Identification of a second linkage group carrying genes controlling resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.)

A sunflower line, XRQ, carrying the gene Pl5, which gives resistance to all French downy mildew races shows cotyledon-limited sporulation in seedling immersion tests; consequently, segregations in crosses with other downy mildew resistance sources were tested both by this method and by a secondary infection on leaves. Pl5 was found to segregate independently of Pl7 (HA338) but to be closely linked, or allelic, with Pl8 (RHA340). F₃ and F₄ progenies from a cross with a line containing Pl2 showed that Pl5 carries resistance to race 100 which segregates independently of Pl2. The Pl5 gene was mapped on linkage group 6 of the Cartisol RFLP map, linked to two RFLP markers, ten AFLP markers and the restorer gene Rf1. Tests with downy mildew race 330 distinguished Pl5 and Pl8, the first being susceptible, the second resistant, whereas both these genes were active against race 304 to which Pl6 (HA335) and Pl7 gave susceptibility. It is concluded that Pl5 and Pl8 are closely linked on linkage group 6 and form a separate resistance gene group from Pl6/Pl7 on linkage group 1. The origins of these groups of downy mildew resistance genes and their use in breeding are discussed. Received: 10 November 2000 / Accepted: 8 February 2001     

RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 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 F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.     

Inheritance and molecular mapping of a downy mildew resistance gene, Pl 13 in cultivated sunflower (Helianthus annuus L.)

The inheritance of resistance to sunflower downy mildew (SDM) derived from HA-R5 conferring resistance to nine races of the pathogen has been determined and the new source has been designated as Pl ₁₃ . The F₂ individuals and F₃ families of the cross HA-R5 (resistant) × HA 821 (susceptible) were screened against the four predominant SDM races 300, 700, 730, and 770 in separate tests which indicated dominant control by a single locus or a cluster of tightly linked genes. Bulked segregant analysis (BSA) was carried out on 116 F₂ individuals with 500 SSR primer pairs that resulted in the identification of 10 SSR markers of linkage groups 1 (9 markers) and 10 (1 marker) of the genetic map (Tang et al. in Theor Appl Genet 105:1124–1136, 2002) that distinguished the bulks. Of these, the SSR marker ORS 1008 of linkage group 10 was tightly linked (0.9 cM) to the Pl ₁₃ gene. Genotyping the F₂ population and linkage analysis with 20 polymorphic primer pairs located on linkage group 10 failed to show linkage of the markers with downy mildew resistance and the ORS 1008 marker. Nevertheless, validation of polymorphic SSR markers of linkage group 1 along with six RFLP-based STS markers of linkage group 12 of the RFLP map of Jan et al. (Theor Appl Genet 96:15–22, 1998) corresponding to linkage group 1 of the SSR map, mapped seven SSR markers (ORS 965-1, ORS 965-2, ORS 959, ORS 371, ORS 716, and ORS 605) including ORS 1008 and one STS marker (STS10D6) to linkage group 1 covering a genetic distance of 65.0 cM. The Pl ₁₃ gene, as a different source with its location on linkage group 1, was flanked by ORS 1008 on one side at a distance of 0.9 cM and ORS 965-1 on another side at a distance of 5.8 cM. These closely linked markers to the Pl ₁₃ gene provide a valuable basis for marker-assisted selection in sunflower breeding programs.     

Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based markers

Resistance of sunflower to the obligate parasite Plasmopara halstedii is conferred by specific dominant genes, denoted Pl. The Pl6 locus confers resistance to all races of P. halstedii except one, and must contain at least 11 tightly linked genes each giving resistance to different downy mildew races. Specific primers were designed and used to amplify 13 markers covering a genetic distance of about 3 cM centred on the Pl6 locus. Cloning and sequence analysis of these 13 markers indicate that Pl6 contains conserved genes belonging to the TIR-NBS-LRR class of plant resistance genes. Received: 9 April 2001 / Accepted: 10 August 2001     

Development of PCR markers for the<Emphasis Type="Italic"> Pl5/Pl8</Emphasis> locus for resistance to<Emphasis Type="Italic"> Plasmopara halstedii</Emphasis> in sunflower,<Emphasis Type="Italic"> Helianthus annuus</Emphasis> L. from complete CC-NBS-LRR sequences

