Genet Resour Crop Evol (2013) 60:953-965 DOI 10.1007/sl0722-012-9891-x RESEARCH ARTICLE Cytoplasmic diversity of Brassica napus L., Brassica oleracea L. and Brassica rapa L. as determined by chloroplast microsatellite markers Shirin Zamani-Nour • Rosemarie Clemens • Christian Möllers Received: 26 January 2012/Accepted: 23 July 2012/Published online: 10 August 2012 © The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Cytoplasmic genomes in most angio-sperms are known to be maternally inherited. Oilseed rape {Brassica napus L.) as a natural amphidiploid species hence may carry the B. oleracea L. or the B. rapa L. cytoplasm, depending on the cross direction. The presence of either the B. oleracea or the B. rapa cytoplasm in oilseed rape has been reported to affect agronomically important traits. However, to date little is known about the cytoplasmic composition and genetic diversity of current winter oilseed rape culti-vars and breeding material. The aim of this study was to assess the usefulness of 40 previously published chloroplast cpSSR markers from Brassica species and Arabidopsis thaliana (L.) Heynh. for distinguishing the cytoplasms of 49 different genotypes of B. napus and its diploid ancestor species. Results showed that only 14 out of the 40 tested primer combinations were suitable to distinguish the cytoplasms of a test set of 8 Brassica genotypes. With the 14 primer pairs 64 different cpSSR alleles were identified in the set of 49 genotypes. Cluster analysis indicated distinct groups for the cytoplasms of B. napus, B. rapa, and B. oleracea. However, an unambiguous identification and classification of the cytoplasm types was not S. Zamani-Nour • R. Clemens • C. Möllers (El) Division of Plant Breeding, Department of Crop Sciences, Georg-August-Universität Göttingen, Von-Siebold-Str. 8, 37075 Göttingen, Germany e-mail: cmoelle2@gwdg.de possible in all cases with the available polymorphic set of cpSSR primer pairs. Keywords Brassica ■ Chloroplast • Cytoplasma • Oilseed rape • Plastome • Resynthesized rapeseed • SSR marker Introduction Oilseed rape {Brassica napus L., AACC-genome, 2n — 38) is an amphidiploid crop species, spontaneously arisen from a cross between Brassica rapa L. (turnip rape, AA-genome, 2n — 20) and Brassica oleraceaL. (cabbage, CC-genome, 2n — 18). Although oilseed rape is considered to be of polyphyletic origin, it is assumed that there have been only a limited number of successful hybridization events. From an evolutionary point of view, oilseed rape is a fairly new crop. First reliable documented records are about 500 years old (Downey and Robbelen 1989; Gomez-Campo and Prakash 1999) and results from molecular phylogenetic analyses indicate that hybridization occurred less than 10,000 years ago (Rana et al. 2004). Unlike, the divergence and separate evolution of the diploid species Brassica rapa and Brassica oleracea began between 3 and 4 million years ago (Inaba and Nishio 2002; Cheung et al. 2009). As cytoplasmic genomes—plastome and mitochondrial genome—are maternally inherited (Bock 2007), Springer 954 Genet Resour Crop Evol (2013) 60:953-965 oilseed rape may carry either the cytoplasm of its diploid ancestors B. oleracea or B. rapa, depending on the direction of the hybridization event. Furthermore, during the past 60 years, oilseed rape has been resynthesized via interspecific crossing of various B. oleracea and B. rapa forms followed by embryo rescue and in vitro plant regeneration, so that depending on the cross direction different cytoplasms are available (Chen and Heneen 1989). However, the parental material and the direction of the crosses have not always been fully documented in the literature. The presence of either the B. oleracea or the B. rapa cytoplasm in oilseed rape has been reported to affect agronomically important traits, like oil quality (Rajcan et al. 2002), oil and protein content (Wu et al. 2005, 2006; Wang et al. 2010) and floral characteristics (Chang et al. 2011). Furthermore, genes relevant to the expression of agricultural traits are located in the plastome. For example, one unit of the heteromeric Acetyl-CoA Carboxylase (with four subunits) which is required for the de novo fatty acid synthesis in the plastids (Sasaki and Nagano 2004; Kode et al. 2005) is encoded by the chloroplast DNA (cpDNA). Furthermore, the cpDNA gene rbcL encodes for the large subunit of ribulose-l,5-bisphosphate carboxylase (Clegg et al. 1994), which is a vital enzyme for carbon fixation in the Calvin cycle. Despite of the obvious importance of chloroplast genes for many metabolic pathways (Wicke et al. 2011), to date little is known about the cytoplasmic composition of current oilseed rape cultivars and breeding material. Preliminary results from early studies with RFLP markers (Palmer et al. 1983; Erickson et al. 1983; Kemble 1987; Song and Osborn 1992; Hallden et al. 1993) were inconsistent, indicating that oilseed rape may have a cytoplasm derived from B. oleracea (ole-type) or B. rapa (rap-type) or may have its own (nap-type). Furthermore, for hybrid breeding programmes, cytoplasmic male sterility (CMS) has been achieved through the introgression of specific cytoplasms from related species. The Polima CMS system possibly is derived from a Polish winter oilseed rape genotype (Liu et al. 1987), and the Ogura CMS system has been introduced from Raphanus sativus L. into oilseed rape genomic background by sexual crossing and back crossing (Pelletier et al. 1983). In a next step, protoplast fusion was performed to replace the original Raphanus chloroplasts by the chloroplasts of the spring oilseed rape cultivar Brutor (Pelletier et al. 1983). Rather recently it has been discovered that chloroplast DNA does also contain microsatellite polymorphisms. Chloroplast specific SSR-markers (cpSSR) have been developed for a number of species including Arabidopsis (Weising