Theor Appl Genet (1996) 93:1017-1025

9 Springer-Verlag 1996

N. F o i s s e t 9 R. D e l o u r m e 9 P. B a r r e t . N. H u b e r t B. S. L a n d r y 9 M . R e n a r d

Molecular-mapping analysis in Brassica napus using isozyme, RAPD and RFLP markers on a doubled.haploid progeny

Received: 15 January 1996 / Accepted: 19 July 1996

We have undertaken the construction of a Brassica napus genetic map with isozyme (4%), RFLP (26.5%)

Abstract

and RAPD (68%) markers on a 152 lines of a doubled-haploid population. The map covers 1765 cM and comprises 254 markers including three PCR-specific markers and a morphological marker. They are assembled into 19 linkage groups, covering approximatively 71% of the rapeseed genome. Thirty five percent of the studied markers did not segregate according to the expected Mendelian ratio and tended to cluster in eight specific linkage groups. In this paper, the structure of the genetic map is described and the existence of non-Mendelian segregations in linkage analysis as well as the origins of the observed distortions, are discussed. The mapped RFLP loci corresponded to the cDNAs already used to construct B. napus maps. The first results ofintraspecific comparative mapping are presented. Brassica napus 9 L. Genetic mapping 9 RFLP and RAPD markers 9 Segregation distortions 9 Comparative mapping Key words

Introduction The expansion of molecular tools has led to the construction of genetic maps for a large number of plant species over the last 10 years. Many species of the Cruciferae faro-

Communicated by G. Wenzel N. Foisset 1 . R. Delourme (~) . R Barret 9M. Renard INRA, Station d'Am61ioration des Plantes, BP 29, 35650 Le Rheu, France N. Hubert 1 - B. S. Landry 1 Agriculture Canada, 430 Boul. Gouin, St-Jean-sur-Richelieu, J3B3E6, Quebec, Canada Present address:

1 DNA LandMarks Inc., RO. Box 6, St-Jean-sur-Richelieu, J3B6Z1, Qu6bec, Canada

fly, a world-wide plant group of agronomical and economical importance, have benefited from this approach, and especially the species constituents of the U (1935) triangle. Thus, genetic maps have been published for the diploid species Brassica oleracea (Slocum et al. 1990; Kianian and Quiros 1992; Landry et al. 1992; Bohuon et al. 1994; Chyi et al. 1994; Le Corre et al. 1994), B. rapa (McGrath and Quiros 1991; Song et al. 1991; Chyi et al. 1992; Teutonico and Osborn 1994), and B. nigra (Truco and Quiros 1994; Lagercrantz and Lydiate 1995). With the exception of B. carinata, the amphidiploid species, B. hapus (Landry et al. 1991; Lydiate et al. 1993; Chyi et al. 1994; Ferreira et al. 1994; Parkin et al. 1995; Sharpe et al. 1995; Uzunova et al. 1995) and B. juncea (Mohapatra et al. 1995) have also been mapped. Most of the genetic maps were constructed from F 2 populations although some recent Brassica maps were derived from doubled-haploid populations (Bohuon et al. 1994; Ferreira et al. 1994; Parkin et al. 1995; Sharpe et al. 1995; Uzunova et al. 1995) since most of them, and especially B. napus, are species amenable to in vitro androgenesis. DH progenies represent a reference population on which genetic mapping can be easily coupled to quantitative trait studies. A DH population, compared to an equal sized F 2 population, is also recommended for genetic mapping when dominant markers are mainly used since a better estimate of the recombination frequency is obtained. For all the published Brassica maps, markers were mainly RFLP-derived from cDNA libraries, therefore mapping coding genomic regions. However, the availability of new types of markers has recently led to a diversification of the markers employed (Truco and Quiros 1994). Each type of marker has its merits and drawbacks (price, ease of use, scoring of co-dominants, availability in large numbers, relation to loci of known function) and their use is dependent on laboratory facilities as well as the aim of the research program. The existing genetic maps of Brassica have already been used for an interspecific comparison of genome organisation (McGrath and Quiros 1991; Lydiate et al. 1993, Chyi et al. 1994; Parkin et al. 1995). Comparisons

