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Theor Appl Genet (2010) 120:921–931 DOI 10.1007/s00122-009-1221-0 ORIGINAL PAPER Extent and structure of linkage disequilibrium in canola quality winter rapeseed (Brassica napus L.) Wolfgang Ecke • Rosemarie Clemens • Nora Honsdorf • Heiko C. Becker Received: 27 May 2009 / Accepted: 12 November 2009 / Published online: 2 December 2009 The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Linkage disequilibrium was investigated in canola quality winter rapeseed to analyze (1) the prospects for whole-genome association analyses and (2) the impact of the recent breeding history of rapeseed on linkage dis- equilibrium. A total of 845 mapped AFLP markers with allele frequencies C0.1 were used for the analysis of linkage disequilibrium in a population of 85 canola quality winter rapeseed genotypes. A low overall level of linkage disequilibrium was found with a mean r 2 of only 0.027 over all 356,590 possible marker pairs. At a significance threshold of P = 2.8 9 10 -7 , which was derived by a Bonferroni correction from a global a-level of 0.1, only 0.78% of the marker pairs were in significant linkage dis- equilibrium. Among physically linked marker pairs, the level of linkage disequilibrium was about five times higher with more than 10% of marker pairs in significant linkage disequilibrium. Linkage disequilibrium decayed rapidly with distance between linked markers with high levels of linkage disequilibrium extending only for about 2 cM. Owing to the rapid decay of linkage disequilibrium with distance association analyses in canola quality rapeseed will have a significantly higher resolution than QTL anal- yses in segregating populations by interval mapping, but much larger number of markers will be necessary to cover the whole genome. A major impact of the recent breeding history of rapeseed on linkage disequilibrium could not be observed. Introduction QTL mapping in segregating populations derived from biparental crosses has become a common tool in the analysis of quantitative traits in plants. Although this approach allows the identification of loci contributing to a quantitative trait and the estimation of their effects, it has some inherent disadvantages. First, mapping populations have to be developed specifically for the QTL mapping. In addition, only the allelic diversity sampled in the two parents of the cross can be analyzed, that is, QTL where both parents have the same allele cannot be detected. Furthermore, due to the limited number of recombination events available in a segregating population, the resolution is somewhat limited, resulting in confidence intervals for the QTL positions in the range of several cM up to several tens of cM (van Ooijen 1992 ; Darvasi et al. 1993 ). An alternative approach could be association analysis or linkage disequilibrium (LD) mapping using natural popu- lations or, in the case of crop plants, collections of varieties and breeding lines. Owing to the higher number of recombination events in such a material, a higher resolu- tion could be achieved than in segregating populations (Ewens and Spielman 2001 ; Jannink et al. 2001 ). In addi- tion, the allelic diversity would not be limited to the diversity occurring between two parental lines. Further- more, this approach could be easily integrated into the breeding process for new crop varieties. For example, Kraakman et al. ( 2004 ) used data from official Danish variety trials and mapped AFLP markers to localize QTL Communicated by M. Kearsey. Electronic supplementary material The online version of this article (doi: contains supplementary material, which is available to authorized users. W. Ecke ( & ) R. Clemens N. Honsdorf H. C. Becker Department of Crop Sciences, Georg-August-University G ¨ttingen, Von-Siebold-Str. 8, 37075 G ¨ttingen, Germany e-mail: wecke@gwdg.de 123 922 Theor Appl Genet (2010) 120:921–931 for yield and yield stability in modern two-row spring barley cultivars. Association analysis is based on the linkage disequilib- rium between linked loci and is strongly dependent on the extent and structure of the linkage disequilibrium in the population analyzed. Linkage disequilibrium, the non-ran- dom association of alleles at different loci, is created by mutation, admixture between genetically distinct popula- tions, selection and genetic drift, and decays by genetic recombination (Flint-Garcia et al. 2003 ). Accordingly, the