First report of genetic relationship and diversity among Moroccan and introduced rapeseed (Brassica napus l.) varieties as revealed by molecular markers

doi:10.21203/rs.3.rs-2129788/v1

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First report of genetic relationship and diversity among Moroccan and introduced rapeseed (Brassica napus l.) varieties as revealed by molecular markers

https://doi.org/10.21203/rs.3.rs-2129788/v1

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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. Breeding is the key to the success of this crop and judicious choice of crossing parents is a prerequisite to the success of the selection program and the development of new varieties that perform better than the old ones. 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 amplified bands varied from 8 to18, with an average of 13 per primer. The diversity index (PIC) 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 coefficient was 0.31, fluctuating 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. Hierarchical classification allowed identifying three groups with some phylogeographic structuring. This is the first 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.

Brassica napus L.

Crossbreeding

Genetic diversity

ISSR markers

Moroccan germplasm

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 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). Rapeseed (Brassica napus L.) is the most important oilseed Brassica species, it’s 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 success in the selection program and the development of new varieties that perform better than the old ones. Traditionally, parents have been selected based on their phenotypic attributes. However, the morphological features do not give the real level of genetic diversity among rapeseed germplasm.

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 hereditary material. Analysis by molecular markers enables choosing parental types for mapping populations, marker-assisted selection, and crossing schemes. Thus, diversity analysis using molecular markers help breeders improve crop species (Paul et al., 2020). ISSRs are generally very effective in genetic diversity studies because of their longer primers compared with RAPD primers (Pradeep Reddy et al., 2002; Tarıkahya-Hacıoğlu, 2016). Likewise, Mahjoob et al. (2016) compared different markers such as ISSR, IRAP, and REMAP for the assessment of the genetic diversity within the Brassicaceae family. They reported that ISSR markers showed to be the most efficient for genetic diversity evaluations. In fact, this technique combines the advantages of the AFLP and the universality of the RAPD (Maraş-vanlioğlu 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. The Rapeseed breeding program conducted in this country relies on hybridization and selection of promising lines based on phenotypic evaluation of parents and segregating generations under field conditions. Consequently, the environmental conditions largely affect the section 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 rapeseed-breeding program in the choice of genetically distant and complementary breeding parents.

2.1. Plant Material

The plant material used in this study consisted of 22 rapeseed varieties, from which six are Moroccan while sixteen are introduced from different countries in the world. Table 1 presents the list of studied varieties, their origins and their types.

Table 1

Origin and type of rapeseed varieties studied.
Varieties	Origin	Type
ADILA	Morocco	Synthetic Variety
LILA	Morocco	Synthetic Variety
MOUFIDA	Morocco	Pure line
NARJISSE	Morocco	Pure line
CZ-H2	Morocco	Pure line
BARAKA	Morocco	Pure line
NAP9	Australia	Pure line
NAP10	Australia	Pure line
AUP1	Pakistan	Pure line
AUP3	Pakistan	Pure line
AUP4	Pakistan	Pure line
AUP13	Pakistan	Pure line
AUP16	Pakistan	Pure line
AUP17	Pakistan	Pure line
ALBA	Unknown	Pure line
KABEL	Spain	Pure line
LUCIA	Spain	Pure line
LYSIDE	Danemark	Pure line
MACRO	Germany	Pure line
SEVEN	Unknown	Pure line
JURA	France	Pure line
TRAPER	Germany	Hybrid Variety

3.2. DNA Isolation

Genomic DNA of each genotype 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).

2.3. Amplification of DNA using ISSR Markers

A set of 32 ISSR primers was tested on a single genotype and through the PCR-gradient to adjust the annealing temperature of each primer. The goal was to adjust the annealing temperature and to select several primers that regenerate very clear and reproducible profiles. Twenty primers were selected due to their molecular profiles with the determination of the optimum hybridization temperature for each primer (Table 2).

Table 2

List of 20 different ISSR primers and annealing temperatures used in the molecular characterization of the 22 rapeseed varieties studied.
ISSR Primers	Type of repetition	Sequence (5'-3')	Annealing temperature (°C)
F1	Di	(CA)₆AT	43
F2	Di	(CA)₆GC	58
F3	Di	(CA)6	43
F4	Tri	(AGC)₄CT	58
F8	Di	(AG)₈CC	56
F9	Di	(AG)₈CG	43
F11	Di	(CA)₈AC	56
F16	Di	(GT)₈CG	58
IMA-5-Z	Di	(CA)₈GT	56
IMA9Z	Di	(GA)₈CG	56
IMA834z	Di	(AG)₈CTT	50
IMA8Z	Di	(GA)₈GT	50
IMA834-2	Di	(AG)₈YT	50
UBC808-2	Di	(AG)₈C	50
UBC810	Di	(GA)₈T	50
UBC850	Di	(GT)₈TYC	50
UBC856	Tri	(GGAGA)3	56
IMA12-2	Di	(CA)₈TC	56
ISS F1	Di	(AG)₈TA	50
ISSR1	Di	(AG)₈CA	56

The mixture for DNA amplification was prepared in a final volume of 25 µl containing 20 ng/µl of DNA, 1x Taq buffer, 1.5mM MgCl2, 0.2 mM of dNTP, 5 µM 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 (Table 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 4V/cm voltage. A 1kb plus-ladder (invitrogen) served as the size marker. The revelation was carried out using ethidium bromide (0.5 µg / ml) for 30–45 min. Agarose gels are photographed by a UV imaging system.

