454 sequencing of Sencecio madagascariensis transcriptome fragment library
subtractive libraries were generated using a  modified version of the Clontech PCR-Select™ cDNA Subtraction Kit. cDNA  amplified from Australian and South African populations of Senecio  madagascariensis were halved. On both samples two double digests were  performed using Mme1 (to remove Poly A tails) and either Rsa1 or Alu1 (for  suppressive subtractive hybridization). Two subtractive libraries were  generated (an Rsa1 library and an Alu1 library) of the Australian Senecio  madagascariensis ecotype subtracted with the corresponding South African  ecotype digests. The two resulting libraries were cleaned up using the  invitrogen PCR cleanup kit and then combined in equal concentrations to make  one library. Another library was generated in the same manner but using the  reverse subtraction, the south Afician ecotype cDNA was subtracted with the  Australian ecotype cDNA. Creating two subtractive libraries each digested with  a different restriction enzymes for both pyrosequencing library 3 and 4,  allowed for the assembly of long contigs.