More QTL for flowering time revealed by substitution lines in Brassica oleracea A. M. RAE, E. C. HOWELL & M. J. KEARSEY* Plant Genetics and Cell Biology Group, School of Biological Sciences, The University of Birmingham, Birmingham B15 2TT, U.K. Seventy-nine recombinant backcross substitution lines from a cross between Brassica oleracea var. italica and Brassica oleracea var. alboglabra were grown in field trials over five years along with the alboglabra recurrent parent. Plants were scored for the days from sowing to the opening of the first flower, and lines that flowered significantly earlier or later than the recurrent parent were identified. Based on the lengths of the substitutions, evidence for 11 QTL on chromosomes O1, O2, O3, O5 and O9 was found, five of which mapped to similar regions to five of the six found in a previous analysis of doubled haploid lines from the same cross. Several of the QTL were linked closely in repulsion. Keywords: Brassica, flowering time, QTL analysis, substitution lines. Introduction Brassica oleracea is an agriculturally important species including many vegetables such as cabbage, broccoli and cauliflower. It is closely related to the model dicoty- ledonous plant Arabidopsis thaliana and so it is expected that information concerning control of basic biological processes in A. thaliana will be transferable to Brassica crops. Information should also be transferable between dierent species within the Brassica genus, as the three diploid genomes, A, B and C, of B. rapa, B. nigra and B. oleracea, respectively, are thought to have derived from the same ancestor. These genomes reveal striking conservation of content, although chromosome duplication and translocation have occurred during divergence (Lagercrantz & Lydiate, 1996). The amphi- diploid B. napus (oilseed rape) genome is made up of the A and C genomes probably from close relatives of B. rapa and B. oleracea. (U, 1935; Parkin et al., 1995). Flowering time is not only of scientific interest because it facilitates the understanding of plant devel- opment, it is also important in agriculture, because its modification may enable the geographical range of the Brassica crop to be extended. For example, cultivation of B. napus is usually restricted to temperate latitudes, but the development of early flowering cultivars would allow the crop to be grown in low-rainfall regions such as the Western Australian wheat belts (Thurling & Depittayanan, 1992), and more northern regions of Canada (Murphy & Scarth, 1994). Genes aecting flowering time have been identified in A. thaliana by mutagenesis (Koornneef et al., 1991). For example, mutations of the CONSTANS gene cause delayed flowering under long days, but not short, and the gene has been cloned by chromosome walking (Putterill et al., 1993, 1995). Two regions influencing flowering time in B. nigra (on LG2 and LG8) have been found to be homologous to the CONSTANS gene region (Lagercrantz et al., 1996). These regions have also been shown to carry quantita- tive trait loci (QTL) in the A genome of B. napus, and show large-scale colinearity between regions on chro- mosomes O2, O3 and O9 of B. oleracea. Previous studies mapping QTL for flowering time in B. oleracea have been reported. For example, Kennard et al. (1994) found significant QTL using single-factor ANOVA ANOVA on an F 2 from a cabbage · broccoli cross, whereas Camargo & Osborn (1996) used F 3 families from a dierent cross between cabbage and broccoli. Because of the lack of both a standardized nomenclature for linkage groups in Brassica species and of common probes, it is not possible to compare the QTL locations found in these studies. Another drawback to these studies is the limited life-span of the populations used. The heterozygosity of individuals prevents their main- tenance by selfing, so trials can only be carried out once in a single environment. Bohuon et al. (1998) overcame this by using doubled haploid lines derived from the F 1 of a cross between a Chinese kale and a hybrid of *Correspondence. E-mail: [email protected]Heredity 83 (1999) 586–596 Received 17 March 1999, accepted 27 May 1999 586 Ó 1999 The Genetical Society of Great Britain.
Transcript
More QTL for ¯owering time revealed bysubstitution lines in Brassica oleracea
A. M. RAE, E. C. HOWELL & M. J. KEARSEY*Plant Genetics and Cell Biology Group, School of Biological Sciences, The University of Birmingham, Birmingham
B15 2TT, U.K.
