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CSG 15
DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
Project title
Identify markers associated with crown rust resistance in ryegrass
DEFRA project code
LS3618
Contractor organisation
and location
IGER
Plas Gogerddan, Aberystwyth, Ceredigion
SY23 1JJ
Total DEFRA project costs
Project start date
£ 226,808
01/07/00
Project end date
30/06/03
Executive summary (maximum 2 sides A4)
The overall aim of this project was to advance our knowledge of the genetics of resistance to crown rust in ryegrass so
that plant breeders can make better use of resistant germplasm. This disease is the most important fungal disease of
perennial ryegrass in southern Britain and Western Europe. There is some concern that it is becoming more prevalent.
This might be a reflection of climate change rather than the pathogen becoming more virulent. The disease reduces both
the nutritional quality of the herbage and fitness of infected plants, and ultimately affects sward composition. Using plant
natural defences is the most cost effective and environmentally acceptable method of controlling crown rust and this is
given a high priority by plant breeders.
The heterogeneous nature of ryegrass has made it difficult in the past to make significant advances in our understanding
of the genetics of resistance to crown rust. The development of molecular genetic marker technologies in recent years
provides us with the tools to extend our knowledge.
Objective 1 Identify quantitative trait loci (QTL) for crown rust resistance in perennial ryegrass.
In the first part of the project a perennial ryegrass mapping family, developed at IGER with BBSRC funding, and which
was segregating for resistance to crown rust was used to identify and locate resistance genes (QTL). Previous work
funded by DEFRA showed that temperature had a significant effect on the resistance of different cultivars, therefore,
firstly we tested the mapping family at 10, 15, 20 and 25°C. The resistance of the plants was estimated on a 0-4 scale
depending on the type of symptoms observed on several leaves on each plant (the reaction types). There were some 245
markers available in the mapping family for linkage to resistance. The statistical package used to analyse the data
estimates the likelihood (lod value) that a gene is linked to specific marker on each chromosome (linkage group) and this
can be plotted as a graph. Over all temperatures QTL were located on four linkage groups and the resistance was of a
quantitative nature. Most of the genes had only a small effect, (responsible for only 7% of the variation) and were
sensitive to temperature, but some were effective at all temperatures. The QTL with the highest lod value was on linkage
group 5 and was identified in all the tests.
CSG 15 (Rev. 6/02)
1
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
Four crown rust isolates with different levels of virulence were isolated and characterised. When replicated sets of the
mapping family were inoculated with these isolates, it was shown that there were QTL segregating in the mapping
population which were sensitive to different isolates. The four isolates could be broadly assigned to two groups based on
the QTL analysis and distribution of R and S plants. For the first group, the same QTL were identified as in the first
experiment, except that one on linkage group 3 differentiated between the two isolates. A number of QTL were also
identified for the more virulent isolates but the effect they had on resistance was, by definition, very small. We suggest
that they are responsible for subtle differences between intermediate and susceptible reaction types. The most noticeable
difference was the ineffectiveness of the QTL at 0cM on linkage group 2 against the more virulent isolates. As in the
previous test the highest lod values were on linkage group 5. This is the first report of pathogen isolate specific QTL in
perennial ryegrass.
Having tested for differences between isolates and temperature individually, we then compared the results of testing the
mapping family with an isolate from each of the two virulence groups at high and low temperatures (10 and 25°C). There
were fewer susceptible plants at 10°C with both isolates but temperature had a greater effect on the virulent isolate. One
QTL on each linkage group 2 and 3 only occurred at the lower temperature. In contrast, the QTL which had previously
given high lod scores on linkage group 5 was only just above the threshold value on one scoring date at 10°C but gave
high values at 25°C. Thus the differential sensitivity of perennial ryegrass resistance genes to temperature appears to be
isolate dependent.
Other mapping families developed at IGER were tested to identify suitable material for future work, but none of these
were segregating for crown rust resistance.
The results from this project supports the work on producing improved varieties of perennial ryegrass by providing
knowledge on the inheritance of resistance to crown rust and how it might be exploited in the future. This will assist in
maintaining an output of new ryegrass varieties which in itself help to maintain the sustainability of UK livestock
production.
