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Rose is a woody perennial shrub popular for its elegant and fragrant flowers. This inspiring and verstyle ornamental belongs to genus Rosa originating in the northern hemisphere i.e. Europe, North America, Asia and the Middle East (Joyaux, 2003). Among ornamental plants, roses are the most popular garden plant, cut flower and pot flower. The world's leading exporters of the cut flowers are Netherlands, Danmark, USA, Columbia und Kenia (Döpper & Unterlercher 2007). Total cut flower imports of Germany include roses as 35%, Tulpe as 10 % und Gerbera as 7 %(ZMP 2006). According to statistischem Bundesamt Germany has spent about 219 Mio. € to import rose cut flowers, 37 Mio. € for Chrysanthemen und 34 Mio. € for Nelken, respectively in year 2005.
The broad expansion and competition in the international trade of floricultural products demand blemish free ornamentals with eye catching visual quality (Hei-ti Hsu 2006). Rose diseases caused by different pathogens are major cause of losses in terms of lower yields, weak growth, poor visual quality and in extreme cases plants death. The economically important pests of roses are mites, aphids, thrips, whiteflies, scale insects, weevils, caterpillar, nematodes and beetles (Shorthouse 2003). Whereas, the main diseases of roses include, powdery mildew (Podosphaera pannosa), black spot (Diplocarpon rosae Wolf), botrytis or grey mold (Botrytis cinerea), downy mildew (Peronospora sparsa Berk), rust (Phragmidium mucronatum, Phragmidium tuberculatum), and crown-gall (Agrobacterium tumefaciens) (Linde 2003; Dreves-Alvarez 2003; Gleason 2003; Xu 2003; Shattock 2003). Currently, these diseases are controlled by intensive spraying of agrochemicals that has many reservations as financial costs, unfavourable environmental consequences, health hazards and legal restrictions. The most devastating fungal diseases of roses are powdery mildew and black spot. Black spot is a problem for field and garden roses grown in humid and moist conditions throughout the world (Horst, 1983). This disease is controlled by continuous spraying of fungicides during spring and summer. In addition to that the use of roses in landscaping, demands carefree type of roses that can survive without protection measures and pruning. To relieve these problems, the safest option is the use of resistant varieties. Now a days the breeding for resistant rose varieties against black spot is an important goal for many rose breeding programmes.
Despite of all beauty and magnetism most of the cultivated roses lack natural resistance against black spot. However, there are numerous wild roses that are resistant to black spot. Rose species reported to be resistant to black spot are R. multiflora, R. rugosa, R. wichuraiana, R. roxburghii, R. virginiana, R. carolina and R. laevigata (Drewes-Alvarez, 2003). The introgression of the natural genetic resistance to modern roses is very suitable strategy for creating resistant cultivars. At this point conventional and molecular methods of breeding possess some limitations to manipulate rose, as its high heterozygosity, polyploidy and limited knowledge of its genetic make-up. In addition to that this exploitation of natural genetic resistance requires understanding the resistance genes in terms of diversity, genomic organization and functionality. Plant pathogen interaction studies with particular reference to functional genomics on one hand will provide the basis for conventional and molecular resistance breeding and on other will help to identify and isolate the functional rose resistance genes. Up to date a single monogenic dominant resistance gene locus (Rdr1) active against black spot (Diplocarpon rosae) has been characterized by phytopathological methods in tetraploid roses (Debener et al. 1998; Malek and Debener 1998) and mapped to a telomeric position of linkage map by means of molecular markers (Debener and Mattiesch 1999; Malek and Debener 2000). The construction of two large insert BAC libraries (Kaufmann et al. 2003) and sequencing of important BAC clones located the gene Rdr1 within a 220kb interval of DNA (Debener Lab. unpublished data). However, this interval contains about 9 copies of “resistance-gene-like” sequences (RGA) of the T1R-NBS-LRR type of resistance genes (Hattendorf and Debener 2007). As the only known function of this class of genes is resistance and no other resistance like sequence could be detected via degenerate PCRs there is a chance that one of the 9 genes is the functional gene. This PhD research is aimed to functionally characterize the Rdr1 resistance gene family of roses and to identify the major single gene that confers resistance to black spot.