Sunflower downy mildew, caused by Plasmopara halstedii, is one of the major diseases of this crop. Development of elite sunflower lines resistant to different races of this oomycete seems to be the most efficient method to limit downy mildew damage. At least two different gene clusters conferring resistance to different races of P. halstedii have been described. In this work we report the cloning and mapping of two full-length resistance gene analogs (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The two sequences were then used to develop 14 sequence tagged sites (STS) within the Pl5/Pl8 locus conferring resistance to a wide range of P. halstedii races. These STSs will be useful in marker-assisted selection programs.Communicated by C. Möllers     

Identification of non-TIR-NBS-LRR markers linked to the <Emphasis Type="Italic">Pl5</Emphasis>/<Emphasis Type="Italic">Pl8</Emphasis> locus for resistance to downy mildew in sunflower

The resistance of sunflower, Helianthus annuus L., to downy mildew, caused by Plasmopara halstedii, is conferred by major genes denoted by Pl. Using degenerate and specific primers, 16 different resistance gene analogs (RGAs) have been cloned and sequenced. Sequence comparison and Southern-blot analysis distinguished six classes of RGA. Two of these classes correspond to TIR-NBS-LRR sequences while the remaining four classes correspond to the non-TIR-NBS-LRR type of resistance genes. The genetic mapping of these RGAs on two segregating F2 populations showed that the non-TIR-NBS-LRR RGAs are clustered and linked to the Pl5/ Pl8 locus for resistance to downy mildew in sunflower. These and other results indicate that different Pl loci conferring resistance to the same pathogen races may contain different sequences.     

Molecular characterization of resistance to Heterodera glycines in soybean PI 438489B

Soybean (Glycine max L. Merr.) plant introduction (PI) 438489B is a newly found germplasm source that has resistance to multiple soybean cyst nematode (Heterodera glycines Ichinohe, SCN) races. We studied the inheritance of resistance to SCN races 1, 2, 3, 5 and 14 in PI 438489B using F₂ and F_2:3 families, which were generated by crossing to the susceptible cultivar ’Hamilton.’ The objectives of this study were to investigate the inheritance for resistance to SCN races in PI 438489B, to find molecular markers associated with resistances, and to study the allelic relationships among resistance loci for different SCN races. The results showed that the responses to SCN races were approximately normally distributed with large environmental effects, and were also highly correlated, which implied that genes giving resistance to different races were similar. The narrow-sense heritabilities of resistance to all five SCN races ranged from 0.55 to 0.88. Fifty one restriction fragment length polymorphism (RFLP) markers and 64 simple sequence repeat (SSR) markers were found to be polymorphic in the F₂ population. Quantitative trait loci (QTLs) associated with resistance to SCN races were anchored on soybean linkage groups (LGs) A1, A2, B1, B2, C1, C2, D1a, E and G. These QTLs explained 47.3%, 45.8%, 51.5%, 34.5% and 37.2% of the total phenotypic variances, respectively, for each race we investigated. Some QTLs for different races encompassed the same region of flanking markers; therefore, QTLs for multiple races may be linked or pleiotropic effects may be involved. Some loci provided resistance in a race-specific manner. Resistance to SCN race 14 had a different pattern compared to other races. Our results indicated that resistance to race 14 did not include loci on LGs A2 and G. These flanking markers associated with QTLs could be used to select for resistance to multiple SCN races in soybean breeding programs. Received: 25 March 2000 / Accepted: 4 August 2000     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

New Races of Sunflower Downy Mildew (Plasmopara Halstedii) in Germany

Plasmopara halstedii, the causal agent of downy mildew of cultivated sunflower (Helianthus annuus), was documented in Germany for the first time in commercial fields. The pathogen was first observed in the Württemberg area, where races 1 and 4 were identified using a set of differential lines. Later, commericial fields near Baden were found to be infected by race 5, which is the first occurrence of that race outside of North America. Withthe discovery of race 5, there are now eight races of the sunflower downy mildew fungus that have been found in Europe. The sunflower cultivars most frequently grown in Germany were investigated for resistance to race 1, 4 and 5; while all were resistant to race 1, none were resistant to either race, 4 or 5.     

