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
Objective 1. A total of 67,194 SNPs from different publicly available sources have been assembled. Of which, 17,291 are from peach and almond from the ESTree database (www.itb.cnr.it/estree/), 40,794 are from peach from GDR (www.rosaceae.org), 109 are from almond and a 9,000 peach SNP genotyping chip from NCBI (www.ncbi.nlm.nih.gov). Validation of these in silico SNPs will be performed by verifying the presence of primer sequences in the genomic DNA sequences of species involved in hybrid production. Currently the genomic libraries of selected diverse Prunus spp. used in hybrid production are under construction and subsequently sequenced using next generation sequencing technologies. Contigs of the genomic sequence from a set of diverse peach, almond, and wild Prunus spp. will be searched for the presence of in silico SNP primer sequences using readily available bioinformatic tools. Objective 2. The RNA profile differences between rootstock genotypes that are contrasting in disease reaction (susceptible vs. resistance) to various soil borne pests and pathogens will provide useful information on the genes differentially expressed in the roots between susceptible and resistant rootstocks. This facilitates identifying SNPs that are located well within the fictional genes called candidate genes thus providing potentially useful SNPs that are linked to genes that confer resistance. These SNPs along with others identified using genomic sequences will allow us for developing fine scale mapping of resistant genes. Sequencing of both genomic and cDNA libraries should be completed by the end of summer 2011. Objective 3. During 2011 Spring season, several thousands of pollinations were made to produce interspecific hybrids using a number wild Prunus spp. that are identified as potential donors of resistance to soil borne diseases. About 150 pollinations yielded hybrid seeds in nine cross combinations. During 2010, 116 hybrid seeds were produced from twenty crosses. The seeds from 2010 crosses were put into culture at the Davis repository after stratification. The immature fruits from 2011 crosses were harvested for embryo rescue to grow them into seedlings. These seedlings will be further propagated to produce 50 clonal copies of each of these hybrids for greenhouse evaluation of resistance to the soil borne diseases. Semi-hardwood cuttings were collected from the parents and F1 hybrid of P. persica ‘Andross’ by P. argentea (DPRU 194) during summer 2011 for clonal propagation by rooting. This population will be evaluated for disease resistance and genotyped using SNPs developed in the project for genetic mapping.