This investigation developed and validated a marker KASP_Vf_0703, linked with rust resistance gene Uvf-2 carried by a single plant selection (#12034) from the faba bean cultivar Doza and KASP_AC×F165 linked with resistance gene Uvf-3 in an exotic genotype V-300 from central Europe accessioned Ac1655 in Australia.  The cultivar Doza was released as a mixture of resistant and susceptible plants and few single rust resistant plants were selected from the mixture (#12034, #12035 and #14916) in the field (J. van Leur, Unpublished data). The phenotypic expression of Doza#12034 insighted a strong chlorosis, onset sporulation, leading the death of infected leaves on 1st and 2nd nodes within 2-3 days post-sporulation. Whereas, accession Ac1655 was reported resistant IT 2+ in greenhouse against rust isolate collected  from Cordoba, Spain (Sillero et al. 2000), but it produced lower IT when we tested against Australian pathotype 24-40 in comparison to the Spanish races (12C VS 2+) (Sillero et al. 2000). This IT’s variability could be due to the differences in specificity among Spanish and Australian U. viciae-fabae pathotypes used for phenotyping because Australian rust isolates showed less virulence when compared with isolates collected from Spain, Syria and Netherland (Barilli et al. 2009a).

The SNPs Vf_0498 and Vf_0146 flanked q_rust_Doza on chromosome-III and AC×F138 and Vf_1318 with q_rust_Ac1655 on chromosome-V (Sudhesh et al. 2016) could be misleading for practical use because the phenotypic classification was performed on Fiord/Doza#12034 and Fiord/Ac1655 F4 populations, respectively. Therefore, when we converted these SNPs Vf_0498, Vf_0146, AC×F138 and Vf_1318 into KASPs, none of these SNPs showed good clusters among parents. We have resolved the location of resistance genes Uvf-2 and Uvf-3 in this investigation and identified robust KASP markers for each resistance gene.

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We have seen the KASP_0703 showed robustness for practical breeding by picking same allele in Doza#14916 like resistant parent Doza#12034. Both these selections were originated from the same background of cultivar Doza. The KASP_AC×F165 picked heterozygosity in three faba bean cultivars Doza, PBA Warda and PBA Nasma. Faba bean is a partially outcrossing species and one-third partial allogamy is acceptable in faba bean (Stoddard and Bond 1987) due to insects driven outcrossing which explains the  resistance segregation in Doza and PBA Nasma during seedling test (Chapter 3; Table 3.2).

Although, genes Uvf-2 and Uvf-3 elucidating resistance against same pathotype 24-40, both are different from each other because we have confirmed the mapping of these genes on two different chromosomes. Moreover, this uniqueness was also confirmed through inheritance studies conducted on Ac1655×Doza#12034 derived F2 and F3 populations (Adhikari et al. 2016; Ijaz and Adhikari 2016). When both resistance genes Uvf-2 and Uvf-3 were present together the additivity of interaction was seen IT ;1= C indicating the potential of pyramiding these resistance gene (Ijaz et al. 2017).

Rust is an obligate parasite that holds a capacity to overcome any widespread singly deployed resistance gene more quickly as compared to more than one genes deployed in a pyramid (Ijaz et al. 2017). Deployment of single resistance gene through conventional breeding is possible, but pyramiding of more than one resistance genes is feasible only when combination of genes produced additive effect (phenotypically) or closely linked molecular markers are available. Until now, three  rust resistance genes Uvf-1 (Avila et al. 2003), Uvf-2  and Uvf-3 (present study) have been identified in faba bean. The gene Uvf-1 conferring hypersensitivity against single isolate 96-Cord-2 (characterized as race 1) of Uromyces viciae-fabae,  was reported from Spain (Avila et al. 2003) in accession 2N52 with unknown chromosome location. The nomenclature of naming rust resistance genes Uvf-2 and Uvf-3 in this study was adopted from Avila et al. (2003). It was unknown to the author(s) at the time when present study was conducted that the gene Uvf-1 is same or different from Uvf-2 and Uvf-3. The faba bean genotype 2N52 is recorded in Australia under accession no Ac1653 (Ijaz et al. 2017), therefore, to recognise the uniqueness of gene Uvf-1 VS both Uvf-2 and Uvf-3, it is recommended to collect and test this genotype with the KASP markers designed in this study.

Breeding disease resistance is among the key objectives to ensure the yield sustainability of improved cultivars in competitive environment. Rust resistance is among the trait of high importance for Australian faba bean breeders. Although, rust resistance sources were identified and characterised in faba bean (Adhikari et al. 2016; Conner and Bernier 1982a, c; Ijaz et al. 2017; Sillero et al. 2000), no molecular markers linked with these resistance genes were available before. The present study represents the first reference in literature in which molecular markers linked with rust resistance gene Uvf-2 and Uvf-3 were designed for practical plant breeding since Avila et al. (2003). We have preferred KASP chemistry because of two mindful reasons: firstly, reducing the cost of low to medium resolution QTL scans and secondly, to outfit for marker-assisted selection in practical breeding programmes, where the marker based genotyping become under budgeted, eliminating other currently available high-throughput technologies. The genes Uvf-2 and Uvf-3 provides resistance against broad range of rust pathotypes prevalent in SA (0-10, 0-46, 40-31 and 40-55) and NSW (24-40) (Chapter 3; Table 3.5). Therefore, these genes Uvf-2 and Uvf-3 can be deployed in any genetic background using MAS in breeding faba bean cultivars targeting these regions. 

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