Mapping of QTL for downy mildew resistance in maize

Quantitative trait loci (QTLs) of maize involved in mediating resistance to Peronosclerospora sorghi, the causative agent of sorghum downy mildew (SDM), were detected in a population of recombinant inbred lines (RILs) derived from the Zea mays L. cross between resistant (G62) and susceptible (G58) inbred lines. Field tests of 94 RILs were conducted over two growing seasons using artificial inoculation. Heritability of the disease reaction was high (around 70%). The mapping population of the RILs was also scored for restriction fragment length polymorphic (RFLP) markers. One hundred and six polymorphic RFLP markers were assigned to ten chromosomes covering 1648 cM. Three QTLs were detected that significantly affected resistance to SDM combined across seasons. Two of these mapped quite close together on chromosome 1, while the third one was on chromosome 9. The percentage of phenotypic variance explained by each QTL ranged from 12.4% to 23.8%. Collectively, the three QTLs identified in this study explained 53.6% of the phenotypic variation in susceptibility to the infection. The three resistant QTLs appeared to have additive effects. Increased susceptibility was contributed by the alleles of the susceptible parent. The detection of more than one QTL supports the hypothesis that several qualitative and quantitative genes control resistance to P. sorghi.     

Rapid mapping of two genes for resistance to downy mildew from Lactuca serriola to existing clusters of resistance genes

Two resistances to downy mildew derived from Lactuca serriola were characterized genetically and mapped using molecular markers. Classical genetic analysis suggested monogenic inheritance; however, the presence of multiple, tightly-linked genes in each case could not be eliminated. Therefore, they were designated resistance factors R17 and R18. Analysis with molecular markers known to be linked to clusters of resistance genes quickly revealed linkage of R18 to the major cluster of resistance genes and provided six linked markers, three RAPD (Random Amplified Polymorphic DNA) markers and three codominant SCAR (Sequence Characterized Amplified Region) markers. The mapping of R17 required the screening of arbitrary RAPD markers using bulked segregant analysis; this provided five linked markers, three of which segregated in the basic mapping population. This demonstrated loose linkage to a second cluster of resistance genes and provided additional linked markers. Two RAPD markers linked to R17 were converted into SCARs. The identification of reliable PCR-based markers flanking each gene will aid in selection and in combining these resistance genes with others.     

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     

Genetic mapping and localization of a major QTL for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis)

Downy mildew caused by the fungus Peronospora parisitica is a serious threat to members of the Brassicaceae family. Annually, a substantial loss of yield is caused by the widespread presence of this disease in warm and humid climates. The aim of this study was to localize the genetic factors affecting downy mildew resistance in Chinese cabbage (Brassica rapa ssp. pekinensis). To achieve this goal, we improved a preexisting genetic map of a doubled-haploid population derived from a cross between two diverse Chinese cabbage lines, 91-112 and T12-19, via microspore culture. Microsatellite simple sequence repeat (SSR) markers, isozyme markers, sequence-related amplified polymorphism markers, sequence-characterized amplified region markers and sequence-tagged-site markers were integrated into the previously published map to construct a composite Chinese cabbage map. In this way, the identities of linkage groups corresponding to the Brassica A genome reference map were established. The new map contains 519 markers and covers a total length of 1,070 cM, with an average distance between markers of 2.06 cM. All markers were designated as A1–A10 through alignment and orientation using 55 markers anchored to previously published B. rapa or B. napus reference maps. Of the 89 SSR markers mapped, 15 were newly developed from express sequence tags in Genbank. The phenotypic assay indicated that a single major gene controls seedling resistance to downy mildew, and that a major QTL was detected on linkage group A8 by both interval and MQM mapping methods. The RAPD marker K14-1030 and isozyme marker PGM flanked this major QTL in a region spanning 2.9 cM, and the SSR marker Ol12G04 was linked to this QTL by a distance of 4.36 cM. This study identified a potential chromosomal segment and tightly linked markers for use in marker-assisted selection to improve downy mildew resistance in Chinese cabbage.     

Induction of resistance against downy mildew on sunflower by rhizobacteria

Abstract

Induction of resistance to downy mildew caused by Plasmopara halstedii in sunflower was studied after treatment with PGPR (plant growth promoting rhizobacteria) strain INR7 (Bacillus spp). Treatment of sunflower seeds with 1×10⁸cfu/ml of PGPR strain INR7 resulted in decreased disease severity and offered 51 and 54% protection under green house and field conditions, respectively. The induction of resistance to P. halstedii by PGPR strain INR7 was accompanied by the accumulation of various host defense-related enzymes in susceptible sunflower seedlings. Enhanced activation of catalase (CAT), phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and chitinase (CHI) was evident at 6, 9, 12, 12 and 12h post inoculation, respectively, in sunflower seedlings raised from seeds treated with PGPR strain INR7. This enhanced and early activation of defense-related responses in the susceptible cultivar after treatment with PGPR strain INR7 was comparable to that in the resistant cultivar. The results indicate that PGPR strain INR7 induced resistance against P. halstedii in sunflower is mediated through enhanced expression of defense mechanism.     

