Assessment of genetic diversity in Fusarium wilt tolerant and susceptible oil palm (Elaeis guineensis Jacq) progenies in Nigeria using inter-simple sequence repeat (ISSR) molecular markers

Background Improving oil palm in Nigeria for food security and subsequent export requires a better understanding of the genetic diversity among oil palm progenies tolerant and susceptible to Fusarium wilt disease. In view of the limitations of the orthodox method used in screening this disease, and the advantages of molecular markers, fourteen (14) Inter-simple sequence repeat (ISSR) DNA markers were applied to evaluate the genetic diversity, population structure and cluster resolutions of alleles responsible for tolerance of 560 Elaeis guineensis Jacq palms representing 8 different progenies distributed across NigeriaResults The amplification product revealed a moderately high level of genetic diversity with a total of 46 alleles identified, resulting in an average of 4.9091 alleles per locus detected between the oil palm progenies. Polymorphic information content (PIC) values varied between 0.3706-0.7861, with a mean value of 0.6829. The genetic diversity values ranged from 0.4063-0.8125 with a mean of 0.7216, while the major allele frequency ranged from 0.2500- 0.7500 with a mean value of 0.3750. Shannon's information index (I), Nei's gene diversity (H), and the effective number of alleles (Ne) had values of 0.6931, 0.5000, and 2.000, respectively. The genetic diversity was highest in progeny 3023, and lowest in progeny 4189. Mean values of the total gene diversity (Ht), gene diversity within the population (Hs) of the progenies, coefficient of gene differentiation among the progenies (Gst) and level of gene flow (Nm) were 0.4899, 0.3520, 0.2815 and 1.2764, respectively. The dendrogram clustered the progenies into six major clusters, while Principal Component Analysis (PCA) grouped the progenies into five clusters. PCA further identified the coordinate positions of tolerant and susceptible alleles of oil palm progeniesConclusion This study confirmed the identification of the coordinate positions of tolerant alleles in the gene loci, which could be exploited by breeders to developing tolerant oil palm seedlings. ISSR of PIC for on radiata Vigna subterranea accessions. PIC revealed that ten of the ISSR markers were able to identify the genetic diversity between the different oil palm progenies, in agreement the study Okoye the PIC inequity between Total ISSR: Inter-simple of alleles; Nm: Level of gene flow; NPL: of loci; PCR: chain Polymorphic information PPL: polymorphic loci; RAPD: polymorphic SSR: Simple Unweighted pair with


Background
Oil palm (Elaeis guineensis Jacq) is a crop that lasts for more than two growing seasons. It is allogamous and of African origin. Oil palm is the highest oil-yielding crop in the world.
Nigeria, the most populous nation in Africa, is the fourth largest world palm oil producing country and number one in Africa. In the humid tropical areas of Africa, there are not less than 43 countries where the crop is cultivated presently (Sheil et al.,, 2009;FAO, 2015). A high percentage, greater than 30% of vegetable oil, is generated from this crop, which makes it stand out in fat supply to the world (USDA, 2015). The plant, apart from being the producer of palm oil which humans take in as a diet, is also essential in body insulation and energy to people domiciled in a lot of developing countries (Goh et al.,, 2016). The problem of maintaining production and development of oil palm establishment has been raising issues not only to the local sphere of plant science but also in the fields of economics (Rival et al.,, 2016).
The crop is vulnerable to many diseases, and the most devastating of them all in Africa is the Fusarium wilt disease whose causative agent is Fusarium oxysporum f.sp elaeidis (Paterson et al.,, 2013;Noumouha et al.,, 2014). The production of oil palm in Africa is cut drastically by the effects of this disease (Ntsomboh et al.,, 2015). The devastating effects of this disease are as high as 70%, and some factors which include areas where the condition had occurred do favour the sudden emergence of the disease (Rival, 2017).
Fusarium as genera includes many species which are capable of causing the physical manifestation of a disease. The causative agent is soil borne; however, it is still possible for the pathogen transmitted by rain and wind (Leslie and Summerell, 2006). There as some saprophytic species of Fusarium which are soil borne and have the ability to live and utilize the dead organic matter of the oil palm trunk, while some species live as endophytes inside the palm and do not cause disease to the palm.
The genetic diversity that exists among plant cultivars makes it easier for breeders to manipulate and enhance traits which are desirable to farmers and suitable to breeders (pest and disease resistance). In carrying out the genetic assessment, the genetic diversity that is possessed by plant progenies may not be at par with the physical observation of traits or essential characters, but has many benefits over the morphological assessment (Govindaraj et al.,, 2015). It is evident to note that varieties that possess high genetic diversity identified by molecular markers could be used and  . The (ISSR) marker system is a PCR based system that makes use of a single amplification primer which is made up of a set of repeated DNA motifs that targets the microsatellite regions (Salis et al., 2017). Inter-simple sequence repeat markers target multiple genomic loci and amplify DNA segments that occur in between two microsatellite regions that face each other in terms of orientation.
The deployment of classical breeding techniques geared at improving beneficial traits to farmers like tolerance to pathogenic organisms requires the assessment of many different progenies. However, to assess progenies for tolerance phenotypically, it requires lots of finance and above all can be affected by environmental factors. The use of classical breeding in classifying individual varieties into various clusters adopts morphological features but has limitations because they do not reveal real genetic relationships and other shortcomings. The advantage of ISSR markers of not requiring sequence data for primer design and the allelic richness it possesses was essential for adopting in this study.
The identification of the genetic relationships, population structure that exists among Fusarium wilt tolerant and susceptible oil palm progenies, and the determination of the coordinate positions of tolerant alleles possessed by oil palm progenies which could be exploited by breeders in developing tolerant oil palm plant lines.