Seventy-nine recombinant backcross substitution lines from a cross between Brassica oleracea var.italica and Brassica oleracea var. alboglabra were grown in ®eld trials over ®ve years along with thealboglabra recurrent parent. Plants were scored for the days from sowing to the opening of the ®rst¯ower, and lines that ¯owered signi®cantly earlier or later than the recurrent parent were identi®ed.Based on the lengths of the substitutions, evidence for 11 QTL on chromosomes O1, O2, O3, O5 andO9 was found, ®ve of which mapped to similar regions to ®ve of the six found in a previous analysis ofdoubled haploid lines from the same cross. Several of the QTL were linked closely in repulsion.
Keywords: Brassica, ¯owering time, QTL analysis, substitution lines.
Introduction
Brassica oleracea is an agriculturally important speciesincluding many vegetables such as cabbage, broccoli andcauli¯ower. It is closely related to the model dicoty-ledonous plant Arabidopsis thaliana and so it is expectedthat information concerning control of basic biologicalprocesses in A. thaliana will be transferable to Brassicacrops.
Information should also be transferable betweendi�erent species within the Brassica genus, as the threediploid genomes, A, B and C, of B. rapa, B. nigra andB. oleracea, respectively, are thought to have derivedfrom the same ancestor. These genomes revealstriking conservation of content, although chromosomeduplication and translocation have occurred duringdivergence (Lagercrantz & Lydiate, 1996). The amphi-diploid B. napus (oilseed rape) genome is made up of theA and C genomes probably from close relatives ofB. rapa and B. oleracea. (U, 1935; Parkin et al., 1995).
Flowering time is not only of scienti®c interestbecause it facilitates the understanding of plant devel-opment, it is also important in agriculture, because itsmodi®cation may enable the geographical range of theBrassica crop to be extended. For example, cultivationof B. napus is usually restricted to temperate latitudes,but the development of early ¯owering cultivars wouldallow the crop to be grown in low-rainfall regions suchas the Western Australian wheat belts (Thurling &
Depittayanan, 1992), and more northern regions ofCanada (Murphy & Scarth, 1994).
Genes a�ecting ¯owering time have been identi®ed inA. thaliana by mutagenesis (Koornneef et al., 1991). Forexample, mutations of the CONSTANS gene causedelayed ¯owering under long days, but not short, andthe gene has been cloned by chromosome walking(Putterill et al., 1993, 1995).
Two regions in¯uencing ¯owering time in B. nigra (onLG2 and LG8) have been found to be homologous tothe CONSTANS gene region (Lagercrantz et al., 1996).These regions have also been shown to carry quantita-tive trait loci (QTL) in the A genome of B. napus, andshow large-scale colinearity between regions on chro-mosomes O2, O3 and O9 of B. oleracea.
Previous studies mapping QTL for ¯owering time inB. oleracea have been reported. For example, Kennardet al. (1994) found signi®cant QTL using single-factorANOVAANOVA on an F2 from a cabbage ´ broccoli cross,whereas Camargo & Osborn (1996) used F3 familiesfrom a di�erent cross between cabbage and broccoli.Because of the lack of both a standardized nomenclaturefor linkage groups in Brassica species and of commonprobes, it is not possible to compare the QTL locationsfound in these studies. Another drawback to thesestudies is the limited life-span of the populations used.The heterozygosity of individuals prevents their main-tenance by sel®ng, so trials can only be carried out oncein a single environment. Bohuon et al. (1998) overcamethis by using doubled haploid lines derived from the F1
of a cross between a Chinese kale and a hybrid of*Correspondence. E-mail: [email protected]
Heredity 83 (1999) 586±596 Received 17 March 1999, accepted 27 May 1999
586 Ó 1999 The Genetical Society of Great Britain.
calabrese, resulting in six QTL for ¯owering time beingmapped to chromosomes O2, O3, O5 and O9.The use of doubled haploid plants still has the
limitation that segregating populations can map QTLonly to a relatively large region of a chromosome. Thispresent paper reports work which reinforces the resultsof Bohuon et al. (1998) by using the same cross toengineer near-isogenic lines. Repeated backcrossing andsel®ng of the progeny from this cross have enabled theproduction of lines that contain small segments of donorparent DNA within a recurrent parent background(Ramsay et al., 1996). Di�erences in phenotype betweenthe lines and the recurrent parent should be caused bythe substituted region. The substitution lines should behomozygous so that the lines can be maintained andmultiplied by sel®ng allowing the same lines to be grownin di�erent environments so that experiments can berepeated over a number of years and conditions. Theprecision to which a QTL can be mapped depends uponthe size of the introgressed region, but comparisonsbetween di�erent substitution lines can narrow theseregions further.
