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.

     

QTL analysis of powdery mildew resistance in cucumber (Cucumis sativus L.)

A population of F₇ recombinant inbred lines (RILs) was made from a cross between susceptible (‘Santou’) and resistant (PI197088-1) lines of cucumber in order to study powdery mildew resistance loci. Susceptibility to powdery mildew in the F₇ RIL individuals showed a continuous distribution from susceptible to resistant, suggesting that powdery mildew resistance is controlled by quantitative trait loci (QTLs). A QTL analysis identified two and three loci for powdery mildew resistance under 26 and 20°C conditions, respectively. One QTL was found in the same position under both temperature conditions. Therefore, it is more likely that one major QTL acts under both temperature conditions and that other QTLs are specific to the two temperature conditions. The above results suggest that the four QTLs are controlled in a different temperature manner, and that their combination played an important role in expressing a high level of resistance to powdery mildew in this cucumber population. Sequence-tagged site (STS) markers associated with each QTL were developed and would be useful for breeding a cucumber line with a high level of powdery mildew resistance. Y. Sakata and N. Kubo contributed equally to this work and are considered as first authors.     

QTL molecular marker location of powdery mildew resistance in cucumber (Cucumis sativus L.)

The cucumber lines, S94 (Northern China open-field type, powdery mildew (PM) susceptible) and S06 (European greenhouse type, PM resistant), and their F6:7 populations were used to investigate PM re-sistance under seedling spray inoculation in 2005/Autumn and 2006/Spring. QTL analysis was under-taken based on a constructed molecular linkage map of the corresponding F6 population using com-posite interval mapping. A total of four QTLs (pm1.1, pm2.1, pm4.1 and pm6.1) for PM resistance were identified and located on LG 1, 2, 4 and 6, respectively, explaining 5.2%-21.0% of the phenotypic variation. Three consistent QTLs (pm1.1, pm2.1 and pm4.1) were detected under the two test conditions. The QTL pm6.1 was only identified in 2005/Autumn. The total phenotypic variation explained by the QTLs was 52.0% and 42.0% in 2005/Autumn and 2006/Spring, respectively. Anchor markers tightly linked to those loci (<5 cM) could lay a basis for both molecular marker-assisted breeding and map-based gene cloning of the PM-resistance gene in cucumber.     

Genetic mapping of the powdery mildew resistance gene in soybean PI 567301B

Powdery mildew (PMD) of soybean [Glycine max (L.) Merr.] is caused by the fungus Microsphaera diffusa. Severe infection of PMD on susceptible varieties often causes premature defoliation and chlorosis of the leaves, which can result in considerable yield losses under favorable environmental conditions for disease development in the field. A total of 334 F(7)-derived recombinant inbred lines (RILs) from a cross of a PMD susceptible soybean cultivar Wyandot and PMD-resistant PI 567301B were used for genetic mapping of PMD resistance in PI 567301B and for development of molecular markers tightly linked to the gene. The result of the PMD screening for each line in the field was in agreement with that in the greenhouse test. The genetic map containing the PMD resistance gene was constructed in a 3.3?cM interval flanked by two simple sequence repeat (SSR) markers on chromosome 16. The PMD resistance gene was mapped at the same location with SSR marker BARCSOYSSR_16_1291, indicating that there was no recombination between the 334 RILs and this marker. In addition, a single nucleotide polymorphism (SNP) marker developed by high-resolution melting curve analysis and a cleaved amplified polymorphic sequence (CAPS) marker with Rsa1 recognition site were used for the genetic mapping. These two markers were also mapped to the same genomic location with the PMD resistance gene. We validated three tightly linked markers to the PMD resistance gene using 38 BC(6)F(2) lines and corresponding BC(6)F(2:3) families. The three marker genotypes of the backcross lines predicted the observed PMD phenotypes of the lines with complete accuracy. We have mapped a putatively novel single dominant PMD resistance gene in PI 567301B and developed three new molecular markers closely linked to the gene. Molecular markers developed from this study may be used for high-throughput marker-assisted breeding for PMD resistance with the gene from PI 567301B.     

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.     

Interactions between mycorrhiza and powdery mildew of cucumber

Interactions between the arbuscular mycorrhizal fungus Glomus intraradices and the powdery mildew fungus Podosphaera xanthii were examined with cucumber as the host plant in pot experiments under greenhouse conditions. Plants were inoculated with mildew two weeks after seedling emergence. Plants were mycorrhizal or not, prior to mildew infection and were harvested two weeks after mildew inoculation. We found no influence of the cucumber — G. intraradices symbiosis on development of cucumber mildew in terms of numbers of colonies per unit area. Similarly, biomass and amount of energy reserves of G. intraradices as examined with signature fatty acids were unaffected by mildew. Both biotrophs caused growth depressions of the host plant. Plant carbon allocation is discussed in relation to biotrophs as sinks.     