New Races of Sunflower Downy Mildew (Plasmopara Halstedii) in Germany

Plasmopara halstedii, the causal agent of downy mildew of cultivated sunflower (Helianthus annuus), was documented in Germany for the first time in commercial fields. The pathogen was first observed in the Württemberg area, where races 1 and 4 were identified using a set of differential lines. Later, commercial fields near Baden were found to be infected by race 5, which is the first occurrence of that race outside of North America. With the discovery of race 5, there are now eight races of the sunflower downy mildew fungus that have been found in Europe. The sunflower cultivars most frequently grown in Germany were investigated for resistance to race 1, 4 and 5; while all were resistant to race 1, none were resistant to either race 4 or 5.     

Candidate disease resistance genes in sunflower cloned using conserved nucleotide-binding site motifs: genetic mapping and linkage to the downy mildew resistance gene Pl1.

Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.     

Consensus mapping of major resistance genes and independent QTL for quantitative resistance to sunflower downy mildew

Major gene resistance to sunflower downy mildew (Plasmopara halstedii) races 304 and 314 was found to segregate independently from the resistance to races 334, 307 and 304 determined by the gene Pl2, already positioned on Linkage Group (LG) 8 of sunflower molecular maps. Using a consensus SSR-SNP map constructed from the INEDI RIL population and a new RIL population FU?×?PAZ2, the positions of Pl2 and Pl5 were confirmed and the new gene, denoted Pl21, was mapped on LG13, at 8?cM from Pl5. The two RIL populations were observed for their quantitative resistance to downy mildew in the field and both indicated the existence of a QTL on LG8 at 20-40?cM from the major resistance gene cluster. In addition, for the INEDI population, a strong QTL on LG10, reported previously, was confirmed and a third QTL was mapped on LG7. A growth chamber test methodology, significantly correlated with field results, also revealed the major QTL on LG10, explaining 65?% of variability. This QTL mapped in the same area as a gene involved in stomatal opening and root growth, which may be suggested as a possible candidate to explain the control of this character. These results indicate that it should be possible to combine major genes and other resistance mechanisms, a strategy that could help to improve durability of sunflower resistance to downy mildew.     

<Emphasis Type="Italic">Pl</Emphasis><Subscript><Emphasis Type="Italic">Arg</Emphasis></Subscript> from <Emphasis Type="Italic">Helianthus argophyllus</Emphasis> is unlinked to other known downy mildew resistance genes in sunflower

The Pl _Arg locus in the sunflower (Helianthus annuus L.) inbred line Arg1575-2 conferring resistance to at least four tested races (300, 700, 730, 770) of downy mildew (Plasmopara halstedii) was localized by the use of simple sequence repeat (SSR) markers. Bulked segregant analysis (BSA) was conducted on 126 individuals of an F₂ progeny from a cross between a downy mildew susceptible line, CmsHA342, and Arg1575-2. Twelve SSR markers linked to the Pl _Arg locus were identified. All markers were located proximal to Pl _Arg on linkage group LG1 based on the map of Yu et al. (2003) in a window of 9.3 cM. Since Pl _Arg was mapped to a linkage group different from all other Pl genes previously mapped with SSRs, it can be concluded that Pl _Arg provides a new source of resistance against P. halstedii in sunflower.     

Molecular mapping of the Pl 16 downy mildew resistance gene from HA-R4 to facilitate marker-assisted selection in sunflower

The major genes controlling sunflower downy mildew resistance have been designated as Pl genes. Ten of the more than 20 Pl genes reported have been mapped. In this study, we report the molecular mapping of gene Pl(16) in a sunflower downy mildew differential line, HA-R4. It was mapped on the lower end of linkage group (LG) 1 of the sunflower reference map, with 12 markers covering a distance of 78.9 cM. One dominant simple sequence repeat (SSR) marker, ORS1008, co-segregated with Pl(16), and another co-dominant expressed sequence tag (EST)-SSR marker, HT636, was located 0.3 cM proximal to the Pl(16) gene. The HT636 marker was also closely linked to the Pl(13) gene in another sunflower differential line, HA-R5. Thus the Pl(16) and Pl(13) genes were mapped to a similar position on LG 1 that is different from the previously reported Pl(14) gene. When the co-segregating and tightly linked markers for the Pl(16) gene were applied to other germplasms or hybrids, a unique band pattern for the ORS1008 marker was detected in HA-R4 and HA-R5 and their F(1) hybrids. This is the first report to provide two tightly linked markers for both the Pl(16) and Pl(13) genes, which will facilitate marker-assisted selection in sunflower resistance breeding, and provide a basis for the cloning of these genes.     

