GENETIC DIVERSITY STUDIES OF TROPICAL IPGRI MAIZE (Zea mays L.) LANDRACES HELD IN IITA BY AGROMORPHOLOGICAL AND SIMPLE SEQUENCE REPEATS MARKER EVALUATION

ABSTRACT
Genetic diversity among maize (Zea mays L.) landraces reveals genetic backgrounds with respect to alleles, polymorphisms and heterozygosities, as well as relationships among genotypes. There has always been the need in Africa to identify useful alleles for maize improvement in a wide genetic base yet little is done to search for diversity among existing maize landraces. The IPGRI landraces of the IITA maize collection has neither record of geographical origin nor information on genetic diversity. The research objective was to estimate the level of genetic diversity, determine relationships among the landraces, and reveal evolutionary processes that have contributed to the genetic status of the population. A total of 60 landraces and a check, „Obatanpa GH‟ were evaluated by agromorphological characterization on 5 qualitative and 24 quantitative traits. Except for cob colour which was least variable with 98.0 % white and 2.1 % red, a large variability was observed for silk and grain colour, kernel texture and kernel arrangement Kernel arrangement with fairly equal distribution of straight, regular, irregular and spiral types was the most variable. On quantitative evaluation, large variability was demonstrated for all traits except number of ears per plant. Earliness ranged from 39 to 74 days with a mean of 54.8 ± 6.2 days to 50 % anthesis while days to 50 % silking covered 44 to 78 days and mean of 57.6 ± 6.3 days. Six early-maturing genotypes identified were TZm-149, TZm-1148, TZm-1150, TZm-1157, TZm-1153, and TZm-1152. Mean anthesis-silking interval revealed genotypes for drought tolerance having 1.2 to 1.4 days of anthesis-silking interval in TZm-1188, TZm-1183, and TZm-1106. Many individual plants of these accessions exhibited protogyny. Mean grain yield ranged from 2.16 ± 0.4 Mgha-1 to 6.19±1.7 Mgha-1 of which the best performers with yield exceeding 4.2 Mgha-1 were TZm-1185, TZm-1142, TZm-1213, TZm-1129, TZm-1143, TZm-1215, TZm-1150, TZm-1211, TZm-1152, TZm-1101, TZm-1123, TZm-1100, TZm-1138, TZm-1112, TZm-1212, TZm-1130, TZm-1190, TZm-1118, TZm-1106, TZm-1144, TZm-1122, TZm-1125, TZm-1117, TZm-1119 and TZm-1139. Low to moderate broad sense heritability estimates of 0.00 for stay green and ear weight to 0.68 and 0.69 for earliness were recorded. The medium to high heritability estimates signify traits are under control of minimal additive and some dominance gene effects for a slow pace in progress in breeding. Besides the strong positive correlation of yield components with grain yield, all other correlation coefficients with grain yield were weak and nonsignificant (P ≤ 0.05). Genetic similarities ranged from 0.00 to 0.80 with a mean of 0.14±0.15 indicating extensive genetic diversity. The UPGMA cluster analysis grouped genotypes into two main heterogeneous clusters, cluster I having early-maturing, short plants with high grain yield and low anthesis-silking intervals whereas cluster II was of tall plants with poor grain yield. The first two principal components explained 85.0 % of the total variance with large contributions from plant height, ear height, anthesis, silking, ear leaf length, grain weight, grain yield, ear position, hundred kernel weight, kernel length, and kernel width. SSR profiling of 64 IPGRI genotypes at 12 loci produced a rate of polymorphism of 85.7 %, a total of 1,826 alleles ranging from 108 to 216. The number of alleles per locus ranged 3 to 10 with mean of 5.64±2.15 indicating lots of variability. The mean observed heterozygosity of 0.36±0.18 was not significantly different from the expected heterozygosity of 0.69±0.08, an indication of substantial mutation rate and polymorphism maintained by balancing selection. The high heterozygosity is also suggestive of a historical admixture event. Genetic distance by means of DICE similarity coefficient was 0.49±0.14. UPGMA clustering grouped the accessions into six clusters from which hybridization could be exploited. The large variability, polymorphism, and heterozygosity identified by both agromorphological and molecular assessments affirm the existence of wide genetic diversity in the IPGRI genotypes and their possible beneficial contributions if exploited in maize improvement programmes.


TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDICES
LIST OF ABBREVIATIONS
ABSTRACT

CHAPTER ONE
1.0 INTRODUCTION

CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 The role of maize in the world‟s agricultural economy
2.2 Maize production and consumption in Africa
2.3 Origin of maize
2.3.1 Cytological evidence
2.3.2 Isozyme evidence
2.3.3 Molecular evidence
2.4 Maize accessions and landraces
2.5 Biology of maize
2.5.1 Morphology of maize plant
2.5.2 Maize inflorescence
2.5.3 Pistillate flower
2.5.4 Fertilization and embryogenesis
2.5.5 Growth stages of maize plant
2.6 Maize research in Ghana
2.6.1 Maize breeding in Ghana (varietal development)
2.7 Genetic diversity in maize
2.8 Estimation of genetic diversity
2.8.1 Agromorphological trait evaluation
2.8.2 Estimation of diversity by coancestry coefficients
2.8.3 Isozyme and storage protein analysis
2.8.4. Assessment of genetic diversity by molecular analysis
2.9 Measures of genetic diversity
2.10 Determination of relationships among genotypes
2.10.1 Genetic distance
2.11 Multivariate techniques for interpretation of genetic distance
2.11.1 Cluster analysis
2.11.2 Bootstrapping
2.11.3 Principal components analysis

CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Plant Material
3.2 Location and conditions of experimental site
3.3 Land preparation, planting and experimental design
3.4 Data Collection
3.4.1 Morphological Data
3.5 Statistical analyses of morphological data
3.5.1 Description of genetic diversity
3.6 Genotypic and phenotypic correlation and their standard error
3.7 Assessment of relationships between genotypes
3.7.1 Data Standardization
3.7.2 Euclidean distance measurement and cluster analysis
3.7.3 Cluster analysis
3.7.4 Bootstrapping
3.7.5 Principal components analysis
3.8 Genetic diversity in maize by means of SSR fingerprinting
3.8.1 SSR primer selection
3.8.2 Amplification and detection of bands
3.9 Statistical analysis of molecular data
3.9.1 Allele scoring and data analysis
3.9.2 Estimation of genetic diversity within populations
3.9.2.1 Rate of polymorphism
3.9.2.2 Average number of alleles per locus (AP) or allele diversity
3.9.2.3 Polymorphic information content or average expected heterozygosity
3.9.3 Estimation of genetic diversity among populations
3.9.3.1 Genetic distance and cluster analysis

CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
4.1 Morphological Description of Qualitative Traits
4.1.1 Qualitative trait description
4.2 Means, standard deviation, range and mean squares of quantitative traits of the IPGRI maize landraces
4.2.1 Earliness in the IPGRI landraces
4.2.2 Plant characteristics
4.2.3 Ear characteristics
4.2.4 Yield and yield components
4.3 Heritability, phenotypic variance and genotypic variance of 24 quantitative traits on the 60 IPGRI genotypes and a check „Obatanpa GH‟
4.4 Phenotypic and genotypic correlation of agro-morphological quantitative traits
4.5 Genetic distance and cluster analysis of 60 IPGRI maize accessions held in IITA and a check, “Obatanpa GH”
4.6 Principal components analysis of morphological traits
4.7 Molecular diversity in IPGRI maize accessions
4.8 Genetic similarity estimates of molecular data

CHAPTER FIVE
5.0 CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusion
5.2 Recommendations
REFERENCES


CHAPTER ONE
1.0 INTRODUCTION
Maize (Zea mays) is a member of the family Poaceae, the group of crops known as the grasses, which includes wheat, barley, and rye. It is generally believed that maize originated from Central America, specifically Mexico where it was domesticated and spread rapidly around the globe through trade routes (Matsuoka et al. 2002; Smith, 1998). Maize introduction to Africa can be traced back to the 1500s by the Portuguese traders (Sinha, 2007). To date, maize is grown in sub-Saharan Africa as the most important economic crop, and is used as food, feed, and a raw material for many industrial products. Maize is used as food for over 1.2 billion people in sub-Saharan Africa and Latin America (FAO, 2011). Presently, maize makes up more than 50 % of the total caloric intake (Sinha, 2007; McCann, 2005) and 53 % of the protein intake of local diets (Bressani, 1991).

Global maize production is estimated to be 785 million metric tons (MMT) with Africa producing about 51 million metric tons (6.5 %). Because this quantity is not enough for the population, Africa imports 28 % deficit of maize from other countries (IITA, 2012). Maize yield in developing countries has been consistently lower than that in developed countries primarily due to factors such as drought, use of landraces and old varieties. In contrast, developed countries cultivate hybrids and improved varieties (Munsch, 2009).

Maize landraces in Africa are adapted to various environments, from cold to hot, humid to drought, and on various elevations (Taba and Twumasi-Afriyie, 2008).

