Isozyme analysis of polymorphism of winter rapeseed Polish cultivars (Brassica napus L. var. oleifera)

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1 Tom XXVIII ROŚLINY OLEISTE OILSEED CROPS 2007 Wiesława Popławska, Alina Liersch, Iwona Bartkowiak-Broda, Krystyna Krótka Instytut Hodowli i Aklimatyzacji Roślin, Oddział w Poznaniu Isozyme analysis of polymorphism of winter rapeseed Polish cultivars (Brassica napus L. var. oleifera) Analiza izoenzymatyczna polimorfizmu polskich odmian rzepaku ozimego (Brassica napus L. var. oleifera) Key words: winter oilseed rape, isozyme markers, polymorphism, genetic distance, cultivars distinetness, MDH, PGM The possibility of differentiation of rapeseed cultivars qualitatively and quantitatively by the use of variability isozyme analysis has been observed. The largest polymorphism of isozyme patterns was present in MDH (ph 6.1) and PGM (ph 7.0) systems. These systems differentiate all investigated winter rapeseed cultivars in the best manner. Limited significance for discrimination of rapeseed cultivars have PGI (ph 7.0), LAP (ph 7.0) and 6 PGD (ph 6.1) systems because of their low level of polymorphism of isozyme patterns. IDH (ph 6.1) isozyme system was useless for identification of investigated cultivars. Monomorphic isozyme banding patterns were characteristic and were recurring in all investigated rapeseed cultivars. Obtained results revealed that for cultivars discrimination on the basis of their protein isozyme polymorphism, it is necessary to use at least two enzyme systems. Słowa kluczowe: rzepak ozimy, markery izoenzymatyczne, polimorfizm, dystans genetyczny, odrębność odmian, MDH, PGM Stwierdzono, że istnieje możliwość ilościowego i jakościowego rozróżnienia odmian rzepaku ozimego za pomocą analizy izoenzymatycznej. Największy polimorfizm we wzorach prążkowych wystąpił w systemach MDH (ph 6,1) i PGM (ph 7,0). Systemy te najlepiej różnicują badane odmiany rzepaku ozimego, występują wyraźne różnice między odmianami oraz zróżnicowanie wewnątrz odmian. Ograniczone znaczenie w rozróżnianiu odmian rzepaku ze względu na niski polimorfizm wzorów izoenzymatycznych mają systemy PGI (ph 7,0), LAP (ph 7,0) i 6 PGD (ph 6,1). Nieprzydatny do identyfikacji badanych odmian okazał się system IDH (ph 6,1). Monomorficzne izoenzymatyczne profile były charakterystyczne i powtarzalne dla wszystkich badanych odmian. Uzyskane wyniki wykazały, że dla odróżnienia odmian na podstawie polimorfizmu izoenzymatycznego białek konieczne jest przebadanie kilku, a przynajmniej dwóch systemów izoenzymatycznych.

