PHENOTYPIC CHARACTERIZATION OF COWPEA (VIGNA UNGUICULATA (L) WALP) ACCESSIONS UNIVERSITY OF GHANA COLLEGE OF BASIC & APPLIED SCIENCES BY KINGSLEY SMITH ARTHUR (10806420) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IN BOTANY DEGREE DEPARTMENT OF PLANT AND ENVIRONMENTAL BIOLOGY DECEMBER 2021 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION I, Kingsley Smith Arthur, declare that this thesis herein presented to the University of Ghana, Legon for the award of Master of Philosophy in Botany is my own investigation and no such work has been presented by me or any other person for a degree at any other University or institution of higher education. 14th January, 2022 …………………………………. …………………………… KINGSLEY SMITH ARTHUR Date (Student) 14th January, 2022 ……………………………………. …………………………... PROF. I. K. ASANTE Date (Supervisor) 14th January, 2022 ……………………………………. …………………………... PROF E. T. Blay Date (Supervisor) University of Ghana http://ugspace.ug.edu.gh iii ABSTRACT Cowpea is indigenous to the African continent and is usually grown for its leaves and grain in different countries of the world. The objective of this study was to evaluate cowpea accessions using morphological descriptors and phytochemical analysis to identify cowpea accessions with a more specific traits that could be used by breeders. Phenotypic characterization of twenty-seven (27) cowpea accessions which consist of twenty-four (24) from the Department of Plant and Environmental Biology as test material and three (3) others, namely ‘Asontem’, ‘Wang Kae’ and ‘Kirkhouse’ which were used as checks, was undertaken. The cowpea population was evaluated based on agro-morphological traits (quantitative and qualitative) and phytochemical traits. STATA version 15.0 was used to carry out descriptive, multivariate analyses whiles ‘R’ software was used for genetic component analysis to determine the extent of variation. Phytochemical analysis was carried out to study the polyphenolic and amino acid contents of the cowpea seeds using standard methods and protocols. Polyphenolic compounds were determined using a spectrometer and amino acid contents were determined with assistance from Ghana Standard Authority. In the morphological qualitative traits studied, erect cowpea plants showed the highest distribution for growth habit with frequencies of 91% in the test material and 84.17% in the entire population. The pale tan pod colour recorded the highest frequency of 62.58% in the entire population, similarly, the pale tan with frequencies 65.48% and 62.58% were the highest frequency in both control and test material respectively. The white seed coat colour had the highest frequency of 66.11%in the entire population as well. The chi-square test of association between qualitative traits showed 86 significant associations in the test materials, with growth habit having the University of Ghana http://ugspace.ug.edu.gh iv highest signicant associations with leaf size (χ² = 220.84, P = 0.000), whereas the control showed 78 significant associations. The overall population exhibited 86 significant associations, with growth habit having the highest positive significant association with terminal leaflet shape (χ² = 243.87, P = 0.000). The phenolic compound had higher mean concentration in the controls than the test material, however higher mean concentration of amino acid was obatined in the test materials than in the control. A total of 145, 113 and 123 significant pairwise correlations were observed in the control, test materials, and entire population respectively for all quantitative traits. In the test material vanillic acid was highly correlated with gallic acid (r = 1.00) and p-coumaric acid (r = 1.00). Principal component analysis showed that, the first seven principal components in the morphological traits accounted for a total variability of 84.00% in the control, while nine principal components explained 81.00% of the total variation in the test material. On the other hand the first five principal components in the phytochemical traits accounted for 100%, 100% and 83% of the total variation in the control, test materials and entire population respectively. The biplot showed that the relationships among accessions and morphological, phytochemical and all traits explained 33% and 51% of the total variance respectively. The cluster analysis for the morphological traits revealed five distinct clusters. Genotypes named Asontem, T3, T5, T22 were grouped in a single cluster and were characterized by least days to germination (3.58), highest mean number of branches (4.93), least mean number of leaves (41.26), highest mean number of nodes (9.83), least mean leaflet width (3.69), the highest mean days to first flowering, least mean number of pods per peduncle (2.81), least average pod width (0.80), highest mean number of pods per plant (49.50), the highest average number of seed per pod (15.97), highest mean days to first matured pod (42.01), highest mean University of Ghana http://ugspace.ug.edu.gh v number of seeds per plants (795.75) compared to other clusters. These genotypes could be used as parents in genetic improvement programs aiming to increase yield. Biometrical analysis revealed that the phenotypic variance (σ2 p) was higher than the genotypic variance (σ2 g) regarding all morphological traits evaluated. The number of seeds per plant reported the highest genotypic (80841.20) and phenotypic (107162.90) variance while the lowest genotypic (0.09) and phenotypic (0.69) value was recorded by the days to germination and average seed thickness respectively. Low (≤20%) heritability broad sense values were observed in all the morphological traits and ranged from 0.33% for days to germination to 0.99% for seed weight. University of Ghana http://ugspace.ug.edu.gh vi DEDICATION This work is dedicated to my parents Mr. King Arthur and Madam Selina Sam, as well as my supervisor Professor Isaac Kwadwo Asante University of Ghana http://ugspace.ug.edu.gh vii ACKNOWLEDGEMENTS I am very much grateful to our gracious Lord for his mercies and grace that sustained me through the Mphil programme successfully. I am extremely thankful to Professor E. T. Blay and Professor Isaac K. Asante for their guidance, support, supervision and intuition. My indebtedness goes to the Head of Department and the whole staff of the Department of Plant and Environmental Biology who supported the accomplishment of my work. My appreciation also go to Mrs. Felicia Oppong for her technical support. I wish to thank the West African Center for Crop Improvement and Ghana Standards Authority for their contributions. To the Genetics Students Group, Nortey, Ralieva Awura-Korkor, Ajara Seidu, Pearl Zoryeku, Henry Mensah and Fredrick Sarkodie Mensah, I say a big thank you for your support. My ultimate appreciation goes to my parents, Mr. King Arthur and Miss Selina Sam, and my siblings, their financial support they offered me through the period of my studies. University of Ghana http://ugspace.ug.edu.gh viii TABLE OF CONTENTS DECLARATION ........................................................................................................... ii ABSTRACT ................................................................................................................. iii DEDICATION .............................................................................................................. vi ACKNOWLEDGEMENTS ......................................................................................... vii TABLE OF CONTENTS ........................................................................................... viii LIST OF TABLES ....................................................................................................... xii LIST OF FIGURES ..................................................................................................... xv LIST OF ABBREVIATION ....................................................................................... xvi LIST APPENDICES ................................................................................................ xviii CHAPTER ONE ............................................................................................................ 1 1.0 INTRODUCTION ................................................................................................... 1 1.1 JUSTIFICATION ................................................................................................. 5 1.2 OBJECTIVES OF STUDY .................................................................................. 6 1.2.1 Main objective ............................................................................................... 6 1.2.2 Specific objectives ......................................................................................... 6 CHAPTER TWO ........................................................................................................... 7 2.0 LITERATURE REVIEW ........................................................................................ 7 2.1 TAXONOMY, ORIGIN AND DISTRIBUTION ................................................ 7 2.2 MORPHOLOGY AND BIOLOGY OF COWPEA ........................................... 10 2.3 PRODUCTION AND PRODUCTION CONSTRAINTS OF COWPEA ......... 12 2.4 COWPEA GERMPLASM CHARACTERIZATION ........................................ 14 2.5 COWPEA CHARACTER ASSOCIATION STUDIES ..................................... 15 2.6 PHYTOCHEMICAL AND NUTRITIONAL CHARACTERIZATION OF COWPEA ................................................................................................................. 17 2.6.1 Phytochemical characterization ................................................................... 17 University of Ghana http://ugspace.ug.edu.gh ix 2.6.2 Nutrition....................................................................................................... 19 2.7 GENETIC VARIABILITY AND HERITABILITY OF COWPEA.................. 19 2.8 THE ROLE OF CLUSTER ANALYSIS AND PRINCIPAL COMPONENT ANALYSIS IN VARIABILITY STUDY ................................................................ 21 CHAPTER THREE ..................................................................................................... 23 3.0 MATERIALS AND METHODS ........................................................................... 23 3.1 SOURCES AND COLLECTION OF COWPEA ACCESSIONS ..................... 23 3.2 EXPERIMENTAL SITE .................................................................................... 24 3.3 EXPERIMENTAL DESIGN.............................................................................. 24 3.4 CULTURAL PRACTICES ................................................................................ 24 3.5. MORPHOLOGICAL CHARACTERIZATION ............................................... 24 3.5.1 Qualitative data ............................................................................................ 25 3.5.2 Quantitative data .......................................................................................... 26 3.6 TOTAL PHENOLIC COMPOUND ANALYSIS .......................................... 31 3.6.7 Amino Acids Analysis ................................................................................. 34 3.7 STATISTICAL ANALYSIS .......................................................................... 35 CHAPTER FOUR ........................................................................................................ 37 4.0 RESULTS .............................................................................................................. 37 4.1 MORPHOLOGICAL QUALITATIVE TRAITS .............................................. 37 4.1.1 Frequency Distribution of Qualitative Traits ............................................... 37 4.1.1.1 Growth pattern .......................................................................................... 