QTL mapping of resistance to Fusarium ear rot using a RIL population in maize

Fusarium ear rot is a prevalent disease in maize, reducing grain yields and quality. Resistance breeding is an efficient way to minimize losses caused by the disease. In this study, 187 lines from a RIL population along with the resistant (87-1) and susceptible (Zong 3) parents were planted in Zhengzhou and Beijing with three replications in years 2004 and 2006. Each line was artificially inoculated using the nail-punch method. Significant genotypic variation in response to Fusarium ear rot was detected in both years. Based on a genetic map containing 246 polymorphic SSR markers with average genetic distances of 9.1 cM, the ear-rot resistance QTL were firstly analyzed by composite interval mapping (CIM). Three QTL were detected in both Zhengzhou and Beijing in 2004; and three and four QTL, respectively, were identified in 2006. The resistant parent contributed all resistance QTL. By using composite interval mapping and a mixed model (MCIM), significant epistatic effects on Fusarium ear rot as well as interactions between mapped loci and environments were observed across environments. Two QTL on chromosome 3 (3.04 bin) were consistently identified across all environments by the two methods. The major resistant QTL with the largest effect was flanked by markers umc1025 and umc1742 on chromosome 3 (3.04 bin), explaining 13–22% of the phenotypic variation. The SSR markers closely flanking the major resistance QTL will facilitate marker-assisted selection (MAS) of resistance to Fusarium ear rot in maize breeding programs.     

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.     

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.

     

Thermal imaging of cucumber leaves affected by downy mildew and environmental conditions

Pathogenesis of Pseudoperonospora cubensis causing downy mildew of cucumber resulted in changes in the metabolic processes within cucumber leaves including the transpiration rate. Due to the negative correlation between transpiration rate and leaf temperature, digital infrared thermography permitted a non-invasive monitoring and an indirect visualization of downy mildew development. Depending on the stage of pathogenesis and the topology of chloroses and necroses, infection resulted in a typical temperature pattern. Spatial heterogeneity of the leaf temperature could be quantified by the maximum temperature difference (MTD) within a leaf. The MTD increased during pathogenesis with the formation of necrotic tissue and was related to disease severity as described by linear and quadratic regression curves. Under controlled conditions, changes in temperature of infected leaves allowed the discrimination between healthy and infected areas in thermograms, even before visible symptoms of downy mildew appeared. Environmental conditions during thermographic measurement, in particular air temperature and humidity, as well as water content and age of the leaf influenced the temperature of its surface. Conditions enhancing the transpiration rate facilitated the detection of changes in leaf temperature of infected leaves at early stages of infection. As modified by environmental conditions, MTD alone is not suitable for the quantification of downy mildew severity in the field.     

Leaf morphology and anatomy of transgenic cucumber lines tolerant to downy mildew

The objective of the present paper was to investigate the reason of increased tolerance to the pathogenic fungus Pseudoperonospora cubensis found in transgenic cucumber (Cucumis sativus L.) lines 210 and 212 bearing 35S:cDNA preprothaumatin II gene construct. The tolerance investigation was accomplished by comparing the morphological and anatomical structure of plant leaves. The results obtained demonstrate that leaves of both lines exhibited some anatomical and morphological characteristics (e.g. wax load and composition, cuticle ultrastructure, ultrastructure of secondary wall, arrangement of mesophylll cells) which may be responsible for enhanced tolerance.     

虎尾兰水提取物对黄瓜霜霉病的防治研究

明确虎尾兰水提取物对黄瓜霜霉病菌无性阶段的抑制作用及对黄瓜霜霉病的防治效果。采用凹玻片法和离体叶五点接种法分别测定该提取物对游动孢子的释放和游动、休止孢的萌发和芽管伸长以及菌丝生长和孢子囊产生的抑制作用,采用离体叶五点接种法测定该提取物的保护作用、治疗作用和持效期,并采用盆栽试验测定该提取物的保护作用和治疗作用。一系列试验结果显示,虎尾兰水提取物对黄瓜霜霉病菌游动孢子的释放和游动,休止孢的萌发和芽管伸长以及菌丝生长和孢子囊产生的EC₅₀值分别为1.19、0.77、0.70、2.13、9.09和13.15 mg/mL。在离体叶片条件下,虎尾兰水提取物对黄瓜霜霉病的保护和治疗作用的EC₅₀值分别为3.69 和14.57 mg/mL,持效期达14 d以上。盆栽条件下,该提取物对黄瓜霜霉病保护作用和治疗作用的防效分别为68.38%和21.33%。虎尾兰水提取物对黄瓜霜霉病具有很好的保护作用和较长的持效期。     

Fine mapping quantitative resistances to downy mildew in lettuce revealed multiple sub-QTLs with plant stage dependent effects reducing or even promoting the infection

Key message

Three regions with quantitative resistance to downy mildew of non-host and wild lettuce species, Lactuca saligna , disintegrate into seventeen sub-QTLs with plant-stage-dependent effects, reducing or even promoting the infection.

