In this study we have analyzed the genetic diversity and the relationships among 32 cultivars of oilseed rape (Brassica
napus) using quantitative analysis and random amplified polymorphic DNA (RAPD) markers. For this purpose we
analyzed four morphological traits (plant height, branch number, pod number and mean number of seeds per pod) at 32
oilseed rape cultivars provided from Center for Genetic Resources Netherlands (CGN). RAPD analysis was perform
with 8 primers chosen after a previous screening. Significant genetic variability among those 32 cultivars was obtained
both at the morphological and molecular level. We obtained a dendrogram for the morphological traits and a
dendrogram for RAPD analysis and we compared them

Burlacu Arsene, Mădălina-Cristina

Calistru, Anca-Elena

Leonte, Constantin

ALSE Repository

Lucrări Ştiinţifice – vol. 54, Nr.1/2011, seria Agronomie      EVALUATION OF THE GENETIC DIVERSITY AMONG SOME OILSEED RAPE BRASSICA NAPUS CULTIVARS REVEALED BY RAPD MARKERS COMPARED WITH MORPHOLOGICAL TRAITS EVALUATION   Mădălina Cristina BURLACU (ARSENE)1, Anca-Elena CALISTRU1,  Constantin LEONTE1 e-mail: burlacucmadalina@yahoo.com   Abstract  In this study we have analyzed the genetic diversity and the relationships among 32 cultivars of oilseed rape (Brassica napus) using quantitative analysis and random amplified polymorphic DNA (RAPD) markers. For this purpose we analyzed four morphological traits (plant height, branch number, pod number and mean number of seeds per pod) at 32oilseed rape cultivars provided from Center for Genetic Resources Netherlands (CGN). RAPD analysis was performwith 8 primers chosen after a previous screening. Significant genetic variability among those 32 cultivars was obtainedboth at the morphological and molecular level. We obtained a dendrogram for the morphological traits and adendrogram for RAPD analysis and we compared them.  Key words: RAPD analysis, dendrogram, genetic similarity                                                    1 University of Agricultural Sciences and Veterinary Medicine from Iaşi Oilseed rape is consider to be one of the most important oil crop worldwide because is a high quality source of vegetable oil for the food industry and suppliers protein to the animal feed market. It is also seen as a key crop for raw material supply in the biodiesel industry (Mabel and Kamundia, 2007). In the breeding process, the quality and production had a significant improvement as well as utilization of rapeseed oil in human nutrition.  The are many techniques available for evaluation of crop genetic variability, such as morphological, biochemical and molecular markers (Marjanovic et al, 2009). Molecular (DNA) markers technique is the most used method for the evaluation of genetic variability because it have many advantages over other techniques (independent of environment and plant growth stage, unlimited number, etc) and the method is more precise (Prasad et al, 2000; Kondic-Spika et al, 2008). In order to estimate the genetic variation among the diverse group of important crops in Brassica genus it have been used a variety of molecular markers such as: Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length polymorphism (AFLP), Random Amplified Length Polymorphism (RAPD) , etc (Hallden et al, 1994;, Diers et al., 1996).  The present study was to estimate the genetic diversity among some oilseed rape cultivars based on morphological and molecular characterization. For this purpose, 32 oilseed rape cultivars were analyzed and the results of genetic distances estimated by morphological and molecular evaluations were compared.   