Genetic Diversity of Moroccan Orobanche crenata Populations Revealed by Sequence-Related Amplified Polymorphism Markers

Orobanche crenata Forsk. is a root holoparasitic plant that affects legume species in Mediterranean basin especially in Northern Africa. This parasitic weed is particularly problematic in faba bean (Vicia faba L.) and lentil (Lens culinaris Medik.) fields. In Morocco, development of legume resistant/tolerant genotypes is considered the most economical and ecological control strategy against O. crenata. Efficient selection of resistant/tolerant cultivars requires prerequisite knowledge of the genetic diversity of the parasite. Thus, the present study focused on the assessment of the genetic diversity among and within Moroccan O. crenata populations, growing in faba bean fields, using Sequence-Related Amplified Polymorphism markers (SRAP). This marker system proved to be a powerful and an efficient tool for the evaluation of the genetic diversity among O. crenata populations. In fact, a total of 101 markers were identified and used for the Analysis of Molecular Variance (AMOVA), among which 98 bands were polymorphic (97.02%), indicating considerable genetic variation of these O. crenata populations. However, at population level, low level of polymorphic loci was observed with a percentage ranging between 41.58% and 67.33%. The Jaccard’s similarity coefficient and Principal Coordinate Analysis (PCoA) showed a clear differentiation among O. crenata samples according to the geographical origin of each population. AMOVA analysis revealed also a large extent of variation among O. crenata populations (60%; p < 0.010). Our outputs on molecular genetics of O. crenata combined with future epidemiological studies of these populations should clarify occurrence of O. crenata pathotypes and thereby validate the relevance of using multisite screening trials during breeding programs.


Introduction
Faba bean (Vicia faba L.) is one of the most important legumes worldwide.According to Food and Agriculture Organization (2015), it is grown in 58 countries, from temperate, tropical to hot and arid conditions.Faba bean is used as a source of protein and minerals in human diet and as a feed crop for animals (Crépon et al., 2010).Furthermore, it provides an important added value on agriculture, improving fertility and soil structure for sustainable yield crop (Herridge, Peoples, & Boddey, 2008).
In Morocco, no information is available on the genetic structure of Moroccan O. crenata populations.Also, as no attempts using SRAP targeting ORFs as function regions to characterize O. crenata populations have yet been undertaken.The present study is aiming to assess levels of genetic diversity at the molecular level using SRAP markers of O. crenata populations on faba bean fields from seven regions in Morocco.

Plant Material
A total of 162 plants (spikes) from seven O. crenata populations were collected during spring of 2014.Spikes of O. crenata populations were sampled from seven highly infested regions of Morocco (Taza, Taounate, Fez, Meknes, Khemissat, Benslimane, and Settat) (Figure 1).In each region, samples were collected from faba bean fields.Each population consisted about twenty-three O. crenata mature plants.The number of O. crenata plants analyzed per population depending on the availability of specimens found in the screened fields.

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Statistical Analyses
The amplification profile for each primer combination was scored according to present (1) or absent (0) of homologous bands to create a binary matrix of the different SRAP phenotypes.Only bands that were reproducible and could be scored unambiguously across all individuals were included in the analysis.
The Polymorphism Information Content values (PICv) were calculated for the most produced primer combinations, using the formula: Where, pi is the frequency of the i th allele (Smith et al., 1997).
Using Gen AlEx ver.6.5, levels of genetic diversity within and between each population were measured by calculating: expected heterozygosity, percentage of polymorphic loci, pairwise distance matrices, and pairwise genetic distances.The number of permutations for significant testing was set at 1000 for analysis.Analysis of molecular variance (AMOVA) based on PhiPT values was carried out using the same program, to calculates Fixation index (F ST ) analogue (PhiPT = AP/(WP + AP) = AP/TOT with AP = Est.Var.Among Populations, WP = Est.Var.Within Populations), which estimates variation among and within populations.These values can range between 0 (no differentiation) and 1 (complete differentiation).
In addition, to assess genetic relationships among populations, the matrix of inter-individual Dice's distance coefficients (Nei & Li, 1979) was used for a principal coordinate analysis (PCoA).Finally, Jaccard's similarity coefficient was estimated using XLSTAT 5.14 software, UPGMA (un-weighted pair-group method with arithmetic average) was performed and a dendrogram was derived from the tree option.