Abstract

Previous studies on the genetic dissection of the complete resistance of wild lettuce, Lactuca saligna, to downy mildew revealed 15 introgression regions that conferred plant stage dependent quantitative resistances (QTLs). Three backcross inbred lines (BILs), carrying an individual 30–50 cM long introgression segment from L. saligna in a cultivated lettuce, L. sativa, background, reduced infection by 60–70 % at young plant stage and by 30–50 % at adult plant stage in field situations. We studied these three quantitative resistances in order to narrow down their mapping interval and determine their number of loci, either single or multiple. We performed recombinant screenings and developed near isogenic lines (NILs) with smaller overlapping L. saligna introgressions (substitution mapping). In segregating introgression line populations, recombination was suppressed up to 17-fold compared to the original L. saligna × L. sativa F ₂ population. Recombination suppression depended on the chromosome region and was stronger suppressed at the smallest introgression lengths. Disease evaluation of the NILs revealed that the resistance of all three BILs was not explained by a single locus but by multiple sub-QTLs. The 17 L. saligna-derived sub-QTLs had a smaller and plant stage dependent resistance effect, some segments reducing; others even promoting downy mildew infection. Implications for lettuce breeding are outlined.     

Oleozon: A novel control strategy against powdery mildew in cucumber

Ozonized sunflower oil (oleozon) is an effective agent for controlling powdery mildew in cucumber. In this study, the mechanisms of oleozon in the control of powdery mildew were determined. The development of Podosphaera xanthii on cucumber leaves treated with oleozon (2%) and water was investigated at different times after inoculation. The germinating conidia, hyphae and conidiophores of the pathogen were severely damaged by oleozon. No visible phytotoxic effect was observed on cucumber after the application of oleozon. This compound had highly preventive effects as well as curative effects against powdery mildew based on in vivo potted seedling assays. The control effects of oleozon were further confirmed in a greenhouse trial. These results may provide a basis for further development of a natural fungicide against cucumber powdery mildew.     

Effects of powdery mildew on cucumber yields, and its chemical control

Effects of Sphaerotheca fuliginea were deduced from experiments in which cucumber foliage, exposed to naturally occurring inocula, was fungicidally sprayed. Yields increased as the incidence of mildew decreased but the relation was sometimes affected by differing amounts of phytotoxicity. The fungitoxicity and phytotoxicity of a range of chemicals applied as high-volume sprays, fumigants or soil drenches, were tested. Non-phytotoxic concentrations of some fungicides adequately protected cucumber foliage from subsequent attack but were often insufficient to eradicate established infections, the increased amounts needed for this being phytotoxic. Powdery mildew was controlled more effectively when drazoxolon and quinomethionate were applied as sprays with 0·1 and 0·02% a.i. respectively, than when used as fumigants at 2–4 and 1·3 g/28·3 m³(= 1000 ft³). Sprays of drazoxolon (0·1% a.i.) increased yields from 7·0 to 11·2 kg/ plant during 8 weeks picking and in another experiment weights of fruit were increased by applying quinomethionate (0·02% a.i.) from 10·8 to 52·2 kg/plot of four plants. In the former experiment appreciable amounts of phytotoxicity and infection were tolerated before yields decreased but, in the latter, yields were inversely proportional to numbers of dead leaves which were directly related to the incidence of mildew. Spraying with quinomethionate, or drazoxolon plus tetradifon (0·012% a.i.) increased numbers of female flowers from 59·5 in the mildewed controls to 93·0 and 142·8 per plant respectively, and of these 17·6, 32·8 and 16·2 % subsequently produced marketable cucumbers. In addition to decreasing yields, severe S. fuliginea infestations were associated with increased numbers of misshapen cucumbers. Increasing sulphur concentrations from 0·5 to 1·0g/28·3m³during nightly fumigations significantly decreased mildew incidence and increased (a) yields from 35·8 to 52·9 kg during 5 weeks picking, and (b) the proportion of high-quality cucumbers from 49 to 63 %. Similar trends occurred in another trial where sulphur concentrations were increased from 0·75 to 1·0 g/28·3 m³, but comparisons with fortnightly sprays of 0·1% drazoxolon suggest that these sulphur concentrations caused some damage.