Some landraces are still being used as local cultivars in West and Central Africa, although the vast majority of the production areas are planted with modern commercial varieties. These landraces, though are believed to possess alleles for many important economic traits (Brandolini, 1969), they have not been utilized as valuable germplasm for breeding modern maize cultivars. For instance, more than 20 cultivars released in Ghana are bred from the superior offspring from „Obatanpa GH‟ derived from Population 63 genotype developed by the Crops Research Institute (CRI), Kumasi, Ghana in collaboration with the International Institute of Tropical Agriculture (IITA), Ibadan; the International Maize and Wheat Improvement Center (CIMMYT), Mexico; and the Sasakawa Global 2000 (Badu-Apraku et al., 2006). Other cultivars in Ghana were derived from International Institute of Tropical Agriculture (IITA) maize lines (Sallah, 1998).

There appears to be dearth of information on the genotypic composition of maize landraces in West and Central Africa, hence they have not been utilized in maize improvement programs. Landraces are important genetic resources which serve as sources of biotic and abiotic stress resistance, yield, and disease resistance genes, quality and many useful agronomic characteristics, and comprise high genetic variability and fitness to the natural and anthropological environments where they have originated (Brandolini, 1969). Consequently, landraces represent a unique and valuable material for improvement of modern varieties adapted to changing environments (Rao, 2004; Heslop-Harrison, 2002). In view of this, efforts must be made to collect and conserve landraces and wild relatives for utilization in future breeding programs.

To promote the efficient use of genetic variation in the collection, information on genetic diversity and relationships within and among cultivars, traditional populations and their wild relatives is essential (Sidkar et al., 2010).

Considering the need to conserve plant genetic resources, more than 800 tropical maize accessions have been collected and deposited at the IITA Genetic Resource Center in Ibadan, Nigeria, with the collaboration of local germplasm institutions in many countries in Africa and the International Plant Genetic Resources Institute (IPGRI), Rome, Italy. This large number presents challenges and demands for more efficient management and cost-effective conservation. Management of germplasm collections encompasses assessment of genetic diversity and construction of a core collection to represent the variation within the group.

Genetic diversity analysis reveals genetic backgrounds and relationships of germplasm, and provides strategies to establish, utilize, and manage crop germplasm (Roussel et al., 2004; Brown-Guedira et al., 2000). It also offers the basis for devising future strategies for crop improvement, cultivar development, conservation, and sustainable use of crop germplasm for long-term crop improvement and reduction of vulnerability in plants to diseases. Measurement of genetic diversity is useful for enhancing genetic variation in base populations.

Despite the many benefits of genetic diversity analyses, there have been few reports of detailed assessment of genetic diversity among the African maize germplasm compared to the collections of other regions. For example, temperate maize genotypes such as the U.S. Corn Belt germplasm (Smith et al., 1997; Hallauer et al., 1988;

Goodman and Stuber, 1983 ), North America (Smith, 1986; Goodman and Stuber, 1983; Kahler et al., 1983), European maize genotypes (Hartings et al., 2008; Messmer et al., 1993;1992 ), France maize genotypes (Dubreuil et al., 1996) and Japanese maize inbred lines (Enoki et al., 2002) are fully classified into heterotic groups. Similarly, thousands of tropical maize germplasm at CIMMYT are listed to be evaluated (Warburton et al., 2005; 2002; Xia et al., 2005, 2004; Reif et al., 2003a, 2003b).

These efforts have led to the assignment of lines into heterotic groups for hybrid maize development, as well as identification of desirable traits for future breeding programs.

Records available on genetic diversity in African maize include assessment of few germplasm from Ethiopia (Legesse et al., 2007; Beyene et al., 2006), Ghana (Obeng-Antwi, 2007), Zimbabwe, Zambia and Malawi (Magorokosho, 2006), and six other countries in West Africa (Sanou et al., 1997). There is therefore the urgent need to study the genetic diversity in the African maize collection.

Since the 1970‟s, African maize has undergone changes arising from hybridization with genotypes of plant introductions from the U.S.A. and CIMMYT, Mexico with the aim of producing improved cultivars (Morris et al., 1999). An example is „Obatanpa GH‟, an open pollinated variety (OPV) and a quality protein maize (QPM) developed by the Crops Research Institute (CRI), Kumasi, Ghana in collaboration with the International

Institute of Tropical Agriculture (IITA), Ibadan; the International Maize and Wheat Improvement Center (CIMMYT), Mexico; and the Sasakawa Global 2000 (SG 2000) (Badu-Apraku et al. 2006). As these practices are carried out, gene flow and genetic erosion are inevitable. Some elite African inbred lines and accessions have also contributed to maize improvement in exotic lines, as they are reported to demonstrate good yield potential, disease resistance, and overall favorable agronomic performance (Mwololo et al, 2012). Among these are few TZi accessions of International Institute of Tropical Agriculture, Ibadan, together with Institut National de la Recherche Agronomique (INRA), Cameroon (Nelson and Goodman, 2008).

