Pim Lindhout

Use of locus-specific AFLP markers to construct a high-density molecular map in barley.

By using 25 primer combinations, 563 AFLP markers segregating in a recombinant inbred population (103 lines, F9) derived from L94/Vada were generated. The 38 AFLP markers in common to the existing AFLP/RFLP combined Proctor/Nudinka map, one STS marker, and four phenotypic markers with known map positions, were used to assign present AFLP linkage groups to barley chromosomes. The constructed high-density molecular map contains 561 AFLP markers, three morphological markers, one disease resistance gene and one STS marker, and covers a 1062-cM genetic distance, corresponding to an average of one marker per 1.9 cM. However, extremely uneven distributions of AFLP markers and strong clustering of markers around the centromere were identified in the present AFLP map. Around the centromeric region, 289 markers cover a genetic distance of 155 cM, corresponding to one marker per 0.5 cM; on the distal parts, 906 cM were covered by 277 markers, corresponding to one marker per 3.3 cM. Three gaps larger than 20 cM still exist on chromosomes 1, 3 and 5. A skeletal map with a uniform distribution of markers can be extracted from the high-density map, and can be applied to detect and map loci underlying quantitative traits. However, the application of this map is restricted to barley species since hardly any marker in common to a closely related Triticum species could be identified.

Naturally Occurring Broad-Spectrum Powdery Mildew Resistance in a Central American Tomato Accession Is Caused by Loss of Mlo Function

The resistant cherry tomato (Solanum lycopersicum var. cerasiforme) line LC-95, derived from an accession collected in Ecuador, harbors a natural allele (ol-2) that confers broad-spectrum and recessively inherited resistance to powdery mildew (Oidium neolycopersici). As both the genetic and phytopathological characteristics of ol-2-mediated resistance are reminiscent of powdery mildew immunity conferred by loss-of-function mlo alleles in barley and Arabidopsis, we initiated a candidate-gene approach to clone Ol-2. A tomato Mlo gene (SlMlo1) with high sequence-relatedness to barley Mlo and Arabidopsis AtMLO2 mapped to the chromosomal region harboring the Ol-2 locus. Complementation experiments using transgenic tomato lines as well as virus-induced gene silencing assays suggested that loss of SlMlo1 function is responsible for powdery mildew resistance conferred by ol-2. In progeny of a cross between a resistant line bearing ol-2 and the susceptible tomato cultivar Moneymaker, a 19-bp deletion disrupting the SlMlo1 coding region cosegregated with resistance. This polymorphism results in a frameshift and, thus, a truncated nonfunctional SlMlo1 protein. Our findings reveal the second example of a natural mlo mutant that possibly arose post-domestication, suggesting that natural mlo alleles might be evolutionarily short-lived due to fitness costs related to loss of mlo function.

Identification of QTLs for partial resistance to leaf rust (Puccinia hordei) in barley.

Abstract The partial resistance to leaf rust in barley is a quantitative resistance that is not based on hypersensitivity. To map the quantitative trait loci (QTLs) for partial resistance to leaf rust, we obtained 103 recombinant inbred lines (RILs) by single-seed descent from a cross between the susceptible parent L94 and the partially resistant parent Vada. These RILs were evaluated at the seedling and adult plant stages in the greenhouse for the latent period (LP) of the rust fungus, and in the field for the level of infection, measured as area under the disease progress curve (AUDPC). A dense genetic map based on 561 AFLP markers had been generated previously for this set of RILs. QTLs for partial resistance to leaf rust were mapped using the “Multiple Interval Mapping” method with the putative QTL markers as cofactors. Six QTLs for partial resistance were identified in this population. Three QTLs, Rphq1, Rphq2 and Rphq3, were effective at the seedling stage and contributed approximately 55% to the phenotypic variance. Five QTLs, Rph2, Rphq3, Rphq4, Rphq5, and/or Rphq6 contributed approximtely. 60% of the phenotypic variance and were effective at the adult plant stage. Therefore, only the QTLs Rphq2 and Rhpq3 were not plant-stage dependent. The identified QTLs showed mainly additive effects and only one significant interaction was detected, i.e. between Rphq1 and Rphq2. The map positions of these QTLs did not coincide with those of the race-specific resistance genes, suggesting that genes for partial resistance and genes for hypersensitive resistance represent entirely different gene families. Also, three QTLs for days to heading, of which two were also involved in plant height, were identified in the present recombinant inbred population. These QTLs had been mapped previously on the same positions in different populations. The perspectives of these results for breeding for durable resistance to leaf rust are discussed.

