ГЕНЕТИКА, 2015, том 51, № 10, с. 1134-1140
ГЕНЕТИКА РАСТЕНИЙ ^
УДК 581.2:582.98
MOLECULAR ANALYSIS OF NEW SOURCES OF RESISTANCE TO Pseudoperonospora cubensis (Berk. et Curt.) Rostovzev IN CUCUMBER
© 2015 W. Szczechura, M. Staniaszek, U. Klosinska, and E. U. Kozik
Research Institute of Horticulture, 96-100 Skierniewice, Poland e-mail: wojciech.szczechura@inhort.pl Received December 01, 2014
Downy mildew of cucumber (Cucumis sativus L.), caused by Pseudoperonospora cubensis (Berk. et Curt.) Rostovzev, is one of the most important foliar diseases of cucurbit crops. Two parental lines resistant PI 197085, susceptible PI 175695 and their F2 generation were used in our study. Inheritance of resistance to Pseudoperonospora cubensis in PI 197085 was quantitative. JoiMap 4.1 and MapQTL 6.0 software was used for a linkage groups construction and QTL mapping. Three QTL were detected: DM1, DM2, DM3. The loci were mapped on chromosome 5 of cucumber genome. Molecular analysis confirmed results of classical quantitative genetics indicating that resistance to Pseudoperonospora cubensis in PI 197085 is polygenic trait.
Keywords: Cucumis sativus, Downy mildew, QTL mapping, DNA markers, disease resistance. DOI: 10.7868/S0016675815090118
Pseudoperonospora cubensis (Berk. et Curt.) Rostovzev, isone of the most destructive and economically important pathogens of cucumber (Cucumis sativus L.) [1]. This pathogen belong to kingdom Chromista, subdivision Peronosporomycotina, class Oomycetes, order Peronosporales and family Peronosporaceae [2]. It is an obligate parasite attacking the leaves of cucurbit plants in many regions worldwide [1]. Symptoms of downy mildew appears on the adaxial leaves surface of cucumber in the form of chlorotic lesions that are limited by veins. The lesions are a response to decrease in photosynthesis and that results in leaves withering [3]. The disease development depends on external condition especially on temperature, humidity, light, inoculum concentration [4—6]. Downy mildew has been a serious problem in Central Europe since 1984 [7]. The pathogen was first reported in Poland in 1985, and since then it has become increasingly prevalent in cucumber growing areas causing serious yield losses [8].Two types of resistance, single- and polygenic were described in cucumber [3, 9, 10]. Doruchowski and Lackowska-Ryk [11] reported that three recessive genes (dml, dm2, dm3) were responsible for resistance to Pseudoperonospora cubensis in cucumber line WI 4783. In cv. Poinsett 76 a single recessive gene was identified [9]. One or two major genes and several minor genes were described in cucumber South Carolina No. 8-63 [12]. Polygenic character with a few major genes controlling resistance to downy mildew was observed in Ames 2354 [13]. In K8 inbred line, resistance to downy mildew was determined by five QTL [14]. In IL52 line derived from crossing between cucumber and Cucumis hystrix four QTLcontrol resistance to downy mildew [15].
Previously, the available USDA Plant Introduction (PI) collection of cucumber germplasm was studied to identify new accessions having a higher level of resistance under natural field epidemics in Poland and North Carolina [16, 17]. Among about 1300 cucumber cultigens tested in Poland, six of them: PI 330628, PI 197088, PI 197086, PI 197085, Ames 2353, and Ames 2354 showed the highest level of resistance to DM over four years of study. These six lines were more resistant than the currently available resistant Polish F1 hybrids Rodos and Aladyn, and American cultivars Poinsett 76 and Slice.
The aim of this study was to identify QTL controlling resistance to Pseudoperonospora cubensis in material derived from crossing between resistant PI 197085 and susceptible PI 175695 lines.
MATERIALS AND METHODS
Plant and fungal material
The parental genotypes used in this study were: resistant PI 197085 (India), and susceptible PI 175695 (Turkey), chosen on the basis of their reaction to Pseudoperonospora cubensis in our previous studies [16]. These lines were obtained from the North Central Regional Plant Introduction Station, in Ames, IA, USA.
The plant material consisted of F1, and 115 plants of F2 populations from the initial cross between PI 197085 and PI 175695. All crosses were made by hand pollination in a greenhouse of the Research Institute of Horticulture, Skierniewice, Poland.
Heavily infected with Pseudoperonospora cubensis cucumber leaves, which had not been sprayed with fungicides, were collected from the experimental field
in Skierniewice, Poland. The infected leaves were soaked in distilled water and rubbed gently with a glass rod to dislodge sporangia. The concentration of sporangia suspension was determined with the use of hemocytometer and adjusted to a final concentration of 5 x 104 sporangia x mL-1.
