Assessment of genetic diversity of some Brassica napus L. cultivars revealed by molecular markers and phenotypic evaluation

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1 Indian Journal of Biotechnology Vol 15, October 2016, pp Assessment of genetic diversity of some Brassica napus L. cultivars revealed by molecular markers and phenotypic evaluation Constantin Leonte and Madalina C Arsene* Ion Ionescu de la Brad University of Agricultural Sciences and Veterinary Medicine Mihail Sadoveanu Alley no.-3, Iaşi , Romania. Received 17 April 2015; revised 20 September 2015; accepted 28 September 2015 The genetic diversity of 50 oilseed rape cultivars was studied using phenotypic evaluation and molecular markers. For the phenotypic evaluation, seven morphological traits were analysed. Euclidian distances among mean values of the obtained data, after the morphological evaluation of genotypes, were used to form a matrix for the construction of a dendrogram. For the molecular studies, RAPD and SSR markers were employed. In the RAPD analysis, 20 decamer primers were used, which gave 301 fragments, where 215 were polymorphic. The SSR analysis used 55 primers, which gave 123 polymorphic fragments. In order to determine the genetic diversity between the oilseed rape cultivars, both RAPD and SSR data were used. With this data, genetic similarity (GS) was estimated, which led to generate two similarity matrices. These two matrices were correlated using the Mantel test and the final matrice was used to design an UPGMA dendrogram using cluster analysis. Among the studied cultivars, significant genetic variability was obtained both at the morphological and molecular level. The obtained dendrogram for the morphological traits and for the RAPD and SSR results were compared. In both the cases, the genotypes were grouped after the geographical origin. Keywords: Cluster analysis, dendrogram, RAPD, SSR, traits, UPGMA Introduction Brassica napus L. is a young species that originated by means of an interspecific hybridization between B. rapa L. and B. oleracea L. 1. Now-a-days, due to intensive cultivation for its seeds and oil quality traits, the spring and winter oilseed forms represent one of the most important sources of vegetable oil worldwide, not only for human nutrition but also for livestock feed and industry 2,3. The basis for the breeding programmes of the oilseed rape relies on the knowledge of the characteristics of the genotypes and their genetic relationship 4. Further, the genetic basis for the elite selection in the breeding programmes is based on an intensive selection of the oilseed rape germplasm considering the main agronomic traits and specific oil and seed quality traits 5. For this reason, breeders collect material from all over the world and use them in their studies. The genetic diversity among individuals can be determined in different ways, such as, by use of morphological, biochemical and molecular markers 6. In order to determine the genetic diversity of the *Author for correspondence Tel: arsenemadalina83@gmail.com Brassica species, many molecular markers are used, such as, restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphism (RAPD), simple sequence repeats (SSR), etc In order to obtain a diverse gene pool accession of oilseed rape material, 50 winter oilseed rape cultivars originating from different countries were obtained. These lines were not characterized earlier either genetically or morphologically. The aim of the present study was to estimate the genetic relationship between 50 oilseed rape cultivars using RAPD and SSR markers, and a set of morphological traits. Material and Methods Plant Material Fifty oilseed rape (B. napus) cultivars provided by the Centre for Genetic Resources, Netherlands were tested in the present study. The names of the cultivars and their country of origin are shown in Table 1. For the phenotypic evaluation, a 3-year experiment was conducted at the Agricultural Research Station Secuieni, Neamţ using a randomized block design with three replications. Each genotype was sown in a 3 m length 5-row plot with a spacing of cm 2 (row and plant). During the growing season, common

