1. 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 % 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