Phytopathological test and disease evaluation
Resistance test was conducted under controlled environment conditions in the growth chamber. Seeds were pre-germinated on petri dishes followed by planting in 10 cm diameter plastic pots (one seed per one pot) filled with a peat substrate Kronen-Klasmann. Seedlings were grown at 24/18°C (day/night) temperatures and 12 hours of light. Plants were inoculated at 1-2 leaves stage by misting the adaxial side of leaves with the sporangial solution until runoff using a hand-sprayer bottle (1L size). The inoculated seedlings were incubated in dark for 48 h at 20°C and 100% relative humidity (RH). After incubation plants were grown at 24°C (day/night) with 12 hours of light in growth chamber. Disease assessments were made 7 days after inoculation using a scale of 0 to 9, where: 0 = no disease, 1-2 = trace, 3-4 = slight, 5-6 = moderate, 78 = severe, 9 = dead [18]. Ratings are based on percentage of infected leaf area, from which a disease severity index (DSI) was calculated.
DNA isolation, PCR and electrophoresis
Genomic DNA was extracted from young cucumber leaves, frozen in -80°C after harvest. Isolation was made with a commercial kit NucleoSpin Plant II (Macherey-Nagel) according to the protocol. DNA concentration and purity were measured spectropho-tometrically and by electrophoresis in 0.8% agarose gel stained with ethidium bromide.
For mapping and identification of quantitative traits loci (QTL) controlling resistance to Pseudoper-onospora cubensis, amplification of random amplified polymorphic DNA (RAPD), sequence characterized amplified region (SCAR) and simple sequence repeat (SSR) markers were tested.Two hundred thirty eight DNA markers were used for molecular analysis. DNA markers used in this study were derived from chromosome 1, 5, 6 Cucumis sativus genome and from chromosome VIII, X, XI, XII Cucumis melo genome. The primer sequences were obtained from: Cucurbit Ge-nomics Database, US DA Cucumber SCAR Marker Database, literatures [14, 19, 20] and seven series of RAPD primers such as OPA, OPB, OPG, OPK, OPL, OPR, OPX (Operon Technologies Inc.) from our study. Twenty nine DNA markers polymorphicin parental genotypes PI 197085 and PI 175695 were used for 115 F2 population analysis (Table 1).
RAPD-PCR was performed in 20 ^L volume containing: 1x PCR buffer, 2.5 mM MgCl2, 0.1 mM dNTP (Thermo Scientific, Fermentas), 0.3 ^M 10-
mer primer, 1 U Taq DNA polymerase (Thermo Scientific, Fermentas), 0.01% gelatin and 20 ng genomic DNA. PCR was conducted using the following conditions: 94°C for 1 min, 45 cycles of: 92°C for 15 s, 36°C for 25 s, 72°C for 74 s and 1 cycle of 72°C for 5 min. Amplification products were separated in 1.4% agarose gel electrophoresis stained with ethidium bromide.
SCAR analysis were carried out in 20 ^L containing: 1x PCR buffer, 0.2 mM each of dNTP (Thermo Scientific, Fermentas), 3 mM MgCl2, 0.4 ^M forward and reverse primers, 2 U Taq DNA polymerase (Thermo Scientific, Fermentas), 20 ng genomic DNA. DNA was amplified under the following thermal conditions: 94°C for 3 min, 40 cycles of 94°C for 30 s, 60°C for 60 s, 72°C for 90 s, and final extension of 72°C for 5 min. PCR products were separated in 1.6% agarose gel electrophoresis, stained with ethidium bromide.
SSR analysis wereconducted in 20 ^L volume consisting of: 1x PCR buffer 0.13 mM each of dNTP (Thermo Scientific, Fermentas), 1.5 mM MgCl2, 0.3 ^M primers, 1 U Taq DNA polymerase (Thermo Scientific, Fermentas) and 40 ng genomic DNA. The PCR parameters were: 94°C for 3 min, 35 cycles of 94°C for 15 s, 55°C for 15 s, 72°C for 30 s and final extension 72°C for 5 min. The amplicons were separated in 8% polyacrylamide gel electrophoresis and visualized in UV radiation after ethidium bromide stained.
All PCR reactions were performed in a thermocy-cler GeneAmp PCR System 9700 (Applied Biosystems).
Data analysis
Linkage group was constructed using JoinMap 4.1 (Kyazma) software. The Kosambi function and minimum logarithm of odds (LOD) likelihood score of 3 were used to construct a map and calculate genetic distance between markers. The QTL's detection were carried out using interval mapping analysis in MapQTL 6.0 (Kyazma) software. The LOD threshold value was set up on 3.0. The determination coefficient (R2) for identified QTL was calculated in MapQTL 6.0 software. The R2 defines the percentage of phenotypic variation determined by quantitative trait loci.
RESULTS AND DISCUSSION
Plants of the resistant parent PI 197085 (P1) showed very low degree of disease symptoms (DSI = 1.3), (Fig. 1). In contrast, plants of PI 175695 (P2) exhibited high susceptibility (DSI = 8.2). The F2 population showed continuous phenotypic segregation in classes 1 to 8, with predominance of moderately resistant (classes 3, 4) and moderately susceptible (classes 5, 6), 37 and 44, respectively. The F2 phenotype class segregation resembling the normal distribution suggested that cucumber resistance against Pseudoperonospora
Table 1. RAPD, ISSR, SCAR and SSR markers used for analysis of F2 population
Markers Localization Primers sequence References
RAPD-OPAS05850 C. sat
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