Identification of quantitative trait loci and candidate genes for specific cellular resistance responses against Didymella pinodes in pea.

KEY MESSAGE: Phenotyping of specific cellular resistance responses and improvement of previous genetic map allowed the identification of novel genomic regions controlling cellular mechanisms involved in pea resistance to ascochyta blight and provided candidate genes suitable for MAS. Didymella pinodes, causing ascochyta blight, is a major pathogen of the pea crop and is responsible for serious damage and yield losses. Resistance is inherited polygenically and several quantitative trait loci (QTLs) have been already identified. However, the position of these QTLs should be further refined to identify molecular markers more closely linked to the resistance genes. In previous works, resistance was scored visually estimating the final disease symptoms; in this study, we have conducted a more precise phenotyping of resistance evaluating specific cellular resistance responses at the histological level to perform a more accurate QTL analysis. In addition, P665 × Messire genetic map used to identify the QTLs was improved by adding 117 SNP markers located in genes. This combined approach has allowed the identification, for the first time, of genomic regions controlling cellular mechanisms directly involved in pea resistance to ascochyta blight. Furthermore, the inclusion of the gene-based SNP markers has allowed the identification of candidate genes co-located with QTLs and has provided robust markers for marker-assisted selection.

Lathyrus diversity: available resources with relevance to crop improvement--L. sativus and L. cicera as case studies.

BACKGROUND: The Lathyrus genus includes 160 species, some of which have economic importance as food, fodder and ornamental crops (mainly L. sativus, L. cicera and L. odoratus, respectively) and are cultivated in >1·5 Mha worldwide. However, in spite of their well-recognized robustness and potential as a source of calories and protein for populations in drought-prone and marginal areas, cultivation is in decline and there is a high risk of genetic erosion. SCOPE: In this review, current and past taxonomic treatments of the Lathyrus genus are assessed and its current status is examined together with future prospects for germplasm conservation, characterization and utilization. A particular emphasis is placed on the importance of diversity analysis for breeding of L. sativus and L. cicera. CONCLUSIONS: Efforts for improvement of L. sativus and L. cicera should concentrate on the development of publicly available joint core collections, and on high-resolution genotyping. This will be critical for permitting decentralized phenotyping. Such a co-ordinated international effort should result in more efficient and faster breeding approaches, which are particularly needed for these neglected, underutilized Lathyrus species.

Identification of Sources of Quantitative Resistance to Fusarium oxysporum f. sp. medicaginis in Medicago truncatula.

The resistance of 267 Medicago truncatula accessions was determined against the soilborne pathogen Fusarium oxysporum, one of the major constraints of forage and grain legumes worldwide. The initial screening of the collection revealed a wide range of disease response from completely resistant to highly susceptible to one strain of F. oxysporum f. sp. medicaginis. As a result, 26 accessions were identified as resistant, 9 as susceptible, and all other accessions as partially resistant. The phenotype of 12 resistant accessions was confirmed in two independent experiments on a subset of 23 accessions. Quantification of F. oxysporum f. sp. medicaginis within plant tissue indicated that the resistance level of the accessions is correlated with the amount of F. oxysporum f. sp. medicaginis within its shoot. Inoculation with a different F. oxysporum f. sp. medicaginis isolate indicated that the resistance phenotype was stable because accession response to both F. oxysporum f. sp. medicaginis strains followed similar trends. However, grouping accessions according to their geographic origin did not reveal foci of resistance, which supports the idea that resistance arose from independent events. The identification of 12 resistant accessions will be useful for further cellular and molecular studies to unravel the basis of resistance to F. oxysporum in this model species and to transfer resistance to legume crop.

Future prospects for ascochyta blight resistance breeding in cool season food legumes.

