Parental screening for QTL pyramiding on multiple-abiotic stress tolerance under irrigated rice condition Objectives Successful QTL pyramiding is a breeding technique to combine multiple tolerance genes from various donors into target rice cultivars without losing the recipients’ desirable traits. The end product of this QTL pyramiding project will be a small number of genotypes with multiple traits that would provide breeders with the opportunity to combine multiple stress tolerance in new rice varieties. Identify cross combination for a more effective QTL introgression by marker-assisted backcrossing (MABC) scheme. 2. Contribute to a wider understanding of “trait-packaging” that is easily transferred to wider genetic backgrounds. 3. Contribute to the development of varieties with multiple stress tolerance, marker validation, and new allele mining. Breeding Strategy Materials and methods QTL pyramiding by MABC scheme Suggested recipients for multiple tolerance Screening methods Anaerobic germination screening Donor Recipient Preference Productivity Ecosystem Market size Donor of multi-gene High-value Traits Genes/QTLs Markers Allele variation Variety selection Variety Target Subspecies NSIC Rc 238 Southeast Asia Indica IR64 Asia Makassane Africa MS 11 Temperate countries Japonica Swarna-Sub1 India Seeds of each line (donor/recipient) were directly sown on trays, with 1seed/hole, at 10 holes per entry Each tray was replicated three times, submerged at 10-cm water depth, and observed for 21 days. Khao Hlan On and IR42 served as tolerant and susceptible checks, respectively. Percentage survival and tolerant lines were identified and characterized. Marker-assisted backcrossing (MABC) Background/recombinant markers Whole genome sequence SNP/SSR whole genome scale Foreground markers QTL mapping Functional genomics High-throughput and low- cost Genotyping Phenotyping Informatics tools Pre-breeding lines Molecular breeding facility DNA extraction Marker sets Rapid generation advancement Effective crossing system Salinity tolerance screening Two-day old pregerminated seeds of each line (donor/recipient) were directly sown on seedling floats with holes. Each hole contains 2 seeds per entry. A total of 5 holes were allotted per entry int three replications. FL478 and IR29 served as tolerant and susceptible checks, respectively. The first three days was provided with SNAP culture solution (1% SNAP A, 1% SNAP B, 98% distilled water) and FeSO4 On the fourth day, the solution was salinized at EC12 ds/m -1, pH 5.0 Two weeks after the 1st salinization, solution was adjusted to EC18 ds/m -1, pH 5.0. Plants were scored using the Standard evaluation Score (SES) for EC 12. Customer’s choice Market formation Seed distribution Government support Business plan – promotion, location, price New variety Breeder’s selection Multi-environmental testing (MET) Yield test Quality test Donors for multiple tolerance trait Trait QTL Donor P-deficiency tolerance Pup1 Kasalath Salinity tolerance Saltol Pokkali Submergence tolerance Sub1 FR13A Heat tolerance qHTSF1.1 N22 Anaerobic germination AG1 KHO Drought tolerance qDTY2.2+qDTY4.1 Adday Sel High-throughput genotyping and phenotyping system Submergence screening Three-day old pregerminated seeds of each line were sown directly on seeding trays at a seeding rate of 12plants/row in three replications. IR49830 served as the tolerant check and IR42 was the susceptible check. At 14 DAS, total number of seedlings of each line was recorded prior to submergence. Trays were submerged at1m depth in the submergence tank for 14 days. Percent survival was recorded 14 days after desubmergence. Parental phenotyping Rapid cross and generation advancement High throughput genotyping Results and discussion I. Phenotyping II. Genotyping NSIC RC238 showed a full Saltol gene, however full expression of the gene was not shown in parental phenotyping. IR64, upon introgression of the QTLs of Sub1, Saltol, and AG1 displayed a full effect on its NILs. Makassane showed no genes for all the stresses currently tested. However, during phenotyping, it showed tolerance for anaerobic germination. Though it showed susceptibility on the first nine days, it had mostly recovered at the 12th day of the experiment. Makassane also exhibited better survival rate than susceptible checks in the submergence experiment and slight tolerance in salinity screening. MS11, a japonica representative, showed no gene for anaerobic screening, but it showed tolerance during phenotyping screening. Conversely, it has full and partial Sub1 and Saltol QTL, respectively, but it did not exhibit tolerance in phenotyping screening. Fig 1. Anaerobic germination (%) of each recipient variety for QTL pyramiding. Fig 2. Submergence survival (%) of each recipient variety for QTL pyramiding. Fig 3. Salinity screening survival (%) of each recipient variety for QTL pyramiding. Variety Target Tolerance QTL pyramiding NSIC Rc 238 Southeast Asia Susceptible Doubling germination frequency at 12 DAS IR64 Asia Full effect of AG QTL Makassane Africa S (6 DAS) -> T (12 DAS) 6-day earlier germination advantage MS 11 Temperate countries Tolerant Subject to allele mining for new gene Variety Target Tolerance QTL pyramiding NSIC Rc 238 Southeast Asia Susceptible Need to check QTL effect on BC1F1 IR64 Asia Full effect of Sub1 QTL Makassane Africa Sl Slightly better than IR42; need to check QTL effect on BC1F1 MS 11 Temperate countries Very susceptible; need to check BC1F1 for varietal improvement Variety Target Tolerance QTL pyramiding NSIC Rc 238 Southeast Asia Susceptible Susceptible but better than IR42 (susc check) IR64 Asia Full effect of Saltol QTL Makassane Africa 33% lower than the Tol check (IR49830) Intermediate tolerance for salinity; needs an increase of 30-50% tolerance MS 11 Temperate countries Need to check BC1F1 for tolerance improvement Conclusions/Recommendations Ongoing activities Multi-tolerant QTL pyramiding lines with IR64 will be used as sources to transfer as many QTLs to other recipient varieties. A marker chipset for multi-tolerance pyramiding will be developed. A rapid and efficient MABC system supported by high-throughput genotyping is being established. In the MABC program for multiple traits tolerance, a parental polymorphism survey using QTL-specific markers with proper phenotyping is important. Some varieties already show tolerance and have conferred tolerance alleles for specific stresses.This information is critical in designing the most desirable cross-combinations for multi-tolerance breeding. Gene-specific markers used in parental screening may not be enough to determine instant QTL presence/introgression. Flanking markers for QTL regions should also be applied for the highly efficient MABC programs. Chenie S. Zamora, Katreena Titong, Ian Paul Navea,Sang-ho Chu, Bertrand Collard, Endang Septiningsih, Michael Thomson, Glenn Gregorio, Eero Nissila, and Joong Hyoun Chin Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, Los Baños, Laguna, Philippines