1. Anther culture for doubled haploid (DH) production
Stem rust resistance in barley: complexity of introgressing rpg4/Rpg5 into TTKSK-susceptible germplasm
Javier Hernandez1, Brian J. Steffenson2, Kelly Vining1, Tanya Filichkin1, Scott Fisk1, Laura Helgerson1, Brigid Meints1, David S. Marshall3 and Patrick M. Hayes1,
1Oregon State University, Corvallis, OR, 2University of Minnesota, St. Paul, MN, 3USDA-ARS, Raleigh, NC
Introduction
Stem rust (Puccinia graminis f. sp. tritici, Pgt) race TTKSK is a major threat to barley because most cultivars are susceptible. Rpg1, a widely-used gene which confers resistance to most North America races, is not effective against TTKSK. The rgp4-mediated resistance locus (RMRL), which confers resistance to TTKSK, was discovered in Q At this complex locus, which maps to 5H, there are at least four genes related with resistance: of these, polymorphism at Rpg5 is most commonly used to identify resistant and susceptible germplasm. Introgression of rpg4/Rpg5 into diverse genetic backgrounds is a first line of defense against the eventual arrival of TTKSK to the Americas. Marker assisted selection (MAS) could accelerate this process.
Materials and Methods
Steffenson et al 2017.
TTKSK Resistant
(rgp4/Rpg5) Lines
N= 119
Cycle 1 Population
F1’s
TTKSK Susceptible
Lines
(Food, Malt, Feed)
Genotyping
Barley 9K iSelect SNP Chip
Genotyping Rpg1 and Rpg5
Re-sequencing Rpg5
Phenotyping
Stem rust race TTKSK rated as Infection Type (IT)
Plant Growth Biosafety Level – 3 Facility, UMN
Seedling stage
IT converted to Coefficient of Infection (CI)
Anther culture for doubled haploid (DH) production
Results
TABLE 1. Parental accessions reaction to seedling inoculation with Pgt race TTKSK
TABLE 3. Chi-square tests for allele-specific markers targeting Rpg5 and Rpg1 in the TTKSK Cycle I population
rpg4/Rpg5
Fig. 1. GWAS using CI means in Cycle I. The continuous line indicates the 5% FDR. The dotted line indicates the coincident threshold levels at FDR 10% using all SNPs and at 5% using a subset of SNPs.
Line
IT-M (GR) CI
Genotype MC
210; (R)
2.5
Rpg1, rpg4/Rpg5 MC
3- (MS)
3.3
SH98076
0; (R)
0.5
SB97197
23- (MR)
3.2
TR02272
1-20; (R)
1.8
Full Pint
2;3- (S)
3.4
------,
04_028_36
33+ (S)
4.1
rpg1, Rpg4/rpg5
Violetta
3 (S)
4.0
------, Rpg4/rpg5
Karma
23 (S)
3.7
Oscar
3- (S)
3.5
------, rpg4/Rpg5
Tibet 37
# DH lines
1:1
Type
Rpg5
rpg5
null χ2
Prob (> χ2)
Rpg1
rpg1
Resistant 21 9 2 10
Susceptible 43 40 13 51 24
Total 64 53
1.03
0.31 60 57
0.1
0.78
5H QTL
7H QTL
TABLE 4. AOV for main and interaction effects for race TTKSK
Source of variation Df
Sum of squares
F value
Rpg5 1
2.18
8.17**
QTL5
8.57
34.31***
QTL7
1.05
4.20*
Rpg5*QTL5
5.73
22.94***
Rpg5*QTL7
2.12
8.48**
Residuals
113
28.22
Resistant donors: all Rpg1 and Rpg5
Overall: 1(R) : 3(S) ratio: Two unlinked loci
All R lines Rpg5. Some S lines are Rpg5
Rpg1 not related to phenotype
Rpg5 + QTL alleles at 5H and 7H: Lowest CI. Fits (S) : 3(R) ratio
Significant interaction between Rpg5 and other two QTL alleles
TABLE 2. Classification of DHs as resistant or susceptible based on a threshold of CI = 2.8. Chi-square tests for CI.
# DH lines
1:1
1:3
Pedigree
Total R S χ2
Prob (>χ2)
All
110 22 88
38.76
0.00
1.35
0.24
MC x__ 13 4 9
1.92
0.16
0.23
0.63
SH98076 x__ 57 15 42
12.79
0.05
0.81
SB97197 x__ 40 3 37
28.90
6.53
0.01
Conclusions
In the Cycle I genetic backgrounds
The rpg4/Rpg5 complex is necessary, but not sufficient, to confer resistance to TTKSK
Rpg1 is not related with resistance to race TTKSK
At least two QTLs interact with rpg4/Rpg5 to confer resistance
Effective MAS for rpg4/Rpg5-mediated TTKSK resistance requires consideration of genetic backgrounds and phenotypic validation
Fig. 2. Multi-locus haplotype at Rpg5 and QTL on 5H and 7H for CI. “+” and “-” refer to resistance and susceptibility alleles, respectively. Bars represent the mean for each haplotype plus or minus standard error.
Bibliography
Arora et al
Brueggeman et al
Mascher et al
Steffenson et al. 2009, 2017.
Wang et al
Financial support: USDA-NACA Awards and