to 50% flowering and flower colour showed negative correlations with grain yield.

5.3 Genetic Diversity Assessment of AYB accessions

The study results exhibited a wide range of diversity among the tested 169 AYB accessions.

Morphogenetic diversity in agronomic traits is of great importance in establishing the ideal method for crop improvement (Fayeun et al., 2015; Ogunbayo et al., 2005). Principal component and cluster analyses were performed on the data to establish the source of variation and group accessions into distinct classes. The PCA exhibited a high discrimination potential for the variables tested in the study. Principal components were established at an eigen scale of greater than 1. Five principal components (PC1, PC2, PC3, PC4 and PC5) were identified to be responsible for explaining the major share of total variation. The observed

PCs were lower than the eleven PCs recorded by Popoola et al. (2011) in their study of interspecific variabilities in AYB accessions. However, the 72.983% total variation accounted for by the five principal components was greater than 66.70% and 63.89% attained by

Akinyosoye et al. (2017) and Aremu and Ibirinde (2012) respectively.

The differential trait loadings on the first two PCs showed that, the delineation of diversity among accessions emanated from seed yield per plant, 100 seed weight, main stem pigmentation, primary branches pigmentation and petiole pigmentation parameters. The high contribution of seed yield parameters was also observed by Akinyosoye et al. (2017) and

Ikhajiagbe and Kwesi-Mensah (2012). Conclusively, the identified parameters can be incorporated in breeding focuses for efficient AYB improvement programmes.

Hierarchical clustering analysis grouped the tested accessions into three major clusters (at

100% similarity level) and five sub-clusters (at 50 % level of similarity) based on seed grain parameters (Fig. 4.6). The recorded clustering pattern was in agreement with that of

Akinyosoye et al. (2017) who classified AYB accessions in three major groups. The data was further classified using the Unweighted Neighbour-Joining method with special emphasis on specific parameters loading to the identified first five principal components (Fig. 4.7). The second method grouped the accessions into four major clusters. The observation was also in discord with Omena et al. (2014) who classified AYB accessions into two major clusters. In conclusion, the cluster analysis showed high homogeneity within the one cluster and high heterogeneity between clusters.

CHAPTER SIX

6.0 CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion

Africa yam bean is an underutilized crop with greater potential to be incorporated into the modern food systems and aid in alleviating the negative influences of food and nutrition insecurity. Despite the recorded potentiality, limited research work has been done on the crop to improve and scale-up its adoption. Consequently, AYB commercial varieties have not been established for uptake by the small-holder farmers. Variability studies on AYB will therefore play a pivotal role in its future advancement and development. The morphological traits observed in this study exhibited a wide and significant (P≤0.05) variation among the tested

169 AYB accessions. ANOVA showed that, number of primary branches, days to 50% flowering, seed yield per plant, petiole length and terminal leaf length were significant across the studied parameters. The descriptive statistics of quantitative and qualitative morphological characters showed that, there is a significant variation between the tested accessions. The deep green leaf colouration dominated as shown by observations in 99.4% of the accessions. Furthermore, pinkish-white flower colouration, ovate leaf shape and non- pigmented plant part characters commanded the majority of observations as depicted by

89.1%, 98.2% and 76.9% proportions of the 169 AYB accessions.

The use of Principal Component Analysis in the research study allowed the identification of traits that explained the existing genetic variation. The major contribution in total variation under PCA was observed in pigmentation parameters and seed yield components. Pearson's correlation analysis observed significant association among traits imply that, there is high probability of improving multiple traits simultaneously. The present research showed that, leaf colour, leaf shape, pigmentation parameters and terminal leaf length had a positive significant correlation with grain yield. The observed inter-character association can be utilized as a guide in the formulation of hybridization procedures for accession selection.

The distribution of AYB accessions into different cluster groups further confirmed the existence of genetic variation. The clusters showed high homogeneity within the same cluster and high heterogeneity between clusters. Accessions in hierarchical cluster V (including TSs

23C, TSs 56, TSs-2015-07 and TSs 357) were found promising for economically important trait selection. The aforementioned accessions can be utilized as a parental source for further improvement and selection in grain yield-oriented breeding programs. The observed variation should be exploited for AYB genetic advancement and conservation since it ensures success of future breeding schemes on the crop. Furthermore, direct selection can be employed in improving the parameters under study.

6.2 Recommendations

i. The sensitivity of morphological characterization to environmental influences limit its

capacity in providing a reliable phenotyping record. It is therefore imperative to,

further the work by incorporating molecular genotypic analysis tools for comparison

and confirmation of the recorded diversity among the collected and studied accessions

of Africa yam bean.

ii. Nutritional and anti-nutritional proximate analysis on the studied AYB accessions

should be also considered so as to establish the economic value of these accessions

and their capacity in ameliorating nutritional insecurities in Africa. The results from

such a study will also give a forecast into the accessions which can be adopted

quickly by consumers. Furthermore, proximate results will also assist in selection of

nutrient-based traits.

iii. Additional morphological characterizations under multi-locational and seasonal trials

should also be done to ascertain the role of environments on accession phenotypic

expressions and reaffirm the findings.