and Gardner 1999; Provan 2000; Jakobsson et al. 2007; Haider 2011) and oilseed rape (Provan 2000; Flannery et al. 2006; Allender et al. 2007). Chloroplast cpSSR repeats may be found in both coding and non-coding regions but more variation were reported in the non-coding regions including introns and intergenic spacer of cpDNA (Provan et al. 2001; Jakobsson et al. 2007). Ebert and Peakall (2009) emphasized that cpSSR repeats are likely more abundant in intergenic spacer regions than in introns. Nearly all of the cpSSR markers detected are of the mononucleotide type (Flannery et al. 2006; Jakobsson et al. 2007). Using a limited set of cpSSR markers for studying cytoplasmic diversity in oilseed rape has led to differing results (Flannery et al. 2006; Jakobsson et al. 2007; Allender and King 2010). The identification of a set of most informative cpSSR primer pairs for the Brassica AA, CC and AACC genomes could be most useful in future work for the characterization and evaluation of the effects of different cytoplasms in oilseed rape. The objective of the present study was to analyse a set of cpSSR primers previously developed for Arabidopsis, Brassica oleracea and for Brassica napus (Provan 2000; Flannery et al. 2006; Allender et al. 2007; Jakobsson et al. 2007) for their suitability to distinguish the cytoplasms of Brassica species. The plant material included in the study comprised wild and cultivated forms of Brassica oleracea, cultivated vegetable and oil forms of B. rapa and cultivated and resynthesized forms of oilseed rape (B. napus). For comparison, oilseed rape genotypes with the Polima, the Ogura cytoplasmic male sterility cytoplasm and with the Brassica B genome cytoplasm were included. Materials and methods Plant material The 49 genotypes comprised resynthesized forms of B. napus L. (AACC, n — 7), current German winter oilseed rape cultivars (B. napus L., AACC, n — 10), spring B. napus cultivar Korall with its normal cytoplasm (AACC, n — 1), with the Polima male Springer Genet Resour Crop Evol (2013) 60:953-965 955 sterility cytoplasm (n — 1), and with the Brassica B genome cytoplasm (n — 1), current winter oilseed rape B. napus hybrid cultivars with the Ogura male sterility cytoplasm (AACC, n — 2), B. rapa L. (AA, n — 9), wild forms of B. oleracea L. (CC, n — 13; B. cretica Lam., B. incana Ten., B. villosa Biv. 3821, B. villosa Biv. subsp. bivoniana 6581, B. bourgeaui Kuntze, B. montana Pourr., B. macrocarpa Guss., B. rupestris Raf., B. taurica Tzvel., B. hilarionis G.E.Post, B. insularis, B. oleracea subsp. oleracea 7695 and B. oleracea var. alboglabra (Bail.) Sun BRA 165), cultivated forms of B. oleracea (CC, n — 4) and one accession of B. carinata A. Braun (BBCC, n — 1) (Table 1, 2). The three Korall genotypes were provided by Bo Gertsson, Svalov (Sweden). For nomenclature see Gladis and Hammer (1990, 1992). Chloroplast SSR primers A total of 40 cpSSR primer pairs were used for the analysis. They included 11 primer pairs previously developed for B. napus by Flannery et al. (2006; MF-1, MF-2, MF-3, MF-4, MF-6, MF-7, MF-8, MF-9) and by Allender et al. (2007; ChloroO, ChloroP, ChloroQ). The remaining 29 primer pairs were developed for Arabid-opsis thaliana by Provan (2000; ATCP7905, ATCP 28673, ATCP30287, ATCP46615, ATCP66701, ATCP 70189), by Allender et al. (2007; Chlal6, Chloro35, Chloro39) and by Jakobsson et al. (2007; 01, 07,08,11, 12, 17, 19, 21, 24, 29, 34, 37, 43, 44, 45, 47, 51, 55, 58, 60). The 20 primer pairs of Jakobsson et al. (2007) were selected from the 60 published ones so that they were representing all regions of the Arabidopsis chloroplast genome. The primer pair names in this publication refer to their names given in the original publications. Primers were ordered from Eurofins MWG Operon (www. eurofinsdna.com). Purification of total DNA Leaf samples were taken from one young plant each of the 49 Brassica genotypes. Total genomic DNA was isolated using the Qiagen DNeasy Plant Mini Kit (The Netherlands) and following basically the procedure described in the manual. DNA concentration was TM determined by using the Bio-Rad VersaFluor Fluo-rometer and the Bio-Rad Fluorescent DNA Quantitation Kit (Bio-Rad, CA, USA) containing the fmorochrome Hoechst 33258 (bisbenzimide), following the instruction manual. PCR reaction A total volume of 20 ul was used for each PCR reaction, containing 0.05 units/ul FIREPol Taq polymerase (Solis Biodyne; Tartu, Estonia), 1 x FIREPol PCR buffer without MgC12, 2.5 mM MgCl2 (Solis Biodyne; Tartu, Estonia), 0.2 mM dNTP-Set (Bio-Budget Technologies GmbH; Krefeld, Germany), 0.05 uM M13-universal primer (23 bp)(Applied Bio-systems), 0.05 uM forward primer with M13 (18 bp) tail at its 5' end (Eurofins MWG Operon; Ebersberg, Germany), 0.05 uM unlabelled reverse primer (Eurofins MWG Operon; Ebersberg, Germany) together with 25 ng of template DNA. The PCR reaction was performed in a Biometra Thermocycler (Biometra GmbH; Gottingen, Germany) using the following two-step touchdown PCR program: 95 °C for 2 min; 5 cycles of 95 °C for 45 s, 68 °C (-2 °C/cycle) for 5 min, 72 °C for 1 min; 5 cycles of 95 °C for 45 s, 58 °C (-2 °C/cycle) for 1 min, 72 °C for 1 min; 27 cycles of 95 °C for 45 s, 47 °C for 30 s, 72 °C for 1 min; and 72 °C for 10 min and then cooled down to 4 °C after the last cycle. Preparation of cpSSR-PCR products for capillary array analysis and identification of cpSSR alleles Three different SSR-PCR products with different TM TM TM colours (FAM , VIC and NED ) (Applied Biosys-tems) were mixed together. 