Theor Appl Genet (1995) 91:802-808

9 Springer-Verlag 1995

D. T o r o s e r 9 C. E. T h o r m a n n 9 T. C. O s b o r n R. M i t h e n

RFLP mapping of quantitative trait loci controlling seed aliphaticglucosinolate content in oilseed rape (Brassica napus L.)

Received: 6 February 1995 / Accepted: 12 May 1995

We report the RFLP mapping of quantitative trait loci (QTLs) which regulate the total seed aliphaticglucosinolate content in Brassica napus L. A population of 99 Fl-derived doubled-haploid (DH) recombinant lines from a cross between the cultivars Stellar (low-glucosinolate) and Major (high-glucosinolate) was used for singlemarker analysis and the interval mapping of QTLs associated with total seed glucosinolates. Two major loci, GSL-1 and GSL-2, with the largest influence on total seed aliphatic-glucosinolates, were mapped onto LG 20 and LG 1, respectively. Three loci with smaller effects, GSL-3, GSL-4 and GSL-5, were tentatively mapped to LG 18, LG 4 and LG 13, respectively. The QTLs acted in an additive manner and accounted for 71% of the variation in total seed glucosinolates, with GSL-1 and GSL-2 accounting for 33% and 17%, respectively. The recombinant population had aliphatic-glucosinolate levels of between 6 and 160 pmoles per g-1 dry wt of seed. Transgressive segregation for high seed glucosinolate content was apparent in 25 individuals. These phenotypes possessed Stellar alleles at GSL-3 and Major alleles at the four other GSL loci demonstrating that low-glucosinolate genotypes (i.e. Stellar) may possess alleles for high glucosinolates which are only expressed in particular genetic backgrounds. Gsl-elong and Gsl-alk, loci which regulate the ratio of individual aliphatic glucosinolates, were also mapped. Gsl-elong-1 and Gsl-elong-2, which control elongation of the o~-amino-acid precursors, mapped to LG 18 and LG 20 and were coincident with GSL loci which regulate total seed aliphatic glucosinolates. A third tentative QTL, which regulates side-chain elongation, was tentatively mapped to LG 12. Gsl-alk, which regulates H3CS- removal and side-chain de-saturation, mapped to LG 20. Abstract

Communicated by G. E. Hart D. Toroser - R. Mithen (~) The John Innes Centre, Brassica and Oilseeds Research, Colney Lane, Norwich NR4 7UH, UK C. E. Thormann 9T. C. Osborn Department of Agronomy, University of Wisconsin, Madison, WI 53706, USA

Key words Aliphatic glucosinolates 9Brassica napus 9 Restriction fragment length polymorphism (RFLP) 9 Genomic mapping

Introduction Oilseed rape (Brassica napus L.) is one of the most important crops in the world. It is grown primarily for its oil which is used both for nutritional and industrial purposes (Fenwick et al. 1983). After oil extraction, the residual seed-meal is limited in its usefulness as a protein source by a group of secondary metabolites known as glucosinolates (Fenwick 1984; Duncan 1991). Various biological functions have been ascribed to glucosinolates and their break-down products (e.g. see Andersen and Muir 1966; Larsen 1981; Larsen et al. 1992). Enzymatic hydrolysis of glucosinolates leads to a complex mixture of products (Benn 1977). Some of these are cytotoxic (e.g. isothiocyanates and their derivatives; Horfikov~ 1966), others cause severe nutritional problems in livestock (e.g./-5-vinyl-2thiooxazolidone; Fenwick et al. 1983). The elimination of glucosinolates could potentially make rapeseed-meal as valuable as the oil. To this end, plant breeders have selected against high seed glucosinolate content in oilseed rape (Kondra and Stefansson 1970; R6bbelen and Thies 1980; Gland et al. 1981) and, through the introgression of alleles from the Polish cultivar "Bronowski", have reduced the seed aliphatic-glucosinolate content from above 100 pmoles g-a to less than 20 gmoles g-l. The reduction in seed glucosinolate content has been entirely due to a reduction in aliphatic glucosinolates, derived from methionine; the levels of indolyl glucosinolates, derived from tryptophan, have remained approximately constant. Despite initial concerns, the reduction in seed aliphatic-glucosinolates has had no discernable effects on the interaction between oilseed rape and pests and pathogens. This is partly because the total level of leaf aliphatic glucosinolates in low seed-glucosinolate cultivars is similar to that in high seed-glucosinolate cultivars for the majority of the

