Genetic Diversity of Groundnut (Arachis hypogaea L.) Accessions using Inter Simple Sequence Repeats (ISSRs) Marker


 Background: Groundnut (Arachis hypogaea L.) belongs to the family Leguminoseae. It is the world’s most important source of edible oil and vegetable protein. To our knowledge, little is known about the genetic variability of cultivated Ethiopian A. hypogaea at a molecular level. It is important to understand the genetic diversity of the crop to collect, conserve and use the germplasm for variety development. In the present study, ISSR markers were used to determine the genetic variability and diversity of 43 accessions of A. hypogaea collected from different regions of Ethiopia.Results: Four ISSR primers were used to generate 56 reproducible bands of which 29 (51.8%) were polymorphic. The band size ranges from 120 bp to 1100 bp. The number of amplified bands varied from 12 in primer UBC841 to 18 in primer UBC 881. The polymorphic bands percentage ranges from 27.8 % of Primer UBC 881 to 84.6 % of primer UBC 857. The polymorphic information content (PIC) value ranges from 0.29 to 0.76 with the average value 0.49. The mean Nei’s gene diversity and Shannon’s information index were 0.25 and 0.33, respectively. Genetic relationship between A. hypogaea accessions based on Jaccard’s pair wise similarity coefficients varies from 44% to 83% with an average value of 63.5%. The UPGMA dendrogram based on cluster analysis grouped A. hypogaea accessions into five distinct clusters at 63.5% similarity coefficient, and the principal coordinate analysis revealed similar grouping. Conclusions: Even though in both UPGMA and PCoA most of the accessions were grouped in separate clusters irrespective of their geographic origins, the ISSR marker-based analysis shows the presence of genetic variability among the accessions. Moreover, the current study demonstrated the potential informativeness of ISSR markers in estimating the extent of genetic variation in A. hypogaea accessions.


Introduction
Groundnut or peanut (Arachis hypogaea L.) belongs to the family Leguminoseae and genus Arachis. Cultivated groundnut (Arachis hypogea L.) is a highly self-pollinated, allotetraploid annual legume with 2n = 4x = 40 with a basic chromosome number of x = 10 [1]. A. hypogaea is cultivated and grown throughout the tropics and sub-tropics between 40° south and 40° north of the equator where the annual rainfall ranges between 500 to 1200 mm and with average daily temperature of higher than 20 °C [2]. Major A. hypogaea growing countries include China, India, the United States and Nigeria [3].
A. hypogaea is the world's 4th most important source of edible oil and 3rd most important source of vegetable protein [4]. A. hypogaea seeds contain 42-50% oil, 26% protein, 18% carbohydrates, and are also rich source of ribo avin, thiamine, nicotinic acid and vitamin E [5]. A. hypogaea is one of the four economically important oilseed crops along with noug, ax and sesame in Ethiopia [2]. Besides, this crop helps small scale producers in getting signi cant revenue and also helps Ethiopia in getting foreign currency earnings through export. Being a legume, this plant improves soil by xing nitrogen biologically without consuming non-renewable energies and without disturbing agro-ecological balance [6].
In Ethiopia, A. hypogaea is grown and covered nearly 80,000 hectares [7] of arable land per annum and the major producing regions are Eastern Hararghe in Oromia and Metekel in Benishangul-Gumuz regional state [7,8]. Despite its importance, the national average yield produced by the farmers in Ethiopia is considerably low, 1.3 tons/ha, indicating the need of maximum effort to improve productivity [9].
Genomic research can provide new tools and resources to revolutionize crop genetic improvement and production. It also provides accurate knowledge at molecular level which is not possible with phenotypic markers [10]. Assessment of genetic diversity is an important step in any crop improvement program [11].
Understanding the molecular basis of the essential biological phenomena in plants is crucial for the effective conservation, management, and e cient utilization of plant genetic resources (PGR) [12]. Collecting DNA marker data to determine whether phenotypically similar cultivars are genetically similar would therefore be of great interest in crop breeding programs [13]. Evaluation of genetic diversity could be based on morphological or molecular markers. Morphological features may not be e cient as they are highly in uenced by environments. Molecular marker technique is an e cient tool for genetic variation evaluation in plants [14].Consequently, the development of marker protocols such as RFLP, AFLP, ISSR, SNP and SSR have revolutionized the genetic analysis by detecting level of polymorphism [15].
Inter Simple Sequence Repeats (ISSR) marker has been reported as a rapid, reproducible, and cheap ngerprinting technique based on the variation found in the regions between microsatellites [21,22]. The ISSR method has several bene ts over other techniques: rst, it is known to be able to discriminate between closely related genotypes and second, it can detect polymorphisms without any previous knowledge of the crop's DNA sequence [23]. In addition, it does not require genome sequence information; it leads to multilocus, highly polymorphous patterns and produces dominant markers [24]. ISSR PCR is a fast, inexpensive genotyping technique based on variation in the regions between microsatellites [23]. ISSRs segregate mostly as dominant markers following simple Mendelian inheritance. ISSR analyses offer breeders and geneticists with competent means to link phenotypic and genotypic variations in various elds of plant improvement [25].
Genetic diversity study in A. hypogaea, based on morphological, biochemical and some molecular markers has been reported [16][17][18][19][20]. However, more molecular marker-based genetic diversity study of A. hypogaea accessions in Ethiopia is demanding. In this paper we report the level and pattern of genetic variability in 43 accessions of A. hypogaea grown in different regions of Ethiopia using ISSR markers. Moreover, we addressed the potential informativeness of ISSR markers for identifying A. hypogaea accessions.

