Genetic dissection of resistance to root-knot nematodes Meloidogyne spp. in plum, peach, almond, and apricot from various segregating interspecific Prunus progenies

Sources of resistance in Prunus spp. exhibit different spectra to the root-knot nematodes (RKN) Meloidogyne incognita, Meloidogyne javanica and Meloidogyne floridensis. In this Prunus genus, two dominant genes, Ma with a complete spectrum from the heterozygous Myrobalan plums P.2175 and P.2980 (section Euprunus; subgenus Prunophora) and RMia with a more restricted spectrum from the peaches Nemared and Shalil (subgenus Amygdalus), have been identified. This study characterizes the resistance spectra of interspecific crosses involving (1) previous Myrobalan and peach sources, (2) two Alnem almonds (subgenus Amygdalus) resistant to M. javanica, and (3) the apricot A.3923, representing a species considered RKN-resistant (section Armeniaca; Prunophora). For both latter species, genetic data could be obtained through F1 crosses with genetically characterized Myrobalans that conferred their rooting ability for clonal multiplication of the hybrids and permitted their simultaneous evaluation to the three RKN. Crosses involving either Ma or RMia or both generated the expected resistance spectra. Nemared confirmed the species-specific resistance to M. incognita conferred by RMia. This rootstock, also previously considered resistant to M. javanica, was susceptible to the M. javanica isolate used, what illustrates an isolate-specific resistance to this species. Alnem accessions were shown homozygous resistant to M. javanica. In the progeny P.2980 × A.3923, Ma markers allowed to distinguish resistant individuals carrying that gene from resistant individuals lacking it. Distribution of non-Ma individuals in this cross suggested, in the apricot parent, (1) the absence of a major gene allelic to Ma and (2) the presence of a non RKN specific polygenic resistance.     

Development of nine polymorphic microsatellite markers for the phytoparasitic nematode Xiphinema index, the vector of the grapevine fanleaf virus

We report isolation, characterization and cross-species amplification of nine microsatellite loci from the phytoparasitic nematode Xiphinema index, the vector of grapevine fanleaf virus. Levels of polymorphism were evaluated in 62 individuals from two X. index populations. The number of alleles varies between two and 10 depending on locus and population. Observed heterozygosity on loci across both populations varied from 0.32 to 0.857 (mean 0.545). The primers were tested for cross-species amplification in three other species of phytoparasitic nematodes of the Xiphinema genus. These nine microsatellite loci constitute valuable markers for population genetics and phylogeographical studies of X. index.     

The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1-TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.     

Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum?×?almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.     

Inheritance of resistance to the root-knot nematode Meloidogyne arenaria in Myrobalan plum

The inheritance of resistance of the self-incompatible Myrobalan plum Prunus cerasifera to the root-knot nematode Meloidogyne arenaria was studied using first a diallel cross between five parents of variable host suitability (including two highly resistant clones P.1079 and P.2175, a moderate host P.2032, a good host P.2646 and an excellent host P.16.5), followed by the G2 crosses P.16.5 × (P.2646 × P.1079) and P.2646 × (P.16.5 × P.1079). A total of 355 G1 and 72 G2 clones obtained from hard-wood cuttings sampled from trees in the field experimental design, then rooted in the nursery and inoculated individually in containers (5–10 replicates per clone) under greenhouse conditions, were evaluated for their host suitability based on a 0–5 gall-index rating under a high and durable inoculum pressure of the nematode. In the crosses involving the resistant P.1079 and P.2175 and the hosts P.2646 and P.16.5: (1) all of the G1 crosses of P.1079 were resistant while the G2 crosses segregated 1 resistant to 1 host, (2) the G1 crosses between P.2175 and either P.2646 or P.16.5 segregated 1 resistant to 1 host, and (3) all of the G1 progeny between P.2646 and P.16.5 were host. These results indicate that resistance is conferred by a single major dominant resistance gene (homozygous) in P.1079, and the same, or an allelic or a different, major dominant gene (heterozygous) in P.2175, and that P.2646 and P.16.5 are recessive for this (these) major resistance gene(s). As expected according to the hypothesis of a recessive genotype for P.2032, all of its hybrids with P.1079 were resistant, all of its hybrids with P.2646 and P.16.5 were host, and its hybrids with P.2175 segregated for resistance. Nevertheless, the 32 segregation ratio of these latter hybrids suggests that clones bearing the P.2175 gene would have a selective advantage. Both resistance genes are completely dominant and confer a non-host behaviour that totally prevents the multiplication of the nematode. This is the first reported evidence of major nematode resistance genes towards M. arenaria in a species of the subgenus Prunophora in the genus Prunus. The symbols Ma1 for the P.2175 gene and Ma2 for the P.1079 gene are proposed.     

RAPD and SCAR markers linked to the Ma1 root-knot nematode resistance gene in Myrobalan plum (Prunus cerasifera Ehr.)