803 g r o w i n g season (Mithen 1992). M a n i p u l a t i o n of leafg l u c o s i n o l a t e content does, h o w e v e r , h a v e significant effects on h e r b i v o r e interactions ( G i a m o u s t a r i s and M i t h e n 1995). T h e s e e d - g l u c o s i n o l a t e content o f o i l s e e d rape is a quantitative trait under c o m p l e x genetic r e g u l a t i o n ( K o n dra and Stefansson 1970; Rticker and R u d l o f f 1991). Recent a d v a n c e s in the construction of R F L P l i n k a g e m a p s h a v e e n a b l e d researchers to detect and m a p quantitative trait loci (QTLs) (Weller 1986; Paterson et al. 1988). Estimates o f Q T L effects on p h e n o t y p e can p r o v i d e i n f o r m a tion on the g e n o t y p i c control o f c o m p l e x traits and D N A m a r k e r s c l o s e l y l i n k e d to Q T L s can be used to select plants with desirable p h e n o t y p e s (Edwards et al. 1992). D e t a i l e d genetic m a p s b a s e d on R F L P s have b e e n constructed for B. n a p u s ( L a n d r y et al. 1991; F e r r e i r a et al. 1994). A B . n a p u s m a p c o n s t r u c t e d b y F e r r e i r a et al. (1994) was b a s e d on a r e c o m b i n a n t d o u b l e d - h a p l o i d (DH) p o p u l a t i o n f r o m a cross b e t w e e n high- and l o w - g l u c o s i n o l a t e parents. These D H lines r e p r e s e n t a p e r m a n e n t p o p u l a t i o n for m a p p i n g and in the p r e s e n t study they were used to identify Q T L s w h i c h r e g u l a t e the aliphatic s e e d - g l u c o s i n o l a t e content of B. n a p u s .

Materials and methods Plant material A single plant from the B. napus cv Major (high in glucosinolate) was crossed with a doubled-haploid line derived from cv Stellar (low in glucosinolate) and a segregating population was obtained by microspore culture of a single F I hybrid plant (Ferreira et al. 1994). Self-pollinated seeds from 105 Fl-derived doubled-haploid (DH) lines were harvested between August and September 1992 from a field trial held in Madison, Wis. Open-pollinated seeds from these lines were obtained during August 1993 from a field trial held in Griffin Ga. Glucosinolate analyses Seed samples from self- and open-pollinated plants were analyzed for total and individual glucosinolate content using the extraction and HPLC procedures described by Heaney et al. (1986). All individuals from the selfed population were analyzed for total glucosinolate content. For comparative purposes, 30 corresponding genotypes, ten individuals from the lower extreme of the frequency distribution, ten from the higher and ten having intermediate total aliphatic glucosinolates, were analyzed from the open-pollinated population. Glucosinolate identification was based on a comparison of retention times with authentic glucosinolate standards. Benzyl glucosinolate (0.8 mM; 50 gl) was used as the internal standard and was included at the beginning of each extraction. Published relative response factors were used for the quantification of individual glucosinolates (Heaney et al. 1986). The calculation of flux through reactions for side-chain modifications giving rise to an alteration in the content of individual glucosinolates were as described by Magrath et al. (1993), The following formulae were used to obtain the phenotypic data used to map the loci controlling the percentage butyls (Gsl-elong) and butenyls (Gs/-alk):