37 4.1.1.2 Growth habit ............................................................................................. 37 4.1.1.3 Plant pigmentation .................................................................................... 37 4.1.1.4 Leaf shape ................................................................................................. 38 4.1.1.5 Leaf colour ................................................................................................ 38 4.1.1.6 Twinning tendency ................................................................................... 38 4.1.1.7 Flower colour ............................................................................................ 38 University of Ghana http://ugspace.ug.edu.gh x 4.1.1.8 Pod tip colour............................................................................................ 39 4.1.1.9 Leaf markings ........................................................................................... 39 4.1.1.10 Leaf size .................................................................................................. 39 4.1.1.11 Pod attachment ....................................................................................... 39 4.1.1.12 Pod thickness .......................................................................................... 39 4.1.1.13 Pod colour ............................................................................................... 41 4.1.1.14 Pod curvature .......................................................................................... 41 4.1.1.15 Pod hairiness ........................................................................................... 41 4.1.1.16 Seed coat colour ...................................................................................... 41 4.2 CHI – SQUARE TEST OF ASSOCIATION AMONG QUALITATIVE TRAITS .................................................................................................................... 43 4.2.1 Chi-square test of association in control material ....................................... 43 4.2.2 Chi-square test of association among qualitative traits in the test materials .............................................................................................................................. 48 4.2.3 Chi-square test of association among qualitative traits in the combined material ................................................................................................................. 53 4.3 VARIABILITY IN QUANTITATIVE MORPHOLOGICAL TRAITS ........... 58 4.4 VARIABLITITY OF PHYTOCHEMICAL TRAITS ............................... 64 4.5 ASSOCIATION AMONG QUANTITATIVE TRAITS OF 27 COWPEA ACCESSIONS ......................................................................................................... 70 4.5.1 Morphological traits .................................................................................... 70 4.5.2 Phytochemical traits .................................................................................... 82 4.6 RELATIVE CONTRIBUTION OF TRAITS TO OBSERVED VARIABILITY .................................................................................................................................. 92 4.6.1 Quantitative agro-morphological traits ........................................................ 92 4.6.2 Phytochemical traits. ................................................................................... 98 4.7 INTER-RELATIONSHIPS AMONG ACCESSIONS .................................... 104 4.7.1 Morphological traits .................................................................................. 104 University of Ghana http://ugspace.ug.edu.gh xi 4.7.2 Phytochemical Traits ................................................................................. 108 4.8 GENETIC VARIABILITY, HERITABILITY AND EXPECTED GENETIC ADVANCE ............................................................................................................ 112 4.8.1 Morphological traits ...................................................................................... 112 4.8.2 Phytochemical traits .................................................................................. 115 CHAPTER FIVE ....................................................................................................... 117 5.0 DISCUSSION ...................................................................................................... 117 5.1 PHENOTYPIC VARIABILITY OF AGRO-MORPHOLOGICAL AND PHYTOCHEMICAL TRAITS OF COWPEA .......................................................... 117 5.1.1 Morphological qualitative characterization ...................................................... 117 5.1.2 Morphological quantitative characterization .................................................... 119 5.1.3 Phytochemical traits .......................................................................................... 121 5.2 ASSOCIATION AMONG AGRO-MORPHOLOGICAL AND PHYTOCHEMICAL TRAITS OF COWPEA .......................................................... 122 5.3 GENETIC VARIABILITY, HERITABILITY ANS RESPONSE TO SELECTION FOR AGRO-MORPHOLOGICAL AND PHYTOCHEMICAL TRAITS COWPEA .................................................................................................... 123 5.4 INTERRELATIONSHIPS AMONG COWPEA ACCESSIONS BASED ON AGRO-MORPHOLOGICAL AND PHYTOCHEMICAL TRAITS ........................ 125 CHAPTER SIX .......................................................................................................... 128 6.0 CONCLUSION & RECOMMENDATIONS ...................................................... 128 6.1 CONCLUSION .................................................................................................... 128 6.2 RECOMMENDATION ................................................................................... 130 REFERENCES .......................................................................................................... 131 APPENDICES ........................................................................................................... 150 University of Ghana http://ugspace.ug.edu.gh xii LIST OF TABLES Table 1: List of Cowpea accessions used for the research ...........................................23 Table 2: Qualitative traits measured in 27 cowpea (Vigna unguiculata) accessions...24 Table 3: Polyphenolic compounds and their standard curves ......................................33 Table 4: Frequency distribution of morphological traits for the 27 cowpea accessions .....................................................................................................................42 Table 5: Chi-square test of association among 27 cowpea qualitative traits of control materials. ......................................................................................................................46 Table 6: Chi-square test of association among 27 cowpea qualitative traits of test materials. ......................................................................................................................51 Table 7: Chi-square test of association among 27 cowpea qualitative traits of the combined ......................................................................................................................56 Table 8: Means and their standard errors of morphological quantitative traits in cowpea accessions .......................................................................................................60 Table 9: Means (mg/ml) and standard error values for polyphenolic compounds in the cowpea. ........................................................................................................................65 Table 10: Means (ppb) and their standard error values for the amino acid concentrations in the cowpea .......................................................................................69 Table 11: Correlation coefficient among morphological quantitative traits of the controls .........................................................................................................................72 Table 12: Correlation coefficient among morphological quantitative traits of the test material ........................................................................................................................76 Table 13: Correlation coefficient among morphological quantitative traits of the combined materials. .....................................................................................................80 University of Ghana http://ugspace.ug.edu.gh xiii Table 14: Correlation coefficient among phytochemical traits of the controls ...........80 Table 15: Correlation coefficient among phytochemical traits of the test ...................86 Table 16: Correlation coefficient among phytochemical traits of the combined material ........................................................................................................................90 Table 17: Principal component analysis for quantitative morphological traits of the controls .........................................................................................................................90 Table 18: Principal component analysis for quantitative morphological traits of the tests material ................................................................................................................95 Table 19: Principal component analysis for quantitative morphological traits of the combined material ........................................................................................................97 Table 20: Principal component analysis for phytochemical traits of the control ........99 Table 21: Principal component analysis for phytochemical traits of the test material ....................................................................................................................................101 Table 22: Principal component analysis for phytochemical traits of the combined material ......................................................................................................................100 Table 23: Major cluster group formed from the dendrogram for 23 morphological traits in cowpea accessions ........................................................................................105 Table 24: Distance between cluster centroids for 23 morphological traits in cowpea accessions ...................................................................................................................105 Table 25: Mean value of morphological traits for 5 groups revealed by cluster analysis .......................................................................................................................106 Table 26: Major cluster group formed from the dendrogram for 18 phytochemical traits in cowpea accessions ........................................................................................109 Table 27: Distance between cluster centroids for 18 phytochemical traits in cowpea accessions ...................................................................................................................109 University of Ghana http://ugspace.ug.edu.gh xiv Table 28: Mean value of phytochemical traits for 4 groups revealed by cluster analysis .......................................................................................................................110 Table 29: ANOVA, Estimates of phenotypic and genotypic variances and coefficient of variations, broad-sense heritability, genetic advance and genetic advance as percent of mean among morphological traits in cowpea ........................................................114 Table 30: ANOVA, Estimates of phenotypic and genotypic variances and coefficient of