Differential susceptibility and suitability of domestic and wild apple species for a florivorous weevil and its parasitoids

Crop plant domestication can change plant resistance to herbivores leading to differences in pest pressure experienced by crop plants and their wild relatives. To compare resistance to herbivores between domesticated and wild fruit trees, we quantified direct resistance and indirect resistance to a pest insect, the florivorous apple blossom weevil Anthonomus pomorum (Coleoptera: Curculionidae), for the cultivated apple Malus domestica and two wild apple species, the European crab apple M. sylvestris and the exotic M. kirghisorum. We measured weevil infestation and performance (weight, sex ratio), and weevil parasitism by parasitoid wasps for different cultivars of M. domestica and for the two wild apple species. To explain weevil and parasitoid responses to different apple species, we quantified tree characteristics including nitrogen content, size of flower buds, bark roughness, tree size, tree phenology and tree position. We found significant differences in susceptibility to weevil infestation between apple species, with lowest infestation (highest apple resistance) in M. domestica and highest infestation in M. kirghisorum. The suitability of apple species also varied significantly: weevils emerging from M. sylvestris were significantly lighter than those from M. kirghisorum. Parasitism of A. pomorum by different parasitoid species was significantly higher in M. sylvestris than in M. domestica. Infestation, weevil weight and parasitism were positively related to tree characteristics: infestation to bud nitrogen content and bark roughness, weevil size to nitrogen content and bud size, and parasitism to tree height and bud density. Our study revealed marked differences between apple species in susceptibility and suitability for the pest herbivore, but also for antagonistic parasitoids. Whereas direct resistance appeared to be higher in cultivated apple, indirect resistance via parasitoids was apparently higher in wild apple trees. Our findings suggest that wild and cultivated apple trees possess different resistance traits that may be combined to optimize resistance in commercial apple cultivars.     

New insight into the history of domesticated apple: secondary contribution of the European wild apple to the genome of cultivated varieties

The apple is the most common and culturally important fruit crop of temperate areas. The elucidation of its origin and domestication history is therefore of great interest. The wild Central Asian species Malus sieversii has previously been identified as the main contributor to the genome of the cultivated apple (Malus domestica), on the basis of morphological, molecular, and historical evidence. The possible contribution of other wild species present along the Silk Route running from Asia to Western Europe remains a matter of debate, particularly with respect to the contribution of the European wild apple. We used microsatellite markers and an unprecedented large sampling of five Malus species throughout Eurasia (839 accessions from China to Spain) to show that multiple species have contributed to the genetic makeup of domesticated apples. The wild European crabapple M. sylvestris, in particular, was a major secondary contributor. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the current M. domestica being genetically more closely related to this species than to its Central Asian progenitor, M. sieversii. We found no evidence of a domestication bottleneck or clonal population structure in apples, despite the use of vegetative propagation by grafting. We show that the evolution of domesticated apples occurred over a long time period and involved more than one wild species. Our results support the view that self-incompatibility, a long lifespan, and cultural practices such as selection from open-pollinated seeds have facilitated introgression from wild relatives and the maintenance of genetic variation during domestication. This combination of processes may account for the diversification of several long-lived perennial crops, yielding domestication patterns different from those observed for annual species.     

Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.)

