Development Of A New Set Of EST-SSR Markers And Their Application To Integrated Linkage Map In Eucalyptus

EST-SSR markers are very limited in the tree genus Eucalyptus. In this sudy, a new set of ESTs were downloaded from GenBank and a total of 266 EST-SSR primer pairs were designed for non-redundant ESTs, of which 203 proved to be successful in PCR optimization with the paternal parent (P2) of a mapping population of E. urophylla×E. tereticornis. Meanwhile, another set of 420 EST-SSRs that were PCR successful but failed in direct sequencing of PCR products in previous work were included in subsequent experiments.Pool-cloning-based sequencing was conducted for the 623 EST-SSRs successful in PCR optimization, and 395 yielded readable traces. Sequence alignment revealed that 295 amplicons were identical to the respective ESTs, whereas the rest 94 were probably resulted from PCR errors.Annotation of putative functionality through BlastX searches against NCBI database of non-redundant protein sequences showed that 191 (64.7%) out of the 295 EST-SSRs were homologous to known genes and 40 (13.6%) corresponded to predicted protein at a threshold of E-value less than e-5 while 64 (21.7%) produced no significant match.All the 295 EST-SSRs were tested for transferability across one species of Corymbia and seven species of Eucalyptus, and 273 scorable EST-SSRs were detected, with a total of 1958 alleles. There were 258 polymorphic markers, with Ho, He and PIC varied from zero to 1.0, 0.0417 to 0.9477 and 0.04 to 0.6234, respectively.The 258 polymorphic markers were used for genetic mapping with an F1 pedigree of E. urophylla×E. tereticornis, and 148 segregated in the mapping populaiton. All of the 273 scorable markers,148 showed separation in 132 sibs. These 148 markers together with pre-mined STS markers were used to construct E. urophylla and E. tereticornis'genetic map separately with the software Mapmaker 3.0. Linkage analysis in the parternal tree (E. urophylla) was based on 227 markers(70 new developed EST-SSRs) and covered 2520.6cM of total map distance; for the E. tereticornis tree a total of 214 markers(66 new developed EST-SSRs) were used for linkage analysis and had a total map distance of 2520.6cM.