Objective 2 - Identify molecular markers associated with crown rust resistance introgressed into Italian ryegrass from
meadow fescue.
Two BC3 introgression lines of Italian ryegrass were developed which contained a segment of chromosome from meadow
fescue carrying resistance to crown rust. The purpose of this project was to determine whether the resistance was on this
segment and to identify molecular markers linked to the resistance gene. The presence of the meadow fescue segment
was identified using a chromosome painting technique. This showed that, in one of the lines, the resistant plants had the
introgressed segment but that it was absent in the susceptible ones. In contrast, in the second line some susceptible
plants had a segment of fescue chromosome but this was physically smaller than in resistant plants. The first line was
clearly not suitable for identifying markers since there was no recombination on the short arm containing the fescue
segment. Also, by intercrossing some BC3 genotypes and testing the progeny, the ratio of resistant to susceptible plants
confirmed the presence of a single resistance gene.
AFLP markers specific to the fescue introgression were identified and a significant proportion mapped close to the
resistance gene (i.e. there was no recombination between the marker and the gene). Other markers were also tested on
the BC3 line which showed that the introgression was on ryegrass linkage group 5. The results of these tests also
provided evidence that suggests that there may be suppressed recombination in this segment. A further generation of
backcrossing would be required to determine if the segment size can be reduced without loss of effectiveness.
This work has demonstrated that a novel form of disease resistance can be utilised in breeding programmes using a
conventional breeding technique in association with molecular markers. It gives plant breeders the choice in the future to
exploit this type resistance should sources present in perennial ryegrass become less effective and new types difficult to
find. Thus future improvements in quality characteristics of new varieties will be protected and assist in maintaining the
sustainability of UK livestock production.
CSG 15 (Rev. 6/02)
2
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
Scientific report (maximum 20 sides A4)
Objective 1 Identify quantitative trait loci (QTL) for crown rust resistance in perennial ryegrass.
Introduction - Crown rust (Puccinia coronata) is the most important fungal disease of perennial ryegrass
(Lolium perenne) in the UK and Western Europe and there are reports that it is becoming more prevalent
across Western Europe. It is known to reduce the nutritive value of herbage, so that it is important that
new cultivars with higher sugar content have adequate resistance to protect that quality improvement.
Although most new cultivars have good levels of resistance, the fungus is continually evolving and new
strains can appear which reduce the effectiveness of resistance genes. To keep one step ahead of the
pathogen, it is necessary to identify those resistance genes currently deployed in existing cultivars and
advanced breeding lines, as well as those in new unexploited sources from wild populations, which are
effective against a wide range of crown rust populations. The resistances may be polygenically inherited
or controlled by single genes, but until recently very little work has been carried out to determine this. The
development of DNA marker technology has made it much easier to study the inheritance of both simple
and complex traits. At IGER, an F2 perennial ryegrass family was developed to study the inheritance of
water soluble carbohydrate contents of herbage, with funding from BBSRC, and a dense linkage map
constructed. This family was found to be segregating for crown rust resistance and has been exploited in
the experiments to identify the potentially more durable forms of resistance.
A previous DEFRA-funded project showed that temperature affected the expression of resistance to crown
rust in perennial ryegrass, with all cultivars being more susceptible at 25˚C than at 10˚C. The genetic
nature of temperature sensitivity was explored using this mapping family. It has also been shown that
crown rust populations from different regions can vary in their pathogenicity, therefore the possibility of
identifying isolates (race) specific resistance genes was tested.
Methods & Results
Plant material
The initial plant material consisted of a family of 183 F2 plants derived from a pair-cross between two plants of
two S3 generation inbred lines. These lines were derived from selections from the early flowering and
crown rust susceptible cv. Aurora and the late flowering and moderately resistant cv. Perma.
