PROJECT RESULTS

Development of Wheat With New Sources of Resistance/Tolerance for Fusarium Head Blight and Barley Yellow Dwarf Virus

Applicant: 

Dr. Jeannie Gilbert

Cereal Research Centre

Agriculture and Agri-Food Canada

Winnipeg, Manitoba  R3T 2M9  Canada

Table of Contents:

• Background and Objectives
• Procedure and Project Activities
• Results and Discussion
• Conclusions

Report Prepared By:

Manika Pradhan

ARDI Project:

Total Approved:

Date Approved:

Project Status:

#99-311

$105,000

February 8, 2000

Completed October, 2005

Background and Objectives:

Fusarium head blight (FHB), a fungal disease caused principally by Fusarium graminearum Schwabe [teleomorph = Gibberella zeae (Schwein) Petch, and barley yellow dwarf (BYD) caused by BYD luteovirus (BYDV) are two serious, worldwide economic threats to small grain cereals.  FHB not only lowers grain yield, but also adversely affects the grain quality.  Mycotoxin (deoxynivalenol or DON) accumulates in the infected grain and may cause a variety of detrimental effects for both human food and livestock feed.  BYDV is vectored by several aphid species, of which the most commonly found species in Manitoba is the oat bird-cherry aphid (Rhopalosiphum padi), which transfers luteovirus subgroup BYDV-PAV.  Barley yellow dwarf reduces yield and grain quality as it causes plant dwarfing, head sterility or grain shriveling.

It would be of great benefit to the field of disease resistance breeding if BYDV tolerance and FHB resistance were combined in spring wheat lines.  The wheat accession Sumai 3 is widely used to derive FHB resistance in wheat breeding programs, but additional new sources of resistance to FHB as well as to other diseases, need to be identified and exploited to enable a strategy for pyramiding independent genes to obtain adequate levels of enduring resistance.  The Chinese wheat line Wuhan, may provide additional resistance genes for FHB, and useful BYDV tolerance has been identified in the Brazilian wheat line, Maringa. 

This research aimed to integrate the two traits and develop spring wheat (Triticum aestivum L.) germplasm that combines fusarium head blight resistance with barley yellow dwarf virus tolerance.  The process involves several objectives.

1. To study the inheritance of FHB resistance and BYDV tolerance using a doubled haploid population.

2. To combine the two traits (FHB resistance and BYDV tolerance) in spring wheat .

3. To characterize and genotype two sister lines of Maringa obtained from Winnipeg and Ottawa.

Procedure and Project Activities:

Population Generation

The previous reports submitted to ARDI reported the generation of DH populations from reciprocal crosses of Wuhan, Maringa and Roblin.  The field test under FHB and BYD disease pressure during summer 2000 showed that the Maringa from Winnipeg responded differently to inoculation with F. graminearum and BYDV from the Maringa line from Ottawa.  The population that we originally planned to use was a cross with Maringa from Ottawa, which had poorer FHB resistance and BYDV tolerance.  The decision to produce new sets of DH lines from three parental lines became a major part of this project.  A total of 553 doubled haploid (DH) lines were generated from three reciprocal crosses: Wuhan\Maringa (WM) - 83, Maringa\Wuhan (MW) - 65, Wuhan\Roblin (WR) - 87, Roblin\Wuhan (RW) – 63, Maringa\Roblin (MR) – 150, and Roblin\Maringa (RM) – 105 lines.

Experiments Under Controlled Environments

Indoor experiments were set up to assess FHB and BYD disease reactions under controlled conditions.  The variation of F₁ and parental generations were used to estimate the non-heritable variation, while the doubled haploid populations were used to estimate heritability.  The mean disease severity in the greenhouse of the three parents and F₁ differed significantly (Table 1).

Table 1.  Mean and standard deviation of fusarium head blight severity for parental and F₁ lines. 