Sunflower resistance to multiple downy mildew pathotypes revealed by recognition of conserved effectors of the oomycete Plasmopara halstedii

Over the last 40 years, new sunflower downy mildew isolates (Plasmopara halstedii) have overcome major gene resistances in sunflower, requiring the identification of additional and possibly more durable broad‐spectrum resistances. Here, 354 RXLR effectors defined in silico from our new genomic data were classified in a network of 40 connected components sharing conserved protein domains. Among 205 RXLR effector genes encoding conserved proteins in 17 P. halstedii pathotypes of varying virulence, we selected 30 effectors that were expressed during plant infection as potentially essential genes to target broad‐spectrum resistance in sunflower. The transient expression of the 30 core effectors in sunflower and in Nicotiana benthamiana leaves revealed a wide diversity of targeted subcellular compartments, including organelles not so far shown to be targeted by oomycete effectors such as chloroplasts and processing bodies. More than half of the 30 core effectors were able to suppress pattern‐triggered immunity in N. benthamiana, and five of these induced hypersensitive responses (HR) in sunflower broad‐spectrum resistant lines. HR triggered by PhRXLRC01 co‐segregated with Pl22 resistance in F3 populations and both traits localized in 1.7 Mb on chromosome 13 of the sunflower genome. Pl22 resistance was physically mapped on the sunflower genome recently sequenced, unlike all the other downy mildew resistances published so far. PhRXLRC01 and Pl22 are proposed as an avirulence/resistance gene couple not previously described in sunflower. Core effector recognition is a successful strategy to accelerate broad‐spectrum resistance gene identification in complex crop genomes such as sunflower.     

A QTL analysis of sunflower partial resistance to downy mildew ( Plasmopara halstedii) and black stem ( Phoma macdonaldii) by the use of recombinant inbred lines (RILs)

Partial resistance to downy mildew (Plasmopara halstedii) and to black stem (Phoma macdonaldii) in sunflower were investigated under natural field infection and a controlled growth chamber respectively. Genetic control for resistance to the diseases was determined in recombinant inbred lines (RILs) and their two parents, ’PAC-2’ and ’RHA-266.’ The experiments were undertaken in a randomized complete block design with two replications, in a field severely infected by downy mildew and in a controlled growth chamber with plants inoculated with an agressive French isolate of P. macdonaldii. Each replication consisted of three rows, 4.6-m long, giving 48 plants per RIL or parent in the field and 15 plants in the growth chamber. Genetic variability was observed among the RILs for resistance to both diseases. When 10% of the selected RILs were compared with the mean of the two parents genetic gain was significant for partial resistance to the diseases. Four putative QTLs for resistance to downy mildew on linkage groups 1, 9 and 17 were detected using composite interval mapping. The QTLs explained 54.9% of the total phenotypic variance. Major QTLs (dmr1–1 and dmr1–2) for resistance were found on linkage group 1 with up to 31% of the phenotypic variability explained by two peaks. QTL analysis of resistance to black stem showed seven QTLs on linkage groups 3, 6, 8, 9, 11, 15 and 17. The detected QTLs together explain 92% of the phenotypic variation of the trait. Crosses between RILs contrasted for their resistance to downy mildew and black stem, and exhibiting molecular polymorphism in detected QTLs, will be made in order to focus more-precisely on the genomic region of interest. Received: 28 February 2001 / Accepted: 14 June 2001     

Discovery and introgression of the wild sunflower-derived novel downy mildew resistance gene <Emphasis Type="Italic">Pl</Emphasis><Subscript><Emphasis Type="Italic">19</Emphasis></Subscript> in confection sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Key message

A new downy mildew resistance gene, Pl ₁₉ , was identified from wild Helianthus annuus accession PI 435414, introduced to confection sunflower, and genetically mapped to linkage group 4 of the sunflower genome.