2. Review of Literature
2.1.1. Taxonomy and Classification:
The genus Rosa comprises of about 200 different species (Wissmann 2003a) with a basic chromosome number of 7. The ploidy levels for wild species range between diploid to octaploid (Gudin 2000), whereas most of the modern cultivars are tetraploid. Cultivated roses comprise a huge genetic variability with more than 20000 varieties as they are heterozygous outcrossers. However, only eight to ten wild species had a major contribution in rose cultivation during more than 2000 years (Chandler and Lu 2005; Gudin 2001).
The genome complexity in terms of various modes of reproduction and character inheritance make the infrageneric taxonomy of this genus very difficult (Wissemann and Ritz 2005 and 2007). The most useful classification in use is the one defined by Alferd Rehder (1960) with modifications. According to this classification genus Rosa is divided in four sub genera i.e. Hulthemia, Hesperrhodos, Platyrhodon and Rosa. Roses belong to subgenera Rosa that is further divided in 10 sections (Wissemann 2003). Some of the important features of these sections are presented in table 1.
The most popular classification for cultivated roses is the one formatted by The American Rose Society and the World Federation of Roses (Cairns 2003). According to that roses can be classified into three groups: the Species (wild roses); Old Garden Roses; and Modern Roses. Wild roses found millions of years before man that bloom once with a flower of four-five petals and varities includes Cherokee Roses, Dog Roses, Gallic Roses, French Roses, Redleaf roses etc. All roses recognized before 1867 are grouped as Old Garden Roses. They usually bloom fragrant white or pastel colour flowers, once per season as summer starts and some varieties include China, Tea, Moss, Damask, Bourbon, Hybrid Perpetual, and Noisette roses. Whereas, all roses recognized after 1867 are grouped as Modern Roses. They bloom colourful flowers many times a year and some important varieties include hybrid tea, floribunda, and grandiflora(Cairns 2003).
Table 1: Subdivision of the subgenera Rosa. This division is based on different physical features of wild roses as stipules, sepals, blooms, styles, leaves, thorns, etc. (Cairns 2003; Wissemann 2003).
Plant geneticists and molecular biologists have paid very little attention to this thorny shrub may be due to the problems related to its polyploid nature, germination, reproduction and/or limited fertility (Linde et al. 2007). However, during last two decades there are continuous attempts to generate valuable genomic and functional resources for these inspiring ornamentals. Up to date eight single gene loci (table 2) and 14 QTLs have been analyzed in roses. Dugo et al. (2005) mapped 13 QTLs in total controlling simple flowers with five petals to double flowers, powdery mildew resistance, leaf size, flowering time and size of the flowers. The gene t4 is a QTL controlling the number of prickles on the stems (Crespel et al. 2002). In addition to that four marker maps are also constructed, 3 in diploid (Debener and Mattiesch 1999; Crespel et al. 2002; Dugo et al. 2005; Yan et al. 2005) and one in tetraploid rose population (Rajapakse et al. 2001).
Rdr1* is the first single dominant resistance gene described in the genus Rosa that confers resistance against black spot and resistance due to Rdr1 is assumed to follow the gene-for-.gene model (Von Malek and Debener, 1998).
2.2. Black Spot:
Black spot of roses was first reported in 1815 by Fries in Sweden (Drewes-Alvarez, 2003). The causative agent of this foliar disease of roses is Diplocarpon rosae, a plant pathogenic ascomycete that belongs to the family Dermateaceae (Nauta & Spooner, 2000). The conidial or imperfect stage of the fungus is known as Marssonina rosae (Lib.) Lind (Baker, 1948). Diplocarpon rosae flourish in humid and wet conditions of spring. During infection it produces ascospores, and conidia that are dispersed by wind and rain splashes to healthy plants. Usually infection starts from lowest leaves and progress upward causing early defoliage, vigour loss and in extrem cases plants death (Bhashkaram et al. 1974). Diplocarpon rosae has been differentiated into six physiological races in Germany and four in United Kingdom using single conidial isolates (Debener et al. 1998, Von Malek et al. 2000, Yokoya et al. 2000). In the research group of Debener at the University of Hannover, AFLP and SSR analyses have been performed to characterise the genetic diversity within a collection of D. rosae isolates. First results showed a lower genetic complexity of populations indicating a low mobility compared to airborne pathogens (Blechert 2005).