Selection from
cv. Aurora

Inbred (S3)
Susceptible to
crown rust
X
Selection from
cv. Perma

Inbred (S3)
Moderate resistance to
crown rust

F1
Single F1 genotype selfed

Segregating F2 family of 183 individuals
Genotyping and linkage map
The F2 plants had been genotyped for Restriction Fragment Length Polymorphisms (RFLPs), Amplified
Fragment Length Polymorphisms (AFLPs), simple sequence repeats (SSRs), single tagged sequences
(STSs), isozyme and functional markers. A final linkage map was constructed for 61 RFLPs, 54 AFLPs,
103 SSRs, 11 STSs, 3 isozymes and 13 functional markers onto seven linkage groups aligned to the
Triticeae map using JoinMap 2.0 software. This genetic marker data was used in the linkage analyses.
Rust isolates
CSG 15 (Rev. 6/02)
3
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
A standard control isolate (isolate 2) was multiplied from a single pustule isolate taken at random from a rust
sample collected in the UK. Other isolates were developed by taking single pustules from a bulk
population collected from several sites in the United Kingdom and one locality in France. These were
tested on various genotypes in the mapping family and two which appeared to be more virulent (isolates 3
& 4) than the control isolate and one which was less virulent (isolate 1) were selected.
Rust inoculation
Plants were grown in plastic trays consisting of 77 cells each containing an individual clonally propagated plant
in a soil-less compost with at least five tillers per cell. A randomised block design was used in all
experiments. There were two replicated sets in the temperature and temperature/isolate experiments and
four replicates in the isolate experiment.
Leaves were inoculated using an aerosol sprayer to spray plants with measured amounts of spores diluted in
odourless kerosene. After inoculation the plants were placed in a growth room at 22°C and 100% humidity
for 20 h, maintaining the same photoperiod. The plants were then either transferred to growth cabinets
(temperature experiments) or to a heated glasshouse set to maintain a minimum temperature of 20°C with
supplementary lighting (isolate experiment).
The reaction of the plants to infection was assessed on a scale of 0-4 (0 = no visible sign of infection
(immune), 0.5 = faint chlorotic spots, 1 = mostly chlorotic spots, 1.5 = chlorotic spots with a few very small
pustules (hardly sporulating), 2 = chlorotic spots and a few small to medium sized pustules, 2.5 = less
chlorosis than with 2, 3 = medium sized pustules, possibly with some chlorotic spots, 3.5 = medium to
large pustules, and 4 = large pustules with hardly any chlorosis surrounding them and profuse sporulation.
QTL analysis
The Map QTL™ computer program was used to conduct interval mapping on the mean crown rust scores.
Both non-parametric and interval mapping were conducted separately on each data set to identify
genomic regions associated with rust resistance. For the non-parametric mapping, the Kruskal-Wallis test
is used in MapQTL, which ranks individuals according to the quantitative trait. A significance level of P <
0.001 was applied in this study. The probability of linkage of crown rust resistance to a marker position is
estimated by calculating a likelihood ratio. This ratio is the likelihood of the observed results if linkage is
assumed over the likelihood of the observed results due to independent assortment and is usually called
the lod score. A lod threshold of 2.0 was used to identify the presence of a putative QTL in a given
genomic region. As the linkage map has been developed significantly during the lifetime of this project, the
data has been re-analysed so that some of the conclusions presented here differ slightly from those in
previous annual reports.
Temperature sensitive QTLs
Four sets of the mapping family, each with two replicates, were inoculated with crown rust (isolate 2) and each
set placed in one of four identical growth cabinets at 10°C, 15°C, 20°C and 25°C. Individual plants were
assessed for crown rust on the first visible sign of infection and at intervals thereafter.
Table 1. Mean crown rust scores of an F2 mapping family at 10, 15, 20 and 25°C
Assessment
1
2
3
4
5
10°C
0.73
1
1.17
1.22
Temperature
15°C
0.82
1.12
1.25
1.35
20°C
1.08
1.34
1.52
1.67
1.71
25°C
1.52
1.73
1.97
The results confirm the previous reports that perennial ryegrass is more resistant at low temperature; overall
there was a gradation in susceptibility from 10 to 25°C (Table 1). There were no major affects of
temperature on the identification of QTL, and the results demonstrate the subtle differences which minor
genes can have on the expression of resistance. The highest lod scores were found on linkage group 5
with the highest values at 25°C at the earliest assessment times and suggest that this QTL might affect
the latent period component of resistance. The lod-likelihood plots were very broad and it was difficult to
CSG 15 (1/00)
4
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
ascertain whether there was more than one QTL on this linkage group. A QTL at 0cM on linkage group 2
also occurred at 10, 15 and 25°C and was just below the threshold value (2.0) at 20°C. Similarly one on
linkage group 3 occurred at 10°C, and was just below the threshold at 20 and 25°C. A QTL on linkage
group 1 only occurred at 10°C, one on linkage group 6 only at 25°C and one on linkage group 4 at 20°C.