1018 b e t w e e n species were enlarged within the Cruciferae through the analysis o f the w e l l - k n o w n Arabidopsis thaIiana ( K o w a l s k i et al. 1994), a useful m o d e l s y s t e m for basic genetic studies, for w h i c h m a n y genetic maps have been p u b l i s h e d ( K o o r n n e e f et al. 1983; Chang et al. 1988; N a m et al. 1989; Reiter et al. 1992; H a u g e et al. 1993; Lister and D e a n 1993; M c G r a t h et al. 1993). B y contrast, to our k n o w l e d g e , no intraspecific c o m p a r i s o n s have been p u b l i s h e d although this m a y be an interesting w a y to d e t e r m i n e g e n o m i c regions associated with c h r o m o s o m a l rearrangements. The present paper reports on the d e v e l o p m e n t o f a Brassica napus genetic m a p on a d o u b l e d - h a p l o i d population. The parental lines were chosen on the basis of two criteria: (1) high p o l y m o r p h i s m level b e t w e e n them, and (2) the occurrence o f m a n y segregating a g r o n o m i c traits. A cross b e t w e e n 'Darmor-bzh' and ' Y u d a l ' was selected since it was segregating for o l i g o g e n i c , as well as p o l y g e n i c , traits concerning plant d e v e l o p m e n t (dwarfism, earliness o f flowering), seed quality (erucic acid and g l u c o s i n o l a t e s contents) and disease resistance (to Phoma lingam, Cylin-

drosporium concentricum).

Isozyme analysis Nine isoenzymatic systems were tested: acid phosphatase (APS), aconitase (ACO), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6 PGD), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), shikimic dehydrogenase (SDH) and triosephosphate isomerase (TPI). The isozyme nomenclature was that of Ch~vre et al. (1995). All systems except APS were tested after electrophoresis on starch gels. Preparation of starch gels and total protein extraction from young leaves have been described in Ch~vre et al. (1995). The gel/electrode buffers proposed by Shields et al. (1983) were used: morpholine citrate pH 6.1 (buffer G) for LAP, MDH, and 6 PGD; histidine tris citrate pH 7.0 (buffer E) for ACO, PGI and PGM; tris citrate borate pH 7.5 (buffer N) for SDH; and tris citrate lithium borate pH 8.3 (buffer C) for TPI. Staining procedures were as described by Arus and Orton (1983) for LAP, Wendel and Stuber (1984) for ACO, and Vallejos (1983) for the others. APS was run on acrylamide gels. Acrylamide gel electrophoresis conditions were as described in Ch~vre et al. (1995). DNA extraction Young leaves were harvested from each DH line (several plants were collected) and kept at -80~ until DNA extraction. Leaves were ground in a mortar with liquid nitrogen. DNA was extracted as described in Doyle and Doyle (1990).

S e g r e g a t i o n studies were m a i n l y done with R A P D markers due to the ease o f the technique and in order to RAPD and PCR-specific analysis m a p r a n d o m l y coding, as well as n o n - c o d i n g , sequences (Williams et al. 1990). I s o z y m e s , c o m m o n l y used in our RAPD reactions were performed in 1.9 mM MgCI2, 150 gM dNTP l a b o r a t o r y were mapped. R F L P markers c o r r e s p o n d i n g (Boerhinger), 0.2 ~tM primer, 0.5 unit Taq polymerase (Eurobio), to selected c D N A probes a l r e a d y used for the construction 1.25 gl lOxTaq polymerase buffer (Eurobio) and 12.5 ng genomic of B. napus m a p s (Landry et al. 1991; D i o n and Landry, DNA. The final volume was adjusted to 12.5 gl with sterile double distilled H20. DNA was amplified in a 480 or 9600 Perkin Elmer u n p u b l i s h e d data) were e m p l o y e d , The R F L P markers represented e v e n l y spaced l a n d m a r k s o f the B. napus ge- Cetus DNA thermocycler, using a cycling profile of 30 s at 94~ followed by 45 cycles of 30 s at 94~ 1 min at 35~ and 3 min at 72~ h o m e and were used to speed up the d e v e l o p m e n t of our 9 At the end of the 45 cycles, the reactions were held at 72~ for 5 rain. map. Amplified products were separated on 1.8% agarose gels prepared in 1 xTAE buffer. Gels were run 5 h at 3 V/cm and then stained in a Here w e report on the m a n a g e m e n t of markers showing distorted segregation ratios in l i n k a g e analysis. The inter- 0.5/,tg/ml of ethidium bromide solution. Random primers were from Operon Technologies Inc. They are est o f using different types o f m a r k e r s is discussed, and designated by an 'OP' prefix, followed by the kit letter and primer first results of intraspecific c o m p a r i s o n s using R F L P m a r k - number. RAPD loci are then specified by adding the size (in base pair) of the corresponding amplified DNA. When RAPD markers are ers are described. co-dominant, the sufix 'cd' followed by a number (indicative of the number of co-dominant markers obtained with the same primer) is added to the name instead of the molecular weight of the two bands. Three specific-PCR markers were also mapped: Slg, a self-incompatibility gene isolated from B. oleracea (Brace et al. 1994), the Materials and methods Fad3.1900 locus, a gene coding for a A15 desaturase (Arondel et al. 1992), and the riga 162 locus, a microsatellite of A. thaliana (Bell Parental lines and mapping progeny and Ecker 1994). A doubled-haploid (DH) population was derived from isolated microspore cultures (as described by Polsoni et al. 1988) of a single F~ hybrid plant obtained from the cross 'Darmor-bzh'x'Yudal'. 'Darmor-bzh' is a dwarf isogenic line (BC3F3) resulting from the introduction of the dwarf Bzh gene (Foisset et al. 1995) in a French Winter cultivar, 'Darmor'. 'Yudal' is a Spring Korean line (F9) that behaves as an early-flowering winter type in temperate climates. More than 400 DH lines were produced either by spontaneous chromosome doubling or colchicine treatment (Foisset et al. 1996). A total of 152 DH lines were randomly selected, from lines with good seed set, for segregation analyses and genetic mapping. A DH population of this size provides estimates of the recombination frequency (7) so that the standard deviation S 7 is < 1/2 7for every 7>0.03 (which is equivalent to an F2 population of 90 individuals scored with co-dominant markers). Isozyme assays on fresh leaves and DNA extraction for RAPD and RFLP markers were performed on HI plants (first generation of selfing of the derived DH lines).

