MOLECULAR CHARACTERIZATION OF SOME NIFOR AND ELITE OIL PALM BREEDING POPULATIONS USING MICROSATELLITE MARKERS

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ABSTRACT


The traditional method of hybrid identification and genetic diversity evaluation based on differences in range of expressions of morphological and agronomical characters has been routinely employed in the assessment of oil palm germplasm and breeding populations at the Nigerian Institute of Oil Palm Research (NIFOR). Considering the limitations of this conventional method and the advantages of molecular markers in complementing conventional breeding methods, this study was conducted to determine the legitimacy of NIFOR oil palm progenies and to assess the genetic variations and relationships existing among the breeding populations using microsatellite (SSR) markers. Ten microsatellite markers were used to screen 226 oil palm samples which included 215 samples from NIFOR and 11 other samples belonging to the Malaysian Palm Oil Board (MPOB) advanced breeding lines and germplasm materials. Results obtained revealed that out of 200 F1 oil palm progeny derived from 11 of the 15 parents evaluated, 57% (114) were true-to-type and 43% (86) were contaminated. Almost all the contamination or illegitimacy detected was due to pollination errors. High genetic diversity was detected among the 15 NIFOR parents with the NIFOR tenera parents recording the highest number of alleles (5), rare alleles (17), and gene diversity (0.650) when compared to the Deli dura NIFOR and NIFOR dura parents. With reference to the MPOB breeding materials, the various oil palm sources showed significant and large value of genetic differentiation (FST= 0.177, P = 0.001) due to variations within the sources of parental materials. Rogers’ dissimilarity coefficient matrix displayed two main clusters, one separating MPOB Madagascar accessions from the rest of the samples. Principal co-ordinates analysis (PCoA) showed that the NIFOR breeding parents clustered closely with the MPOB Nigeria and Angola derived materials indicating a common origin of mainland genotypes. A comparative assessment of molecular and morphological methods of describing genetic relationships in the NIFOR oil palm progenies showed that SSR analysis recorded the highest level of polymorphism (100%) in all the progenies. Although the correlation between agronomic and genetic distance matrices was very low and insignificant (r = 0.2989), both matrices discriminated the progenies effectively into two groups as per their agronomic performance and pedigree or origin, respectively.


TABLE OF CONTENTS

Title page
List of Co-authored publications
Table of contents
List of tables
List of figures
List of abbreviations
Abstract

INTRODUCTION

LITERATURE REVIEW
Classification of oil palm
Biology of oil palm
Economic importance
Oil palm germplasm resources in NIFOR
NIFOR oil palm breeding programme
NIFOR Extension Work Seeds
Legitimacy of planting materials supplied to oil palm growers
Molecular marker technologies in oil palm
Evolutionary forces affecting genetic diversity
Molecular marker studies in oil palm
Isozymes
Restriction fragment length polymorphism (RFLP)
Random amplified polymorphic DNA (RAPD)
Amplified fragment length polymorphism (AFLP)
Microsatellites or simple sequence repeats (SSRs)

MATERIALS AND METHODS
Plant Material
DNA Isolation
DNA quality assessment and quantification
Microsatellite analysis
Elaeis guineensis Jacq. microsatellite markers
PCR Amplification
Scoring of banding patterns
Resolution of PCR products
Dilution of PCR products for Capillary Electrophoresis
Band sizing and sample genotyping
Statistical analysis
Hybrid legitimacy using microsatellite markers
Hybrid Purity
Genetic Diversity
SSR Polymorphism
Allele Frequency
Percentage of polymorphic alleles
Number of Alleles per Locus
Effective Number of Alleles per Locus
Observed Heterozygosity
Expected Heterozygosity
Genetic differentiation of populations
Wright’s Fixation Index ( )
Wright’s Co-ancestry Coefficient (   )
Genetic Relatedness among Parents and Progenies
Genetic Relatedness based on the Genetic Distance
Genetic Relatedness by PCoA
Genetic Diversity Structure in Populations
Morphological Data Analysis