Isolate specific resistance
Overall the mapping family was more resistant to isolates 1 and 2 (Table 2) with which the distribution of rust
scores tended to be bimodal with resistant plants predominant, whereas with isolates 3 and 4 it was
skewed towards susceptibility.
Table 2. Mean crown rust scores of an F2 mapping family infected with four isolates on three scoring
dates
Isolates
Assessment
1
2
3
4
1
1.18
1.57
2.80
2.26
2
1.60
1.59
3.44
3.06
3
1.61
1.57
3.48
3.12
As with the previous experiment, interval mapping with isolates 1 and 2 located putative QTL on the proximal
end of linkage group 2 and one or two on linkage group 5: another QTL on linkage group 3 was only
effective against isolate 2. The lod-likelihood plots on linkage group 5 showed very broad peaks for both
isolates but the percentage phenotypic variation accounted for by this QTL was highest at the earlier
scoring date and the higher for isolate 1 (17.3%) than for isolate 2 (8.6%). The estimated phenotypic
values associated with co-dominant markers suggest that the QTL was recessive against both isolates.
The QTL on linkage group 2 associated with an SSR (M4136) accounted for a maximum of 8.5% of the
variation.
The skewed distribution towards susceptibility against isolates 3 and 4 showed that there were very few
effective QTL segregating and despite the presence of statistically significant QTL on linkage group 5, it
had only a small effect on the level of resistance. The lod-likelihood plots suggest that there were a
number of minor QTL on other linkage groups, but none were significant in the Kruskall-Wallis test.
Isolate x temperature interaction
Although this was not in the original workplan, the results from the previous tests indicated that it was
necessary to determine whether temperature sensitivity could be isolate dependent.
Table 3. Mean crown rust scores of an F2 mapping family infected with two isolates and incubated at
10°C and 25°C
Assessment
1
2
3
Isolate 1/10°C
0.70
1.36
1.53
Isolate/Temperature
Isolate 3/10°C Isolate 1/25°C
1.60
1.34
2.76
1.90
2.93
2.48
Isolate 3/25°C
2.18
2.96
3.37
As with the previous tests, the mean rust scores were lower for isolate 1 than 3 and lower at 10°C than at 25°C
(Table 3). There was a large QTL on linkage group 2 with isolate 3 at 10°C, but not at 25°C. Similarly,
there were other temperature-sensitive QTL showing the same response on linkage group 3 and, to a
lesser extent, on linkage group 1. With isolate 3, a very high lod score was achieved for a QTL on linkage
group 5 at 25°C, but it was only just above the threshold value (2.0) at 10°C and only on the first
assessment date. As was characteristic with this linkage group, the lod plots showed a broad peak. High
lod values on linkage group 5 were also obtained with isolate 1 but they were significant at both
temperatures.
Screening other mapping families
A further three mapping families developed at IGER were screened to identify suitable material for future
evaluation, but none showed segregation for resistance.
CSG 15 (1/00)
5
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
Discussion
The crown rust resistance genes segregating in the F2 mapping family are of a quantitative nature, some
having only a small effect on the expression of resistance, and are therefore probably more sensitive to
the effect of temperature and possibly other environmental or physiological variables. The resistance
genes were largely ineffective against some isolates and of little use for plant breeding, but could be used
to test marker-assisted selection. A QTL on linkage group 5 gave consistently high lod scores in all tests
Interestingly, there are also QTL for water soluble carbohydrate on the same linkage group. When the role
of temperature was looked at with a more virulent isolate the QTL were more temperature sensitive.
