Genetic linkage maps of eucalyptus grandis and eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers

Abstract

We have used a “two-way pseudo-testcross” mapping strategy in combination with the random amplified polymorhic DNA (RAPD) assay to construct two moderate density genetic linkage maps for species of Eucalyptus. In the cross between two heterozygous individuals many singledose RAPD markers will be heterozygous in one parent, null in theother and therefore segregate 1:l in their F, progeny followinga testcross configuration. Meiosis and gametic segregation iena ch individual can be direcatlnyd efficiently analyzed usingR APD markers. We screened 305 primers of arbitrary sequence, and selected 151 to amplify a total of 558 markers. These markers were grouped at LOD 5.0, 0 = 0.25, resulting in the maternal Eucalyptus grandis map having a total of 240 markers into 14 linkage groups (1552 cM) and the paternal Eucalyptus urophylla map with 251 markers in 11 linkage groups (1101 cM) ( n = 11 in Eucalyptus). Framework maps ordered with a likelihood support ?1000:1 were assembled covering 95% of the estimated genome size inb oth individuals. Characterizationo f genome complexity of a sample of 48 mapped random amplified polymorphic DNA (RAPD) markers indicate that 53% amplify from low copy regions. These are the first reported high coverage linkage mapfso r any species of Eucalyptus and among the first for any hardwood tree species. We propose the combined use of RAPD markers and the pseudo-testcross configuration as a general strategy for the construction of single individual genetic linkage maps in outbred forest trees as well as in any highly heterozygous sexuallrye producing living organism. A survey of the occurrence of RAPD markers in different individuals suggests that the pseudo-testcross/RAPD mapping strategy should also be efficieant the intraspecific level and increasingly so with crosseso f genetically divergent individuals. The ability to quickly construct single-tree genetic linkage maps ina ny forest species opens the way for a shift fromt he paradigm of a species index map ttoh e heterodox proposaol f constructing several maps for individual trees of a population, therefore mitigating the problem of linkage equilibrium between marker and trait loci for the application of marker assisted strategies in tree breeding.