V. Sarri

A consensus list of microsatellite markers for olive genotyping

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.

Obtainment of inter-subspecific hybrids in olive (Olea europaea L.)

To enrich the source of germplasm of cultivated olive (Olea europaea subsp. europaea L.), inter-subspecific hybrid plants have been produced by experimental crosses between several varieties of cultivated olive and Asian and African accessions of the wild related subspecies cuspidata. Germination of putative hybrid seeds was enhanced by using in vitro embryo culture. The genetic make-up of germinated seedlings was assayed with the aid of both AFLP and SSR molecular markers and their hybrid nature was proved by the presence of male-specific alleles in their molecular patterns. Most of the parent specific alleles showed segregation among F1 progenies indicating high heterozygosity content of the parental lines. The majority of the hybrids derived from crosses in which an African accession of cuspidata was used as female parent. The overall morphological aspect of hybrids resembled that of the female parent. The production of inter-subspecific hybrid plants in Olea is discussed in relation to the genetic improvement of cultivated olive.

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.

Cryptic Introgression of Dasypyrum villosum Parental DNA in Wheat Lines Derived from Intergeneric Hybridization

Cytogenetic and DNA molecular analyses have been carried out in 3 wheat introgression lines (ILs; CS×V58, CS×V59, and CS×V60) derived from Triticum aestivum cv. ‘Chinese Spring’ (CS) × Dasypyrum villosum(Dv) intergeneric hybridization. All lines, which showed several phenotypic differences compared to CS, had the same chromosome number (2n = 42) and structure as CS, and neither chromosomes nor chromatin from Dv were apparently added to their complement. However, Feulgen/DNA cytophotometry showed that there was more nuclear DNA in the lines than in the parental wheat (by 1.85%, 2.76%, and 1.26% in CS×V58, CS×V59, and CS×V60, respectively). Molecular investigation indicated the presence of Dv DNA in the ILs. AFLP analysis of genomic DNA from the ILs, CS, and Dv detected a total of 120 polymorphic bands, of which 7 (5.8%) were present in some or all the ILs and Dv but were absent in CS. PCR amplification, sequence analysis of amplicons, and Southern blot hybridization confirmed the presence of Dv-specific sequences in each of the ILs. These results indicate cryptic introgression of Dv DNA sequences into the genome of the ILs. Some implications of this finding are discussed.

Characterization, evolution and chromosomal distribution of two satellite DNA sequence families in Lathyrus species.

DNA clones containing highly repetitive DNA sequences were selected from partial libraries of Lathyrus sativus and L. sylvestris. Two satellite DNA sequence families were isolated from the genome of the former species. A first family was made up of repeats that varied in length from 54–56 bp, and shared 51.7–94.8% nucleotide sequence similarity. The repeats of the second sequence family were 52–62 bp in length, and shared a 58.5–78.5% nucleotide sequence similarity. All the repeat units contained in a clone from L. sylvestris were 41 bp in length and showed an almost perfect structural conservation (95.1–100% nucleotide sequence similarity). The evolution of the first sequence family from L. sativus and of that isolated from L. sylvestris was studied by dot-blot hybridization to the genomic DNA of these species and 3 other Lathyrus species, L. clymenum, L. latifolius and L. odoratus. The former repeats were found to be species-specific and their redundancy was calculated to be 2.9 × 107. The satellite DNA sequence isolated from L. sylvestris was present also in L.latifolius, with a redundancy of 1.4 × 107 and 1.1 × 107, respectively. Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal distribution of the two sequence families and of 45S and 5S ribosomal genes. The species-specific sequences of L. sativus were located around the centromere of chromosome pair IV, where they occupied a very broad region, and, in a much smaller amount, close to the centromere in the short arm of pair II. Sequences related to the repeat units isolated from L. sylvestris were found, both in this species and L. latifolius, in all of the chromosome pairs at terminal and interstitial regions, where they co-localize with the vast majority of DAPI bands. The pattern of distribution of the satellite DNA sequences investigated, together with that of DAPI bands and ribosomal DNA, allowed each chromosome pair of the 3 complements studied to be identified unambiguously.