The findings that there were isolate specific QTL segregating in this mapping family is the first time this has
been reported in perennial ryegrass.
The results from this project supports the work on producing improved varieties of perennial ryegrass by
providing knowledge on the inheritance of resistance to crown rust and how it might be exploited in the
future. This will assist in maintaining an output of new ryegrass varieties which in itself help to maintain the
sustainability of UK livestock production.
Future plans
Although it is possible that mapping families developed to look at the genetics of traits other than disease
resistance might be suitable to identify resistance genes, to make further progress it will be necessary to
develop mapping families specifically for crown rust using the cultivars and lines from wild populations.
Lines have been developed from wild populations with effective sources of resistance. Similarly, there
have been further advances in the development of marker technology which make it more efficient to
develop mapping families. Rather than use marker-assisted selection as part of a breeding programme it
is envisaged that genes will be identified and markers used to introgress resistance into suitable parents
including ones that may already be carrying effective genes. Such an approach increases the number of
barriers which the pathogen has to overcome and is likely to provide durability of resistance.
Objective 2 - Identify molecular markers associated with crown rust resistance introgressed into Italian
ryegrass from meadow fescue.
Introduction
Many grasses are hosts of the crown rust fungus including agriculturally important species such as meadow
fescue (Festuca pratensis), and other Festuca spp. The fungus exists in different physiological forms and
this manifests itself both at the formae speciales level - its capacity to infect certain grass species - and as
physiological races - specificity to certain genotypes within the species. However, the relationship between
different hosts and physiological forms of the fungus is complex. Therefore, within any host species some
susceptibility to P. coronata from an unrelated species can occur especially under artificial conditions in a
glasshouse or growth cabinet. Consequently, within a population of F. pratensis, individual plants can
differ in their reaction to infection by P. coronata originating from Lolium spp., with some populations
having a relatively high proportion of susceptible plants, while others are very resistant. Thus, F. pratensis
could be a potential source of durable resistance. In addition, there is some evidence that resistance
derived from Festuca spp. is more stable at high temperature and therefore more effective in warmer
regions.
Meadow fescue and the two cultivated ryegrass species, Italian ryegrass (L. multiflorum) and perennial
ryegrass (L. perenne), are closely related with a degree of homology in their chromosomes. On the other
hand, the relationship is sufficiently distant that L. multiflorum (Lm) and F. pratensis (Fp) chromosomes
can be distinguished in hybrid plants using genomic in situ hybridisation (GISH), sometimes called
chromosome painting. The potential of Lolium/Festuca introgression mapping to develop physical and
genetic maps, to determine the genetic control of important characters and to develop new germplasm for
plant breeders has been explored for some time and it should be possible to use it to identify the region of
the Fp chromosome carrying genes for disease resistance.
Methods & Results
Plant and rust material
CSG 15 (1/00)
6
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
Two BC3 families (P193/88 and P193/104) from a cross between L. multiflorum4 x F. pratensis along with the
parent plants, developed in a previous DEFRA project (LS1405) were selected as potential plant material
to identify molecular markers associated with crown rust resistance derived from the meadow fescue
parent. These were tested using standard methods and the same crown rust population that was used to
screen each generation during the development of the BC line.
Screening plants material
The two BC3 generation lines gave very clear-cut segregation of R:S plants, with almost all of the resistant
plants showing immunity. Both lines were derived from the same F. pratensis parent and F1. But, as with
the BC2 generation, one line had significantly more susceptible plants than the expected 1:1 ratio.
A sample of mitotically dividing cells from crown rust resistant and susceptible plants in the two families was
examined using GISH to identify the introgressed fescue chromosome segment. All resistant plants
carried a fescue segment on a single chromosome, which in one of the lines (P193/88) was confined to
the short arm of the chromosome. None of the susceptible plants of P193/104 contained a visible
introgression whereas those of P193/88 either contained no segment or one which appeared to be
physically smaller than those in resistant plants. Using GISH, the resistance locus could be physically
mapped to the mid-point of a short arm. The P193/104 family was therefore unsuitable for mapping the
introgression as there was no recombination within the fescue segment. Some additional work was carried
out to investigate why this had occurred. Inflorescences from the BC2 parents were harvested and
chromosome pairing at the metaphase 1 stage of meiosis examined in pollen mother cells. In the BC
parent of the P193/104 family there were fewer bivalents (ratio of 4.08 bivalents: 2.92 univalents) than in
the parent of P193/88 (5.72 bivalents: 1.28 univalents). Further analysis using a chromosome painting
technique to trace the fate of the fescue segment during meiosis would be necessary to confirm the
significance of this.
