Genetic relationship and diversity among some Moroccan and introduced rapeseed ( Brassica napus L.) varieties as revealed by molecular markers ☆

– Rapeseed ( Brassica napus L.) crop can be a lever for the development of oilseed sector in Morocco due to its adaptation to local conditions and its major economic and food importance. Genetic diversity and selection of valuable crossing parents are the key to successful breeding and improvement of this crop. In this regard, genetic variation within the existing germplasm must be explored and characterized. Therefore, the present study was carried out to investigate the genetic diversity among 22 varieties from Morocco as well as other origins, using twenty ISSR primers. The selected primers have generated a total of 319 markers. Polymorphic ampli ﬁ ed bands varied from 8 to18, with an average of 13 per primer. The diversity index (PIC value) ranged from 0.295 to 0.509, with a mean value of 0.37 per primer, indicating a good genetic diversity level for the primers used. The average similarity coef ﬁ cient was 0.31, ﬂ uctuating between 0.176 and 0.456, and the pairwise comparison of the studied varieties showed a great discriminating power of primers and a large genetic diversity among accessions. A total of eight ISSR primers could be identi ﬁ ed as key to rapeseed varietal determination. Hierarchical classi ﬁ cation allowed identifying three groups with some phylogeographic structuring. This is the ﬁ rst report of molecular characterization of rapeseed germplasm in Morocco and Africa. The obtained results have important implications for management of this germplasm to conserve the existing genetic diversity and use it properly in breeding programs in Morocco as well as in other Mediterranean and African countries.


Introduction
Over the world, the Brassicaceae family has an important diversity of plant species including more than 3700 species and 338 genera. Brassica species have many uses such as oil, forage, green manure, biodiesel, and other uses (Sharma et al., 2022). Various species of Brassica have been domesticated in different parts of the world depending on ecology and human needs. Generally, Brassica constitute a potential pool of genetic material with many desirable agronomic and horticultural traits (Sharma et al., 2022).
Oilseed Brassica crops collectively contribute about 15% of the world's total supply of vegetable oils and became the third-leading source of edible oil in the world after soybean and palm (McVetty et al., 2016;Guirrou et al., 2023). Rapeseed (Brassica napus L.) is the most important oilseed Brassica species, which is a profitable and evolving crop thanks to the dynamism and relevance of scientific and agronomic research, mainly in genetic breeding, improvement of crop management and molecular biology.
Rapeseed is a crop that presents a broad adaptation to different environmental conditions (climate, soil, etc.). The main objective of the rapeseed-breeding program is the development of highly productive and stable varieties with high seed oil and good oil quality. Throughout the past few decades, rapeseed (Brassica napus L.) has known an intensified varietal selection based on agronomic and economic traits, which led to a reduced genetic base. Crossing is actually the main strategy adopted to create and broaden the existing genetic diversity and to develop the desired varieties. Nevertheless, the judicious choice of crossbreeding parents remains a big challenge for successful selection program and development of new varieties that perform better than the old ones. Traditionally, parents are selected based on their phenotypic attributes; however, the morphological features do not give the real level of genetic diversity among rapeseed germplasm. Since their domestication, oilseed Brassica species have undergone progressive transformation. Efforts to breed B. napus have primarily focused on improving seed yield, oil quality, as well as disease resistance, abiotic stress tolerance, and herbicide resistance (Ton et al., 2020).
The revolution in plant biotechnology, including molecular markers, genomic tools, and genetic mapping, has made it possible to understand the complex genetic makeup and genes function (Ton et al., 2020). Plant breeders rely on a diversity of crop genetic resources, breeding tools and methods to incorporate genetic diversity into commercial cultivars or to develop new cultivars with improved agronomic characteristics, such as high yield, tolerance to biotic and abiotic stresses, and high nutritional quality (Swarup et al., 2021).
Molecular markers are very powerful DNA-based biotechnology tools that can reveal genetic polymorphism not influenced by the environment conditions and, thus, present a great potential to compensate for the low discriminative power of morphological traits. Molecular markers allow identifying and characterizing plant genotypes through direct access to genetic information. Analysis by molecular markers enables choosing parental types for mapping populations, markerassisted selection, and crossing schemes. Thus, diversity analysis using molecular markers help breeders to improve crop species (Paul et al., 2020). Inter simple sequence repeat (ISSR) are generally very effective in genetic diversity studies because of their longer primers compared with random amplified polymorphic DNA (RAPD) primers (Pradeep Reddy et al., 2002;Tarıkahya-Hacıo glu, 2016). Likewise, by comparing different markers, such as ISSR, Interretrotransposon amplified polymorphism (IRAP), and retrotransposon microsatellite amplified polymorphism (REMAP), for the assessment of genetic diversity within the Brassicaceae family, it was found that ISSR markers were the most efficient (Mahjoob et al., 2016). In fact, this technique combines the advantages of the Amplified Fragment Length Polymorphism (AFLP) and the universality of the RAPD (Maras-vanlio glu et al., 2020).
In Morocco, the development of oilseed crops, such as rapeseed, can reduce import dependence, improve the trade balance and strengthen the economic activity of this vital sector. Rapeseed area has increased from nearly 500 ha in 2012/2013 to around 10 000 ha in 2020/2021, ensuring a production of about 9000 tons (DDFP, 2022). Nevertheless, the new agricultural strategy "Green Generation" has just strengthened the sector to reach 30 000 ha by 2030. One of the main production basins for oilseed crops in Morocco is the region of Fez-Meknes.
Rapeseed-breeding program conducted in this country relies on hybridization and selection of promising lines, where crossbreeding parents' choice is based on phenotypic evaluation under field conditions. Consequently, the environmental conditions largely affect the selection process. Therefore, the objective of this research is to characterize the genetic diversity among a set of rapeseed varieties, using the ISSR genetic markers, and to identify the level of genetic phylogeographic structure. Knowledge of genetic distances among the genotypes studied and identification of homogeneous genetic pools will be helpful for the ongoing rapeseedbreeding program in the selection of genetically distant and complementary breeding parents.