Phylogenetic relationships between annual and perennial species of Helianthus: evolution of a tandem repeated DNA sequence and cytological hybridization experiments.

The amplification and chromosomal localization of tandem repeated DNA sequences from Helianthus annuus (clone HAG004N15) and the physical organization of ribosomal DNA were studied in annual and perennial species of Helianthus. HAG004N15-related sequences, which did not show amplification in other Asteraceae except for Viguiera multiflora, were redundant in all the Helianthus species tested, but their frequency was significantly higher in perennials than in annuals. These sequences were located at the ends and intercalary regions of all chromosome pairs of annual species. A similar pattern was found in the perennials, but a metacentric pair in their complement was not labelled. Ribosomal cistrons were carried on two chromosome pairs in perennials and on three pairs in annuals except for H. annuus, where rDNA loci were on four pairs. No difference was observed between cultivated H. annuus and its wild accessions in the hybridization pattern of the HAG004N15 and ribosomal probes. These findings support the hypothesis that the separation between annual and perennial Helianthus species occurred through interspecific hybridization involving at least one different parent. However, GISH in H. annuus using genomic DNA from the perennial Helianthus giganteus as blocking DNA failed to reveal different genomic assets in annual and perennial species.

Characterization of two families of tandem repeated DNA sequences in Potamogeton pectinatus L.

DNA sequences belonging to two families of tandem repeats, PpeRsa1 (362-364 bp in length, 62% A+T residues) and PpeRsa2 (355-359 bp in length, 59% A+T residues), have been isolated from the Potamogeton pectinatus L. genome. The two sequence families do not share significant nucleotide sequence similarity, even if an evolutionary relationship between them could be assumed. The comparison of the cleaving activity of isoschizomeres that are either sensitive or insensitive to methylation of cytosine residues in the target sequence revealed high methylation in both sequence families. The copy number per 1C DNA of PpeRsa1- and PpeRsa2-related sequences is estimated to be 4.92 x 10(4) and 7.96 x 10(4), respectively. Taken together, these sequences account for about 7.5% of the entire genome of P. pectinatus. The chromosomal organization of these sequences was investigated by fluorescent in situ hybridization. PpeRsa1 and PpeRsa2 repeats found related sequences in 52 chromosomes of the P. pectinatus complement (2n = 78). The related sequences were localized around the centromeres and at the chromosome ends in three pairs of chromosomes, while they were found only at the chromosome ends in the remaining pairs. Twenty-six chromosomes did not show any hybridization signal. The hypothesis that the species is a hybrid between a diploid parent and an allotetraploid parent is put forward.

Ogre retrotransposons in Lathyrus species

Abstract A repeated DNA element (pLsat10) 225 bp in length, whose nucleotide sequence was partly identical to that of the PBS of Ogre retrotransposons in Pisum sativum and in part 78.3% similar to that of the adjoining portion of the 5′ LTR of these retroelements, was isolated from a partial genomic library of Lathyrus sativus. By using this DNA sequence in dot-blot and Southern blot hybridizations, Ogre retrotransposons were traced and studied in four Lathyrus species (L. latifolius, L. odoratus, L. sativus, and L. sylvestris). The copy number per ng of DNA of pLsat-related sequences in L. sativus turned out to be 1.5×107, and an upper limit estimate of the proportion of the genome made up by Ogre may be 10–16%. The results of Southern blot hybridization of pLsat10 to genomic DNAs indicated the occurrence, in each Lathyrus genome, of distinct subfamilies of Ogre and suggested a higher activity of these retrotransposons in annual than in perennial species. Fluorescent in situ hybridization of pLsat10 showed Ogre to be widely dispersed in all chromosomes, but poorly represented or absent in heterochromatic chromosome regions.