2.2.1. Symptoms and Infection Pattern:
During 20th century, the infection cycle of fungus was described in detail (Aronescu 1934 and Frick 1943). The disease symptoms include brown or dark black spots usually surrounded by chlorotic areas on leaves leading early defoliage of the host (Horst, 1983).
D. rosae is a hemibiotroph having a biotrophic phase characterized by haustoria and a necrotrophic phase characterized by necrotrophic intracellular hyphae (Gachomo et. al, 2007). In nature this hemibiotrophic fungus produces a two celled conidia asexually that under favourable conditions germinates and penetrates the cuticle of the leaf producing hyphae and appressoria followed by haustoria and within 10-12 days of infection the damage of the host can be visualized macroscopically. Blechert and Debener (2005) have characterized the morphology of different interactions of D. rosae and roses in 8 different types; 5 types as susceptible and 3 types as resistant interactions.
2.2.2. Disease Control
Black spot is controlled by integration of different approaches e.g. planting under sun-shine to keep foliage dry, good sanitation, no overhead watering and intensive application of fungicides throughout the growing season. Effective fungicides propiconazole, mancozeb, chlorothalonil, benomyl, or a copper-sulphur dust are sprayed shortly after bud break and continue at regular intervals until the first hard frost (Bowen and Roark 2001). These regular sprays are very important for proper disease control as black spot may develop at any time during wet and humid conditions. The continuous use of fungicides poses serious legal restrictions and environmental concerns. Therefore, rose growers are very much interested to exploit the most effective and safe option of growing roses i.e. growing black spot resistant rose varieties.
2.3. Rose Resistance Breeding for Black spot
Utilization of conventional methods of breeding to introduce natural resistance of wild species to cultivated roses is a lengthy process that will add many undesired traits in existing regular varieties (Drewes-Alvarez, 2003). Recent developments in biotechnology and genetic engineering, with particular reference to the transfer of foreign genes into plants, gene isolation, identification and functional analysis have opened new opportunities for the alterations of single traits in already successful varieties (Chandler and Lu 2005). There are reports describing the transfer of genes for pathogenesis-related proreins (Marchant et al., 1998), ribosome inactivating proteins (Dohm et al. 2007) and phytoalexins (Lorito et al., 2002) to increase the plant resistance against pathogen. Chitinases and glucanases are produced by plants in response to pathogen attack, and their over-expression could improve resistance to fungi. A rice gene for chitinase was transferred into rose callus showing 13-43% reduction of black spot severity (Marchant et al., 1998). In addition to that gene for ribosome inactivating proteins and barley genes for chitinases and glucanases were transferred into rose embryos by Agrobacterium and their over expression reduced the black spot infection upto 60% (Dohm et al. 2002; 2007); that is not enough to have a resistant variety. The transfer of a disease resistance gene active against black spot provides an effective option of producing black spot resistant rose varieties and the first step towards this landmark is to find the sources of black spot resistance in wild and cultivated roses. Many rose varieties, both in cultivated and natural species are reported as resistant against many major diseases. Field and laboratory evaluations proved many rose species highly resistant to black spot as R. multiflora, R. rugosa, R. wichuraiana, R. roxburghii, R. virginiana, R. carolina and R. laevigata (Drewes-Alwarez, 2003).
Rdr1 is the first resistance gene described in the genus Rosa and active against black spot (von Malek and Debener 1998). However, the exploitation of natural genetic resistance and transfer of the resistance to modern roses requires understanding the resistance genes in terms of diversity, genomic organization and functionality.