Materials and methods
Production of experimental lines
The parent plants were descended from microspore-derived doubled haploid lines. The recurrent parent isA12DHd, derived from Brassica oleracea var. albogla-bra, and the donor is GDDH33, which is Brassicaoleracea var. italica, derived from a commercialcalabrese F1 hybrid variety, Green Duke (Bohuon et al.,1996). For simplicity, the doubled haploid lines used asparents will be referred to as A12 and GD, respectively.The substitution lines were produced (as described in
Ramsay et al., 1996) by using a single F1(A12 ´ GD) topollinate three A12 plants to produce the ®rst backcrossgeneration (BC1). A12 was used as the female to ensurethat all cytoplasm from the ®rst backcross generationand subsequent generations was A12 in origin. Lineswere selected for a high number of purely recurrentlinkage groups, wide coverage of the genome by thedonor introgression and duplications of introgressedregions. Lines were backcrossed again and then selfedtwo or three times to produce lines with homozygousregions of introgressed GD DNA in an A12 back-ground.Seventy-eight RFLP probes selected from those
previously described by Bohuon et al. (1996) toconstruct a linkage map from doubled haploid linesproduced from the F1 of this cross, were used to mapthe substituted regions in the lines together withan additional 119 AFLP markers (Sebastian et al.
1999). This gave an average spacing between markersof 4.5 cM. The positions of the introgressed GD DNAin the 79 substitution lines are shown in Appendix 1.The minimum substitution indicates the region knownto be GD DNA, whereas the maximum substitutionincludes the region between A12 and GD markers inwhich recombination has occurred. It is not yetpossible to de®ne the proportion of this region whichis GD, so the whole section must be taken intoaccount.
Trial plan
Eight ®eld trials were carried out over a period of ®veyears; one each in 1994 and 1995, two each in 1996,1997 and 1998. Lines were used in trials as theybecame available from the breeding programme. Overthe ®ve years, 79 substitution lines, with varyingnumbers of replicates, were grown alongside therecurrent and the donor parents. Table 1 shows thesowing dates and the number of lines and replicatesfor each trial.Seeds were sown in 5 cm plastic pots, using John
Innes compost no. 3, in the glasshouse and placed in asingle block with complete individual plant randomiza-tion, surrounded by guard plants. This design was usedto maximize the power to detect QTL (Kearsey & Pooni,1996). The glasshouse was unheated and unlit so thattemperature and daylength depended on natural condi-tions.After nine days the number of germinated seeds was
noted, and plants were thinned from two to one perpot. Any pots in which no seed germinated werereplaced with guard plants from the same line. Thepots were moved to an open-ended polythene tunnelafter 15 days for acclimatization. After 23 days theseedlings were planted in the ®eld in the same randomposition. The ®eld was set out in rows of 100 plants,
Table 1 The sowing dates for the eight Brassica oleraceatrials over the ®ve years, with the number of lines and thenumber of replicates. A12 and GD are the parent lines andSL denotes the substitution lines
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spaced 76 cm between rows and 31 cm between plantsin the rows. Guard plants were grown around theperimeter and in the spaces left by dead plants. Thewhole trial area was protected by nylon netting toprevent bird damage. Plants were irrigated during dryperiods, and chemical treatments were used to controlpests.
Data collection
Flowering was checked daily, and scored as the numberof days from sowing to the ®rst ¯ower opening on eachplant. Data from plants that were badly a�ected bycabbage root ¯y or grey aphid were removed. Othertraits were also recorded, but their analysis will bepublished elsewhere.
Data analysis
1994, 1995 and 1996 trials Four one-way analyses ofvariance (ANOVAANOVAs) were carried out to test for variationbetween lines within each trial in 1994, 1995 and 1996.The within-lines mean squares from these ANOVAANOVAs wereused to carry out Tukey±Kramer multiple comparisontests (Dunnett, 1980), to determine which substitutionlines ¯owered signi®cantly earlier or later than the A12recurrent parent.
