Abstract:

The increasing evolution and aggressiveness of wheat rust pathogens with high virulence
against widely deployed resistance genes constitutes a major threat to the global wheat
production and consequently food security. The changes in the pathogenicity and
environmental adaptation of these fungi have resulted in serious epidemics and in some cases
total crop loss. In order to develop appropriate rust control measures, there is a need to
understand the current pathogen-environment interactions; mechanisms of pathotype
evolution, and pathogenic (virulence/avirulence) dynamics against the existing resistance
genes. Host resistance is the major and most economically viable rust control strategy, having
the capacity to significantly reduce wheat yield loss and fungicide use if effectively utilised.
The challenge posed by changing rust pathogens calls for the application of advanced
techniques, so that efforts to breed for durable resistance can be sped up. However, the need
to expand the wheat rust resistance gene pool is crucial today. Therefore, the aims of this
research were to study and understand the basis of genetic variability among selected
Australian pathotypes of Puccinia graminis f. sp. tritici (Pgt) and to identify new sources of
leaf rust, stem rust and stripe rust resistance in two wheat germplasm collections, one from
Africa and one comprising mainly spring wheat landraces.
These studies examined genetic variability among 157 Pgt isolates collected over a 39 year
period in Australia, that were chosen to represent two putative closely related clonal lineages
derived from two founding pathotypes (pts.) 326-1,2,3,5,6 (founder of Lineage 3), and 194-
1,2,3,5,6 (founder of Lineage 4). Representative isolates from Lineages 1 (pt. 126-5,6,7,11), 2
(pt. 21-0), and Lineage 5 (pts. 34-2,4,5,6,7,11 and 34-2,3,5,7), were also included as controls.
Pathogenicity tests of all purified isolates on standard differential genotypes showed general
consistency in avirulence and virulence patterns with previous research reports implying that
they were actual representatives of the original pathogen cultures. Via cluster analysis, the 99
isolates representing Lineage 3 and Lineage 4 grouped together, indicating similarity in their
pathogenicity against the known resistance genes. The level of variation in pathogenicity
observed among the subgroups of the 99 isolates resulted from the pathotypes with
uncommon or additional virulence. Because single-step mutation is believed to be the major
mechanism via which most of these pathotypes have evolved in Australia, it was expected
that pathotypes within Lineages 3 and 4 should show very little genetic diversity.
Assessments with six SSR markers clearly showed the genetic similarity of isolates within
Lineages 2, 3 and 4, and their distinctiveness from isolates from Lineage 1, consistent with
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Many African wheat lines also carried varying levels of APR against Pt, Pgt and Pst in field,
and based on molecular marker analysis, selected lines were found to carry both characterised
and uncharacterised APR genes. Apart from the Lr68 and Lr74, the pleiotropic linked genes
{Lr34, Lr46 and Lr67 for leaf rust; Sr2, Sr55, Sr57 and Sr58 for stem rust and Yr 18, Yr29 and
the independent origins of each founding isolate. A further analysis of Lineage 3 and Lineage
4 using seven SSR markers with higher polymorphism revealed some level of genetic
diversity among the pathotypes. The 34 SSR genotypes detected among the 142 isolates were
characterised by very low genetic variation (F$r= 0.042), which implied that these pathotypes
were genetically related, further confirming that they were clonal thus evolving from each
other via mutation. However, the existing genetic variations among the genotypes originate
from the different forms and rates of mutation which vary per locus.
