Theor Appl Genet (1995) 91:733-737

9 Springer-Verlag 1995

S. M o u z e y a r 9 P. R o e c k e l - D r e v e t 9 L. G e n t z b i t t e l J. P h i l i p p o n 9 D. T o u r v i e i l l e D e L a b r o u h e 9 F. V e a r P. N i c o l a s

RFLP and RAPD mapping of the sunflower PI1 locus for resistance to Plasmopara halstedii race 1

Received: 19 January 1995 / Accepted: 31 March 1995

The Pll locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F 2 progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pll. Two RFLP markers and one RAPD marker linked to the Pll locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pll. The RAPD marker is situated at 43.7 cM from Pll. The significance and applications of these markers in sunflower breeding are discussed.

Abstract

Helianthus annuus 9Plasmopara halstedii 9 Disease resistance 9Bulked segregant analysis 9 Molecular markers Key words

Introduction Downy mildew of sunflower (Helianthus annuus L.) is caused by the obligate parasite Plasmopara haIstedii. This disease is found in most parts of the world where sunflowers are grown and can cause severe losses in this crop

Communicated by J. W. Snape S. Mouzeyar 9J. Philippon. D. Tourvieille De Labrouhe 9F. Vear Institut National de la Recherche Agronomique, Domaine de Crouelle, 63039 Clermont-Ferrand Cedex, France R Roeckel-Drevet ([])) 9R Nicolas Unit6 associ6e Universit6 Blaise Pascal-INRA, Organisation et Variabilit6 des Gdnomes Vdg6taux, 4 rue Ledru, 63038 Clermont-Ferrand Cedex, France L. Gentzbittel G.I.E. CARTISOL, 4 rue Ledru, 63038 Clermont-Ferrand Cedex, France

(Sackston 1981). At least seven different races have been described according to their reaction on different sunflower lines (Gulya et al. 1991 a, b). Resistance is controlled by single dominant genes designated as Pl (Vranceanu 1970) and has been found for all known downy mildew races. Some P1 genes, such as Pll and Pl2 were found in cultivated sunflower (Vranceanu 1970; Zimmer and Kinmann 1972) while others were derived from interspecific crosses, such as Pl4 derived from H. tuberosus (Vear 1974), Pl7 from H. praecox, and Pl8 from H. argophyllus (Miller and Gulya 1991). The Pll gene was the first downy mildew resistance gene described in the sunflower (Vranceanu 1970). It confers resistance to race 1, the prevalent race in Europe. In France, resistance to this race is obligatory for the registration of commercial varieties. Although genetical studies on the inheritance of resistance to downy mildew in sunflower are numerous (Vranceanu 1970; Zimmer and Kinmann 1972; Vranceanu et al. 1981; Miller and Gulya 1991), the relationship between the different Pl genes remains unclear. Some P1 genes such as Pl2 and Pl4 were first described as independent (Vear 1974), but have now been reported not to segregate (Sackston 1992). Some sunflower lines are resistant to more than one race, possibly because they possess more than one Pl gene (Sackston et al. 1990). It would therefore be beneficial to locate Pl loci on a sunflower linkage map. Recently, the use of near-isogenic lines (Young et al. 1988) or bulked segregant analysis (Michelmore et al. 1991), together with molecular markers, has accelerated the mapping of many resistance genes in different plant species. Examples are the Pto gene in tomato (Martin et al. 1991), the Din5~8 gene in lettuce (Michelmore et al. 1991) and the Are gene in French bean (Adam-Blondon et al. 1994). RFLP and RAPD markers have already been shown to be useful in studies of polymorphism in the sunflower (Gentzbittel et al. 1994; Teulat et al. 1994). This paper reports the mapping of the Pll locus for resistance to downy mildew race 1 on the consensus linkage RFLP map of cultivated sunflower (Gentzbittel et al. 1995) using bulked segregant analysis.