To unravel the relationship between the European wild apple, Malus sylvestris (L.) Mill., and its domesticated relative M. domestica Borkh., we studied chloroplast DNA variation in 634 wild and 422 domesticated accessions originating from different regions. Hybridization between M. sylvestris and M. domestica was checked using 10 nuclear microsatellites and a Bayesian assignment approach. This allowed us to identify hybrids and feral plants escaped from cultivation. Sixty-eight genotypes belonging to 12 other wild Malus species, including 20 M. sieversii (Ledeb.) Roem. accessions were also included in the analysis of chloroplast diversity. Marker techniques were developed to type a formerly described duplication and a newly detected transversion in the matK gene. Chloroplast DNA variation was further investigated using PCR-RFLP (Polymerase Chain Reaction-Random Fragment Length Polymorphism), and haplotypes were constructed based on all mutational combinations. A closer relationship than presently accepted between M. sylvestris and M. domestica was established at the cytoplasmic level, with the detection of eight chloroplast haplotypes shared by both species. Hybridization between M. sylvestris and M. domestica was also apparent at the local level with sharing of rare haplotypes among local cultivars and sympatric wild trees. Indications of the use of wild Malus genotypes in the (local) cultivation process of M. domestica and cytoplasmic introgression of chloroplast haplotypes into M. sylvestris from the domesticated apple were found. Only one of the M. sieversii trees studied displayed one of the three main chloroplast haplotypes shared by M. sylvestris and M. domestica. This is surprising as M. sieversii has formerly been described as the main maternal progenitor of the domesticated apple. This study hereby reopens the exciting discussion on the origin of M. domestica.     

Isolation and characterization of resistance gene analogues from Psilanthus species that represent wild relatives of cultivated coffee endemic to India

Biotic or abiotic stress can cause considerable damage to crop plants that can be managed by building disease resistance in the cultivated gene pool through breeding for disease resistance genes (R-genes). R-genes, conferring resistance to diverse pathogens or pests share a high level of similarity at the DNA and protein levels in different plant species. This property of R-genes has been successfully employed to isolate putative resistance gene analogues (RGAs) using a PCR-based approach from new plant sources. Using a similar approach, in the present study, we have successfully amplified putative RGAs having nucleotide-binding-site leucine-rich repeats (NBS-LRR-type RGAs) from seven different sources: two cultivated coffee species (Coffea arabica L. and Coffea canephora Pierre ex. A. Froehner), four related taxa endemic to India (wild tree coffee species: Psilanthus bengalensis (Roem. & Schuttles) J.-F. Leroy, Psilanthus khasiana , Psilanthus travencorensis (Wight & Arn.) J.-F. Leroy, Psilanthus weightiana (Wall. ex Wight & Arn.) J.-F. Leroy), and a cDNA pool originally prepared from light- and drought-stressed Coffea arabica L. leaves. The total PCR amplicons obtained using NBS-LRR-specific primers from each source were cloned and transformed to construct seven independent libraries, from which 434 randomly picked clones were sequenced. In silico analysis of the sequenced clones revealed 27 sequences that contained characteristic RGA motifs, of which 24 had complete uninterrupted open reading frames. Comparisons of these with published RGAs showed several of these to be novel RGA sequences. Interestingly, most of such novel RGAs belonged to the related wild Psilanthus species. The data thus suggest the potential of the secondary gene pool as possible untapped donors of resistance genes to the present day cultivated species of coffee.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

高温对苹果花粉在花柱内萌发和生长的影响

以‘长富2号’红富士苹果为母本,‘红星’苹果为父本,研究35℃高温对苹果花粉在花柱内萌发和生长的影响。结果表明：温度影响花粉在柱头和花柱内的萌发及生长,与对照相比,高温促进花粉在柱头表面快速萌发,并加速花粉管在花柱内的伸长生长,但是在授粉72hN,高温处理下培养的花粉管形态出现花粉管变粗、弯曲并有瘤状小结的异化现象,同时,花柱发生褐变,并产生胼胝质,最终导致花粉管不能进入胚珠完成受精。     

Species-specific accumulation of interspersed sequences in genus Saccharum

The genus Saccharum consists of two wild and four cultivated species. Novel interspersed sequences were isolated from cultivated sugar cane S. officinarum. These sequences were accumulated in all four cultivated species and their wild ancestral species S. robustum, but were not detected in the other wild species S. spontaneum and the relative Erianthus arundinaceus. The species-specific accumulation of interspersed sequences would correlate to the domestication of sugar canes.     

