Development of microsatellite markers from loquat, Eriobotrya japonica (Thunb.) Lindl.

Loquat (Eriobotrya japonica) is a minor fruit which has become an interesting alternative into the European fruit industry. This interest resulted in a loquat germplasm collection established at the Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain. Currently, it is the main reservoir of this species outside Asia. We developed and characterized the first 21 polymorphic microsatellite loci from a CT/AG-enriched loquat genomic library. The observed heterozygosity ranged between 0.20 and 1.00, expected heterozygosity ranged between 0.17 and 0.81, three markers were multilocus and eight loci departed significantly from Hardy-Weinberg equilibrium. These markers will facilitate diversity and genetic studies into the species.

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.).

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.

Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes.

The gametophytic self-incompatibility (GSI) system in Rosaceae has been proposed to be controlled by two genes located in the S -locusan S-RNase and a recently described pollen expressed S -haplotype specific F-box gene (SFB). However, in apricot (Prunus armeniaca L.) these genes had not been identified yet. We have sequenced 21 kb in total of the S -locus region in 3 different apricot S -haplotypes. These fragments contain genes homologous to the S-RNase and F-box genes found in other Prunus species, preserving their basic gene structure features and defined amino acid domains. The physical distance between the F-box and the S-RNase genes was determined exactly in the S2-haplotype (2.9 kb) and inferred approximately in the S 1-haplotype (< 49 kb) confirming that these genes are linked. Sequence analysis of the 5' flanking regions indicates the presence of a conserved region upstream of the putative TATA box in the S-RNase gene. The three identified S-RNase alleles (S1, S2 and S4) had a high allelic sequence diversity (75.3 amino acid identity), and the apricot F-box allelic variants (SFB1, SFB2 and SFB4) were also highly haplotype-specific (79.4 amino acid identity). Organ specific-expression was also studied, revealing that S1- and S2-RNases are expressed in style tissues, but not in pollen or leaves. In contrast, SFB1 and SFB2 are only expressed in pollen, but not in styles or leaves. Taken together, these results support these genes as candidates for the pistil and pollen S-determinants of GSI in apricot.

An apricot (Prunus armeniaca L.) F2 progeny linkage map based on SSR and AFLP markers, mapping plum pox virus resistance and self-incompatibility traits.

A genetic linkage map of apricot ( Prunus armeniaca L.) was constructed using AFLP and SSR markers. The map is based on an F(2) population (76 individuals) derived from self-pollination of an F(1) individual ('Lito') originated from a cross between 'Stark Early Orange' and 'Tyrinthos'. This family, designated as 'Lito' x 'Lito', segregated for two important agronomical traits: plum pox virus resistance (PPV) and self-incompatibility. A total of 211 markers (180 AFLPs, 29 SSRs and two agronomic traits) were assigned to 11 linkage groups covering 602 cM of the apricot genome. The average distance (cM/marker) between adjacent markers is 3.84 cM. The PPV resistance trait was mapped on linkage group G1 and the self-incompatibility trait was mapped on linkage group G6. Twenty two loci held in common with other Prunus maps allowed us to compare and establish homologies among the respective linkage groups.

Construction and application of a bacterial artificial chromosome (BAC) library of Prunus armeniaca L. for the identification of clones linked to the self-incompatibility locus.

To facilitate gene discovery in the Rosaceae, a bacterial artificial chromosome (BAC) library was constructed using high-molecular-weight (HMW) DNA from apricot leaves (Prunus armeniaca L.). The library contains 101,376 clones (264, 384-well plates) with an average insert size of 64 kb, equivalent to 22-fold genome coverage. In the first application of this library, high-density filters were screened for self-incompatibility genes using apricot DNA probes. Eight positive BAC clones were detected and fingerprinted to determine clone relationships and assemble contigs. These results demonstrate the suitability of this library for gene identification and physical mapping of the apricot genome.

Genetic linkage maps of two apricot cultivars ( Prunus armeniaca L.), and mapping of PPV (sharka) resistance.