2 10 Wiesława Popławska... Introduction Rapeseed (Brassica napus L.) is an amphidiploid plant which came into being from a crossing of turnip rape (Brassica campestris L. syn. B. rapa) and cabbage (Brassica oleracea L.) and has been domesticated relatively recently. In Europe rapeseed was domesticated in early Middle Ages (Gupta, Pratap 2007). Considering high fat and protein content in seeds for the last half century this plant has been subjected to strong breeding pressure aimed at increasing its yielding abilities and morphological traits decisive about seed yield and at obtaining zero-erucic forms (Stefansson et al. 1961) and low glucosinolate forms (Krzymański 1970). Intensive breeding of zero-erucic and low glucosinolate cultivars of canola type has resulted in narrowing of the variability of B. napus species, also in respect to morphological traits. Therefore it is very difficult to distinguish the licensed cultivars on the basis of morphological traits, although 13 morphological traits are evaluated in the process of evaluation of rapeseed cultivars in COBORU in Poland (Heimann and Lewandowski 2005). Therefore other descriptive traits are needed, such as molecular and biochemical markers. Isozyme markers, which have been widely used in genetic investigations of plant populations until recently, are currently being replaced by DNA markers. However, isozyme markers supplement molecular markers in genetic studies due to their properties, such as Mendelian way of heredity, almost always codominant expression, presence in all organs and tissues in the course of plant s whole life development, lack of pleiotropic and epistatic interactions, lack of genotypic and environmental interactions, quick, simple and non expensive technique of analysis (Wolko, Kruszka 1997). These markers have been used not only to distinguish the cultivars and evaluation of their purity, but also for evaluation of genetic distance between genotypes and the presence of genes conditioning qualitative and quantitative traits. Evaluation of genetic distance is especially important in the breeding of hybrid cultivars which make use of heterosis effect whose scope is often related to genetic distance of hybrid parental lines. Isozymes have turned out to be useful in investigating rapeseed and other plants from Brassica type, especially for identification of breeding cultivars of rapeseed (Mündges et al. 1990, Delourme and Foisset 1991, Polus 1998), identification of interspecies hybrids (Quiros et al. 1988, Truco and Arús 1987), purity of F 1 hybrids (Arús et al. 1985), evaluation of genetic variability of rapeseed cultivars (Delourme and Foisset 1991), investigation of phylogenetic links within Brassica (Truco and Arús 1987, Quiros et al. 1988), differentiation of species of Brassica genus (Arús et al. 1987, Mündges et al. 1989), evaluation of genetic distance and the degree of open- and self-pollination in different rapeseed lines (Hackenberg, Köhler 1996).

3 Isozyme analysis of polymorphism of winter rapeseed The usage of isozymes is however limited by low frequency of their occurrence. This influences relatively smaller range of detected polymorphism than in the case of DNA markers whose number is theoretically unlimited (Lee et al. 1996). The research reported in this paper aimed at evaluating the possibility of identification of polymorphism and genetic distance of Polish winter rapeseed cultivars by means of analysis of polymorphism of enzymatic seedling protein. Materials and methods The tested material included double improved cultivars: six Polish cultivars of winter rapeseed Bor, Kana, Leo, Mar, Polo, Marita and, for comparison, a French cultivar of winter rapeseed, Samourai. The investigated Polish cultivars are open pollinated cultivars made by pedigree breeding method. Scheme 1 shows detailed origins of Polish cultivars. Bor cultivar was bred in the Experimental Station of IHAR in Borowo and the other cultivars were bred in the Experimental Station of IHAR in Małyszyn (at present Divisions of Plant Breeding Company Strzelce). The French cultivar Samourai is an inbred line. BOR (1994) BNW 1.63 {[(1509/ /81) 1515/81] 1509/81} KANA (1997) (Jet Neuf 722/77) Cobra LEO (1993) Jet Neuf 722/77 MAR (1991) Jet Neuf 17/7 MARITA (1994) [(Garant Jet Neuf) (Jet Neuf Seger)] (2295 7/25) POLO (1993) (Jet Neuf 17/7) (Brink 0750) Zero erucic cultivars Odmiany zeroerukowe: Jet Neuf, Brink, Garant, BNW 1.63, Seger Double low lines and cultivars Linie i odmiany podwójnie ulepszone: Cobra, 722/77, 17/7, 7/25, 0750, 1509/81, 1515/81 Scheme 1. Pedigree of investigated cultivars Rodowody badanych odmian