Plant material
The plant material used in this study consisted of 22 rapeseed varieties of which six are Moroccan while sixteen are introduced from different countries in the world. The Moroccan varieties were developed by the National Institute of Agronomic Research (INRA-Morocco) and registered in the Official Catalogue as high-performing inbred lines and synthetic varieties in terms of seed yield and oil content (Guirrou et al., 2023). Table 1 presents the list of the varieties studied, their origins, their types, their pedigree, and their phenotypic description. Information on pedigree and phenotypic description is only available for the six Moroccan varieties.

DNA isolation
Genomic DNA of each variety was extracted from a sample of 40 mg of lyophilized young leaves using the Cetyl Trimethyl Ammonium Bromide (CTAB) modified method (Saghai-Maroof et al., 1984). The extracted DNA was then quantified using a spectrophotometer (Bio-drop Touch Spectrophotometer, USA).

Amplification of DNA using ISSR markers
A set of 32 ISSR primers was tested on a single genotype and through the PCR-gradient (Eppendorf Mastercycler Gradient, Germany) to adjust the annealing temperature of each primer and to select primers that regenerate very clear and reproducible profiles. Finally, twenty primers were selected due to their molecular profiles with the determination of the optimum hybridization temperature for each primer (Tab. 2).
The mixture for DNA amplification was prepared in a final volume of 25 ml containing 20 ng/ml of DNA, 1x Taq buffer, 1.5 mM MgCl2, 0.2 mM of dNTP, 5 mM of ISSR primer, and 1U of Taq polymerase. The PCR amplifications were carried out in a 96-well thermal cycler (Eppendorf model, Mastercycler gradient) according to the following program: Denaturation step at 94°C for 5 min, followed by 35 cycles with a denaturation step at 94°C for 1 min, hybridization at a primer specific temperature (Tab. 2) for 1 min, and an elongation step at 72°C for 1 min. Amplification is sealed off by an elongation step at 72°C for 10 min. Electrophoresis of PCR products was performed on a 2% agarose gel prepared with a 1x TBE buffer and at 4 V/cm voltage. A 1 kb plus-ladder (invitrogen) served as the size marker. The revelation was carried out using ethidium bromide (0.5 mg/mL) for 30-45 min. Agarose gels are photographed by a UV imaging system.