linkage disequilibrium in a population is dependent on the population history and the mating system of the species. Linkage disequilibrium has been analyzed in a number of plant species, either globally, using molecular markers, or locally by sequencing specific genomic segments of up to several hundred kilobytes. In maize, an allogamous spe- cies, Tenaillon et al. ( 2001 ) observed a rapid decay of linkage disequilibrium within 100–200 bp in a genetically broad material of inbred lines and exotic landraces. On the other hand, in Arabidopsis thaliana, an autogamous spe- cies, linkage disequilibrium extended over about 1 cM or 250 kb in a global population of 20 accessions from dif- ferent parts of the world and the decay of linkage dis- equilibrium with distance was even lower in local populations (Nordborg et al. 2002 ). In sugar cane, where elite varieties are propagated clonally, Jannoo et al. ( 1999 ) observed linkage disequilibrium between RFLP markers to extend over up to 10 cM. Nevertheless, analyzing only inbred lines of maize Remington et al. ( 2001 ) found link- age disequilibrium extending over 1.5 kb and Rafalski ( 2002 ) reported that linkage disequilibrium in elite germ- plasm of maize extends over more than 100 kb. Analyzing linkage disequilibrium with SSR markers in the flint and dent germplasm groups used in maize hybrid breeding in Europe Stich et al. ( 2005 ) observed very high levels of linkage disequilibrium with 55 and 48% of the linked and unlinked marker pairs, respectively, in significant LD in the flint germplasm group and an average length of LD blocks of 26 cM. The levels of linkage disequilibrium were even higher in the dent germplasm group. Conversely, Kim et al. ( 2007 ) observed a rapid decay of linkage disequilibrium within 10 kb in a sample of 19 A. thaliana accession. The results from the different populations indicate that popu- lation history may be more important than the mating system in determining the level of linkage disequilibrium in a population. A strong influence of population history was also observed in barley and soybean (Caldwell et al. 2006 ; Hyten et al. 2007 ). In both crop plants, linkage dis- equilibrium extends farthest in elite breeding materials while decaying the most rapidly in wild relatives with landraces taking an intermediate position. When comparing SSR and AFLP markers, Stich et al. ( 2006 ) also found a strong influence of the marker type on the detection of linkage disequilibrium. The level of linkage disequilibrium detected in European maize inbred lines was much higher with SSR markers than with AFLP markers, presumably because the former distinguish between more alleles than the latter. Rapeseed is a partially allogamous species that is bred like an autogamous species with controlled crosses fol- lowed by several generations of selfing to develop new varieties. It gained its current importance as a major oil crop in temperate regions only after two rounds of intense selection for two new quality traits: zero erucic acid and low glucosinolate content, which were initially introduced into the breeding material from one donor genotype each in the 1960s and 1970s, respectively. Current elite breeding materials produce seed oil free from erucic acid and a meal low in glucosinolates—a quality termed ‘canola’—and are supposed to be derived from a limited number of crosses between the original genotypes with these quality traits and breeding lines of that time (Becker et al. 1999 ). Accord- ingly, the introduction of the two traits may have consti- tuted a genetic bottleneck in the breeding history of rapeseed that, together with the following intense selection for the new traits, could have had a major impact on the level and structure of linkage disequilibrium in current canola quality rapeseed materials. In rapeseed, QTL mapping in segregating populations is well established and has been used in a number of studies to analyze quality traits such as oil content (Ecke et al. 1995 ; Zhao et al. 2005 ; Delourme et al. 2006 ; Qiu et al. 2006 ; Zhao et al. 2006 ), glucosinolate content (Toroser et al. 1995 ; Uzunova et al. 1995 ), tocopherol content (Marwede et al. 2005 ), phytosterol and sinapate ester content (Amar et al. 2008 ), and the fatty acid composition of the seed oil (Thormann et al. 1996 ; Zhao et al. 2008 )as well as disease resistances such as blackleg (Pilet et al. 1998 ) or heterosis (Radoev et al. 