The Tomato Sequencing Project, the First Cornerstone of the International Solanaceae Project (SOL)

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the ‘International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation’. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae—and beyond—achieves a context that will facilitate a systems biology approach.

Development of AFLP markers in barley

Abstract To investigate the application of amplified fragment length polymorphism (AFLP) markers in barley, 96 primer combinations were used to generate AFLP patterns with two barley lines, L94 and Vada. With seven primer combinations, only a few intense bands were obtained, probably derived from repeated sequences. With the majority of the remaining 89 primer combinations, on average about 120 amplification products were generated, and the polymorphism rate between the two lines was generally over 18%. Based on the number of amplified products and the polymorphism rate, the 48 best primer combinations were selected and tested on 16 barley lines, again including L94 and Vada. Using a subset of 24 primer combinations 2188 clearly visible bands within the range from 80 to 510 bp were generated; 55% of these showed some degree of polymorphism among the 16 lines. L94 versus Vada showed the highest polymorphism rate (29%) and Proctor versus Nudinka yielded the lowest (12%). The polymorphism rates per primer combination showed little dependence on the barley lines used. Hence the most efficient and informative primer combinations identified for a given pair of lines turned out to be highly efficient when applied to others. Generally, more than 100 common markers (possibly locus specific) among populations or crosses were easily identified by comparing 48 AFLP profiles of the parent lines. The existence of such a large number of markers common to populations will facilitate the merging of molecular marker data and other genetic data into one integrated genetic map of barley.

QTLs for tomato powdery mildew resistance (Oidium lycopersici) in Lycopersicon parviflorum G1.1601 co-localize with two qualitative powdery mildew resistance genes.

Tomato (Lycopersicon esculentum) is susceptible to the powdery mildew Oidium lycopersici, but several wild relatives such as Lycopersicon parviflorum G1.1601 are completely resistant. An F2 population from a cross of Lycopersicon esculentum cv. Moneymaker x Lycopersicon parviflorum G1.1601 was used to map the O. lycopersici resistance by using amplified fragment length polymorphism markers. The resistance was controlled by three quantitative trait loci (QTLs). Ol-qtl1 is on chromosome 6 in the same region as the Ol-1 locus, which is involved in a hypersensitive resistance response to O. lycopersici. Ol-qtl2 and Ol-qtl3 are located on chromosome 12, separated by 25 cM, in the vicinity of the Lv locus conferring resistance to another powdery mildew species, Leveillula taurica. The three QTLs, jointly explaining 68% of the phenotypic variation, were confirmed by testing F3 progenies. A set of polymerase chain reaction-based cleaved amplified polymorphic sequence and sequence characterized amplified region markers was generated for efficient monitoring of the target QTL genomic regions in marker assisted selection. The possible relationship between genes underlying major and partial resistance for tomato powdery mildew is discussed.

