Loblolly Pine EST Sequencing Information

We have generated ESTs from four different pine cDNA libraries. In total, we have generated >83,000 ESTs which are all available in Genbank. Descriptions of the four cDNA libraries used in the EST sequencing are listed below. All cDNA libraries were provided by Dr. John Cairney. Clones are available upon request to TIGR Pine Group (pine@tigr.org).

Library A (Prefixed with PIA,PIC, PID, RPI)

ORGANISM: Loblolly Pine (Pinus taeda L.)
CULTIVAR: 7-56 mother tree, open-pollinated tree from, Lyons, Georgia, USA
ORGAN: Zygotic Embryo and Megagametophyte, Somatic Embryo
TISSUE: Whole megagametophytes isolated from pine seeds, Whole embryos excised from these megagametophytes, whole somatic embryos and suspensor tissue from tissue culture, isolated from cell line A12
HOST: E.coli DH10B-TonA
VECTOR: pCMV-SPORT 6.1 Cloned into the NotI and EcoRV sites
DESCRIPTION: Pooled RNA from zygotic embryos, megagametophytes, and somatic embryos was used for library construction. Pine cones were harvested weekly from open-pollinated 7-56 mother trees, collections occurred from 7/01/02 until 10/15/02. Whole megagametophytes were first isolated from pine seeds, and whole embryos excised from these megagametophytes each was flash frozen. Embryo development was assessed using the system of Pullman et al. (Pullman GS, Johnson S, Peter G, Cairney J, Xu N. 2003. Loblolly Pine somatic embryogenesis: development of a maturation medium and resulting embryo quality. Plant Cell Reports 21:747-758 (http://link.springer.de/link/service/journals/00299/contents/03/00586/). For photographs see Ciavatta et al. 2001. (Ciavatta VT, Morillon R, Pullman GS, Chrispeels M, Cairney J. 2001. An aquaglyceroporin is abundantly expressed early in the development of the suspensor and the embryo proper of loblolly pine (Pinus taeda L.). Plant Physiol. 127: 1556-1567 (http://www.plantphysiol.org/cgi/content/full/127/4/1556)).

Library B (Prefixed with PSA)

Library C (Prefixed with PTA)

ORGANISM: Loblolly Pine (Pinus taeda L.)
CULTIVAR: 7-56 mother tree, open-pollinated tree from, Lyons, Georgia, USA
ORGAN: Zygotic Embryos
TISSUE: Whole embryos excised from megagametophytes
HOST: JM109
VECTOR: pGEM-T Easy cloned into the EcoRI and SpeI sites
DESCRIPTION: Library enriched in transcripts present in late phase of loblolly pine embryo maturation. A subtractive library was made using the protocols of Clontech (Palo Alto,CA). RNA from loblolly pine embryos eight weeks after cotyledon emergence (Stage 9.8, see Ciavatta VT, Morillon R, Pullman GS, Chrispeels M, Cairney J. 2001. An aquaglyceroporin is abundantly expressed early in the development of the suspensor and the embryo proper of loblolly pine (Pinus taeda L.). Plant Physiol. 127: 1556-1567 [http://www.plantphysiol.org/cgi/content/full/127/4/1556]) was subtracted from RNA from pine embryos collected nine weeks after cotyledon emergence (Stage 9.9).

Library D (Prefixed with PWA)

ORGANISM: Loblolly Pine (Pinus taeda L.)
CULTIVAR: 7-56 mother tree, open-pollinated tree from, Lyons, Georgia, USA
ORGAN: Suspension Culture Somatic Embryos (Stages 1-3)
TISSUE: Suspension Culture Somatic Embryos (Stages 1-3)
CELL_TYPE: 31 from Mother Tree 7-56
HOST: DH10B
VECTOR: pGADT7-RecAB cloned into the EcoRI and ClaI sites
DESCRIPTION: Library enriched in transcripts present in suspension culture of Loblolly Pine (Pinus taeda L.), 7-56. Suspension culture was established from fertilized megagametophytes, according to method of Pullman et al. 2003 (Pullman, G., S. Johnson, G. Peter, J. Cairney, and N. Xu. 2003. Improving loblolly pine somatic embryo maturation: Comparison of somatic and zygotic embryo morphology, germination, and gene expression. Plant Cell Reports 21:747-758. ) This suspension cell culture, which contains early developing embryos and undifferentiated embryogeneic tissue, was the source of the RNA used for the suspension culture library. Subtracted from this was cDNA made from a pool of RNA from zygotic embryos and female gametophytes from all stages of development plus somatic embryos from developmental stages beyond susepension culture (see Ciavatta VT, Morillon R, Pullman GS, Chrispeels M, Cairney J. 2001. An aquaglyceroporin is abundantly expressed early in the development of the suspensor and the embryo proper of loblolly pine (Pinus taeda L.). Plant Physiol. 127: 1556-1567 [http://www.plantphysiol.org/cgi/content/full/127/4/1556]) .

Other ESTs

Other ESTs have been deposited into Genbank and represent clones that were selected for the microarray and were re-sequenced as part of our validation process. These clones are names with a "PIM" prefix for Pine Microarray. Nomenclature for these rearrayed clones are as follows:
Prefix of PIMA were derived from Library A
Prefix of PIMC or PIMD were derived from Library B
Prefix of PIME were derived from Library C
Prefix of PIMF, PIMG, or PIMH were derived from Library D

Total ESTs Generated: 83,655

* Clones are available upon request to TIGR Pine Group.

The NSF Genomics of Loblolly Pine Embryogenesis Project at IPST and TIGR is funded by the National Science Foundation.