variations, broad-sense heritability, genetic advance and genetic advance as percent of mean among phytochemical traits in cowpea ........................................................116 University of Ghana http://ugspace.ug.edu.gh xv LIST OF FIGURES Figure 1: Dendrogram showing the relationship among 27 cowpea accessions based on morphological traits. .............................................................................................107 Figure 2: Dendrogram showing the relationship among 27 cowpea accessions based on phytochemical traits. .............................................................................................111 University of Ghana http://ugspace.ug.edu.gh xvi LIST OF ABBREVIATION DTG = Days to germination PTL = Petiole length NB = Number of branches NL = Number of leaves NN= Number of nodes NPPPD = Number of pod per peduncle PH = Plant height TLL = Terminal leaflet length TLW = Terminal leaflet width DFF = Days to first flowering APDLN = Average pod length APDWD = Average pod width ANLPP = Average number of locules per pod NSPPL = Number of seeds per plant ANSPP = Average number of seeds per pod DFMP = Days to first matured pod NSAB = Number of seeds aborted PA = Percentage abortion ASDLT = Average seed length ASDWT = Average seed width ASDTH = Average seed thickness SDWG = Seed weight per plant Lhis = L-Histidine Llys = L-Lysine University of Ghana http://ugspace.ug.edu.gh xvii Isoleu = Iso-Leucine Llue = L-Leucine Lmet = L-Methionine Lval = L-Valine Ltryp = L-Tryptophan Dlbp = D-L-B-Phenylalanine Ltyr = L-Tyrosine Lasp = L-Aspartic Acid Lser = L-Serine Lcyst = L-Cysteine Gly = Glycine Gal = Gallic Acid Vani = Vanillic Acid Pca = P-Coumeric Acid Rut = Rutin Que = Quercetin % = Percentage cm = Centimeter mm = Millimeter g = Grams ≥ = greater than or equal to IBPGR = International Board for Plant Genetics and Resources GSA = Ghana Standard Authority ppb = Part per billion University of Ghana http://ugspace.ug.edu.gh xviii LIST APPENDICES Appendix 1: Marginal analysis showing accessions that significantly (P<0.05) performed below and above the grand mean for morphological traits ......................150 University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION Cowpea [Vigna unguiculata (L.) Walp.] is one of the most grown and consumed pea in West Africa as well as some other parts of the world. It is usually grown in semi- arid areas intercropped or in rotation with other plant crops (Menéndez et al., 1997). Cowpea is believed to be indigenous to West Africa, but is part of the daily food of about 110 million people worldwide (Osondu, 1997). It is widely grown and consumed in sub Saharan African countires like Ghana, Nigeria and Niger (Aryeetey, 1971; Bennett-Lartey and Ofori, 1999). Cowpea is mostly grown by peasant farmers and in most cases, these farmers intercrop cowpea with different cereal plant and leagumes such as maize, sorghum, rice, millet as well as other crops including vegetables and plantain. Cowpea is a general-purpose crop, which grown for food for man and animal and serving as an important commodity that generate revenue for farmers and those who trade the grain (Singh 2002; Langyintuo et al. 2003). Cowpea that are grown are classified into five (5) main groups based solely on pod, seed and ovule traits (Pasquet, 1999; 1998). Cowpea plays an essential part in the livelihood of great number of individual worldwide, more especially in the emerging economies. It provide them dietary protein that nutritionally balances low-protein tuber crop staples and cereals (Timko and Singh, 2008). The protein content in the foliage of the cowpea ranges from 15 to 21% and in crop residues; it is 6% to 8%, but the total seed protein content also ranges from 23% to 32% (Nielsen et al., 1997). Cowpea plant is also enriched with vitamin B and vitamin B9 needed during pregnancy to decrease birth defect in the brain and spine according to various researches; (Hall et al., 2003; Timko and Singh 2008). Generally, cowpea are purposely cultivated for their seeds but few individual also grow them for their green leafy vegetables and for animal feed in University of Ghana http://ugspace.ug.edu.gh 2 Africa and for their fresh pods in eastern part of Asia (Boukar et al., 2015). In the cowpea plant, the seed, is the most essential part for human consumption. They are mostly harvested and preserved by drying and consumed when needed. Cowpea can be consumed by cooking or milled into product of flour and used in numerous delicacy (Ahenkora et al. 1998). Cowpea has the capacity to use its root tubers to fix atmospheric nitrogen. They also grow well on barren soils with low levels of phosphorus, more than 85% sand, and less than 0.2% organic matter (Singh, 2003). The crop is known to be a shade tolerant however, its ability to be used in mixed cropping with other crops helps to prevent buildup of weeds, insects and diseases. Globally, cowpea production has increased to about 230 M.P as reported by Singh, (2003), from a yearly average of about 1.2mmt in the 1970s to about 3.6mmt per annum. This rise in production can partially be attributed to the prolonged drought in the West Africa Sahelian zone that made several farmers in sub sarhan of Africa moved away from the production of other crops, to cowpea due to its drought tolerance ability (Duivenbooden et al. 2002). Research has suggested that cowpea is one of the most extensively grown crops in Africa and statistics goes a long way to confirmed that, about 90% of the world’s cowpea grain production of 5.7 million tonnes is cultivated and harvested in Africa (FAOSTAT, 2008). Cowpea is among the most useful crops grown in the hot and semi-arid area of Africa where other crops may not grow due to drought, low soil fertility conditions and poor adaptation to heat. (Ehlers & Hall, 1997; Singh et al., 1999a; Hall, 2004). Five (5), out of the six (6) leading producers of cowpea in the world are found in Africa and they are Burkina Faso, Mali, Nigeria, Niger and Senegal (Fery 2002). Subsequently, improvement of seed quality constitutes a major objective of almost all breeding programs; and the presence of diverse source material has been associated with progress in plant University of Ghana http://ugspace.ug.edu.gh 3 breeding (Mangova and Rachovska, 2004). The development of cowpea varieties that has early ripeness, resistance to some important diseases and pests, and acceptable grain quality have significantly improved yield and acreage (Ehlers and Hall, 1997). Yield is a complex characteristic and is affected by several other significant characteristics that are influenced by environmental factors and polygenes. The total effect of plant breeding on genetic diversity has long been an issue in the evolutionary biology of cultivated plants (Simmonds, 1962). Programmes for cowpea breeding has recognised some germplasm which are beneficial in advancement of the crop, the discoveries includes identifying populations of wild Vigna in the Kalahari Desert that resists bruchid beetle in the cowoea storage (Mathodi, 1992). Selection of plants or crops that have desirable traits to develop new varieties depends mainly on genetic diversity. Genetic diversity is a significant component to the reduction of crop vulnerability by promoting long-term selection and gains in genetic enhancement and encourages judicious use of genetic resources (Barrett and Kidwell, 1998; Messmer et al., 1993). Understanding the genetic diversity in a germplasm is helpful for plant breeders as it supports their decision about choosing a parent genotype and is important for increasing the genetic base of the crop (Prasanthi et al., 2012). The valuation of diversity also enables effective sampling, particularly when the core samples are well developed, which allows for adequate management of the germplasm (Van Hintum et al., 2000). According to Mayes et al., (2009) Some accessions are evaluated primarily on the basis of morphological traits, which are based on few genes and may not necessarily reflect the actual variation in agronomic traits present in the crop. In order to select improved cowpeas varieties that are better adapted to a dry environment, morphological, physiological and phenological criteria could be used to aid in the selection (Blum, 1988). University of Ghana http://ugspace.ug.edu.gh 4 Traditionally, diversity is valued by measuring the variation in phenotypic traits, starting with flowering, days to maturity, plant type, flower colour, seed type, seed colour, seed size, hilum colour, and quantitative morphological characteristics. However, this approach is often limited and the expression of quantitative characteristics which is subjected to strong environmental influences (Kameswara, 2004). To increasing the main components of the cowpea that affect yields, such as pods per plant, seed size, pod length and seeds per pod, will allow for an improvement in the cowpea's yield potential. Morphological variability of cowpea traits has been reported by various authors such as Patil and Baviskar (1987), Sardana et al. (2001), Mishra et al. (2002), Carnide et al. (2007). Knowing about morphological and phytochemical variations and their relationships among cowpeas genotypes can be helpful for breeders to develop appropriate breeding techniques and programs that result in the production of adaptable and productive varieties. The presence of genetic diversity among cowpea genotypes for the different minerals and protein is vital to improve its nutritional quality through breeding (Ng’uni et al. 2012; Shegro et al. 2012; Gerrano et al. 2015; 2017) towards contributing to food and nutritional security. According to Carnovale et al. (1990), the nutritional characteristics in cowpea and they found that the mean concentrations for Ca, Zn, P and K contents in grains were 37.00, 4.70, 430.00 and 125.00 mg 100 g−1, respectively. Therefore, cowpea genotypes can be selected with desirable nutritional contents through characterisation of the available genetic resources. University of Ghana http://ugspace.ug.edu.gh https://www.tandfonline.com/doi/full/10.1080/09064710.2018.1520290 https://www.tandfonline.com/doi/full/10.1080/09064710.2018.1520290 https://www.tandfonline.com/doi/full/10.1080/09064710.2018.1520290 https://www.tandfonline.com/doi/full/10.1080/09064710.2018.1520290 https://www.tandfonline.com/doi/full/10.1080/09064710.2018.1520290 5 1.1 JUSTIFICATION Cowpea is one of the most important grain legumes in Sub-Saharan Africa. Ngalamu et al., (2011) reported that West Africa produces over 80 % of the quantity produced in Africa, with Nigeria, Niger and Burkina Faso as leading producers in the sub- region. Cowpea is a crop cultivated for it grain seeds, immature pods, fresh and dried leaves, as well as hay for livestock feed (Xu et al., 2009). The crop provides strong support to the livelihood of small-scale farmers through its contributions to their nutritional security, income generation and soil fertility enhancement. According to Boukar et al., (2016), about 6.5 million metric tons of cowpea are produced annually on about 14.5 million hectares worldwide. Generally, cowpea production and productivity is low due to insufficient availability of improved varieties and locally adapted cultivars (Ishiyaku et al., 2005), as a results cowpea farmers also have limited access to quality seeds of improved varieties for planting. Some progress has been made through conventional breeding at international and national research institutions in the last three decades (Ishiyaku et al., 2005). Despite all the previous efforts, there is still a big gap in cowpea improvement to identify the ideal varieties that have high yielding potential, high productivity and enhanced nutrition. Cowpea improvement could also benefit from modern breeding methods based on