Blackberry breeders have implement morphological marker-assisted selection for thornlessness using Rubus L, which trigger development of molecular marker for blackberry cultivation. However, no genetic map and molecular marker exist for cultivation purpose. Therefore, the purpose of this study is to develop genetic map, molecular markers, or first blackberry expressed sequence tag (EST) library. The newly release of two blackberry (Rubus L) cultivars with new trait, primocane fruiting has the potential to expand the industry by combining primocane fruiting with useful trait such as thornlessness.
To compensate lacking of molecular linkage map and molecular marker, simple sequence repeat (SSR) has been generated from strawberry EST sequences from Genbank. Strawberry is a related species with blackberry by possessing the same sub-family, Rosoideae. The primer pair (SSR) has detected 68% and 43% polymorphisms for sequences with six or more and less than six repeats respectively. Thus, it was expected that useful SSR can be generated from blackberry and sufficient EST for construction of genetic map.
Blackberry cultivars that produce fruit in summer is called floricanes, canes that go dormant and come back in second spring. Meanwhile, primocanes fruiting cultivars produce fruit together with floricanes and subsequently produce a smaller second batch in late summer and early fall. This can prolonged period of harvesting for farmer, marketer, and consumers.
Development of EST was started by choosing blackberry cultivars (Merton Thornless). Reason of choosing is because it is the progenitor. Firstly, the leaves of blackberry is been harvested at farm field. The leaves were wrapped in aluminium foil and then placed in liquid nitrogen for transportation to laboratory for RNA extraction. 3.5 g of leaves is been used for total RNA extraction using RNeasy Plant Mini Kit (Qiagen, Inc., Valencia, Calif). Successful RNA extraction from seven extraction were pooled together to increase the total yield. Afterwards, separation of mRNA from RNA is done using Poly(A) PuristTM Kit (Ambion, Austin, Tex.).
mRNA was then been used for cDNA synthesis using reverse transcription and cDNA library was cloned in Gateway system (Invitrogen Corp., Carlsbad, Calif.) with pDONR222 vector and ElectronMAXTM DH10BTM T1 Phage Resistant Cells (Invitrogen Corp.) by following manufactures’s instruction. Before transformation, kanamycin is used to select colonies containing vectors. A total of 18432 clones was selected and arrayed into 96 –well plates for sequencing. For the generation of EST, the M13F primer and the ABI PRISM BigDyeTM Terminator v3.1 reaction mix were used to sequence from the 5’ end of 3884 clones. The cycle sequencing is as follow: 96 °C 5 minutes, and 35 cycle of (96 °C for 45 second, 50 °C for 5 second, and 60 °C for 4 minutes). The sequence is generated using ABI 3730xl Genetic Analyzer (Applied Biosystems).
The sequence trace...