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.

Phylogeny of the genus Pistacia as determined from analysis of the chloroplast genome.

Classification within the genus Pistacia has been based on leaf morphology and geographical distribution. Molecular genetic tools (PCR amplification followed by restriction analysis of a 3.2-kb region of variable chloroplast DNA, and restriction fragment length polymorphism analysis of the Pistacia cpDNA with tobacco chloroplast DNA probes) provided a new set of variables to study the phylogenetic relationships of 10 Pistacia species. Both parsimony and cluster analyses were used to divide the genus into two major groups. P. vera was determined to be the least derived species. P. weinmannifolia, an Asian species, is most closely related to P. texana and P. mexicana, New World species. These three species share a common origin, suggesting that a common ancestor of P. texana and P. mexicana originated in Asia. P. integerrima and P. chinensis were shown to be distinct whereas the pairs of species were monophyletic within each of two tertiary groups, P. vera:P. khinjuk and P. mexicana:P. texana. An evolutionary trend from large to small nuts and leaves with few, large leaflets to many, small leaflets was supported. The genus Pistacia was shown to have a low chloroplast DNA mutation rate: 0.05-0.16 times that expected of annual plants.

Phylogenetic relationships of cultivated Prunus species from an analysis of chloroplast DNA variation.

Chloroplast DNA (cpDNA) restriction-site mutations in seven cultivated Prunus species were compared to establish the phylogenetic relationships among them. Mutations were detected in 3.2-kb and 2.1-kb amplified regions of variable cpDNA, cut with 21 and 10 restriction endonucleases, respectively, to reveal polymorphisms. Parsimony and cluster analyses were performed. The species pairs P. persica-P. dulcis, P. domestica-P. salicina, and P.cerasus-P. fruticosa were completely monophyletic. All of the species were grouped with conventional subgenus classifications. The subgenus Cerasus was the most diverged. Cerasus ancestors separated from the remainder of Prunus relatively early in the development of the genus. P. persica-P. dulcis, P. domestica-P. salicina and P. armeniaca formed a second monophyletic group. Prunophora species were less diverged than Amygdalus species. The results also suggest that the rate of mutation in Cerasus spp. chloroplast genomes is significantly greater than for the other subgenera sampled.