S-RNases from self-incompatible and -compatible apple cultivars: purification,cloning, enzymic properties,and pollen tube growth inhibitory activity

Four S-RNases (RNase associated with self-incompatibility) were purified from the styles of two apple cultivars (Malus domestica), a self-incompatible cv., Starking Delicious (SD), and a self-compatible cv., Megumi (MG). Each cultivar produced two S-RNases and their enzymatic properties such as specific activity, pH optimum, thermal stability, and molecular mass, were characterized. The four S-RNases inhibited the tube growth of apple pollen in an in vitro bioassay at 25 microg/ml (1.0 microM), but did not distinguish self from non-self pollen. The cDNAs of four S-RNases were cloned, and the nucleotide and deduced amino acid sequences were analyzed. The nucleotide sequence of SD-Se RNase was a new one and the other was identical to that of Sc-RNase of cv. Fuji. In MG one was identical to the sequence of SD-Sc RNase and the other to that of Sa-RNase of cv. Golden Delicious except for one base. From results of the isolation amounts and the Western blot analysis for stylar crude extracts the amount of S-RNases in MG was apparently less than that in SD.     

Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1–12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. Received: 2 May 2001 / Accepted: 3 August 2001     

Application of a high-speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection

Breeding of apple (Malus × domestica) remains a slow process because of protracted generation cycles. Shortening the juvenile phase to achieve the introgression of traits from wild species into prebreeding material within a reasonable time frame is a great challenge. In this study, we evaluated early flowering transgenic apple lines overexpressing the BpMADS4 gene of silver birch with regard to tree morphology in glasshouse conditions. Based on the results obtained, line T1190 was selected for further analysis and application to fast breeding. The DNA sequences flanking the T-DNA were isolated and the T-DNA integration site was mapped on linkage group 4. The inheritance and correctness of the T-DNA integration were confirmed after meiosis. A crossbred breeding programme was initiated by crossing T1190 with the fire blight-resistant wild species Malus fusca. Transgenic early flowering F(1) seedlings were selected and backcrossed with 'Regia' and 98/6-10 in order to introgress the apple scab Rvi2, Rvi4 and powdery mildew Pl-1, Pl-2 resistance genes and the fire blight resistance quantitative trait locus FB-F7 present in 'Regia'. Three transgenic BC'1 seedlings pyramiding Rvi2, Rvi4 and FB-F7, as well as three other BC'1 seedlings combining Pl-1 and Pl-2, were identified. Thus, the first transgenic early flowering-based apple breeding programme combined with marker-assisted selection was established.     

Evolutionary meta-analysis of solanaceous resistance gene and solanum resistance gene analog sequences and a practical framework for cross-species comparisons

Cross-species comparative genomics approaches have been employed to map and clone many important disease resistance (R) genes from Solanum species-especially wild relatives of potato and tomato. These efforts will increase with the recent release of potato genome sequence and the impending release of tomato genome sequence. Most R genes belong to the prominent nucleotide binding site-leucine rich repeat (NBS-LRR) class and conserved NBS-LRR protein motifs enable survey of the R gene space of a plant genome by generation of resistance gene analogs (RGA), polymerase chain reaction fragments derived from R genes. We generated a collection of 97 RGA from the disease-resistant wild potato S. bulbocastanum, complementing smaller collections from other Solanum species. To further comparative genomics approaches, we combined all known Solanum RGA and cloned solanaceous NBS-LRR gene sequences, nearly 800 sequences in total, into a single meta-analysis. We defined R gene diversity bins that reflect both evolutionary relationships and DNA cross-hybridization results. The resulting framework is amendable and expandable, providing the research community with a common vocabulary for present and future study of R gene lineages. Through a series of sequence and hybridization experiments, we demonstrate that all tested R gene lineages are of ancient origin, are shared between Solanum species, and can be successfully accessed via comparative genomics approaches.     