Segregation ratios of the progeny of BC3 plants, when crossed as R x S and R x R, were in agreement with the
hypothesis that the resistance was controlled by a single gene or very closely linked genes (Table 4). No
resistant plants were produced by crossing S x S plants, confirming that the resistance is carried on the
introgressed segment.
Table 4. Segregation ratios in progenies of pair crosses between crown rust resistant and susceptible
BC3 plants of crosses between Festuca pratensis and Lolium multiflorum4
Cross
Resistant
Rust reaction
Intermediate†
Expected ratio
Χ2 values
Susceptible
SxS
Cross A
Cross B
0
0
0
0
59
66
-
-
RxR
Cross C
Cross D
44
50
0
3
15
21
3:1
0.005 NS
0.45 NS
RxS
Cross E
Cross F
72
62
0
0
57
51
1:1
1.74 NS
1.07 NS
† intermediate types were grouped with resistant class for analysis
NS = no significant difference
AFLP analysis
DNA was extracted from all the parent and 79 BC3 plants, followed by standard procedures for PCR reactions.
EcoR1 and Mse1 primer combinations were used. AFLP analysis was carried out using an ABI gene
scanner. Initially, only parental DNA was analysed and AFLP markers present on the F. pratensis and BC
parents but absent in the Lolium parents were identified. Because the mapping line was multi-parental,
fescue-specific markers were rare events. Those primer pair combinations giving fescue specific markers
CSG 15 (1/00)
7
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
were repeated for the entire BC3 population and plants were scored for the presence or absence of the
marker. The data output was viewed using ABI Genotyper™ software package.
Genetic map
A genetic map was developed from the linkage relationships between AFLP markers and crown rust
resistance using JoinMap 2.0 software (Fig.1). There was no recombination between some markers and
crown rust resistance suggesting that theoretically these markers were very close to or part of the
resistance gene. In addition, Restriction Fragment Length Polymorphisms (RFLP), simple sequence
repeats (SSR), and single tagged sequence (STS) markers were identified which segregated with the
crown rust resistance. There is a strong linkage between the markers CDO127 and e51174a and weak
linkage with CDO412a. This shows that the introgression is located on Lolium chromosome 5. The three
STS markers (rust665393f/4r, APO077529.5/6 and APO066757.4/5) are based on sequences that come
from one end of the rice chromosome 12 and therefore show a syntenic relationship. On another Lolium
perenne map there is approximately 10 cM distance between two of the STS markers (I. Armstead, pers.
comm.). This, along with the AFLP data, suggests that there is a degree of suppressed recombination in
the introgressed region where the crown rust resistance is located.
Fig. 1 Linkage map of the chromosome (left) carrying crown rust resistance in Lolium multiflorum (Bc3 line
P193/88) in comparison with the linkage map (right) for chromosome 5 in the F2 Lolium perenne
mapping family.
0
1
2
CR5
5
6
8
9
10
11
E35M5101
E35M5102
E35M6101
AP066757
E35M5901
E41M5401
CR
E37M6101
E38M4901
E41M4901
E41M4902
E35M5201
E37M4701
17
E51174S
24
CDO127
4
50
CDO412
LP F2 C5
0
AP077529
E37M6001
E38M5901
rust66539
7
10
11
13
16
APO77529
20
21
22
24
25
28
30
32
37
39
43
47
H11G05
APO66757
rv0757
GSY60.2
E51174S rv0814
CDO127 CDO1380.2
F29-1
rv0184
RZ206
BCD1087
rv0495
PSR574
rv0950
rv1258
CDO412
rv0340
B6103
rv1112
E40M5907
66
69
E36M5512
E40M5905
74
R2710
83
RZ404
17
Although the map indicates some recombination between those markers close to the crown rust gene, the
actual data does not show this in all cases, which is a reflection of the sample size.