QTL mapping for downy mildew resistance in cucumber via bulked segregant analysis using next-generation sequencing and conventional methods

Key message

QTL mapping using NGS-assisted BSA was successfully applied to an F ₂ population for downy mildew resistance in cucumber. QTLs detected by NGS-assisted BSA were confirmed by conventional QTL analysis.

Abstract

Downy mildew (DM), caused by Pseudoperonospora cubensis, is one of the most destructive foliar diseases in cucumber. QTL mapping is a fundamental approach for understanding the genetic inheritance of DM resistance in cucumber. Recently, many studies have reported that a combination of bulked segregant analysis (BSA) and next-generation sequencing (NGS) can be a rapid and cost-effective way of mapping QTLs. In this study, we applied NGS-assisted BSA to QTL mapping of DM resistance in cucumber and confirmed the results by conventional QTL analysis. By sequencing two DNA pools each consisting of ten individuals showing high resistance and susceptibility to DM from a F₂ population, we identified single nucleotide polymorphisms (SNPs) between the two pools. We employed a statistical method for QTL mapping based on these SNPs. Five QTLs, dm2.2, dm4.1, dm5.1, dm5.2, and dm6.1, were detected and dm2.2 showed the largest effect on DM resistance. Conventional QTL analysis using the F₂ confirmed dm2.2 (R ² = 10.8–24 %) and dm5.2 (R ² = 14–27.2 %) as major QTLs and dm4.1 (R ² = 8 %) as two minor QTLs, but could not detect dm5.1 and dm6.1. A new QTL on chromosome 2, dm2.1 (R ² = 28.2 %) was detected by the conventional QTL method using an F₃ population. This study demonstrated the effectiveness of NGS-assisted BSA for mapping QTLs conferring DM resistance in cucumber and revealed the unique genetic inheritance of DM resistance in this population through two distinct major QTLs on chromosome 2 that mainly harbor DM resistance.

     

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.     

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.     

DNA markers for downy mildew resistance genes in sorghum.

The random amplified polymorphic DNA technique was used to find markers for a downy mildew resistance gene in sorghum. Of the 674 random primers screened for polymorphism, 2 amplified fragments were linked to a downy mildew resistance gene in sorghum line SC414. Utilization of an existing restriction fragment length polymorphism mapping population (IS3620C x BTx623) also revealed two markers that are linked to a different resistance gene in another sorghum line, BTx623.     

Mapping quantitative trait loci for downy mildew resistance in pearl millet

Quantitative trait loci (QTLs) for resistance to pathogen populations of Scelerospora graminicola from India, Nigeria, Niger and Senegal were mapped using a resistant x susceptible pearl millet cross. An RFLP map constructed using F₂ plants was used to map QTLs for traits scored on F₄ families. QTL analysis was carried out using the interval mapping programme Mapmaker/QTL. Independent inheritance of resistance to pathogen populations from India, Senegal, and populations from Niger and Nigeria was shown. These results demonstrate the existence of differing virulences in the pathogen populations from within Africa and between Africa and India. QTLs of large effect, contributing towards a large porportion of the variation in resistance, were consistently detected in repeated screens. QTLs of smaller and more variable effect were also detected. There was no QTLs that were effective against all four pathogen populations, demonstrating that pathotype-specific resistance is a major mechanism of downy mildew resistance in this cross. For all but one of the QTLs, resistance was inherited from the resistant parent and the inheritance of resistance tended to be the result of dominance or over-dominance. The implications of this research for pearl millet breeding are discussed.     

Single and multiple phenotype QTL analyses of downy mildew resistance in interspecific grapevines

Key message

Downy mildew resistance across days post-inoculation, experiments, and years in two interspecific grapevine F₁ families was investigated using linear mixed models and Bayesian networks, and five new QTL were identified.

Abstract

Breeding grapevines for downy mildew disease resistance has traditionally relied on qualitative gene resistance, which can be overcome by pathogen evolution. Analyzing two interspecific F₁ families, both having ancestry derived from Vitis vinifera and wild North American Vitis species, across 2 years and multiple experiments, we found multiple loci associated with downy mildew sporulation and hypersensitive response in both families using a single phenotype model. The loci explained between 7 and 17% of the variance for either phenotype, suggesting a complex genetic architecture for these traits in the two families studied. For two loci, we used RNA-Seq to detect differentially transcribed genes and found that the candidate genes at these loci were likely not NBS-LRR genes. Additionally, using a multiple phenotype Bayesian network analysis, we found effects between the leaf trichome density, hypersensitive response, and sporulation phenotypes. Moderate–high heritabilities were found for all three phenotypes, suggesting that selection for downy mildew resistance is an achievable goal by breeding for either physical- or non-physical-based resistance mechanisms, with the combination of the two possibly providing durable resistance.