Resistance gene analogue markers are mapped to homeologous chromosomes in cultivated tetraploid cotton

Degenerate primers designed from conserved motifs of known plant resistance gene products were used to amplify genomic DNA sequences from the root-knot nematode (Meloidogyne incognita) resistance genetic source, Upland cotton (Gossypium hirsutum) cultivar Auburn 634 RNR. A total of 165 clones were isolated, and sequence analysis revealed 57 of the clones to be novel nucleotide sequences, many containing the resistance (R)-protein nucleotide-binding site motif. A cluster analysis was performed with resistance gene analogue (RGA) nucleotide sequences isolated in this study, in addition to 99 cotton RGA nucleotide sequences already deposited in GenBank, to generate a phylogenetic tree of cotton R genes. The cotton RGA nucleotide sequences were arranged into 11 groups and 56 sub-groups, based on genetic distances. Multiple sequence alignments were performed on the RGA sequences of each sub-group, and either the consensus sequences or individual RGA sequences were used to design 61 RGA-sequence-tagged site primers. A recombinant inbred line (RIL) population of cultivated tetraploid cotton was genotyped using RGA-specific primers that amplified polymorphic fragments between the two RIL parents. Nine RGA markers were mapped to homeologous chromosomes 12 and 26, based on linkage to existing markers that are located on these chromosomes.     

Isolation and linkage mapping of NBS-LRR resistance gene analogs in red raspberry (<Emphasis Type="Italic">Rubus idaeus</Emphasis> L.) and classification among 270 Rosaceae NBS-LRR genes

Plant R genes confer resistance to pathogens in a gene-for-gene mode. Seventy-five putative resistance gene analogs (RGAs) containing conserved domains were cloned from Rubus idaeus L. cv. ‘Latham’ using degenerate primers based on RGAs identified in Rosaceae species. The sequences were compared to 195 RGA sequences identified from five Rosaceae family genera. Multiple sequence alignments showed high similarity at multiple nucleotide-binding site (NBS) motifs with homology to Drosophila Toll and mammalian interleukin-1 receptor (TIR) and non-TIR RNBSA-A motifs. The TIR sequences clustered separately from the non-TIR sequences with a bootstrap value of 76%. There were 11 clusters each of TIR and non-TIR type sequences of multiple genera with bootstrap values of more than 50%, including nine with values of more than 75% and seven of more than 90%. Polymorphic sequence characterized amplified region and cleaved amplified polymorphic sequence markers were developed for nine Rubus RGA sequences with eight placed on a red raspberry genetic linkage map. Phylogenetic analysis indicated four of the mapped sequences share sequence similarity to groupTIR I, while three others were spread in non-TIR groups. Of the 75 Rubus RGA sequences analyzed, members were placed in five TIR groups and six non-TIR groups. These group classifications closely matched those in 12 of 13 studies from which these sequences were derived. The analysis of related DNA sequences within plant families elucidates the evolutionary relationship and process involved in pest resistance development in plants. This information will aid in the understanding of R genes and their proliferation within plant genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library

Resistance to grapevine powdery mildew is controlled by Run1, a single dominant gene present in the wild grapevine species, Muscadinia rotundifolia, but absent from the cultivated species, Vitis vinifera. Run1 has been introgressed into V. vinifera using a pseudo-backcross strategy, and genetic markers have previously been identified that are linked to the resistance locus. Here we describe the construction of comprehensive genetic and physical maps spanning the resistance locus that will enable future positional cloning of the resistance gene. Physical mapping was performed using a bacterial artificial chromosome (BAC) library constructed using genomic DNA extracted from a resistant V. vinifera individual carrying Run1 within an introgression. BAC contig assembly has enabled 20 new genetic markers to be identified that are closely linked to Run1, and the position of the resistance locus has been refined, locating the gene between the simple sequence repeat (SSR) marker, VMC4f3.1, and the BAC end sequence-derived marker, CB292.294. This region contains two multigene families of resistance gene analogues (RGA). A comparison of physical and genetic mapping data indicates that recombination is severely repressed in the vicinity of Run1, possibly due to divergent sequence contained within the introgressed fragment from M. rotundifolia that carries the Run1 gene.     