Legume cultivation is strongly hampered by the occurrence of ascochyta blights. Strategies of control have been developed but only marginal successes achieved. Breeding for disease resistance is regarded the most cost efficient method of control. Significant genetic variation for disease resistance exists in most legume crops with numerous germplasm lines maintained, providing an excellent resource for plant breeders. Fast and reliable screening methods have been adjusted to fulfill breeding program needs. However, the complex inheritance controlled quantitatively by multiple genes, has been difficult to manipulate. Successful application of biotechnology to ascochyta blight resistance breeding in legume crops will facilitate a good biological knowledge both of the crops-pathogen interaction and of the mechanisms underlying resistance. The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to speed up the delivery of improved cultivars to the farmer. To date, however, progress in marker development and delivery of useful markers has been slow in most legumes. The limited saturation of the genomic regions bearing putative QTLs in legume crops makes difficult to identify the most tightly linked markers and to determine the accurate position of QTLs. The application of next generation sequencing technologies will contribute to the development of new markers and the identification of candidate genes for ascochyta blight resistance.

Transcription factor profiling leading to the identification of putative transcription factors involved in the Medicago truncatula-Uromyces striatus interaction.

Understanding the host response to Uromyces sp., the causal agent of rust in many crop species, is crucial in elucidating the specific biology of rust resistance. In an attempt to unravel the Medicago truncatula-U. striatus interaction, we performed a global analysis of transcription factor (TF) expression in resistant and susceptible accessions of the model plant M. truncatula during infection with U. striatus. For this purpose, an established qPCR platform was applied, consisting of specific primer pairs for more than 1,000 predicted TF genes. A total of 107 putative TF genes out of the 1,084 studied were differentially expressed. Thirteen of the TFs that were differentially expressed between resistant and susceptible genotypes are known to be relevant in cellular defense. These data suggest that resistance could be mediated both by genes that are constitutively expressed and by genes, which are activated/repressed when plants are inoculated. These defense related TFs sequences were amplified in chickpea DNA with the aim of determining the location of these genes on the genetic map of this crop and identifying possible DNA regions involved in resistance mechanisms.

Recognition of root exudates by seeds of broomrape (Orobanche and Phelipanche) species.

BACKGROUND AND AIMS: The long co-existence of broomrapes and their hosts within the same environment has culminated in a strong adaptation and effective parasitism. As a first step of specialization in the parasitic process, seed receptors of parasitic plant species vary in their ability to recognize compounds released by their hosts. This work aims to investigate potential patterns for the reception requirements needed to activate germination within Orobanche and Phelipanche species. METHODS: Induction of the germination of seeds of nine Orobanche and Pheliphanche species by root exudates of 41 plant species was studied and subjected to biplot multivariate analysis. KEY RESULTS: A high level of specialization in root exudate recognition was found in Orobanche densiflora, O. gracilis and O. hederae, which germinated almost exclusively in contact with root exudates from the plants they infect in nature. At the opposite extreme, Phelipanche aegyptiaca, P. ramosa and O. minor were highly generalist, germinating when in contact with the root exudates of most plant species. Orobanche crenata, O. cumana and O. foetida showed intermediate behaviour. CONCLUSIONS: A universal germination stimulant for all broomrape species has not being identified to date. The synthetic stimulant GR24 is active against most of the weedy broomrape species, but fails with the non-weedy species tested in this study and with the very recent weedy species O. foetida. In addition, germination behaviour of broomrape species depends on the crop plant tested. Weedy broomrapes with a broad host spectrum respond better to the different exudates released by a wide range of crops and wild species than do non-weedy broomrapes, which have a narrow host spectrum and are more restricted to their host range. Root exudates of many plant species were active in stimulating germination of seeds of Orobanche and Phelipanche species for which they are not described as hosts, representing interesting examples of potential trap crops.

First Report of Crenate Broomrape (Orobanche crenata) on White Lupine (Lupinus albus) Growing in Alkaline Soils in Spain and Egypt.