734

Materials and methods Sunflower genotypes An F 2 population of 135 plants was derived from a cross between two sunflower lines, GH and RHA266, GH is susceptible to downy mildew race 1, with no characterised resistance genes. RHA266 contains the resistance gene Pll (Fick and Zimmer 1974) and is resistant to downy mildew race 1. These lines are maintained at INRA by self pollination under paper bags. Fungal isolate An isolate of downy mildew race 1 (European race) was used. It is maintained at INRA (Clermont-Ferrand) on HA89, a susceptible sunflower genotype. Evaluation of disease reaction The downy mildew resistance genotype of each F2 plant was determined by testing their F 3 progenies, obtained from selfing under paper bags. For each progeny, at least 20 seedlings were infected and grown as described previously by Mouzeyar et al. (1993). Seedlings were scored as susceptible if fungal sporulation was evident on cotyledons and true leaves, and resistant if no sporulation, or only a slight sporulation, was observed on cotyledons (Mouzeyar et al. 1993, 1994). The corresponding F z plants were then classified as homozygous susceptible, homozygous resistant, or heterozygous for

PI1. DNA extraction and bulk preparation About five fully grown leaves were collected from each of the 135 F 2 plants at flowering. DNA extraction was conducted as described by Gentzbittei et al. (1992). Equal quantities of DNA were bulked from 12 homozygous-resistant and from 12 homozygous-susceptible F 2 plants, according to the method Of Michelmore et al. (1991), to give two DNA bulks. RFLP and RAPD procedures

RFLP analysis DNA digestion and Southern hybridization were performed as described previously (Gentzbittel et al. 1992) using four restriction enzymes: BglII, EcoRI, EcoRV and HindIII (Amersham). The RFLP probes used were produced and mapped by Gentzbittel et al. (1995).

ed into centiMorgans (cM) by applying the Haldane function. For consensus mapping, the Gmendel package was used (Holloway and Knapp 1993) with the same initial options as for Mapmaker.

Results B u l k e d segregant analysis The d o w n y m i l d e w resistance tests on the ( G H x R H A 2 6 6 ) F 3 p r o g e n i e s , d e r i v e d f r o m the 135 F 2 plants, g a v e 24 hom o z y g o u s - r e s i s t a n t , 76 s e g r e g a t i n g and 35 h o m o z y g o u s s u s c e p t i b l e p r o g e n i e s . This s e g r e g a t i o n fitted the e x p e c t e d ratio for a single s e g r e g a t i n g gene ( 1 : 2 : 1 , ) 5 2 = 3 . 9 , 0 . 1 0 < P < 0 . 2 5 ) . T w e l v e h o m o z y g o u s - r e s i s t a n t and 12 hom o z y g o u s - s u s c e p t i b l e F 2 plants were then s e l e c t e d for p r e p a r a t i o n o f the two bulks. R A P D p r i m e r s and R F L P p r o b e s were screened for p o l y m o r p h i s m against the two bulks and the parental lines G H and R H A 2 6 6 . P o l y m o r phic m a r k e r s were then s c r e e n e d against the 135 F 2 plants i n c l u d i n g those used for b u l k preparation.

RAPD primers. O f the 350 d e c a m e r p r i m e r s tested, 48 (14%) r e v e a l e d p o l y m o r p h i s m b e t w e e n the p a r e n t a l lines but none r e v e a l e d p o l y m o r p h i s m b e t w e e n the two bulks. H o w e v e r , the p r i m e r OPD13 a m p l i f i e d a 1600-bp b a n d f r o m the D N A o f R H A 2 6 6 and the resistant bulk, w h e r e a s this b a n d was faint in the s u s c e p t i b l e b u l k and absent f r o m G H (Fig. 1) Thus, this m a r k e r was n a m e d OPD13160o.

RFLP probes. A total o f 33 loci distributed on the first seven l i n k a g e groups o f the R F L P m a p o f c u l t i v a t e d sunf l o w e r were tested. Two p r o b e s that d e t e c t e d p o l y m o r p h i s m b e t w e e n the bulks w e r e identified. S U N 0 1 7 is a d o m i n a n t m a r k e r w h i c h r e v e a l e d a 2.1-kb b a n d p r e s e n t o n l y in R H A 2 6 6 and resistant b u l k D N A . S U N 1 2 4 rev e a l e d two bands (9 and 7.8 kb) in G H and the s u s c e p t i b l e b u l k D N A , and a 4.6-kb b a n d in R H A 2 6 6 and the resistant b u l k D N A (Fig. 2).