Plant Materials
Seeds of 43 A. hypogea accessions collected from different regions of Ethiopia were obtained from Ethiopian Biodiversity Institute (EBI), Addis Ababa, Ethiopia. The accessions were collected from different geographical locations of Ethiopia (Fig. 1). The seeds of all the 43 A. hypogaea accessions were planted in plastic pots containing sandy loamy (composted) soil and maintained in a greenhouse under controlled temperature (30 °C) for about four weeks at Awash Melkasa Agricultural Research Center. Watering was done once a day regularly. Fresh young leaves from four week old plants were collected from plants of each accession in tubes containing silica gel for genomic DNA extraction. These accessions, their gene bank number, code, collection region, locality and geographical location (latitude and longitude) are listed in Table 1. dried leaves for each accession were ground with mix and miller machine. Genomic DNA extraction was done based on the CTAB method [26] with minor modi cation in the amount of CTAB solution used (1000 µl), as well as incubation and centrifugation time, to get optimal amounts of DNA. The yield of DNA isolated was measured/quanti ed using a Nano Drop ND-8000 UV spectrophotometer. Moreover, the purity of DNA was visually determined by agarose gel electrophoresis by running the samples on 1% agarose gel ( Fig. 2). The samples were stored at 4 o C until subsequent analysis is carried out.

Primer Selection and Optimization
For PCR optimization and screening of primers, the concentration of extracted DNA from each accession were adjusted to 50 ng/µl. A total of nine ISSR primers obtained from the Genetic Research Laboratory (Primer kit UBC), originally bought from University of British Columbia, were used for the initial testing of polymorphism and reproducibility. All the 9 primers were screened for reproducibility and polymorphism. Finally, three di-nucleotide primers (UBC810, UBC841 and UBC857), and one penta-nucleotide primer (UBC881) which shows polymorphic and reproducible bands were selected for ISSR ampli cation (Table 2).

Electrophoresis
Agarose gel (1.67%) was prepared using 300 ml TBE mixed with 5.01 g agarose using 500 ml Erlenmeyer ask and then boiled in micro oven for 3 minutes. After it was cooled for about 20 min at room temperature, 12 µl Ethidium Bromide (10 mg/ml) was added and the gel was poured on gel casting tray to solidify.
The ampli ed products were run on to ISSR gel using 1.67% agarose, with 1 X TBE using gel electrophoresis chamber. Eight micro litter ISSR ampli cation products and 2 µl (6X) loading dye (0.12% bromo-phenol blue and 30% glycerol) were mixed thoroughly and loaded on the gel. A 1200 bp ladder (molecular marker) was used to estimate the molecular size of the DNA fragments. The gel was run on electrophoresis machine for 2 h at constant voltage of 100 V. The ISSR band patterns were visualized and photographed under UV light using Biometra Biodoc analyzer.
Data scoring and statistical analysis ISSR bands were scored as present (1) and absent (0) representing the ISSR pro le of each sample. For each ISSR marker, total ampli ed bands, number of polymorphic bands, and percentage of polymorphic bands (PPB) were determined. The 0/1 matrix data was analyzed using Free Tree 0.9.1.50 [27] and NTSYSpc version 2.02 Rohlf (2000) software to calculate the Jaccard's similarity coe cient for all possible pairs of samples. Jaccard's similarity coe cient was calculated as: where, S ij is Jaccard's similarity coe cient, a is the total number of bands shared between individuals i and j, b is the total number of bands present in individual i but not in individual j and c is the total numbers of bands present in individual j but not in individual i. The resulting similarity matrices were employed to construct UPGMA-based dendrogram. The unweighted pair group method with arithmetic mean (UPGMA) was used in order to determine the genetic relationship among accessions using NTSYS-pc version 2.02 [28]. The matrix of genetic similarity was also used in a principal coordinate analysis (PCoA) to resolve the patterns of clustering among the accessions based on Jaccard's coe cient.
Percent of polymorphism, Nie's pairwise gene diversity and Shannon's Weaver pairwise diversity index (I) were determined with POPGENE software 1.32 [29]. The binary data generated were used to determine levels of polymorphism by dividing the polymorphic bands by the total number of scored bands. Genetic diversity measures were tested using Nei's gene diversity statistics. Shannon's diversity index (I) was also used to examine partitioning of genetic diversity within and among populations.
To measure the informativeness of the ISSR markers to differentiate between the A. hypogaea accessions, polymorphism information content (PIC), effective multiplex ratio (EMR), marker Index (MI) and resolving power (RP) were calculated. The value of polymorphism information content (PIC) was calculated using software Power Marker version 3.2 [30]. The PIC was calculated by the formula: PIC = 2Pi (1-Pi), where, Pi is the frequency of occurrence of polymorphic bands in different priers. EMR is the product of the fraction of polymorphic bands and the number of polymorphic bands [31]. MI was determined according to Powell et al. [32] as the product of PIC and EMR. RP was calculated using the formula RP=∑Ib, where Ib is band informativeness and Ib = 1-[2 × (0.5-p)], where p is the proportion of genotypes containing the band [33].