2 ul of each of three PCR products were mixed and diluted 1:100 using HPLC water. Afterwards 2 ul of diluted PCR product was TM added to a loading mixture of 12 ul Hi-Di Formam-ide and 500 ROXTM size standard. The mixture was denatured for 2 min at 90 °C in a Thermocycler. The electrophoresis was then carried out automatically in Genetic Analyzer 3130x, a 16-capillary instrument, using POP7, 36 cm capillary and 23 s injection time. Fluorescently labelled fragments were interpreted using GeneMapper software v.3.7 (Applied Biosys-tems). Each locus was represented by one peak. If more than one peak occurred, unspecific binding of primers were anticipated and those primer pairs were excluded from further analysis. The maximum and minimum sizes of markers were selected in the range of 50-500 (bp). An allele height of more than 500 was Springer 956 Genet Resour Crop Evol (2013) 60:953-965 Table 1 B. rapa, B. oleracea, B. napus, resynthesized B. napus and B. carinata genotypes used in this study Species Subspecies/Genotype or accession no. Comments/Origin B. rapa L. subsp. trilocularis/Yellow Sarson 59 Prof. Fu, Wuhan, China var. nipposini ca/Mi zuna www.nelson.se; Art. No. 60233 subsp. oleifera/Rex NPZ Lembke KG, DE subsp. oleifera/Lzrgo Lantmännen SW Seed/DE subsp. oleifera/Toú Brown Sarson/L. Hassan, Bangladesh subsp. o/ez/ěra/Steinacher/B AZ1 8101 BAZ Braunschweig, DE subsp. oleifera/Perko Tetraploid/KWS SAAT AG, DE subsp. oleifera/Orbit Lantmännen SW Seed, DE subsp. oleifera/Salut Winter turnip rape Br. cretica Lam. subsp. aegaea 6344 Genbank Spain (ES) B. incana Ten. 6564 Genbank Spain (ES) B. villosa Biv. 3821-75 Genbank Spain (ES) Br. villosa Biv. subsp. bivoniana 6581 Genbank Spain (ES) B. bourgeaui Kuntze BRA 2998 (=K 9825) Genbank Gatersleben (IPK), DE B. montana Pourr. BRA 1644 (=K5457) Genbank Gatersleben (IPK), DE B. macrocarpa Guss. 3819-75 Genbank Spain (ES) B. rupestris Raf. subsp. hispida 6580-84 Genbank Spain (ES) B. taurica Tzvel. BRA 2947(=K9238) Genbank Gatersleben (IPK), DE B. hilarionis G.E.Post HRIGRU 12483 Genbank Great-Britain (GB) B. insularis BRA 3050 (=K 9321) Genbank Gatersleben (IPK), DE B. oleracea L. subsp. oleracea 7695 Genbank Spain (ES) B. oleracea L. var. alboglabra BRA 165 Genbank Gatersleben (IPK), DE var. botrytis L./Super Regama/BRA 1381 Cauliflower/Genbank Gatersleben (IPK), DE var. botrytis L/Vasco Cauliflower/Novartis Seeds, CH var. capitata L./Reliant Red cabbage/Novartis Seeds, CH var. gongylodes L./Azur Turnip, stem cabbage/Novartis Seeds, CH Switzerland B. napus L. Ha 699/91-4 WOSR, Breeding line/GAU Göttingen, DE Komando-5 WOSR, Line cultivar/KWS Saat AG, DE Oase-3 WOSR, Line cultivar/DSV AG, DE Krypton WOSR, Line cultivar/KWS Saat AG, DE Charly-7 WOSR, Line cultivar/DSV AG, DE Favorite-6 WOSR, Breeding Line/DSV AG, DE DSV 2-08-1 WOSR, Breeding Line/DSV AG, DE ES Alienor WOSR, Line cultivar/Euralis Semences, FR NK Beauty-10 WOSR, Line cultivar/Syngenta Seeds GmbH, DE Express 617-4 WOSR, NPZ Lembke KG, DE Triangle with Ogura (a) CMS cytoplasm WOSR, Ogura CMS hybrid/KWS Saat AG, DE Germany Flash with Ogura CMS cytoplasm WOSR, Ogura CMS hybrid/DSV AG, DE Korali SOSR, Line cultivar/SE Korali with Polima CMS cytoplasm SOSR, Line cultivar/SE Korali with B genome cytoplasm SOSR, Line cultivar/SE H123-1 Dept. of Crop Sciences, GAU Göttingen, DE HI 0-3 Dept. of Crop Sciences, GAU Göttingen, DE H61 Dept. of Crop Sciences, GAU Göttingen, DE S3 Dept. of Crop Sciences, GAU Göttingen, DE S13 Dept. of Crop Sciences, GAU Göttingen, DE H48 Dept. of Crop Sciences, GAU Göttingen, DE L239 Dept. of Crop Sciences, GAU Göttingen, DE B. carinata A. Braun BRA 1151/90 Genbank Gatersleben (IPK), DE WOSR winter oilseed rape, SOSR spring oilseed rape Springer Genet Resour Crop Evol (2013) 60:953-965 957 Name Female parent Table 2 Resynthesized B. napus genotypes with reported cytoplasmic origin 1 H10-1 H61 S3 Origin Department of Crop Sciences, Gottingen, S13 Germany. For more information about H10-3 see Kraling (1986) and for H48 the rest of the samples see L239 Girke (2002) - B. oleracea L. convar. capitata (L.) Alef. var. sabauda L. B. oleracea L. convar. capitata f.capitata L. B. napus L. em. Metzg. ssp. napus var. pabularia (DC.) Rehb. B. rapa L. em. Metzg. ssp. rapa B. rapa subsp. oleifera (DC.) Metzg. 4x B. oleracea convar. capitata var. sabauda B. oleracea convar. gemmifera DC. Male parent B. rapa ssp. nipposinica (Bail.) Hanelt B. rapa ssp. pekinensis (Lour.) Hanelt B. rapa ssp. pekinensis B. oleracea convar. acephala (DC.) Alef. var. sabellica L. B. oleracea convar. acephala var. medullosa Thell. 4x B. rapa ssp. nipposinica B. rapa ssp. chinensis (L.) Hanelt preferred and of less than 100 was ignored. The presence and absence of microsatellite alleles were scored manually as 1 and 0, respectively, and data were stored as binary data in a matrix. Cluster analysis The binary matrix file was used to calculate genetic similarities and to perform a cluster analysis with the software NTSYSpc v2.1 (www.exetersoftware.com) using the Dice coefficient (equal to Nei-Li equation; GSij = 2Ni,j/(2Ni,j + Ni + Nj) where GSi,j represents the similarity between the genotypes i and j, Ni,j is the total number of loci common in i and j, and Ni and Nj correspond to the number of loci found in genotypes i and j) (Nei and Li 1979). The analysis was performed as Unweighted Pair Group Method with Arithmetic mean (UPGMA). The cluster matrix was then compared to a cophenetic value matrix of the original data to produce a cophenetic correlation value as a measure of goodness of fit. Value less than 0.7 indicates very poor fit, 0.7-0.8 poor fit, 0.8-0.9 good fit and 0.9-1.0 very good fit (Rohlf 1997). Bootstrap analysis was performed using Winboot program (Yap and Nelson 1996) to verify if the number of markers was good enough to provide an accurate approximation (Hallden et al. 1994). The strength of the cluster was determined in 2,000 replicates. Results From the 40 chloroplast microsatellite primer pairs that were used to analyse each four B. oleracea and B. rapa genotypes and two B. napus genotypes, only 14 showed clear polymorphism (Table 3). The remaining ones were either monomorphic, showed no amplification (null alleles) or showed