Identification and molecular tagging of two Arabidopsis resistance genes to Botrytis cinerea

Abstract

To map Arabidopsis resistance genes to Botrytis cinerea, Arabidopsis Col-0 ecotype resistant to B. cinerea BC18 isolate and Arabidopsis Ler ecotype susceptible to B. cinerea BC18 isolate were crossed. According to the resistant responses of the F₁, BC₁ and F₂ populations to B. cinerea, we identified two genes, named BC1 and BC2, responsible for the resistance of Arabidopsis Ler ecotype to B. cinerea. Through the method of map-based cloning, BC1 was linked to DNA markers CCR1 and DHS1 on the fourth chromosome of Arabidopsis with genetic distances of 1.2 cM and 1.6 cM for CCR1 and DHS1, respectively, and BC2 was linked to DNA markers CA72/NGA151 and NGA106 on the fifth chromosome with genetic distances of 1.4 cM and 2.4 cM for CA72/NGA151 and NGA106, respectively. Our results are beneficial for chromosome walking so that we can obtain the whole gene sequences, which will facilitate the understanding of their roles and manners of resistance to B. cinerea.

Keywords

Arabidopsis thaliana / Botrytis cinerea / resistant genes / linkage analysis / gene mapping

Introduction

Botrytis cinerea, an important plant pathogenic fungus, causes a severe disease in a wide range of plant species both in the field and in postharvest situations and results in significant economic losses. The molecular and cellular mechanisms involved in plant resistance to B. cinerea and their genetic control have been poorly understood up to now. Having a small genomic DNA, short growth cycle and the completion of sequencing, Arabidopsis thaliana has been widely used as a model organism for researching cellular and molecular interactions between fungi and plants (The Arabidopsis Genome Initiative, 2000). In 2003, Mengiste et al. (2003) identified one Botrytis-susceptible loci (BOS1) encoding an R2R3MYB transcription factor that was required for biotic and abiotic stress responses in Arabidopsis. Ferrari et al. (2003) found that salicylic acid and camalexin were involved in the local resistance of Arabidopsis to B. cinerea and needed genes EDS4 and PAD2 to be turned on but genes SID2, EDS5 or PAD4 to be turned off. Veronese et al. (2004) identified three other Arabidopsis-susceptible genes, BOS2, BOS3 and BOS4, which were necessary for B. cinerea infection. Two years later, they identified a membrane-anchored Botrytis-induced kinase 1 which played distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens (Veronese et al., 2006). Expression profiling and mutant analysis of ZFAR1 and WRKY70 showed an increase in Arabidopsis resistance to B. cinerea (AbuQamar et al., 2006). Dhawan et al. (2009) showed that Arabidopsis HUB1 (HISTONE MONOUBIQUITINATION1) was a regulatory component of the plant defense from B. cinerea.

Previously, we examined the responses of 11 different Arabidopsis ecotypes and genotypes to the infection with 21 B. cinerea isolates. We found that various Arabidopsis ecotypes infected by the same B. cinerea isolate showed different symptoms. Among them, Col-0 ecotype appeared immune to the B. cinerea BC18 isolate infection, while Ler ecotype exhibited susceptible symptoms (Xing et al., 2010). In this study, the resistant Col-0 ecotype and susceptible Ler ecotype were crossed. On the basis of the responses of the F₁ and F₂ populations, we identified two genes mediating the resistance of Arabidopsis against infection from B. cinerea. Through the method of map-based cloning, two disease-resistant genes were located on the fourth and fifth chromosome of Arabidopsis.

Materials and methods

Arabidopsis thaliana ecotypes and B. cinerea isolate BC18

Arabidopsis ecotypes Col-0 and Ler were kindly provided by Dr Xi (Donald Danforth Plant Science Center, USA). Ecotype Col-0 appeared immune to the B. cinerea isolate BC18, but ecotype Ler appeared susceptible to it. The B. cinerea isolate BC18 was provided by the Molecular Plant Pathology Laboratory, Agricultural University of Hebei, China.

Primers of SSLP (simple sequence length polymorphism)

The sequences of primers were obtained from http://www.arabidopsis.org/home.html. We selected 3 to 4 molecular primers that were distributed symmetrically on each chromosome (Table1). Primers were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd.