ISSR polymorphism
Four of nine ISSR primers used (Table 3) could produce reproducible bands ranging in size from 120 to 1100 bp (Fig. 2). Fifty-six bands were generated across the four ISSR primers, of which 29 (51.8%) were polymorphic ( Table 3). The ampli ed bands by the primers range from 12 (UBC841) to 18 (UBC881) across the accessions. The number of polymorphic bands of the primers ranged from 5 in primer UBC841 and UBC881 to 11 in primer UBC857. The percentage of polymorphism for primers ranged from 27.8% in primer UBC881 to 84.6% in primer UBC 857, with an average polymorphism percent of 51.8% (Table 3). In the present study, the di-nucleotide primers, namely UBC810, UBC841 and UBC857 were observed to have 61.5%, 41.7% and 84.6% of polymorphism, respectively. The penta-nucleotides primer UBC881 was observed to have 27.8% polymorphism. A representation of the ISSR band pro le obtained with primer UBC857 is shown (Fig. 3). In the present study, the di-nucleotide ISSR primers UBC857 with AC repeats and UBC810 with GA repeats, detected higher polymorphism among accessions compared with pentanucleotide primer.

Polymorphism Information Content (PIC)
Polymorphism information content (PIC) is the probability of detection of polymorphism by a primer/primer combination between two randomly drawn genotypes and depends on the number of detectable alleles and the distribution of their frequency. In the present study, the PIC value varied from 0.29 (primer UBC881) to 0.76 (primer UBC857) with an average value of 0.49 (Table 3).
The effective multiplex ratio (EMR), the number of polymorphic fragments detected per assay, varied from 5 to 26 with a mean value of 15.94.

Resolving power (RP)
The resolving power (RP) is a parameter that speci es the discriminatory potential of the primers (the ability of a primer to generate optimally informative bands). Many studies have indicated RP index as an important feature of a good marker system [34,35,36,37]. In the present study, the estimated RP for primers varied from 2.65 (UBC881) to 18.34 (UBC857 ) with an average value of 10.46. The highest RP values suggesting the capacity of the primers used to distinguish among different accessions. RP was positively correlated with total ampli ed bands, number of polymorphic bands, MI and EMR at P < 0.01. MI and EMR were positively correlated with RP (r = 0.924 and r = 0.738, respectively, P < 0.01) and also positively correlated with PIC.
Cluster Analysis UPGMA clustering analysis put the accessions into one cluster at 59.2% similarity. However, the accessions were clustered in to ve clusters based on the cut-off point of 63.5% similarity (Fig. 4). The dendrogram for the accessions (Fig. 4)  Principal Coordinate Analysis (PCoA) All the data obtained using four ISSR primers were used for PCoA using Jaccard's coe cients of similarity.
The rst two components of the coordinates of the PCoA having eigen values of 16.3 and 8.2 with variance of 33.2% and 16.4%, respectively, and together 49.6% used to show the grouping of individuals using two co-ordinates. Similar to the UPGMA clustering pattern, the 43 accessions of A. hypogaea were grouped into ve groups (clusters) based on the principal co-ordinate analysis (Fig. 5).
The PCoA plot indicated that most of the accessions did not group together with other accessions originated from the same geographical location (Fig. 5). This result is in line with the result obtained in UPGMA. However, most of the accessions that show geographical proximity were found to form distinct groups and spread all over the plot (Fig. 5). In addition, accessions from distant geographical locations tend to form similar group or cluster. As the result, low coe cient of variation was observed in A. hypogaea accessions considered in this study.