 The Myrobalan plum (Prunus cerasifera) is a self-incompatible species in which the clones P.2175, P.1079 and P.2980 are highly resistant to all root-knot nematodes (RKN), Meloidogyne spp. Each clone bears a single major dominant gene, designated Ma1, Ma2 and Ma3 respectively, that controls a high and wide-spectrum resistance. Bulked segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) analysis were both performed to detect markers linked to the Ma1 gene using three segregating progenies from P.2175 (Ma1 ma1) crossed by three host parents (ma1 ma1). Four dominant coupling-phase markers were identified from a total of 660 10-base primers tested. The resulting linkage map spans 14.7 cM and comprises three markers located on the same side of Ma1 and one marker located on the other side. The nearest markers (OPAL19₇₂₀ and OPA16₁₄₀₀) are located at 3.7 and 6.7 cM, respectively, on each side of the gene. Among the three markers that could be successfully converted into sequence characterized amplified region (SCAR) markers, two of them (SCAL19₆₉₀ and SCAN12₆₂₀) were scored as dominant markers whereas the third (SCAO19₇₇₀) failed to produce any polymorphism. SCAL19, and to a lesser extent SCAN12, can be used reliably in the marker-assisted selection of Prunus rootstocks. These markers are adequate to identify the Ma1 RKN resistance gene in intraspecific segregating progenies and will be suitable for the creation of interspecific rootstocks involving Myrobalan plum. Some of the RAPD and SCAR markers for Ma1 were also recovered in clones P.1079 and P.2980, but not in additional host clones, suggesting that Ma1, Ma2 and Ma3 are either allelic or at least closely linked. Received: 22 September 1998 / Accepted: 19 December 1998     

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.     

High-resolution mapping and chromosome landing at the root-knot nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library

The Ma gene for root-knot nematode (RKN) resistance from Myrobalan plum (Prunus cerasifera L.) confers a complete-spectrum and a heat-stable resistance to Meloidogyne spp., conversely to Mi-1 from tomato, which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designated SCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175, Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175, totaling 30,720 clones with a mean insert size of 145 kb and a 14–15× Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing. In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species.     

Biotin-Avidin ELISA Detection of Grapevine Fanleaf Virus in the Vector Nematode Xiphinema index

The value of biotin-avidin (B-A) ELISA for the detection of grapevine fanleaf virus (GFLV) in Xiphinema was estimated with field populations and greenhouse subpopulations. Samples consisted of increasing numbers of adults ranging from 1 to 64 in multiples of two. Tests with virus-free X. index populations reared on grapevine and fig plants as negative controls did not reveal a noticeable effect of the host plant. ELISA absorbances of virus-free X. index samples were greater than corresponding absorbances of X. pachtaicum samples. Differences occurred between two X. index field populations from GFLV-infected grapevines in Champagne and Languedoc. In most tests, 1-, 2-, 4-, and 8-nematode samples of virus-free and virus-infected populations, respectively, could not be separated. Consequently, B-A ELISA was not a reliable method for GFLV detection in samples of less than 10 X. index adults, but comparison of the absorbances obtained with increasing numbers may allow differentiation of the viral infectious potential of several populations.     

Molecular analysis and comparative morphology to resolve a complex of cryptic Xiphinema species

Gutiérrez‐Gutiérrez, C., Palomares‐Rius, J.E., Cantalapiedra‐Navarrete, C., Landa, B.B., Esmenjaud, D. & Castillo, P. (2010). Molecular analysis and comparative morphology to resolve a complex of cryptic Xiphinema species. —Zoologica Scripta, 39, 483–498. During nematode surveys in cultivated and natural environments in southern Spain nine populations of parthenogenic Xiphinema species tentatively identified as Xiphinema cf. pyrenaicum and one population morphologically close to Xiphinema turcicum were detected. Surveys in southern France also identified one population resembling X. pyrenaicum. We developed a comparative study among these related Xiphinema species, including topotypes of two species of this group previously synonymized, viz. Xiphinema hispanum and Xiphinema sphaerocephalum, by considering morphological and morphometrical features together with molecular data from nuclear ribosomal RNA genes (D2‐D3 expansion segments of 28S, ITS1, and partial 18S). Morphological and morphometrical results identified eight of the Spanish populations as Xiphinema nuragicum (previously synonymized with X. pyrenaicum) whereas the ninth population was identified as Xiphinema adenohystherum (also synonymized with X. pyrenaicum). The species X. adenohystherum, X. nuragicum, X. pyrenaicum, and X. sphaerocephalum were shown to be morphologically almost indistinguishable but clearly separated by phylogenetic analyses, thus constituting a complex of cryptic species. Consequently, X. adenohystherum, X. nuragicum, and X. sphaerocephalum were re‐established as valid species. Similarly, X. hispanum (morphologically similar to X. aceri) was also shown as a valid species. Xiphinema turcicum, morphologically related to X. pyrenaicum complex by its rounded tail, uterus with a pseudo‐Z‐differentiation and small spines, was phylogenetically distant to these species based on D2‐D3 expansion segments of 28S and ITS1, which suggests a morphological convergence in their evolution.