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Development of a molecular marker for rust resistance genes SR39 and LR35 in wheat breeding lines Julie Gold Don Harder Fred Townley-Smith Taing Aung James Procunier* *Corresponding author
Traditionally, plant breeders have selected plants based upon their visible or measurable characteristics. One of the most powerful and useful tools of biotechnology for plant selection is marker assisted selection (MAS). Progeny plants from a breeding program are selected by using a molecular marker. The marker is a molecular signature near the DNA sequence of the desired gene. MAS reduces both the time and greenhouse space needed to develop a new variety. MAS simply speeds- up the breeding process which breeders have been using for centuries. Incorporating natural , resistance genes into varieties is the most effective, economic and enviromentally safe means of controlling the disease. This is the response to the demand for costeffective, "green" solutions since it eliminates the need for expensive chemicals to control plant diseases. The resistance genes are natural, being found in many wild relatives of the plant. The use of user-friendly molecular markers for these resistance genes has many advantages over conventional phenotypic testíng. The markers can be used on seed material thus eliminating the lengthy growing time for many adult specific resistance genes. Seed testing requires no growth cabinet space. The markers are cost effective, híghly refiable/accurate and many independent resistance genes can be diagnosed simultaneously. Introduction The wheat stem and leaf rust pathogens can potentially devastate wheat crops. Resistant cultivars have long been depended upon to control disease epidemics. All spring wheat cultivars reconimended for westem Canada are fairly resistant to the prevalent races of rust and this resistance has been effective since the last stem rust epidemic in 1954 . However, most resistant cultivars generally succumb to virulent rust strains within 5-7 years or sooner afier widespread cultivar adoption by farmers. The potential for new virulent pathotypes to arise makes it important to continue to breed new stem rust resistance genes into commercial wheat cultivars. The rust resistance genes , Sr39 (stem rust) and Lr35 (leaf rust) were transferred to the susceptible, hexaploid wheat cultivar Thatcher from the wild wheat relative Aegilops speltoides L. These genes are on a translocated chromosome segment from A. speltoides. The resistant line demonstrated highly resistant infection type reactions to approximately 1200 isolates of the pathogen. These gene have not yet been used in wheat breeding. The Sr39 and Lr35 genes normally co-segregate in crosses, but the síze of the translocated segment and the true linkage between these genes is unknown. Diploid progenitors of durum and bread wheats have been exploited as sources of new resistance genes, although genetic determination of rust resistance can be complicated by difficulties in obtaining appropriate pathogen isolates for testing purposes. Moreover, while pathologists consider multigenic resistance to be rnore durable, a single gene of interest in a complex background of other resistance genes may be difficult to detect through traditional phenotypic analysis. Hence, specific genetic markers can be a valuable tool to allow gene pyramíding in MAS breeding programs. Moreover, not only can several resistance genes be tracked by simultaneous testing for the presence of appropriate molecular markers (multiplexing), but markers for other traits of agronornic ímportance can be monitored at the same time In wheat, comparisons between nearisogenic lines (NILs) and their recurrent parents have been used to identify molecular markers linked to pathogen resistance genes . This study was undertaken to identify a molecular marker for the Lr35/Sr39 resistance genes because of their potential value as rust resistance sources and the specific marker would facifitate the transfer of these genes to elite wheat lines. Materials and methods Plant material The rust resistant line RL6082 was crossed to the susceptible line (Tc) and the resulting F, population was selfed . The F2 population was used for genomic DNA extraction. The F2 plants were grown to maturity and selfed; infection types from F3 families homozygous for resistance or susceptibility, or segregating for resistance to rust were used to deduce F2 genotypes. Infection types were scored 14 days after inoculation. DNA isolation, PCR conditions, andgel electrophoresis Genornic DNAs from freeze-dried wheat leaf tissue from Thatcher(Tc) , R1,6082, and each of the F2 segregating RL6082 x Tc plants were extracted . Individual homozygous resistant and susceptible F2 plant DNA sainples were initially screened for polymorphic bands . The F2 segregating population was used to detemúne the polymorphic band's inheritance pattern and linkage to the resistance Sr39/Lr35 genes. The inter-simple sequence repeat (1SSR) primers (Set #9) used for screening were obtained from the Biotechnology Laboratory, University of British Columbia, Vancouver, B. C., Canada.. The 'hot start' polymerase chain reaction (PCR) techáque was used . Primer reaction products were resolved by gel electrophoresis carried out in polyacrylamide gels or in agarose gels. Diagnostic fragment cloning, sequencing and primer design. One ISSR primer showed a band that was tight1y linked to the Sr39/Lr35 genes. The band was present in resistant plants and absent in the Tc parent and other susceptible plants. The polymorphic band was excised from an agarose gel, cloned and DNA sequenced. Fom the polymorphic band DNA sequence, primers were designed to allow a more robust PCR reaction and eliminate the multiple banding pattern. Results and discussion One hundred ISSR primers were screened to identify polymorphic bands between the homozygous rust resístant and susceptible F2 plants. Those prímers which generated polymoiphic bands were then used to amplify DNA extracted from al] plants of the F2 segregating population in order to establish linkage of a polymorphic band with the Sr3 9/Lr3 5 resistance genes. One primer amplified a polymorphic band identified on both agarose and polyacrylamíde gels which was tight1y linked to the resistance genes in the segregating population. This frequency of finding a marker was comparable to other studies. The polymorphic band was cloned, sequenced, and a number of forward and reverse primers were designed from the band's sequence. One primer set allowed a more robust PCR reaction and eliminated the multiple band pattem found in ISSR primer reactions. This primer set amplified a single band from DNA of the resistant plants, but no amplification occurred with DNA from susceptible plants. The marker will allow the detection of the Sr39 stem rust resistance, as well as Lr35 leaf rust resistance genes. The marker has already provided a valuable tool for the detection of the Lr35 rust resistance gene. Using marker assisted selection, the Lr35/Sr39 genes have been successfully backcrossed into Canada Prairie Spring (CPS) and Canada Western Extra Strong (CWES) elite classes of wheat. |
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