Discussion
In the present study ISSR pro le was used for diversity analysis and relationship among different A. hypogaea accessions. Several studies on populations indicate the percentage of the polymorphic locus as an important measure of genetic diversity [14]. A. hypogaea showed moderate genetic diversity as indicated by percent polymorphic loci (p = 51.8%). A study reported 54% of polymorphism among 13 A. hypogaea accessions using ISSR markers [15]. The observed highest Nei's gene diversity (H) value (0.38 ± 0.174), and the highest Shannon's indices (0.41 ± 0.194) shows presence of genetic diversity among the studied accessions. The Shannon index vary from 0 to 1, and lower genetic diversity is represented by values closer to zero [39].
In the present study, the PIC value varied from 0.29 (primer UBC881), less informative to 0.76 (primer UBC857), high informative with an average value of 0.49. PIC value of 0.70 and above is highly informative whiles a value of ~ 0.44 is moderately informative. PIC is a statistic that measures the usefulness of a genetic marker for linkage analysis [38].
The genetic differentiation of a species re ects the interactions of various evolutionary processes including long-term evolutionary history, such as shifts in distribution, habitat fragmentation and population isolation, mutation, genetic drift, mating system, gene ow and natural selection [42]. The coe cient of gene differentiation (Gst) for the entire accessions of A. hypogea was 0.29, suggesting a restricted genetic differentiation between accessions. The average value of differentiation might re ect the interactions of various factors including reduced geographic range in most of the accessions, inter and intra-regional climates, their breeding system and limited genetic drift or genetic isolation of the samples.
Based on the Gst value, the mean estimated number of gene ow (Nm) for the entire accessions was found to be 0.827 (Table 4). In owering plant, the level of Nm is divided into three grades: high, Nm equal to or larger than 1.0; moderate, Nm ranging from 0.250 to 0.99; and low, Nm ranging from 0.00 to 0.249 [43]. Gene ow is generally considered as the main factor that could homogenize the genetic structure of populations in their distribution area. According to Wright (1931) [44], Nm¼ 1 is su cient to overcome the effects of genetic drift. Also, species with low gene ow have higher genetic differentiation than species with high gene ow. However, our results indicated that virtually moderate gene ow occurred between the accessions of A. hypogaea.
Based on Jaccard's similarity coe cient, highest genetic similarity observed among accessions suggests that the existence of genetic similarity among the accessions possibly due to gene ow. On the other hand, the least genetic similarity observed between some accessions is useful in broadening genetic base of A. hypogaea accessions in Ethiopia and these accessions should be considered as the primary/valuable sites in conservation and breeding program of the crop. Both PCoA and UPGMA cluster analysis shows the clustering of all 43 accessions into ve clusters without clear geographical differentiation. This might be due to gene ow caused by the exchange of seeds by farmers in Ethiopia.
Knowledge on the genetic diversity of the selected individuals is of ultimate importance, since it contributes to the information on the species and allows the selection of genotypes to be included in future conservation programs. Thus, the most divergent genotypes can be selected to maintenance the level of genetic diversity of a species to keep its ability to adapt to novel environmental changes. The present nding also contributes valuable information on the genetic diversity of A. hypogaea accessions grown in Ethiopia.
Parameters such as MI and EMR have been used for assessing the informative potential of molecular markers in various genetic diversity studies [31,34]. In the present study, the primers that generated high number of bands had higher MI and EMR values. MI and EMR were positively correlated with RP (r = 0.924 and r = 0.738, respectively, P < 0.01) and PIC. The resolving power (RP) is a parameter that speci es the discriminatory potential of the primers (the ability of a primer to generate optimally informative bands).
Many studies have indicated RP index as an important feature of a good marker system [34,35,36,37]. In the present study, the highest RP value 18.34 (UBC-857 ) suggesting the capacity of the primer used to distinguish among different accessions. RP was positively correlated with total ampli ed bands, number of polymorphic bands, MI and EMR at P < 0.01, suggesting the informativines of the ISSR primers used in the present study.
ISSR markers have demonstrated their e ciency in the study of genetic variability for several other species. Many studies have proved the effectiveness of this marker in articles on genetic diversity and characterization of accessions between and within populations, such as those with, Capparis spinosa L. [45], Pitcairnia ammea [46] Erythrina velutina [47] and Croton tetradenius [48].

Conclusions
The study has revealed genetic polymorphism (51.8%) among the A. hypogaea accessions. The highest genetic similarity observed among the accessions of A. hypogaea and the observed ve clusters without clear geographical differentiation suggest the existence of genetic similarity among the accessions possibly due to gene ow caused by seed exchange. The ISSR based ngerprinting of A. hypogaea accessions demonstrated the usefulness of the marker in estimating the extent of genetic variation and genetic relationships among A. hypogaea accessions. To conclude, the present nding is an important milestone for future germplasm collection, sound conservation, improvement and breeding of the crop.
Further study with more geographic range and the use of additional molecular markers would give additional picture of the genetic diversity of A. hypogaea accessions in Ethiopia.