2 LEONTE & ARSENE: GENETIC DIVERSITY IN B. NAPUS CULTIVARS 569 cultivation practices were applied. For the morphological traits analysis, 10 plants on each cultivar were analyzed once they reached the maturation stage. The observations were recorded on seven morphological traits, viz., plant height (PH), the number of primary branches (NB), the number of pods per plant (NPP), pod length (PL), number of seed per pod (NSP), the 1000 grain weight (GW) and yield per plant (YP). DNA Extraction and RAPD Analysis The leaf samples were collected from 10 individuals of each cultivar and immediately frozen in liquid nitrogen. DNA was extracted using the CTAB procedure, modified according to Doyle and Doyle protocol 11. DNA content was measured using a Nano Drop spectrophotometer. Based on the data, DNA was diluted to a concentration of 5 ng/µl for RAPDs and 20 ng/µl for SSRs. Polymerase chain reaction (PCR) mixture (25 µl) for RAPDs contained: 5 ng genomic DNA, 10 µm of each dntp, 25 mm MgCl 2, 5 pmol/µl decamer primer (ROTH) and 0.1 Units Taq DNA-polymerase (Go Taq Polymerase, Promega) and 10 respective reaction buffer. Amplification was performed in a Palm Cycler Corbett. PCR conditions were: 94 C for 4 min, followed by 45 cycles consisting of 1 min at Table 1 The oilseed cultivars used in the study No. Accessions Country of origin No. Accessions Country of origin 1 Diadem Germany 26 Lecor - 2 Diamant Germany 27 Ledos Germany 3 Doral Germany 28 Lesira Germany 4 Doublol Germany 29 Lester Germany 5 Eka Germany 30 Libelle Germany 6 Elena Germany 31 Liberator Germany 7 Elvira Germany 32 Kombainer Ukraine 8 Erra Germany 33 Liborius Germany 9 Enrol France 34 Librador Germany 10 Falcon Germany 35 Libraska Germany 11 Fertodi Hungary 36 Libravo Germany 12 Fiona Germany 37 Cascade USA 13 Gesunder Germany 38 Bridger USA 14 Girita Germany 39 Kromerska Czech Republic 15 Glacier Germany 40 Slapska Czech Republic 16 Gundula Germany 41 Mestnji Russia 17 Hambourg France 42 Trebieckska Krajova Czech Republic 18 Hambourger - 43 Niemierczanski Russia 19 Heimer Sweden 44 Jet Neuf France 20 Herkules Sweden 45 Rafal France 21 Hunnia - 46 Expander Germany 22 Jade - 47 Mansholts Hamburger Netherlands 23 Janetzkis Austria 48 Primor Netherlands 24 Jupiter Sweden 49 R-33 France 25 Kurander Germany 50 Rapol France 94 C, 1 min at 36 C, and 2 min at 72 C. The amplified products were separated by electrophoresis in 2% agarose gel and stained with ethidium bromide (0.5 µl/ml). SSR Analysis For the SSR analysis, 50 SSR primers were selected, which had previously been used and reported to reveal polymorphism in several studies on B. napus 10,12,13. The PCR reactions were carried out in 10 µl containing 20 ng of DNA template, 0.75 pmol of each primer, 0.2 mm dntp mix, 1 mm MgCl 2, 10 PCR reaction buffer and 5 unit of Taq DNA polymerase (Qiagen). Amplifications were performed using a standard amplification cycle in a GeneAmp PCR System 9700 thermal cycler, and SSR polymorphisms were separated and visualized using a LI-COR GeneReadir 4200 (MWG Biotech, Ebersberg). To reduce primer-labeling cost, PCR products were labeled with the M13-tailing technique. In this method, the fluorescently labeled universal M13 primer 5 -AGGGTTTTCCCAGTCACGACGTT- 3 was added to the PCR reaction, and the forward primer of each SSR is appended with the sequence 5 -TTTCCCAGTCACGACGTT-3. The amplification was performed after a touch-down PCR cycle was modified by the procedure described by Xu et al 14 as