The second study focused on the identification of wheat rust seedling and APR genes in
African wheat germplasm via multi-pathotype tests, DNA marker analyses and genetic
analysis of APR inheritance in selected genotypes. Through multi-pathotype tests with
characterised Australian Puccinia triticina {Pt), Puccinia graminis f. sp. tritici {Pgt) and
Puccinia striiformis f. sp. tritici {Pst) pathotypes, both known and unknown seedling and
APR genes functioning either singly or in combinations were detected. Some of the widely
deployed genes detected in this study, for example Sr9e, Sr30, Sr36 and Sr42, for which
virulent pathotypes exist in some regions worldwide, were highly effective especially in
wheat lines where they existed in combination. These results implied that such genes can be
best utilised in combination with other effective resistance genes in wheat cultivars for
increased synergistic efficacy and longevity. Apart from stripe rust resistance genes Yr3, Yr4,
Yr6 and Yr8, which were the most common genes postulated, Yr 17, Yr24 and Yr27 were
effective against many pathotypes apart from the currently aggressive members of the Pst-134
lineage, which have gained virulence to Yr 17 and Yr27. Lines such as Beladi 132, IYN
68/9.44, Kenya Kifaru and Kenya Mbweha were postulated to carry resistance against the
three wheat rust pathogens. Lines that carried seedling resistance genes that were effective
against all Pt, Pgt and Pst pathotypes used in this study were also detected. IAR/W/163-3 was
resistant to all Pt, Pgt and Pst pathotypes, while Grano Di Moggio Tipo 44 was resistant to all
Pst and Pgt pathotypes, and Trigo 48 was resistant to all Pt and Pgt pathotypes. The presence
of multiple resistance genes that are effective against different rust pathogens is very
important because these lines are potential candidates for development of durable resistance
through gene pyramiding.
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A panel of international spring wheat landraces plus old cultivars were evaluated for seedling
and APR to Australian isolates of Pt, Pgt and Pst, and by applying genome-wide association
studies (GWAS), SNP markers that were significantly associated with rust resistance genes/
QTLs were mapped on the wheat chromosomes based on the genome consensus maps. Many
landraces were postulated with the common ineffective seedling resistance genes like Sr6,
Sr7b, Sr9b, Sr 17 and Sr 15 but important genes in breeding like Sri2, Sri3, Sr23, Sr30 and
Sr36 were also postulated as single or in combination with other genes. A high number of
landraces carried seedling resistance genes that could not be identified, either because they are
new or because they are combinations of genes that could not be determined with the
pathotypes used. Varying levels of APR to all three rust pathogens were detected in the
landraces, and molecular analysis of the selected landraces using markers for known APR
genes confirmed the presence of leaf rust, stem rust and stripe rust APR genes including the
pleiotropic linked genes such as Lr34/Yrl8/Sr57; Lr46/Yr29/Sr58; Lr67/Yr46/Sr55 and
Sr2/Lr27/Yr30 as well as Lr68 and Lr74. The landraces carrying uncharacterised resistance
are of great potential value to future efforts to develop cultivars with resistance to rust.
Through a genome-wide association study, 77 SNP markers that were significantly associated
with resistance to leaf rust, stem rust and stripe rust were identified and mapped. SNP markers
The genetic study of inheritance of APR detected one or two resistance genes for Pst, Pt and
Pgt in bi-parental (resistant by susceptible) F3 populations developed from African wheat
lines, which were found to cany high levels of APR. One of the two APR genes detected in
the parents of F3 populations developed for stripe rust (West Savla 68 and Kazouria) and leaf
rust (AUS 12568 and AUS 19685) resistance were detected via molecular marker analyses as
Yr46 and Lr46, respectively, but the most important resistance genes are those that appear to
be new. Besides the unidentified APR genes in the five stem rust F3 populations, the
resistance genes identified in Kenya Governor, AUS 12578 and Restamacao were probably
Sr55, Sr57 and Sr58. The other APR genes in Restamacao and AUS 12578 were not
identified
Yr49 for stripe rust) were identified. The accumulation of three to four APR genes in the
wheat lines such as Kenya Leopard II (Lr34, Lr46 and Lr68), Laetissimum 2661 (Lr34, Lr68
and Lr74), AUS 12000 (Lr34, Lr46, Lr68 and Lr74) and AUS 19630 (Lr34, Lr46, Lr67 and
Lr68) indicated potential sources of effective and durable resistance in the African wheats.
The lines detected to carry either new uncharacterised or unknown seedling and APR are
considered potential new sources of resistance.
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located in chromosome positions where known APR genes were previously mapped further
indicated the presence of these genes in this panel of landraces. This study also detected SNP
markers associated with multiple rust pathogens that probably indicate the presence of new
pleiotropic linked genes or QTLs. The most important SNPs detected were those located in
the chromosome positions that have not been reported to carry rust resistance genes, implying
that they may represent new sources of resistance in wheat that need to be comprehensively
studied.