Development Of SNPs In EST Of Eucalyptus And Construction Of EST Linkage Maps For Eucalyptus

Four procedures for purifying EST-PCR products, were compared for their yield and effects on subsequent sequencing. Further, five dosages of Bigdye Terminator V3.1, the manufacture's standard, were compared for their sequencing results. The final conclusion of the optimization : ethanol-NaAc purification and Terminator V3.1with 1/16, or 1/32 of the manufacture's standard. The protocol optimized here is advantageous in low cost and technical simplicity and suitable for large-scale sequencing based on 96 or 384well plates.A total of 1684 EST were amplified successfully, which elucidated that the general application of the EST of Eucalyptus is great. About 71.08% EST (1197EST)were sequenced successfully. Also, it proved us the optimized protocol for sequencing EST-PCR products in Eucalyptus was passed well. A total of 1456 SNPs were discovered from the sequences of the resulting amplicons, giving an estimated frequency of 1 SNP per 404 bp in the Eucalyptu transcriptome. In contrasted the sequence of the amplicons and original EST, the number of EST with the indentity over 80% is 719. Including the SNPs found in the extron. There were 472 SNP in the extron. The relationship were identified by comparing the frequency of SNP in different Eucalyptus. That elucidated the potential of developing the SNPs in the EST of Eucalyptus is great.132 individuals of mapping population, the F1 hybrid population between E.urophylla×E.tereticornis . 526 EST primer pairs were used for CAPS. Finally 62 ESTs could lead to allele polymorphisms across the mapping population, which were used for maping. The makers also includingEST-SSR,EST-CAPS(G)., 27 EST-CAPS were used on the linkage map of maternal E.urophylla . 39 EST-CAPS were used on the linkage map of paternal E.tereticorn. For E.urophylla×E.tereticorni, the linkage map contained 62 EST-CAPS makers. This frame genetic map of E.urophylla and E.tereticornis would provide good reference for exploring the genetic mechanism of Eucalyptus and mapping QTLs that affect important traits in Eucalyptus. It also established a fine platform for the comparison research within Eucalyptus.