1997 trials Seeds for each substitution line in both trialsin 1997 were taken from two di�erent parent plantswhich had been grown in two completely randomizedblocks in glasshouses. This was to allow maternal e�ectsin the progeny to be estimated. The two ®eld trialscontained the same number of plants from the sameparent plants so that variation between trials could alsobe examined.
The data for both trials were standardized to meanzero and variance one, to minimize genotype-by-environment e�ects arising from di�erent ranges in¯owering time, so allowing the trials to be analysedtogether. A cross-classi®ed hierarchical ANOVAANOVA wascarried out for the substitution lines on the standardizeddata to test for interactions between genotypes andenvironments. This was repeated for the A12 parents inthe two trials. The within-lines mean square for theseANOVAANOVAs were combined for use in a Tukey±Kramermultiple comparison test.
1998 trials The lines used in the 1998 ®eld trials wereselected as being earlier or later ¯owering than A12based on the analysis of lines in previous years. Thisallowed the use of one-tailed tests to compare each lineagainst the recurrent A12 parent. The data from eachtrial were standardized permitting the trials to beanalysed together.
Results
1994, 1995 and 1996 trials
The ANOVAANOVAs for the four trials in 1994, 1995 and 1996indicated signi®cant variation between lines withintrials, implying that the di�erent substitutions of GDa�ected the ¯owering time of the lines.
The Tukey±Kramer test showed the lines SL112,SL113, SL140, SL175 in 1994; SL113, SL129, SL140,SL172 in 1995; SL102 in 1996 trial 1; and SL102, SL128,SL137, SL157, SL172, SL175 and SL178 in 1996 trial 2were signi®cantly later ¯owering (at the 5% level) thanthe A12 parent. Line SL108 was signi®cantly earlier¯owering than A12 in 1995.
1997 data
The mean and standard deviation for ¯owering timefor trial 1 and trial 2 (�x� 64.889, s� 2.527 and �x�65.476, s� 4.041, respectively) were used to standardizethe data.
The cross-classi®ed hierarchical ANOVAANOVA (Table 2a)showed that the variation between lines was highlysigni®cant (P < 0.001), and that replicate familieswithin lines also di�ered (0.01 > P > 0.05). The latterindicates the importance of maternal e�ects. The inter-action of lines with trials, and the families within linesinteraction with trials, were not signi®cantly di�erentfrom each other (1.67/1.12� 1.49, P > 0.05), althoughfamilies within lines did interact with trials signi®cantlywhen compared to the error (P < 0.01). As expected,the variation between trials was very small (P > 0.05),any di�erence from zero being attributed to unequalnumbers of observations in each trial.
A similar ANOVAANOVA was carried out for A12 recurrentparent plants (Table 2b), showing that the variationbetween di�erent families within the A12 line and thevariation between trials were not signi®cant. However,interaction between trials and families was just signi®-cant despite the scalar transformation.
In order to carry out the Tukey±Kramer multiplecomparison, the mean square `between families withinsubstitution lines' combined with the mean square`between families' for the A12 was used as the errorvariation. The Tukey±Kramer test showed that linesSL115, SL134, SL141 and SL175 were signi®cantly later¯owering than the A12 recurrent parent.
1998 data
Flowering data for the two trials in 1998 were stan-dardized, using the mean and standard deviation for thetwo trials (�x� 68.417, s� 3.742 and �x� 64.257, s� 3.551,
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respectively). ANOVAANOVAs were carried out using the com-bined data from the two trials. Lines SL115, SL121,SL127, SL133, SL134, SL138, SL141, SL142, SL158,SL172, SL175 and SL177 were shown to be signi®cantlylater ¯owering than the A12 parent, whereas SL108 andSL122 were signi®cantly earlier ¯owering.