Discussion
The objective of this part of the project was to identify AFLP markers which were common to the introgressed
segment and provide supporting evidence, alongside the cytological assessment, that the crown rust
resistance was derived from the fescue parent. The results obtained support this hypothesis and the
project has been successful as a proof of principle for this procedure. Markers associated with the fescue
parent and closely linked to the crown rust resistance have been identified and some additional work has
identified the Lolium linkage group carrying the introgression. However, there is evidence of suppressed
CSG 15 (1/00)
8
Project
title
Identify markers associated with crown rust resistance in
ryegrass
DEFRA
project code
LS3618
recombination in the introgressed region. If this is correct then it will be difficult to recombine the
resistance into a ryegrass background without also carrying many other fescue derived genes, some of
which might be deleterious. Although the two individual objectives of this project were independent of each
other, it is interesting that crown rust resistance genes identified in both ryegrass and fescue have been
located on linkage group 5.
This work has demonstrated that a novel form of disease resistance can be utilised in breeding programmes
using a conventional breeding technique in association with molecular markers. It gives plant breeders the
choice in the future to exploit this type resistance should sources present in perennial ryegrass become
less effective and new types difficult to find. Thus future improvements in quality characteristics of new
varieties will be protected and assist in maintaining the sustainability of UK livestock production.
Future plans
This project was initiated to prove the hypothesis that fescue-derived crown rust resistance could be
transferred into a ryegrass background. Another generation of backcrossing will be necessary to see whether
the fescue segment can be reduced in size without losing the crown rust resistance. To exploit this resistance
it will also be necessary to follow a precautionary principle and test it with a range of crown rust cultures to
demonstrate that it is an effective source. Some of the flanking AFLP markers could be used to incorporate the
resistance into perennial ryegrass breeding lines, but there is evidence that these unmodified markers are not
fescue specific, being only specific to the parental material, so that only potential ryegrass parents without the
marker(s) can be used. The development of fescue specific markers at IGER could be exploited in the future
to develop this further.
Publications
Refereed Journals
1. Thorogood, D., Paget, M., Humphreys, M., Turner, L., Armstead, I. & Roderick, H. (2001) QTL
analysis of crown rust resistance in perennial ryegrass - implications for breeding. International Turfgrass
Society Research Journal 9, 218-223.
2. Roderick, H.W., Morgan, W.G., Harper, J. A. & Thomas, H.M. Introgression of crown rust (Puccinia
coronata) resistance from meadow fescue (Festuca pratensis) into Italian ryegrass (Lolium multiflorum)
and physical mapping of the locus. Heredity (accepted Feb 2003).
Proceedings of conferences
1. Thorogood, D., Paget, M., Humphreys, M., Turner, L. & Roderick, H. (1999) A QTL analysis of crown
rust resistance in perennial ryegrass. In: New Approaches and Techniques in Breeding Sustainable Fodder
Crops and Amenity Grasses. Proceedings of the 22nd Eucarpia Conference, Fodder crops and amenity
grass section meeting, St Petersburg, Russia, pp 200-207
2. Humphreys, M., Abberton, M., Cook, R., Turner, L., Roderick, H., & Thorogood, D. (2001) Molecular
breeding in pasture species and its application to improving pest and disease resistance. Proceedings of
the 3rd International conference on harmful and beneficial microorganisms in grassland, pastures and turf.
Soest (Germany) 26 September 2000, Eds V.H. Paul and P.D. Dapprich, pp 77-87.
3. Roderick, H.W., Humphreys, M.O., Turner, L., Armstead, I. & Thorogood, D. (2002) Isolate specific
quantitative trait loci for resistance to crown rust in perennial ryegrass. Grass for Food & Grass for Leisure.
Proceedings of the 24th Eucarpia Conference, Fodder crops and amenity grass section meeting, FAL
Braunschweig, Germany 22-26 September 2002.
CSG 15 (1/00)
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CSG 15 (Rev. 6/02)
10