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

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.

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. In

fact, oil palm  has to be grown several  years (3 to 4  years) before producing  fresh  fruit bunches to confirm authenticity of the hybrid. A reliable method for hybrid identification in oil palm at an early stage is therefore essential.

Another feature of oil palm breeding programmes is the very narrow genetic base of some  of  the  ancestral  populations  (Rosenquist,  1986;  Corley,  2005).  This  can  lead  to reduction in productivity and increased susceptibility to diseases and pests, thus threatening the  profitability  of  the  crop.  Also,  the  reduction  in  genetic  variability  and  increase  in homozygosity is often followed by inbreeding depression due to the limited number of parent palms  for  subsequent  selection  cycle.  Consequently,  selection/genetic  progress  in  inbred populations  become  slow  and  limited  than would  be expected  based  on heritability  and selection  intensity.  Genetic  diversity evaluation  based on agro-morphological  information which  has  been  routinely  used  in  the  assessment  of  oil  palm  germplasm  and  breeding populations  in  NIFOR  is  no  longer  sufficient.  Agro-morphological  markers  are  often unreliable  and  ambiguous  due  to  (i)  long  juvenile  phase,  (ii)  confounding  effects  of developmental  stage  of  the  plant,  (iii)  low  polymorphism  due  to  predominating  one environment-one phenotype relation, (iv) long term field evaluation, and (v) vulnerability to environmental effects. An additional and objective measure of genetic variation which would allow the use of germplasm materials with precision to widen the genetic base of breeding populations  is  necessary.  Until  recently,  the  only  information  linking  different  breeding populations was pedigree, inadequate in many cases and sometimes incorrect (Mayes et al.,

2000).

The  genetic  improvement  in  oil  palm  bunch  and  oil  yields  has  generally  been achieved through conventional breeding in NIFOR. Unfortunately, the method is a slow (≥10 years)  and  expensive  process  fraught  with the  limitations  of long  generation interval  of approximately 19 years for phenotypic evaluation of testcrosses and inter-crossing of the best palms to initiate a new cycle, large planting areas for evaluation of testcrosses and enormous manpower requirement to manage and conduct  oil palm breeding trials (Soh et al., 1990; Rance et al., 2001). The application of DNA/molecular marker technology in the NIFOR oil palm breeding programme would  not only reduce the time taken for conventional breeding but ensure greater  precision  in  the production of planting materials  (Mohan et al., 1997; Mayes et al., 2008).

Molecular   markers   provide   an  opportunity   to   characterize   genotypes,   and   to determine  genetic  diversity of natural or breeding  populations  more precisely than  agro- morphological markers (Brumlop and Finckh, 2010). Furthermore, they have been considered

an  indispensable   tool  to  complement  reciprocal  recurrent  selection  scheme;   allowing monitoring of the genetic variability of the original and selected populations, identification of contaminants and selection of families to be recombined to maximize heterosis effect ( Pinto et al., 2003; Tardin et al., 2007).

Among the available molecular markers, microsatellites  or simple sequence  repeats (SSRs) are considered as ideal genetic marker for plant genetic and breeding studies due to (i) their high polymorphism,  (ii) co-dominant  inheritance,  reproducibility and (iii) abundance throughout  the genome  when compared  to restriction  fragment  length polymorphic  DNA (RFLP),  random  amplified  polymorphic  DNA  (RAPD),  and  amplified  fragment  length polymorphism  (AFLP)  (Powell et al., 1996; Gupta et  al., 1999; Philips and Vasil, 2001; McCouch et al., 2002; Mohammadi and Prasanna, 2003; Schlotterer, 2004; Varshney et al.

2004; Bindu et al., 2004; Singh and Cheah, 2005; Feng et al., 2009). In addition, they are readily transferable,  and easily assayed using polymerase chain reaction (PCR) (Peakall et al., 1998). Multiplexing PCR and multiloading PCR products on single gels also reduces the workload for studies requiring a large number of samples (Saghai-Maroof et al., 1994).

SSRs has proven its advantage and suitability in oil palm population  genetics  and breeding  studies (Billotte et al., 2001; Billotte et al., 2005; Bakoumé et al., 2007; Singh et al., 2008; Arias et al., 2012; Bakoumé et al., 2015), varietal identification (Rajinder et al.,

2007; Norziha et al., 2008; Thawaro and Te-chato, 2010; Bakoume et al., 2011; Hama-Ali et al., 2014), pedigree analysis, genome mapping and quantitative trait loci (QTL) detection for molecular marker-assisted selection (Billotte et al., 2010; Montoya et al., 2013; Ting et al.,

2013).

Considerable amount of molecular markers studies on oil palm have already been carried out in  Malaysia   (MPOB),  France  (CIRAD),   Colombia   (CENIPALMA),   to  name   a  few. Unfortunately, not as much works have emerged from the proposed centre of distribution of the species i.e. Nigeria as well as from the West Africa oil palm belt.  Essentially, authors have either attempted evaluation of within species genetic variability, molecular marker-trait association (QTLs) or establishment of phylogenetic relationship using populations of some African origin while others have tried to  characterize the trait potentials of the varieties at molecular level.

Despite  the  importance  of  information  on  genetic  diversity  in  improvement  and efficient conservation of crops, no studies, to date, have been specifically conducted on the genetic  diversity  of  the  current  NIFOR  breeding  populations  using  SSR.  Therefore,  the objectives of the present study were:

1.   To confirm the legitimacy of progenies derived from 11 parents of the NIFOR Main

Breeding Programme.

2.   To determine the genetic diversity and genetic relatedness among the NIFOR oil palm breeding parents using microsatellite (SSR) analysis.

3.   To compare the genetic diversity of the present parental populations of the NIFOR oil palm breeding programme with some elite breeding populations from Malaysia. 4.   To compare  the genetic  diversity of 10 D x T oil palm progenies  determined  by microsatellite markers to that revealed by agronomic markers.

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