1a. Objectives (from AD-416):
Objective 1. Assemble and validate in silico discovered SNPs of peach. Objective 2. SNP discovery based on EST resources developed from a set of diverse peach, almond and wild species Objective 3. Molecular characterization of peach and almond wild relatives, and production and testing of interspecific hybrids to identify novel sources of resistance to soil borne diseases.
1b. Approach (from AD-416):
Objective 1. Assemble and validate in silico discovered peach SNPs. Both GDR and ESTree databases contain high quality, annotated peach, almond and peach x almond EST sequences assembled into contigs using CAP3 program with associated sequence and assembly quality (phred and phrap) information. Putative SNPs have been identified using autoSNP program and mapped on to these contigs with associated quality and reliability information. In silico discovered SNPs will be validated by either of the following methods: (1) resequencing of a diverse panel of peach and almond genotypes using primers designed flanking regions of the SNPs and sequences aligned with those predicted and position of SNP checked and recorded; or (2) High-throughput SNaPshot multiplex system (Applied Biosystems) followed by capillary electrophoresis for SNP visualization. Objective 2. SNP discovery based on EST resources developed from a set of diverse peach, almond and wild species. Normalized root specific cDNA libraries for six diverse genotypes including peach, almond, and peach x almond hybrid will be constructed using Creator SMART™ cDNA Library Construction Kit (Takara Clontech, Palo Alto, CA) by following the manufacturer’s protocol. A pooled library will be sequenced using Illumina GAII platform to generate ESTs and for SNP discovery. The sequence data will be processed to assess quality and reads are aligned and analyzed using the short-read alignment tool ELAND and GenomeStudio data analysis softwares to detect and confirm SNPs. Confirmed SNPs will be assessed for functionality, designability and high quality SNPs will be selected for Oligonucleotide Pool Assay (OPA) with the Illumina Assay Design Tool. The OPAs also known as GoldenGate Assay will be used to perform SNP genotyping. Objective 3. Molecular characterization of peach and almond wild relatives and production and testing of interspecific hybrids (see Table 1) to identify novel sources of resistance to soil borne diseases. A set of selected germplasm of peach, almond and some of the wild relatives involved in the production of interspecific hybrids (hereafter called association mapping population) will be genotyped using the Illumina GoldenGate Assay and the same set of germplasm accessions and hybrids will be clonally propagated and subjected to extensive disease-pest screening in conjunction with the SCRI project funded by the CDFA block grant. The genotypic and phenotypic data will be subjected to association genetic (linkage disequilibrium) analysis to indentify markers linked to disease-pest resistance.
3. Progress Report:
A total of 67,194 SNPs from publicly available sources have been assembled. Of the total, 17,291 are from peach and almond from the ESTree database, 40,794 are from peach from GDR, 109 are from almond and a 9,000 peach SNP genotyping chip from NCBI. The data will be evaluated for duplicate SNPs, which will reduce the total number of putative SNPs to between 40,794 and 67,194. Of the 17,291 ESTree and 8709 NCBI SNP-containing peach and almond sequences, the Mosaik sequence assembler (version 1.1.0014) has mapped 46.8 and 68.6 percent of the sequences uniquely and 12.0 and 27.4 percent of sequences non-uniquely to the peach reference genome sequence. This suggest that many SNP containing sequences in the database may be originating from the duplicate regions of the genome. However, the uniquely mapping SNP-containing sequences are useful for genotyping peach-almond hybrids. We are in the process of mapping peach and almond EST sequences to identify new SNPs. The progress applies to Objective 3 of the parent project, "Strategically characterize (“genotype”) and evaluate (“phenotype”) priority vine, tree fruit, and nut crop genetic resources adapted to Mediterranean-like climates for molecular markers and key horticultural traits such as adaptation and product quality".