RFLP analysis A total of 56 RFLP probes representing loci evenly spaced on the B. napus maps established by Landry et al. (1991) and Dion and Landry (unpublished data), were selected, and the corresponding cDNA probes used. Most of them correspond to anonymous clones from a cDNA library from a B. napus embryo (Harada et al. 1988), and were designated as 1ND 1 to 6NF3. Four seedling-specific cDNA clones named pCal5, pCa25, pGs43 and pCot44 (Harada et al. 1988) and a cruciferin cDNA clone, pC1 (Simon et al. 1985), corresponding to a major storage protein in Brassica, were also used as probes. Probe preparation and Southern blot analyses were as described in Cloutier et al. (1995). Only one restriction enzyme, HindIII, was used. Duplicated loci detected by a single clone were designated by the same name followed by a different lower case letter as described in Landry et al. (1991).

1019 Genetic linkage analysis Goodness of fit to expected Mendelian ratios for each segregating locus was tested by chi-square analysis (a=l%). Markers with too great distortion values (chi-square>50) were excluded. Linkage analyses were performed on Mapmaker/exp version 3.0 (Lincoln et al. 1992). Linkage groups were first established with a minimum LOD score of 4.0 and a maximum recombination frequency of 0.4. Markers within each linkage group were then ordered using only highly informative and evenly spaced markers ("Compare" command, LOD>3.0). The remaining markers were sequentially placed ("Try" command, LOD>2.0) using first the most informative markers. The framework was verified with the "Order" command (LOD>3.0). The best one was confirmed by permuting all adjacent triplets of markers ("Ripple" command, LOD>2.5). Arrangements of the linkage groups with distorted segregation ratios were confirmed by a chisquare (Mather 1957) to test the independence of two segregations, conditional on their marginal frequencies; tests (c~=1%) were applied between pairs of markers within each group and between groups. Distances were re-estimated with the product formula of Bailey (1949). Centimorgan distances were expressed with the Kosambi function (Kosambi 1944).

RFLP markers From the 56 clones directly tested on both the parental and the progeny DNA cut with only one enzyme (HindIII), nine were monomorphic; that is to say, 84% of them gave at least one segregating band in the DH progeny. Each probe revealed on average 1.6 markers (1 to 3 per probe). Sixty nine RFLP loci were used for mapping. Fifty one loci gave a co-dominant scoring. The others were analysed as dominant due to the overlapping of bands with a monomorphic locus or the absence of the alternate band. Nineteen probes detected two to three polymorphic loci. Thus, the majority of the probes detected only one polymorphic locus while hybridizing to additional monomorphic bands. Only one probe, named 1NH6, displayed a hybridization profile that would correspond to a single-copy D N A sequence.

Segregation analysis in the doubled-haploid progeny

Results Polymorphism observed between 'Darmor-bzh' and 'Yudal' and marker characteristics Only markers that were reproducible, and consequently could be scored unambiguously on the corresponding DH progeny, were analysed. The polymorphism level was expressed as a function of the isozyme system, the RAPD primer or the RFLP probe that detected at least one polymorphic locus.

In a DH population, a 1:1 Mendelian segregation ratio is expected when no selection occurs. Out of the 266 markers assayed, 34.6% did not segregate according to this expected Mendelian ratio (~=1%), with an equal number of loci favouring the alleles of each parental line. The three types of genetic markers revealed some loci with biased segregation [isozyme (30%), RFLP (30%) and RAPD (36.6%)] and the proportions of loci with a distorted segregation ratio were not significantly different between the three types of marker (chi-square test value for contingency--2.1, P=35.1%).

