Detection of genetic variations in orobanche crenata using inter simple sequence repeat (ISSR) markers

This recent investigation aimed to detect and characterize the genetic variabilities and genetic similarity in Orobanche crenata collected from naturally Orobanche-infested field at Gemmeiza Research Station, Agriculture Research Center (ARC), Egypt. Five different ISSR primers were used in this study. Obtained results showed that a huge genetic variation has been detected by these ISSR primers; moreover, there were clear relationship/genetic similarity between and within all collected O. crenata parasite plants. However, the results revealed that there were high relationships where similarity matrix ranged from 0.837 (the highest) between R4 and R17 followed by 0.831 for R4/R25 and in the end, the lowest one was R1/R29 similarity matrix 0.655. It was noticed that the relationship and similarity did not reach 100% but the maximum was 83.7% (there were no identical plants); therefore, each Orobanche plant in this study was different genotype than neighboring plant within location.


Introduction
Faba bean (Vicia faba L.) is an important pulse crop grown worldwide as a source of protein both for human food and animal feed. However, its cultivation is strongly hampered in Mediterranean and Middle East farming systems by the parasitism of broomrape causing important yield losses (Rubiales et al. 2006;Joel et al. 2007;Parker 2009). Legumes are parasitized mainly by two different species of broomrapes, namely, crenata broomrape (Orobanche crenata Forsk) and foetida broomrape (Orobanche foetida Poir). O. crenata has threatened the legume cultivation in the Mediterranean Basin and Middle East crops since antiquity. On the contrary, O. foetida has been reported to damage faba bean in Tunisia only (Kharrat et al. 1992 andDiego Rubiales 2014).
Orobanche spp. seeds germinate in response to chemical signals exuded from host roots (Fernández-Aparicio et al., 2009, 2011. Subsequently, the seedling of the parasite develops into an appressorium, a specialized structure that penetrates the host root and then into a haustorium, which forms a connection between the host vascular tissue and the parasite. Then, the parasite develops into a tubercle, a bulbous structure from which a shoot arises to emerge from the soil to flower and set seeds (Joel et al. 2007).
Legume breeding for broomrape resistance is difficult considering the scarce and complex nature of resistance in legumes in general (Rubiales et al. 2006). This contrasts with the success experienced in other crops such as sunflower (Helianthus annuus L.), in which single genes governing resistance against Orobanche cumana Wallr. have been identified and exploited (Fernández-Martínez et al. 2008). This limitation has made selection more difficult and slowed down the legume breeding programs.
Recent investigations supported by molecular taxonomy analyses have resulted in re-definition of Orobanchaceae family. According to this classification, Orobanchaceae consists of 89 genera, containing 2061 species. On the Balkans, the family Orobanchaceae is represented by 3 Orobanche which includes 25 species: three of which are present in Egypt (crenata, aegyptiaca, and minor), and they are widely spread in Bulgaria Southern Europe, Russia, Middle East, and Northern Africa. They cause losses in crop productivity estimated at hundreds of millions of dollars annually that affect the livelihoods of around 100 million farmers (Gevezova et al. 2012).
The genetic nature of broomrape resistance/ tolerance is not clear at now and requires more studies on Egyptian faba bean genotypes. Sources of resistance to broomrape are scarce and of complex nature. However, several tolerant cultivars are known to farmers in Egypt under the commercial names "Giza"*. An acceptable level of resistance was found in Vf1071, an inbred line selected from cv. Giza 402 in Southern Spain. This line has been used in breeding programs to develop the welladapted, high yielding cv. Baraca (Gnanasambandam et al. 2012).
ISSR is a popular marker system, owing to its ability to detect polymorphisms without requiring the sequence information necessary for primer design. The main advantage of ISSRs is that no sequence data for primer construction are needed. This is mostly dominant marker. It is widely used for characterization of genetic relatedness among populations (Tomar et al. 2014).
ISSR markers are suitable for investigating genetic diversity among O. aegyptiaca genetic groups and able to discriminate between individuals (Abedi et al. 2014). These markers have several benefits over other techniques: first, it is known to be able to discriminate between closely related genotypes (Fang andRoose 1997 andHodkinson et al. 2002) and second, it can detect polymorphisms without any previous knowledge of the crop's DNA sequence. ISSR markers are also quick and easy to handle and more informative for the evaluation of genetic diversity (Korbin et al. 2002, Rakoczy-Trojanowska et al. 2004,and Abdalla et al. 2016).
So, this study aimed to distinguish and determine the differences and similarity among O. crenata plants (spikes) in infested field using ISSR molecular markers. *Agric. Research Center (ARC) and "Cairo" (Cairo University).

Plant material
Thirty plants from O. crenata Forsk were individually collected from naturally Orobanche-infested field at Polymerase chain reaction (PCR) was carried out in a volume of 15 μl volume containing DNA (40 ng), Master Mix (Gene Direx one PCR TM), template DNA, and primer (five different ISSR primers were used) ( Table 1).
The PCR mixture was subjected to 35 cycles in PCR with intial denaturation 94°C for 30 s and annealing 52°C for 45 s followed by a final extension step at 72°C for 7 min and store 4°C. Amplified products were stained with ethidium bromide and electrophoresed in a horizontal gel electrophoresis unit using 1.5% agarose gel (Maniatis et al. 1982).
Each experiment was repeated twice with each primer, and those primers which gave reproducible fingerprints were considered for data analysis.

Statistical analysis
A cluster analysis based on the similarity matrix was performed using the neighbor-joining method (Satiu and Nei 1987).