molecular genetic tools, also nutritional analysis of diverse cowpea genotypes on the basis of grain mineral elements and crude protein content is essential in the identification of potential parental genotypes. Improvement of nutritional quality of cowpea genotypes, however, requires information on the genetic variability that exists among available germplasm. The knowledge of genetic variability, heritability, genetic advance as well as correlation among yield and its associated traits in the advance breeding lines is a pre-requisite for selection and development of well-adapted cowpea varieties (Addisu University of Ghana http://ugspace.ug.edu.gh https://www.sciencedirect.com/science/article/pii/S2405844021019939#bib53 https://www.sciencedirect.com/science/article/pii/S2405844021019939#bib53 https://www.sciencedirect.com/topics/earth-and-planetary-sciences/burkina https://journals.ashs.org/hortsci/view/journals/hortsci/50/10/article-p1435.xml#B18 https://journals.ashs.org/hortsci/view/journals/hortsci/50/10/article-p1435.xml#B8 https://journals.ashs.org/hortsci/view/journals/hortsci/50/10/article-p1435.xml#B8 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/genetic-divergence https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/heritability https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/breeding-line https://www.sciencedirect.com/science/article/pii/S2405844021019939#bib1 6 and Shumet, 2015). The efficiency with which genotypic variability can be exploited by selection depends upon heritability, genetic advance and correlation among the individual traits (Bilgin et al., 2010). This present study, report on the characterization of more than 27 cowpea accession from the department of plant and environmental biology (DPEB) was done. The present study was to analyse the morphological and phytochemical traits of these cowpea accessions to assess their genetic diversity. This study may assist in the selection process of cowpea for cultivation based on their characteristic features and draw more attention to the variations that differentiate consumer-preferred cowpea grains from the others. Superior accessions which may be identified during the study can aslo be used as parent to enhance cowpea improvement programmes. 1.2 OBJECTIVES OF STUDY 1.2.1 Main objective The main objective of this study is to evaluate 27 accessions of cowpea using morphological descriptors and phytochemical analysis to identify cowpea accessions with a more specific traits that could be used by breeders. 1.2.2 Specific objectives The specific objectives of the study were to; 1. Determine agro-morphological characterization of cowpea genotypes 2. Evaluate the phytochemical contents of 27 cowpea accessions. 3. Determine association among the cowpea traits. University of Ghana http://ugspace.ug.edu.gh https://www.sciencedirect.com/science/article/pii/S2405844021019939#bib1 https://www.sciencedirect.com/science/article/pii/S2405844021019939#bib13 7 4. Estimate genotypic and phenotypic variances and broad-sense heritability of the morphological traits CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 TAXONOMY, ORIGIN AND DISTRIBUTION Cowpea (Vigna unguiculata (L.) Walp) is a member of the kingdom plantae, division Magnoliophyta, class Magnoliopsida, order Fabales, family Fabaceae, subfamily Faboideae, genus Vigna and species unguiculata (Singh et al., 1993). The cowpea is an herbaceous plant, which grows well under high temperature and drought throughout all development stages and have an optimal growth temperature of around 28 ° C (Craufurd et al. 1997). There are two main cultivated groups of cowpea and they are: unguiculata and sesquipedalis (Xu et al., 2011). Unguiculata is the largest group of varieties and the most diverse of the grown subspecies. These varieties have different growth habit which range from semi-erect, erect, climbing to prostrate. However, growth habit of this variety also ranges from indeterminate to determinate in this cultivar. Cultivar group Sesquipedalis (variously known as “asparagus bean”, “yardlong bean”, “long bean” or “snake bean”) has over 16 ovules and seeds spaced within the pod. Current molecular studies have revealed that the cultivar group Sesquipedalis is a subspecies (Xu et al., 2012; 2010). Faris (1965) concluded that cowpea cultivars were domesticated from subspecies dekindtiana in West Africa, the only centre of cowpea diversity. The variation in West Africa cultivars of subspecies unguiculata included the entire flower, pod and testa colour. And the range of variation within species is greatest in Southern Africa (Marechal, 1978). However, greater diversity does not necessarily mean centre of origin since agriculture is known University of Ghana http://ugspace.ug.edu.gh 8 to have spread from the north to the south through migration. According to Simmonds, (1976) cowpea is now grown throughout most parts of the world. Its use is recorded in Jamaica as early as 1675. It has a documented use in Florida in 1700 and in North Carolina in 1714. The introduction and spread of cowpea into the new tropics from West Africa are alleged to have occurred along the slave trade route through the Spanish (Simmonds, 1976). The crop was first grown in southern United States in the early eighteenth century. Cowpea is believed to be one of the primeval crop plants known to human (Mejia 2004) and one of the first food source for humans since the Neolithic era. (Summerfield, Huxley, and Steel, cited by Agyemang et al., 2014). The exact genesis of cultivated cowpeas has been an issue of speculation for such a long time (Timko & Singh, 2008). As assessed by Asante (1996), the origin of cowpea is unknown but believed to have originated from West Africa particularly, due to its abundance of most wild and cultivated species. Few archaeological evidences have revealed a counter argument supporting that Africa, Asia, and South America as the origin of the crop (Doumbia, 2012). One view is that, the origin and domestication of the cowpea took place in Africa near Ethiopia and was subsequently developed mainly in the farms of the African savannah. (Duke, quoted by UC SAREP, 2017). Girei (2013) also notes that the origin and subsequent domestication of cowpea correlates with pearl millet and sorghum in Africa. The slave trade in West Africa resulted in the harvest reaching the southern United States in the early 18th century (Doumbia, 2012). Other evidence suggests that the Transvaal region in the Republic of South Africa was the center of speciation for cowpeas due to the presence of the most primitive wild species (Padulosi and Ng, cited by Doumbia, 2012). University of Ghana http://ugspace.ug.edu.gh 9 Cowpea is grown extensively in 16 African countries, with the continent producing two-thirds of the world total (Winrock, cited by Agyeman et al., 2014). West Africa produces the most diverse cultivated cowpea in an area including southern Niger, the savanna region of Nigeria, Togo, northern Benin, the northwestern part of Cameroon and part of Burkina Faso (Doumbia, 2012). In Nigeria, cowpea is known to be cultivated mostly in the North West and North Central geo-political zones also substantial quantities are also grown in the other four geo-political zones identified in the country, (Davis, cited by Girei, 2013). In South America, Brazil is the only major country which grows cowpea. (Gomez, 2004). There are three recognized specific groups of cultivated cowpeas. Two of these are grown in Australia with most varieties grown for grain, forage and green manure and the other type, the yard-long bean, is a minor vegetable. (Imrie, cited by Mejia, 2004). The origin of cowpea had been related to its morphological and cytological proof and data on the geographical distribution and cultural practices (Ng, 1995; Ng and Maréchal, 1985). It is most probable that cowpea was first introduced to Asia during the Neolithic period (Pant et al. 1982) and prior to the Christian era, since it has a Sanskrit name in writings dated to 150 BC (Ng and Maréchal 1985). Therefore, Asia is often considered a secondary domestication site for the crop. Cowpea is geographically distributed worldwide on a large scale throughout the western and central African, countries such as Nigeria, Mali, Niger, Burkina Faso, Tanzania, Senegal and Uganda. Outside Africa, India, Myanmar, Sri Lanka, Australia, the United States, Brazil, Bosnia and Herzegovina all have significant distribution. University of Ghana http://ugspace.ug.edu.gh 10 2.2 MORPHOLOGY AND BIOLOGY OF COWPEA Morphological variations in different cowpea accessions is very high. In terms of use, there are three main types: for grain, feed or dual purpose. Cowpea is an herbaceous pendulous, climbing bushy, prostrate, or semi-upright annual plant that can grow to 15cm-80cm tall. The leaves are alternately three-leaved with a petiole length of 5- 25cm. The lateral leaflet is asymmetrical and opposite, while the middle leaflet is ovoid and symmetrical. The leaves show noteworthy differences in shape (linear, hasty, spherical, and lanceolate to ovate), size (6-16 x 4-11) and are typically dark green in colour. The stems are straight, smooth or slightly hairy and sometimes purple in colour. The flowers come in alternate pairs that mostly have only two flowers on each inflorescence. These are self-pollinating, conspicuous, borne on short pedicels and the corollas may be white, pink, cream, pale, yellow, blue or purple. The flowers close at about midday after opening early in the morning. They wilt and collapse after blooming/ anthesis. Growth pattern is usually indeterminate under favorable conditions but can be determinate as well. The pods of the fruit vary in shape, size, texture and colour. They plant can be crescent-shaped, erect or coild. They are commonly slightly yellow when ripe, but ca be purple or brown in colour. Seeds may weigh 5-30 g/100 seeds and are relatively large (0.2-1.2 cm long) in size. They are variable in shape such Crowder, kidney, rhomboid, ovoid, and globose; and may also vary in size (IBPGR, 1983). The testa can be either smooth or wrinkled, green, white, buff, brown, red, black, blotched, speckled, eyed or mottled in color. The seed shape dpends on pod shape. The average pod length of the cowpea is between 8-22 cm and it contains 10-20 seeds per pod (Chevalier, 1944). Cowpea is a short day plant and many cowpea accessions show photoperiodic sensitivity to flower bud initiation and University of Ghana http://ugspace.ug.edu.gh 11 development, but others are day neutral (Ehlers and Hall 1996; Craufurd et al. 1997). In some genotypes, the degree of sensitivity to the photoperiod is changed by temperature (Wein and Summerfield 1980; Ehlers and Hall 1996). In West Africa, different levels of light sensitivity was selected in different climatic zones, so that pod ripening at a certain location coincides with the end of the rainy season, regardless of the planting date, which is often variable due to the variability at the beginning of the rainy season (Steele and Mehra 1980). These characteristics allows pods to be protected againts harm from pathogens and excessive moisture. Photoperiod sensitivity, when well done, can be vital to guarantee crop maturity before drought, after wet seasons or cold weather limits that could impair crop growth. Cultivated cowpeas can be classified into five cultivar groups depending chiefly on their seed characteristics and pod (Pursglove 1968; Pasquet 1999). Cultivar group Unguiculata is the largest and includes most medium- and large-seeded African grain and forage- type cowpeas. Members of cultivar group Biflora abound in India and categorized by their relatively small smooth seeds borne in short pods that are held erect until maturity. Cultivar group Textilis is a rather rare form of cowpea with