Crenate broomrape (Orobanche crenata Forsk.) is a parasitic weed known to threaten legume crops since antiquity. It is mainly restricted to the Mediterranean Basin, Southern Europe, and the Middle East where it is an important pest in grain and forage legumes and in some apiaceous crops such as carrot and celery (1). White lupines are cultivated in acid soils, which usually are free of O. crenata infestations. However, breeders are attempting to develop white lupine cultivars adapted to alkaline soils (2). We report here findings of O. crenata infection in field trials of this new lupine germplasm in alkaline soils in experimental farms with a known history of faba bean cultivation and heavy infestation of O. crenata in Kafr El-Sheikh, Egypt and Córdoba, Spain in the spring of 2009. Symptoms were typical of O. crenata infection with reduced growth and emergence of typical O. crenata nonbranched spikes close to the lupine plants. Infection was confirmed by digging up the plants to verify the attachment of the broomrape plant to the lupine. O. crenata plants growing on lupines were fully fertile, producing viable seeds. Plant morphology was typical of O. crenata (1). Voucher specimens were deposited at the Herbarium of the Botanic Department of the University of Córdoba. To our knowledge, this is the first report of O. crenata infecting lupine and is relevant because the expected introduction of alkaline-tolerant lupine cultivars will extend its area of cultivation into fields heavily infested with Orobanche. O. crenata is highly polymorphic and could easily adapt to, recognize, and infect this new host. Development of lupine-adapted O. crenata populations should be monitored because it could represent a major constraint on lupine introduction into alkaline soils. References: (1) D. M. Joel et al. Biology and Management of Weedy Root Parasites. Page 267 in: Horticultural Reviews. Vol. 33. John Wiley and Sons, Inc. Hoboken, NJ, 2007. (2) M. Vishnyakova and A. Mikic, White lupin (Lupinus albus L.) landraces and the breeding for tolerance to alkaline soil reaction. Page 142 in: Second GL-TTP Workshop: Integrating Legume Science and Crop Breeding. Novi Sad, Serbia, 2008.

First Report of Crenate Broomrape (Orobanche crenata) on Lentil (Lens culinaris) and Common Vetch (Vicia sativa) in Salamanca Province, Spain.

Broomrapes (Orobanche spp.) are obligate parasites that infect roots of dicotyledonous plants. Orobanche species are particularly important in southern and eastern Europe, the Middle East, and North Africa. O. crenata (crenate broomrape) has been known to threaten legume crops since antiquity. This parasitic weed is mainly restricted to the Mediterranean Basin, southern Europe, and the Middle East and is an important pest in grain and forage legumes as well as in some Apiaceous crops such as carrot and celery (2,3). In Spain, O. crenata is a well-known problem on grain legumes in the south (Andalucía and Extremadura regions) and along the entire east coast north to the border with France. However, it has never been reported as a problem in central Spain. Castilla-León is the major pea-, vetch-, and lentil-producing area of Spain and was believed to be free of O. crenata. However, widespread and heavy infections of O. crenata (as many as 20 broomrapes per m2) were identified in the spring of 2007 on lentil and common vetch growing in several fields in Salamanca Province, between La Vellés, Palencia de Negrilla, and Aldeanueva de Figueroa, covering an area of approximately 80 km2. Infection of lentil and vetch plants was confirmed by digging up the plants to verify the attachment of the broomrape plant to the lentil or vetch roots. Morphology was typical of O. crenata (2), i.e., large erect plants with single, nonbranched spikes that may reach a height of up to 1 m, bearing many flowers of diverse pigmentation from yellow through white to pink and violet. The calyx is 13 to 18 mm with segments free and bidentate. The corolla is 18 to 28 mm, glandular pubescent, the lips often with lilac veins, lips divergent, large, and not ciliate. The anthers are brown, glabrous, or subglabrous. The filaments insert 2 to 3 mm above the base of the corolla and are hairy at the base with glandular hair at the apex. Voucher specimens were deposited at the Herbarium of the Botanic Department of the University of Córdoba. The heavy and widespread level of infection observed in several fields is most likely explained by an inadvertent introduction of O. crenata seed mixed with seed of lentil and vetch. To our knowledge, this is the first report of O. crenata infecting lentil and common vetch in Salamanca Province and is relevant because the area was considered free of the plant. Remarkably, a recent modeling study suggested that O. crenata might become a problem in central and northern Spain (1) since climatic conditions are suitable for its establishment. The spread of this infestation should be monitored because it could represent a major constraint on legume production in this region. References: (1) J. H. Grenz and J. Sauerborn. Agric. Ecosyst. Environ.122:275, 2007. (2) D. M. Joel et al. Biology and Management of Weedy Root Parasites. Page 267 in: Horticultural Reviews. Vol. 33. John Wiley and Sons, Inc. Hoboken, NJ, 2007. (3) D. Rubiales et al. Euphytica 147:187, 2006.

Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues.

BACKGROUND AND AIMS: The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. METHODS: Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. KEY RESULTS: Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. CONCLUSIONS: The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.

Plant resistance to parasitic plants: molecular approaches to an old foe.

Parasitic weeds pose severe constraint on major agricultural crops. Varying levels of resistance have been identified and exploited in the breeding programmes of several crops. However, the level of protection achieved to date is either incomplete or ephemeral. Resistance is mainly determined by the coexistence of several mechanisms controlled by multigenic and quantitative systems. Efficient control of the parasite requires a better understanding of the interaction and their associated resistance mechanisms at the histological, genetic and molecular levels. Application of postgenomic technologies and the use of model plants should improve the understanding of the plant-parasitic plant interaction and drive not only breeding programmes through either marker-assisted selection (MAS) or transgenesis but also the development of alternative methods to control the parasite. This review presents the current approaches targeting the characterization of resistance mechanisms and explores their potentiality to control parasitic plants.

Differential effects of phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and energetic metabolism inhibition on resistance of appropriate host and nonhost cereal-rust interactions.

ABSTRACT Effects of phenylpropanoid and energetic metabolism inhibition on resistance were studied during appropriate host and nonhost cereal-rust interactions. In the appropriate barley-Puccinia hordei interaction, phenylalanine ammonia lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) inhibition reduced penetration resistance in two genotypes, suggesting a role for phenolics and lignins in resistance. Interestingly, penetration resistance of the barley genotype 17.5.16 was not affected by phenylpropanoid biosynthesis but penetration resistance was almost completely inhibited by D-mannose, which reduces the energy available in plant host cells. This suggests a parallel in the cellular basis of penetration resistance between 17.5.16 rust and mlo barleys powdery mildew interaction. Results revealed differing patterns of programmed cell death (PCD) in appropriate versus nonhost rust interactions. PAL and CAD inhibitors reduced PCD (hypersensitivity) in appropriate interactions. Conversely, they had no effect in PCD of wheat to P. hordei; whereas D-mannose dramatically reduced nonhost resistance and allowed colony establishment. The differential effects of inhibitors in the expression of the different resistances and the commonalities with the cereal-powdery mildew interaction is analyzed and discussed.

Genetic mapping of QTLs controlling horticultural traits in diploid roses.

A segregating progeny set of 96 F1 diploid hybrids (2n = 2x = 14) between "Blush Noisette" (D10), one of the first seedlings from the original "Champneys' Pink Cluster", and Rosa wichurana (E15), was used to construct a genetic linkage map of the rose genome following a "pseudo-testcross" mapping strategy. A total of 133 markers (130 RAPD, one morphological and two microsatellites) were located on the 14 linkage groups (LGs) of the D10 and E15 maps, covering total map lengths of 388 and 260 cM, respectively. Due to the presence of common biparental markers the homology of four LGs between parental maps (D10-1/E15-1 to D10-4/E15-4) could be inferred. Four horticulturally interesting quantitative traits, flower size (FS), days to flowering (DF), leaf size (LS), and resistance to powdery mildew (PM) were analysed in the progeny in order to map quantitative trait loci (QTLs) controlling these traits. A total of 13 putative QTLs (LOD > 3.0) were identified, four for FS, two for flowering time, five for LS, and two for resistance to PM. Possible homologies between QTLs detected in the D10 and E15 maps could be established between Fs1 and Fs3, Fs2 and Fs4, and Ls1 and Ls3. Screening for pairwise epistatic interactions between loci revealed additional, epistatic QTLs (EQTLs) for DF and LS that were not detected in the original QTL analysis. The genetic maps developed in this study will be useful to add new markers and locate genes for important traits in the genus providing a practical resource for marker-assisted selection programs in roses.