RAPD analysis

M a p p i n g o f the P l l gene

Random decamer primers (kits A to T, Operon Technologies, Calif., USA) were used to identify polymorphic DNA between the two bulks. Amplification was conducted according to Williams et al. (1990) with minor modifications: 40-50 ng DNA were used as template in a 25-gl reaction volume containing 10 mM Tris-HC1 pH 9, 200 gM of each dNTP (Pharmacia), 50 mM KC1, 1.5 mM MgC12, 0.1% TritonX100, 0.2% gelatin, 0.2 gM of primer and l unit of Taq DNA Polymerase (Appligbne). Amplification was performed in a 9600 Perkin Elmer Cetus thermal cycler as follows: initial denaturation for 5 min at 91~ then 40 cycles of 1 min at 91~ 1 min at 36~ and 2 min at 72~ The 40 cycles were followed by final extension for 5 rain at 72~ Amplification products were analysed by electrophoresis on 1.4% agarose gels in Tris-acetic acid-EDTA (TAE) buffer and stained with ethidium bromide.

S u b s e q u e n t s e g r e g a t i o n analysis using the 135 F 2 plants with the M a p m a k e r 3.0 p r o g r a m m e s h o w e d that S U N 0 1 7 , S U N 1 2 4 and OPD1316oo are l i n k e d to the PI1 locus. S U N 0 1 7 is 5.6_+2.0 c M ( L O D = 2 2 . 1 ) from the Pll locus whilst S U N 1 2 4 is situated at 7.1_+1.5 c M (LOD=8.4). OPD1316oo is 43.7+3 c M ( L O D = 2 . 2 ) f r o m the P l l locus. A n a l y s i s using G m e n d e l software with S U N 0 1 7 and S U N 1 2 4 as anchor loci a l l o w e d l o c a t i o n o f the P l l locus on l i n k a g e group 1 o f the R F L P consensus m a p o f the cultivated s u n f l o w e r (Fig. 3). (Gentzbittel et al. 1995).

Lirikage analysis

Discussion

Linkage analysis was conducted using the software Mapmaker 3.0 (Lander et al. 1987). Markers were ordered with a minimum LOD score of 3,0 and rmax=0.35. Recombination fractions were convert-

The p u r p o s e o f this study was to locate the d o w n y m i l d e w resistance locus P l l on the s u n f l o w e r R F L P map. A b u l k e d

735 Fig. 1 Amplification of genomic DNA from RHA266 (resistant parent), RB (resistant bulk), SB (susceptible bulk), GH (susceptible parent) and 12 segregating F 2 plants with the OPD 13 primer (operon technologies). The 1600-bp band is the OPD 13160o product linked to the Pll locus. Downy mildew phenotypes: R homozygous resistant, S homozygous susceptible and H heterozygous resistant

Fig. 2 Autoradiograph showing linkage between the PlI locus and the RFLP locus SUNI24. Probe SUN124 was hybridized onto blots of EcoRIdigested DNA from the two bulks, the parental lines, and 135 F 2 plants (only 12 F 2 plants are shown). The 9.0-kb and 7.8kb bands are the GH (susceptible) allele, the 4.6-kb band is the RHA266 (resistant) allele

Fig. 3 RFLP map of sunflower linkage group 1 containing the Pll locus for resistance to P. halstedii race 1. Map distances in cM are based on the analysis of 135 F 2 plants derived from the cross between the susceptib•e line GH and the resistant line RHA266. Loci nomenclature is as follows: SXXXYY-Z with SXXX the SUN probe number, YY the restriction enzyme used for mapping (El EcoRI, H3 HindIII). For those probes that hybridized to more than one site in the genome, the suffix - Z (-1, -2, - 3 ) indicates each duplicate locus

Markername

Dist (cM)

OPD1316oo

38.1

S017H3-3 5.6

Pll 7.1 S124EI-2 6.7 S003H3 13.4

S109H3-2 7 S075H3-1

0.0 17.6

S039H3 S145H3-2 S095E1 S029H3 S097H3 S037H3 S031H3

1.1 1.2 1.1 5,2 3.1 5.5

segregant analysis as described by Michelmore et al. (1991) was used to identify two RFLP markers, one on each side of the PI1 locus, and also a linked RAPD marker. Recently, this strategy has also been used for other agronomic characters, such as photosensitive genic male sterility in maize (Zhang et al. 1994). Bulked segregant analysis was found to be useful in the present study, since it was possible to detect a RAPD marker 43.7 cM from the Pll locus (30+5 % recombination). Although the susceptible bulk included three recombinant individuals, with the OPD1316oo band, it showed only a weak amplification of this band, in comparison with the resistant bulk. This agrees with the report of Michelmore et al. (1991), who considered that segregating markers within a 30% recombination range would be detectable by bulked segregant analysis. The RFLP probes used in this study were initially selected by Gentzbittel et al. (1994, 1995) and they proved to be useful. In contrast, only 14% of the RAPD primers tested showed polymorphism between GH and RHA266. This result may be explained by the low level of polymorphism which has been found in cultivated sunflower using isozymes (Quillet et al. 1992), RFLP (Gentzbittel et al. 1994) and RAPD markers (Teulat et al. 1994). Progenies from five crosses involving four inbred lines were used to construct a consensus RFLP map of the cultivated sunflower and SUN017H3-3 and SUN124E1-2 were found to be separated by 52 cM (Gentzbittel et al. 1995). In the present paper, we present a new calculation for this linkage group, in which the number of Segregating families was increased (six instead of five crosses and 795 plants compared with 560 plants for the first version of the map). Also, the loci SUN075H3-1 and SUN109H3-2 were found to be identical. This led us to propose a new map of