     

Characterization and mapping of a downy mildew resistance gene,Pl36, in sunflower (Helianthus annuus L.)

Molecular Breeding - Leaf thickness is an important trait in rice (Oryza sativa L.). It affects both photosynthesis and sink-resource efficiency. However, compared to leaf length and length width,...     

Linkage between genes for resistance to downy mildew (Bremia lactucae) in lettuce

Data are presented for the joint segregation of the gene combinations: Dm-2/Dm-3, Dm-3/Dm-6, Dm-2/Dm-6 and Dm-6/Dm-8, following inoculation with five races of B. lactucae. It is postulated that Dm-2, 3 and 6 comprise a tight linkage group but that Dm-6 and Dm-8 are not linked as has been proposed previously. Information on the reaction of some resistant lettuce cultivars to certain B. lactucae races and some new postulated host genotypes is also presented.     

Haplotyping and mapping a large cluster of downy mildew resistance gene candidates in sunflower using multilocus intron fragment length polymorphisms

Downy mildew (Plasmopara halstedii (Farl.) Berlese et de Toni) is a serious foliar pathogen of cultivated sunflower (Helianthus annuus L.). Genetic resistance is conditioned by several linked downy mildew resistance gene specificities in the HaRGC1 cluster of TIR-NBS-LRR resistance gene candidates (RGCs) on linkage group 8. The complexity and diversity of the HaRGC1 cluster was assessed by multilocus intron fragment length polymorphism (IFLP) genotyping using a single pair of primers flanking a hypervariable intron located between the TIR and NBS domains. Two to 23 bands were amplified per germplasm accession. The size of the included intron ranged from 89 to 858 nucleotides. Forty-eight unique markers were distinguished among 24 elite inbred lines, six partially isogenic inbred lines, nine open-pollinated populations, four Native American land races, and 20 wild H. annuus populations. Nine haplotypes (based on 24 RGCs) were identified among elite inbred lines and were correlated with known downy mildew resistance specificities. Sixteen out of 39 RGCs identified in wild H. annuus populations were not observed in elite germplasm. Five partially isogenic downy mildew resistant lines developed from wild H. annuus and H. praecox donors carried eight RGCs not found in other elite inbred lines. Twenty-four HaRGC1 loci were mapped to a 2-4 cM segment of linkage group 8. The multilocus IFLP marker and duplicated, hypervariable microsatellite markers tightly linked to the HaRGC1 cluster are powerful tools for distinguishing downy mildew resistance gene specificities and identifying and introgressing new downy mildew resistance gene specificities from wild sunflowers.     

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.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Positional cloning of a candidate gene for resistance to the sunflower downy mildew, Plasmopara halstedii race 300

The resistance of sunflower to Plasmopara halstedii is conferred by major resistance genes denoted Pl. Previous genetic studies indicated that the majority of these genes are clustered on linkage groups 8 and 13. The Pl6 locus is one of the main clusters to have been identified, and confers resistance to several P. halstedii races. In this study, a map-based cloning strategy was implemented using a large segregating F2 population to establish a fine physical map of this cluster. A marker derived from a bacterial artificial chromosome (BAC) clone was found to be very tightly linked to the gene conferring resistance to race 300, and the corresponding BAC clone was sequenced and annotated. It contains several putative genes including three toll-interleukin receptor-nucleotide binding site-leucine rich repeats (TIR-NBS-LRR) genes. However, only one TIR-NBS-LRR appeared to be expressed, and thus constitutes a candidate gene for resistance to P. halstedii race 300.     

Variation in seedling susceptibility to Bremia lactucae (lettuce downy mildew) in the absence of effective major resistance genes

In the absence of effective major genes the importance of interactions between cultivar genotype, isolate genotype and environment was investigated in experiments using seedlings of eight lettuce cultivars inoculated with three isolates of Bremia lactucae and grown under a number of different environmental conditions. The outcome of the cultivar-isolate association was measured using four criteria and the data were examined by analysis of variance and correlation. The relative susceptibility of cultivars was generally independent of environment and there was no evidence that isolates were adapted to particular cultivars. Although significant cultivar X isolate interactions were found in individual experiments they were not consistent between experiments, even where these were conducted under apparently identical conditions. Variation resulted from either cultivar X environment or isolate X environment interaction with the environment always the dominant variable.