2.3.1. Plant pathogen interplay
Plants have evolved multiple defense strategies to counteract biotic stresses. These include passive or pre-existing defenses as waxes, cuticle, apoplastic space, stable cell wall etc. and inducible defenses (Goehre and Robatzek, 2008). Pathogen overcome some of the passive defenses by secreting hydrolytic enzymes or utilizing natural openings as stomata, hydathodes or wound sites to invade the apoplast space (Jones and Dangl, 2006). Some pathogens exclusively stay in apoplast; other pathogens such as bacteria use a type III secretion system (T3SS) to inject effector proteins through the cell wall and plasma membrane whereas fungi and oomycetes penetrate their hyphae through the cell wall and form haustoria (feeding structures) surrounded by the host plasma membrane. The inducible or active line defenses can detect microbe associated molecular patterns (MAMPs or PAMPs) as bacterial flagellin or fungal chitin by pattern recognition receptors (PRRs) usually residing in plasma membrane and trigger MAMPs-triggered-immunity (MTI or PTI) (Bent and Mackey, 2007). MTI activates signaling cascades involving Ca++ fluxes and mitogen activated protein kinases (MAPKs) leading to defense reactions involving production of reactive oxygen species (ROS), deposition of callose in the cell wall, expression of pathogenesis related proteins and defensins (Zipfel, 2009; Bolton, 2009; Pitzschke et al., 2009). However, some pathogens are able to suppress MTI through injecting effector proteins; these can also reinforce plants defenses by encoding resistance proteins (RP). RPs recognize specific effectors directly (gene-for-gene hypothesis) or indirectly (guard hypothesis) resulting in effector-triggered-immunity (ETI). The typical symptoms of ETI are hypersensitive response (HR) or programmed cell death (PCD) and systemic acquired resistance (SAR) (Shah, 2009; Zipfel, 2009; Goehre and Robatzek, 2008; Heath, 2000).
220.127.116.11 Plant Resistance Genes
As plants solely depend on innate immunity, they amplify R-genes in high number and position them in the genome in a way that favors their rapid evolution (Fluhr, 2001). RPs are classified in different families or groups depending on their structural domains (table 3). Most of the RPs contain tandem leucine-rich repeats (LRRs) that play an important role in specific recognition (McDowell, 2003).
18.104.22.168. NBS LRR Gene Family
Until recently, more than 40 plant resistance genes have been cloned from different plant species, majority of which belong to the NBS-LRR resistance gene family (Lukasik and Takken 2009; Jiang et al., 2007). These genes are found as single genes and/or as tight gene clusters and the only known function of this gene family in plants is elicitor recognition and activation of downstream signal pathways leading to disease resistance (Lorang et al., 2007) . This family is further subdivided in two groups based upon the structural differences at the amino terminus. Group 1 contains a TIR motif with homology to the toll/interleukin-1-receptor (TIR), whereas group 2 has a coiled coil (CC) domain, also sometimes referred as leucine zipper (LZ) (Pan et al,. 2000). In addition to that TIR group has an aspartic acid (D) as the final amino acid of the kinase 2 in the NBS domain whereas non-TIR group has a tryptophan (W) on this place (Pei et al. 2007).
The NBS is a part of a nucleotide binding (NB)-ARC domain that belongs to the STAND (signal transduction ATPases with numerous domains) family of NTPases. These proteins are proposed to regulate signal transduction as NB domain hydrolyzes NTP and changes its conformational states (reviewed in Takken et al., 2006). The structure of NB-ARC domain of RPs was derived from the crystal structures of APAF-1 or CED-4 by Takken and colleagues (2006). The alignment APAF-1 with RPs revealed three subdomains conserved in NBS-LRR proteins: a P-loop NTPase fold forming a parallel b-sheet flanked by a-helices, an ARC1 consists of a four-helix bundle and an ARC2 adopting a winged-helix fold that is connected to LRR domain by a short linker. LRR domains contain various numbers of tandemly repeated leucine-rich motifs with conserved core consensus of L-x-x-L-x-L-x-x-N that form a series of b-strands (Jiang et al., 2007;Wroblewski et al., 2007; Fluhr, 2001). The arc-shaped structure of LRR domain suggests its role in different intra and intermolecular interactions such as this domain is capable of direct recognition of pathogen effectors, regulating protein activation and signal transduction (Padmanabhan et al., 2009).. However, the mechanisms that make these dynamic functions possible await exploration. The N-terminus of NBS-LRR proteins is structurally diverse having homology to TIR or CC domain, as described earlier. The proposed functions of N-terminal domain are downstream/upstream signaling and pathogen recognition (Fluhr, 2001; Takken et al., 2006; Lukasik and Takken, 2009).