Interaction between Orobanche crenata and its host legumes: unsuccessful haustorial penetration and necrosis of the developing parasite.

BACKGROUND AND AIMS: Orobanche species represent major constraints to crop production in many parts of the world as they reduce yield and alter root/shoot allometry. Although much is known about the histology and effect of Orobanche spp. on susceptible hosts, less is known about the basis of host resistance to these parasites. In this work, histological aspects related to the resistance of some legumes to Orobanche crenata have been investigated in order to determine which types of resistance responses are involved in the unsuccessful penetration of O. crenata. METHODS: Samples of resistance reactions against O. crenata on different genotypes of resistant legumes were collected. The samples were fixed, sectioned and stained using different procedures. Sections were observed using a transmission light microscope and by epi-fluorescence. KEY RESULTS: Lignification of endodermal and pericycle host cells seems to prevent parasite intrusion into the root vascular cylinder at early infection stages. But in other cases, established tubercles became necrotic and died. Contrary to some previous studies, it was found that darkening at the infection site in these latter cases does not correspond to death of host tissues, but to the secretion of substances that fill the apoplast in the host-parasite interface and in much of the infected host tissues. The secretions block neighbouring host vessels. This may interfere with the nutrient flux between host and parasite, and may lead to necrosis and death of the developing parasite. CONCLUSIONS: The unsuccessful penetration of O. crenata seedlings into legume roots cannot be attributed to cell death in the host. It seems to be associated with lignification of host endodermis and pericycle cells at the penetration site. The accumulation of secretions at the infection site, may lead to the activation of xylem occlusion, another defence mechanism, which may cause further necrosis of established tubercles.

Infection Structures of Host-Specialized Isolates of Uromyces viciae-fabae and of Other Species of Uromyces Infecting Leguminous Crops.

A study was made of the morphology of urediniospores and primary infection structures of 12 isolates of six legume-infecting species of Uromyces. Infection structures were sufficient to distinguish among species. Isolates of Uromyces viciae-fabae proved to be specialized with respect to host, because each isolate infected only cultivars of the species from which it was collected. Host-specialized isolates of U. viciae-fabae also were morphologically distinct, differing in both spore dimensions and infection structure morphology. In particular, the shape and dimensions of the substomatal vesicle were distinctive. These results support the view that U. viciae-fabae sensu lato is a species complex.

Sources of Resistance to Crenate Broomrape Among Species of Vicia.

Crenate broomrape is a parasitic weed that represents a major constraint for pulse and forage legume production in the Mediterranean and West Asia regions. Control strategies have centered around agronomic practices and the use of herbicides, although success has been marginal. Resistance breeding is hampered by scarcity of proper sources of resistance and of a reliable and practical screening procedure. A germ plasm collection of 208 accessions of vetch belonging to 42 Vicia spp. was screened for resistance to crenate broomrape under field conditions. High levels of resistance were found in several species. Resistance of selected accessions was confirmed by a low induction of broomrape seed germination in pot and in vitro experiments. This was followed by a scarce establishment of broomrape radicles in contact with host roots and a limited development of established tubercles. In addition, a hypersensitive-like necrosis occasionally was observed, but at low frequency.

First Report of Orobanche foetida on Common Vetch (Vicia sativa) in Morocco.

