Frances G Eliott

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 x Q165). The sequencing yielded 96,755 (IJ76-514) and 86,241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP--an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent--with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.

Novel microsatellite markers for the endangered Australian rainforest tree Davidsonia jerseyana (Cunoniaceae) and cross-species amplification in the Davidsonia genus

Davidsonia jerseyana is an endangered rainforest tree endemic to far north-eastern New South Wales, Australia. The species occurs in small fragmented populations and has an edible plum-like fruit important to the Australian Native Food industry. Twenty one novel microsatellite markers were developed for D. jerseyana of which 13 were polymorphic for the species. Markers were characterised using 28 individuals, representing six populations from across the species geographic range. Species level analysis of the polymorphic markers revealed the mean number of alleles per locus was 3.154 (range 2–7) and mean expected and observed heterozygosities were 0.437 (range 0.035–0.725) and 0.044 (range 0–0.250) respectively. The heterozygote deficiency may indicate a predominantly selfing breeding system. All markers cross amplified in the other two Davidsonia species. These markers will be used to assess the genetic diversity, population structure and breeding systems in D. jerseyana and related taxa which will facilitate conservation management strategies.

Contrasting breeding systems revealed in the rainforest genus Davidsonia (Cunoniaceae): can polyembryony turn the tables on rarity?

Plant breeding systems can have a profound effect on a species ability to persist, colonise new areas and adapt to environmental change. Determining the breeding systems in rare and common congeners may shed light on factors influencing rarity. Endemic to Australia, the Davidsonia genus comprises three species of rainforest trees. The two sympatric, subtropical species, Davidsonia jerseyana (F.Muell. ex F.M.Bailey) G.Harden & J.B.Williams, and D. johnsonii G.Harden & J.B.Williams, are endangered whereas the tropical D. pruriens F.Muell. is widespread. Other than exclusive clonality in D. johnsonii, the reproductive systems in the genus are unknown. We used segregation analysis of microsatellite loci in open-pollinated progeny arrays to investigate the breeding systems in D. jerseyana and D. pruriens. Reproductive success, under glasshouse conditions, was measured by the proportion of viable seeds, germination rate and seedling growth and survival over 12 months. Davidsonia jerseyana appears to be predominantly selfing with high fecundity. In D. pruriens, polyembryony was common and 89% of the progeny were identical to the heterozygous maternal parent, implying likely apomixis. Overall, fecundity was significantly lower than for D. jerseyana, although survival was higher from D. pruriens polyembryonic than from monoembryonic seed. The high fecundity in D. jerseyana indicates a lack of inbreeding depression and also suggests that it would be less likely to be endangered than the less fecund D. pruriens. This raises the possibility that polyembryony and likely apomixis may provide a reproductive advantage to D. pruriens, which could otherwise share rarity with its congeners.

SNP discovery by ecotilling using capillary electrophoresis

Ecotilling (derived from targeting-induced local lesions in genomes (TILLING)) is a high-throughput method of detecting naturally-occurring DNA polymorphisms in specific gene sequences. Sugarcane, a complex polyploid species, was utilized as a model to develop and test new protocols for high throughput ecotilling using capillary electrophoresis (CE) systems. Segregating single nucleotide polymorphisms (SNPs) were easily identified in the progeny of the sugarcane population using the ABI 3730 CE system. In the SPS gene family 1, a cluster of SNP peaks formed a distinctly different pattern in each of the parents Q165 and IJ76-514, and these two patterns clearly segregated in the progeny. In ecotilling or TILLING using gel-based systems, polymorphisms are typically localized to within ±10 bases. In the analyses using CE, SNPs were typically localized to within ±3 bases of that determined through sequence information. A higher level of precision in the assignment of fragment sizes based on the size standards was achieved with the sensitivity of a CE system compared with gel electrophoresis systems. In the two sugarcane genotypes, Q165 and IJ76-514, SNP loci in SPS gene family III, identified through ecotilling, corresponded precisely with SNPs identified in and through the respective alignment of the 58 and 86 sequences amplified with the same set of primers used in ecotilling. An ecotilling strategy utilizing the sensitivity of a CE system has been shown as a robust means for genetic mapping in sugarcane. In a number of instances, multiple single-dose SNPs belonging to homoeologous genes were detected and mapped to separate locations on the Q165 map. A summary of the optimized protocol for ecotilling using CE is also presented.