Extending the rapeseed genepool with resynthesized Brassica napus L. I: Genetic diversity

Andreas Girke 0 1 Antje Schierholt 0 1 Heiko C. Becker 0 1 0 A. Schierholt H. C. Becker (&) Department of Crop Sciences, Georg August Universitat Gottingen , von Siebold Strasse 8, 37075 Gottingen, Germany 1 A. Girke Norddeutsche Pflanzenzucht Hans-Georg Lembke KG, Inselstrasse 15, 23999 Malchow/Poel, Germany Resynthesized Brassica napus L. could be employed to increase the genetic variation in the narrow oilseed rape genepool and to establish a genetically distant genepool for hybrid breeding. One important criterion for selecting appropriate resynthesized lines is their genetic distance to adapted breeding material. In this study we estimated the genetic distances in a wide collection of 142 resynthesized lines and 57 winter and spring rapeseed cultivars from Europe, North America and Asia using RFLP marker data. The highest in-group genetic distance (0.36) was observed in 142 resynthesized lines compared to 0.21 in winter, 0.23 in summer, and 0.28 in Asian genotypes. The group of adapted breeding material clustered into three groups of winter-, spring-type, and Asian genotypes. Resynthesized lines did not form distinct subgroups in the cluster analysis, but large differences were revealed in the genetic distance of resynthesized lines to the adapted winter oilseed rape genepool. The highest distance to winter oilseed rape was found in resynthesized lines with parental lines from the Asian genepool as B. oleracea convar. botrytis var. alboglabra (Bail.) Sun or B. rapa ssp. chinensis (L.) Hanelt. - The narrow genetic base of oilseed rape (Brassica napus L.; 2n = 38; AACC) limits the prospects for hybrid breeding since the optimal utilization of heterosis requires complementing genepools. The present breeding material was derived from very few interspecific crosses between cabbage (Brassica oleracea L.; 2n = 18; CC) and turnip (B. rapa L.; 2n = 20; AA) that occurred spontaneously some centuries ago. Hybridizations between different subspecies of B. rapa and B. oleracea allow the construction of resynthesized B. napus with favorable alleles from the unadapted ancestral parents. Examples for this strategy of introgression of single traits are reviewed by Qiong et al. (2009). Apart from single traits, the utilization of artificially resynthesized B. napus could be one option for increasing genetic variation by means of the development of genetically distinct genepools for hybrid breeding. Becker et al. (1995) investigated genetic distances between resynthesized lines and B. napus varieties with RFLP and allozyme markers and suggested that resynthesized lines might be a valuable source for broadening the genetic base of the present breeding material of B. napus. Seyis et al. (2003) analysed genetic distances of six families of resynthesized lines, spring oilseed and fodder rape cultivars with AFLP-marker. Udall et al. (2004) described the introgression of resynthesized B. napus into hybrid spring canola as a source of novel alleles for the improvement of canola spring hybrids. However, the resynthesis lines poor agronomic performance and undesired seed quality traits such as high seed erucic acid and glucosinolate content complicated the direct introduction of resynthesized lines into hybrid breeding programs. Backcrossing genetically diverse resynthesized lines into well adapted breeding material to establish a heterotic genepool for hybrid breeding is a labor intensive approach and a long-term perspective, and appropriate resynthesized lines would have to be selected carefully. One important criterion for the selection of such lines is their genetic distance to adapted breeding material. Therefore, in this study, we have evaluated the genetic distance in a wide collection of 142 resynthesized lines, which are based on nearly all cultivated types of B. oleracea and B. rapa as parents, and in 57 winter and spring rapeseed cultivars from Europe, North America and Asia using molecular markers. Materials and methods Plant material A collection of 199 B. napus genotypes included 142 resynthesized B. napus lines originating from the Georg August Universitat Gottingen, Freie Universitat Berlin, Dansk Planteforaedling and Svalof Weibull AB (Online Resource 1). The B. napus resyntheses were obtained from hybridizations of different B. oleracea and B. rapa subspecies (Table 1, Online Resource 1). The set of genotypes was completed with 32 winter- and 13 spring-type B. napus varieties and 12 genotypes of Asian origin (Online Resource 2, Fig. 2). The taxonomic classification was carried out according to Hanelt (2001). RFLP analyses Plants for DNA extraction were cultivated in a greenhouse where a mixed leaf sample (10 g) of 10 plants per genotype was harvested. DNA was extracted in accordance with Rogers and Bendich (1988). Genomic DNA was restricted with the enzymes EcoRI and HindIII (Pharmacia Biotech) and transferred onto nylon membranes (Southern 1975). RFLP analyses were performed as described by Uzunova et al. (1995) using 71 informative DNA probes (Knaak 1996). Twenty-three probe-enzymecombinations were selected which revealed in 199 genotypes 127 (98.5%) out of 129 RFLP-fragments as polymorphic. On average, 5.5 informative fragments were evaluated per probe-enzyme-combination. Genetic distances among genotypes based on RFLP markers were estimated in accordance with Nei and Li (1979) using the similarity index of Dice (1945). Genotypic groups were differentiated in a cluster analysis as described by Backhaus et al. (1990; average linkage), and dendrograms were verified by cophenetic correlations as a measure of goodness of fit (Sneath and Sokal 1973). The principle coordinates analysis was performed according to Backhaus et al. (1990). All statistical analyses were performed using NTSYS-pc 2.1 software (Rohlf 2000). Genetic distances within the three groups of winter, spring-type, and Asian B. napus were significantly smaller than between these groups (Table 2). Mean genetic distances were 0.21 in winter and 0.23 in spring-type B. napus. Maximum genetic distances of 0.39 (between Accord and Mansholts) and 0.35 (between Tanto and Iris), respectively, were estimated. The highest in-group genetic variation (0.36) was observed in 142 resynthesized lines. The principle coordinates analysis (Fig. 1) of 142 resynthesized lines and 57 B. napus varieties and lines from Asia and Europe revealed high genetic variation in the resynthesized lines, which were almost evenly distributed over all quadrants. Winter and spring-type B. napus were most easily differentiated by the second principle coordinate, which explained 6.8% of the variation. Winter B. napus varieties mainly clustered Table 1 Overview of the parental Brassicaceae (taxonomic classification according to Hanelt 2001) and the extent of use as maternal or paternal genotype in 142 resynthesized lines (for details see Online Resource 1) maternal genotype paternal genotype Brassica oleracea L. convar. oleracea L. convar. (...truncated)