ambiguous results with two or three different alleles. In those cases, the primer pairs were not considered for the further analysis. From the eight primer pairs developed by Flannery et al. (2006) for Brassica napus, only MF-1, MF-2, MF-3, MF-4, MF-7 and MF-9 revealed polymorphism. The three primer pairs developed by Allender et al. (2007) for B. napus gave in two cases no amplification and in one case only a monomorphic peak was observed. From the six primer pairs developed by Provan (2000) for Arabidopsis thaliana, only one (ATCP28673) showed a useful polymorphism. Two out of the three primer pairs from Allender et al. (2007) and only five out of the 20 primer pairs from Jakobsson et al. (2007) for Arabidopsis revealed useful polymorphism in the test set of eight genotypes. By applying the 14 primer pairs to the whole collection of 49 genotypes, altogether 64 polymorphic cpSSR alleles were generated (Table 4). The number of detected alleles per primer pair ranged from 2 to 10. Primer pair MF-7 produced the largest number of polymorphic alleles (10 alleles) followed by MF-3 (8 alleles) and MF-4 (7 alleles). Primer pairs ATCP28673, Chlal6 and Chloro35 produced the lowest number of polymorphic allele (2 alleles for each primer combination). B. oleracea represented the most diverse cytoplasmic group with 13 different haplotypes and 1-6 alleles per locus (Table 5), followed by the B. napus group with 12 haplotypes and 1-7 alleles per cpSSR locus (Table 6). The B. rapa group represented the least diverse group with a total number of 6 haplotypes and 1-3 alleles per cpSSR locus (Table 7). Only one of the B. napus Springer Table 3 Allele sizes obtained with 40 cpSSR primer pairs applied to a test set of 8 genotypes for checking the specificity and degree of polymorphism Primer name Largo Yellow Sarson 59 Tori B. montana Vasco B. hilarionis NK Beauty-10 Express 617-4 Considered Flannery et al. 2006 MF-1 187 MF-2 199 MF-3 309 MF-4 168 MF-6 183 MF-7 175 MF-8 2 peaks MF-9 333 Provan (2000) ATCP7905 161 ATCP28673 165 ATCP30287 2 peaks ATCP46615 124 ATCP66701 Null ATCP70189 146 Jakobsson et al. (2007) 01 07 08 11 12 17 19 21 24 29 34 37 43 44 45 Null 173 279 238 301 Null 303 326 205 310 310 390 Null 476 Null 187 199 309 167 183 178 2 peaks 332 161 165 2 peaks 124 2 peaks 146 Null 173 279 238 301 Null 303 326 205 310 310 Null 335 476 Null 187 198 309 167 183 179 2 peaks 332 161 165 2 peaks 124 Null 146 Null 173 279 238 301 Null 303 326 205 310 310 390 335 476 477 195 195 310 163 183 172 2 peaks 333 161 164 2 peaks 124 2 peaks 146 Null 173 280 238 301 Null 303 327 205 310 310 390 335 476 477 195 195 310 163 183 172 2 peaks 333 161 164 2 peaks 124 2 peaks 146 133 173 279 238 301 Null 303 327 205 310 310 390 335 476 477 198 198 313 165 183 174 2 peaks 333 161 164 2 peaks 124 2 peaks 146 Null 172 279 238 301 Null 304 326 205 310 310 390 Null Null Null 195 195 310 166 183 174 2 peaks 331 161 164 2 peaks 124 Null 146 Null 173 278 239 301 Null 304 326 205 310 310 390 335 476 477 195 195 309 167 183 174 Null 332 161 164 2 peaks 124 68 146 Null 173 278 239 301 Null 303 326 205 310 310 390 335 476 477 Yes Yes Yes Yes No Yes No Yes No Yes No No No No No Yes Yes Yes No No Yes Yes No No No No No No No Genet Resour Crop Evol (2013) 60:953-965 959 o U 3 m 2; 05 a S 05 o H 01} — o o o o o Z z z z z H >h Z Z Z Z m ex, ^ Z m ex, ^ z m ex, ^ Z m Z m m ex, 3 Z m m ex, ^ Z Z cn 3 a Z cn 3 a Z cn 3 z Oh in oo o IT) IT) ^ ^ m o On m o 0< o O o a o o o o o o Chi Chi Chi Chi Chi " a .2 'd o & « £ O "P .g o S -2 3 2 Z " haplotypes (AC7) was identical with a B. oleracea haplotype (C12). B. carinata as BBCC genome species was unique in its haplotype (Table 8), but showed high similarity with the haplotype of the B. napus cultivar Korall, carrying the Brassica B genome cytoplasm (AC12; Table 6). The results from the UPGMA cluster analysis showed that at a genetic distance of 0.49 (Dice) the 49 genotypes form six major clusters (Fig. 1). Genetic similarity coefficients for the genotypes based on pairwise comparisons of cpSSR marker alleles ranged from 0.21 to 1.00. The cophenetic correlation value indicated with r — 0.92 a high goodness of fit. Bootstrap values ranged from 51.4 to 100 %. Cluster 1 comprised all B. rapa genotypes (A genome) and in addition the wild species B. cretica accession 6344 (C genome). B. oleracea genotypes were apparently more diverse and were found in clusters 2, 3 and 5. Cluster 2 did contain also the three B. napus genotypes Korall with Polima CMS cytoplasm, DSV-2-08-1 and resynthesized B. napus L239. Cluster 5 included mostly wild and cultivated B. oleracea genotypes but also the three resynthesized B. napus genotypes H48, H123-1 and H10-3. The majority of the B. napus genotypes were found in cluster 4. The two B. napus cultivars Flash and Triangle carrying the Ogura cytoplasm and the three resynthesized B. napus genotypes H61, S3 and S13 were also found in this group. Cluster 6 consisted of the two genotypes B. carinata Bral 151/90 and B. napus cultivar Korall with the B genome cytoplasm (bootstrap value — 99.8 %). Cluster 6 was most distantly related to the other clusters with the promising bootstrap value of 61.2 %. Discussion The ultimate aim of the present work was to identify a set of chloroplast microsatellite markers that could be used to unambiguously distinguish between the B. rapa and the Brassica oleracea chloroplast genomes and hence could be used as diagnostic markers to determine the cytoplasmic origin of amphidiploid Brassica napus. However, screening of 40 previously published microsatellite primer pairs for Brassica species and Arabidopsis chloroplast DNA showed that only 14 of them were useful to detect polymorphism in a test set of each four Brassica oleracea and Brassica rapa genotypes and two Brassica napus genotypes (Table 3). <£} Sprin: ger 960 Genet Resour Crop Evol (2013) 60:953-965 