2.4. Scoring Data and analysis

The size of the obtained bands was calculated using the Mesurim Pro software v3.4 (Madre, 1998–2013) 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 genotypes were studied based on a similarity matrix using all polymorphic bands. The genetic distances were calculated using the Clustering Calculator program developed by Brzustowski (2002) through Simple Matching Coefficient. 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 follow.

$${P}{I}{C}{i}= 1-\sum _{{j}}{{f}}_{{i}{j}}^{2}$$

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:

$${P}{I}{C}=\frac{1}{{n}}\sum _{{i}=1}^{{n}}{P}{I}{C}\text{i}$$

The Effective multiple ratio (EMR) was obtained according to Powell et al. (1996) as follow:

EMR = np (np/n)

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.

MI = PIC x EMR.

The Marker index (MI) is a statistical parameter used to estimate the total utility of the maker system.

Finally, a dendrogram was drawn based on the UPGMA algorithm using the NTSYS-pc program version 2.11a (Rohlf, 2000).

3.1. Polymorphism and diversity parameters

The 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. 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-Hacıoğlu, 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 (Table 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 from 250 to 2800 bp (Pradeep et al., 2005).

Table 3

ISSR primers, and amplification results of 20 ISSR primers in the rapeseed genotypes studied.
Primer	AT (°C)	SB	TNB	NPB	NMB	PPB	EMR	MI	PIC
F1	43	184–1122	19	18	1	94.74	17.05	6.41	0.376
F2	58	192–808	14	9	5	64.29	5.79	2.07	0.357
F3	43	270–934	15	11	4	73.33	8.07	3.40	0.422
F4	58	167–1329	20	16	4	80	12.80	4.57	0.357
F8	56	161–1249	22	18	4	81.82	14.73	6.08	0.413
F9	43	237–765	13	9	4	69.23	6.23	2.43	0.39
F11	56	176–718	17	16	1	94.12	15.06	4.44	0.295
F16	58	238–1220	18	14	4	77.78	10.89	3.82	0.351
IMA-5-Z	56	171–966	18	16	2	88.89	14.22	4.72	0.332
IMA9Z	56	185–726	11	11	0	100	11.00	3.70	0.336
IMA834z	50	175–911	16	13	3	81.25	10.56	4.54	0.43
IMA8Z	50	166–935	15	14	1	93.33	13.07	4.90	0.375
IMA834-2	50	175–1122	15	10	5	66.67	6.67	3.39	0.509
UBC808-2	50	193–984	19	11	8	57.89	6.37	2.28	0.358
UBC810	50	190–929	16	13	3	81.25	10.56	3.35	0.317
UBC850	50	265–1311	14	11	3	78.57	8.64	2.78	0.322
UBC856	56	137–985	15	14	1	93.33	13.07	4.48	0.343
IMA12-2	56	210–1085	16	15	1	93.75	14.06	5.23	0.372
ISS F1	50	299–1213	14	13	1	92.86	12.07	3.69	0.306
ISSR1	56	183–717	12	8	4	66.67	5.33	2.13	0.399
AT: Annealing temperatures; SB: Size of bands; TNB; Total number of bands; NPB: Number of polymorphic bands; NMB: Number of monomorphic bands; PPB: Percentage of polymorphic bands; EMR: Effective multiple ratio; MI: Marker index; PIC: polymorphic information content.

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 (Fig. 1). The total polymorphism rate of the 20 primers is 81.5%. This number reflects the high level of polymorphism among the varieties revealed by the set of selected primers. This value is very close to that reported by Abdelmigid et al. (2012) (87%) and higher than that that reported by Paul et al. (2020) who found a polymorphism percentage 71.5% in the characterization of rapeseed genotypes by ISSR markers.

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 (Nagarju et al., 2002; Wiesner and Wiesnerová, 2003).

When compared with other species, the percentage of polymorphic bands revealed by ISSR primers is higher in Asparagus acutifolius L. (100%) (Sica et al., 205), and Lupinus spp. (99%) (Talhinhas et al., 2003), comparable in Oryza sativa (80.9%) (Nagarju et al., 2002), and lower in Bombyx mori (64%) (Pradeep et al., 2005).

The polymorphism information content (PIC) values indicate the ability of each primer to distinguish among genotypes. 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 Maraş-vanlioğlu 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 genetic characterization of rapeseed genotypes. 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 small 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 are attributed to the type of germplasms investigated, the number of varieties analyzed and their multiple geographical origins.

The average similarity between the 22 varieties of rapeseed studied is 69% and the pairwise comparison shows that all varieties are genetically distinct by a minimum of 56 markers for the case of (Seven / Narjisse) and a maximum of 145 markers for the case of (AUP4 / Adila) (Fig. 2). This clearly indicates the high level of genetic diversity among the varieties studied.

3.2. 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 other genotypes and consequently classified as a single branch (Group D).

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 study on rapeseed germplasm characterization through ISSR markers and isozymes markers. However, the 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 the interest of identifying genetically distant genotypes to use as crossing parents for the widening of genetic diversity and the reinforcement of genetic progress in future selected varieties.

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 Australian origin, Moroccan synthetic varieties and a German hybrid variety.

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. It was shown that there was a phylogeographic structuring the analyzed accessions 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 pedigrees parents with a close genetic background. The results of our study 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 germplasm.

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No competing interests reported.

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First report of genetic relationship and diversity among Moroccan and introduced rapeseed (Brassica napus l.) varieties as revealed by molecular markers

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Plant Material

3.2. DNA Isolation

2.3. Amplification of DNA using ISSR Markers

2.4. Scoring Data and analysis

3. Results And Discussion

3.1. Polymorphism and diversity parameters

3.2. Cluster analysis

4. Conclusion

References

Additional Declarations

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