736 l i n k a g e group 1, on w h i c h the locus Pll is l o c a t e d b e t w e e n S U N 0 1 7 H 3 - 3 and S U N 1 2 4 E 1 - 2 . The d i s t a n c e b e t w e e n these 2 loci is n o w e s t i m a t e d at 12 cM. It should be p o s s i b l e to use the two R F L P m a r k e r s to a c c e l e r a t e the i n t r o d u c t i o n o f P l l into s u s c e p t i b l e lines b y b a c k - c r o s s p r o c e d u r e s , as p r o p o s e d b y M e l c h i n g e r (1990). The use o f these markers, l i n k e d to the Pll locus at 5.6 and 7.1 cM, should r e d u c e the n u m b e r and size o f f a m i l i e s that need to be tested, since less than 0.5% o f plants should s h o w d o u b l e c r o s s i n g - o v e r such that the two m a r k e r s are p r e s e n t and the resistant allele absent. In addition, this could, with a h i g h d e g r e e of probability, h e l p to c l a r i f y the r e s i s t a n c e or s u s c e p t i b i l i t y o f plants s h o w i n g c o n s i d e r a b l e amounts o f fungus s p o r u l a t i o n on the c o t y l e d o n s . This is o f p a r t i c u l a r i m p o r t a n c e since c o t y l e d o n - l i m i t e d infection is v e r y frequent in the p r e s e n c e o f the Pll gene (Vear 1978). Perhaps the m o s t interesting a p p l i c a t i o n o f the m a r k e r s l i n k e d to Pll will be to d e t e r m i n e l i n k a g e r e l a t i o n s h i p s bet w e e n Pl genes, as it has b e e n s u g g e s t e d that certain Pl genes are tightly l i n k e d ( M o u z e y a r et al. 1992). Studies with S U N 0 1 7 and S U N 1 2 4 , and other l i n k e d R F L P m a r k ers, should m a k e it p o s s i b l e to d e t e r m i n e w h e t h e r other Pl loci are l o c a t e d in the s a m e l i n k a g e group as the Pll locus. The u l t i m a t e o b j e c t i v e o f this w o r k is the m o l e c u l a r c l o n i n g o f PII and the analysis of its m o d e of action. This and the other Pl genes induce a h y p e r s e n s i t i v e - l i k e reaction w h i c h is rather unusual in that this occurs, after fungal p e n e t r a t i o n o f the h y p o c o t y l , within the cortical p a r e n c h y m a ( M o u z e y a r et al. 1994). Recently, two r e s i s t a n c e genes o f p l a n t species h a v e been c l o n e d b y a m a p - b a s e d c l o n i n g strategy: the Pto gene for resistance against Pseudomonas syringae in t o m a t o (Martin et al. 1993) and RPS2 in Arabidopsis thaliana against the s a m e p a t h o g e n carrying the a v i r u l e n c e gene avrRpt2 (Bent et al. 1994; M i n d r i nos et al. 1994). In both cases, m a r k e r s c l o s e l y l i n k e d to the r e s i s t a n c e gene were l o c a t e d and these served either to identify YAC clones c o n t a i n i n g the resistance gene (Pto) or for c h r o m o s o m e w a l k i n g (RPS2). In our case, it will be n e c e s s a r y to find other m a r k e r s m o r e c l o s e l y l i n k e d to Pll than are S U N 0 1 7 H 3 - 3 and S U N 1 2 4 E 1 - 2 , in o r d e r to initiate such a strategy to clone this gene. Acknowledgements This work was supported, in part, by the Centre Technique Interprofessionnel des Oldagineux M6tropolitains (CETIOM) and a State/Region contract "Semences et plants".

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