Typically, RPs activate a HR/PCD to halt the growth of a pathogen (Goehre and Robatzek, 2008). However, sudden activation of these proteins can damage plants themselves which suggests a tight regulation of their activation. This inactivation of RPs is achieved by intramolecular interactions between the various domains (Autoinhibition). According to the proposed model for the activation of NBS-LRR proteins (Lukasik and Takken, 2009; 2006; Bent and Mackey, 2007) in the absence of a pathogen these proteins are in resting (ADP) or off state. Detection of pathogen elicitors releases this tight negative control by conformational changes in LRR and ARC2 subdomain (Induced state) followed by exchange of a nucleotide that triggers active state of RPs ready to interact with downstream signaling components and activate defense responses. The perception of pathogen elicitors is accomplished by direct (gene-for-gene model) or indirect manner (guard or decoy model) (Hoorn and Kamoun, 2009). However, it is reported that the TIR or CC domain mediates indirect recognition in majority of such cases whereas LRR domain mediates direct recognition (reviewed in Padmanabhan et al., 2009).
Rdr1 resistance locus of roses has high similarity to the TIR type of NBS-LRR gene family (N from tobacco) and this locus contains 9 copies of such genes (Hattendorf 2005). There could be a many possibilities to transfer resistance genes in roses may be inter or intra species but to exploit the full potential of natural resistance it is very important to screen the real resistant genes candidates active against black spot through genetic and functional analysis.
2.3.2. Positional Cloning of Resistance Genes
One of the most traditional and unbiased approaches for the identification of genes governing important heritable traits is the positional or map based cloning. Typically, the procedure can be summarized as:
This unbiased method of discovering genes searches whole genome without any prior knowledge of the physiology, biology and/or the role of the genes. However, this approach is limited by the genome size, number of the genes within the locus of interest, presence of the transposons/ repetitive sequences in the species being investigated and time required for the complementation test. An alternative strategy could be the candidate gene approach based on the assumption that the loci controlling the trait of interest are carrying the genes of biologically known function. Human, animal and plant geneticists have successfully utilized this approach since the 1990s to reduce the number of candidate genes (Rothschild and Soller 1997; Byrne and Mc-Mullen 1996). These candidate genes can be classified as functional CGs when based on molecular or physiological studies or as positional CGs when based on linkage data of the locus of interest. The final validation of a CG is usually provided through physiological analyses, genetic transformation and/or sexual complementation (Byrne and McMullen 1996; de Vienne 1999). Several genes have been screened and mapped using this approach, the CO (constans) gene of Arabidopsis, involved in late flowering (Putterill et al. 1995), CGs for fruit quality in peach (Etienne et al. 199) and in tomato (Causse et al. 1999), plant height QTLs in maize (Beavis et al. 1991), QTLs affecting flowering time in Arabidopsis (Koornneef et al. 1998) for review), cloning and isolation of major disease resistance genes in several species (Lamb et al. 1989; Staskawicz et al; 1995; Bent 1996; Hammond-Kosack and Jones 1996 and 1997; Gebhardt 1997). Structural similarities between resistance genes isolated from different plants made it easier to clone R-genes using candidate gene approach (Mindrinos et al., 1994). The cloning of potato Gro1 gene, resistance to nematodes, was reported without prior construction of a physical map (Paal et al., 2004). They assumed that the resistance gene like marker St322, co-localized with Gro1 and based on a high resolution genetic map of Ballvora et al., (1995) and Leister et al., (1996), was identical or highly homologous to the nematode R gene. This marker isolated 15 candidate genes from genomic libraries. Inheritance analysis, linkage mapping and sequencing reduced the number of candidates to three. Stable Genetic complementation of potato validated that the gene Gro1-4 provided resistance against G. rostochiensis pathotype Ro1. However, the most successful and updated strategy for the identification of R-genes is the combination of positional cloning and candidate gene approaches.