3 570 INDIAN J BIOTECHNOL, OCTOBER 2016 follows: an initial denaturation was performed at 95 C for 2 min, followed by five cycles of denaturation for 45 sec at 95 C, annealing for 5 min beginning at 68 C and decreasing by 2 C in each subsequent cycle, and extension for 1 min at 72 C. Then five cycles were performed with 45 sec denaturation at 95 C, 1 min annealing beginning at 58 C and decreasing 2 C in each subsequent cycle, and 1 min of extension at 72 C. The PCR was then completed with an additional 27 cycles of 45 sec denaturation at 94 C, 2 min of annealing at 47 C, and 30 sec of extension at 72 C, with a final extension at 72 C for 10 min. Statistical Analysis The analysis of the morphological traits of the studied 50 cultivars was performed using factorial ANOVA and significance of differences was determined using LSD test. The obtained data after morphological evaluation of 50 cultivars were used to form a matrix using Euclidian distances among mean values of the genotypes for the construction of a dendrogram 15. The statistical calculation was done by the SYSTAT ver. 13. RAPD amplification products were scored using the software RFLP scan 2.1, assuming that each band of different size reflects a single locus. Only unambiguously scored fragments were used for the estimation of genetic similarity according to Nei and Li 16. The visualization of the SSR amplified fragments was made using Saga generation software version 1. Each primer was scored manually for the presence of the band using 1 and 0 for the absence of band. Based on the molecular data obtained with the SSR and RAPD analysis UPGMA (unweighted pair group method using arithmetic averages) clustering was carried out using the software package NTSYS-pc Results and Discussion The 3-year average values of the data collected on seven morphological traits studied in the 50 oilseed rape samples are presented in Table 2. The study shows significant differences between the cultivars regarding the traits studied. The most important agronomical trait, seed yield per plant, showed a range of 15.1 (Janetkis) to g (Libraska).Using Euclidian distances among mean values of the morphological traits and the cluster analysis, a dendrogram was constructed (Fig. 1). In an earlier study, Marjanovic-Jeromela et al 5 used a similar method for determining the genetic diversity of some oilseed rape genotypes. The obtained dendrogram showed a high phenotypic variation in the studied cultivars. Based on the morphological traits, the studied cultivars are grouped into three clusters. The first cluster (A) comprised of 25 cultivars with different origins, such as, France, Netherlands, Russia and Germany. The genotypes from this cluster represent small to medium size plants, with heights ranging 58.6 (Mansholts Hambourger) to cm (Elena), small to good seed yield per plant with recorded values of 15.1 (Janetkis) to 30.2 g (Primor). The 1000 grain wt of the cultivars from this cluster reached values ranging 3.2 (Janetkis) to 5.0 g (Elena), while the number of siliques ranged (Elena) to (Niemierczanski). The second cluster (B) had 21 genotypes, most of them originating from Germany. The genotypes from this cluster were characterized by average values of the studied traits. The seed yield per plant of the genotypes from this cluster ranged 35.8 (Kromeska) to 8.5 g (Libravo), the 1000 wt grain ranged from 5.1 (Jet Neuf) to 5.9 g (Gesunder) and the number of pods per plant ranged from (Kombainer) to 567 (Fiona). The last cluster (C) comprised five cultivars originating from Germany. These cultivars were characterized by the best value of the seed yield, 1000 grain wt and number of pods per plant. It was observed that cultivar Libraska grouped separately from the other cultivars. This cultivar reached values of g of the seed yield, for the number of silique and 4.89 g for the 1000 grain wt. Thus the obtained results demonstrate that the use of cluster analysis is a good tool to determine the phenotypic differences among the cultivars, which agrees with the results from other studies 6,19,20. Genetic diversity between cultivars has proved to be a useful tool for breeders in their attempt to identifying the cultivars with special traits that can be further used to improve the agronomic characteristics 21. Within national breeding programmes, the study of genetic relationships (diversity) is important for the selection of suitable diverse parents to obtain heterotic hybrids, predict progeny performance, conserve and characterize the used germplasm. In addition, knowledge about relatedness between parents in canola breeding programmes could be used to avoid the possibility of elite germplasm to become genetically uniform, thereby endangering long-term selection gains and conserved genetic resources. In order to determine the genetic relationships between the cultivars studied, SSR and RAPD analyses

