FruitBreeding - Rubus genomics research | The James Hutton Institute

Rubus genomics research

Genetic linkage maps in Rubus


Breeding methods used in raspberry (Rubus) have changed very little over the last 40 years or so. Little novel germplasm has made its way into commercial cultivars. However, with the narrowing genetic base coupled with the increasing demands from consumers, new breeding methods are required to meet changing demands. The speed and precision of breeding can be improved by the deployment of molecular tools for germplasm assessment and the development of genetic linkage maps. Such genetic linkage maps can facilitate the development of diagnostic markers for polygenic traits and the identification of genes controlling complex phenotypes. View some of the maps we have developed.

Understanding the genetic control of commercially and nutritionally important traits and the linkage of these characteristics to molecular markers on chromosomes is the future of plant breeding. Red raspberry (Rubus idaeus) is a good species for the application of such techniques, being diploid (2n=2x=14) with a very small genome (275 Mbp). Indeed, the haploid genome size of raspberry is only twice the size of Arabidopsis, making it highly amenable to complete physical map construction, thereby providing a platform for map-based gene cloning and comparative mapping with other members of the Rosaceae (Dirlewanger et al 2004).

The availability of abundant genetic variation in natural and experimental populations of Rubus and adaptation to a range of diverse habitats (Graham et al 1997b; Marshall et al 2001; Graham et al 2003) offers researchers a rich source of variation in morphology, anatomy, physiology, phenology and response to a range of biotic and abiotic stress. The ability to vegetatively propagate individual plants provides opportunities to capture genetic variation over generations and replicate individual genotypes to partition and quantify environmental and genetic components of variation of genetic linkage maps. These are necessary to develop diagnostic markers for polygenic traits and in the future, possibly identify the genes behind the traits.

Mapping in Rosaceae

The Rosaceae is an economically important family of perennial fruit bearing crops that includes members of the following genera: Malus (apple), Pyrus (pear), Rubus (raspberry, blackberry), Fragaria (strawberry) and Prunus (stone fruits). In addition the family also includes a number of important ornamental plants such as roses, flowering cherry, crab apple and quince. Molecular marker applications have been reviewed in Rubus (Antonius-Klemola 1999) and in the small fruits (Hokanson 2001). Linkage maps have been generated in other woody species (Ritter et al 1990; Grattapaglia & Sederoff 1994; Bradshaw et al 1994; Bradshaw & Stattler 1995) and in the small (soft) fruit crops a few maps exist. In the diploid strawberry (Fragaria vesca) and diploid blueberry (Vaccinium spp.) 445 cM and 950 cM or 1288 cM long linkage maps based on RAPD markers have been constructed (Davis & Yu 1997; Rowland & Levi 1994; Qu & Hancock 1997). Maps of other Rosaceous crops include Prunus maps (Joobeur et al 1998; 2000; Ballester 2000; Dirlewanger et al 1997,1998; Dettori et al 2001; Aranzana et al 2003) apple (Hemmat et al 1994; Maliepaard et al 1998; Liebhard et al 2003).

Resources are being developed in strawberry to enhance maps based on RAPD markers (Sargent et al 2003; Graham 2005). In raspberry the first genetic linkage of raspberry has recently been constructed (Graham et al 2004b). This 789 cM genetic linkage map was constructed utilising a cross between the phenotypically diverse European red raspberry cultivar Glen Moy and the North American cultivar Latham. SSR markers were developed from both genomic and cDNA libraries from Glen Moy. These SSRs, together with AFLP markers, were utilised to create a linkage map. Work is underway to enhance the map by the addition of further SSR, EST-SSR, SNiP and gene markers.

Mapping of genes underlying commercial traits

Mapping in raspberry is at an early stage. Preliminary work is underway to map genes underlying a number of commercially important traits. Gene H in raspberry has recently been mapped to Group 2 of the raspberry map (Graham et al 2005).

Raspberry breeders in general have limited resources and rarely include a primary screen for fungal diseases. It has been reported that some disease resistances are associated with distinctive morphological traits, most notably cane pubescence (fine hairs). Pubescence is determined by gene H (genotype HH or Hh), the recessive allele of which gives glabrous canes (genotype hh). Gene H is rarely homozygous because it is linked with a lethal recessive gene (Jennings 1988).

Raspberry cultivars and selections with fine hairs (pubescent canes) are more resistant to cane botrytis (Botrytis cinerea), cane blight (Leptosphaeria coniothyrium) and spur blight (Didymella applanata) than non-hairy ones (Knight & Keep 1958; Jennings & Brydon 1989) but more susceptible to cane spot (Elsinoe veneta), powdery mildew (Sphaerotheca macularis) and yellow rust (Phragmidium rubi-idaei) (Keep 1968, 1976; Jennings & McGregor 1988; Anthony et al 1986; Williamson & Jennings 1992).

How Gene H influences the large increase or decrease in disease resistance has not been determined. It has been suggested that it is due to linkage with major resistance genes or minor gene complexes that independently contribute to the resistance or susceptibilities of the six diseases affected. An alternative explanation is that the gene itself is responsible through pleiotrophic effects on each of the resistances (Williamson & Jennings 1992).