Rin4 Causes Hybrid Necrosis and Race-Specific Resistance in an Interspecific Lettuce Hybrid

Some inter- and intraspecific crosses may result in reduced viability or sterility in the offspring, often due to genetic incompatibilities resulting from interactions between two or more loci. Hybrid necrosis is a postzygotic genetic incompatibility that is phenotypically manifested as necrotic lesions on the plant. We observed hybrid necrosis in interspecific lettuce (Lactuca sativa and Lactuca saligna) hybrids that correlated with resistance to downy mildew. Segregation analysis revealed a specific allelic combination at two interacting loci to be responsible. The allelic interaction had two consequences: (1) a quantitative temperature-dependent autoimmunity reaction leading to necrotic lesions, lethality, and quantitative resistance to an otherwise virulent race of Bremia lactucae; and (2) a qualitative temperature-independent race-specific resistance to an avirulent race of B. lactucae. We demonstrated by transient expression and silencing experiments that one of the two interacting genes was Rin4. In Arabidopsis thaliana, RIN4 is known to interact with multiple R gene products, and their interactions result in hypersensitive resistance to Pseudomonas syringae. Site-directed mutation studies on the necrosis-eliciting allele of Rin4 in lettuce showed that three residues were critical for hybrid necrosis.

An RFLP linkage map of Lycopersicon peruvianum

In order to map genes determining resistance to bacterial canker in tomato, backcrosses were made between a resistant and a susceptible Lycopersicon peruvianum accession. The linkage study with RFLP markers yielded a genetic map of L. Peruvianum. This map was compared to that derived from a L. esculentum x L. pennellii F2 population, based on 70 shared RFLP markers. The maps showed a good resemblance in both the order of markers and the length of the chromosomes, with the exception of just one relocated marker on chromosome 9. Because backcrosses were made with the F1, either as the pollen parent or as the pistil parent, linkage maps from male and female meioses could be estimated. It was concluded that recombination at male meiosis was reduced, and that gametophytic selection for parental genotypes at more than one locus per chromosome might be partly responsible for the reduction of the estimated male map length.

Isolate-specific QTLs for partial resistance to Puccinia hordei in barley

Abstract By using a high-density AFLP marker linkage map, six QTLs for partial resistance to barley leaf rust (Puccinia hordei) isolate 1.2.1. have been identified in the RIL offspring of a cross between the partially resistant cultivar ’Vada’ and the susceptible line L94. Three QTLs were effective at the seedling stage, and five QTLs were effective at the adult plant stage. To study possible isolate specificity of the resistance, seedlings and adult plants of the 103 RILs from the cross L94×’Vada’ were also inoculated with another leaf rust isolate, isolate 24. In addition to the two QTLs that were effective against isolate 1.2.1. at the seedling stage, an additional QTL for seedling resistance to isolate 24 was identified on the long arm of chromosome 7. Of the eight detected QTLs effective at the adult plant stage, three were effective in both isolates and five were effective in only one of the two isolates. Only one QTL had a substantial effect at both the seedling and the adult plant stages. The expression of the other QTLs was developmental-stage specific. The isolate specificity of the QTLs supports the hypothesis of Parlevliet and Zadoks (1977) that partial resistance may be based on a minor-gene-for-minor-gene interaction.

The development of lettuce backcross inbred lines (BILs) for exploitation of the Lactuca saligna (wild lettuce) germplasm

Backcross inbred lines (BILs) were developed in which chromosome segments of Lactuca saligna (wild lettuce) were introgressed into L. sativa (lettuce). These lines were developed by four to five backcrosses and one generation of selfing. The first three generations of backcrossing were random. Marker-assisted selection began in the BC4 generation and continued until the final set of BILs was reached. A set of 28 lines was selected that together contained 96% of the L. saligna genome. Of these lines, 20 had a single homozygous introgression (BILs), four had two homozygous introgressions (doubleBILs) and four lines had a heterozygous single introgression (preBILs). Segregation ratios in backcross generations were compared to distorted segregation ratios in an F2 population, and the results indicated that most of the distorted segregations can be explained by genetic effects on pollen- or egg-cell fitness. By means of BIL association mapping we were able to map 12 morphological traits and hundreds of additional amplified fragment length polymorphic (AFLP) markers. The total AFLP map now comprises 757 markers. This set of BILs is very useful for future genetic studies.