Table 4 Total number of polymorphic alleles among 49 genotypes using 14 selected cpSSR primer pairs Name MF-1 MF-2 MF-3 MF-4 MF-7 MF-9 ATCP28673 07 08 11 19 21 Chlal6 Chloro35 No. of alleles 4 6 8 7 10 3 2 443452 2 Table 5 Haplotypes and allele sizes (bp) detected in B. oleracea (C genome) cpSSR haplotype (n) Name MF-1 MF-2 MF-3 MF-4 MF-7 MF-9 ATCP 28673 07 08 11 19 21 Chlal6 Chloro35 CI B. cretica 187 199 310 167 173 333 165 172 280 238 303 327 110 111 C2 B. incana 187 195 311 163 171 333 165 172 280 238 303 327 109 110 C3 B. villosa 3821 187 196 307 164 178 332 164 171 280 238 307 323 110 111 C4 B. v. subsp. bivoniana 187 196 307 164 177 332 164 171 280 238 307 326 110 111 C5 B. montana 195 195 310 163 172 333 164 173 280 238 303 327 110 111 C6 B. macrocarpa 187 194 307 164 178 332 164 171 281 238 307 323 110 111 C7 B. rupestris 195 195 307 164 178 332 164 171 280 238 307 323 110 111 C8 B. taurica 187 195 310 163 172 332 164 172 279 238 303 327 109 110 C8 B. bourgeaui 187 195 310 163 172 332 164 172 279 238 303 327 109 110 C9 B. oleracea 187 195 309 163 172 332 164 172 279 238 303 327 109 110 C10 B. insularis 187 199 309 163 174 332 164 172 279 238 238 327 110 111 Cll B. hilarionis 198 198 313 165 174 333 164 172 279 238 304 326 110 111 C12 Reliant 195 195 310 163 173 332 164 173 279 238 303 327 109 110 C12 Super Regama 195 195 310 163 173 332 164 173 279 238 303 327 109 110 C12 B. alboglabra 195 195 310 163 173 332 164 173 279 238 303 327 109 110 C12 Azur 195 195 310 163 173 332 164 173 279 238 303 327 109 110 C13 Vasco 195 195 310 163 172 333 164 173 279 238 303 327 109 110 Total no. of alleles 3 5 5 4 6 2 2 3 3 1 4 3 2 2 The remaining 26 primer pairs were either monomor-phic, did not give any amplification (null alleles) or they produced doubtful results by showing two or more peaks in the capillary electrophoresis. Although the test set comprised quite diverse genotypes, it is possible that those primer pairs showing no amplification or monomorphism, could detect polymorphism in the complete set of 49 genotypes. The number of alleles detected per cpSSR locus using the primer pairs MF-1 to MF-9 ranged from 3 to 10 in the present study (Table 4), whereas Flannery et al. (2006) reported a range from 5 to 11 alleles for those primer pairs. As in the study of Flannery et al. (2006) primer pair MF7 proved to be the most polymorphic one in this study (Table 4). Surprisingly, for most of the primer pairs there was no overlap in the fragment size range of the amplicons reported by Flannery et al. (2006) and found in the present study. For locus MF-6, e.g. Flannery et al. (2006) reported 5 different alleles with a fragment size of 155 to 164 bp, whereas in the present study only one monomorphic allele was found with an allele size of 183 bp (Table 3). Furthermore, the three primer pairs ChloroO, ChloroP, and ChloroQ (Table 3) designed by Allender et al. (2007) for Brassica species, did not show amplification in PCR or produced a monomorphic band in the present test set. However, two of the three primer pairs designed by Allender et al. (2007) for A. thaliana detected each two alleles per cpSSR locus among the 49 Brassica genotypes of the present study (Table 4), although allele size ranges did not overlap. Provan (2000) demonstrated cross-species amplification in Brassica species using Arabidopsis chloroplast microsatellite primers but no allele sizes were reported. Cross-species amplification of the chloroplast microsatellite primer pairs identified in Arabidopsis was not tested by Jakobsson et al. (2007). Although not a single primer pair or a few primer pairs were useful to distinguish between the cytoplasms of Brassica rapa, B. oleracea and B. napus, the cluster analysis performed with the marker data from Springer Genet Resour Crop Evol (2013) 60:953-965 961 Table 6 Haplotypes and allele sizes detected in B. napus (AC genome) cpSSR Name MF-1 MF-2 MF-3 MF-4 MF-7 MF-9 ATCP 07 08 11 19 21 Chlal6 Chloro35 haplotype (n) 28673 AC1 Oase-3 195 195 309 166 175 AC1 Favorite-6 195 195 309 166 175 AC1 Charly-7 195 195 309 166 175 AC1 H61 195 195 309 166 175 AC1 S3 195 195 309 166 175 AC1 Korali 195 195 309 166 175 AC2 Triangle 195 195 309 166 175 AC2 Flash 195 195 309 166 175 AC2 ES Alienor 195 195 309 166 175 AC2 Krypton 195 195 309 166 175 AC3 Komando-5 195 195 309 166 174 AC3 Ha 699/91-4 195 195 309 166 174 AC4 H48 195 195 310 163 172 AC5 NK Beauty-10 195 195 310 166 174 AC6 Express 617-4 195 195 309 167 174 AC7 (CI2) H123-1 195 195 310 163 173 AC7 (CI2) H10-3 195 195 310 163 173 AC8 DSV 2-08-1 187 198 309 169 179 AC9 S13 187 195 309 166 175 AC10 L239 187 198 309 169 180 AC11 Korali" 187 198 308 169 179 AC12 Korallb 189 197 296 168 183 Total no. of alleles 3 3 4 5 7 a With Polima CMS cytoplasm b With B genome cytoplasm the 14 polymorphic chloroplast microsatellite markers revealed the existence of clearly separated groups. All individuals of B. rapa clustered together in one group (Cluster 1; Fig. 1). However, unexpectedly B. cretica as a member of the B. oleracea cytodeme clustered within the same group. The remaining B. oleracea genotypes were found in clusters 2, 3, and 5, thus confirming earlier reports describing B. oleracea as an 'incredible diverse' species (Mei et al. 2010; and references therein). Cluster 2 is linked to the B. rapa cluster 1 with a weak bootstrap value of less than 50 % (data not shown). Cluster 2 contains Brassica insularis and B. hilarionis, the spring oilseed rape cultivar Korall with the Polima male sterility cytoplasm, the resynthesized B. napus line L239 and the winter oilseed rape breeding line DSV2-08-1. The origin of the Polima cytoplasm is still unknown, but Erickson et al. (1986a) classified this cytoplasm as rap-type. According to Liu et al. (1987) the Polima cytoplasm is of Polish origin, but Erickson et al. (1986a, b) stated 331 164 173 278 239 303 326 110 111 331 164 