Percentage butyls =

100 x (butenyl + hydroxybutenyl + methylthiobutyl + methylsulphinylbutyl)/ total aliphatic glucosinolates

Percentage pentyls = 100 x (pentenyl + hydroxypentenyl + methylthiopentyl + methylsulphinylpentyl)/total aliphatic glucosinolates Percentage butenyls = 100 x (butenyl + hydroxybutenyl)/ total butyl glucosinolates RFLP linkage analysis One-hundred-and-five DH lines were used previously to construct a linkage map of 138 RFLP loci (Ferreira et al. 1994). A revised map containing 21 linkage groups plus five pairs of loci covering 1343 cM was used for the present study. The map contained a total of 196 RFLP loci: 131 loci were detected by 92 genomic DNA clones from either a B. napus cv Westar library ("wg" prefix) or a B. rapa cv Tobin library ("tg" prefix) and 59 loci were detected by cDNA clones from a B. napus cv Westar library ("ec" prefix) (Ferreira et al. 1994; Thormann et al. 1994). The revised map was constructed as described previously (Ferreira et al. 1994). The linkage group numbers in the present paper correspond to those previously published by Ferreira et al. (1994). Subsequent analysis has combined LG16 and LG18 into a single linkage group. QTL analysis Phenotypic data was analysed by a single-marker QTL analysis, as described by Magrath et al. (1994), and by interval-mapping analysis (Lander and Botstein 1989). Interval-mapping was carried out using the MAPMAKER/QTL computer program (Lincoln and Lander 1990). Multipoint linkage map distances were calculated using the Kosambi (1944) transformation function of MAPMAKER. A LOD (lOgl0 of the likelihood odds ratio) score of 3 was taken to indicate QTLs which had a significant effect on the phenotype (Lander and Botstein 1989), and a LOD score of 2 was used as the lower threshold for QTLs which had tentative effects on the phenotype.

Results P h e n o t y p e and f r e q u e n c y distribution Total a l i p h a t i c g l u c o s i n o I a t e s

Seeds f r o m the parental lines M a j o r and Stellar had 109 g m o l e s g-1 and 18 g m o l e s g - t of aliphatic glucosinolates, respectively. The p h e n o t y p i c distribution o f total aliphatic g l u c o s i n o l a t e s in seeds from self p o l l i n a t i o n o f D H lines is shown in Fig. 1. Six lines had e q u i v a l e n t or l o w e r lev-

14

..................................................................................................................... Major ..........................................................

12 ~10

............................................................................................................................. V ........................................................

~8

g6 2 0

0

20 z,0 60 80 100 120 1/~0 GIucosino[ate content (~mo[es g-1 dr. wt.)

160

Fig. 1 Phenotypic distribution of recombinant DH lines of B. napus for total seed aliphatic-glucosinolates. The parental phenotypes are indicated by arrows

804 Table 1 Associations between RFLP markers and total seed aliphatic-glucosinolate content LGa 20

1

18

4 13

Locus

tglhl2 Wg3f7a wg2dlla wg7a8a wgSa6 wg6c6a ec2b3 wg9e9 wg8h5

Segragation ratio b

t-Value d

3.89 : 1.00~ 2.77 : 1.00c

4.02*** 6.48***

1.00 : 1.04 1.10:1.00 1.00 : 1.09

3.67*** 2.94** 3.18"*

1.00 : 1.53

2.62**

1.00 : 1.27 1.00 : 1.10

2.84** 3.15"*

1.33 : 1.00

2.76**

Table 2 Genotypes of transgressive lines (i.e. lines with higher amounts of glucosinolates than Major, Fig. 1) at individual GSL loci controlling total seed aliphatic-glucosinolate content DH line

a LG, linkage group b Segregation ratios of DH lines for marker genotypes Major:Stellar c Z2 test, significantly different from 1:1 ratio (P