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APPENDICES

Appendix 1. 1: Descriptive Statistics for the Morphological Trait Data

Descriptive Statistics

Range Minimum Maximum Mean

Statistic Statistic Statistic Statistic Std. Error

100 Seed Weight 17.00 14.00 31.00 21.3580 .17866

Branch Intensity 5.00 .00 5.00 .5148 .06133

Branch Pigmentation 1.00 .00 1.00 .2308 .02295

Days to 50% Flowering 35.00 102.00 137.00 118.7130 .44564

Flower Colour 2.00 1.00 3.00 1.9793 .01799

Leaf Colour 1.00 2.00 3.00 2.9970 .00296

Leaf Shape 2.00 1.00 3.00 1.0296 .01315

Main Stem Intensity 5.00 .00 5.00 .6450 .06661

Main Stem Pigmentation 1.00 .00 1.00 .2308 .02295

Number of Primary Branches 3.00 2.00 5.00 2.9882 .02841

Peduncle Intensity .00 .00 .00 .0000 .00000

Peduncle Pigmentation .00 .00 .00 .0000 .00000

Petiole Intensity 3.00 .00 3.00 .2367 .02426

Petiole Length 4.00 4.00 8.00 5.6506 .04481

Petiole Pigmentation 1.00 .00 1.00 .2308 .02295

Seed Yield per plant 32.00 22.00 54.00 36.1716 .32813

Terminal Leaf Length 7.00 9.00 16.00 12.1186 .07167

Terminal Leaf Width 10.00 3.00 13.00 4.5825 .05496

Valid N (listwise)

Appendix 1.2 Principal Component Analysis Matrix

Component Matrixa

Component

1 2 3 4 5

Main Stem Pigmentation .986 -.101 .016 .034 -.013

Petiole Pigmentation .986 -.101 .016 .034 -.013

Branch Pigmentation .986 -.101 .016 .034 -.013

Petiole Intensity .974 -.116 .021 .032 -.011

Main Stem Intensity .972 -.073 .032 .035 -.017

Branch Intensity .881 -.103 .030 -.058 .013

Seed Yield per plant .191 .806 -.410 .111 -.014

100 Seed Weight .230 .769 -.464 .150 .040

Terminal Leaf Length .152 .753 .164 -.009 .027

Terminal Leaf Width .157 .578 .355 -.121 -.059

Petiole Length -.049 .389 .662 .033 -.154

Days to 50% Flowering -.144 -.333 -.402 .355 -.208

Number of Primary Branches -.067 -.035 .124 .807 .007

Leaf Shape -.077 .103 .328 .453 .038

Leaf Colour .043 .068 -.049 -.006 .891

Flower Colour -.051 -.224 .211 .141 .377

Extraction Method: Principal Component Analysis.a a. 5 components extracted.

Appendix 1.3 Rotated Component Matrix

Rotated Component Matrixa

Component

1 2 3 4 5

Main Stem Pigmentation .990 .054 .018 -.020 .002

Petiole Pigmentation .990 .054 .018 -.020 .002

Branch Pigmentation .990 .054 .018 -.020 .002

Petiole Intensity .981 .037 .012 -.021 .004

Main Stem Intensity .973 .065 .045 -.013 -.003

Branch Intensity .882 .014 .045 -.098 .026

100 Seed Weight .096 .933 .064 .002 .015

Seed Yield per plant .053 .919 .135 -.014 -.040

Petiole Length -.079 -.061 .718 .263 -.146

Terminal Leaf Width .074 .265 .650 .011 -.063

Days to 50% Flowering -.085 -.008 -.608 .207 -.207

Terminal Leaf Length .038 .533 .573 .059 .015

Number of Primary Branches -.016 .034 -.153 .805 .021

Leaf Shape -.058 -.030 .180 .541 .049

Leaf Colour .014 .113 .004 -.039 .888

Flower Colour -.007 -.268 -.012 .190 .389

Extraction Method: Principal Component Analysis. Rotation Method: Varimax with Kaiser Normalization.a a. Rotation converged in 5 iterations.

Appendix 1.4 Component Transformation Matrix

Component Transformation Matrix

Component 1 2 3 4 5

1 .986 .140 .074 -.056 .012

2 -.155 .801 .577 .030 -.024

3 .039 -.555 .765 .324 .026

4 .049 .173 -.277 .944 .014

5 -.017 .029 -.003 -.020 .999

Extraction Method: Principal Component Analysis. Rotation Method: Varimax with Kaiser Normalization.

Appendix 1.5 Component Score Coefficient Matrix

Component Score Coefficient Matrix

Component

1 2 3 4 5

Leaf Colour -.013 .068 -.014 -.034 .878

Flower Colour .012 -.128 .033 .168 .381

Leaf Shape .011 -.033 .096 .485 .048

Main Stem Pigmentation .177 -.010 -.010 .024 -.009

Branch Pigmentation .177 -.010 -.010 .024 -.009

Petiole Pigmentation .177 -.010 -.010 .024 -.009

Main Stem Intensity .174 -.008 .006 .029 -.013

Branch Intensity .155 -.033 .020 -.054 .016

Petiole Intensity .176 -.017 -.010 .023 -.007

Number of Primary Branches .032 .067 -.146 .747 .020

Petiole Length -.008 -.157 .467 .201 -.143

Terminal Leaf Length -.016 .172 .272 .040 .023

Terminal Leaf Width -.003 .015 .373 -.013 -.058

Days to 50% Flowering .004 .114 -.405 .217 -.206

100 Seed Weight -.015 .471 -.132 .025 .026

Seed Yield per plant -.024 .451 -.082 .005 -.027

Extraction Method: Principal Component Analysis. Rotation Method: Varimax with Kaiser Normalization. Component Scores.

Appendix 1.6 Component Plot in Rotated Space

Appendix 1.7 Predictor Space