MATERIAL AND METHOD  Plant material: the plant material for this study comprised 32 genotypes of rapeseed cultivars proceeded from the Centre for Genetic Resources Netherlands. The plant were sown in the field in the year 2009 in order to obtain the morphological data and to collect the fresh tissue material for DNA extraction. Each genotype was sown in the field at 50 cm distance between rows and 50 plant on each row. Measurements and counts were made at each genotype and comprised 20 plants per genotype. The following morphological trait were analyzed: plant height, number of branches per plant, number of pods per plant, mean number of seed per pod.  The average values of the morphological data are presented in table 1.  RAPD analysis In order to estimate the genetic similarity above the 32 oilseed rape genotypes, a number of 8 RAPD primers (tab. 2) were used to see the DNA polymorphism in these genotypes. DNA extraction: Leaf samples were taken from all plants of each cultivar, bulked and immediately frozen in liquid nitrogen.  Universitatea de Ştiinţe Agricole şi Medicină Veterinară Iaşi  Table 1 The average of the values for morphological traits at 32 rapeseed genotypes Nr.  Crt Genotype Country of origin Plant high Branch number No.of pods per plant No. of seeds per pod1 Matador Sweden 134 9 351 25 2 Mirander unknown 120 8 289 23 3 Niederarnbacher Germany 111 5 248 20 4 Norli Germany 117 7 342 28 5 Octavia unknown 124 10 485 30 6 Olimpiade Italy 111 4 218 18 7 Olymp Germany 110 8 385 22 8 Panter Sweden 101 5 256 21 9 Perle Germany 126 10 498 29 10 Andol France 98 11 255 25 11 Arabella Germany 116 11 415 28 12 Bienvenu France 98 6 341 31 13 Brilland Pollen 132 11 265 26 14 Bristol France 106 12 239 23 15 Buko unknown 104 10 377 32 16 Capricorn Great Britain 110 10 412 33 17 Cobra Germany 97 7 252 20 18 Collo Germany 128 10 318 27 19 Planet Germany 107 6 255 24 20 Prominj Former-URSS 125 6 232 22 21 Ridana Germany 125 14 378 35 22 Samourai France 101 7 219 18 23 Score Great Britain 108 7 252 23 24 Silesia Former- Czechoslovakia 106 10 362 36 25 Silex Germany 119 8 285 29 26 Silvia Germany 137 10 468 38 27 Sollux Germany 120 8 378 36 28 Susana Germany 118 7 391 33 29 Tamara Germany 116 6 362 31 30 Tapidor France 106 10 421 39 31 Tor Sweden 120 9 374 25 32 Veronika Germany 121 8 503 37 DNA was extracted using the CTAB procedure modified according to Doyle and Doyle (1987). DNA content was measured using a Nano Drop 3300 florospectrophotometer. Based on these data DNA was diluted to a concentration of 5ng/µl for RAPDs and 25ng/ µl  for RAPD. Polymerase chain reaction (PCR) mixture (20 µl) for RAPDs contained 5 ng genomic DNA, 10 µM of each dNTP, 25 mM MgCl2, 5pmol/ μl decamer primer (ROTH) and 0,1 Units Taq DNA-polymerase (Go Taq Polymerase – Promega ) and 10X 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 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 strained with Ethidium bromide (0,5 µl/ml). Statistical analysis With the values obtained we formed a matrix of Euclidian distances among the mean values of the genotypes for the construction of a dendrogram (Fox and Rosielle, 1982). The statistical calculations were done by the System of Statistic SYSTAT 13, software modules STATS and CLUSTER.  RAPD amplification products were scored using the software RFLPscan 2.1 assuming that each band of different size reflect a single locus.  Only unambiguously scored fragments were used for the estimation of genetic similarity according to Nei and Li (1979). Based on these data UPGMA-clustering (unweighted pair group method using arithmetic averages) was carried out using the software package NTSYS-pc 2.1 (Rohlf 1992). Table 2 Primers used for generating RAPDs in Brassica napus  genotypes No. Primer’s name Sequence (5’-3’)1. ROTH A08 GTG ACG TAG G 2. ROTH A10 GTG ATC GCA G 3. ROTH A13 CAG CAC CCA C 4. ROTH B04 GGA CTG GAG T 5. ROTH B07 GGT GAC GCA G 6. ROTH B10 CTG CTG GGA C 7. ROTH B11 GTA GAC CCG T 8. ROTH B18 CCA CAG CAG T  RESULTS AND DISSCUSIONS  Based on the morphological traits measured on each genotype, we constructed a dendrogram (fig.1) that divided the genotype into three clusters (cluster A, cluster B, cluster C). Looking at the morphological dendrogram it can be observed that the cultivars were grouped in three clusters: cluster A, B and C. The biggest group is Cluster B that includes 15 genotypes originating from different counties, while cluster B comprised 13 genotypes and Cluster C only four.   