22.214.171.124 Rdr1 Characterization
The Genetic Characterization of Rdr1 was started investigating the interaction of single conidial isolates of black spot on wild and cultivated roses. This interaction resulted in the identification of five different physiological races of black spot, Diplocarpon rosae, on roses (Debener et al. 1998). In this study a so called quadratic check implied as first evidence for the presence of a gene-for-gene relationship between black spot and roses. Meanwhile extensive phytopathological analyses in tetraploid rose populations were performed and the segregation ratios of the resistance reaction against DortE4, a black spot isolate, indicated the presence of a single dominant resistance locus in the duplex configuration (RRrr), which they called Rdr1.
The first linkage map for diploid roses was constructed using RAPD and AFLP markers in a population of 60 F1 plants (Debener and Mattiesch 1999). The hybrid population was resulted from a cross between the diploid rose genotypes 93/1–117 and 93/1–119 (a double pseudo-test-cross). In addition to molecular markers the map also shows the location of two genes controlling important morphological traits, petal number and flower colour. During the following year 7 AFLP markers linked to Rdr1 within a distances of 1.1 and 7.6 cM were developed using a tetraploid progeny-95/3 segregating for the presence of the blackspot resistance gene Rdr1 (von Malek et al. 2000). The most closely linked AFLP marker M10 was converted into a SCAR marker and screened in a larger population. The SCAR marker was found to be linked at a distance of 0.76 cM.
The closely linked markers developed by von Malek should enable the localisation Rdr1 on the rose linkage map developed by Debener and Mattiesch 1999. The direct mapping of Rdr1 in this population was not possible because no clear segregation of blackspot resistance could be observed. Moreover, the direct integration of the markers was not possible since none of the AFLP markers could be detected in the parental lines (93/1–117 and 93/1–119) and the SCAR marker SCM10 did not show any polymorphism between both genotypes even when applied as CAPS marker. The development of the RFLP markers BMA 1-4 from the AFLP marker M10 afforded the indirect localisation of Rdr1 to the distal ends of linkage groups A1 and B1 of 93/1–117 and 93/1—119, respectively (von Malek et al. 2000).
The first crucial step to start map based cloning of Rdr1 was the construction of a BAC library for R. rugosa genotype (Kaufmann et al. 2003). This BAC library comprised about 27,300 clones with an average insert size of 102 kb, containing 5.2 genome equivalents and the probability of recovering any given sequence of rose genomic DNA from this library is greater than 99%. Meanwhile, to faciliate positional cloning the mapping resolution in the Rdr1 region was improved by bulked segregant analysis using 538 plants of three diploid sister backcross populations segregating for Rdr1 (Kaufmann et al. 2003). The SCAR marker SCM10 and the other Rdr1-linked AFLP markers that were identified in the diploid populations could not be analyzed in the tetraploid population due to the lack of polymorphism between the parents (von Malek et al. 2000). Three new AFLP markers were located on one side of Rdr1, the closest of which was at a distance of 0.18 cM to Rdr1 and one AFLP marker co-segregated with black spot resistance in the 538 plants in addition to that one CAPS marker was located on the telomeric side of Rdr1 at a distance of 0.93 cM in this way the gene was bracketed between two closely linked markers (Kaufmann et al. 2003).
