Progress 09/15/04 to 09/14/07
Outputs
OUTPUTS: Coffee (Coffea arabica L.) is an economically important crop in Hawaii. The cultivar Tall Mokka (MA2) is one of the genotypes producing high quality beans with superior flavor but low yield due to smaller bean size. Based on AFLP marker analyses, MA2 is very similar to 'Kona-Typica' (KO34), which produces a larger bean size. All parts of MA2 plants are smaller than those from KO34. Examination of leaf epidermis indicated that the difference in organ size is probably due to the difference in cell size rather than cell number. In order to identify genes related to organ size in coffee, we compared gene expression patterns in MA2 and KO34 using the potato cDNA microarray from TIGR. This microarray was chosen as a cross species platform based on sequence comparison analyses of coffee with potato and arabidopsis. Forty-five genes with 2-fold or greater difference in expression level were identified using the potato microarrays hybridized with cDNA probes synthesized from shoot
tip RNAs of MA2 and KO34. Among these genes, 28 homologous coffee sequences were found in the publicly available databases. Only 1 out of these 28 homologs was confirmed to be differentially expressed via qRT-PCR with an approximately 4 fold higher expression in MA2. The difference in expression of this gene was also observed in the small bean cultivars 'Laurina' and 'Mokka' as compared to KO34. Similar difference also occurred between MA2 and 'Catimor', another larger size cultivar. This gene is homologous to an arabidopsis gene encoding prolyl oligopeptidase (POP). Efforts are underway to clone the full-length cDNA and genomic clones of the POP homolog from coffee. POP cuts short peptides at the proline residue. In animals, POP controls the activities of several short peptide hormones. There are also short peptide growth regulators present in plants. However, the exact function of POP in plants is not clear. We have obtained and planted arabidopsis mutant seeds with reduced POP
expression. An expression casette is under construction to transform arabidopsis and create POP overexpression lines. The phenotypes of the wild-type, mutant, and POP overexpression arabidopsis plants will be compared. The expression of potential downstream genes of POP will also be monitored to characterize POP function.
PARTICIPANTS: Partner institutions include USDA ARS Pacific Basin Agricultural Research Center and the Hawaii Agricultural Research Center.
TARGET AUDIENCES: Hawaii coffee growers.
Impacts
In 2005, 6.6 million pounds of green beans with a value of $37.31 million were produced from 8,000 acres of farmland. The value of coffee production ranked 5th of all crops in the state. Hawaiian farm revenue from coffee has increased over the years due to nationally and internationally recognized premium quality. In order to maintain this worldwide reputation for high quality coffee, the development of Hawaiian coffee cultivars with new and better flavor profiles while simultaneously producing high yields is required. The gene(s) found to be responsible for organ size can be utilized in the coffee breeding program, established in 1997 at Hawaii Agriculture Research Center (HARC) with the cooperation of University of Hawaii (UH) and support from the Hawaii Coffee Growers' Association and the State of Hawaii, and contribute to improving yields of high-quality coffee. Furthermore, the new knowledge on genetic control of organ size may be applied to other crops to improve
productivity.
Publications
- Singh, R., B. Irikura, H. H. Albert, C. Nagai, M. Kumagai, R. E. Paull and M.-L. Wang. Coffee genomics: isolation and characterization of genes involved in organ size. CTAHR Student Research Symposium, April 5-6, 2007, University of Hawaii at Manoa.
- Singh, R., B. Irikura, C. Nagai, H. H. Albert, M. Kumagai, R. E. Paull and M.-L. Wang. Identification of gene(s) differentially expressed between 'Kona-Typica' and 'Tall Mokka'. Abstract submitted to Solanaceae Genome Workshops, September 10-13, 2007, Jeju Island, South Korea.
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Progress 10/01/05 to 09/30/06
Outputs
MA2 'Mokka-Typica Hybrid' cultivar, present in our coffee breeding germplasm collection appears to be very similar to 'Kona-Typica' (KO32) based on AFLP marker analysis but retains the small organ characteristics. Our goal is to identify genes related to organ size in coffee (C. arabica L.) by comparing gene expression patterns in these two varieties that differ significantly in organ size. Our preliminary observations revealed that the reason for the difference in organ size lies in cell size rather than cell number. Endoreduplication, multiplication of chromosomes without cell division, is one of the factors controlling cell size. We have ruled out the possible role of endoreduplication in this case through flouresence activated cell sorting (FACS) analysis of coffee nuclei (UH SOEST FACS Facility). Interspecies microarray hybridization is proving to be a useful tool to study species for which microarray chips are not available. Hybridization of a coffee probe to
>51% of the genes on a potato chip during initial experiments supports the use of this system for coffee gene expression analysis. Potato and coffee have also been shown to share high homology between many genes. We have completed hybridization for 4 out of the 8 replicates for the microarrays. Analyses will be performed after all the hybridizations are completed. Primers will be synthesized for the differentially expressed genes. The expression patterns will be confirmed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR).
