Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762
Performing Department
Food & Animal Sciences
Non Technical Summary
Advancements in genomic tools and genetic engineering are providing unprecedented opportunities to equip food crops with endogenous capacities to enable improved yield and enhanced food quality. Yam (Dioscorea spp) is an ideal candidate to benefit from the progress in genetic and genomics to harness its full potential as a major staple food crop worldwide. From the 600 known yam species, only 6 are edible among which D. rotundata, D. cayenensis and D. alata are the most cultivated in the world. Yam production has increased steadily in the last decade, from 18 million metric tones in 1990 to recent estimates of over 39 million. This increase has been achieved mainly through the use of traditional landraces and the rapid increase in acreage of yam fields into marginal lands and in non-traditional yam growing areas. With a Continued population growth worldwide, particularly an annual growth rate of 2.8 of the African population, the utilization of yams is rapidly increasing, and per capita consumption of yam is expected to grow from the current 35.5 kg/yr to 38.2 kg/yr within the next 10 years. In the U.S., Hawaii is one of the most yam producing areas, and the US importation of yam is increasing due to the increasing number of immigrants from the tropical and sub-tropical regions. Yam breeding is tedious and slow, hence cultivated yam is suffering from severe losses to abiotic and biotic stresses due the limited possibilities for genetic improvement. Yam yield and agronomical performance are hindered by intrinsic biological constraints such as long growth cycle, dioecy, poor to non-flowering, polyploidy, vegetative propagation, prolonged dormancy, and a heterozygous genetic background. Despite its important role in feeding the world population, yam is still an "orphan" from the genomic revolution. Intellectual Merit: Expressed Sequence tags (ESTs) provide valuable information on a significant fraction of the transcripts encoded in a genome. The isolation and sequencing of yam ESTs will enable identification of genes involved in specific metabolic pathways, unraveling metabolic networks in simply-inherited and polygenetically-controlled traits in yams. A better understanding of the molecular mechanisms underlying these processes could lead to novel genetic strategies to equip yam with endogenous capabilities to tolerate stresses and to enhance agronomic performances. Broader Impacts This project will provide an EST data set for yam that will be available in the public domain in the GenBank, to augment the DNA sequence data base, very useful in gene discovery. 2) ESTs will allow faster identification of genes involved in yam metabolic pathways, and thus facilitate improvement of the crop using genetic modification, and 3) a postdoctoral researcher and a graduate student will be trained and mentored to successfully embrace their academic and/or research career.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Objectives of the project: 1. To construct a cDNA library from different yam tissues and organs at various growth stages, 2. To screen the library, to sequence and to analyze yam expressed sequence tags and/or full-length cDNA clones. 3. All EST sequences will be submitted to the GenBank. 4. To mentor and train a postdoctoral researcher and a MS student to prepare them to successfully embrace their scientific career in Academia or in Industry. Expected outcomes 1) This project will develop yam EST data base to unravel gene expression profiles and metabolic parthways in yams, 2) will facilitate genetic mapping in yam for easy identification and isolation of genes governing simply-inherited and polygenically-controlled traits in yam, 3) establish interdisciplinary and collaborative work between the departments of Food Science and the Natural Resources and Env. Science, and 4) train and prepare minority graduate students for successful career in science and/or academia
Project Methods
Objective 1: Task 1: Plant materials and treatments Yam tubers from the genotypes D. alata, D. cayenensis and D. rotundata will be used. Minisetts will be prepared and put in soil to initiate sprouting and plant growth in an Enconair A60 growth chamber. Yam plants will be prepared under various developmental and environmental conditions. Three week-old plantlets will be exposed to a variety of stresses (drought, salt,cold). Plants will be harvested at 0, 1, 5, 10 and 24 h after the initiation of the stresses. Each plant sample will be stored at minus 80 degree Celcius until use. Task 2: cDNA library construction procedures will utilize the protocole outlined by Gu et al. (2002). Ten μg of RNA from each sample will be loaded onto 1.2 percent formaldehyde-agarose gel and electrophoresed to determine the quality of total RNA. cDNA libraries will be constructed using the SMARTTM technology. Libraries will be constructed by ligation of double stranded, normalized cDNA into pDNR-LIB and transformed into GC5 High Eff Competent Cells. Task 3: Construction of a full-length-enriched cDNA library Library construction will be accomplished by the biotinylated CAP trapper method and trehalose thermoactivated reverse transcriptase technique. The resultant double strand cDNAs will be ligated into a lFLC-III vector.23 Objective 2: Task 1: PCR amplification and End sequencing of cloned cDNA. The DNA of each clone will be amplified from 384 bacterial cultures of a glycerol stock plate by the RCA method using a TempliPhi HT DNA amplification kit. End sequencing of clones will be carried out using the Forbes DNA sequencer. The M13-21 primer (5'-TGTAAAACGACGGCCAGT-3') and the 1233 primer (5'-AGCGGATAACAATTTCACACAGGA-3') will be used for forward and reverse sequencing, respectively. Task 2: Sequence data trimming and assembly After assessing the quality of each library, gene discovery rate and the level of redundancy, about 100,000 ESTs for each of the library will be sequenced using the ABI3100 sequencer. The entire collection of reads generated by this project will be clustered both as a discrete dataset and together as one dataset. About 100,000 ESTs generated will be assembled into ~30,000 tentative contigs (TCs) and singletons, a subset of which would be represented in the existing Gene Indeces for other species (www.tigr.org). ESTs will be assembled by the 454 Assembler and CAP3 program with 40 bp overlap and 90% sequence identity. Task 3 Full-length sequencing for yam cDNAs One representative clone from each contig will be selected, and the DNA of each clone will be amplified. Full-length sequencing using the Forbes DNA sequencer will be performed. The Phred/Phrap/Consed system will be used to assemble sequences. All full-length sequences will be submitted to the GenBank. Task 4: Full-length cDNA library quality The quality of yam full-length cDNA library will be evaluated, and clones fulfilling the established conditions will be considered full-length yam cDNAs containing 5'-UTR, CDS and 3'-UTR