Field Experiments - Summer 2003

Field experiments were carried out in a single year (summer 2003) with two replications and multiple checks in a randomized complete block design at four locations for FHB (Carman, Glenlea, Ottawa, Portage La Prairie FHB nurseries) and one site for BYD testing (Glenlea ).

Field data were transformed with square root transformation to stabilize variances.  Analysis of variance was done using SAS Proc GLM for both disease severity (% spikelets infected) and FHB Index (%incidence X %severity/100) by cross and location.  Chi-Square analysis of DH lines was used to determine the minimum number of genes that control FHB/BYD.  Broad sense heritability (H²) on a plot basis was estimated from the equation H²= σ²_G / (σ²_G + σ²_E/) where σ²_G and σ²_E are the genotypic variance and environmental variance, respectively.  Correlations between greenhouse and the field experiments were calculated using SAS Proc Corr.   

Results and Discussion:

The three parents, Roblin, Maringa and Wuhan differed significantly in mean disease severity for FHB (Table 1).  Under FHB disease pressure the F₁ progenies of reciprocal crosses of Wuhan (FHB-resistant) and Maringa (FHB-moderately susceptible) showed the phenotype of the parents, indicating resistance is dominant (Table 1).  Based on reduction in plant height and head mass after inoculation with BYDV, and percent infection after inoculation with FHB, no significant reciprocal differences were observed among the F₁ populations studied, indicating that no maternal inheritance is involved and that inheritance is predominantly under nuclear genetic control.

Indoor (greenhouse/growth cabinet) and the field experiments, showed three genes (ratio 1:6:1) for disease spread and three genes for FHB index with additive effects for FHB resistance in Wuhan (X². = 0.37), and the involvement of three minor genes in ‘Maringa’ (X².  = 0.389) with a fit of greater than 90%.

Frequency distribution for disease spread (disease severity) in the greenhouse and in the field provided evidence of transgressive segregation for both resistant and susceptible types (Figures 1 and 2).  DH lines segregated with values that covered the entire parental range.  The fusarium head blight index (FHB Index: product of percent disease incidence x percent disease severity) ranged from as low as 0.25 to 100, or total infection, from the field experiments.  The mean scores for severity and FHBI are presented in Table 2.

Table 2.  Mean disease spread and FHB Index of 503 DH lines of the three reciprocal crosses from Maringa/Wuhan/Roblin following inoculation with Fusarium graminearum.

Similar frequency distributions were observed for the other reciprocal populations studied for disease severity and FHB Index.

Inheritance of barley yellow dwarf virus tolerance was studied with Roblin/Maringa reciprocal crosses.  Two dominant nuclear genes control the BYDV tolerance in Maringa.

Experiments were conducted in the greenhouse (GH) at the Cereal Research Centre, Winnipeg, and at field locations in Carman, Glenlea, and Portage, Manitoba, and in Ottawa.

Analysis of variance showed significant genetic variation among doubled haploid lines in all populations across all locations.  The variance associated with replicates within location was not significant in any analysis.  The mean scores for disease severity and FHB Index for each location are presented in Table 2.  Mean disease severity of DH lines averaged over the three Manitoba field experiments ranged from 26.7% to 90% for Maringa/Roblin, 17.5% to 93.9 % for Maringa/Wuhan, 22.5% to 80.8% for Roblin/Maringa, 14.2% to 85% for Roblin/Wuhan, 14.5% to 81.7% for Wuhan/Maringa and 17.5% to 85% for Wuhan/Roblin populations, showing large phenotypic variations.  Mean disease severity for the Ottawa FHB nursery ranged from 15% to 82.5% for Maringa/Roblin, 10% to 90% for Maringa/Wuhan, 7.5% to 67.5% for Wuhan/Maringa and 7.5% to 70% for Wuhan/Roblin populations which was lower than the Manitoba average.  

The environmental variances were small and the genotypic variances were large for the FHB Index at all three locations in Manitoba for Roblin/Wuhan, Roblin/Maringa and reciprocal DH populations; as a result, the broad sense heritability estimates were high, ranging from 0.88 to 0.94.  The heritabilities for four DH populations that were tested in the FHB nursery in Ottawa ranged from 0.71 to 0.89.  