4 12 Wiesława Popławska... Seeds of cultivars used in electrophoretic investigations were sown into pots. Seedlings were kept in phytotron in temperatures +20 o C at day and night length of 16/8 h. plants between the second and eighth week of growth were analysed. Analyses were performed for plants obtained from certified seeds. Enzymatic proteins were extracted from young tissue of cotyledon or leaf. Separation of isozymes was conducted with the use of horizontal electrophoresis on 10.5% starch gel in two buffer systems: morpholine citric (ph 6.1) and L-histidine (ph 7.0). Extraction and electrophoretic separation of enzymatic proteins, and also staining of 6 enzymatic systems (Table 1) were conducted following methods developed by Shilds et al. (1983) and Vallejos a (1983). Table 1 Characteristics of enzyme systems used in investigations of polymorphism of winter rapeseed cultivars (according to Johnson 1974, Kephart 1990) Charakterystyka systemów enzymatycznych wykorzystanych w badaniach polimorfizmu polskich odmian rzepaku ozimego (wg Johnson 1974, Kephart 1990) Enzyme system System enzymatyczny E.C. number Numer E.C. Abbreviation Skrót Quaterary structure Budowa czwartorzędowa Subcellular localization Lokalizacja* Reaction (ph) of gel and electrolyte buffers Odczyn (ph) roztworu żelu i elektrolitu Malate dehydrogenase MDH dimer c, mt ph 6.1 Dehydrogenaza jabłczanowa Phosphoglucomutase PGM monomer c, p ph 7.0 Fosfoglukomutaza Phosphoglucoisomerase PGI dimer c, p ph 7.0 Fosfoglukoizomeraza Leucine aminopeptidase LAP monomer c, p ph 7.0 Aminopeptydaza leucynowa 6-Phosphogluconate dehydrogenase PGD dimer c, p ph 6.1 Dehydrogenaza 6-fosfoglukonianowa Isocitrate dehydrogenase Dehydrogenaza izocytrynianowa IDH dimer c, p ph 6.1 *c cytosol cytozol, mt mitochondria mitochondria, p plastids plastydy E.C. number of enzyme classification numer klasyfikacji enzymatycznej On the basis of obtained zymograms electrophoretic phenotypes and frequency of their occurance have been determined. A homogeneity test for factors creating classification of s r type has been used double cross classification of n factors based on s independent trials and r mutually exclusive categories of a given varying trait (Oktaba 1966).

5 Isozyme analysis of polymorphism of winter rapeseed Obtained results have allowed for qualitative classifications (typical of cultivars banding patterns) and for quantitative classification (frequency of occurrence of a given banding pattern) for different loci of enzymatic markers. Two investigated cultivars were considered qualitatively different if one of them was characterized by one banding pattern which was not observed in the other cultivar in at least 5% of individual trials. With the use of homogeneity test Chi-square (χ 2 ) calculated for each combination of pairs of cultivars, significance of differentiation of cultivars by a given izozymatic system has been observed. Calculated values χ 2 have been compared to table data at the level of significance P < 0.001, 0.01, 0.05 at one degree of freedom for the systems PGI (ph 7.0) and IDH (ph 6.1), two degrees of freedom for the system LAP (ph 7.0), six degrees of freedom for the system MDH (ph 6.1). Degrees of freedom have been calculated according to the formula: v = (s 1) (r 1) s number of obtained electrophoretic phenotypes, r compared paires of cultivars. Values of genetic distance and dendrogram resulting from isoenzymatic polymorphism of cultivars have been obtained on the basis of a formula developed by Nei (1972) in the packet PHYLIP 3,5 (programmes Seqboot, Gendist, Neighbor, Consense, Drawgram) (Felsenstein 1993). Data were recorded in zero / one (0 lack of a band for a given cultivar, 1 occurrence of a band). The dendrogram has been built using the method UPGMA (unweighted pair group method using arithmetic averages) Results System MDH (ph 6.1) Isozyme system MDH (ph 6.1) revealed the greatest variability of band profiles (Table 2). For 7 investigated cultivars 12 different banding patterns have been obtained. Intra- and inter-cultivar qualitative and quantitative differentiations were observed. Samourai cultivar had monomorphic isoenzymatic profile and only phenotype 3 occurred. Phenotypes of other cultivars were polymorphic. The greatest polymorphism occurred in Marita cultivar. In this cultivar 4 different banding patterns (patterns 4, 5, 6, 7) appeared. s Bor, Kana and Polo had 3 banding patterns (patterns 1, 2, 12). Leo cultivar was characterized by 2 patterns (patterns 8 and 9), and Mar cultivar by patterns 11 and 12, and pattern 11 occurred in 99% of investigated plants. The obtained electrophoretic phenotypes and their frequency are shown in Table 2.

