| Summary: | Stripe rust, caused by Puccinia striiformis West. f. sp. tritici is one of the most damaging
diseases of wheat (Triticum aestivum L.) globally. The South African wheat cultivar Kariega
expresses APR and has retained yield levels acceptable for commercial production, which is
of great importance to plant breeders. A Kariega x Avocet S partial linkage map has made a
significant contribution to understanding the genetics underlying APR to stripe rust (Yr) in
Kariega. Two major YrQTL with indications of different resistance mechanisms were
identified on chromosomes 2B and 7D.
In this study we investigated the effectiveness of identifying AFLP markers closely linked to
the YrQTL using a targeted bulk segregant analysis (tBSA) approach in F1 doubled haploid
(DH) individuals. Individual Kariega x Avocet S DH lines were characterised and bulked
based on stripe rust phenotypes and DNA marker allele profiles. In agreement with standard
BSA, an extreme resistant bulk (both QTL present: +7D+2B) and extreme susceptible bulk
(both QTL absent: -7D-2B) were constructed based on phenotypic data and verified with
marker allele data. Three additional bulks (+7D-2B; -7D+2B and narrow down +7D±2B with
marker recombinations in 7D QTL interval) were constructed based on a combination of
phenotypic and marker data, with a strong emphasis on the presence or absence of marker
alleles representing a specific QTL interval as required by a specific bulk.
A total of 184 AFLP primer combinations (SseI and MseI) were tested on the two parental
lines and five bulks. Thirty-one of these primer combinations detected 32 putative markers
that could discriminate between the extreme resistant and susceptible bulks and that were
putatively linked to either the 7D or 2B QTL regions. After validation of these markers on all
individuals used in the extreme resistant and extreme susceptible bulks, nine markers were
identified that were present in the extreme resistant and the specific -7D+2B bulk, but absent
in the extreme susceptible bulk. Another two markers were identified that were present in the
extreme resistant, +7D-2B and narrow down +7D±2B bulks, but absent in the extreme
susceptible bulk. These markers were mapped onto the existing Kariega x Avocet S partial linkage map using Map Manager QTXb20. Six AFLP markers mapped within or close to the
QYr.sgi.2B and one close to the QYr.sgi.7D QTL regions.
The tBSA approach was efficient since 10 of the 11 markers (91%) putatively identified after
screening of the individuals constituting the bulk samples mapped to either chromosome 2B
or 7D. AFLP analysis in combination with tBSA was shown to be reproducible, faster and a
more cost effective approach compared to a traditional BSA since tBSA lead to a reduction
of 28.2% of primers that need to be tested. Following the tBSA approach, marker s23m53d
mapped 3 cM from marker gwm148 previously shown to be significantly associated with
mean host reaction type for final field data as well as leaf area infected of the QYr.sgi-2B
QTL region. This resulted in an increase in LOD score from 20.1 to 23.9 using interval
mapping. Even though two markers were added to the 7D chromosome, both mapped outside
the QYr.sgi-7D QTL region. Marker s20m38b mapped 9 cM from the SSR marker gwm295
and 20 cM from the Ltn gene previously shown to be associated with the trait of interest on
chromosome 7D.
In summary, the combination of AFLP analysis and a tBSA approach has proved to be useful
in the identification of QTL, the placement of closely linked markers to known QTLs and
targeting chromosome areas with low marker numbers. Indications are that a large number of
AFLP primer combinations need to be screened to successfully target a specific QTL interval
for increased map resolution.
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