These point to the fact that there is useful distribution of genes in the African maize germplasm awaiting to be utilized to transform maize improvement in Africa. Assessment of the extent and distribution of genetic variation within plant populations has the capacity to increase the understanding of the historical processes underlying the genetic diversity. It can reveal both novel genes waiting to be exploited, as well as identify heterotic groups. This information finds uses in breeding for trait improvement and for management of the large number of germplasm in repositories.

Little is known about the genetic backgrounds and relationships including the geographical origins of the accessions collected by IPGRI and held by the Genetic Resource Center of IITA. In response to the lack of information on the geographical distribution of the IPGRI accessions, this research project was designed to reveal its potential exploitation in breeding programs.

In order to reveal the genetic backgrounds and relationships including variability within and among the IPGRI accessions, it is required that a combination of approaches such as morphological trait evaluation and molecular genotyping be applied to identify genes, reveal the richness of allelic polymorphisms, partition the population into heterotic groups, and identify a set of genotypes which maximize their diversity.

Information regarding genetic diversity of breeding materials especially landraces is indispensable for maize improvement. Genetic diversity of maize has usually been assessed based on morphological data characterization using descriptors (Goodman and Bird, 1977), and pedigree analysis through estimation of coancestry coefficients (Mal├ęcot, 1948).

Collecting and analyzing data by this technique is inexpensive in developing countries where labour cost is considerably low. Morphological evaluation is relatively simple and does not require sophisticated technology. Despite the simplicity, these descriptors alone present several limitations such as high demand of time and labour intensiveness.

Again, morphological characters are often influenced by environment, hence are limited in their reliability. In contrast, molecular markers such as SSRs (Warburton et al., 2002), AFLP (Beyene et al., 2006), RFLPs (Dubreuil et al., 1999) and SNPs (Yu et al., 2011) have proven to be powerful in discriminating among accessions. They are immune to environmental effects and have high heritability. Among these, SSRs have been widely used for the study of diversity including population structure and demographic history of domesticated species because of their high level of allelic diversity over RFLPs, AFLPs, or SNPs loci (McGregor et al., 2000; Powell et al., 1996). They are highly polymorphic, reliable (Smith et al., 1997), easy to generate, have low cost, are highly repeatable (Warburton et al., 2002), and are suitable for large-scale investigations as needed for the characterization of genetic resources (Powell et al., 1996). Molecular markers are therefore superior to morphological and biochemical markers because they are more efficient and sensitive in detection of distinct differences arising from mutations among genotypes at DNA level (Melchinger et al., 1991). They are however expensive and demand sophisticated equipment.

In a morphological study involving twenty-two traits Ruiz de Galaretta and Alvarez (2001) evaluated 100 landraces of maize from Northern Spain and came up with seven groups having promising breeding values. Beyene et al. (2005) researched into 62 traditional Ethiopian highland maize using morphological traits and molecular profiling by encompassing AFLPs and SSRs and concluded that variability existed among the selected genotypes. Hartings et al. (2008) reported a large genetic heterogeneity among 54 maize landraces originating from Italy on the basis of morphological and AFLP analyses and revealed four major clusters relating to their geographical origin.

Rebourg et al. (2001) examined genetic variation among 130 European traditional maize populations and split them into six groups on the basis of morphological and molecular analyses. Analysis of 294 landraces originating from Malawi, Zambia, and Zimbabwe using 34 phenotypic traits partitioned the set into three non-overlapping groups by cluster analysis (Magorokosho, 2006). Obeng-Antwi (2007) performed genetic diversity study on 92 maize landraces from Ghana and observed a large variability among accessions within groups (96 %) rather than among groups using AFLPs and agromorphological traits. Studies by the various researchers confirm the effectiveness of the combined use of morphological evaluation and molecular genotyping.

Therefore for a comprehensive study of the IPGRI genotypes held in IITA with little passport data the combined techniques must be applied to reveal their useful characteristics in terms of allele diversity, unique genotypes worth incorporating in breeding programs, relationships among the genotypes, as well as their evolutionary history.

The main goal of this study was to estimate the level of genetic diversity and relationships among the tropical IPGRI maize landraces in the IITA germplasm repository.

The specific objectives are:
(1)        To determine genetic variation in the IPRGI population by means of agromorphological traits evaluation

To investigate the heritability, genotypic and phenotypic correlations among the IPGRI maize genotypes

To estimate genetic diversity of the IPGRI genotypes using SSR profiling

To assemble the IPGRI population into groups on the basis of genetic distance

To determine the allele diversity and heterozygosity among the genotypes

The research is driven by the hypothesis given below:
That, the IPGRI maize landraces in IITA repository with little passport data are genetically diverse and contain alleles that can be exploited for maize improvement especially in Sub-Saharan Africa.

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Item Type: Ghanaian Project Material  |  Attribute: 147 pages  |  Chapters: 1-5
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