enlongated peduncles that were used in Africa as a source of fiber. Cultivar group Melanophthalmus includes “blackeye pea”-type cowpea with large, fragile pods, and somewhat elongated seeds with wrinkled seed coats (Pasquet 1999). Cultivar group Sesquipedialis (known as “yardlong bean,” “long bean,” “Asparagus bean,” or “snake bean”) is commonly grown in Asia for production of its very long (40 to 100 cm) green pods that are used as “snap” beans. Regardless of the differences in the morphological characteristics seen among the different cultivar groups, there are no known hurdle to recombination between members of the different cultivar groups. University of Ghana http://ugspace.ug.edu.gh 12 2.3 PRODUCTION AND PRODUCTION CONSTRAINTS OF COWPEA Rachie and Rawal (1975) reported that bulk of world-wide production of cowpea is mainly in Africa which produces about 75% with Nigeria being the principal producer (58.6%), Burkina Faso (5.6%), Niger (2.4%) and Senegal (2.0%). According to the Food and Agriculture Organization of the United Nations FAO / UN (2012), the average cowpea yield in West Africa in 2012 was an estimated 483 kg/m, which is 50% below the estimated potential production yield. Cowpea production is grown on over 10 million hectares of land worldwide, more than 85% of which is in Africa (FAOSTAT, 2008). Asia, Middle East, Southern Europe, South USA and Central and South America are also known to be huge producers of cowpea (Singh et al., 2002). In Ghana, the crop is usually cultivated under rainfed conditions predominantly in the savannah and transitional agro ecological zones, but the bulk of the grain is produced in the northern part of the country (CSIR-SARI, 2012). Cowpea yields in Ghana are among the lowest in the world, averaging 310 kg ha-1 (Ofosu-Budu et al., 2007) and efforts made to improve the production of the crop include the introduction of high yielding varieties (Addo-Quaye et al. 2011). According to FAOSTAT (2017), cowpea was grown on an estimated 11 million ha in Africa in 2017 with most of production confined to West Africa (10.6 million ha), especially in Niger, Nigeria, Burkina Faso, Mali, and Senegal. More than 7.4 million tons of cowpeas are produced worldwide, with Africa producing nearly 5.2 million tons. Second to the most important pulse after pigeon pea, cowpea contributes to about 13% to 17% of food pulses for subsistence in the tropics. For good plant stand and high yields, seeds must be free of insects and diseases (Dauost et al., 1985). Planting preferably should be planned in relation to the maturity period of the variety University of Ghana http://ugspace.ug.edu.gh 13 such that the crop is harvested in bright dry weather. According to (SARI, 2012) early light sensitive types can be planted depending on the patterns of rainfall. Cowpea cultivars with creeping growth habit are shade tolerant, and can be grown with maize, millet, and sorghum in mixed cropping system. This quality makes cowpea an essential component in the mixed cropping systems, more importantly in dry savanna areas. In these areas, dried stalks of cowpea are valuable animal feed and are sold to livestock farmers. Most cowpea varieties form root nodules freely. But recent studies in northern Ghana have shown that cowpea will fix more nitrogen when farmers inoculate the soil with rhizobia. Cowpea production faces challenges and thus affects its productivity worldwide. According to (ICRISAT, 2013), the major constraints in Africa’s high yield cowpea production are mainly infertile soil, diseases, drought and insect pest. The major challenge to growing cowpea in Ghana is insect pests and weeds such as Striga and Alectra. Insects and weeds can cause losses of 15 to 100%, depending on the level of infestation, drought, soil fertility, and the susceptibility or resistance of the variety. A few cowpea varieties are resistant to striga. Many areas with a high population of insect infestation can lead to zero crop output when appropriate steps are not implemented (Asiwe et al, 2005). Aphid (Acyrthosiphon pisum) is one of the most destructive insect pest of cowpea plant. In large numbers, aphids can lead to a lot of losses and early infestation can result in enation mosaic virus (ICRISAT, 2013). The insect sucks sap from the cowpea plant and through this feeding habit spreads viral diseases. University of Ghana http://ugspace.ug.edu.gh 14 2.4 COWPEA GERMPLASM CHARACTERIZATION Germplasm characterization refers to recording of traits that are highly heritable. These heritable traits and their attributes identified in germplasm characterization are known as descriptors. These descriptors can either be quantitative or qualitative. Qualitative refers to descriptive traits, whereas quantitative refers to measured or numerical scoring of characteristics. Cobbinah et al. (2011) studied approximately one hundred and thirty-four (134) accessions of cowpea which were collected from eight (8) topographical origins in Ghana. These accessions were grown at two (2) different locations, Bunso, a semi- deciduous forest and Pokuase, a coastal savanna to analyse their performance and to select the genotype with desirable agro-morpholical characters for further improvement. By 1986, about a total of 35,000 accessions of more than crop types have been multiplied and characterized. Like any other crop, cowpea breeding programmes comprise important procedures. The first step involves, breeders collecting and evaluating germplasm to select parental lines or cultivars for crosses. A germplasm characterization is carried out for the following reasons: Classification of accessions into their natural groups, describe accessions and determine their characteristics, estimate the extent of the variation in the genebank collection, assess relationships between accessions or between features and between geographical groups of collections, monitor possible genetic changes in the germplasm collection and identifying duplicates in a collection. University of Ghana http://ugspace.ug.edu.gh 15 2.5 COWPEA CHARACTER ASSOCIATION STUDIES Correlation coefficients, is the measure of relationship that exist among different plant characters or traits, which facilitate selection. The degree of identified association between two characters is referred to as, the total phenotypic correlation. However, environmental correlation is the measure of ecological impact on the co-variation between the two traits being referred to. Correlation studies provide knowledge regarding association of traits among themselves and with economic traits on which indirect selection could be made for improvement. It helps to simultaneously select for more than one character of importance at a time. However, correlation is inadequate to elucidate the exact association between traits, as yield is dependent on many components characters. The relative magnitude of several traits is therefore more desirable to be considered in order for a stronger representation of yield components for effective breeding programs (Ajayi et al., 2014; Sadras et al., 2019). Moreover, significant correlations among a number of traits and seed yield of different genotypes of cowpea across numerous locations are valuable in planning an effective selection and breeding program for cowpea. According to Falconer (1993), the amount of relationship between two traits that is observed directly is the phenotypic value. Many works on cowpea have revealed significant variability and degree of association of traits among yield and yield contributing traits in cowpea (Adewale et al., 2010; Ajayi et al., 2014). According to Patil et al. (1989) grain yield was much strongly influenced by number of pods per plant, 100 seed weight, pod length and days to 50 % flowering. Siddique and Gupta (1991b) identified an extensive correlation of seed yield per plant with days to 50 % flowering and number of pods per plant. Oseni et al. (1992) found a strong positive relationship between seed yield and pods per plant, between number of University of Ghana http://ugspace.ug.edu.gh 16 days to 50% flowering and 100-seed weight; however, negative association occurred between days to 50% flowering and seed yield and between 100 seed weight; seed yield, days to maturing, 100-seed weight, and days to pod maturity and pod length were the significant factors adding to yield. Altinbas and Sepetoglu (1993) suggested that days to flowering had no influence on seed yield. On the contrary100 seed weight was associated with number of pods per plant and seeds per pod. According to Sawant (1994) seed yield was significantly and particularly corresponded with number of branches per plant, inflorescence per plant, pod per plant, pods length, seeds per pod, 100-seed weight and harvest index. Tamilselven and Das (1994) suggested that number of pods per plant and 100 seed weight must be utilized as a determination model in the progression of high yielding cowpea genotypes. In a review including three F2 populations Birader et al. (1996) detailed that there was solid connection existed in pod weight per plant and seed yield, number of seeds per pod and pod length, number of pods per plant and number of clusters and pod weight per plant. Gowda (1996) revealed a positive and huge relationship between seed yield and number of pods per plant, number of seeds per pod and 100-seed weight and further reported a critical and negative relationship with 100-seed weight and number of seeds per plant. Singh and Singh (1997) showed that the number of seeds per pod and number of pods per plant made most vital direct contribution to seed yield in 45 accessions of cowpea. Venkatesan et al. (2003) observed that the number of branches per plant, number of pods per cluster, number of pods per plant and pod yield were highly associated with seed yield at the genotypic and phenotypic levels when he considered the association and path analysis studies in 20 genotypes of cowpea. The extent of genotypic relationship was higher than that of phenotypic relationship. University of Ghana http://ugspace.ug.edu.gh 17 2.6 PHYTOCHEMICAL AND NUTRITIONAL CHARACTERIZATION OF COWPEA 2.6.1 Phytochemical characterization 2.6.1.1. Polyphenolic compound According to Mtolo, et al. (2017), Awika, and Duodu (2016), Phenolic compounds are the most important group of bioactive compounds in cowpea, are concentrated in the seed coat and have the potential to protect the body against chronic diseases. According to Pandey & Rizvi (2009), polyphenolic compounds are secondary plant metabolites which are usually used in defense against ultraviolet rays and aggression by plant pathogens. These phenolic compounds are responsible for most of the colouration, flavor, bitterness, odor and oxidative stability observed in diverse cowpea seeds (Pandey and Rizvi, 2009). The most vital phenolic compounds identified to be in cowpea are the flavonoids and phenolic acids (Awika, & Duodu 2016, and Zhao, et al., 2014). Important polyphenolic compounds which are predominant in cowpea include trans-ferulic acid, trands-p-courmaric and the derivatives of flavonoids (Pandey & Rizvi, 2009). Nassourou et al., (2016) stated that flavonoids are also have an essential role in plant protection. Epidemiological studies have revealed the consumption of phenolic antioxidants-rich foods protect humans against chronic non- communicable diseases such as cancer, ageing, diabetes, and cardiovascular disease, again they also act as scavengers of radicals, reducing agents, and metal ion chelators (Zhao, et al., 2014). According to Kapravelou et al. (2015), cowpea seeds have some beneficial health effects related to their antioxidant, hypoglycaemic, hypolipidaemic, University of Ghana http://ugspace.ug.edu.gh 18 and antihypertensive properties. Phenolic content composition and bioactive properties of cowpea may vary greatly depending on the cultivar or variety. 