Broomrapes (Orobanche spp.) are obligate parasites that infect roots of dicotyledoneous plants. Orobanche spp. are particularly important in southern and eastern Europe, the Middle East, and north Africa. O. crenata causes severe damage to legume crops, O. cumana threatens sunflower, O. ramosa attacks potato, tobacco, tomato, and hemp, O. aegyptiaca is severe on legumes and vegetables, and other broomrapes such as O. minor are widespread on forage legumes (3). O. foetida Poir. is considered important as an agricultural parasite of faba bean (Vicia faba) and common vetch (V. sativa) crops in the Beja Region of Tunisia (1). Aside from that, it has never been found infecting crops, even where it is widely distributed in the western Mediterranean area (Portugal, Spain, Morocco, Algeria, and Tunisia) parasitizing wild herbaceous leguminosae in the genera Anthyllis, Astragalus, Ebenus, Lotus, Medicago, Ononis, Scorpiurus, and Trifolium (2). In May 2004, we found O. foetida commonly occurring in Morocco infecting wild leguminosae. It was not seen infecting legume crops such as faba beans, chickpeas, or lentils, even where O. foetida was abundant in the close proximity to the crop fields, or in the field itself. In these instances, we verified that O. foetida was infecting weeds such as Scorpiurus spp. and not the crop plants themselves by gently pulling out the plants and examining the roots. However, O. foetida was identified infecting common vetch (V. sativa) on a small farm in Taounate, Saiss Region, 50 km north of Fes. Infection of vetch plants was confirmed by digging up the plants to verify the attachment of the broomrape plant to the vetch roots. The level of infection was moderate (0.1 to 0.2 broomrape plants per vetch plant). The farmer stated that he had observed this problem only in the last 3 years. Morphology was typical of O. foetida, i.e., dark reddish plant, 20 to 50 cm tall, corolla 12 to 20 mm, dark, purplish-red, lower lip not ciliate, filaments inserted 3 to 7 mm above base of corolla, and stigma deep yellow at anthesis (2). Voucher specimens were deposited in the Herbarium of the University of Córdoba. To our knowledge, this is the first report of O. foetida infecting common vetch and it is relevant since it indicates the first introduction of this parasite into crops in Morocco. The spread of this new crop parasite population should be monitored because it could represent a further constraint for legume production in this area. References: (1) M. Kharrat et al. FABIS Newsl. 30:46, 1992. (2) A. J. Pujadas-Salvá. Pages 187-193 in: Resistance to Orobanche: The State of the Art. Junta de Andalucía, Spain, 1999. (3) D. Rubiales. Grain Legumes 33:10, 2001.

Isolate and organ-specific QTLs for ascochyta blight resistance in faba bean ( Vicia faba L).

The main objective of the present study was to locate the genomic regions responsible for ascochyta blight resistance in faba bean. Six QTLs were identified with the help of a linkage map constructed from a F(2) population from the cross between the inbred lines 29H (resistant) and VF136 (susceptible). Two pathogenically distinct Ascochyta isolates were used to study the genetic control against them and disease evaluations were performed separately on leaves and stems to investigate whether different genetic systems control resistance in each plant organ, as previously suggested. The six QTLs detected were named Af3 to Af8. Af3 and Af4 were effective against both Ascochyta isolates, Af5 was only effective against isolate CO99-01 while Af6, Af7 and Af8 were only effective against isolate LO98-01. Af3, Af4, Af5 and Af7 were revealed in both leaves and stems. By contrast, Af6 was only effective in leaves and Af8 only in stems. The validity and application of these results in a MAS program is discussed.

Mildew-resistant mutants induced in North American two- and six-rowed malting barley cultivars.