Scoring data and analysis
The size of the obtained bands was calculated using the Mesurim Pro software v3.4 (Madre, 2013) based on an algorithm produced by the bands of the size marker. The data obtained was transformed into a binary matrix (0-1) with only clear bands being considered.
Genetic relationships among the varieties were studied based on a similarity matrix using all polymorphic bands. The genetic distances were estimated by the Simple Matching coefficient (Sokal and Michener, 1958). The histogram of pairwise comparison among genotypes based on the number of allelic differences was established.
The polymorphism information content (PIC) related to the genetic diversity for each primer used was determined according to Anderson et al. (1993) as follows: where f ij is the frequency of the pattern j in the band i.
Since dominant markers have two models for a group: present and absent. Then, the PIC of each primer was calculated as follows: PICi: The Effective multiple ratio (EMR) was obtained according to Powell et al. (1996) as follows:

EMR ¼ npðnp=nÞ;
where np is the number of polymorphic loci and n is the total number of loci.
Moreover, the Marker index (MI) was estimated according to Chesnokov and Artemyeva (2015) as the product of PIC value and EMR value: The Marker index (MI) is a statistical parameter used to estimate the total utility of the maker system. Script AMaCAID "Accurate Marker Choice for Accession Identification and Discrimination" (Caroli et al., 2011) designed for execution in R program was used to identify the minimum number of markers necessary to distinguish the different varieties studied and to define a set of markers maximizing the number of varieties distinguished.
Finally, a dendrogram was drawn based on the unweighted pair group method with arithmetic averages (UPGMA) algorithm using the NTSYS-pc program version 2.11a (Rohlf, 2002).

Polymorphism and diversity parameters
Among the 32 ISSR primers tested using the annealing temperature gradient and revelation on 2% agarose gel, 20 primers (62.5%) generated readable and reproducible profiles (Fig. 1). The annealing temperatures of the retained primers varied between 43°C and 58°C. In a similar study on genetic characterization of rapeseed genotypes, the hybridization temperature of ISSR markers ranged from 45 to 60°C (Tarıkahya-Hacıo glu, 2016). The size of the bands generated by these 20 primers is highly variable, oscillating between 137 bp for UBC856 and 1329 bp for F4 (Tab. 3). According to Zietkiewicz et al. (1994), the ISSR technique generates amplified DNA fragments ranging from 200 to 2500 bp; however, more recent studies reported DNA fragment sizes ranging from 150 to 1100 bp (Sica et al., 2005), 140 to 1500 bp (Nagaraju et al., 2002), and 250 to 2800 bp (Pradeep et al., 2005).
These ISSR primers revealed a total of 319 markers and a number of bands ranging from 11 bands for the IMA9Z primer to 22 bands generated by F8 primer, with an overall average of 16 fragments per primer (Tab. 3). The total polymorphism rate of the 20 primers is 81.5%. This number reflects the high level of polymorphism among the evaluated varieties revealed by the set of selected primers. This value is very close to that reported by Abdelmigid (2012) (87%) and higher than that reported by Paul et al. (2020) who found a polymorphism percentage of 71.5% in their rapeseed genetic diversity characterization using ISSR markers. When compared with other species, the percentage of polymorphic bands revealed by ISSR primers is higher in Asparagus acutifolius L. (100%) (Sica et al., 2005) and Lupinus spp. (99%) (Talhinhas et al., 2003), and comparable in Oryza sativa (80.9%) (Nagaraju et al., 2002).
The number of ISSR markers generated is positively correlated with the number of primers used. However, this number can be greatly influenced by the plant species and the nature of the migration gel used (Wiesner and Wiesnerová, 2003).
The polymorphism information content (PIC) values indicate the ability of each primer to distinguish among varieties. All the primers have generated very polymorphic profiles, with an index varying between a minimum value of 0.295 for F11 and a maximum value of 0.509 for IMA834-2, with an average of 0.37. This is in agreement with Safari et al. (2013) who found a PIC of 0.346 for RAPD and 0.35 for ISSR markers in their study on rapeseed genetic diversity. However, our values are higher than those of Maras-vanlio glu et al.
(2020) who reported PIC values generated by the ISSR primers varying between 0.14 and 0.26, with an average of 0.18 in their study on rapeseed genetic assessment. However, in our study, most primers have PIC values around 0.4, indicating the high discriminating power of the used primers.
The pairwise comparison of the 22 varieties according to the Simple Matching coefficient showed distances ranging from a minimum of 0.176 for (Seven/Narjisse) to a maximum of 0.456 for (AUP4/Adila), with an average of 0.31. This range of variation is relatively smaller, compared with that reported by Safari et al. (2013) who found distances varying between 0.26 and 0.95. In addition, based on 15 ISSR primers, Abdelmigid (2012) indicated that within 10 accessions of rapeseed from different countries, the highest genetic similarity was 0.72 while the lowest one was 0.41. These differences might be attributed to the nature/type of accessions investigated, the number of these accessions and their multiple geographical origins.
The average similarity among the 22 varieties herein studied is 69% and the pairwise comparison shows that all varieties are genetically distinct by a minimum of 56 markers in the case of (Seven/Narjisse) and a maximum of 145 markers in the case of (AUP4/Adila) (Fig. 2). This clearly indicates the high level of genetic diversity among the varieties studied. Similar previous studies in rapeseed showed an average level of similarity of 67.3% using AFLP markers (Yu et al., 2007) and 64% using RAPD markers (Ana et al., 2009). In another work on canola, the percentage of similarity among 10 genotypes of different geographical origins, ranged from 0.47 to 0.73 using a combination of ISSR and RAPD markers (Abdelmigid, 2012).

