Development Of EST-CAPS Markers And Their Application To Linkage Map Construction In Eucalyptus

A genetic map for the genome of Eucalyptus was constructed using EST-CAPS markers and an interspecific cross of E. urophylla×E. tereticornis including two parents and 124 sibs. Initially 4037 EST sequences were downloaded from GenBank, from which 3095 uni-sequences were formed after sequence assembly and 2500 pairs of primers were designed and synthesized. By primer screening 1684 primer pairs could result in single PCR fragment, from which 622 primer pairs with PCR product more than 400bp were used for CAPS marker screening with six restriction endonucleases. Finally 61 ESTs could lead to allele polymorphisms across the mapping population. In addition, 12 EST-CAPS markers developmed previously were included in this mapping work. Thus a total of 73 EST-CAPS markers were generated for mapping analysis.Theχ2 analyses on the 73 markers indicated that 24 (32.9%) were abnormally segregated (α=0.05). As the number of makers was not large, all the makers were applied to mapping analysis.Three software packages, Mapmaker 3.0, JoinMap 4.0, and FsLinkageMap 1.0, were used for linkage analyses. By Mapmaker 3.0 using a threshold of minimum LOD of 3.0, 48 markers were ordered into18 linkage groups, with 2-5 markers per group and a total length of 537.3 cM. By JoinMap 4.0 using a threshold of minimum LOD 3.0, 49 makers were ordered into 18 linkage groups, with 2-6 markers per group and a total length 654.0 cM. By FsLinkageMap 1.0 using a threshold of minimum LOD 3.0, 63 makers were ordered into 17 linkage groups, with 2-10 markers per group and a total length 993.4 cM. Comparison among the three maps indicates that the map construted by Mapmaker 3.0 is similar with that of JoinMap 4.0 as nine identical linkage groups were found, but much different from that of FsLinkageMap 1.0 as there were not any identical linkage groups.Based on the total linkage length (537.3 cM) by Mapmaker 3.0 and the genome length previously estimated, the map constructed had a coverage of 33.9%-35.6% of the Eucalyptus genome.