Maurizio Rossetto

Analysis of SSRs derived from grape ESTs

Abstract One hundred and twenty four microsatellites were isolated from analysis of 5000 Vitis expressed sequence tags (ESTs). A diversity of dinucleotide and trinucleotide simple sequence repeat (SSR) motifs were present. Primers were designed for 16 of these SSRs and they were tested on seven accessions. Ten of the sixteen primer pairs resulted in PCR products of the expected size. All ten functional primers were polymorphic across the accessions studied. Polymorphisms were evident at the level of cultivars, Vitis species, and between related genera. SSRs that were from the 3′ untranslated region (3′UTR) were most polymorphic at the cultivar level, the 5′ untranslated region (5′ UTR) SSRs were most polymorphic between cultivars and species, and those SSRs within coding sequence were most polymorphic between species and genera. These results show that EST-derived SSRs in Vitis are useful as they are polymorphic and highly transferable. With EST SSRs being applicable to studies at several taxonomic levels, the large number of SSRs (approximately 1000) that will be available from an expanded EST database of 45 000 will have many potential applications in mapping and identity research.

Evaluating the potential of SSR flanking regions for examining taxonomic relationships in the Vitaceae

Abstract  Three EST-derived microsatellite loci from Vitis vinifera were amplified and sequenced across eight species of Vitaceae from four different genera. Phylogenetic analysis of the microsatellite’s flanking regions produced informative results in congruence with previous studies. Generic relationships were respected and the data produced sufficient inter-specific variation to distinguish between Cayratia acris and Cayratia saponaria, two very closely related species. Overall, the sequence alignments showed that priming sites were conserved, whereas microsatellite repeats were present in most cases but structurally variable. The sequence data provided information on the evolutionary patterns of various microsatellite repeats and their correlation to evolutionary relationships among taxa.

The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.

Genetic variation and island biogreography: Microsatellite and mitochondrial DNA variation in island populations of the Australian bush rat, Rattus fuscipes greyii

To understand the impact of various factors on the maintenance of genetic variation in natural populations, we need to focus on situations where at least some of these factors are removed or controlled. In this study, we used highly variable, presumably neutral, microsatellite and mtDNA markers to assess the nature of genetic variation in 14 island and two mainland populations of the Australian bush rat, where there is no migration between islands. Thus we are controlling for selection and gene flow. Both marker sets revealed low levels of diversity within the small island populations and extreme differentiation between populations. For six microsatellite loci, all of the small island populations had less genetic variation than the mainland populations; reduction in allelic diversity was more pronounced than loss of heterozygosity. Kangaroo Island, the large island population, had similar levels of diversity to the mainland populations. A 442 base pair (bp) section of the mtDNA control region was screened for variation by outgroup heteroduplex analysis/temperature gradient gel electrophoresis (OHA/TGGE). Only three of the 13 small island populations showed haplotypic diversity: Gambier (2), Waldegrave (2), and Eyere (3). The level of haplotypic diversity in the small island populations was similar to that on the mainland, most likely reflecting a recent population bottleneck on the mainland. In contrast, Kangaroo Island had 9 mtDNA haplotypes. The dominant factor influencing genetic diversity on the islands was island size. No correlation was detected between genetic diversity and the time since isolation or distance form the mainland. The combination of genetic drift within and complete isolation among the small island populations has resulted in rapid and extreme population divergence. Population pair-wise comparisons of allele frequency distributions showed significant differences for all populations for all loci (Fst = 0.11–0.84, Rst = 0.07–0.99). For the mtDNA control region, 92.6% of variation was apportioned between populations; only the Pearson islands shared a haplotype. Mantel tests of pair-wise genetic distance with pair-wise geographic distance showed no significant geographical clustering of haplotypes. However, population substructuring was detected within populations where sampling was conducted over a broader geographical range, as indicated by departures from Hardy-Weinberg equilibrium. Thus substructuring in the ancestral population cannot be ruled out. The dominant evolutionary forces on the islands, after the initial founder event, are stochastic population processes such as genetic drift and mutation.

Sourcing of SSR markers from related plant species.

As microsatellites, or simple sequence repeats (SSRs), have been discussed at length in previous chapters, an in-depth description of this technique is not necessary. Their abundant and uniform distribution throughout the genome, codominant inheritance, simple screening requirements and reproducibility ensure that microsatellites are valuable for a multitude of applications. One of the greatest advantages of these markers is a rate of polymorphism which is higher than in other common techniques such as restriction fragment length polymorphism and random amplified polymorphic DNA (Powell et al., 1996b). Slippedstrand mispairing during DNA replication is thought to be the predominant mode of mutation for microsatellites (Levinson and Gutman, 1987; Schlotterer and Tautz, 1992; Wierdl et al., 1997). Such mutational events accumulate more rapidly than point mutations and insertions/deletions (indels); rates are estimated at 10−2 to 10−3 per locus, per gamete, per generation (Weber and Wong, 1993). Some questions remain about the predominant mutation pattern of these rapid modifications, the main alternatives being single-step or random mutation events. A number of theoretical studies supporting one model or the other (as well as combinations of both) have been suggested (see Shriver et al., 1993; Di Rienzo et al., 1994; Estoup et al., 1995; Goldstein and Pollock, 1997 for example). The potential of microsatellites as useful markers for plant studies was promptly recognized, resulting in their successful characterization and application in a number of species. Most early studies were based on crops such as rice (Wu and Tanksley, 1993), barley (Saghai Maroof et al., 1994) and wheat (Roder et al., 1995). More recently, the ability of these hypervariable regions to reveal high allelic diversity and delimit fine genetic structure has resulted in an