Variation for SRAP Markers
The objective of the study was to determine the genetic relationships among 162 Moroccan O. crenata populations from different regions using SRAP analysis.Among thirty primer combinations tested, only six (F3R4, F4R2, F4R3, F4R6, F5R3, F5R5) generated robust and reproducible amplification products.The number of polymorphic fragments detected by each primer combinations varied from 13 bands (F4R2) to 26 bands (F4R3), and fragments sizes ranged between 50 bp to 2000 bp.A total of 101 bands amplified were scored, 3 were monomorphic, and 98 bands were Polymorphic, discriminating therefore between the seven analyzed O. crenata populations.The average number of total bands and polymorphic bands per primer were 16.83 and 16.33, respectively.Three of the SRAP primer combinations analyzed (F4R3, F4R6, and F5R3) yielded 100% polymorphic bands.On the other hand, the minimum proportion of polymorphic bands (92.30%) was obtained by F4R2 (Table 2).
In order to measure the efficiency of polymorphic loci for detecting the genetic diversity among the studied populations, the PICv was calculated.For the 6 primer combinations, PICv ranged from 0.91 (F4R2) to 0.98 (F4R3, F4R6, and F5R3) with an average of 0.95 (Table 2).Note.F: forward of SRAP primers; R: reverse of SRAP primers.

Genetic Diversity
The SRAP marker analysis of the seven O. crenata populations revealed a high level of genetic variation with 97.02% of polymorphic bands.Nevertheless, a low genetic diversity within populations was shown with a variation ranging from 41.58% to 67.33%.The mean percentage of polymorphic loci was 53.18%.At the intra-population levels, the highest percentage of polymorphism was found in Taounate population, whereas the lowest value was that of Meknes population (Table 3).The diversity analysis within the populations using Shannon's index (I) as well as the expected heterozygosity (He) ranked the populations based on their region of origin from the most diverse to the least diverse as follows of Taounate, Benslimane, Khemissat, Taza, Meknes, Fez, and Settat region, with an average of 0.238 (I) and 0.154 (He) respectively.The estimated allele frequency with number of different alleles (Na) of Taounate population was the highest (1.574) and that of Meknes and Fez populations was the lowest (1.307).For the estimated allele frequency with number of effective alleles (Ne), the Taounate population was the highest one (1.323) and the Settat population was the lowest one (1.144)(Table 3).Note.P (%) = percentage of polymorphic loci; N = number of individuals; Na = number of different alleles; Ne = number of effective alleles; I = Shannon's diversity index; He = expected heterozygosity.

Genetic Structure of Global O. crenata Populations Based on Geographic Origin
Genetic differentiation between populations was relatively high, and all populations were significantly different (P < 0.05; Table 4).Genetic distance and identity coefficient were calculated by the method (Nei, 1972).These values were practically high with the mean genetic identities among populations varying from 0.553 to 0.806.Considering the genetic distance, the values ranged from 0.216 to 0.592.Genetic identity and genetic distance among the seven regions demonstrated that the Benslimane and Taza population pair had the highest genetic similarity (0.806) and the lowest genetic distance (0.216; P = 0.377), while Meknes and Benslimane had the least similarity (0.553) and the highest genetic distance (0.592; P = 0.628) (Table 5).Analysis of Molecular Variance (AMOVA) was used to evaluate diversity structure between the seven O. crenata populations (Table 6).Considerable internal variation was observed within O. crenata populations (60%).Furthermore, significant divergence (40%; Ø = 0.597; p = 0.010) among the seven populations was also detected.Note.Df = degree of freedom; SS = Sums of squares; MS = mean squares; Est.var = estimate of variance; % = percentage of total variation; PhiPT = Phi-statistics probability level after 1000 permutations; P = is based on 1000 permutation.