Lucrări Ştiinţifice – vol. 54, Nr.1/2011, seria Agronomie     Figure 1 Dendrogram constructed for 32 oilseed rape cultivars based on morphological traits  RAPD analysis lead to the amplification of 166 scorable fragments ranging from 101 to 1310 bp resulting in 139 polymorphic bands. The number of amplicons ranged between 13 (ROTH A13) to 27 (ROTH A10) and presented molecular weight between 100-1116 bp. The distance matrices were made based on the two similarity coefficients according to Nei and Li (1979). Similarly matrices were used to cluster analysis using UPGMA method and a dendrogram was constructed. Results of UPGMA cluster analysis is presented in figure 2.  Figure 2 Dendrogram illustrating genetic relationships between 32 rapeseed cultivars generated by UPGMA cluster analysis of polymorphic RAPD fragments  Looking at the UPGMA dendrogram we can observe that there are  five major clusters (cluster A, cluster B, cluster C, cluster D and cluster E). The biggest group (cluster A) contain 20 Brassica napus  cultivars and it is divided in five sub clusters. In all of these sub clusters we can observe that most of the grouped cultivars are originating from the same country and made from the same breeder and this demonstrate a uniformity of the germplasm. In this group we can observe that the cultivars „Mirander” and „Niederarnbacher”are the most related cultivars and we can suppose that between these two cultivars is a very close similarity. These two are linked to genotype “Matador” meaning that the three cultivars may have common ancestry.  The other clusters are smaller Cluster B having four genotypes three of them originating from Germany, cluster D five genotypes and Cluster E one genotype.  Most of the cultivars ere grouped after the country of origin. The most different cultivar seems to be “Buko” that is alone in cluster E which means that had different ancestry than the other cultivars.  Looking at the two dendrograms based on the morphological traits and on the molecular markers we can observe that both dendrograms shows a genetic variation among the cultivars. Both RAPD and morphological characters were sufficient to assess variability among 32 oilseed rape cultivars. No correlation was found between the matrices obtained by molecular and morphological traits. Despite the low correlation between those two dendrograms it can be observed that some cultivars were grouped in the same cluster in both dendrograms. For example the cultivars “Tapidor”, “Tor’’ and” Veronica” appear to be grouped in the same cluster in both dendrograms. The same results were obtained by other authors in different studies (Maric et al.,2004). The low correlation between RAPD dendrogram and morphological dendrogram had been also reported in other studies in European barley varieties (Schut et al, 1997), synthetic hexaploid wheat and their parents (Lage et al., 2003) and Squash (Ferriol et al., 2004). Normally until now, germplasm has been classified on the basis of morphological and agronomical traits, but recently the use of molecular markers to study diversity and characterization of the plants has become more common. This differences between the two dendrograms can be due to the fact that the morphological traits can be influenced by many factors such as: environmental conditions, the sample size, the time of making the measures, etc.   