2008 ). So far, no study has been published on the application of association anal- ysis in rapeseed or about linkage disequilibrium in rape- seed populations. The objective of this study was to determine the extent and structure of linkage disequilib- rium in canola quality winter rapeseed to (1) analyze the prospects for association analysis in current elite breeding materials of this crop plant and (2) to elucidate the impact the introduction of the ‘canola’ quality has had on the linkage disequilibrium in this material. Materials and methods Plant materials Linkage disequilibrium was analyzed in a set of 85 Northern European canola quality winter rapeseed varieties 123 Theor Appl Genet (2010) 120:921–931 923 and breeding lines (Table 1 ), further called LD population. For the analysis, one individual plant per variety was used. For genetic mapping, a mapping population of 94 doubled haploid lines derived from one F 1 plant of a cross between the winter rapeseed variety ‘Express’ and a resynthesized rapeseed, ‘R53’, was used. This population had already been used to develop a genetic map in rapeseed comprised mainly of SSR markers (Radoev et al. 2008 ). DNA preparation and AFLP analysis Table 1 Origin of the 85 canola quality varieties and breeding lines used in the analysis of linkage disequilibrium in rapeseed Variety Breeder Variety Breeder DNA was prepared from 0.1 g of leaf material of 3 weeks old greenhouse grown plants using Nucleon PhytoPure extraction kits (RPN8510, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) following the manufacturer’s instructions. The EcoRI primers used in AFLP analysis were labeled with one of the following four fluorescent dyes: (6, 5) FAM, NED, VIC, or PET (Applied Biosystems, Darmstadt, Germany). AFLP analyses were carried out following the protocol of Vos et al. ( 1995 ) modified for multiplexing in the PCR according to F. Kopisch-Obuch (personal communication): 250 ng DNA were digested in 30 llRL buffer (10 mM Tris–acetate, 10 mM Mg–acetate, 50 mM K–acetate, 5 mM DTT, pH 7.5) with 4 U EcoRI (Fermentas, St. Leon-Rot, Germany) and 4 U MseI(New England Biolabs, Frankfurt, Germany) for 1.5 h at 37. After adding 10 ll of a mix containing 5 pmol EcoRI adapter, 50 pmol MseI adapter, 1 mM ATP and 1 U T4-DNA ligase (Promega, Mannheim, Germany) in RL buffer, DNA and adapters were ligated in a time series of different temperatures (3 h 10 min 37, 3 min 33.5, 3 min 30, 4 min 26 and finally 15 min 22). The final restriction–ligation product (RL) was diluted 1:5 with HPLC grade water. For pre-amplification, 8 ll of the diluted RL was added to 12 ll of a reaction mixture, giv- ing final concentrations of 19 Taq buffer (Solis Biodyne, Tartu, Estonia, Reaction buffer B), 3.125 mM MgCl 2 , 0.45 mM dNTPs, 10 pmol EcoRI?1 primer, 9 pmol MseI?1 primer and 2.5 U Taq DNA polymerase (FIREPol, Solis Biodyne). The pre-amplification was carried out in a Biometra T1 Thermocycler (Biometra GmbH, G ¨ ttingen, Germany) with the following program: 94 for 30 s, 20 cycles of 94 for 30 s, 56 for 30 s and 72 for 2 min, and a final 5 min at 72. The pre-amplification product was diluted 1:10 with HPLC grade water. The final AFLP amplification used 6 ll of the diluted pre-amplification product in a total reaction volume of 20 ll containing 19 Taq buffer, 0.36 mM dNTPs, 3.125 mM MgCl 2 ,1UTaq polymerase, 7 pmol MseI?3 primer, 2 pmol of (6, 5)FAM labeled EcoRI?3 primer, 2 pmol of VIC labeled EcoRI?3 primer, 4 pmol of NED labeled EcoRI?3 primer, and 6 pmol of PET labeled EcoRI?3 primer. The protocol for the Thermocycler was as follows: 1 cycle of 94 for 1 min, 65 for 30 s, and 72 for 2 min, 12 cycles of 94 for 30 s, 64.2 for 30 s and 72 for 2 min, 25 cycles of 94 for 30 s, 56 for 30 s and 72 for 2 min, and finally 72 for 5 min. Alesi KWS Magnum Syngenta Remy KWS Madrigal Syngenta Robust KWS Laser Syngenta Alaska KWS Fortis Syngenta Pirola KWS Smart Syngenta Adder KWS Roxet Syngenta Milena KWS NK Bravour Syngenta Allure KWS NK Fair Syngenta Agalon KWS Aviso SW Seed K615 KWS Sansibar SW Seed KW1519 KWS SWGospel SW Seed Picasso KWS Verona SW Seed Lord KWS Tenor SW Seed KW3077 KWS Expert SW Seed Rodeo KWS Musette SW Seed Rapid Limagrain-Nickerson Kvintett SW Seed Boston Limagrain-Nickerson Falstaff SW Seed Escort Limagrain-Nickerson SW Sinatra SW Seed Montego Limagrain-Nickerson Viking NPZ Ontario Limagrain-Nickerson Aragon NPZ Pacific Limagrain-Nickerson Aurum NPZ Savannah