2.6.1.2 Amino acids Amino acids are the building blocks of proteins and, based on their synthesis, are divided into essential and non-essential amino acids in humans. The essential amino acids are only synthesized by plants, while non-essential amino acids are synthesized by both plants and humans. The essential amino acids must be ingested from external sources in animal or vegetable food. Legumes are the most important staple food worldwide. Research has shown that their seeds contain the following nutritional content; Carbohydrates and proteins (Auestad & Fulgoni, 2015; Burch et al., 2007; Millward et al., 2008; Tessari et al., 2016). Essential amino acids are those that are very important to build proteins in humans and animals, but cannot be synthesized by these organisms themselves. For this reason essential amino acids must be provided in the diet. In humans, there are nine essential amino acids, these are; histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. Non-essential simply means our bodies produce these amino acids, and must not necessary to be added to the food we eat. These are examples of non-essential amino acids; alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine. The legumes most commonly used worldwide are beans, chickpeas, lentils, peas, broad beans, soybeans (Guillon & Champ, 2002). University of Ghana http://ugspace.ug.edu.gh 19 2.6.2 Nutrition Cowpea (Vigna unguiculate (L.) Walp.) is a widespread food crop in the tropics and sub-Saharan regions that is used as human food because of its nutritional value (Alidu et al., 2020). According to Addo-Quaye et al., (2011) cowpea seeds serve as a good source protein (19-35%) and an essential source of carbohydrate (50-60%). Quaye et al. (2009) reported that, cowpea may contains substantial quantity of micronutrients such as, iron, calcium, zinc and vitamin A. The high quantities of Ca, Mg, K, and Zn in the leaves and stems have also been identified, suggesting that they could used as a mineral source for animals and soil improvers to improve fertility and increase crop yield and also used as supplement (Alidu et al., 2020). Anele et al., (2010) reveal in their study that the haulms of cowpea could be used to support milk production in lactating dairy cattle and the growth of animals. The chemical constitution of cowpea and its nutritional properties vary greatly between different variety (Giami, 2005; Rangel et al., 2004). Hence, they are a very important components of cowpea improvement. Therefore an evaluation of their nutritional-physiological properties is very important for a good use of newly developed cowpea varieties for human and / or animal nutrition (Giami, 2005). 2.7 GENETIC VARIABILITY AND HERITABILITY OF COWPEA As with all crops, the likelihood of improvement in cowpea depends on the amount of genetic variability. Studies initiated by Johanssen (1909) and East (1916) have resulted in the division of total variability into genetic and environmental components. In all cases, both heritable and non-heritable factors influence variation in segregating populations, while variation in pure line is solely due to the environment (Johanssen, 1909). These heritable genetic characteristics vary among cowpea in diverse manner. University of Ghana http://ugspace.ug.edu.gh 20 According to Inuwa et al. (2012) and Gerrano et al. (2015), the existence of wide- ranging genetic variability is essential to effective selection for traits of interest in a plant breeding improvement program. According to Amoatey (1987), variability studies in seed colour, pod length, number of seed per pod have been conducted in some cowpea accessions in Ghana. In cultivated plants, the adaptability of the genotypes varies due to their genetic differences. As an expression of yield and other quantitative characteristics, the environmental conditions have a significant influence. Previous studies performed by Comstock and Robinson (1952), Athwal and Singh (1966) and Mital et al. (1969), all revealed that the evaluation of genotypes under several environmental conditions provided an interesting information on the relative magnitude of phenotypic and genotypic variability and the extent of genetic advance. Robinson et al., (1949), Grafius, (1959), and Nikell and Grafius, (1969) paid more emphasis on the improvement of crop yield by studying the variability and heritability of yield and yield contributing components of crops. Asante (1991) also studied variability in pigmentation of immature pod tip, flower bud tip and sepal, seed coat colour and eye pattern, flower colour and paleness in chlorophyll. Spillman (1913) noticed that all varieties having coffee-colour or white or cream-colour seed had white flowers and devoid of anthocyanin in stem and leaves. Grafius (1964), observed that the major yield components in cowpea in the accessions he studied were; number of seeds per pod, number of pods per plant and 100g seed weight. He further noted that any change in yield is as a results of change in any one or more of the component listed. The heritability score of a trait shows the effectiveness of selecting the trait based on phenotypic expression. In almost all cases, quantitative traits are determined by a University of Ghana http://ugspace.ug.edu.gh https://journals.ashs.org/hortsci/view/journals/hortsci/50/10/article-p1435.xml#B7 https://journals.ashs.org/hortsci/view/journals/hortsci/50/10/article-p1435.xml#B5 21 larger number of genes and are further influenced by the environment, some of the observed values of which are inheritable. 2.8 THE ROLE OF CLUSTER ANALYSIS AND PRINCIPAL COMPONENT ANALYSIS IN VARIABILITY STUDY Morphological traits have been used comprehensively to establish phylogenetic relationships between and within species. Again, they have been used to study variability and correlated traits in different accessions of cowpeas. Environmental factors has a strong influence on morphological traits that may result in varied relationship pattern outlines (Smith & Smith 1989, Selvi et al. 2003), but they are nevertheless a good source of selecting materials. (Magloire 2005). Quantitative and qualitative traits have been used by many researchers for various purposes in cowpeas and related plants (Pasquet 1993, Obute 2001). Some multivariate statistical tools that have been used extensively include principal component analysis (PCA), and the cluster analysis (CA), these techniques identify plant traits that characterise the distinctness among selected genotypes. Principal component analysis is used to analyze multivariate data and generate new sets of variables that are linear combinations of the original variables (Zagrodzki et al., 1995). The fundamental idea of Principal Component Analysis (PCA) is to reduce the dimensionality of a data set that consists of a large number of interrelated variables and at the same time, an attempt is made to preserve as much as possible of the variation present in the data set. This is achieved by converting it into a new set of variables, the Principal Components (PC), which are not correlated and are arranged in such a way that the first variables retain most of the variation that is present in all of the original variables (Jolliffe, 2002). University of Ghana http://ugspace.ug.edu.gh 22 The Principal Component Analysis (PCA) has been used to partition observed agronomic variations in genotypes of many crops such as rubber (Omokhafe and Alika, 2000), rice (Nassir, 2002), and sesame (Mponda et al., 1997). Principal component analysis is an adaptive data analysis technique which is effectively used to visualize the similarity and difference between the genotypes and helps in identifying the quantitative characters contributing maximum towards genetic divergence (Jindal et al., 2018, Ringnér 2008). The use of cluster analysis as an effective tool in classifiying cowpea accessions with similar characteristics cannot be over emphasized, as it has now become very widespread in research studies (Sabater 2004, Vural & Karasu 2007). Furthermore, analysis of phylogenetic tree has most distinguished afford to classify the different clusters by the dendrogram construction for identifying the greater variability, which is more useful to the trait of interest for future crop plant breeding enhancement Cluster analysis and Principal component analysis could be used to evaluate the degree of morphological variability betweem cowpea accessions and also to know the exact relationships between yield and yield component of these accessions. Hence, these will be useful tool for breeders for breeding purposes. University of Ghana http://ugspace.ug.edu.gh 23 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 SOURCES AND COLLECTION OF COWPEA ACCESSIONS Seeds of accessions of cowpea were collected from Department of Plant and Environmental Biology (DPEB), University of Ghana. The cowpea germplasm had been collected, separated according to seed colour, assigned accession numbers and put under cold storage. Twenty-seven (27) cowpea accessions were selected from the germplasm for the study, out of which three were used as controls (Asontem, Kirk House and Wang Kae) and the remaining 24, the test materials. These cowpea accessions were evaluated for various agro-morphological and phytochemical characteristics of interest to plant breeders and growers. A list of the 27 cowpea accessions showing their origin is shown in Table 1. Table 1: cowpea accessions used for the research Accessions Sources Accessions Sources Asontem SARI T 12 DPEB Kirk House SARI T 13 DPEB Wang Kae SARI T 14 DPEB T 1 DPEB T 15 DPEB T 2 DPEB T 16 DPEB T 3 DPEB T 17 DPEB T 4 DPEB T 18 DPEB T 5 DPEB T 19 DPEB T 6 DPEB T 20 DPEB T 7 DPEB T 21 DPEB T 8 DPEB T 22 DPEB T 9 DPEB T 23 DPEB T 10 DPEB T 24 DPEB T11 DPEB University of Ghana http://ugspace.ug.edu.gh 24 3.2 EXPERIMENTAL SITE The experimental field was the Experimental farm of the West Africa Centre for Crop Improvement (WACCI), University of Ghana, Legon, 3.3 EXPERIMENTAL DESIGN The experimental design used for the field experiment was a randomised complete block design (RCBD) with three (3) replications. Each accession was represented by 30 plants. The spacing between and within rows was maintained at 50 cm X 50 cm. One seed was planted per a hole. 3.4 CULTURAL PRACTICES Plants were watered two times a day and weeding was done with cutlass every 2 weeks. No fertilizer was applied because the WACCI field was used. Cowpea plants were sprayed against insect pests using an insecticide called “Attack” at a rate of 600 mls/ha at 14, 28, and 42 days after sowing 3.5. MORPHOLOGICAL CHARACTERIZATION Agro-morphological data on the plants were recorded after 6 weeks of planting. Cowpea Descriptor by International Board of Plant Genetic Resources (IBPGR) (1983) was used in collecting both quantitative and qualitative data on the following traits. Thirty–nine (39) morphological triats (qualitative and quantitative) were scored comprising 16 qualitative and 23 quantitative traits. The sixteen (16) qualitative traits analysed were, growth pattern, growth habit, twinning tendency, attachment of pods to peduncle, flower colour, plant pigmentation, pod pigmentation, pod shape, pod thickness, terminal leaflet shape, leaflet markings, leaflet size, leaflet size, leaf colour, seed colour and plant hairiness. University of Ghana http://ugspace.ug.edu.gh 25 The twenty-three (23) quantitative traits were studied. These comprised; plant height, days to germination, number of branches, number of nodes per main stem, number of leaves, petiole length, days to first flowering, days to pods maturity, pod length, pod width, leaflet length, leaflet width, seed length, seed width, seed thickness, number of seeds aborted, percentage seed abortion, pods per peduncle, locules per pod, seeds per pod, pods per plant, 100 seed weight, number of seed per plant and peduncle length. 