6 Electrophoretic phenotypes and their frequencies for MDH system (ph 6.1) Fenotypy elektroforetyczne i ich frekwencje dla systemu MDH (ph 6,1) Table 2 Phenotype pattern for MDH ph 6.1 Fenotyp układ prążków dla MDH ph 6,1 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) Bor Kana Marita Mar 99 1 Leo 96 4 Polo Samourai

7 Isozyme analysis of polymorphism of winter rapeseed Homogeneity test χ 2 revealed that out of 21 combinations of pairs, 20 combinations can be quantitatively differentiated at a significant level with probability P < Only a combination of Bor Kana cultivars did not differe significantly. The largest number of electrophoretic phenotypes, 7, differentiate cultivars Polo and Marita, and only two patterns differentiate Leo and Mar and both of these cultivars are distinguished from Samourai cultivar. Table 3 includes, in brackets, numbers of isozyme phenotypes generated in MDH system, which differentiate in a highly significant manner, pairs of investigated cultivars. Table 3 s differentiation obtained by χ 2 test using MDH (ph 6.1) banding patterns Zróżnicowanie odmian uzyskane testem χ 2 na podstawie zymogramów MDH (ph 6,1) Bor Kana Marita Mar Leo Polo Samourai Bor - Kana - Marita *** (1,4,5,6,7,12) Mar *** *** (1,4,5,6,7,12) - *** (3,4,5,6,7) - *** *** *** *** *** (1,11,12) (1,11,12) (4,5,6,7,11) (8,11) (8,9,10,11) (3,11) Leo *** *** *** - *** *** (1,8,9,12) (1,8,12) (4,5,6,7,8) (8,9,10) (3,8) Polo *** *** *** - *** (1,8,9,10,12) (1,8,10,12) (4,5,6,7,8,9,10) (3,8, 9,10) Samourai *** (1,3,12) *** (1,3,12) v - s discrimination significant at Zróżnicowanie odmian istotne przy P < 0,001 (***) System PGM (ph 7.0) In isozyme system PGM the investigated cultivars revealed large differentiation which was manifested in polymorphism of banding patterns, especially among cultivars (Table 4). Isozyme PGM has a monomeric structure, and its large polymorphism results from the presence of three regions of activity, that is three genes and their alleles. There have been observed 7 electrophoretic phenotypes (Table 4). s Bor, Kana, Marita, Leo and Mar have been monomorphic in respect to electrophoretic phenotype. It was pattern 1 for cultivars Bor and Kana, pattern 4 for Marita cultivar, pattern 7 for Leo and Mar cultivar. Samourai cultivar was characterized by 2 banding patterns: 2 and 3. Polo was also characterized by two isozyme phenotypes, patterns 5 and 6. It has been proved, with the use of significance test χ 2, that out of 21 combinations of pairs of cultivars 19 pairs (90.4%) can be distinguished with

8 16 Wiesława Popławska... probability P < Among them 8 pairs (42%) can be distinguished on the basis of 2 banding patterns, 10 pairs (52,6%) on the basis of 3 banding patterns, while 1 pair of cultivars (5.4%) Samourai Polo, could be distinguished on the basis of 4 patterns (Table 4). Pairs Bor and Kana and Leo and Mar are not distinguishable by the system PGM (ph 7.0). Table 4 Electrophoretic phenotypes and their frequencies for PGM system (ph 7.0) Fenotypy elektroforeyczne i ich frekwencje dla systemu PGM (ph 7,0) Phenotype pattern for PGM ph 7.0 Fenotyp układ prążków dla PGM ph 7,0 Bor Kana Marita Mar Leo Polo Samourai (1) (2) (3) (4) (5) (6) (7) Table 5 s differentiation obtained by χ 2 test using PGM (ph 7.0) banding patterns Zróżnicowanie odmian uzyskane testem χ 2 na podstawie zymogramów PGM (ph 7,0) Bor Kana Marita Mar Leo Polo Samourai Bor - Kana - Marita *** (1,4) *** (1,4) - *** (2,3,4) Mar *** (1,7) *** (1,7) *** (4,7) - *** (5,6,7) *** (2,3,7) Leo *** (1,7) *** (1,7) *** (4,7) - *** (5,6,7) *** (2,3,7) Polo *** (1,5,6) *** (1,5,6) *** (4,5,6) - *** (2,3,5,6) Samourai *** (1,2,3) *** (1,2,3) - s discrimination significant at Zróżnicowanie odmian istotne przy P < 0,001 (***)