Current mapping work at The James Hutton Institute

Gene H has now been mapped and further mapping of other disease resistance genes is underway. This includes identifying the gene(s) responsible for resistance to raspberry root rot caused by Phytophthora spp (Graham & Smith 2002). Four regions across 4 linkage groups have been identified and further research aimed at confirming these in a second population through glasshouse and field trials is underway (Graham, Smith and Tierney unpublished data).

Preliminary QTL mapping has been carried out in raspberry using the recently developed genetic linkage map (Graham et al 2004b). Morphological data based on the segregation of cane spininess, and root sucker density and diameter were quantified in two different environments. Breeding for spinelessness is a major concern for breeders and there are several major genes that confer this trait (Jennings & Ingram 1983; Jennings 1988). The mapping parents differ for spine morphology with Glen Moy having a spine-free phenotype (being homozygous for gene s (Jennings 1988) whereas Latham is a densely spiny cultivar, the genetics of which has not been determined. The progeny generated from the cross were all spiny, though the extent of spines varied continuously from a very sparsely spiny cane to the densely spiny phenotype of the Latham parent. From the phenotypic data it was proposed that two or more genes are involved. This was supported by the mapping data where a number of markers were identified, linked to the spiny phenotypes. These markers mapped onto linkage group 2, and there appeared to be two linked regions within this group accounting for 98% of the variation.

Large differences exist in the extent of root sucker production in cultivated raspberries. Control measures based on the chemical burning of early canes produced from suckers are required in commercial plantations to optimise fruit yield (Jennings pers. comm). Roots of red raspberry have adventitious buds, which develop on most roots. The number, density and distance from the mother plant of the root suckers varies between genotypes. Only a proportion of the buds normally develop into suckers. Knight & Keep (1960) have shown that the ability to produce suckers in red raspberry is determined by the recessive gene skI or by the complementary genes sk2 and sk3. Interestingly, and probably not surprisingly, the measurements of density and spread map to the same linkage group (Group 8) with an overlap in the location of the QTLs for the two traits (Graham et al 2004b).

Map based cloning and genetic engineering

Map based cloning has yet to be carried out in raspberry. However, genetic engineering technologies, if they become widely acceptable to customers, could allow high quality cultivars to be transformed with genes conferring resistance to a range of pests and diseases (Watt et al 1999) thus offering the prospect of reduced pesticide application.

Potential of genetic engineering to confer pest and disease resistance

An example that shows the potential for genetic engineering is some recent research in strawberry. In this work the Cowpea protease trypsin inhibitor (CpTi) gene was introduced into strawberry and its expression resulted in promising levels of control in glasshouse feeding trials and field trials against larvae of vine weevil (Otiorhynchus sulcatus) (Graham et al 1997a, 2002b).

Use of gene transfer technologies to improve resistance to mites, insects and nematodes would be especially valuable because of the toxicity of acaricides, insecticides and nematicides, many of which are likely to be withdrawn from use in minor crops in the future. Fruit quality and other stress resistance genes would be valuable.

Risk assessment

However, it is vitally important that these genetically engineered crops are not toxic or pose a serious allergenic risk to humans, do not harm beneficial organisms (e.g. natural enemies of pests, crop pollinators, soil micro-organisms) or affect the wider environment. Large scale 'risk assessments' of genetically engineered, crops such as the Farm-Scale Evaluation of Oil Seed Rape, Sugar Beet and Maize are currently being undertaken in the UK, to ensure that on release they are environmentally benign. However, at present it is unlikely that genetic engineering will be used for soft fruit, but in future the availability of the appropriate technologies could be useful to allow us to respond to a new pest or disease situation.

DNA Marker Systems in Raspberry

DNA markers have a number of potential applications in raspberry. These include genotyping/fingerprinting, development of linkage maps, marker assisted selection. Rubus DNA based marker systems have been developed by Antonius-Klemola 1999; Hokanson 2001; Graham et al 2002a.

Use of marker systems to study genome variation

Genetic markers have been used widely to examine genetic variation within and between Rubus spp. An M13 bacteriophage probe has been used to examine different Rubus spp. and a number of red raspberries (Nybom et al 1990). A minisatellite probe was used by Kraft et al. 1996 to demonstrate that fingerprints of out-crossing species vary considerably compared to vegetative and apomictic clones. Chloroplast DNA sequence probes were used by Waugh et al 2000; Howarth et al 1997 to examine genotypic and taxonomic relatedness in raspberry. Ribosomal DNA ITS region has been used to construct a phylogenetic tree with representatives from 20 species (Alice & Campbell 1999). RAPD markers have been widely used to examine the relatedness of raspberry cultivars and species (Graham et al 1997b; Graham & McNicol 1995; Coyner 2000). A genetic linkage map has been constructed in raspberry using AFLP and SSR markers Graham et al (2004).

Use of marker systems for breeding

Marker-assisted selection is developing into a powerful tool for plant breeding, through its ability to select plants with the desired trait(s) accurately and at an early stage of growth. Rather than screening for a particular phenotype (trait), a breeder can screen for a marker tightly linked to the gene of interest that is identified through the construction of a linkage map in a population segregating for that trait. Alternatively, bulked segregant analysis can be used to identify markers linked to a particular trait, the position of which can then be determined on a linkage map (Graham & Smith 2002). QTL locations for resistance gene locations have been determined .


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