Genotyping systems for Eucalyptus based on tetra-, penta-, and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests

Abstract

Eucalypts are keystone species in their natural ranges and are extensively planted worldwide for highquality woody biomass. A novel set of 21 polymorphic and interspecifically transferable microsatellite markers based on tetra-, penta-and hexanucleotide repeats were developed and tested for high-precision genotyping of species of Eucalyptus. These microsatellites were characterized in population samples of four species, Eucalyptus grandis, Eucalyptus globulus, Eucalyptus urophylla, and Eucalyptus camaldulensis, representing three phylogenetic sections of subgenus Symphyomyrtus. These markers provide a clear advantage for accurate allele calling due to their larger allele size difference. Two multiplexed microsatellite combinations, a 14-locus/four-dye and an 18-locus/fivedye set, analyzable in single lanes were designed, providing resolution and throughput analogous to those routinely used in human DNA profiling. This set of microsatellites was shown to have high resolution for clone fingerprinting, inter-individual genetic distance estimation, species distinction, and assignment of hybrid individuals to their most likely ancestral species. These systems will be particularly useful for comparative population genetics and molecular breeding applications that require consistent allele calling across different points in time or laboratories.

Transferability of microsatellite markers located in candidate genes for wood properties between Eucalyptus species

“…Furthermore, these loci were physically aligned to a unique position on the chromosome scaffolds 3, 7, 8 and 11 (Table 1). The polymorphism rate was similar to those described by Torales et al (2012), in Nothofagus (19%) but lower than those described by Faria et al (2011), (39%) and Acuña et al (2012a), (46%) in Eucalyptus.…”

“…Estimates of average shared allele distance (DAS) among individuals within species ranged from 0.48 to 510 C. V. Acuña et al / Forest Systems (2014) Faria et al (2011). As expected, these values increased when considering DAS between individuals of different species (82% of pairwise data were higher than 0.7).…”

Genetic mapping provides evidence for the role of additive and non-additive QTLs in the response of inter-specific hybrids of Eucalyptus to Puccinia psidii rust infection

“…A total of 106 microsatellite markers, chosen to provide even genome coverage, were selected based on the reference linkage map (Brondani et al 2006) and on the microsatellites developed by CSIRO (Thamarus et al 2002). An additional set of eight EST derived markers (Embra915, 954, 979, 1468(Embra915, 954, 979, , 1851(Embra915, 954, 979, , 1928(Embra915, 954, 979, , 1977(Embra915, 954, 979, and 2014 recently developed (Faria et al , 2011 were also used, totalizing 114 loci. Genotyping was carried out in multiplexed systems with four to six co-amplified loci, each one labeled with a specific fluorochrome (6-FAM, HEX or NED).…”

Research Article Analysis of Mendelian inheritance and genetic linkage in microsatellite loci of Eucalyptus urophylla S.T. Blake.

“…In PT, we sampled 605 seedlings (denominated seeds) from 23 mother trees. The extraction and purification of the DNA were performed based on the CTAB method (Doyle and Doyle, 1987) and amplification was performed using PCR (polymerase chain reaction), with minor modifications to the protocol (see Faria et al, 2011). For the genotyping of individuals, we used EMBRA microsatellite markers (Brondani et al, 1998(Brondani et al, , 2006: 15 SSR loci for SO2 and PT; and 13 common SSR loci for SO1.…”