9 Isozyme analysis of polymorphism of winter rapeseed System PGI (ph 7.0) System PGI demonstrated little differentiation within and among investigated cultivars. In the second region of activity of phosphoglucoizomeraze two banding patterns have been observed. The obtained banding patterns are typical of enzyme with dimeric structure while the first region of activity of investigated isoenzyme PGI was 1-band, monomorphic for all cultivars. Phenotype 2 occurred in investigated cultivars more often. Phenotype 1 was dominant only in cultivars Samourai and Kana (Table 6). s Samourai, Leo and Mar were monomorphic in respect to electrophoretic phenotype. Samourai was characterized by pattern 1, and cultivars Leo and Mar pattern 2. Statistical analysis with χ 2 test showed that in 21 combinations of pairs of cultivars, 19 cultivars (90.4%) were distinguishable on the basis of the frequency of occurrence of individual allozymes (Table 7). Out of these cultivars 4 pairs (21.1%) were distinguished with probability P < 0.01, 2 pairs (10.5%) with probability P < 0.05, and 13 pairs (68.4%) with probability P < Isozyme system PGI made it impossible to distinguish qualitatively combinations of pairs of cultivars Bor Polo and Leo Mar. Table 6 Electrophoretic phenotypes and their frequencies for PGI system (ph 7.0) Fenotypy elektroforetyczne i ich frekwencje dla systemu PGI (ph 7,0) Phenotype pattern for PGI ph 7.0 Fenotyp układ prążków dla PGI ph 7,0 Bor Kana Marita Mar Leo Polo Samourai (1) (2)

10 18 Wiesława Popławska... Table 7 s differentiation obtained by χ 2 test using PGI (ph 7.0) banding patterns Zróżnicowanie odmian uzyskane testem χ 2 na podstawie zymogramów PGI (ph 7,0) Bor Kana Marita Mar Leo Polo Samourai Bor - Kana *** (1,2) - Marita *** (1,2) *** (1,2) - *** (1,2) Mar ** (1,2) *** (1,2) * (1,2) - *** (1,2) *** (1,2) Leo *** (1,2) ** (1,2) * (1,2) - *** (1,2) *** (1,2) Polo *** (1,2) ** (1,2) - *** (1,2) Samourai *** (1,2) *** (1,2) - s discrimination significant at Zróżnicowanie odmian istotne przy P < 0,001 (***); P < 0,01 (**); P < 0,05 (*) System LAP (ph 7.0) Isozyme system LAP (ph 7.0) has monomeric structure. In the investigated cultivars occurred three electrophoretic phenotypes of this enzyme (Table 8). In the course of isozyme analysis of 7 cultivars of rapeseed it was stated that in cultivars Bor and Kana banding pattern no. 2 is more frequent, which makes them distinct from other cultivars with prevailing phenotype 3 (Table 8). It occurred in % plants of cultivars Samourai, Polo, Leo, Mar and in 96% plants of Marita cultivar. Electrophoretic phenotypes and their frequencies for LAP system (ph 7.0) Fenotypy elektroforetyczne i ich frekwencje dla systemu LAP (ph 7,0) Table 8 Phenotype pattern for LAP ph 7.0 Fenotyp układ prążków dla LAP ph 7,0 Bor Kana Marita Mar Leo Polo Samourai (1) (2) (3)