4 LEONTE & ARSENE: GENETIC DIVERSITY IN B. NAPUS CULTIVARS 571 Table 2 Three-year average values for the morphological traits in 50 rapeseed genotypes No. Cultivar PH (cm) NB NPP PL (cm) NSP GW (g) YP (g) 1 Diadem * 8.2 * 25.8 * Diamant * * 7.2 * Doral * * 7.3 * 28.7 * Doublol * Eka * * 8.2 * 32.8 * Elena * * 8.3 * 28.4 * Elvira * Erra * 5.67* * Enrol 73.24* * * 10 Falcon 80.76* * 7.6 * * Fertodi * 6.6 * 24.7 * Fiona 95.90* * * 13 Gesunder * Girita * 7.7 * Glacier 98.05* * 8.6 * 36.2 * Gundula * * 7.4 * 27.0 * Hambourg 96.12* * * Hambourger * * * Heimer * 7.0 * * 20 Herkules 95.82* * Hunnia 88.25* * * 22 Jade * * 7.2 * * 23 Janetzkis 77.76* * 5.8 * * Jupiter 86.27* * * Kurander * 12.33* * 6.1 * Lecor * * * * 27 Ledos * * * * 28 Lesira * * 7.6 * * 53.3 * 29 Lester * * 6.9 * Libelle * * 6.2 * * 31 Liberator * * 8.0 * 35.8 * 3.95* Kombainer * * * * 33 Liborius * * * * 34 Librador * * 7.6 * 28.6 * Libraska * * 8.0 * 31.2 * * 36 Libravo * * 8.6 * 28.7 * 5.85* 83.5 * 37 Cascade * 5.00* * Bridger 98.90* * 6.3 * * Kromerska * * 6.9 * Slapska * * 6.2 * * Mestnji * * * Trebieckska Krajova * * 6.5 * 27.0 * Niemierczanski * 11.33* * * Jet Neuf * * * * 45 Rafal * * 7.15 * * Expander * * 5.12 * * 4.11* 44.3 * 47 Mansholts Hamburger 59.99* * * Primor 81.86* * 7.2 * * R * 12.00* * 7.3 * * 50 Rapol 88.73* * Arithmetic Mean Standard Deviation Coefficient of Variation PH, Plant height; NPP, No. of pods per plant; NB, No. of primary branches; GW, 1000 wt grain; PL, Pod length; NSP, No. of seeds per pod; YP, Yield per plant *Significant at 0.05 probability level

5 572 INDIAN J BIOTECHNOL, OCTOBER 2016 Fig. 1 A dendrogram constructed using cluster analysis based on Euclidian distances among mean values of the morphological traits of 50 oilseed rape cultivars. were performed. For the RAPD analysis, 20 decamer primers were used, which led to the amplification of 301 scorable fragments ranging 288 to 961 bp, in which 215 were polymorphic. The level of polymorphism ranged 29 to 90% and the number of polymorphic fragments per marker ranged 5 to 18. For the SSR analysis, 52 markers gave 123 polymorphic fragments ranging 80 to 340 bp. Thus the results from the present study demonstrate the suitability of RAPD and SSR techniques for analyzing the genetic diversity between B. napus genotypes. The high level of polymorphism indicates a high genetic variability of the analyzed cultivars that, in this case, can be explained by means of the different genetic inheritance and origin. The genetic diversity from oilseed rape germplasm was described earlier in different studies, where a 76, 14 and 6% level of polymorphism was reported 7,22,23. With both RAPD and SSR data, the genetic similarity (GS) between the oilseed rape cultivars was estimated, which generated two similarity matrices. These two matrices were correlated using the Mantel test and the final matrix was used to design an UPGMA dendrogram using cluster analysis 18. The UPGMA dendrogram representing the genetic similarity among the studied accessions is shown in Fig. 2. As expected, the cultivars were grouped according to their origin into two major clusters (cluster A & B). The first cluster grouped 26 oilseed