Signi®cant substitution lines
Table 3 shows the mean of the A12 parent in each year,together with the means for each line that di�eredsigni®cantly in ¯owering time from the A12. Over theeight trials it can be seen that only two substitution lines(SL108 and SL122) were signi®cantly earlier ¯owering,whereas 21 were signi®cantly later ¯owering than theA12 parent (SL102, SL112, SL113, SL115, SL121,SL127, SL128, SL129, SL133, SL134, SL137, SL138,SL140, SL141, SL142, SL157, SL158, SL172, SL175,SL177, SL178).
Discussion
Seventy-nine lines were grown over the ®ve years. Ofthese, only two lines showed evidence for earlier¯owering than A12, whereas 21 lines were signi®cantlylater ¯owering. This bias was to be expected becausesections of chromosomes from the late ¯owering GDparent had been substituted into the early ¯owering A12recurrent parent.
Of the 23 lines that showed signi®cant variation in¯owering behaviour from the A12 parent, 10 have asingle substitution, four have two substitutions, eighthave three regions and one has four regions substitutedwith GD. The following interpretation should be read inconjunction with Fig. 1
Lines with one substitution
Any variation between the A12 and the 10 linescontaining just one substitution must result from GDDNA in this region, therefore such lines enable theGD DNA region to be treated as a single segregatingfactor. Because it is not possible to de®ne theproportion of GD DNA in the region in whichrecombination has occurred, it was assumed that anyQTL present may be anywhere within the maximumsubstitution.The late ¯owering line SL102 had a maximum GD
substitution in the region between 0.0 and 35.7 cM onchromosome O1, but the early ¯owering line SL108 hada minimum substitution between 30.3 and 35.7 cM, so itis unlikely that a late ¯owering QTL was present in thisregion (Fig. 1, O1). It is therefore most likely that therewas a late ¯owering QTL between 0.0 and 30.3 cM andan early ¯owering QTL between 30.3 and 38.1 cM onchromosome O1.Lines SL128 and SL133 both showed late ¯owering
behaviour. Assuming the presence of just one QTL on
Table 2 (a). The cross-classi®ed hierarchical ANOVAANOVA for the Brassica oleracea substitution lines in 1997 trials 1 and 2. TheF-value is taken to be nonsigni®cant (NS) when P > 0.05; *indicates 0.05 > P > 0.01, **indicates 0.01 > P > 0.001;***indicates P < 0.001 (b). The cross-classi®ed ANOVAANOVA for the A12 recurrent parent in 1997 trials 1 and 2(a)
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chromosome O3, it is likely to be located in the region inwhich the substitutions in these lines overlap, i.e.between 30.5 and 43.3 cM, although the presence of asecond QTL in this region is discussed later (Fig. 1, O3).
Line SL157 suggested the presence of a late ¯oweringQTL between 0.0 and 34.2 cM on chromosome O5 inGD (Fig. 1. O5).
The ®ve late ¯owering lines with single substitutionson chromosome O9 suggest three separate regions towhich QTL may be mapped. Lines SL142, SL175 andSL178 have substitutions which overlap between 70.8and 106.8 cM; line SL172 has a substitution between 0.0and 43.0 cM; and line SL177 has a substitution between43.0 and 64.4 cM (Fig. 1, O9).
If these seven QTL are assumed to be present in theregions described, inferences can now be made aboutthose lines which contain more than one QTL.
Lines with more than one substitution
Of the 13 signi®cant lines with more than one GDsubstitution, the behaviour of 11 can be explained by
QTL found in lines with single substitutions. Inferencesfor QTL in the two lines SL122 and SL127 cannot bemade using substitution line data alone, but are discus-sed below.
Comparison to previous work
Previous work carried out on this A12 ´ GD cross byBohuon et al. (1998) analysed data from ®eld trials usinga mapping method based on marker regression (Kearsey&Hyne, 1994). Thismethod showed evidence for sixQTLover the two trials analysed in 1994. Single QTL werefound on chromosomes O2 and O3, whereas chromo-somes O5 and O9 both showed evidence for two QTL.
Figure 1 provides a comparison of the doubledhaploid and substitution line data. The QTL are`named' according to their chromosome and positionin the two doubled haploid trials simply to facilitatecomparisons (see Bohuon et al., 1998).