Limagrain-Nickerson Lorenz NPZ Missouri Limagrain-Nickerson Baros NPZ Manitoba Limagrain-Nickerson Rasmus NPZ Ladoga Limagrain-Nickerson Gefion NPZ Atlantic Limagrain-Nickerson Nugget NPZ Cooper Limagrain-Nickerson Zephir NPZ Licapo DSV SLM 0413 NPZ Capitol DSV SLM 0512 NPZ Idol DSV LSF 0519 NPZ Vivol DSV HSL 1032 NPZ Bristol DSV Campari NPZ Lirajet DSV Caramba NPZ Lisabeth DSV Express 617 NPZ Lipid DSV Prince NPZ Lipton DSV Wotan NPZ Lisek DSV Amor Petersen/Raps GbR Contact DSV Orlando Saaten Union Columbus DSV Pollen Adrien Momont Lion DSV Ascona SW Seed Oase DSV Duell Raps GbR Apex Syngenta Jessica – Recital Syngenta KWS KWS SAAT AG; DSV Deutsche Saatveredelung AG; NPZ Nord- deutsche Pflanzenzucht Hans-Georg Lembke KG 123 924 Theor Appl Genet (2010) 120:921–931 The AFLP products were separated on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) using 50-cm capillary arrays and GeneScan-500 LIZ size standard (Applied Biosystems). GeneMapper v3.7 software (Applied Biosystems) was used for a semi-automatic marker scoring. Since in GeneMapper v3.7’s output, AFLP primer combinations are written as markers and the actual AFLP markers as alleles of these markers a Perl script, ‘Extract_marker’, was developed to transform GeneMap- per’s output into a marker matrix. The primer combinations used, the labels of the EcoRI primers and the numbers of markers identified with the different primer combinations are listed in Table S1. analyses were carried out in Microsoft Office Excel 2007. The threshold for declaring linkage disequilibrium between two markers significant was derived by a Bonferroni cor- rection from a global a-level of 0.1, resulting in a per test threshold of P = 2.8 9 10 -7 . Results Marker analysis and map construction By using 132 primer combinations 2161 AFLP markers could be scored in the mapping population. In the LD population, 1,463 of these markers were also polymorphic and 898 showed allele frequencies equal to or larger than 0.1. Of the markers with allele frequencies C0.1 in the LD population 845 could be mapped in the mapping popula- tion. The AFLP markers were mapped within a framework of 167 markers from the earlier map that had been estab- lished in the mapping population by Radoev et al. ( 2008 ). After the initial map construction, the markers were dis- tributed across 21 linkage groups. By mapping some additional markers from the full set of 2,161 markers, the map could be consolidated in 19 linkage groups, a number corresponding to the 19 chromosomes of the haploid rapeseed genome. Based on map alignments using the SSR markers from the earlier map, 18 of the linkage groups could be named according to the N-nomenclature of rapeseed linkage groups. The last linkage group was des- ignated as N8 by exclusion. The final map (Table 2 ) has a length of 2,473 cM and comprises 1,032 markers distrib- uted across 551 map positions. Included are 865 new AFLP markers that cover 2,345 cM (95%) of the total map. Individual linkage groups range in length from 77 to 242 cM, holding between 27 and 132 markers. The full map is listed in Table S2. Genetic mapping Genetic mapping of the AFLP markers was based on a framework map previously established in the mapping population by Radoev et al. ( 2008 ). Using the program MAPMAKER/EXP V.3.0b, the new markers were assigned to linkage groups by the ‘near’ command with an LOD threshold of 4.0 and a maximum recombination frequency of 0.4. Linkage groups were then reanalyzed using the ‘order’ command. Finally, markers that could not be placed by the ‘order’ command were manually placed using the ‘try’ command. Double crossovers were identified using MAPMAKER’s ‘genotype’ command and were rechecked in the trace files and, if necessary, corrected, followed by a remapping of the affected markers. Markers with high numbers of double crossovers and markers with strongly disturbed segregations where one class was represented by fewer than 25 genotypes were excluded from the mapping. Linkage groups were named according to the N-nomen- clature proposed by Parkin et al. ( 1995 ). Recently, a new nomenclature was proposed by the Steering Committee of the Multinational Brassica Genome Project ( . In this nomenclature, A1–A10 correspond to N1–N10 and C1–C10 to N11–N19. Levels of linkage disequilibrium Analysis of linkage disequilibrium Linkage disequilibrium in canola quality winter rapeseed was analyzed using pairwise combinations of the 845 