173 278 239 303 326 110 111 331 164 173 278 239 303 326 110 111 331 164 173 278 239 303 326 110 111 331 164 173 278 239 303 326 110 111 331 164 173 278 239 303 326 110 111 331 164 172 278 239 303 326 110 111 331 164 172 278 239 303 326 110 111 331 164 172 278 239 303 326 110 111 331 164 172 278 239 303 326 110 111 331 164 173 278 239 303 325 110 111 331 164 173 278 239 303 325 110 111 332 164 172 279 238 303 327 109 110 331 164 173 278 238 304 326 110 111 332 164 173 278 239 303 326 109 110 332 164 173 279 238 303 327 109 110 332 164 173 279 238 303 327 109 110 331 164 171 279 238 304 326 110 111 331 164 173 278 239 303 326 110 111 331 164 172 279 238 303 326 110 111 332 164 172 279 238 303 326 110 111 331 164 172 278 248 303 329 109 110 2 1 3 2 3 2 4 2 2 that it is probably derived from B. juncea (AABB, n — 18). And Palmer et al. (1983) and Erickson et al. (1983) indicated that B. rapa (AA) likely is the ancestral maternal parent of amphidiploid B. juncea. Resynthesized B. napus line L239 reportedly has a B. oleracea genotype as maternal parent (Table 2; Girke 2002). However, this report is doubtful, since L239 is low in erucic acid content of the seed oil (Girke 2002) and hence L239 may be semi-synthetic derived from a cross with oilseed rape. Brassica villosa 3821, B. macrocarpa, B. rupestris and B. villosa subsp. bivoniana Mazzola et Raimondo 6581 were found in cluster 3 which is in support of Snogerup et al. (1990) who have mentioned that these species form a unique group together with B. incana and B. insularis with its origin in Sicily/Italy. However, in this study B. incana and B. insularis were separated from the Sicilian group. Brassica incana grouped together with B. oleracea subsp. oleracea 7695 and cultivated B. oleracea forms in cluster 5. The Springer 962 Genet Resour Crop Evol (2013) 60:953-965 Table 7 Haplotypes and allele sizes (bp) detected in B. rapa (A genome) cpSSR haplotype (n) Genotype MF-1 MF-2 MF-3 MF-4 MF-7 MF-9 ATCP 28673 07 08 11 19 21 Chlal6 Chi Al Rex 187 198 310 167 175 333 165 172 280 238 303 326 110 111 Al Steinadler 187 198 310 167 175 333 165 172 280 238 303 326 110 111 Al Salut 187 198 310 167 175 333 165 172 280 238 303 326 110 111 Al Perko 187 198 310 167 175 333 165 172 280 238 303 326 110 111 A2 Mizuna 187 198 310 168 179 332 165 172 280 238 303 326 110 111 A3 Tori 187 198 309 167 179 332 165 173 279 238 303 326 110 111 A4 Largo 187 199 309 168 175 333 165 173 279 238 303 326 110 111 A5 Yellow Sars 59 187 199 309 167 178 332 165 173 279 238 303 326 110 111 A6 Orbit 187 199 310 168 175 333 165 172 280 238 303 326 110 111 Total no. 1 2 2 2 3 2 1 2 2 1 1 1 1 1 of alleles Table 8 Allele sizes (bp) detected in B. carinata BRA 1151/90 (BC genome) Allele name MF-1 MF-2 MF-3 MF-4 MF-7 MF-9 ATCP28673 07 08 11 19 21 Chlal6 Chloro35 Allele size 189 197 295 168 183 331 164 160 278 248 303 329 109 110 rapa) Yellow Sarson rapa) Tori rapa) Largo rapa) Mizuna rapa) Rex . rapa) Steinadler rapa) Perko rapa) Orbit oleracea) B. cretica Dleracea) B. insularis □leracea) B. hilarionis ■ (Resyn. B. napus) LZ39 napus) DSV-2-08-1 lapus) Korall with Polirr íleracea) B. rupestris ileracea) B. v. spp. bivon íapus) Komando íapus) Ha 699/91 íapus) Oase íapus) Charly lapus) Korall (Resyn. B. napus) HS1 (Resyn. B. napus) 53 íapus) Favorite íapus) Krypton íapus) Flash with Ogura íapus) ES Alienor íapus) Triangle with Ogii ■(Resyn. B. napus) S13 íapus) N K Beauty íapus) Express 617 54.3 |~ -(Resyn. B. napus) I 3. oleracea) B. ole i. oleracea) Relia Dice coefficient (Resyn. E (Resyn. E \apus) H123 íapus) H10 ■(B. oleracea) Vasc ■ (B. oleracea) B. mo :arinata) BRA r íapus) Korall wi "~1 1.00 Fig. 1 UPGMA dendrogram of 49 different Brassica species based on cpSSR markers. Each cluster is separated with the line within the gray box at the right hand side and numbered from 1 to 6. Bootstrap values >50 % are indicated above the corresponding branch. B. r. B. rapa, B. n. B. napus, Resyn. B. n. Resynthesized B. napus, B. o. B. oleracea, CMS cytopl. Cytoplasmic Male Sterility cytoplasma Springer Genet Resour Crop Evol (2013) 60:953-965 963 separation of B. incana from the Sicilian group and its close relationship to B. oleracea subsp. oleracea has also been documented by other authors (Lazaro and Aguinagalde 1998a,b; Allender et al. 2007; Mei et al. 2010). In the present study, Brassica insularis grouped together with B. hilarionis and B. napus genotypes in cluster 2. Reports in the literature about the relationship of B. insularis to the Sicilian group are ambiguous (Lanner 1998; Lazaro and Aguinagalde 1998a,b). Most of the B. napus winter oilseed rape cultivars grouped together in cluster 4. Furthermore, two of the three resynthesized B. napus lines (S3 and S13, Table 2) with B. rapa as maternal parent clustered in this group. And as the two hybrid cultivars Flash and Triangle with the Ogura male sterility cytoplasm contain the chloroplasts of spring oilseed rape cultivar Brutor (Pelletier et al. 1983), which has previously been found to carry a rap-type cytoplasm (Song and Osborn 1992), it is not surprising that the two Ogura hybrids also cluster in group 4. Cluster 5 contains many of the wild and cultivated forms of B. oleracea. The close relationship between B. taurica, B. bourgeaui and B. oleracea subsp. oleracea 7695 is in agreement with results of Lanner et al. (1997) and Lanner (1998). Brassica montana which is also found in cluster 5 has previously been considered to be an intermediate taxon between the Sicilian group and B. oleracea subsp. oleracea (Lazaro and Aguinagalde 1998b), although RFLP-cpDNA analysis has shown that B. montana is related to both, the B. rapa and the B. oleracea cytoplasm (Song and Osborn 1992). Interestingly, cultivated forms including B. oleracea Reliant (var. capitata, Red cabbage), Super Regama (var. botrytis, Cauliflower) and Azur (var. gongylodes L., Turnip/stem