The correlation among green house experiments and from the four field experiments ranged from 0.51 to 0.65 (P < 0.01).  The most resistant and most susceptible lines correlated more highly than the intermediate lines.

A significant positive correlation was found between FHB Index and FDK (fusarium damaged kernels) calculated for Carman and Glenlea populations (r value ranged from 0.62 to 0.89). 

Correlation analysis between 2004 FHB Index and DON levels, conducted on selected DH lines of Wuhan/Maringa reciprocal crosses, showed a positive relationship, r = 0.48.

Genotyping of Two Maringa Lines

Molecular markers reveal polymorphism at the DNA level and serve as a tool for genotypic characterization and estimation of genetic diversity.  The two Maringa lines originating from Winnipeg and Ottawa were examined alongside five other parental lines using 24 microsatellite, PCR-based co-dominant DNA markers on six loci on five chromosomes (Table 3).  

Table 3.  Primers used for genotyping two Maringa lines.

From electrohoretic gel fragment image analysis, it is evident that the Maringa line from Winnipeg is different from the Maringa line from Ottawa and is closely related to Frontana as indicated by different primer pairs (Figures 3 and 4).  This is also supported by the physical characteristics of the two lines, for example, Maringa W is awned whereas Maringa O is not.  These data support the decision for generating new reciprocal working populations.  Based on the marker analysis and plant phenotypes, the two Wuhan lines appear to be the same.

Of the DH lines, 89 were randomly selected from the Wuhan/Maringa crosses and also screened with the 24 microsatellite markers as a result of the allelic polymorphisms observed in Wuhan and Maringa.  A 7% variation was explained by the primer pair gwm 493 at chromosome 3BS.  This may be due to Wuhan’s susceptibility at this locus and the resistance is likely conferred by the Maringa (moderately susceptible) allele.  This may also indicate that the resistance gene in Wuhan is different from Sumai 3, as Sumai 3 is known to carry FHB resistance at 3BS.  GWM 533, an important primer for Sumai 3 QTL identification on chromosome 4B proved to be monomorphic (Figure 3 gwm 533) for Wuhan and the Winnipeg Maringa which implies this primer cannot be used for QTL analysis on the new population.

Maringa (Winnipeg) and Wuhan are monomorphic.

Fourteen out of 147 Wuhan/ Maringa DH lines were equal to or better than the resistant parent, Wuhan for FHB resistance and 12 were equal to or better than the resistant parent, Maringa in the case of BYDV.  Three Wuhan/Maringa (WMB29, WME41 and WMB21) and three Maringa/Wuhan (MWC27, MWB55 and MWB35) doubled haploid lines showed high resistance to FHB and tolerance to BYDV.  These DH lines are considered to have combined both traits.

Conclusions:

In conclusion, the DH technology used in the breeding program has the advantages of reducing the time required to obtain homozygous genotypes and increases selection efficiency.  However, a larger DH population is required to increase the probability of selecting desirable genotypes when using F1 hybrids as they contain the largest genetic variation (greatest difference between resistant and susceptible parents).  Doubled haploid populations allow assessment of genotypes individually and simultaneously to determine if the desired trait(s) are present in the progeny.

Transgressive segregation represents the quantitative nature of inheritance and these desirable resistant segregants can become valuable parental sources of resistance, since resistance is heritable in this case.

The DH populations generated in this research can be used in FHB/BYDV QTL mapping projects when the resources are available, but the main goal of this project was achieved; to transfer FHB resistance and BYDV tolerance from Wuhan and Maringa, respectively.  This research shows that resistance to two different diseases, BYD requiring seedling evaluation and FHB involving adult plant response evaluation can be combined successfully.  Six lines selected from this research project can be used as resistant/tolerant germplasm for FHB/BYDV in future breeding programs since there is a growing need to develop cultivars resistant to multiple diseases. 

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