Chromosome O1
The doubled haploid data showed no evidence for QTLon chromosome O1, whereas the substitution line datashowed the presence of two QTL; one late ¯oweringbetween 0.0 and 30.3 cM and one early ¯owering between30.3 and 38.1 cM. As the regions to which these twoputative QTLs map are adjacent to each other, they arelikely to cancel each other in the doubled haploid lines.
Chromosome O2
The QTL FTO2.1, which had a late ¯owering allelefrom GD, was located at 78 � 9 cM on chromosomeO2. Eight of the substitution lines have introgressedGD DNA at this position. Of these, four ¯oweredsigni®cantly late, one ¯owered early and three did notshow signi®cant variation from A12. The ®ve thatshowed signi®cant ¯owering variation all have substi-tutions on other chromosomes which may be a�ecting¯owering time. SL112 and SL113 are thought tocontain a region coding for late ¯owering on chro-mosome O3; SL121 contains late ¯owering regions onchromosomes O1 and O9; and the early ¯owering ofSL122 may be caused by a QTL mapped in the regionof FTO9.1 in the doubled haploid lines. It is possiblethat SL127 is re¯ecting the late ¯owering e�ect ofFTO2.1. It may also be possible that lines SL112 andSL113 show this e�ect as well as the late ¯oweringQTL on chromosome O3, because the mean ¯oweringdates for lines SL112 and SL113 were 17 and 16 dayslater than A12, respectively, in 1994, whereas theother signi®cantly late lines in this year ¯owered just12±13 days later than A12.
Table 3 The mean ¯owering date for the Brassica oleracearecurrent parent, A12, and the substitution lines thatshowed signi®cantly di�erent ¯owering behaviour fromA12 over the ®ve years. Bold type denotes those lines thatwere signi®cant and `Ð' indicates that the line was notraised in that year
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Fig. 1 (see caption on p. 593)
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Fig. 1 (see caption on p. 593)
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Chromosome O3
The late ¯owering lines SL128 and SL133 have singlesubstitutions which overlap, suggesting that FTO3.1 isin the overlapping region, but the maximum substitutionin the late ¯owering line SL115 only just overlaps themaximum substitution in SL133. There is no evidencethat the GD substitutions on chromosome O1 or O6 inSL115 a�ect the ¯owering behaviour of this line,therefore there may be two QTL a�ecting ¯oweringtime on this chromosome. This view is supported by thesize of the e�ects of the QTL on this chromosome. Thelines SL115 and SL133 ¯owered 2 and 1.5 days laterthan the A12 parent, respectively, in 1998, whereas lineSL134, which overlaps both regions, ¯owered 6 dayslater than the A12 parent, implying the presence of twoQTL in this line. The presence of a second QTL onchromosome O3 may explain the large con®denceinterval of FTO3.1 in the doubled haploid data.
Chromosome O5
FTO5.1 maps to 16 � 13 cM, whereas FTO5.2 maps to40 � 10 cM. The substitution line data suggest that
there is a QTL between 0.0 and 34.2 cM. This regionoverlaps with the con®dence intervals for both FTO5.1and FTO5.2, so the substitution lines neither con®rmnor reject the two-QTL hypothesis.
Chromosome O9
The doubled haploid data indicated the presence of twoQTL on chromosome O9; an early QTL (FTO9.1) at46 cM in trial 1 and 36 cM in trial 2, and a late QTL(FTO9.2) at 74 cM in trial 1 and 94 cM in trial 2. Themarker regression approach used on these doubledhaploid data does not give con®dence intervals whenmore than one QTL is mapped to a chromosome. Thesubstitution-line data map a late ¯owering QTL between70.8 and 106.8 cM, which coincides with the doubledhaploid QTL FTO9.2.The doubled haploid data did not reveal the two late
QTL found in the substitution lines in the regions of0.0±43.0 cM and 43.0±64.4 cM, but did detect an earlye�ect at 46 cM in trial 1 and 36 cM in trial 2. This maybe the cause of early ¯owering in SL122, which has aGD substitution between 23.1 and 47.0 cM on chromo-some O9. The late ¯owering line SL129 completely
Fig. 1 The positions of introgressed regions in the Brassica oleracea substitution lines on chromosomes O1, O2, O3, O5 and O9.Minimum GD regions are shown as a box, whereas the maximum region, including where recombination occurred, is shown asa single line. The number of substitutions contained by each line is noted. The signi®cantly late ¯owering lines are shaded in black,
whereas those that were signi®cantly early ¯owering are shaded in grey. The regions to which the substitution lines best map QTLare shown by vertical, dotted lines. For comparison, the regions to which the doubled haploid data mapped QTL are also shown.