AFLP markers with allele frequencies C0.1 in the LD population. With a mean r 2 value of only 0.027 over all 356,590 possible pairwise combinations, the overall level of linkage disequilibrium in the rapeseed genome is very low (Table 3 ). This conclusion is reinforced by the obser- vation that only 0.78% of marker pairs are in significant LD. With a mean r 2 of 0.122 linkage disequilibrium among physically linked marker pairs, that is pairs where both markers are on the same linkage group, is nearly five times higher than the overall mean. Furthermore, 11.58% of these marker pairs are in significant LD and with a count of 2,658 represent the vast majority of marker pairs in For the analysis of linkage disequilibrium, only markers with allele frequencies in the LD population of 0.1 or larger were used. This discrimination against rare alleles is jus- tifiable because the information from them is neither useful in the analysis of linkage disequilibrium nor in association analysis. R 2 values of linkage disequilibrium for all pair- wise marker combinations and the corresponding signifi- cance levels (P values) were calculated using the program TASSEL V.2.0.1 (Zhang et al. 2006 ). Recombination fre- quencies between marker pairs were calculated by a Perl script and added to the corresponding rows of the LD table generated by TASSEL. All further statistical and graphical 123 Theor Appl Genet (2010) 120:921–931 925 Table 2 Summary of the genetic map with the AFLP markers used in the analysis of LD in rapeseed Linkage group No. of markers No. of map pos. Length (cM) a New AFLP markers Framework markers b N1 33 25 118 26 7 N2 28 21 141 20 8 N3 46 26 122 39 7 N4 32 19 78 27 5 N5 50 30 174 43 7 N6 54 26 135 45 9 N7 41 17 77 37 4 N8 27 16 77 24 3 N9 65 26 133 55 10 N10 37 21 83 27 10 N11 132 49 132 122 10 N12 52 30 161 45 7 N13 82 52 242 67 15 N14 63 28 147 53 10 N15 51 30 142 39 12 a Recombination frequencies were transformed to centimorgan according to the Kosambi mapping function b Markers from the map of Radoev et al. ( 2008 ) N16 44 28 130 35 9 N17 54 34 124 43 11 N18 74 32 140 62 12 N19 67 41 117 56 11 Sum 1032 551 2473 865 167 Table 3 Number of marker pairs and average level of LD (mean r 2 ) in different classes of marker pairs Class a All pairs in the class Pairs in significant LD at P = 2.8 9 10 -7 n (%) b Mean r 2 n (%) c Mean r 2 All 356,590 (100.00) 0.027 2,775 (0.78) 0.722 Linked 22,951 (6.44) 0.122 2,658 (11.58) 0.729 Unlinked 333,639 (93.56) 0.020 117 (0.04) 0.544 a All: all marker pairs, linked: pairs of markers from the same linkage group, unlinked: pairs where the markers are on different linkage groups b Percentage from all (356,590) marker pairs c Percentage from all marker pairs in the class significant LD indicating that the major determinant of linkage disequilibrium in the rapeseed genome is genetic linkage. Accordingly, only 117 of the unlinked marker pairs are in significant LD and at 0.544, the mean r 2 of distance (Fig. 1 b). Closely linked marker pairs at recom- bination frequencies of 0–2% show high levels of linkage disequilibrium with mean r 2 values ranging from 0.566 to 0.374, but at a recombination rate of 5%, the mean r 2 is already down to 0.1 and at high distances, it is not sig- nificantly different from the overall mean of 0.027. Like- wise, the fraction of marker pairs in significant LD decays from 65 to 48% for closely linked marker pairs to 6% at a recombination rate of 5% and 1–3% at intermediate recombination rates (6–20%). With the exception of two marker pairs at 24 and 27%, no marker pairs in significant LD are found at higher recombination rates. The rapid decay of linkage disequilibrium is also apparent when looking at the distribution of linkage dis- equilibrium across individual linkage groups (Fig. 2 a). Colors indicative of high LD are close to the diagonal these marker pairs is still lower than the mean r 2 of 0.729 of the linked marker pairs in significant LD. Structure of linkage disequilibrium To investigate the structure of linkage disequilibrium in the rapeseed genome, the dependency of linkage disequilib- rium on distance was analyzed among the physically linked marker pairs. The number of marker pairs at recombination rates from 0 to 50% ranges from 126 to 1,554 (Fig. 1 a) providing a solid base for this analysis. Among the linked marker pairs, linkage disequilibrium decays rapidly with 123 [ Pobierz całość w formacie PDF ] |