3.5.1 Qualitative data Qualitative characters were scored based on visual evaluation in accordance with the cowpea descriptor and with the aid of banana colour chat, where colours were involved. The qualitative traits were scored by using relevant scales as shown in Table 2. Table 2: Qualitative traits measured in 27 cowpea (Vigna unguiculata) accessions. Traits Score Code Growth habit 1=Acute erect, 2=Erect, 3=Semi-erect, 4=Intermediate, 5=Semi– prostrate, 6=Prostrate, 7=Climbing Growth pattern 1=Determinate, 2=Indeterminate Twinning tendency 0=None, 3=Slight, 5=Intermediate, 7=Pronounced Plant hairiness 3=Glabrescent, 5=Short appressed hairs, 7=Pubescent Leaf colour 3=Pale Green, 5=Variegated, 7=Dark green Leaf marking 0=Absent, 1=Present Flower colour 1=White, 2=Violet, 3=Mauvie-pink, 4=White with violet patches Leaf size 1=Narrow (<10 cm), 3=Small (10 - 20 cm), 5=Medium (21 - 30 University of Ghana http://ugspace.ug.edu.gh 26 cm), 7=Big (31-40 cm) Terminal leaflet shape 1=Globose, 2=Sub–globose, 3=Sub–hastate, 4=Hastate Plant pigmentation 0=None, 1=Very slight, 3=Moderate at the base and tips of petioles, 5=Intermediate, 7=Extensive, 9=Solid Pod colour 1=Straw, 2=Dark tan, 3=Reddish straw, 4=Tan, 5=Greenish tan Podtip colour 0=Absent, 1=Present Pod attachment to peduncle 3=Pendant, 5=30o – 90o down from erect, 7=Erect Pod curvature 0=Straight, 3=Slightly curved, 5=Curved, 7=Coiled Pod wall thickness 3=Thin, 5=Intermediate, 7=Thick Pod hairiness 3=Glabrescent, 5=Short appressed hairs, 7=Pubescent seed coat colour 1=Reddish brown, 2=white, 3=Cream, 4=Cream with brown patches 3.5.2 Quantitative data Three plants per accession were selected randomly and analysed for quantitative assessment. Measurements were taken and recorded in SI units. 3.5.2.1 Mean days to germination Number of days from date of planting to germination was recorded for each accession and the mean was estimated and recorded. University of Ghana http://ugspace.ug.edu.gh 27 3.5.2.2 Mean plant height Plant height measurements were taken as the distance between the soil level or collar and apex of the plant. This measurement was done by using a 30-centimetre rule. Plant height was recorded for each plant and the mean was estimated and recorded. 3.5.2.3 Mean number of nodes The number of nodes of each plants were counted and the mean was estimated and recorded. 3.5.2.4 Mean leaf length The leaf length was taken by using a 30-centimetre rule. Measurement was done by taking the distance from the leaf base of the lamina to leaf apex for each plant. The mean was then estimated and recorded. 3.5.2.5 Mean leaf width Leaf width measurement were taken using a 30-centimetre rule. The broadest part of the leaf of each plant was measured and the mean was estimated and recorded. 3.5.2.6 Mean number of leaves The number of leaves of three plants per accession was obtained by counting both the matured and young leaves and the mean number of leaves of three plants per accession was estimated and recorded. University of Ghana http://ugspace.ug.edu.gh 28 3.5.2.7 Mean number of branches The number of branches of three plants per accession was counted and the mean of two plants per accession was estimated and recorded. 3.5.2.8 Mean petiole length The length of two petiole per plant for three plants per accession was measured using a 30-centimetre rule from the node to the tip of the petiole. The average petiole length of three plants per accession was estimated and recorded. 3.5.2.9 Mean days to first flowering The mean number of days of flowering per accession from days of germination to flowering were estimated and recorded. 3.5.2.10 Mean number of pods per peduncle Number of pods per peduncle for three plants per accession was obtained by counting both the matured and the young pods per peduncle. The mean of three plants per accession was estimated and recorded. 3.5.2.11 Mean number of pods per plant Two of pods per plant of three plants per accession was obtained through counting both matured and the young pods and the mean number of pods of three plant per accession were estimated and recorded. University of Ghana http://ugspace.ug.edu.gh 29 3.5.2.12 Mean pod length Two fully matured pods per plants of the three accession were measured using a string from the stylar end to the point of attachment of the pod to the stalk. The string was stretched on a 30-centimetre rule and the pod length was determined in centimeters. The mean pod length and its standard errors of three accesions were estimated and recorded. 3.5.2.13 Mean pod width The pod width was taken by measuring the width of the same three pods which was used to measure the pod length. The measurement was done using a 30-centimetre rule. The mean pod width of three pods was calculated and recorded. 3.5.2.14 Mean number of locules per pod Three pods were studied for this traits. Pod locules were recorded by counts. Three pods were broken along the dorsal sutures by the hand and ovules were counted starting from the stylar end to the basal end. Counts were taken for the total number of locules per pod. The measurement was done using the three matured pods for each accession and the mean locules per three pods were calculated and recorded. 3.5.2.15 Mean days to first mature pod The mean number of days from germination to the first matured pod per accession was estimated and recorded. University of Ghana http://ugspace.ug.edu.gh 30 3.5.2.16 Mean number of seeds per pod Two pods per plant of the three accessions were threshed and the number of seeds in each pod was counted and mean number of seeds of two pods was estimated and recorded. 3.5.2.17 Mean number of seeds per plant Two plants per three accessions were selected and the number of seeds in each pod per accession was counted. The mean number of seeds of two plants per accession was calculated and recorded. 3.5.2.18 Mean number of aborted seeds Three pods were studied for the mean percentage seed set for each pod, the total number of seeds and the total number of locules were calculated and recorded. The mean number of aborted seeds of three pods were calculated and recorded 3.5.1.19 Mean percent seed set (PA) Mean number of seeds per pod was expressed as a percentage over the mean total number of locule per pod. This was recorded as the mean percentage seed set per accession. 3.5.2.20 Mean seed length A micrometre screw gauge was used to measure length of each of 10 randomly chosen seeds from each accession. The mean seed length was calculated and recorded. University of Ghana http://ugspace.ug.edu.gh 31 3.5.2.21 Mean seed width A micrometre screw gauge was used to measure the width of each of 10 randomly selected seeds from each accession. The mean seed width was calculated and recorded. 3.5.2.22 Mean seed thickness A micrometre screw gauge was used to measure the thickness of each of 10 randomly selected seeds from each accession. The mean seed thickness was calculated and recorded. 3.5.1.23 Mean seed weight The seeds of two plants per three the accessions were weighed separately using an electronic balance and mean seed weight for each accession was calculated and recorded. 3.6 TOTAL PHENOLIC COMPOUND ANALYSIS 3.6.1 Preparation of cowpea flour 100g of the cowpea seeds from each accessions were collected and pulverized by using an electronic blender into cowpea flour. 3.6.2. Preparation of sodium carbonate solution A 50ml volumetric flask was filled with 20ml distilled water. A mass of 6.25g of sodium carbonate was weighed and dissolved in the distilled water. The solution was boiled, allowed to cool and then few crystals of sodium carbonate were added. The University of Ghana http://ugspace.ug.edu.gh 32 solution was made to stand for 24 h and then filtered. Distilled water was added up to the 25ml mark. 3.6.3 Extraction of samples for phytochemical studies A mass of 0.5g of the cowpea flour was weighed using the electronic balance and poured into McCartney bottles. Twenty millilitres of 100% methanol was added and shaken. The bottles were covered and allowed to stand for 24 hours. After the 24 hour period, the sample in solution was filtered and the filtrate stored in tightly covered McCartney bottles at a temperature of 4 °C in the fridge. 3.6.4 Determination of phenolic acid content Extract of the cowpea flour for each accession were analysed for phenolic compounds using the Folin-Ciocalteu method (Singleton and Rossi, 1965). After the 24 hour period, 1 ml of each extract was measured with a measuring cylinder and then diluted to 10 ml with distilled water in test tubes. Twenty microliters (20μl) of diluted samples were pipetted into cuvettes. A volume of 1.58 ml of distilled water and 100 μl Folin-Ciocalteu reagent was measured with a measuring cylinder and a 100 μl micropipette respectively and was added to the solution. The solution was shaken to mix. A volume of 300μl of sodium carbonate was pipetted and added to the solution after 5 min and shaken. The solution was placed in the oven for 30 min at a temperature of 40 °C. The cuvettes were taken out after the 30 min and allowed to stand for 90 min. The absorbance at 765 nm was determined against the blank methanol using the visible spectrophotometer. The concentration for the phenolic compounds for each accession was determined from the standard curves of linear equations (Table 2). University of Ghana http://ugspace.ug.edu.gh https://www.sciencedirect.com/science/article/pii/S2405844020302516#bib33 33 3.6.5 Determination of flavonoid content A modified aluminium chloride colorimetric procedure was used for the determination of flavonoid content in the cowpea sample. A volume of 100μl of samples extract was pipetted and added to 500μl of distilled water and 30 μl of 5% sodium nitrite in cuvettes. The solutions were made to stand for 5 min after which 30 μl of aluminium chloride was added. The solutions were allowed to stand again for 6 min after which 200 μl of sodium hydroxide and 110μl of distilled water were added to the solutions and vortexed. The absorbance was taken at a wavelength of 425 nm for rutin and 415 nm for quercetin using the spectrophotometer. The concentration for individual flavonoid compounds were calculated according to their respective standard curves and the results expressed as mg/l of extract. 3.6.6 Standard curves The standard curves used for the polyphenolic compounds are presented in Table 3. Table 3: Polyphenolic compounds and their regression equation Polyphenolic compound Regression equation Gallic acid y = 0.2263x + 0.1606 Vanillic acid y = 0.1246x + 0.0794 p-coumaric acid y = 0.0726x + 0.0874 Rutin y = 0.0229x + 0.0301 Quercetin y = 0.1548x + 0.0301 University of Ghana http://ugspace.ug.edu.gh 34 3.6.7 Amino Acids Analysis The following eighteen amino acids were analysed: L-Aspartic acid, Glutamine, L- Histidine, Isoleucine, L-Lysine, L-Cysteine, L-Methionine, DL-Beta-Phenyl-Alanine, D-Proline, LSerine, L-Threonine, L-Tyrosine, L-Valine, DL-Alpha-Alanine, B- Threonine, Trans-4- ydroxyl-Proline, L-Asparagine and L-Tryptophan. 3.6.7.1. Sample preparation A standard solution was prepared by weighing 2.5 mg of the standard compound in 25 ml of ethanol and diluting to a concentration of 100 ppm. A mixed standard solution of 10 ppm was prepared from the stock solution, which was diluted to 1 ppm as a working solution. A weight of 0.5 g of cow pea flour was extracted into 20 ml of 100% methanol. The extracts were filtered through filter paper and a funnel into McCarntey tubes. The extracts were latter taken to Ghana Standard Authority for amino acid analysis. 