Cluster Analysis
The data obtained from SRAP analysis of 162 O. crenata samples was used in the frame of a cluster analysis.
The estimated Jaccard's differences between O. crenata populations varied from 0 to 0.996 (p = 0.001) between different pairs of individuals and the UPGMA method showed a good fit to the matrix on which it was based.UPGMA separated O. crenata populations into three main clusters: The first group encompassed the populations from North-central area including Fes, Meknes, Khemissat, and six samples of the Benslimane region.The second group consisted on 2 populations (Taounate and Settat populations).The third group included Taza population and the rest of the Benslimane population (10 samples).In this dendrogram, most samples formed clusters according to the the geographical scope (Figure 2).

Discuss
The aim geographic were poly (97.02%) primers to from differ Parameters (Botstein, in (Tajdoost, Khavari-Nejad, Meighani, Zand, & Noormohammadi, 2013) with 53.6% and 81%, respectively.In fact, population genetic structure is affected by a number of evolutionary factors including gene flow, seed dispersal, and mode of reproduction (Tajdoost, Khavari-Nejad, Meighani, Zand, & Noormohammadi, 2013).At the opposite, (Román, Rubiales, Torres, CuberoI, & Satovic, 2001) based on the RAPD markers, found high intra-population variability (94%) within six southern Spain O. crenata populations.Furthermore, our previous study on genetic diversity, using RAPD markers, within six O. crenata populations collected from faba bean and lentil fields in three highly infested regions of Morocco (Taza, Meknes and Settat) showed 81% and 82% of intra-population variability respectively (Ennami et al., 2017).These dissimilarities between our current study and former RAPD analysis may be due to the type of markers used.In fact, previous studies reported the presence of artifactual bands (false positives and false negatives) on RAPD markers.This may seriously restrict the reliability of this marker for genetic diversity studies (Semagn, Bjornstad, & Ndjiondjop 2006;Li & Quiros, 2001).At the opposite, SRAP results seam more trustworthy, as they have the highest average discriminating power among the four systems AFLP, SSR, ISSR, and RAPD (Budak, Shearman, Parmaksiz, Gaussoin, & Riosdan, 2004).Furthermore, SRAP markers have the asset to amplify coding regions of the genome with primers targeting ORFs, elucidating therefore regions with inherent biological significance (Robarts, & Wolfe, 2014).

Conclusions
In this study, SRAP markers revealed sufficient polymorphism and provided adequate information for the assessment of genetic diversity of O. crenata populations.In fact, AMOVA of this parasitic weed showed a high level of inter-population variation (60%; p < 0.010).Furthermore, cluster analysis illustrated a clear differentiation among O. crenata samples according to the geographical origin.These results may suggest the existence of pathovars.Further epidemiological studies of these seven O. crenata populations should clarify occurrence of O. crenata pathotypes.In this case, multisite screening trials during breeding programs should be considered for the development of faba bean resistant/tolerant genotypes. jas.ccsenet.

Table 2 .
Polymorphism number and rate and PIC values of six SRAP primers pairs used to amplify 162 genomic DNA templates of seven Moroccan O. crenata populations

Table 3 .
Summary of molecular data of SRAP primer combinations used for O. crenata populations from seven regions of Morocco

Table 5 .
Pairwise comparison matrix of Nei genetic identity (Above diagonal) and Nei genetic distance (Below diagonal) for O. crenata populations from Taounate, Benslimane, Khemissat, Taza, Meknes, Fez, and Settat regions based on their geographic origin

Table 6
. Analysis of molecular variation (AMOVA) for O. crenata population from seven regions were reported among O. cumana populations from Eastern Bulgaria (Pineda-Martos et al., 2014) and among Cuscuta campestris populations from Iran