cabbage) are sharing the same haplotype (C12) together with the wild species B. alboglabra. The cultivated forms are also closely related with the wild B. oleracea subsp. oleracea. Song et al. (1990) pointed out that B. alboglabra along with the wild B. oleracea subsp. oleracea can be the ancestors of cultivated forms of B. oleracea. The two resynthesized B. napus lines H123-1 and H10-3 are also found in cluster 5. This fits well to their reported origin with B. oleracea being their maternal parent (Table 2). Finally, cluster 6 is quite distantly related to all other clusters. It contains Brassica carinata BRA1151/90 and the spring oilseed rape cultivar Korall with the Brassica B genome cytoplasm. This finding is in line with early reports by Uchimiya and Wildman (1978), Erickson et al. (1983) and Palmer et al. (1983) and the recently published work of Allender and King (2010) indicating that B. carinata with the nuclear genome BBCC harbours the B. nigra (BB genome) cytoplasm. In conclusion, the results of the present study show for a new set of Brassica rapa, Brassica oleracea and Brassica napus winter oilseed rape genotypes that even with a comparatively large number of chloroplast microsatellite markers, an unambiguous differentiation of the cytoplasm types is not possible. As in previous work, oilseed rape was found to form its own cluster separated from B. oleracea and B. rapa (e.g. Erickson et al. 1983; Palmer et al. 1983; Hallden et al. 1993; Flannery et al. 2006; Allender and King 2010). Allender and King (2010) concluded that multiple hybridization events including different maternal genotypes may be the reason for this. Results from the present study also show that transferability of primer pairs from different material groups is limited, because of lack of amplification (null alleles), lack of polymorphism and the occurrence of doubtful results. Furthermore, ranges of allele sizes found in the present study deviated partly from allele sizes reported in other work using the same primer pairs. This could also be indicative for multiple hybridization events and/or for an increased mutation rate of chloroplast microsatellite markers (for discussion, see Jakobsson et al. 2007; Allender and King 2010). An increased mutation rate of chloroplast microsatellite markers could perhaps explain to some extend the development of a separate B. napus cytoplasm within a relative short time of evolution (c.f. Introduction). Bootstrap values of lower than 50 % obtained in this study indicate that relationships may change if results from more polymorphic chloroplast markers would be included in the study. However, considering the results published so far, it is questionable if chloroplast microsatellite markers are the right choice for quickly determining the cytoplasmic origin of oilseed rape genotypes. The recently established 'DNA barcode of land plants' initiative (CBOL Plant Working Group 2009; Hollingsworth et al. 2011) might point towards an easier method to distinguish among the Brassica cytoplasms by sequencing conserved regions of plastidic genes, like e.g. the rbcL- and the matK gene. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Springer 964 Genet Resour Crop Evol (2013) 60:953-965 References Allender CJ, King G (2010) Origins of the amphiploid species Brassica napus L. investigated by chloroplast and nuclear molecular markers. BMC Plant Biol 10:54-66 Allender CJ, Allainguillaume J, Lynn J, King GJ (2007) Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of Brassica oleracea L. and (n = 9) wild relatives. Theor Appl Genet 114:609-618 Bock R (2007) Structure, function, and inheritance of plastid genomes. In: Bock R (ed) Topics in current genetics. Vol 19. Cell and molecular biology of plastids. Springer, Berlin, pp 29-63 CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Natl Acad Sei USA 31:12794-12797 Chang CT, Kakihara F, Hondo K, kato M (2011) The cytoplasm effect comparison between Brassica napus and Brassica carinata on floral characteristics of Brassica oleracea. Plant Breed 130:73-79 Chen BY, HeneenWK (1989) Resynthesized Brassica napus L.: a review of its potential in breeding and genetic analysis. Hereditas 111:255-263 Cheung F, Trick M, Drou N, Lim YP, Park J-Y, Kwon S-J, Kim J-A, Scott R, Pires JC, Paterson AH, Town C, Bancroft I (2009) Comparative analysis between homoeologous genome segments of Brassica napus and its progenitor species reveals extensive sequence-level divergence. Plant Cell 21:1912-1928 Clegg MT, Gaut BS, Learn GH, Morton BR (1994) Rates and patterns of chloroplast DNA evolution. Proc Natl Acad Sei USA 91:6795-6801 Downey RK, Röbbelen G (1989) Brassica species. In: Röbbelen G, Downey RK, Ashri A (eds) Oil crops of the World. McGraw-Hill Publishing Company, New York, pp 339-362 Ebert D, Peakall R (2009) Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species. Mol Ecol Resour 9:673-690 Erickson LR, Strauss NA, Beversdorf WB (1983) Restriction patterns reveal origins of chloroplast genomes in Brassica amphidiploids. Theor Appl Genet 65:201-206 Erickson L, Grant I, Beversdorf WB (1986a) Cytoplasm male sterility in rapeseed (Brassica napus L.); 1. Restriction patterns of chloroplast and mitochondrial DNA. Theor Appl Genet 72:145-150 Erickson L, Grant I, Beversdorf W (1986b) Cytoplasmic male sterility in rapeseed (Brassica napus L.); 2. The role of a mitochondrial plasmid. Theor Appl Genet 72:151-157 Flannery ML, Mitchell FJG, Coyne S, Kavanagh TA, Burke JI, Salamin N, Dowding P, Hodkinson TR (2006) Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs. Theor Appl Genet 113:1221-1231 Girke A (2002) Neue Genpools aus resynthetisiertem Raps (Brassica napus L.) für die Hybridzüchtung. Dissertation, Georg-August-Universität Göttingen, Germany, http://webdoc. sub.gwdg.de/diss/2002/girke/girke.pdf Gladis T, Hammer K (1990) Die Gaterslebener Brassica-Kol-lektion - eine Übersicht. - Kulturpflanze 38:121-156 Gladis T, Hammer K (1992) Die Gaterslebener Brassica-Kol-lektion—Brassica juncea, B. napus, B. nigra und B. rapa. Feddes Repert 103:469-507 Gömez-Campo C, Prakash S (1999) Origin and domestication. In: Gomez-Campo C (ed) Biology of Brassica Coenospe-cies. Elsevier, Amsterdam, pp 33-58 Haider N (2011) Chloroplast-specific universal primers and their uses in plant studies. Biol Plant 55:225-236 Hallden C, Gertsson B, Sali T, Lind-Hallden C (1993) Characterization of organellar DNA in alloplasmic lines of Brassica napus L. Plant Breed 111:185-191 Hallden C, Nilsson NO, Rading IM, Sali T (1994) Evaluation of RFLP and RAPD markers in comparison of Brassica napus breeding lines. Theor Appl Genet 88:123-128 Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS ONE 6(5): el9254. doi: 10.1371/journal.pone.0019254. Accessed 26 Jan 2012 Inaba R, Nishio T (2002) Phylogenetic analysis of Brassiceae based on the nucleotide sequences of the S-locus related gene, SLR1. Theor Appl Genet 105:1159-1165 Jakobsson M, Sail T, Lind-Hallden C, Hallden C (2007) Evolution of chloroplast mononucleotide microsatellites in Arabidopsis thaliana. Theor Appl Genet 114:223-235 Kemble RJ (1987) A rapid, single leaf, nucleic acid assay for determining the cytoplasmic organelle complement of rapeseed and related Brassica species. Theor Appl Genet 73:364-370 Kode V, Mudd EA, Iamtham S, Day A (2005) The tobacco plastid accD gene is essential and is required for leaf development. Plant J 44:237-244 Kräling K (1986) Nutzung Genetischer Variabilität von resynthetisiertem Raps. Dissertation, Georg-August-Universität Göttingen, Germany Lanner C (1998) Relationships of wild Brassica species with chromosome number 2n = 18, based on comparison of the DNA sequence of the chloroplast intergenic region between trnL (UAA) and trnF (GAA). Can J Bot 76: 228-237 Lanner C, Bryngelsson T, Gustafsson M (1997) Relationships of wild Brassica species with chromosome number 2n = 18, based on RFLPs. Genome 40:302-308 Läzaro A, Aguinagalde I (1998a) Genetic diversity in Brassica oleracea L. (Cruciferae) and wild relatives (2n = 18) using RAPD markers. Ann Bot 82:829-833 Lazaro A, Aguinagalde I (1998b) Genetic diversity in Brassica oleracea L. (Cruciferae) and wild relatives (2n = 18) using Isozymes. Ann Bot 82:821-828 Liu H, Fu T, Yang S (1987) Discovery and studies on Polima CMS line. In: Proceeding of the 7th international rapeseed congress, Poznan, Poland, pp 69-78 Mei J, Li Q, Yang X, Qian L, Liu L, Yin J, Frauen M, Li J, Qian W (2010) Genomic relationships between wild and cultivated Brassica oleracea L. with emphasis on the origination of cultivated crops. Genet Resour Crop Evol 57: 687-692 Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sei USA 76:5269-5273 Palmer JD, Shields CR, Cohen DB, Orton TJ (1983) Chloroplast DNA evolution and the origin of amphidiploid Brassica species. Theor Appl Genet 65:181-189 Pelletier G, Primard C, Vedel F, Chetrit P, Remy R, Rousselle P, Renard M (1983) Intergeneric cytoplasmic hybridization in Springer Genet Resour Crop Evol (2013) 60:953-965 965 Cruciferae by protoplast fusion. Mol Gen Genet 191: 244-250 Provan J (2000) Novel chloroplast micro satellites reveal cytoplasmic variation in Arabidopsis thaliana. Mol Ecol 9:2183-2185 Provan J, Powell W, Hollingsworth PM (2001) Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends Ecol Evol 16:142-147 Rajcan I, Kasha KJ, Kott LS, Beversdorf WD (2002) Evaluation of cytoplasmic effects on agronomic and seed quality traits in two doubled haploid populations of Brassica napus L. Euphytica 123:401^109 Rana D, van den Boogaart T, O'Neill CM, Hynes L, Bent E, Macpherson L, Park JY, Lim YP, Bancroft I (2004) Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives. Plant J 40: 725-733 Rohlf FJ (1997) NTSYS-pc: numerical taxonomy and multivariate analysis system. Version 2.00. Exeter software, Setauket, New York Sasaki Y, Nagano Y (2004) Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding. Biosci Biotechnol Biochem 68: 1175-1184 Snogerup S, Gustafsson M, von Bothmer R (1990) Brassica sect. Brassica (Brassicaceae) I. Taxonomy and variation. Willdenowia 19:271-365 Song KM, Osborn TC (1992) Polyphyletic origins of Brassica napus: new evidence based on organelle and nuclear RFLP analyses. Genome 35:992-1001 Song K, Obsorn TC, Williams PH (1990) Brassica taxonomy based on nuclear restriction fragmentlength polymorphisms (RFLPs); 3. Genome relationships in Brassica and related genera and the origin of B. oleracea and B. rapa (syn. campestris). Theor Appl Genet 79:497-506 Uchimiya H, Wildman SG (1978) Evolution of fraction I protein in relation to origin of amphidiploid Brassica species and other members of Cruciferae. Heredity 69:299-303 Wang X, Liu G, Yang Q, Hua W, Liu J, Wang H (2010) Genetic analysis on oil content in rapeseed (Brassica napus L.). Euphytica 173:17-24 Weising K, Gardner RC (1999) A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. Genome 42:9-19 Wicke S, Schneeweiss GM, dePamphilis CW, Müller KF, Quandt D (2011) The evolution of the plastid chromosome in land plants: gene content, gene order, gene function. Plant Mol Biol 76:273-297 Wu JG, Shi CH, Zhang HZ (2005) Genetic analysis of embryo, cytoplasmic, and maternal effects and their environment interactions for protein content in Brassica napus L. Aust J Agric Res 56:69-73 Wu JG, Shi CH, Zhang HZ (2006) Partitioning genetic effects due to embryo, cytoplasm and maternal parent for oil content in oilseed rape (Brassica napus L.). Genet Mol Biol 29:533-538 Yap I and Nelson RJ (1996) Winboot: a program for performing bootstrap analysis of binary data to determine the confidence limits of UPGMA-based dendrogram. IRRI. Discussion paper series no 14, International Rice Institute, Manila, Philippines, http://www.irri.org/science/software/ winboot.asp Springer