Sample Collection and DNA Extraction
A total of 560 palms representing eight progenies were sampled from the fields of oil palms containing susceptible and tolerant oil palm progenies. The progenies were selected based on their use for the basis of screening purposes (Table 1) (Figure 1). For each progeny, 70 palms were sampled based on the availability of the samples. Approximately 100 mg of fresh young unopened leaves were harvested from each palm. The leaves were subsequently cleaned and maintained at -80 0 C. The total genomic DNA extraction was carried out using the CTAB method with little modifications.

Polymerase Chain Reaction and Agarose Gel Electrophoresis
Polymerase chain reaction (PCR) amplification was accomplished by mixing together 1.

Data Analyses
The data matrix of ISSR profiles obtained from fragments of each amplicon was scored as 1 (presence of alleles) and 0 (absence of alleles). The data generated from the scoring of the ISSR amplicons were employed for phylogenetic reconstruction using Unweighted Pair Group Mean with Arithmetic (UPGMA) and dissimilarity index in Jaccard"s option (Ojuederie et al., 2013). The analysis was carried out using NTSYSpc software version 2.02. Furthermore, genetic diversity, allele frequency, and the polymorphic information content (PIC) were analysed using PowerMarker Version 3.25. Genetic diversity and population structure analyses of the oil palm progenies were analyzed using POPGENE software version 1.32. Also, total gene diversity (Ht), gene diversity within the population (Hs), the level of gene flow (Nm), and the coefficient of gene differentiation (Gst) were calculated with POPGENE software version 1.32 (Yeh et al., 1999).    (14) ISSR molecular markers were verified. Ten (10) out of the 14 markers produced scorable bands and used in the diversity investigation (Plate 1).
A dendrogram using Unweighted Pair Group Mean Arithmetic (UPGMA) and dissimilarity index clustered the progenies into six significant clusters based on their genetic similarities and divergence ( Figure 2). Cluster 1 grouped two susceptible progenies (3023 and 2211) at a booth trap of 10% between them. Apart from they are both susceptible progenies, they were grouped based on shared ancestry/ parentage of the progenies.
Taking a close examination at the first cluster, progeny 3023 is an Angola Dura, while progeny 2211 is a cross between Calabar and Aba Dura. However, progeny 3023, which is an Angola Dura, has parentage of (Ufuma and Angola) which is (P8). Ufuma itself has a link with P1 (Ufuma and Aba). Progeny 2211 has a parent of Calabar and Aba which is P3.
Cluster II consists of just one susceptible progeny (120) with a booth trap of 11%. Cluster III contained only one tolerant progeny (1621) with a booth trap of 12%. Cluster IV equally included one susceptible progeny (2478) with a booth trap of 13%. Cluster V contained one susceptible (3456) together with progeny (1723) which is a tolerant progeny with a booth trap of 14% but clustered together due to a common origin. Taking an insight into this cluster, progeny 1723 is an Ecuador Dura, while progeny 3456 is a Malay Deli Dura.
Progeny 1723 has parentage of Ecuador Deli (BB4). The clustering was a result of the common origin, Deli, which is an ethnic group in Indonesia. Cluster VI contained just one susceptible progeny (4189) with a booth trap of 16% all from the Nigerian ( Figure 2 and Table 3).   The ten (10) ISSR primers amplified a total of 46 alleles. The amplified alleles from each marker ranged from 3-6, with a mean of 4.9091 (

Discussion
The inability of farmers to access healthy seedlings has resulted in farmers looking elsewhere and collecting seedlings from doubtful local and foreign sources. Some have had to import seedlings to meet their planting targets, where such imported seedlings which failed in the field due to susceptibility to Fusarium wilt disease may plunge investors into financial crisis. The above raises the issue of unrestricted importation of oil palm seedlings. It has also drawn attention for the need to thoroughly screen the oil palm seedlings to curb the menace of Fusarium wilt disease. However, the revelation of molecular markers in screening has made the work easier in identifying the coordinates and positions of alleles, genetic sequences responsible for Fusarium wilt and tolerance in oil palm progenies. Therefore, genetic diversity study using informative markers is essential in genetic improvement and also in the identification of unique alleles. In this study, ISSR markers were used to assess the level of genetic diversity, relationships among the different progenies of oil palm from various growing fields and cluster resolutions of tolerant and susceptible alleles. In some of the clusters, there was a clustering of a susceptible (3456) progeny with a tolerant progeny (1723). In this case, the grouping was not due to genetic similarities, but because they share a common origin. Taking a look at their relationship implies that progeny 1723 is an Ecuador Dura, while progeny 3456 is a Malay Deli Dura. Progeny 1723 has a parent of an Ecuador Deli (BB4) which led to the clustering; this, however, agrees with the report of Okoye (2016) which stated that some oil palm progenies were grouped with a parent-progeny because they share a common origin.
The Principal Component Analysis (PCA) amplified by ISSR molecular markers, resolved the progenies into five clusters, which was different from the dendrogram, which grouped the oil palm progenies into six distinct clusters. The principal component analysis clustered some of the progenies 120, which is an Aba Dura; progeny 3023 (Angola Dura), and progeny 2211 (Calabar and Aba Dura) because they share a familiar parent P1, which is a combination of Calabar and Aba Dura. This study disagrees with the study of Okoye   Figure 1 The GIS map site of susceptible and tolerant oil palm sample collection in Nigeria Dendrogram of oil palm progenies amplified with ISSR molecular markers