Mildew-resistant mutants were induced with sodium azide in three North American malting barley cultivars, two in the six-rowed Ursula (URS1 and URS2), one in the six-rowed Gertrud (GER1), and one in the two-rowed Prudentia (PRU1). Two of the mutants, URS1 and PRU1, showed complete resistance and were shown to have two new alleles at the mlo locus; these were designated, respectively, mlo31 and mlo32. Mutant URS2, showing partial resistance, was inherited as a dominant gene, but was not an allele at the Mla locus. The mean yield of each mutant was higher than that of its parental line, but yield levels varied across environments, although this was independent of the severity of the mildew attack. Other reasons, for example, the severity of the necrotic lesions in the mutants, may account for yield variations. The malting quality of the GER1 mutant proved similar to that of Gertrud, but both URS1 and URS2 showed lower malt extract than Ursula. This lower extract might be due to the smaller grain size of the mutants that could, in turn, result from necrotic lesions in the leaves, as implied by the effects on grain yield.

QTL mapping provides evidence for lack of association of the avoidance of leaf rust in Hordeum chilense with stomata density.

In cereals, rust fungi are among the most harmful pathogens. Breeders usually rely on short-lived hypersensitivity resistance. As an alternative, "avoidance" may be a more durable defence mechanism to protect plants to rust fungi. In Hordeum chilense avoidance is based on extensive wax covering of stomata, which interferes with the induction of appressorium formation by the rust fungi. High avoidance levels are associated with a higher stoma density on the abaxial leaf epidermis. The avoidance level was assessed as the percentage of germ tube/stoma encounters that did not result in appressorium differentiation by Puccinia hordei, the barley leaf rust fungus. One hundred F(2) individuals from the cross between two H. chilense accessions with contrasting levels of avoidance showed a continuous distribution for avoidance of the rust fungus and for stoma density, indicating quantitative inheritance of the traits. No significant correlation was found between avoidance and stoma density in the segregating F(2) population. In order to map quantitative trait loci (QTLs) for both traits, an improved molecular marker linkage map was constructed, based on the F(2) population. The resulting linkage map spanned 620 cM and featured a total of 437 AFLP markers, thirteen RFLPs, four SCARs, nine SSRs, one STS and two seed storage protein markers. It consisted of seven long and two shorter linkage groups, and was estimated to cover 81% of the H. chilense genome. Restricted multiple interval mapping identified two QTLs for avoidance and three QTLs for stoma density in the abaxial leaf surface. The QTLs for avoidance were mapped on chromosome 3 and 5; those for stoma density on chromosomes 1, 3 and 7. Only the two QTLs regions located on chromosome 3 (one for avoidance and the other for stoma density) overlapped. The wild barley H. chilense has a high crossability with other members of the Triticeae tribe. The knowledge on the location of the QTLs responsible for the avoidance trait is a prerequisite to transfer this favourable agronomic trait from H. chilense to cultivated cereal genomes.

Genetic relationships among Orobanche species as revealed by RAPD analysis.

RAPD markers were used to study variation among 20 taxa in the genus OROBANCHE: O. alba, O. amethystea, O. arenaria, O. ballotae, O. cernua, O. clausonis, O. cumana, O. crenata, O. densiflora, O. foetida, O. foetida var. broteri, O. gracilis, O. haenseleri, O. hederae, O. latisquama, O. mutelii, O. nana, O. ramosa, O. rapum-genistae and O. santolinae. A total of 202 amplification products generated with five arbitrary RAPD primers was obtained and species-specific markers were identified. The estimated Jaccard's differences between the species varied between 0 and 0.864. The pattern of interspecific variation obtained is in general agreement with previous taxonomic studies based on morphology, and the partition into two different sections (Trionychon and Orobanche) is generally clear. However, the position in the dendrogram of O. clausonis did not fit this classification since it clustered with members of section TRIONYCHON: Within this section, O. arenaria was relatively isolated from the other members of the section: O. mutelii, O. nana and O. ramosa. Within section Orobanche, all O. ramosa populations showed a similar amplification pattern, whereas differences among O. crenata populations growing on different hosts were found. Orobanche foetida and O. densiflora clustered together, supporting the morphological and cytological similarities and the host preferences of these species.