Project Number: 3602-22000-016-04
Project Type: Specific Cooperative Agreement

Start Date: Aug 30, 2007
End Date: Jun 30, 2011

Objective:
The most economical and environmentally sound method of control for Hessian fly is genetic resistance in wheat. However, as with other gene-for-gene resistances new genotypes of the pest appear in populations that can survive on formerly resistant cultivars. The current SCA will create a high density physical and genomic map of the X2 chromosome of the Hessian fly. The objectives of this SCA will investigate and reveal physical and genomic locations of three virulence genes in the Hessian fly genome and also give us insights into the genes themselves. Deliverables include location and characterization of three of the genes responsible for virulence against the most widely deployed wheat resistance genes in the United States. It will also allow us to study the molecular characters of said genes and ideally an understanding in the evolution of virulence in the fly as a whole.

Approach:
To develop a high-density genetic & physical map of the Hessian fly chromosome X2, DNA polymorphisms will be used. These will include simple sequence repeats (SSRs), sequenced tagged sites (STSs), and DNA sequence derived from Hessian fly genomic DNA cloned into bacterial artificial chromosomes (BACs), and secreted salivary gland proteins (SSGPs). They also may include AFLPs, and randomly amplified fingerprints (RAFs). To place DNA polymorphisms on the same genetic map, related Hessian backcross (BC) mapping populations already available from previous work will be used. Sister F1 females will then be backcrossed separately to a single male or his brother to generate genetically related BC populations. Each BC population will consist of 50 to 120 individuals. The DNA of each F1 female and each BC offspring will be isolated and maintained separately. The genetic positions of DNA polymorphisms segregating in this population will be determined by scoring each BC individual for the alleles associated with each marker. Genetic maps will be constructed from these data using statistical computer software (MapMaker and JoinMap). To determine the chromosomal positions of DNA markers on the genetic map, we will take full advantage of the bacterial artificial chromosome (BAC) library in the posession of Dr. Jeff Stuart. The BAC library consists of 6144 genomic clones arrayed on 16 384-well plates consisting of 24 columns and 16 rows. It represents approximately 4.5 genome equivalents. The BAC clones in this library will be pooled in 3-dimensions: by plate, by columns within plates and by rows within plates. Polymerase chain reaction (PCR) experiments will be performed using primers specifically designed to amplify the DNA corresponding to each DNA marker separately. DNA isolated form each pool of BAC clones will be used as template in these experiments so that positive reactions will determine the correspondence between each marker and the BAC clones that contain the marker sequence. The location of the marker on the polytene chromosome of the Hessian fly will then be determined by positioning the corresponding BAC clones directly on the chromosomes by fluorescence in situ hybridization (FISH). Hessian fly chromosomes will be prepared for these experiments using standardized methods. BAC clone DNA (~1 µg) will be labeled by nick translation using either biotin- or digoxigenin-conjugated dUTP (Roche). This labeled DNA will be used as a probe in FISH experiments. Hybridizations will be performed with 40-100ng of denatured probe DNA in 10 µl of hybridization solution (10% dextran solution, 2x SSC, 40% formaldehyde, and 20 ug of Herring sperm DNA) at 37°C for 12 to 15 hours. Detection will be performed using Alexa Fluor (Molecular Probes) conjugated anti-biotin and rhodamin conjugated anti-digoxigenin. Digital images will be taken under UV optics using an ORCA-ER (Hammamatsu) digital camera mounted on an Olympus BX51 microscope, and Meta Morph (Universal Imagine Corp.) imaging software.