Abstract

Wild Helianthus annuus accession PI 435414 exhibited resistance to downy mildew, which is one of the most destructive diseases to sunflower production globally. Evaluation of the 140 BC₁F_2:3 families derived from the cross of CMS CONFSCLB1 and PI 435414 against Plasmopara halstedii race 734 revealed that a single dominant gene controls downy mildew resistance in the population. Bulked segregant analysis conducted in the BC₁F₂ population with 860 simple sequence repeat (SSR) markers indicated that the resistance derived from wild H. annuus was associated with SSR markers located on linkage group (LG) 4 of the sunflower genome. To map and tag this resistance locus, designated Pl ₁₉ , 140 BC₁F₂ individuals were used to construct a linkage map of the gene region. Two SSR markers, ORS963 and HT298, were linked to Pl ₁₉ within a distance of 4.7 cM. After screening 27 additional single nucleotide polymorphism (SNP) markers previously mapped to this region, two flanking SNP markers, NSA_003564 and NSA_006089, were identified as surrounding the Pl ₁₉ gene at a distance of 0.6 cM from each side. Genetic analysis indicated that Pl ₁₉ is different from Pl ₁₇ , which had previously been mapped to LG4, but is closely linked to Pl ₁₇ . This new gene is highly effective against the most predominant and virulent races of P. halstedii currently identified in North America and is the first downy mildew resistance gene that has been transferred to confection sunflower. The selected resistant germplasm derived from homozygous BC₂F₃ progeny provides a novel gene for use in confection sunflower breeding programs.

     

Acclimation of Plasmopara halstedii (sunflower downy mildew) in relationship with trade-off between virulence and aggressiveness in a local pathogen population

The acclimation in relationship with virulence cost was analysed for seven Plasmopara halstedii (sunflower downy mildew) isolates including five progeny isolates of several races descending from two parental isolates of races 100 and 710. Aggressiveness criteria were analysed in one sunflower inbred line showing a high level of quantitative resistance. Isolates of races 100 and 3xx were characterised with shorter latent period and higher sporulation density than isolates of races 7xx. All isolates showed high percentage infection values and caused a large reduction in seedling size except for one isolate involved in dwarfing. The seven isolates were divided, according to their virulence and aggressiveness, into two main groups as more aggressive isolates of the 100 and 3xx races which do not overcome the sunflower differential host D3, and less aggressive isolates of 7xx races which can overcome D3. Consequently, the 100 and 3xx avirulent races had a virulence cost measured by differences in aggressiveness (from 45.5 to 76.3%) compared to 7xx virulent races carrying unnecessary virulence gene.     

Interaction between sunflower plants and five isolates of Plasmopara halstedii on the level of pathogenicity

The interaction between sunflower plants showing a high level of quantitative resistance and five Plasmopara halstedii (the causal agent of downy mildew) isolates of several races were studied using five single zoosporangium isolates per pathogen isolate. Aggressiveness criteria were analyzed for 25 P. halstedii single zoosporangium isolates. Based on the reaction for the P. halstedii isolates to four sunflower hybrids H1–H4 varying only in their downy mildew resistance genes, there were differences in virulence spectrum in pathogen isolates. Analysis of five single zoosporangium isolates for P. halstedii isolates showed significant variability within pathogen isolate for all aggressiveness criteria but not for all pathogen isolates. The hypothesis explaining the interaction between P. halstedii and its host plant was discussed on the level of pathogenicity.     

Virulence cost in Plasmopara halstedii (sunflower downy mildew)

Virulence cost (trade-off between virulence and aggressiveness) was studied in seven Plasmopara halstedii (sunflower downy mildew) isolates of races 100, 300, 304, 314, 710, 704 and 714. The seven isolates were divided, according to their virulence and aggressiveness, into two main groups as more aggressive isolates of the 100 and 3xx races that do not overcome the sunflower differential host D3, and less aggressive isolates of 7xx races that can overcome D3. Consequently, the 100 and 3xx avirulent races had a virulence cost measured by differences in aggressiveness (from 58.3 to 78.2%) compared to 7xx virulent races carrying unnecessary virulence gene.