Impacts
Yield and quality are the major concern in today's agriculture. One of the methods for getting higher yield is by increasing the size of the harvested organ(s). Coffee, the 7th major crop in Hawaii (2005) is famous internationally for its quality. In order to maintain this worldwide reputation for high quality coffee, the development of Hawaiian coffee cultivars with new and better flavor profiles while simultaneously producing high yields is required. One of the genetic resources for high quality is the variety 'Mokka', which produces high quality beans with superior flavor but low yield due to smallest beans among all Coffea arabica varieties. We proposed to compare gene expression profiles in 'Mokka Hybrid' MA2 and 'Typica' KO32 in order to identify genes related to control of organ size. The gene(s) found to be responsible for organ size can be utilized in the coffee breeding program and contribute to improving yields of high-quality coffee. This project is a
complement of our existing breeding program established in 1997 at Hawaii Agriculture Research Center (HARC) with the cooperation of University of Hawaii (UH) and support from the Hawaii Coffee Growers' Association and the State of Hawaii. Furthermore, the new knowledge on genetic control of organ size may be applied to other crops to improve productivity. We will also determine if molecular tools developed for model plants, potato, can be used to study relatively less characterized plant systems, such as coffee
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
We are constructing coffee (Coffea arabica L.) cDNA libraries from varieties that were planted at the HARC Kunia experimental station in 1988. High quality mRNA was isolated from Typica KO34 and Mokka MA2 coffee cherries and shoot apices. We are in the process of cloning the shoot apices cDNAs into the plasmid pOTB7. We have established a centralized database and biological depository for the cDNA libraries. We have developed a semi-automated system to analyze the cDNA clones. In this process, colonies containing cDNA sequences are selected on LB chloramphenicol 34 ug/ml. Recombinant XL-1 Blue MRF' cells are picked by hand and transferred to a 96-well flat bottom block containing terrific broth (TB) chloramphenicol 34 ug/ml. Plasmid DNAs are isolated from overnight cultures using a Qiagen BioRobot. Arrayed E. coli colonies from the libraries have been given unique identifiers (ID) that will be used to track, catalogue, and retrieve samples and information. The
glycerol stocks (E. coli) are being stored in barcoded microtiter plates in a 96-well format. We are currently analyzing the putative coffee clones by dideoxy sequencing to determine the quality of the cDNA library. Mr. Ratnesh Singh joined the project in August 2005 as a graduate assistant. We have isolated total RNA from shoot tips and mature leaves of Mokka Hybrid MA2 and Typica KO32 in duplicates. We are preparing to test the efficacy of using potato cDNA microarrays to identify differentially expressed genes in coffee. We are making N. benthamiana custom microarrays (Combimatrix). We have selected over 6100 unigene sequence and are using bioinformatics programs to design unique 35-mer sequences. We are also negotiating with IRD in France for the use of their coffee expressed sequence tags (ESTs) to produce custom coffee microarrays. We will test the efficacy of using coffee microarrays to identify and characterize sequences that show differential expression between two coffee
varieties (MA2 and KO32).
Impacts
Coffee is a major agricultural commodity in Hawaii. Hawaiian coffee farm revenue has increased over the years due to nationally and internationally recognized premium quality. In order to maintain this worldwide reputation for high quality coffee, the development of Hawaiian coffee cultivars with new and better flavor profiles while simultaneously producing high yields is required. This project is a complement of our existing breeding program established in 1997 at Hawaii Agriculture Research Center (HARC) with the cooperation of University of Hawaii (UH) and support from the Hawaii Coffee Growers' Association and the State of Hawaii. Our breeding program emphasizes the development of high yielding, excellent bean and cupping quality cultivars which are adapted to specific growing conditions in Hawaii. One of the genetic resources for high quality is the variety Mokka, which produces high quality beans with superior flavor but low yield due to small bean size. It
produces the smallest beans among all Coffea arabica L. varieties. We proposed to compare gene expression profiles in Mokka Hybrid MA2 and Typica KO32 in order to identify genes related to control of organ size. The results of this research will provide new tools for our coffee breeding program to maintain coffee yields while breeding for new improved flavor characteristics. Furthermore, the new knowledge on genetic control of organ size may be applied to other crops to improve productivity.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
We are currently constructing several coffee cDNA libraries from clonally propagated Mokka Hybrid MA2 and Kona-Typica KO32 varieties that were planted at the HARC Kunia experimental station in 1988. Cherries from Coffea arabica L. plants were harvested at two different stages of development (early and late). Each group of tissue was flash frozen in liquid nitrogen and ground to a fine powder with a pre-chilled coffee grinder. Total RNA was isolated from each group separately using the hot borate method (Larry Smart and Thea Wilkins, 1995). Messenger RNA was purified from total RNA using an Poly(A)Pure kit (Ambion, Austin TX), following the manufacturer's instructions. A reverse transcription reaction was used to synthesize cDNA from the mRNA template, using a Stratagene (La Jolla, CA) kit. The cDNA was then subcloned into the plasmid pOTB7 at the EcoRI/XhoI sites.
Impacts
We are developing rigorous methods for the isolation and characterization of genes involved in the molecular genetics of coffee. The methods utilize robotics, barcodes, databases, and microarrays. Information involved in genetic control of organ size may be applied to other crops to improve productivity.
Publications
- No publications reported this period
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