Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.     

Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

In the potato, Kunitz-type enzyme inhibitors are abundant and highly polymorphic small proteins found in tubers. DNA sequence analysis of 1596 unselected ESTs (expressed sequence tags) from mature tubers of the cultivars Provita and Saturna resulted in the identification of 55 different DNA sequences with high sequence similarity to Kunitz-type enzyme inhibitors. The frequency of Kunitz-type inhibitor ESTs in Provita was four times higher than in Saturna tubers, and none of the Provita ESTs was identical to any of the Saturna ESTs. A phenogram constructed from the deduced amino acid sequences of the inhibitors revealed three major homology groups-A, B and C. Group A inhibitors were all derived from Provita ESTs. Inhibitor groups A and B were more similar to each other than to group C inhibitors, and for most members within-group similarity was at least 90%. Non-conservative amino acid substitutions and insertion/deletion polymorphisms suggest functional differentiation between members of the gene family. A minimum of 21 genes for Kunitz-type enzyme inhibitors (six for group A, nine for group B and six for group C) was estimated to exist in the potato genome. Genetic mapping and the identification of BAC (bacterial artificial chromosome) clones containing more than one member of the gene family indicated that most inhibitor genes of groups A, B and C are organized in a cluster that maps to a single region on potato chromosome III.     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

Multiple alleles for resistance and susceptibility modulate the defense response in the interaction of tetraploid potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) with <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis> pathotypes 1, 2, 6 and 18

The obligate biotrophic, soil-borne fungus Synchytrium endobioticum causes wart disease of potato (Solanum tuberosum), which is a serious problem for crop production in countries with moderate climates. S. endobioticum induces hypertrophic cell divisions in plant host tissues leading to the formation of tumor-like structures. Potato wart is a quarantine disease and chemical control is not possible. From 38 S. endobioticum pathotypes occurring in Europe, pathotypes 1, 2, 6 and 18 are the most relevant. Genetic resistance to wart is available but only few current potato varieties are resistant to all four pathotypes. The phenotypic evaluation of wart resistance is laborious, time-consuming and sometimes ambiguous, which makes breeding for resistance difficult. Molecular markers diagnostic for genes for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 would greatly facilitate the selection of new, resistant cultivars. Two tetraploid half-sib families (266 individuals) segregating for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 were produced by crossing a resistant genotype with two different susceptible ones. The families were scored for five different wart resistance phenotypes. The distribution of mean resistance scores was quantitative in both families. Resistance to pathotypes 2, 6 and 18 was correlated and independent from resistance to pathotype 1. DNA pools were constructed from the most resistant and most susceptible individuals and screened with genome wide simple sequence repeat (SSR), inverted simple sequence region (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Bulked segregant analysis identified three SSR markers that were linked to wart resistance loci (Sen). Sen1-XI on chromosome XI conferred partial resistance to pathotype 1, Sen18-IX on chromosome IX to pathotype 18 and Sen2/6/18-I on chromosome I to pathotypes 2,6 and 18. Additional genotyping with 191 single nucleotide polymorphism (SNP) markers confirmed the localization of the Sen loci. Thirty-three SNP markers linked to the Sen loci permitted the dissection of Sen alleles that increased or decreased resistance to wart. The alleles were inherited from both the resistant and susceptible parents.     

A genetic map of cassava (Manihot esculenta Crantz) with integrated physical mapping of immunity-related genes

Background

Cassava, Manihot esculenta Crantz, is one of the most important crops world-wide representing the staple security for more than one billion of people. The development of dense genetic and physical maps, as the basis for implementing genetic and molecular approaches to accelerate the rate of genetic gains in breeding program represents a significant challenge. A reference genome sequence for cassava has been made recently available and community efforts are underway for improving its quality. Cassava is threatened by several pathogens, but the mechanisms of defense are far from being understood. Besides, there has been a lack of information about the number of genes related to immunity as well as their distribution and genomic organization in the cassava genome.

Results

A high dense genetic map of cassava containing 2,141 SNPs has been constructed. Eighteen linkage groups were resolved with an overall size of 2,571 cM and an average distance of 1.26 cM between markers. More than half of mapped SNPs (57.4%) are located in coding sequences. Physical mapping of scaffolds of cassava whole genome sequence draft using the mapped markers as anchors resulted in the orientation of 687 scaffolds covering 45.6% of the genome. One hundred eighty nine new scaffolds are anchored to the genetic cassava map leading to an extension of the present cassava physical map with 30.7 Mb. Comparative analysis using anchor markers showed strong co-linearity to previously reported cassava genetic and physical maps. In silico based searching for conserved domains allowed the annotation of a repertory of 1,061 cassava genes coding for immunity-related proteins (IRPs). Based on physical map of the corresponding sequencing scaffolds, unambiguous genetic localization was possible for 569 IRPs.

Conclusions

This is the first study reported so far of an integrated high density genetic map using SNPs with integrated genetic and physical localization of newly annotated immunity related genes in cassava. These data build a solid basis for future studies to map and associate markers with single loci or quantitative trait loci for agronomical important traits. The enrichment of the physical map with novel scaffolds is in line with the efforts of the cassava genome sequencing consortium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1397-4) contains supplementary material, which is available to authorized users.     

Genetic components of root architecture and anatomy adjustments to water-deficit stress in spring barley

Roots perform vital roles for adaptation and productivity under water-deficit stress, even though their specific functions are poorly understood. In this study, the genetic control of the nodal-root architectural and anatomical response to water deficit were investigated among diverse spring barley accessions. Water deficit induced substantial variations in the nodal root traits. The cortical, stele, and total root cross-sectional areas of the main-shoot nodal roots decreased under water deficit, but increased in the tiller nodal roots. Root xylem density and arrested nodal roots increased under water deficit, with the formation of root suberization/lignification and large cortical aerenchyma. Genome-wide association study implicated 11 QTL intervals in the architectural and anatomical nodal root response to water deficit. Among them, three and four QTL intervals had strong effects across seasons and on both root architectural and anatomical traits, respectively. Genome-wide epistasis analysis revealed 44 epistatically interacting SNP loci. Further analyses showed that these QTL intervals contain important candidate genes, including ZIFL2, MATE, and PPIB, whose functions are shown to be related to the root adaptive response to water deprivation in plants. These results give novel insight into the genetic architectures of barley nodal root response to soil water deficit stress in the fields, and thus offer useful resources for root-targeted marker-assisted selection.     

Chromosome landing at the tomato Bs4 locus

The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.     

Functional complementation analysis in potato via biolistic transformation with BAC large DNA fragments

Gene isolation from plants by positional cloning frequently requires several rounds of transformation. To reduce the resources invested and to accelerate the process, we have used large DNA fragments in transformation experiments, followed by analysis of transgenic plants to assess functional complementation. Specifically, the transformation of potato with DNA from the 106 kb BAC plasmid BA87d17 is described. The large fragment was introduced into the potato genome by biolistic transformation, while attempting to clone the R1 gene conferring a race specific resistance to Phytophthora infestans. Thirty-one kanamycin resistant plants were regenerated of which thirteen showed the necrotic lesions typical for the hypersensitive response after infection with the incompatible P. infestans race 4, which carries the avirulence gene Avr1. The successful complementation supported the location of the R1 gene in the BAC insertion of the BA87d17 plasmid. Based on PCR and Southern gel blot analysis, both complete and incomplete integrations of the large construct into the recipient genome were demonstrated.