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:
This project relates to inhouse parent project objective 4, "In collaboration with other NPGS genebanks and research projects, develop novel genetic marker systems for characterizing genetic resources of grape, tree fruit, tree nut, and other specialty crops adapted to Mediterranean climates. Apply those markers to more efficiently and effectively manage the site's germplasm collections and to facilitate their use in germplasm acquisition, breeding and research projects". Development of improved rootstocks with tolerance/resistance to soil borne and replant diseases is the key for sustainable production of almond and therefore, it is one of the top priorities for the California Almond Industry. With restriction on the use of fumigants and with aim to attain sustainable production, the industry reliance on rootstocks with field resistance to soil borne pests and diseases is increasing. Though widely used rootstocks (e.g. ‘Nemaguard’, other peach and peach x almond hybrids) resist the attack of root knot nematodes, they are susceptible to other soil borne pests and diseases, such as lesion and ring nematodes, bacterial canker, crown gall, Phytophthora and Armillaria. In this project, an attempt has been made to produce novel interspecific hybrids involving peach, wild almond species, and diploid plums that are potential donors of resistance to soil borne diseases and in some cases drought tolerance. While hybrids are being screened for disease resistance, genomic tools and methods are being developed to assist in efficient selection of resistance hybrids. Genome resequencing of a dozen wild Prunus spp. that have been used in hybrid generation (P. dulcis, P. argentea, P. kanuensis, P. davidiana, P. bucharica, P. kuramica, P. arabica, P. persica, and P. fenzliana) are being currently performed at the Beijing Genome Institute using Illumina HiSeq 2000 platform. The sequence data equivalent of 30x the average size of Prunus genome is expected soon and will be aligned to discover SNPs. A second approach to discover SNPs and genotype concurrently has also been undertaken using a new method called genotyping-by-sequencing (GBS). A set of all the available rootstocks that are currently being used or under test along with our novel hybrids have been included in the GBS approach.