454 SEQUENCING FOR WATERMELON CDNAS

• Sponsoring Institution: Agricultural Research Service/USDA
• Project Status: COMPLETE
• Funding Source: USDA COOPERATIVE AGREEMENT
• Reporting Frequency: Annual
• Accession No.: 0415165
• Project Start Date: Jan 26, 2009
• Project End Date: Jun 1, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF OKLAHOMA
(N/A)
NORMAN,OK 73019

Performing Department
(N/A)

Non Technical Summary
(N/A)

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1420	1080	50%
202	1440	1080	50%

Knowledge Area
202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1440 - Cole crops; 1420 - Melons;

Field Of Science
1080 - Genetics;

Keywords

watermelon

Goals / Objectives
The main objective of this work is to perform high throughput sequencing of genes that are expressed during the development and ripening of the watermelon fruit. Then, use the data to assemble the gene sequences and identify the genes expressed in watermelon fruit, and develop DNA markers that will be useful for constructing a detailed genetic linkage map for watermelon.

Project Methods
1) cDNA (gene sequences) libraries were prepared using mRNA isolated from watermelon fruits at sequential development and ripening stages (0, 10, 20, 30, and 40 days post pollination). 2) The cDNA libraries will be prepared for massive sequencing using high throughput 454 sequencing procedure. The cDNA libraries will be prepared for 454 sequencing by the University of Oklahoma Team at the Biochemistry and Chemistry Department (Bruce Roe Lab.). This stage will involve testing quality and quantity of the cDNA libraries. Then, equal amounts of cDNA from each developmental stage of the watermelon fruit will be used to have total of 10 ug cDNA for each of the watermelon genotypes used in this study (¿Charleston Gray¿ versus ¿Black Diamond¿). 3) If the cDNAs are of high quality, they will be fragmented by sonication to break the DNA down to the size compatible for 454 sequencing (~300-500bp). 4) Adaptors used for 454 sequencing will be attached to cDNA clones in a ligation reaction, and cDNA concentrations will be optimized for 454 sequencing. 5) The high throughput 454 sequencing procedure will be performed with watermelon cDNAs using the FLX Titanium Platform to produce at least 500, 000 reads per plate run with an average minimum length of 300 bases per read. 6) Data analysis and assembly of the raw cDNA sequences will be conducted in FASTA format, along with quality scores, and the sequences will be assembled and prepared for BLAST analysis or for any other bioinformatics analysis.

Progress 01/26/09 to 06/01/10

Outputs
Progress Report Objectives (from AD-416) The main objective of this work is to perform high throughput sequencing of genes that are expressed during the development and ripening of the watermelon fruit. Then, use the data to assemble the gene sequences and identify the genes expressed in watermelon fruit, and develop DNA markers that will be useful for constructing a detailed genetic linkage map for watermelon. Approach (from AD-416) 1) cDNA (gene sequences) libraries were prepared using mRNA isolated from watermelon fruits at sequential development and ripening stages (0, 10, 20, 30, and 40 days post pollination). 2) The cDNA libraries will be prepared for massive sequencing using high throughput 454 sequencing procedure. The cDNA libraries will be prepared for 454 sequencing by the University of Oklahoma Team at the Biochemistry and Chemistry Department (Bruce Roe Lab.). This stage will involve testing quality and quantity of the cDNA libraries. Then, equal amounts of cDNA from each developmental stage of the watermelon fruit will be used to have total of 10 ug cDNA for each of the watermelon genotypes used in this study (�Charleston Gray� versus �Black Diamond�). 3) If the cDNAs are of high quality, they will be fragmented by sonication to break the DNA down to the size compatible for 454 sequencing (~300-500bp). 4) Adaptors used for 454 sequencing will be attached to cDNA clones in a ligation reaction, and cDNA concentrations will be optimized for 454 sequencing. 5) The high throughput 454 sequencing procedure will be performed with watermelon cDNAs using the FLX Titanium Platform to produce at least 500, 000 reads per plate run with an average minimum length of 300 bases per read. 6) Data analysis and assembly of the raw cDNA sequences will be conducted in FASTA format, along with quality scores, and the sequences will be assembled and prepared for BLAST analysis or for any other bioinformatics analysis. This project is related to Objective 2 of this in-house project: To utilize genomic tools to develop genetic linkage maps for watermelon and diagnostic DNA-based markers for host-plant resistance to viruses and key watermelon fruit traits. Genomic DNA of the watermelon cultivar Charleston Gray was sequenced using 454 technology. The sequence assembly yielded: 13,176 contigs covering 9470.9 kb and 22474 singlets covering 10,867,313 kb. The data are currently being used for assembly of the �Charleston Gray� genome that we have recently sequenced using Solexa Technology. The ARS scientists and cooperators have been communicating frequently via telephone calls and emails.

Impacts
(N/A)

Publications