Linkage analysis with markers showing non-Mendelian segregation

Isoenzymatic markers From the nine systems tested, all but one (PGI) were polymorphic between the two parental lines, which represented an isozyme polymorphism level of 89%. Two loci for the M D H and TPI systems were polymorphic. A total of ten isoenzymatic loci were then mapped. Isozyme markers corresponded to co-dominant allozymes but six were scored as dominant due to unresolved alleles that migrated to the same location as a band of a monomorphic locus. Duplicated loci have been demonstrated in rapeseed for all systems studied (Delourme and Foisset 1991).

RAPD markers A total of 266 RAPD primers, randomly chosen, were tested. Most of them (89.5%) were polymorphic between 'Darmor-bzh' and 'Yudal'. Each primer gave on average 1.9 markers (1 to 5 per primer). Then, 183 RAPD markers revealed with 95 primers were analysed on the DH progeny, of which ten were scored as co-dominant.

Linkage groups and frameworks were first established with the Mapmaker program. Loci with biased segregation were included in the analysis. The use of a LOD>4.0 generated 17 linkage groups containing at least four markers, from a raw data file of 266 markers. One of these groups was in fact a spurious linkage group that was split into three distinct groups - DY6, DY7 and DY11. These three groups are quite exclusively made up with markers that show segregation distortion in favour of 'Yudal' alleles. This phenomenon arose early during the construction of the map when less markers were available. During these preliminary analyses, two groups were established: one corresponding to DY7 and another one to the association of DY6 and DY11. Through the addition of new markers, DY6 and DY11 could be split, but three markers (3NF1 la, 1NG2a and 1NH6a) of the final DY6 group remained at the end of DY 11 (Fig. 1A). Chi-square tests of independence were in fact not significant ( P > l % ) between those three markers and the other markers of the DY11 group, implying independence. Bailey's estimate confirmed the inaccuracy of the distances given by Mapmaker (Fig. 1A). Then, linkage was detected by chi-square test between those three mark-

1020 Fig. 1 Linkage group construction with distorted markers and correction. A Linkage group DY11 constructed with Mapmaker without considering the existence of distortions. Comparison to distances derived by the estimate of Bailey. Inconsistancies between both estimates, as well as doubtful markers, are indicated in bold face type. B Structure of linkage groups DY11 and DY6 constructed by integrating distortion. Distances are expressed in Kosambi cM

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Genetic linkage map The genetic linkage map derived from the cross 'Darmorbzh' x 'Yudal' comprised 254 markers assembled into 19 linkage groups (Fig. 2). From the 266 segregating loci tested, 12 remained unlinked. The linked loci covered 1765 cM; linkage groups varied in size from 6.5 to 206 cM, with an average spacing of 7 cM between loci. Spacing between markers ranged from 0 to 37.5 cM. Markers with distorted segregation ratios tended to cluster in eight specific linkage groups, each of the clusters comprising loci exclusively favouring the alleles of the same parental line (Fig. 2). Four of these groups contained loci with an excess of 'Yudal' alleles (DY6, DY7, DY 11 and DY 15) whereas the four others were skewed towards 'Darmor-bzh' alleles (DY4, DY5, DY13 and DY18).

The genetic map was constructed mostly with RAPD markers (68% of the segregating markers) but also included isozyme (4%) and RFLP (26.5%) loci. A microsatellite derived from Arabidopsis thaliana (Bell and Ecker 1994) was also mapped (nga 162, linkage group DY18). The distribution of each kind of tested marker was generally even on the linkage groups, although DY3, DY7, DY17 and DY18 were essentially composed of RAPD markers. If the markers were randomly distributed throughout the genome, the number of markers within each linkage group should be directly correlated with the length of the group; the longer a linkage group is, the more markers it should have. The correlation coefficient r (calculated without the loci positioned on the map by targeted mapping), between the size of the linkage groups and their number of loci (r=0.79; P=10 -4) confirmed this hypothesis. However, DY2, DY3 and DY4 deviate slightly from the linear regression model; the number of markers per 10 cM varied from 0.7 for DY2 and DY3 to 2.1 for DY4. Most markers were of unknown function except for the isozyme loci that are involved in the metabolic pathway of plant cells and five cDNA clones corresponded to the specific genes described in Materials and methods. A morphological marker (Bzh, group DY6) which corresponds to a dwarf B. napus gene (Foisset et al. 1995) was also localized on the linkage map, as well as two specific PCR markers including Slg (linkage group DY10), a B. oIeracea selfincompatibility gene (Brace et al. 1994) and Fad3.1900 (linkage group DY14; Jourdren et al. 1996a), a A15 desatIb Fig. 2 Linkage map of B. napus. Linkage groups are named DY1 to DY 19 in decreasing order of size. Map distances, in Kosambi cM, are indicated on the right side of the linkage groups and locus name (according to the nomenclature described in Materials and methods) is given on the left. Names of loci with biased segregation are preceded by an asterisk and the letter D (for 'Darmor-bzh') or Y (for 'Yudal') according to the favoured parental line

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