Genetic linkage maps for two apricot cultivars have been constructed using AFLP, RAPD, RFLP and SSR markers in 81 F1 individuals from the cross 'Goldrich' x 'Valenciano'. This family segregated for resistance to 'plum pox virus' (PPV), the most-important virus affecting Prunus species. Of the 160 RAPD arbitrary primers screened a total of 44 were selected. Sixty one polymorphic RAPD markers were scored on the mapping population: 30 heterozygous in 'Goldrich', 19 heterozygous in 'Valenciano', segregating 1:1, and 12 markers heterozygous in both parents, segregating 3:1. A total of 33 and 19 RAPD markers were mapped on the 'Goldrich' and 'Valenciano' maps respectively. Forteen primer combinations were used for AFLPs and all of them detected polymorphism. Ninety five markers segregating 1:1 were identified, of which 62 were heterozygous in the female parent 'Goldrich' and 33 in the male parent 'Valenciano'. Forty five markers were present in both parents and segregated 3:1. A total of 82 and 48 AFLP markers were mapped on the 'Goldrich' and 'Valenciano' maps. Twelve RFLPs probes were screened in the population, resulting in five loci segregating in the family, one locus heterozygous for 'Valenciano' and four heterozygous for both, segregating 1:2:1. Of the 45 SSRs screened 17 segregated in the mapping family, resulting in seven loci heterozygous for the maternal parent and ten heterozygous for both, segregating 1:2:1 or 1:1:1:1. A total of 16 and 13 co-dominant markers were mapped in the female and male parent maps respectively. A total of 132 markers were placed into eight linkage groups on the 'Goldrich' map, defining 511 cM of the total map-length. The average distance between adjacent markers was 3.9 cM. A total of 80 markers were placed into seven linkage groups on the 'Valenciano' map, defining 467.2 cM of the total map-distance, with an average interval of 5.8 cM between adjacent markers. Thirty six marker loci heterozygous in both parents revealed straightforward homologies between five linkage groups in both maps. The sharka resistance trait mapped on linkage group 2. The region containing sharka resistance is flanked by two co-dominant markers that will be used for targeted SSR development employing a recently constructed complete apricot BAC library. SSRs tightly linked to sharka resistance will facilitate MAS in breeding for resistance in apricot.

Pear blister canker viroid: sequence variability and causal role in pear blister canker disease.

The sequences of several cDNA clones of pear blister canker viroid (PBCVd) P1914T and P47A isolates have been determined. Seven out of eight P1914T clones analysed have a constant sequence which differs at six positions from that of the P2098T isolate reported previously. The remaining P1914T clone (8) has a single nucleotide substitution. The same six changes have been also observed in most of the ten P47A clones sequenced. However, some P47A clones show additional variability in positions on both strands of the central conserved region (CCR) and in another conserved sequence at the left-terminal region. This is the first report of a change affecting the upper strand of a viroid CCR. Reasons why such a change is tolerated are discussed. Infectivity bioassays have demonstrated that PBCVd is the causal agent of PBC disease.

Identification of a new viroid as the putative causal agent of pear blister canker disease.

A small RNA species with the structural and functional properties characteristic of viroids has been isolated from three different pear sources each of which induced symptoms of the pear blister canker (PBC) disease when indexed in the pear indicator A 20. A close association between this RNA and PBC disease was established, since two of the three studied sources were known to be affected by this malady only, and the viroid was not detected in healthy pear tissue. Moreover, the PBC-associated viroid (PBCVd) replicated when purified preparations were inoculated into cucumber and pear plants. PBCVd behaved in denaturing polyacrylamide gels as a circular RNA with a molecular size of approximately 315 nucleotide residues. Analysis by dot blot hybridization indicated that PBCVd shares similarities in sequence with peach latent mosaic viroid and hop stunt viroid, and to a lesser extent with apple scar skin viroid.

Some properties of the viroid inducing peach latent mosaic disease.

Analysis by polyacrylamide gel electrophoresis of nucleic acid extracts from different peach samples, healthy or infected with the peach latent mosaic (PLM) disease, demonstrated the association of this disease with an RNA exhibiting the electrophoretic properties typical of circular viroid molecules. This RNA was called peach latent mosaic viroid (PLMV), since a purified preparation of it, when inoculated into GF 305 peach seedlings induced characteristic symptoms of PLM disease. PLMV was estimated to have a molecular size in the range of 330-340 bases, by comparison of its electrophoretic mobility under denaturing conditions with those of several viroid RNA. Dot-blot analysis showed that PLMV had a sequence clearly different from other viroids, including citrus exocortis viroid, apple scar skin viroid (ASSV), hop stunt viroid (HSV) and avocado sunblotch viroid. The possible significance of the limited sequence homology shared by PLMV with HSV, and especially with ASSV, is discussed.