However, the R. rugosa used to construct the BAC library does not possess the black spot resistance allele Rdr1 and this gene cannot be isolated directly from the R. rugosa BAC contig. Nevertheless, the reason for selecting R. rugosa was the small genome size (2 x) and using this valuable contig for the establishment of a syntenic contig in the genotype harbouring Rdr1. Therefore, a second library was established from the R. multiflora genotype 88/124-46 which is homozygous for Rdr1. This genotype obtained the resistance by introgression from the wild rose species R. multiflora and is homozygous for the resistant allele (Kaufmann et al. 2003). The estimated genome size of Rosa multiflora is almost twice as large as of Rosa rugosa. This BAC library was constructed in the transformation competent vector pcLD04145 with a smaller average insert size (46 kb) compared to the R. rugosa library. The multiflora library consists of 60,000 clones with an average insert size of 46 kb providing genome coverage of 4.8. Markers from the rugosa contig were taken to identify the respective multiflora clones via hybridisation and the contig was constructed via hybridisation and PCR analyses of clone end sequences. Although the new 88/124-46 contig differed in physical distances between some of the molecular markers, it turned out to be co-linear and the ends of both contigs could be linked to each other. The new contig is represented by a minimum of six clones with a maximum size of about 400 kb. The contig borders were determined by one plant among 538 showing recombination to the BAC-end derived markers 20T and 55T, respectively. Several markers co-segregating with Rdr1 among 538 plants were found. Without recombination to the target locus a family of resistance gene analogues with high similarity to the TIR-NBS-LRR gene N from tobacco was identified with 9 copies on this contig (Hattendorf 2005).
The contig is not yet finished; while the left end was determined early the right end is not closed yet since there is still a gap between the clones fished with the right end of the rugosa contig and the rest of the contig. A strategy to close the R. multiflora contig and improving the map resolution is to analyse additional plants of population 97/10 to detect any recombination event at the right side of Rdr1 to get an evidence of having spanned the gene. There are additional plants available that are in process of screening.
Based on the observation that the contig contains at least nine resistance gene analogues of the TIR-NBS-LRR class, initially one of the clones in the centre of the contig harbouring three RGAs (155F3) was completely sequenced to obtain full sequence information on the RGAs. Two complete and one partial TIR-NBS-LRR genes with highly significant similarity to the N-gene from tobacco and the potato Gro1 genes were identified along with some copia like retrotransposons and three microsatellites. Sequences obtained for the TIR-NBS-LRRs from both the R. multiflora (88/124-46) BAC and the R. rugosa BAC show that within an amplified region of around 3 kb of the RGA similarities varied between 86 and 96 % even across the two species which shared similarities of 92%-96%. Based on this consensus primers were designed that reliably amplified all the RGA elements from diverse BAC clones. This was verified with hybridisation and sequencing experiments. A total number of 9 RGAs could be sequenced on three to five of the BAC clones, with overlaps, of the R. multiflora contig (figure 1; Table 4). With the conserved primers expression of the RGAs was analysed in rose leaves. A total of six different expressed copies of the RGAs could be identified. One of the expressed RGAs is located on the fully sequenced clone 155F3. First analyses with 3´RACE have identified 3´ends of three of the six expressed genes of 140-220 additional base pairs of sequence. Several degenerate primer combinations that amplify diverse NBS-LRRs and serin threonin kinase candidate sequences from rose DNA were employed to check the contig clones for the presence of other candidate genes. However no amplification apart from the TIR-NBS-LRR gene family could be amplified. Although we cannot exclude that Rdr1 belongs to a class of genes other than TIR-NBS-LRRs it is highly probable that one of the RGA copies on the contig represents Rdr1. The big challenge will now be the identification of the functional resistance gene..
BAC clones spanning Rdr1 resistance locus of R. multiflora
The above diagram is based on the previous molecular and sequence analysis of three BAC clones (29O3, 94G8, 20F5) which are part of the genomic library constructed for R. multiflora genotype 88/124-46, homozygous for Rdr1 (Debener Lab.). Green arrows represent RGAs that are found to be expressed in leaves of rose resistant genotypes whereas; red arrows are for non-expressed RGAs. The yellow triangles are representing copia elements within this interval of DNA. The arrows and triangles are pointing the orientation RGAs and copia elements in the contig, respectively. Table 4 summarizes the exact positions of these elements on single BAC and on contig. In addition to that Table 4 also provides some insights in previous functional data available for Rdr1 candidates.
3. Aims and Objectives of the Project:
The main objective of the project was the genomic and functional characterisation Rdr1 candidates (9-CGs) for the identification of a single major rose gene that confers resistance to black spot (diplocarpon rosae). To reduce the number of candidates following strategic plan was followed.