Construction of mapping population and disease assay

Crosses between ecotypes of Col-0 (male parent) and Ler (female parent) were performed, and the F₁ plants were selfed independently. F₁ plants, BC₁ and F₂ segregating population were used as the mapping population and the inheritance of their resistance to B. cinerea was documented. To infect plants, a 5 μL spore suspension (4×10⁵-8×10⁵ spores/mL) of B. cinerea isolate BC18 was dropped on individual leaves of 4-week-old plants. After inoculation, plants were kept under a transparent cover to maintain 100% relative humidity (RH) and kept in the dark for 24 h before being transferred to a growth chamber in the temperature range of 18°C to 22°C and a photoperiodic regime of 16 h light and 8 h dark. Disease course was scored between 1 and 5 d after inoculation. Susceptible plants were characterized by extensive tissue damage at an early stage after B. cinerea infection compared with resistant plants.

Selection of codominant primers

Genomic DNA was isolated from the leaves of individual plants using a modified CTAB procedure (Stewart and Via, 1993). The procedure of SSLP was taken from a CSHL course with an appropriate modification. The reaction system (25 μL) included 10 × PCR reaction buffer 2.5 μL, dNTP Mix (10 mmol/L) 2.5 μL, positive and negative primers (20 μmol/L) 0.5 μL respectively, template DNA 1.0 μL (DNA quantity: 500 ng/μL), Taq DNA polymerase 0.5 μL (enzyme quantity: 5 U/μL). The program for amplification was as follows: 95°C denature for 30 s; 55°C anneal for 30 s; 72°C extend for 1 min; and for 50 cycles. PCR product was examined by 4% agarose gel electrophoresis. The electricity parameters were 1-3 h, voltage 80-120 V, and 1 × TAE buffer solution.

Construction of resistant and susceptible pools of F₂ segregation population

The DNA of 10 resistant and 10 susceptible plants from the F₂ segregation population were mixed respectively to form a resistant gene pool and a susceptible gene pool for bulk segregation analysis (Michelmore et al., 1991).

Molecular tagging of resistance genes to B. cinerea

Codominant primers were selected to analyze the pooled DNA of the F₂ segregation population for the initial linkage test. The SSLP primers that could distinguish between the resistant gene pool and the susceptible gene pool were linked with markers (Lukowitz et al., 2000). Once the linkage between the resistant gene and genetic markers was established, 144 individual F₂ resistant plants were further analyzed by PCR with at least two flanking markers to score the distance between the resistant gene and the corresponding markers.

The genetic distances (centimorgan, cM) between the resistant gene and markers were determined using MAPMAKER/EXP 3.0 (Lander et al., 1987) based on the segregation data of the plant resistance and the molecular markers of the F₂ mapping population, LOD≥3.0. Genetic mapping was carried out using the MapChart software.

Results

Morphologic observation of hybrid plant

The morphologic observation of 1493 hybrid plants of the cross between Col-0 (male parent) and Ler (female parent) indicated that 520 hybrid plants were similar to the female parent (Ler), while 973 hybrid plants with light leaf color and long leafstalk were similar in character to the male parent (Fig. 1).

Selection of codominant primers

Taking the individual genomic DNA of the parents (Col-0 and Ler) and 10 hybrid plants which were different from the female parent as templates, PCR was performed to select codominat SSLP primers that could identify hybrid plants (Fig. 2). Primers distributed on the first and second chromosomes could not distinguish parents and hybrid plants. These primers were unlinked to the cross site. The primers, including NGA162, NGA6, NGA172, NGA8, NGA1107, NGA151 and NGA76, could identify the hybrid plants. NGA162, NGA6 and NGA172 were distributed on the third chromosome, NGA8 and NGA1107 were distributed on the fourth chromosome, NGA151 and NGA76 were distributed on the fifth chromosome. PCR amplification results using the primers above showed that hybrid plants displayed characteristic bands of male and female parents. These primers were codominant primers which could identify parents and hybrid plants.