11 Isozyme analysis of polymorphism of winter rapeseed Homogeneity test χ 2 revealed that out of 21 combinations of pairs of cultivars, 10 pairs (47.6%) can be distinguished with probability P < The remaining pairs of cultivars (52.4%) were distinguishable neither qualitatively nor quantitatively (Table 9). Table 9 s differentiation obtained by χ 2 test using LAP (ph 7.0) banding patterns Zróżnicowanie odmian uzyskane testem χ 2 na podstawie zymogramów LAP (ph 7,0) Bor Kana Marita Mar Leo Polo Samourai Bor - Kana - Marita *** (1,2,3) *** (1,2,3) - Mar *** (1,2,3) *** (1,2,3) - Leo *** (1,2,3) *** (1,2,3) - Polo *** (1,2,3) *** (1,2,3) - Samourai *** (1,2,3) *** (1,2,3) - s discrimination significant at Zróżnicowanie odmian istotne przy P < 0,001 (***) System 6 PGD (ph 6.1) Isozyme system 6 PGD (ph 6.1) has dimeric structure. There are 2 regions of activity in this system, and consequently there are more alleles conditioning individual phenotypes (Table 10). Isozyme marker 6 PGD (ph 6.1) allowed to observe the genetic distinctness of Samourai cultivar. The genotype of this cultivar includes alleles conditioning the emergence of isozyme phenotype number 3. This pattern did not occurred in other investigated cultivars. Also Bor cultivar is genetically distinct. In this cultivar patterns 1 and 2 occur, pattern 2 is present in 96% plants and pattern 1 only in 4%. These patterns did not occur in other investigated cultivars. In cultivars Kana, Marita, Polo, Leo, Mar only profile 4 occurred (Table 10). With the use of 6 PGD system out of 21 combinations of pairs of cultivars 11 pairs of cultivars (52.3%) can be distinguished significantly with probability P < The combination of cultivars Bor-Samourai was distinguished with patterns 2 and 4 while Samourai cultivar was distinguished from other cultivars by patterns 3 and pairs of cultivars (47.7) could be distinguished neither qualitatively nor quantitatively on the basis of obtained electrophoretic pictures of isozyme phenotype 6 PGD because these cultivars had alleles conditioning the same isozyme phenotype (Table 11).

12 20 Wiesława Popławska... Electrophoretic phenotypes and their frequencies for 6 PGD system (ph 6.1) Fenotypy elektroforetyczne i ich frekwencje dla systemu 6 PGD (ph 6,1) Table 10 Phenotype pattern for 6 PGD ph 6.1 Fenotyp układ prążków dla 6 PGD ph 6,1 Bor Kana Marita Mar Leo Polo Samourai (1) (2) (3) (4) 4 96 Table 11 s differentiation obtained by χ 2 test using 6 PGD (ph 6.1) banding patterns Zróżnicowanie odmian uzyskane testem χ 2 na podstawie zymogramów 6 PGD (ph 6,1) Bor Kana Marita Mar Leo Polo Samourai Bor - Kana *** (2,4) - Marita *** (2,4) *** (3,4) Mar *** (2,4) - *** (3,4) Leo *** (2,4) - *** (3,4) Polo *** (2,4) - *** (3,4) Samourai *** (2,3) *** (3,4) - s discrimination significant at Zróżnicowanie odmian istotne przy P < (***); P < 0.01 (**); P < 0.05 (*) System IDH (ph 6.1) In isozyme system IDH (ph 6.1) the investigated cultivars revealed 2 banding patterns. The differences occurred in isozyme patterns among cultivars; within cultivars the profiles were monomorphic (Table 12). s Bor, Samourai, Marita possessed phenotype 1, and cultivars Kana, Polo, Leo i Mar possessed pattern 2.