RESULTS
Quality and Concentration of DNA Extracted
Screening the parents and progenies using SSR markers
Hybrid Legitimacy
Genotyping the parents and the progeny using SSR markers
Parent-Progeny relationships of legitimate NIFOR oil palm hybrids revealed by microsatellite markers
Genetic diversity in the progenies and their parents
Genetic relatedness among parents and their progenies
Cluster Analysis
Principal Co-Ordinate Analysis (PCoA)
Genetic diversity and relationship among NIFOR oil palm main breeding parent genotypes
Overall Microsatellite Diversity
Allelic frequencies
Polymorphism of SSR loci
Number of alleles and effective number of alleles
Observed heterozygosity and expected heterozygosity
Intra-population genetic diversity
Allelic diversity
Observed heterozygosity and Expected heterozygosity
Genetic differentiation (Analysis of molecular variance)
Genetic relatedness among 15 parental palms of the NIFOR Main Breeding
Programme
Genetic distance
Cluster analysis
Principal co-ordinate analysis
Genetic diversity in NIFOR elite breeding materials and in MPOB elite
breeding materials detected by SSR markers
Allelic frequencies
Polymorphism of SSR loci
Number of alleles and effective number of alleles
Observed heterozygosity and expected heterozygosity
Genetic differentiation
Analysis of Molecular Variance (AMOVA)
Genetic relatedness among the 26 oil palm samples from NIFOR and MPOB
Genetic distance
Cluster analysis
Principal coordinates analysis
Genetic diversity and relatedness of D x T oil palm progenies determined by
microsatellite markers against that revealed by agronomic markers
Variability in progenies of NIFOR parents of the Main Breeding Programme
revealed by SSR markers and morphological traits
Relatedness of progenies of NIFOR parents of the Main Breeding Programme
revealed by agronomic markers and SSR markers
Principal Coordinate Analysis

DISCUSSION
DNA Quality Assessment
Microsatellite analysis
Hybrid Identification
Applying SSR markers for hybrid identification
Improved assessment of hybrids’ legitimacy for breeding programmes
Implications of SSR based hybrid identification to NIFOR
Oil palm varietal improvement
Parent-progeny relationships of some NIFOR oil palm breeding populations revealed by microsatellite markers
Genetic diversity in the progenies and their parents
Parent-Progeny Relationships
Genetic diversity and relationship among NIFOR oil palm main breeding parent genotypes
Allelic frequencies
Polymorphism Information Content
Number of alleles and effective number of alleles
Observed heterozygosity and expected heterozygosity (genetic diversity)
in the entire parental populations
Observed heterozygosity and expected heterozygosity in the three
groups of NIFOR oil palm main breeding parents
Fixation indices
Genetic differentiation among the three groups of parental genotypes
Genetic Relatedness among Parents
Genetic diversity in NIFOR and MPOB breeding materials based on SSR markers
Allelic frequencies
Polymorphism Information Content of SSR loci
Mean number of alleles and effective number of alleles
Observed heterozygosity (   and expected heterozygosity )
Genetic differentiation and relatedness among NIFOR and MPOB breeding and germplasm materials
Genetic diversity and relatedness of D x T oil palm progenies determined by
microsatellite markers against those revealed by agronomic markers
Agronomic and microsatellite variability in the 10 D x T progenies
Relatedness of 10 D x T progenies based on agronomic markers and SSR markers
CONCLUSION
REFERENCES


INTRODUCTION

The oil palm, Elaeis guineensis Jacq is a diploid (2n = 32) monocotyledonous and perennial crop of the humid tropics. Fossil, archaeological, historic, and linguistic evidence indicate that oil palm originated in Africa (Hartley, 1977 and1988; Corley and Tinker, 2003). Fossil pollen similar to oil palm was extracted from Miocene sediments of Nigeria (Zeven, 1964), while Raynaud et al. (1996) reported oil palm pollen found in lake sediments of south-east Cameroon (Hartley, 1977; Corley and Tinker, 2003). Sowunmi (1999) discovered oil palm nut shells in a rainforest site and speculated that an increase in late Holocene times (5000 years ago) connotes the beginning of oil palm cultivation and its importance in the subsistence economy of Africa. Historical records of African origin of oil palm were traced to the major landmarks of Portuguese and English exploration and trade in Africa (Rees 1965). The short and direct translation of the West African vernacular names of oil palm is strong linguistic evidence supporting a West African origin of oil palm (Zeven, 1965; Hartley, 1988). A high concentration of natural/semi-natural groves estimated at about 2.1 million hectares occur in Nigeria and studies have shown that the Nigerian groves have the highest level of polymorphism with respect to the number of alleles, indicating that Nigeria is likely to be the centre of distribution for oil palm (Maizura et al., 2001; Rajanaidu, 2002; Maizura et al., 2006; Bakoumé et al., 2015).