Identification of hybrid plants

According to the morphological identification, 120 of the 973 hybrid plants that were different from the female parent were selected. Taking individual genomic DNA of the hybrid plants as template, PCR amplification was performed using codominant primers NGA172 and NGA8. The real hybrid plant has two bands, which both male and female parents have, whereas non-hybrid plants has only one band which belongs to either the female or the male parent. Plants with other bands also were non-hybrid plants (Fig. 3). Through the identification of SSLP molecular markers, 119 of the 120 plants displayed two characteristic bands of male and female parents simultaneously and were considered real hybrid plants.

Resistance identification of F₁, F₂ and BC₁ progenies

After being inoculated with B. cinerea isolate BC18, 944 of 975 F₁ hybrid individuals from the cross Col-0 × Ler were found resistant, while the other individuals showed susceptibility to B. cinerea. Among the 3270 F₂ individuals, 2631 displayed resistance and 639 individuals displayed susceptibility, and the ratio between resistant and susceptible plants fit the 13∶3 segregation on the basis of the χ² test. Among the 821 BC₁ individuals, 550 individuals displayed resistance and 271 individuals displayed susceptibility, and the ratio fit the 2∶1 segregation. These results indicate that the resistance of Arabidopsis against B. cinerea infection is controlled by two genes. These two genes are suppressor genes. Only when these two genes are expressed at the same time can Arabidopsis display resistance to B. cinerea.

Selection of linked markers

Seven codominant markers located on different chromosomes of Arabidopsis were selected for screening of polymorphism between two bulks at first. Results showed that NGA1107 located on the fourth chromosome and NGA151 located on the fifth chromosome were polymorphic between two parents and two pools (Fig. 4), suggesting that two disease-resistant genes, namely BC1 and BC2, might be located on the fourth and fifth chromosome, respectively.

Chromosomal location of the resistance genes

According to the published CAPS and SSLP markers available on the internet (http://www.arabidopsis.org/home.html), markers CCR1, DHS1, R89998, NGA249, CA72, NGA106, and CIW8 closely linked to NGA1107 or NGA151 were selected to map two disease-resistant genes in 144 F₂ individuals. The analysis of CAPS and SSLP markers was performed as described previously (Konieczny and Ausubel, 1993; Bell and Ecker, 1994). The electrophoresis bands of some F₂ plants with partial molecular markers are shown in Fig. 5.

The linkage relationship between the markers and resistant genes was analyzed using the Mapmaker/Exp Version 3.0 software. The order of the three markers and the BC1 locus was NGA1107-CCR1-BC1-DHS1 with genetic distances of 1.0 cM, 1.2 cM, and 1.6 cM, respectively, for the three intervals. The order of the five markers and the BC2 locus was NGA249-NGA151-BC2-NGA106-R89998-CIW8 with genetic distances of 5.9 cM, 1.4 cM, 2.4 cM, 5.3 cM, and 5.1 cM for the five intervals, respectively. The genetic map of two resistance genes was constructed (Fig. 6).

Discussion

Map-based cloning, also called positional cloning, is a method to isolate genes of interest based on an intensive genetic and physical mapping. Arabidopsis ecotypes Wassilewskija (Ws) and Niederzenz have both successfully been used in positional cloning projects. The most commonly used combination for mapping purposes, however, is Landsberg erecta × Columbia (Ler × Col). Many valuable resources that can facilitate mapping Ler × Col populations have been accumulated. According to early reports, more than 10 disease-resistant genes of Arabidopsis have been cloned using the positional cloning approach, including NPR1 (Cao et al., 1997), RPS2 (Bent et al., 1994), RPM1 (Bisgrove et al., 1994), RPP5 (Parker et al., 1997), RPS4 (Gassmann et al., 1999), and RAC1 (Borhan et al., 2004).

Map-based cloning technology also has its own limitations. When analyzing spontaneous mutations, the most frequent complex situation is a certain given character determined by more than one gene locus. For example, resistance to powdery mildew in a cross between Kashmir-1 (resistant) and Columbia (susceptible) was found to involve at least three genetic loci acting in an additive fashion (Wilson et al., 2001). In our experiment, resistance to B. cinerea in a cross between Col-0 (resistant) and Ler (susceptible) types was found to involve at least two genetic loci acting in an inhibitive fashion. In these cases, fine mapping of these resistant genes requires the reduction of the genetic complexity of the mapping population, for example, by creating recombinant-inbred lines in which only one of the loci remains polymorphic.

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