Microsatellite variation and assessment of genetic structure in tea tree (Melaleuca alternifolia– Myrtaceae)

Analysis of five microsatellite loci in 500 Melaleuca alternifolia individuals produced 98 alleles that were useful for population genetic studies. Considerable levels of observed heterozygosity were recorded (HO = 0.724), with ≈ 90% of the variability being detected within populations. A low level of selfing (14%) was suggested to be the principal cause of excess homozygosity in a number of populations (overall FIS = 0.073). This study showed low levels of inbreeding in certain populations as well as a significant isolation‐by‐distance model. Only two groups of populations (Queensland and New South Wales) constituted different genetic provenances as a result of geographical isolation. The M. alternifolia data suggest that microsatellite loci did not always arise by a stepwise mutation process but that larger jumps in allele size may be involved in their evolution.

Conservation genetics and clonality in two critically endangered eucalypts from the highly endemic south-western Australian flora

Extensive agricultural development has endangered species diversity in the highly endemic south-western Australian flora. Many relict species with restricted distribution are now on the brink of extinction. Eucalyptus phylacis and E. dolorosa are both known from single locations within pockets of native vegetation surrounded by agricultural land. The development of appropriate conservation strategies required the appraisal of the extent of clonality within both species as well as the definition of species identity for E. phylacis. The use of RAPD analysis revealed contrasting stories for these two threatened species. E. phylacis, for which species identity was successfully established, was shown to be a single clone and as a result, possibly the rarest, largest, potentially oldest mallee eucalypt known. Whereas, despite its isolation, E. dolorosa still retained sufficient genetic variability to enable the production of highly outcrossed seed. Different conservation strategies are suggested for the two species and the importance of understanding basic genetic variability in rare clonal species is discussed.

Abundance and polymorphism of microsatellite markers in the tea tree (Melaleuca alternifolia, Myrtaceae)

Abstract The sequencing of 831 clones from an enriched microsatellite library of Melaleuca alternifolia (Myrtaceae) yielded 715 inserts containing repeat motifs. The majority of these (98%) were dinucleotide repeats or trinucleotide repeats averaging 22 and 8 repeat motifs respectively. The AG/GA motif was the most common, accounting for 43% of all microsatellites. From a total of 139 primer pairs designed, 102 produced markers within the expected size range. The majority of these (93) were polymorphic. Primer pairs were tested on five selected M. alternifolia genotypes. Loci based on dinucleotide repeats detected on average a greater number of alleles (4.2) than those based on trinucleotide repeats (2.9). The loci described will provide a large pool of polymorphisms useful for population studies, genetic mapping, and possibly application in other Myrtaceae.

Isolation and characterization of microsatellite loci from the bush rat, Rattus fuscipes greyii

.Ninety-four putative positive recombinants were pickedfrom agar plates, individually suspended in 100 µ L of 10 m m Tris (pH 8.0), and boiled for 5 min to lyse the cells andrelease the DNA. Following settling of the cell debris, 1 µ Lof supernatant was used as template for PCR amplificationusing universal M13 forward (5 ′ -CGCCAGGGTTTTCCCAG-TCACGAC-3 ′ ) and reverse (5 ′ -AGCGGATAACAATTTCACA-CAGGA-3 ′ ) primers (Pacific Oligos).PCR reaction mixtures contained 1 µ L of clone DNA,50 m m KCl, 10 m m Tris (pH 8.0), 0.25 units of

Conservation genetics and implications for restoration of Hemigenia exilis (Lamiaceae), a serpentine endemic from Western Australia

Hemigenia exilis (Lamiaceae) is a rare plant endemic to serpentine soils of the Goldfields of Western Australia. The species was presumed extinct until 1995, when it was re-discovered on a nickel ore deposit. To delineate the origin and extent of seed collection for rehabilitation after mining, and to identify the impact of removing one population due to mining, we assessed level and partitioning of genetic variation and differentiation. Twelve populations were sampled for DNA fingerprinting using the random amplified polymorphic DNA (RAPD) technique. Ten primers produced 89 bands, 97% being polymorphic. Genetic diversity within populations ranged from 0.197 to 0.409, averaging 0.38 at the species level, which is high compared with most other endemic species. Heterozygosity within populations ranged from 0.355 to 0.431, averaging 0.27 over the species. AMOVA partitioned over 80% of the total variation within populations. Multidimensional scaling revealed weak but significant differentiation into a northern and southern provenance. Despite selective sampling, the genetic data provided useful information for the management of Hemigenia exilis. For restoration, seed should be collected from a range of habitats of several populations, while keeping the two provenances separate. This strategy is likely to maintain high genetic diversity and locally adapted populations.