Universitatea de Ştiinţe Agricole şi Medicină Veterinară Iaşi  CONCLUSIONS  This study has shown that significant genetic variability was found among the genotypes on both morphological and molecular analysis, but the molecular analysis is more precise, due to the fact that the genotype is not influenced by the environmental conditions. Also, this study shows that PCR-based techniques such RAPD can be successfully used for detecting genetic variability in rapeseed. This study demonstrated that for determining the genetic variability among some cultivars, the molecular markers technique is more precise than the morphological traits.   ACNOWLEDGEMENTS  The authors would most respectfully like to acknowledge the financial support from the Romanian Ministry of Education, Research, Youth and Sport (Project ID 1230, contract no 1079/2009).  BIBLIOGRAPHY  Diers, BW, McVetty, PBE, Osborn, TC,1996 - Relationship between heterosis and genetic distance based on restriction fragment length polymorphism markers in oilseed rape (Brassica napus L.), Crop Sciences 36: 79-83. Doyle, J. J. and Doyle, J. L., 1990 - Isolation of plant DNA from fresh tissue,  Focus, 12, 13—15. Ferrio, l M., Pico, B., Fernandez de, CP, Nuez, F, 2004 - Molecular diversity of germplasm collection of Squash (Cucurbita moschata) determined by SRAP and AFLP markers, Crop Scences , 44:653-644. Fox, PN, Rosielle, AA, 1982 - Reducing the influence of environmental main-effects on pattern analysis of plant breeeding environments, Euphytica, 31: 645-656. Halldén, C, Nilsson, NO, Rading, IM, Säll, T, 1994 - Evaluation of RFLP and RAPD markers in comparison of Brassica napus breeding lines, Theor. Appl. Genet. 88: 123-128. Kondic-Spika, A, Kobiljski, B, Dencic, S, Mladenov, N, Hristov, N, Kacavenda, D, Brbaklic, Lj, 2008 - DNA Fingerprinting of Wheat (Triticum aestivum L.) Varieties Using Microsatellite Markers, In: Proceedings of Int. Conference: Conventional and Molecular Breeding of Field and Vegetable Crops (Breeding 08), 24-27 November, Novi Sad, Serbia, pp. 149-152 (CD Ed.). Lage, j, Warburton, ML, Crossa, J, Skovmad, B, Andersen SB, 2003 - Assessment of genetic diversity in synthetic hexaploid wheat ann their Triticum dicoccum and Aegilops tauschii parents using AFLPs and agronomic traits, Euphytica 134:305-317.  Marijanovic, J.A., Kondic, S.A., Saftic-Pankovic, D, Marinkovic, R. and Hristov, N., 2009- Phenotypic and molecular evaluation of genetic diversity of rapeseed (Brassica napus L.) genotypes, African Journal of Biotechnology, 8, 4835-4844. Maric, S., Bolaric, S., Martincic, J., Pejic, I. and. Kozumplik, V, 2004 - Genetic diversity of hexaploid wheat cultivars estimated by RAPD markers, morphological traits and coefficient of parentage, Plant Breeding, 123:366-369. Mehdi – Younessi, H., Ali, Izadi, Nejat, P., Mohamad, T., Abbas, M., 2011 - Phenotypic and molecular analysis of soybean mutant lines through random amplified polymorphic DNA (RAPD) marker and some morphological traits, African Journal of Agricultural Research Vol 6(7), 1779-1785.  Nei, M. and Li, W., 1979 - Mathematical model for studying genetic variation in terms of restriction endonuclease, Proceedings of the National Academy of Sciences USA, 79, 5269-5273 Prasad, M, Varshnez, RK, Roy, JK, Balyan, HS, Gupta, PK, 2000 - The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat, Theor. Appl. Genet. 100: 584-592. Rohlf, F.J., 1992 - NTSYS-pc. Numerical taxonomy and multivariate analysis system, version 1.70, Applied Biostatistic, New York. Schut, JW, Qi, X, Stam, P, 1997 - Association between relationships mesaures based on AFLP markers, pedigree data and morphological traits in barley. Theor. Appl. Genet. 95: 1162-1168.   

Evaluation of the genetic diversity among some oilseed rape Brassica napus cultivars revealed by rapd markers compared with morphological traits evaluation

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Evaluation of the genetic diversity among some oilseed rape Brassica napus cultivars revealed by rapd markers compared with morphological traits evaluation

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