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overlaps the region to which this early ¯owering QTL ismapped, but it is possible that this line contains the twolate ¯owering QTLs at this end of chromosome O9,which mask the e�ect of early ¯owering.
There is some evidence that the doubled haploid linesshow segregation distortion in favour of GD in theregion of the early QTL, which may explain why theearly QTL alone is detected in the doubled haploid data.
Precision of mapping
The doubled haploid data mapped QTL with 95%con®dence intervals that varied between 18 and 52 cM.Usually a con®dence interval of 30 cM is thought to bereasonable for analysis of segregating populations. Inthe substitution lines the interval to which a QTL can bemapped relies on the size of the substitution and also thedistance between markers on the linkage map. Substi-tution lines that showed signi®cant e�ects for ¯oweringtime contained introgressed GD DNA that ranged inlength from 8 cM to 43 cM, showing that this method ofmapping QTL does enable e�ects to be mapped moreprecisely. The substitution line approach identi®ed atleast 11 QTL compared to the six detected by thedoubled haploids. Substitution lines identi®ed moreQTL, because they detected e�ects that had beenmasked in the doubled haploid population throughdispersion and close linkage.
It is possible that there are still more QTL for¯owering time segregating in this cross. Some of thesubstitution lines contained introgressed regions as longas 84 cM, so it is quite feasible that such large regionsmay contain more than one QTL, which mask eachother's e�ect. The substitution lines grown over the ®veyears represent 62% of the entire GD genome, althoughthe actual proportion may be larger because thiscalculation does not include the region within whichrecombination has occurred. Only 9.0% of the GDgenome is de®nitely not present in the substitution lines.This implies that there may well be QTL in the regionsthat are not represented in the substitution lines.
Comparison to other genomes
The use of the linkage map produced by Bohuon et al.(1996) in this study has enabled a comparison of resultsto be made. This map has also been used to compare theC genomes of B. oleracea and B. napus, and colinearityof the large segments of the A and C genomes inB. napus (Parkin et al., 1995). Field experiments involv-ing Brassica napus populations have indicated thatregions on linkage groups in the A genome which showhomology to the regions in O2, O3 and O9 also carryQTL for ¯owering time (Keith, 1996; Salinas-Garcia,
1996; Osborn et al., 1997). These regions have also beenshown to be homologous with regions on LG2 and LG8of B. nigra, to which a ¯owering time QTL has beenmapped (Lagercrantz et al., 1996) and to the regionaround the CONSTANS gene and several other ¯ower-ing candidate genes on chromosome 5 in A. thaliana.These regions of homology have been found to map tothe regions of B. oleracea chromosomes O2, O3 and O9,and to show similar e�ects of late ¯owering in GD(Bohuon et al., 1998). These appear to be the same asthe substituted regions in the present study that alsoshowed evidence of QTL for late ¯owering in GD.
The substitution line data showed similar results to thedoubled haploid data, but were not as easily interpreted.To maximize the bene®ts of the use of substitution lines,it would be advantageous to backcross lines further toreduce the size and number of introgressed GD regionsin each line, and to mapmore markers to the linkage mapto enable reduction of the regions in which recombina-tion is presumed to have occurred.
Other measurements
Measurements for height and number of nodes at¯owering were also taken in the eight substitution linetrials, as these have been found to have a positivecorrelation with ¯owering time (Bohuon, 1996). Late¯owering tends to be a result of extended vegetativegrowth rather than stunted or delayed growth (Kowal-ski et al., 1994). Future work will involve the analysis ofthese traits and a comparison to ¯owering time data.
Acknowledgements
The authors particularly thank Judith Craft and SueBradshaw for technical support. Thisworkwas supportedby the UK Biotechnology and Biological Sciences Coun-cil (BBSRC) research grant number 6/G02511.
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