Genetic clues to the origin of the apple

Molecular genetic markers complement archaeological, breeding and geographical investigations of the origins, history and domestication of plants. With increasing access to wild apples from Central Asia, along with the use of molecular genetic markers capable of distinguishing between species, and explicit methods of phylogeny reconstruction, it is now possible to test hypotheses about the origin of the domesticated apple. Analyses of nuclear rDNA and chloroplast DNA (cpDNA) sequences indicate that the domesticated apple is most closely related to series Malus species. Moreover, the occurrence of a shared 18-bp duplication in the cpDNAs of wild and cultivated apple supports the close relationship between them. Hypotheses about the hybridization and the origin of the domesticated apple cannot be rejected completely until more variable, phylogenetically informative markers are found.     

Linked vs unlinked markers: multilocus microsatellite haplotype-sharing as a tool to estimate gene flow and introgression

We have explored the use of multilocus microsatellite haplotypes to study introgression from cultivated (Malus domestica) into wild apple (Malus sylvestris), and to study gene flow among remnant populations of M. sylvestris. A haplotype consisted of alleles at microsatellite loci along one chromosome. As destruction of haplotypes through recombination occurs much faster than loss of alleles due to genetic drift, the lifespan of a multilocus haplotype is much shorter than that of the underlying alleles. When different populations share the same haplotype, this may indicate recent gene flow between populations. Similarly, haplotypes shared between two species would be a strong signal for introgression. As the expected lifespan of a haplotype depends on the strength of the linkage, the length [in centiMorgans (cM)] of the haplotype shared contains information on the number of generations passed. This application of shared haplotypes is distinct from using haplotype-sharing to detect association between markers and a certain trait. We inferred haplotypes for four to eight microsatellite loci on Linkage Group 10 of apple from genotype data using the program phase, and then identified those haplotypes shared between populations and species. Compared with a Bayesian analysis of unlinked microsatellite loci using the program structure, haplotype-sharing detected a partially different set of putative hybrids. Cultivated haplotypes present in M. sylvestris were short (< 1.5 cM), indicating that introgression had taken place many generations ago, except for two Belgian plants that contained a haplotype of 47.1 cM, indicating recent introgression. In the estimation of gene flow, F(ST) based on unlinked loci indicated small (0.032-0.058) but statistically significant differentiation between some populations only. However, various M. sylvestris haplotypes were shared in nearly all pairwise comparisons of populations, and their length indicated recent gene flow. Hence, all Dutch populations should be considered as one conservation unit. The added value of using sharing of multilocus microsatellite haplotypes as a source of population genetic information is discussed.     

Mechanisms of haplotype divergence at the <Emphasis Type="Italic">RGA08</Emphasis> nucleotide-binding leucine-rich repeat gene locus in wild banana <Emphasis Type="Italic">(Musa balbisiana)</Emphasis>

Background  

Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW).     

Isolation and diversity analysis of resistance gene analogues (RGAs) from cultivated and wild strawberries

Degenerate oligonucleotide primers, designed based on conserved regions of Nucleotide Binding Site (NBS) domains from previously cloned plant resistance genes, were used to isolate Resistance Gene Analogues (RGAs) from wild and cultivated strawberries. Seven distinct families of RGAs of the NBS-LRR type were identified from two related wild species, Fragaria vesca and F. chiloensis, and six different Fragaria × ananassa cultivars. With one exception (GAV-3), the deduced amino acid sequences of strawberry RGAs showed strong similarity to TIR (Toll Interleukin I Receptor)-type R genes from Arabidopsis, tobacco and flax, suggesting the existence of common ancestors. GAV-3 seemed to be more closely related to the non-TIR type. Further studies showed that the recombination level and the ratio of non-synonymous to synonymous substitutions within families were low. These data suggest that NBS-encoding sequences of RGAs in strawberry are subject to a gradual accumulation of mutations leading to purifying selection, rather than to a diversifying process. The present paper is the first report on RGAs in strawberry.Communicated by M.-A. Grandbastien