Source: WASHINGTON STATE UNIVERSITY submitted to
AN APPLE GENOME SEQUENCING INITIATIVE
 
PROJECT DIRECTOR: Dhingra, A. Kalyanaraman, A.
 
PERFORMING ORGANIZATION
HORTICULTURE & LANDSCAPE ARCHITECTURE
WASHINGTON STATE UNIVERSITY
PULLMAN,WA 99164
 
NON TECHNICAL SUMMARY: The agricultural efficiency and sustainability of an economically important crop like apple is expected to be enhanced by a comprehensive understanding of its genome structure, function and organization. The apple industry stakeholders, cognizant of this vital genomic resource gap, have enabled establishment of a public initiative to sequence the 750 Mb apple genome in partnership with WSU. Activities pursuant to the project aim to achieve the overall goal of developing a fundamental genomic resource for the Rosaceae research community. We plan to accomplish this goal by creating an apple genome scaffold with paired-end BAC sequences. The uniqueness of this project lies in the utilization of a genetically distinct double haploid apple that enables cost-efficient access to sequence information, streamlining downstream assembly and annotation. Knowledge gained from this project is expected to serve as a foundation for developing functional markers for genomics-assisted apple improvement while also providing an insight into the genomic organization of the Rosaceae. The framework developed here is expected to serve as a model for future genome projects in this family. The ability of the apple breeding programs to develop desirable new cultivars will be enhanced by a comprehensive understanding of the apple genome thereby ensuring the international competitiveness of American agriculture. The activities proposed in this project align with the priority mission area of US agriculture in agricultural genomics and human nutrition.
 
OBJECTIVES: There are several genomic resources available for apple in including over 260,000 ESTs, physical maps, BAC libraries and transformation systems. Despite the availability of these important resources, what is not understood is, how an apple tree responds to its environment both, in a physiological and genetic context. This information is extremely useful for efficient development of new and improved varieties necessary for the sustenance of the apple industry. Thus, there is an urgent need to obtain a reference genome of apple. Continued existence of this need is an important problem, because it prevents us from taking the next vertical step, of having a more comprehensive understanding of its genome's function. Our long-term goal is to unravel the structure, function and organization of the apple genome and utilize this information in efficient crop improvement strategies. The objective of this project is to generate a foundational framework for obtaining a reference genome sequence for apple. The rationale behind the proposed research is that the sequence information and the foundational infrastructure established within this project will facilitate the efficient development of functionally-validated molecular markers for genomics-assisted breeding of superior apple varieties, thereby enhancing the international competitiveness of American agriculture. The main objective of the proposal is to develop an apple genome scaffold. A genome scaffold is vital for de-novo assembly of shotgun reads obtained from 454 technology and other high-throughput sequencing strategies like Solexa and ABI SOLiD. We propose to develop a 6X BAC library from a genetically simpler double haploid apple. End sequencing of 36,864 BACs will facilitate development of a genome scaffold for assembling the shotgun sequences, complementing the 4X genome coverage that is being generated under the aegis of the public initiative for apple genome sequencing that we recently initiated with support from Washington State University (WSU) and the Washington Tree Fruit Research Commission. The genome scaffold developed from a double haploid apple is expected to serve as a foundational framework for overlaying the existing genomic information in the public domain like ESTs and the full length cDNAs being generated in our program. This scaffold is expected to provide a foundation for rapid discovery of alleles and multiple types of polymorphisms. The BAC library will be a tangible deliverable available to the public directly through the contracted company Amplicon Express at a minimal cost covering shipping charges. Obtaining clones from Amplicon Express is expected to reduce human error in individual clone identification as the clones are inventoried and recalled robotically.
 
APPROACH: Activity 1. Development of a BAC library from double haploid apple and sequencing of 36,864 BAC ends: We will provide frozen leaf material from double haploid apple to Amplicon Express and they will isolate high molecular weight DNA (HMW) to prepare large insert BAC library using proprietary procedures. The genomic inserts will be cloned in the pECBACI vector that we are currently modifying to include attB (attachment sites) flanking the multiple cloning site. This is being done to make the BAC clones compatible with Gateway technology (Invitrogen). This will enable mobilization of the large inserts into binary BAC vectors for functional testing of QTLs in the non-juvenile apple. The BAC library representing 36,864 clones will be directly shipped to Agencourt by Amplicon Express at no additional cost for sequencing the ends of all the BAC clones. Sequence information will be provided to the PI via a secure FTP site. The charges are $1.65 for bidirectional reads. Agencourt ensures at least 90% of the reads correspond to cored clone-pairs. Activity 2. Develop an assembly pipeline and create a publicly available framework: The preprocessed 454 sequences already being generated will be passed through the PaCE assembly framework, which will generate a set of assembled contigs. The assembled contigs will have to be ordered and oriented. The mate-pair information generated from BAC end sequencing (Activity 1) will be used for this purpose. Scaffolding will be achieved using the program Bambus (Pop et al., 2004). To provide additional scaffolding, the synteny expected between the apple genomic contigs and other tree species will be exploited - BAC and other fully assembled genomic contigs of Populus if available from previous sequencing projects will be used as reference to guide the order and orientation. Annotation: Contigs containing genic regions will be identified by running BLAST (Altschul et al., 1997) of contigs on known protein and EST databases. In addition, each contig will be examined for gene content using the Fgenesh program (Salamov et al., 2000), Genemark.hmm (http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi), Genscan+ (http://genes.mit.edu/GENSCAN.html), and TwinScan. Exon/introns splice sites will be identified using GeneSplicer (http://www.tigr.org/tdb/GeneSplicer/index.shtml) and tRNAscan-SE will be used to predict tRNA. Visualization and data management: Visualization for the results of the assembly and annotation project will be enabled through the GBrowse (Stein et al., 2002) and Apollo annotation viewers available through the Generic Model Organism Database (GMOD) project (http://www.gmod.org). All the results will be incorporated into a database and made publicly available on a web server with support for BLAST and visualization services. Additionally the data will be hosted on Genome Database for Rosaceae (GDR, www.bioinfo.wsu.edu/gdr).
 
CRIS NUMBER: 0215178 SUBFILE: CRIS
PROJECT NUMBER: WNP07677 SPONSOR AGENCY: CSREES
PROJECT TYPE: NRI COMPETITIVE GRANT PROJECT STATUS: NEW MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Sep 1, 2008 TERMINATION DATE: Aug 31, 2010

GRANT PROGRAM: PLANT GENOME
GRANT PROGRAM AREA: Plant Systems

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
201111010802.2100%

CLASSIFICATION HEADINGS
KA201 - Plant Genome, Genetics, and Genetic Mechanisms
S1110 - Apple
F1080 - Genetics
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 75%
APPLIED 25%
DEVELOPMENTAL (N/A)%

KEYWORDS: rosaceae~apple~double haploid~genome scaffold~bacterial artificial chromosome~pyrosequencing~apple genome project~maloideae

PROGRESS: TO
(N/A)

IMPACT: TO (N/A)

PUBLICATION INFORMATION: TO


PROJECT CONTACT INFORMATION
NAME: Amit Dhingra
PHONE: 509-335-3625
FAX: 509-335-8690