13 Isozyme analysis of polymorphism of winter rapeseed Electrophoretic phenotypes and their frequencies for IDH system (ph 6.1) Fenotypy elektroforetyczne i ich frekwencje dla systemu IDH (ph 6,1) Phenotype pattern for IDH ph 6.1 Fenotyp układ prążków dla IDH ph 6,1 Table 12 Bor Kana Marita Mar Leo Polo Samourai (1) (2) Homogeneity test χ 2 revealed that 12 pairs of cultivars (57.2%) can be distinguished by banding patterns 1 and 2 with probability P < 0.001, and 9 pairs (42.8%) cannot be distinguished either quantitatively or qualitatively on the basis of obtained electrophoretic pictures (Table 13). Table 13 s differentiation obtained by χ 2 test using IDH (ph 6.1) banding patterns Zróżnicowanie odmian uzyskane testem χ 2 na podstawie zymogramów IDH (ph 6,1) Bor Kana Marita Mar Leo Polo Samourai Bor - Kana *** (1,2) - Marita *** (1,2) - Mar *** (1,2) *** (1,2) - *** (1,2) Leo *** (1,2) *** (1,2) - *** (1,2) Polo *** (1,2) *** (1,2) - *** (1,2) Samourai *** (1,2) - s discrimination significant at Zróżnicowanie odmian istotne przy P < 0,001 (***) The largest genetic variability of rapeseed cultivars was demonstrated with the use of isozyme systems PGI (Table 14). The dendrogram obtained on the basis of generated isozyme markers determines similarities of investigated winter rapeseed cultivars and ranked them according to their origin. s Mar and Leo whose origins differ the least are in one genotype group while cultivar Bor which differs the most in respect to origin constitutes a separate group (Fig. 1).

14 22 Wiesława Popławska... Isozyme systems differentiating investigated winter rapeseed cultivars Systemy izoenzymatyczne różnicujące badane odmiany rzepaku ozimego Table 14 Bor Kana Marita Leo Polo Samourai Kana PGI, 6 PGD - Marita Mar Leo Polo Samourai LAP, PGI, 6 PGD LAP, PGI, 6 PGD LAP, PGI, 6 PGD LAP, 6 PGD LAP, PGI, 6 PGD LAP, PGI LAP, PGI LAP, PGI LAP, PGI LAP, PGI, 6 PGD - PGI, 6 PGD MDH, PGM MDH, PGM MDH, PGM, PGI MDH MDH, PGM, PGI - MDH, PGM, PGI PGI, 6 PGD PGI, 6 PGD - PGI, 6 PGD - Fig. 1. Dendrogram of relationship among winter rapeseed cultivars on the basis of izosyme polymorphism Dendrogram określający podobieństwo odmian rzepaku ozimego w oparciu o polimorfizm izoenzymatyczny Discussion Rapeseed is an amphidiploid species (AACC, 2n = 38) which originated as a result of spontaneous crossing between two diploid species: turnip rape (Brassica rapa, syn. campestris; genomaa, 2n = 20) and cabbage (Brassica oleracea; genom CC, 2n = 18). Therefore interpretation of pictures of electrophoretic division is not clear-cut considering different activity of allelic variants of enzymes occurring in each of these species (Chévre i in. 1995). For that reason it is impossible

