Current Applications and Future Potential of High Resolution Melting at the National Clonal Germplasm Repository in Corvallis, Oregon Nahla Bassil 1, Michael Dossett 2, Vidyasagar Sathuvalli 2, Chad Finn 3 and Shawn Mehlenbacher 2 1 USDA-ARS, NCGR, Corvallis, OR 97333 2 OSU, Dept of Horticulture, Corvallis, OR, 97331 3 USDA-ARS, HCRL, Corvallis, OR 97330
NPGS National Holdings 535,853 accessions 10,428 species 1,641 genera Modern & obsolete cultivars Landraces Wild relatives of cultivated species Improved breeding lines Genetic stocks
NPGS Mission Acquisition Documentation Preservation Evaluation Enhancement Distribution Plant Genetic Resources for Improving the Quality and Production of Economic Crops Important to U.S. and World Agriculture
Corvallis Collection Summary Crop Clones Corylus (hazelnut) 842 Cydonia (quince) 171 Fragaria (strawberry) 1,825 Humulus (hops) 1,578 Pyrus (pear) 2,182 Rubus (blackberry, raspberry) 2,287 Vaccinium (blueberry, cranberry) 1,719 11,076 accessions from 54 genera
Germplasm Characterization Currently Microsatellite markers for identity validation, diversity assessment, linkage mapping Illumina sequencing of chloroplast genomes for phylogenetic analysis Challenges Lack of robust markers linked to traits of economic importance in most of our crops Limited resources
Characterization of Germplasm Using HRM: Two Case Studies Map-based cloning of eastern filbert blight resistance gene Detection of sequence polymorphism in black raspberry monomorphic microsatellite markers
Hazelnut Breeding Program: OSU Eastern Filbert Blight (EFB) resistance: Important objective of hazelnut breeding program at OSU (Shawn Mehlenbacher) Gasaway resistance: dominant allele at single locus Most advanced selections Gasaway resistance Jefferson recently released EFB resistance cultivar Gasaway Jefferson Barcelona Susceptible Jefferson Resistant
MAB for EFB Resistance UBC152-800 + _ + _ + + UBC268-580 + + + + New sources of resistance that map to LGs were identified
High Resolution Melting in Hazelnut Breeding HRM used to assist in cloning the Gasaway R gene HRM used to develop markers close to the R gene for MAB
HRM in Map-Based Cloning of EFB Resistance 94 BACs close the EFB resistance were identified by marker hybridization 188 BAC end sequences were obtained and 629 primers combinations were designed to map the BACs in the resistance region Initial screening for blind mapping by HRM was carried out using the resistant parent (RP), susceptible parent (SP), 2 resistant and 2 susceptible seedlings and then in 87 recombinants
HRM in Map-Based Cloning of EFB Resistance 18 HRM markers from BAC end sequences were polymorphic between parents and segregate accordingly 7 out of 18 HRM markers were successfully mapped in the resistance region TBE_II_14
Physical Map of EFB R Region HRM Markers
Conversion of RAPD Markers to HRM Markers W07_350 X01_800 RAPD marker bands were cloned and sequenced to convert them to SCARs All except for OPH04-850 were not polymorphic between the parents Two marker sequences, OPW07-350 and OPX01-800 close to the resistance were polymorphic by HRM These HRM based RAPD markers will be used as part of MAS in Hazelnut breeding program for Gasaway resistance
HRM-Based Markers HRM-based RAPD marker X01-850 HRM-based RAPD marker W07-350 Susceptible meltcurve Resistance melting curve Susceptible meltcurve
Detection of Polymorphism in Black Raspberry Monomorphic SSRs Large numbers of SSR primers from Rubus species were monomorphic in black raspberry (R. occidentalis) Used HRM to identify sequence variants from these monomorphic PCR products
Black Raspberry Population for Linkage Mapping ORUS 3021-2 x ORUS 4153-1 Segregates for aphid resistance and is expected to segregate for geographic adaptability, fruit size and other valuable traits Few informative SSRs have been found to date RhM011 Gen 003
Scanning Rubus SSRs by HRM RhM011 SSRs confirmed to have no size polymorphism by capillary electrophoresis Mapping population parents were analyzed in duplicate by HRM using the LightScanner to identify sequence polymorphisms Gen 003
Scanning Rubus SSRs by HRM Identification of sequence differences in two parental genotypes can be expanded to progeny genotypes using same technique Gen 003 Segregation in 24 progeny of the mapping population is 1:1 and can be used for linkage mapping with no genotype data needed
Sequencing of PCR fragments in parental genotypes can be useful Identify genotype of parents (haplotypes) Confirm segregation if 1:1 or reasons for segregation other than 1:1 Identify number and nature of SNPs present to redesign primers targeting specific mutations ORUS 3021-2 Gen 003 ORUS 4153-1
HRM will be used to map SSR markers that are not polymorphic in the hazelnut and black raspberry mapping populations Potential RGAs identified from the BACs will be mapped through HRM RAPD markers closely linked to self-incompatibility locus in hazelnut will be converted to HRM markers A similar HRM based mapping approach will be carried out for map-based cloning of the self-incompatibility locus in hazelnut HRM markers will be used in MAB and germplasm characterization for these and other important traits for the fruit crops in our collections
NCGR Lab: April Nyberg for technical assistance Idaho Technologies: Jason McKinney and Cameron Gundry for the LightScanner and for technical assistance and troubleshooting USDA-ARS Northwest Center for Small Fruits Research USDA-ARS CRIS Project 5358-21000-038-00D Oregon Raspberry and Blackberry Commission for financial support