Genetic similarity
Genetic similarity matrix among all Orobanche samples was obtained from amplified fragments shown by five ISSR markers using Jaccard coefficients (Jaccard 1908). Moreover, cluster analysis based on similarity matrix was performed using UPGMA (unweighted pair group method with arithmetic mean). All measurements have been made using the NTSYS-pc version 2.1 (Rohlf 2010).

Results
The significant differences among selected Orobanche samples induced using the five ISSR primers were clearly shown and summarized in (Fig. 1). However, multiple bands varied in their molecular weight were detected in all tested plants using these different primers. Moreover, UPGMA dendrogram shows genetic relationship between all collected Orobanche plants using ISSR markers (Table 1). However, there were clear relationships or genetic similarity between and within all selected Orobanche; otherwise, a huge genetic variation has been detected by these ISSR primers.
Similarity and relationship between all tested Orobanche plants are shown in Fig. 1 and dendrogram (Fig. 2), and the comparison revealed that there were high relationship where similarity matrix ranged from 0.837 (the highest) between R4 and R17 followed by 0.831 for R4/R25 and at the end, the lowest one was R1/ R29 similarity matrix 0.655 (Fig. 2).
The dendrogram (Fig. 2) based on neighbor joining method clearly showed that there are five major clusters (A, B, C, D, and E). The cluster (A) has 10 samples that distributed in two sub clusters; each one has 5 samples. Moreover, the highest similarity matrix (0.837) was recorded between R4 and R17 followed by R4/R25 in the first sub cluster and 0.824 between R19 and R23 in the second sub cluster. However, remaining clusters (B, C, and D), every cluster has the same numbers of samples (5), and similarity ratio differed between their individuals and between different clusters (Fig. 2). Therefore, each Orobanche plant in this study is considered as different genotype than neighboring plant within location. Table 2 and Fig. 3 illustrated the genetic similarity or genetic relationship among all collected Orobanche plants in the same plot. However, it is noticed that there were no any identical plants; in other means, every plant has a unique genotype. However, there were many relationships between all samples and ratio of similarity scored as a percentage (0-100%) ( Table 2). Moreover, these relationships or similarity ratio differed from one to another among samples, and these ratios reached more over than 80%, where as it recorded its maximum (83.7%) between plants R4 and R17, followed by (83.1%) between R4 and R25 (Table 2 and Fig. 3) which these plants consider the most related plants or the less distant plants in their genetic material. On the other hand, these ratios were gradually decreased among plants and reached the lowest (65.5%) between R1 and R29 which consider the more distant plants in this study.

Discussions
In this, study there were many plants closely related in its genotype to more than one plant with variable similarity ratios, for example, plant R4 was closely related to both R17, R25, and R8 with similarity ratio of 83.7, 83.1, and 77.8%, respectively. Furthermore, plant R1 was more similar to  Fig. 3). Abedi et al. (2014) found similar results by using ISSR markers for investigating genetic diversity among O. aegyptiaca in Iran. The fact that only five clusters emerged from the 30 samples of Orobanche (5%) indicates that the virulence/ aggressiveness of the parasite may not be that huge compared to its wide genetic diversity investigated by ISSR. Moreover, Abdalla et al. (2016) determined the genetic diversity of Orobanche in two locations (Giza and Sids). Molecular variance indicated greater variation within locations (97%) than between locations (3%); ISSR markers produced fragments covered 218 to 980 bp of the total Orobanche genome. The authors found cluster analysis which divided 96 Orobanche samples into five sub clusters and concluded that there was wide genetic variation among O. crenata plants from Egypt collected from faba bean naturally infested field. ISSR markers were suitable to study identifying genetic diversity among O. crenata individuals. The breeders have to consider this high genetic variation in O. crenata when they breed faba bean for tolerance/ resistance to Orobanche.
The gene-for-gene relationships appear either to prevent the establishment of a compatible relationship or to destroy a compatible relationship once it is established. On the other hand, the data from studies of the blight of oats caused by H. victoriae strongly suggest that the specific interactions are needed for the parasite to successfully invade the host plant (George 1960). The data suggest that the parasite must alter the host in order to develop. In some host-parasite combinations, there appears to be no gene-for-gene relationships that can be demonstrated. Kado and Innan (2018) sequenced genomes of five parasite species in family Orobanchaceae to explore the evolutionary role of horizontal gene transfer in plants.
Orobanche minor and Aeginetia indica are obligate parasites with no photosynthetic activity, whereas the other three (Pedicularis keiskei, Phtheirospermum japonicum, and Melampyrum roseum) are facultative parasites. Their results showed that by using reference genome sequences and/or transcriptomes of 14 species from Fabaceae and Poaceae, their major host families detected 106 horizontally transferred genes (HTG genes), only in the genomes of the two obligate parasites (22 and 84 for Oro. minor and Ae. indica, respectively), whereas none in the three facultative parasites. Moreover, they found that almost all HGT genes retained introns at the same locations as their homologs in potential host species, indicating a crucial role of DNA-mediated gene transfer, rather than mRNA mediated retro transfer. Furthermore, some of the HGT genes might have transferred simultaneously because they located very closely in the host reference genome, indicating that the length of transferred DNA could exceed 100 kb. They confirmed that almost all introns are spliced in the genome of the parasite species, and that about half HGT genes do not have any missense mutations or frame shift-causing indels, suggesting that some HGT genes may be still functional.

Conclusion
From previous results, it noticed that the relationship and similarity did not reach 100% but the maximum was 83.7%, and this indicated that every Orobanche plant has its unique genotype which was partially similar to other plants in the same plot, and there are no any identical plants.