6 LEONTE & ARSENE: GENETIC DIVERSITY IN B. NAPUS CULTIVARS 573 Fig. 2 An UPGMA dendrogram constructed using cluster analysis based on RAPD and SSR data on genetic diversity between 50 oilseed rape cultivars. rape cultivars and had five subclusters. In this cluster, 15 cultivars originated from Germany, 2 from France, 3 from Sweden, 1 from Austria and 4 were of unknown origin. The first subcluster A1, grouped 12 genotypes. It was observed that the cultivars Gundula originating from Germany and Hambourg from France showed identical SSR and RAPD fingerprints, suggesting a very close pedigree relationship. The Doral and Girita, originating from Germany, grouped together had a similarity coefficient of 0.77, suggesting that they are interrelated. The second subcluster, A2 comprised of 5 genotypes. In this subcluster, it was observed that Heimer and Herkules both from Sweden grouped together with the same similarity coefficient of The subcluster A3 had only one cultivar Fiona and thus prove to be the most genetically diverse in this cluster. The subcluster A4 grouped 3 genotypes, Jupiter, Kurander and Lecor. Jupiter originating from Sweden is genetically different compared to Kurander, while Lecor had the same similarity coefficient. The last subcluster A5 was comprised of four genotypes. The genotypes Elvira and Gesunder grouped separately in the last subcluster showing a similarity coefficient of 0.71 and 0.67, which demonstrates that they are not genetically related. It was observed that all the genotypes in this cluster are from Germany. The second cluster comprised of 24 cultivars and also had five sub-clusters. Within this cluster, 10 cultivars are from Germany, 4 from France, 3 from the Czech Republic, 2 are from Russia, 2 from the USA and 2 from the Netherlands. It was observed that, in the first sub-cluster B1, most of the genotypes are from Germany, such as, Ledos, Lesira, Librador, Libraska and Liborius. Ledos and Lesira shared the same similarity coefficient of An interesting fact was that the cultivars form Russia and Czech Republic grouped together in a separate branch in this subcluster. Slapska and Mestnji had the same similarity coefficient but one originated from Russia (Mestnji) and the other from Czech Republic, demonstrating a genetic similarity between them. In the second subcluster B2, it was observed that cultivars Jet Neuf and Rapol, both from France,

7 574 INDIAN J BIOTECHNOL, OCTOBER 2016 grouped together and are genetically closer with a similarity coefficient of This subcluster also had Primor from Netherlands and Expander from Germany. The third subcluster B3 grouped the genotypes Libravo from Germany together with Cascade and Briger originating from USA. The cultivar Liberator from Germany grouped alone in the fourth subcluster B4 and is the most different genotype not sharing the similarity coefficient to any other cultivar. The last subcluster B5 grouped the most diverse genotypes Lester and Libelle, both from Germany, with a similarity coefficient of 0.70, while Kromeska from Czech Republic is genetically different from theses two. In the present study, the similarity index for the 50 studied oilseed rape cultivars oscillated from 0.63 to Similar results were also obtained by Hasan et al 7 in determining the genetic relationship between some winter and spring oilseed rape varieties by means of the SSR technique. The similarity index in this case was 0.38 to Analyzing the obtained dendrogram, it was observed that most of the cultivars grouped according to their origin. Similar observation was those obtained by Yuan et al 24 while studying the genetic diversity of some oilseed rape cultivars originating from different countries. Using the AFLP and SSR techniques, Seys et al 25 and Hasan et al 10 analyzed a large number of genotypes and constructed dendrograms showing their distribution in clusters based on their type (spring & winter), geographic origin and pedigree. Similar results were also obtained in other species of Brassica 26,27 and oilseed rape cultivars 28 originating from China, Great Britain and France. The diversity of the 50 oilseed rape genotypes identified in this study represents a useful resource for improving the heterotic potential in winter oilseed rape cultivars. 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