15 Isozyme analysis of polymorphism of winter rapeseed to determine the frequency of alleles at different loci. Heritability of isozymes in this species is not known so far. Identification of cultivars on the basis of isozyme loci is possible in grass, for example Stuczyński et al. 1997, Nielsen However, investigations of polymorphic loci of isozyme markers allow to determine genetic variability in Brassica napus as well as differentiation among varieties. In the performed investigations it was shown that there is a possibility of qualitative and quantitative differentiation of winter rapeseed varieties by means of the analysis of isozyme variability. The largest polymorphism of isozyme alleles was generated by MDH and PGM systems. These systems differentiate the investigated cultivars of winter rapeseed in the best way. Systems PGI, LAP and 6 PGD display limited value in differentiation of investigated rapeseed cultivars because of low polymorphism of isozyme patterns. System IDH turned out to be of little use for identification of investigated rapeseed cultivars. The investigated cultivars were characterized by one of the two monomorphic profiles of this isozyme. These cultivars revealed within and among cultivars polymorphism to a different degree, depending on an examined enzymatic system. Analysed varieties were characterized by a relatively large genetic variability, especially that these were only double low cultivars. However, these were open pollinaed cultivars which were bred with pedigree method while the pedigree of each cultivar was different. This fact has a significant influence on observed differentiation of isozyme variability within and between varieties. The French cultivar Samurai, also subjected to investigation, is an inbred line and the results of analyses performed by means of six isozyme systems proved entirely a high degree of homozygocity of this cultivar. Only isozyme system PGM revealed some intra-cultivar polymorphism. Similar studies conducted by Mündges et al. (1990) made it possible to identify 10 rapeseed varieties by means of four polymorphic enzyme loci: LAP, PGI, ACO and SDH. Obtained results were the basis for qualitative and quantitative classification of different loci. System PGI revealed cultivar polymorphism greater than in the reported study. However, the examined varieties were more differentiated in respect to their origin and qualitative traits; these were zero erucic and low glucosinolate cultivars, 4 cultivars were high erucic. System LAP also differentiated cultivars to a small degree, similarly to the above mentioned work. Also Hackenberg and Köhler (1996) investigated the differentiation of inbred rapeseed lines with the use of 7 polymorphic isozyme systems, two of which allowed for qualitative and quantitative differentiation of all lines. The usefulness of isozyme systems for research on genetic variability of cultivars was observed also in other species, e.g. lupine (Wolko 1995), alfalfa (Morales Corts, Crespo Martinez 2000), wild service tree (Sorbus torminalis) (Bednorz i in. 2004).

16 24 Wiesława Popławska... Isozyme markers reveal lower level of polymorphism than molecular markers, however, due to analysis speed, low cost, possibility to examine large numbers of objects in a short time, make them attractive and they are in standard use by a French organization, GEVES Groupe d Etude et de Contrôle des Variétés et des Semences, to describe new rapeseed varieties and to evaluate cultivar purity of hybrid seeds (Lombard i in. 2002). Performed investigations showed that isozyme markers allow to detect polymorphism of rapeseed varieties and its importance is in accordance with the origin of the varieties. For example, Leo and Mar varieties, accordingly to their pedigree, are closely related. Hence, majority of the examined isozyme systems do not reveal any genetic differences among them. They can be differentiated qualitatively with the use of LAP system (ph 7.0) and MDH system (ph 6.1) with high polymorphism. The more isozyme systems may be determined for a given genotype the more detailed its characteristics and greater possibilities of discovering alleles which differentiate the genotype from others. It can be concluded from the obtained results that in order to classify qualitatively all 7 investigated cultivars, the analysis of patterns of at least two isozyme systems may be sufficient (Table 14). References Arús P., Shilds C.R., Orton T.J Application of isozyme electrophoresis for purity testing and cultivar identyfication of F 1 hybrids of Brassica oleracea. Euphytica, 34: Arús P., Beladrón J., Ordás A Species identification of cultivated Brassicas with isozyme electrophoresis. Cruciferae Newsletter, 12: Bednorz L., Myczko Ł., Kosiński P Isozyme polymorphism and genetic structure of the population of Sorbus torminalis (L.) Crantz from the Bytyń Forest (Poland). J. Appl. Genet., 45 (3): Chévre A.M., Delourme R., Eber F., Margale E., Quiros C. F., Arus P Genetic analysis and nomenclature for seven isozyme systems in Brassica nigra, B. oleracea and B. campestris. Plant Breeding, 114: Delourme R., Foisset N Isozyme variability in Brassica napus. In: D.I. McGregor (ed.), Proc. 8th International Rapeseed Conf., Saskatoon, Canada, vol. 4: Felsenstein J PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. Gupta S.K., Pratap A History, Origin and Evolution in: Advences in Botanical Research Rapeseed Breeding. Elsevier, edited by Gupta S.K. (ed.), vol. 45: Hackenberg E.M., Köhler W Use of isozyme analysis in the breeding of synthetic rapeseed cultivars. Plant Breeding, 115:

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