3.6.7.2. Chromatography The column used was Agilent Poroshell 120 Bonus RP 2.7 µm, 2.1 x 100 mm; the flow rate was 0.15 ml / min. Mobile phases were: 100% water, 0.1% formic acid (A) and 100% methanol, 0.1% formic acid (B). The gradient was 0 to 5% (B) over 5 minutes and 5 to 50% (B) over 15 minutes. The injection volume was 621. Mass spectrometry: MS-Aligent Triple Quadrupole 6420 was used to identify and quantify the compounds. University of Ghana http://ugspace.ug.edu.gh 35 3.7 STATISTICAL ANALYSIS STATA version 15 software was used for the following analyses: the percent frequency distribution of qualitative traits, summary statistics, Pearson correlation analysis, principal component analyses and cluster analyses. Quantitative traits data taken were expose to analysis of variances (ANOVA) using ‘R’ software. This analysis was done to identify the uniqueness among the accessions and estimate the effects of environment and genes on morphological and phytochemical traits. Genotypic variance, phenotypic variance and environmental variance (Burton & De vane, 1953) with their corresponding coefficients of variation (Singh & Chaudhary, 1985) were used to estimate variability among the cowpea genotypes. The following formulae were adapted from (Belay & Fischa, 2020). 1. Genotypic Variance GV= (MSg −MSe)r, Where MSg = Mean Square of genotypes, MSe = Mean Square of error (environmental variance or δ2e), r = number of replications; 2. Phenotypic Variance PV= GV +MSe, Where GV = Genotypic Variance MSe = Mean Square of error; 3. Genotypic Coefficient of Variation GCV = √𝐺𝑒𝑛𝑜𝑡𝑦𝑝𝑖𝑐 𝑣𝑎𝑟𝑖𝑎𝑛𝑐𝑒 𝐺𝑟𝑎𝑛𝑑 𝑚𝑒𝑎𝑛 𝑥 100 4. Phenotypic Coefficient of Variation PCV = √𝑃ℎ𝑒𝑛𝑜𝑡𝑦𝑝𝑖𝑐 𝑣𝑎𝑟𝑖𝑎𝑛𝑐𝑒 𝐺𝑟𝑎𝑛𝑑 𝑚𝑒𝑎𝑛 𝑥 100 University of Ghana http://ugspace.ug.edu.gh 36 5. Broad sense heritability H2 = [(σ2g) / (σ2p)] × 100, Where, σ2g and σ2p are genotypic and phenotypic variances respectively. 6. Genetic Advance GA = KσpH2, Where, GA = expected genetic advance, K = the standardized selection differential at 5% selection intensity (K=2.063), σp = is phenotypic standard deviation on mean basis H2 = heritability in broad sense. 7. Genetic advance as percentage of population means GAM = KH2 (PCV) Where GCV = Phenotypic Coefficient of Variation K = the standardized selection differential at 5% selection intensity (K=2.063) H2 = heritability in broad sense. University of Ghana http://ugspace.ug.edu.gh 37 CHAPTER FOUR 4.0 RESULTS 4.1 MORPHOLOGICAL QUALITATIVE TRAITS 4.1.1 Frequency Distribution of Qualitative Traits The frequency distributions of different classes of qualitative traits are shown in Table 4. 4.1.1.1 Growth pattern All accessions showed indeterminate growth pattern. 4.1.1.2 Growth habit There were three different phenotypic classes for growth pattern as follows: erect, intermediate and semi – erect. For the controls 67.86% exhibited intermediate growth habit, 32.14% showed erect. In the case of the test materials 91.04 % exhibited erect growth habit, 4.25% showed intermediate habit and 4.72% showed semi – erect growth habit. 4.1.1.3 Plant pigmentation Plant pigmentation exhibited three different phenotypic classes. They were intermediate, moderate and very slight. For the control 10.72% showed intermediate pigmentation, while 54.76% showed very slight. 34.52% showed moderate pigmentation. In the test materials 0.31% were intermediate and 59.91% showed very slight while 39.78% were moderate. University of Ghana http://ugspace.ug.edu.gh 38 4.1.1.4 Leaf shape Terminal leaflet shape had three different phenotypic classes, namely; globose, hastate and sub-globose. For the control 66.67% showed globose, 33.33% showed hastate. In the test material 76.10% exhibited globose and 23.90% showed a sub- globose. 4.1.1.5 Leaf colour Leaf colour was classified into two character states as dark green and intermediate colourations. Out of the 84 control plants, 64 of them were dark green representing 76.19% and 20 of them also had intermediate leaf color with a percentage of 23.81%. In the case test material, the least population frequency of 35.53% was recorded for dark green and the highest frequency of 64.47% was recorded for intermediate. 4.1.1.6 Twinning tendency Twinning tendency had three different phenotypic classes, these were; slight, intermediate and none. For the control 19.05% showed intermediate, 55.95% showed slight and 25% showed none. The frequency scored for twinning tendency recorded for the test material were 0.31% for intermediate, 10.69% for slight and 88.99% for none. 4.1.1.7 Flower colour Flower colour showed three character states: white, violet and white with patches of violet. For the control 66.67% was white and it was the dominant colour and 33.33% showed violet. In the test material, the flower colour, white with patches of violet showed 86.79%, 10.38% showed white, and 2.83% showed violet. University of Ghana http://ugspace.ug.edu.gh 39 4.1.1.8 Pod tip colour Two phenotypic classes of the pod tip colour were observed and recorded. For the control 33.33% of population showed no colour at the tip of the pod, whilst 66.67% had a colouration at the tip of their pod. In the case of the test material 37.89% showed no colour at the tip of the pod whereas 62.11% exhibited colouration at tip of the pod. 4.1.1.9 Leaf markings All accessions showed leaf markings. 4.1.1.10 Leaf size There were four different phenotypic class of leaf size recorded (big, medium, small and narrow). For the controls 23.81% showed narrow leaf size, 32.14% showed big and 44.05% showed medium. In the test materials 11.79% showed big, 16.19% showed small and 72.01% showed medium. 4.1.1.11 Pod attachment Pod attachment to peduncle was classified into three character states namely; pendant, erect and 30 - 90° down from erect. 30 - 90° down from erect was predominant in the control (34.52%). However, pendant was dominant in both the test material (41.51%) and combined material (40.56%). 4.1.1.12 Pod thickness There were three different phenotypic classes of pod thickness. They were intermediate, thick and thin. In the case of the control 67.86% showed intermediate University of Ghana http://ugspace.ug.edu.gh 40 and 32.14% showed thick. For the test materials, 51.26% showed thin, 41.51% showed intermediate and 7.23% showed thick. University of Ghana http://ugspace.ug.edu.gh 41 4.1.1.13 Pod colour Two phenotypic classes for pod colours were observed, these were: dark tan and pale tan. For the control 34.52% showed dark tan and 65.48% exhibited pale tan. In the case of test materials 37.42% showed dark tan and 62.58% showed pale tan. 4.1.1.14 Pod curvature There were three phenotypic class observed for pod curvature, they were curved, slightly curved and straight. For the control 65.48% showed straight, 34.52% showed slightly curved. In the test material 44.03% showed slightly curved and 55.97% showed curved. 4.1.1.15 Pod hairiness Two phenotypic classes of pod hairiness were observed these were glabrescent and short appressed hair. All the control exhibited glabrescent. In the case of the test material 82.70% exhibited glabrescent whereas 17.30% exhibited short appressed hair. 4.1.1.16 Seed coat colour Three different phenotypic classes of the seed coat colour were recorded, they were: reddish brown, white and cream. For the control, 33.33% showed reddish brown 66.67% showed white. In the test material scored 33.96% showed cream colouration, 66.04 showed white. University of Ghana http://ugspace.ug.edu.gh 42 Table 4: Frequency distribution of morphological traits for the 27 cowpea accessions Traits Phenotypic Class Control (%) Test (%) Combined (%) Growth pattern Indeterminate 100.00 100.00 100.00 Growth habit Erect 32.14 91.04 84.17 Intermediate 67.86 4.25 11.67 Semi-erect 0.00 4.72 4.17 Plant pigmentation Intermediate 10.71 0.31 1.53 Moderate 34.52 39.78 39.17 Very slight 54.76 59.91 59.31 Leaf shape Globose 66.67 76.10 75.00 Hastate 33.33 0.00 3.89 Sub-globose 0.00 23.90 21.11 Leaf colour Dark green 76.19 35.53 40.28 Intermediate green 23.81 64.47 59.72 Twinning tendency Intermediate 19.05 0.31 2.50 None 25.00 88.99 81.53 Slight 55.95 10.69 15.97 Flower colour Violet 33.33 2.83 6.39 White 66.67 10.38 16.94 White with patches of violet 0.00 86.79 76.67 Pod tip colour Absent 33.33 37.89 37.36 Present 66.67 62.11 62.64 Leaf markings Present 100.00 100.00 100.00 Leaf size Big 32.14 11.79 14.17 Medium 44.05 72.01 68.75 Narrow 23.81 0.00 2.78 Small 0.00 16.19 14.31 Pod attachment 30 - 90° down from erect 34.52 28.77 29.44 Erect 32.14 29.72 30.00 Pendant 33.33 41.51 40.56 Pod thickness Intermediate 67.86 41.51 44.58 Thick 32.14 7.23 10.14 Thin 0.00 51.26 45.28 Pod colour Dark tan 34.52 37.42 37.08 Pale tan 65.48 62.58 62.92 Pod curvature Curved 34.52 55.97 49.44 Slightly curved 0.00 44.03 42.92 Straight 65.48 0.00 7.64 Pod hairiness Glabrescent 100.00 82.70 84.72 Short appressed hair 0.00 17.30 15.28 Seed coat colour Reddish brown 33.33 0.00 3.89 White 66.67 66.04 66.11 Cream 0.00 33.96 30.00 University of Ghana http://ugspace.ug.edu.gh 43 4.2 CHI – SQUARE TEST OF ASSOCIATION AMONG QUALITATIVE TRAITS 4.2.1 Chi-square test of association in control material Chi-square test of associations among the control is presented in Table 5. Seventy- eight (78) significant associations were observed among the qualitative traits. Growth habit had a significant associated with plant pigmentation (χ² = 33.77, P = 0.000), terminal leaflet shape (χ² = 19.89, P = 0.00), leaf colour (χ² = 12.43, P = 0.000), twinning tendency (χ² = 13.76, P = 0.001), flower colour (χ² = 19.89, P = 0.000), pod tip colour (χ² = 19.89, P = 0.000), leaf size (χ² = 84.00, P = 0.000), pod attachment (χ² = 84.00, P = 0.000), pod thickness (χ² = 84.00, P = 0.000), pod colour (χ² = 20.98, P = 0.000), pod curvature (χ² = 20.98, P = 0.000), and seed coat colour (χ² = 19.84, P = 0.000). Significant associations were observed between plant pigmentation and the following traits: terminal leaflet shape (χ² = 34.70, P = 0.00), leaf colour (χ² = 21.68, P = 0.000), twinning tendency (χ² = 19.54, P = 0.001), flower colour (χ² = 34.70, P = 0.000), pod tip colour (χ² = 34.70, P = 0.000), leaf size (χ² = 71.43, P = 0.000), pod attachment (χ² = 101.0435, P = 0.000), pod thickness (χ² = 33.77, P = 0.000), pod colour (χ² = 84.00, P = 0.000), pod curvature (χ² = 84.00, P = 0.000), and seed coat colour (χ² = 34.70, P = 0.000). Significant associations were observed between terminal leaflet shape and the following traits: leaf colour (χ² = 52.50, P = 0.000), twinning tendency (χ² = 49.84, P = 0.000), flower colour (χ² = 84.00, P = 0.000), pod tip colour (χ² = 84.00, P = 0.000), leaf size (χ² = 55.78, P = 0.000), pod attachment (χ² = 84.00, P = 0.000), pod thickness University of Ghana http://ugspace.ug.edu.gh 44 (χ² = 19.89, P = 0.000), pod colour (χ² = 22.15, P = 0.000), pod curvature (χ² = 22.15, P = 0.000), and seed coat colour (χ² = 84.00, P = 0.000). High significant association was observed between leaf colour and; twinning tendency (χ² = 35.74, P = 0.000), flower colour (χ² = 52.50, P = 0.000), pod tip colour (χ² = 52.50, P = 0.000), leaf size (χ² = 84.00, P = 0.000), pod attachment (χ² = 52.50, P = 0.000), pod thickness (χ² = 12.43, P = 0.000), pod colour (χ² = 13.84, P = 0.000), pod curvature (χ² = 13.84 P = 0.000), and seed coat colour (χ² = 52.50, P = 0.000). Twinning tendency was significantly associated with flower colour (χ² = 49.84, P = 0.000), pod tip colour (χ² = 49.84, P = 0.000), leaf size (χ² = 38.10, P = 0.000), pod attachment (χ² = 49.94, P = 0.000), pod thickness (χ² = 13.76, P = 0.001), pod colour (χ² = 11.26, P = 0.000), pod curvature (χ² = 11.26, P = 0.000), and seed coat colour (χ² = 49.84, P = 0.000). Significant associations were observed between flower colour and the following seven qualitative traits: pod tip colour (χ² = 84.00, P = 0.000), leaf size (χ² = 55.78, P = 0.000), pod attachment (χ² = 84.00, P = 0.000), pod thickness (χ² = 19.89, P = 0.001), pod colour (χ² = 22.15, P = 0.004), pod curvature (χ² = 22.15, P = 0.004), a