Cluster analysis
The dendrogram obtained by analysis of the 319 ISSR markers has allowed dividing the varieties into four distinct groups at a similarity index of 69% (Fig. 3).
The group A contains a mixture of ten Moroccan and introduced varieties. The group B includes all varieties of Pakistani origin. The group C contains the Moroccan varieties Adila and Lila, the Australian varieties Nap 9 and Nap 10, and the German hybrid Traper. However, the variety Alba is very distinct from all the rest of genotypes and consequently classified as a single branch (Group D). Among the 20 primers used, 17 primers were sufficient to distinguish the three groups A, B and C having a number of markers of 160, 127 and 231, respectively. Group D is distinguished with a single genotype. However, no markers are specific to the different groups.
The dendrogram clearly separated the varieties according to their origins, indicating that there was an association between genetic similarity and geographic distance. Similar results were reported by Paul et al., 2020 in their work on rapeseed germplasm characterization through ISSR markers and isozymes markers. However, our study shows that the Moroccan varieties are genetically close to a number of introduced varieties, which would indicate that they might derive from the same pedigree or belong to close genetic backgrounds. This is plausible since rapeseed remains a species characterized by a limited genetic diversity in comparison with other agricultural species (Nabloussi, 2015). As a result, the genetic progress achieved for this crop during the last 15-20 years is also limited. Hence the usefulness and interest of identifying genetically distant genotypes to use as crossing parents to broaden genetic diversity and enhance genetic progress in future selected varieties. This strategy is relevant for rapeseed which is, in fact, a partially allogamous plant, with 1/3 cross-pollination and 2/3 self-pollination (Nabloussi, 2015).
A concrete example that shows how effective the used markers are in structuring genotypes is the composition of group C. It turns out that this group, established by a very small coefficient of similarity, brought together pure lines of

Conclusion
This study has confirmed that ISSR markers perform well for analyzing genetic diversity in rapeseed. The selected primers were found to be effective in discriminating the 22 varieties investigated, revealing a high genetic variability. Eight ISSR primers were identified as a key to rapeseed varietal determination and authentication. There was a phylogeographic structuring the analyzed varieties as their separation was done generally according to their geographical origins. However, we found that some Moroccan varieties are genetically close to a number of introduced varieties, which would indicate that these Moroccan and foreign varieties would have in their pedigree parents with a close genetic background. Our findings provide important clues in understanding the relationship between the rapeseed varieties studied, which will contribute to the success of breeding program by targeting crosses between genotypes belonging to genetically distinct groups and benefiting from heterosis effect. This will enable expanding the genetic variability and developing high-performing rapeseed cultivars for the development of the Moroccan oilseed sector.