Oil palm is cultivated for the oil that is extracted from the mesocarp and the kernel. It produces more than five times oil/year/hectare of any annual oil crop (Basri-Wahid et al., 2005). Palm oil is the most valuable natural oil in the diets of Nigerians both as crude red palm oil and as refined oil (olein). It has played a very significant role in the socio-economic and political life of the people of Nigeria. With the recognition of the economic potential of the crop, both as dietary and industrial fats, concerted efforts towards the genetic improvement of the plant and management practices started at the turn of the last century in Nigeria (Okwuagwu and Ataga, 1992). Today, this genetic improvement strategy using traditional breeding methods has been very successful. The most spectacular achievements are the development of E. guineensis hybrids with one or a combination of the following attributes (i) early bearing, (ii) short stem, (iii) drought tolerance, and (iv) high and stable yield (Okwuagwu and Ataga, 1985; Okwuagwu, 1989; Okwuagwu et al., 2001 and 2005). In spite of these compelling results, oil palm yield potential is yet to be fully realized coupled with the slow and expensive procedure involved in the breeding and selection programme at NIFOR. Should this trend continue, genetic gain expected through conventional breeding would not be able to keep pace with the increasing domestic demand for palm oil, let alone the competition from other vegetable oils and fats. Against this backdrop, the Federal Government of Nigeria has made a firm commitment to restore Nigeria’s agriculture notably oil palm agriculture to its past eminent position in the economy. A memorandum of understanding was then signed with the Nigerian Institute for Oil Palm Research (NIFOR) on the implementation of the oil palm transformation value chain. Subsequently, NIFOR is expected to annually produce 9 million improved tenera planting materials which will be delivered to recommended nursery operators in the various oil palm growing states of the country for distribution to farmers. This is in a bid to meet the growing domestic demand for palm oil, create employment for the youths and possibly re-enter the international market for vegetable oil and fats (Obibuzor, Personal Comm.).

To accommodate the anticipated increase in the demand for palm oil by the year 2020, the area under cultivation will need to increase from the current 470,000 hectares in 2013 (Oil World 2014) with additional 200,000 hectares. Presently, the commercial variety (tenera) yields 20 -25 mt of fresh fruit bunch ha-1 yr-1 and 3 3.5 t oil ha-1 yr-1 (Okwuagwu et al., 2001). The tenera shows great variations in yield with the best yielding about 40% more than the average. Steady breeding progress has been made, with yield being doubled between 1950 (2.5 – 5.0 mt of fresh fruit bunch ha-1 yr-1) and year 2000 (20 – 25 mt of fresh fruit bunch ha-1 yr-1). Accordingly, future breeding progress will rely increasingly on family selection and progeny testing, which require a high degree of legitimacy of both the parents and the progenies.

A notable threat to the oil palm industry in Nigeria is the conformity of planting materials. Oil palm is naturally out-breeding and controlled pollination is not always effective. Anomalous genetic segregation and contamination with unexpected fruit forms do occur from time to time (Corley, 2005). It is therefore crucial to select true hybrids/progenies in a crossing programme for breeding and production of planting materials. This situation is further aggravated by the impurity of planting materials as a result of high patronage of illegal sprouted seeds/seedling producers who pose as NIFOR agents to give credence to their illegal dealing. It has become very necessary to safeguard the industry by using markers by which NIFOR elite tenera hybrids could be characterized and distinguished at the seedling stage.
The traditional method of hybrid identification based on morphological traits is influenced by environmental factors (Murphy et al., 1996; Chakravarthi and Naravaneni, 2006) and most importantly lack means to identify hybrids at the seed or seedling stage.....


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