Breeding

The stem rust resistance genes Sr31 and Sr50 in wheat were both derived from translocations of the short arm of chromosome 1 from rye and conferred resistance to all field isolates of Puccinia graminis f. sp tritici (Pgt) for many years, preventing their distinction as different resistance specificities. We now show that Sr50 confers resistance against the Ug99 strain that overcomes Sr31, whereas a mutant Pgt strain virulent towards Sr50 is avirulent towards Sr31. Because lack of recombination between wheat and rye chromosome arms precludes genetic mapping and so map-based cloning of Sr50, we used a combination deletion mutagenesis and large DNA fragment cloning in bacterial artificial chromosome (BAC) vectors to define this resistance locus. Sequence analysis of a BAC contig spanning the smallest deletion detected with DNA markers at the Sr50 locus identified six coiled coil nucleotide binding site leucine-rich repeat (CC-NB-LRR) genes and a chymotrypsin inhibitor gene closely related to genes at the orthologous barley powdery mildew resistance locus, Mla. Sequencing of these genes from two EMS-induced mutants that had lost no DNA markers revealed mutation in one of the CC-NB-LRR orthologs of Mla. Transgenic complementation tests in stem rust susceptible wheat proved this gene to be Sr50. A survey of a set of rye accessions identified several carrying the gene but occurring in different Mla gene haplotypes based on DNA gel blot patterns and copy number of Mla orthologs. Several different powdery mildew and rust resistance genes including TmMla1 from T. monococcum, 23 Mla alleles from barley and stem rust resistance genes Sr33 from Aegilops tauschii and Sr50 from rye are all members of the Mla clade of cereal R genes. The gene Sr50,was initially thought to be allelic to Sr31, however, appearance of Ug99 showed that this is a different gene and is rye ortholog of barley Mla powdery mildew resistance gene. The cloning of Sr50 gives us an opportunity to screen the rye germplasm for presence of Sr50 and allows us to now do functional analysis of the various domains and understand the mechanism of resistance. The cloning also helps to add very effective resistance to gene cassette. Sr50 is effective against all the stem rust isolates around the globe

The identification of R-genes using traditional map-based approaches is a long, laborious process, not to mention the time required for subsequent development of cultivars incorporating the new resistances. Breeders seek to reduce the length of breeding cycles, and researchers require new tools to accelerate discovery and understanding of mechanisms associated with durable resistance, especially adult plant resistance (APR). A new method for rapid generation advancement, known as ‘speed breeding’, significantly reduces the length of breeding cycles, provide increased recombination during line development and enable selection in early generations. The speed breeding protocol uses controlled temperature regimes and 24h light to accelerate plant growth and development. Phenotyping methods adapted for use in the speed breeding system permit year-round evaluation of APR to rust pathogens within 5 weeks from time of sowing. RNA sequencing (RNA-Seq) technology has revolutionized gene expression profiling in plants. We previously used RNAseq to identify novel transcripts and miRNAs associated with seedling resistance (Lr28) leading to identification of transcription factors and miRNA families (e.g. miR36, miR37 and miR39) involved in signalling and defense response (Kumar et al. J. Nuc. Acids 2014:570176). In this study we report the application of speed breeding and RNAseq technologies for the purpose of rapidly identifying transcripts and miRNA associated with APR. Wheat landraces harbouring novel sources of resistance were grown under speed breeding conditions and sampled for RNA at key growth stages, before and after inoculation, which enabled discovery of differentially expressed miRNAs. Our next steps are aimed at validating these genetic factors associated with APR in order to better understand the signalling pathways and deliver tools to assist the assembly of robust wheat cultivars for the future.

A wheat genotype PBW343+Gpc-B1+LR24 containing the high grain protein content (GPC) gene Gpc-B1 linked to marker Xucw108 was used as the donor parent to transfer Gpc-B1 and Lr24 into Eastern Gangetic Plains (EGP) cv. HUW234 and HUW468 that were released in 1986 and 1999, respectively. The backcrossing program involved the following steps: (i) foreground selection, (ii) marker selection, and (iii) recovery of recipient parent genome. Grain protein contents were recorded for all selected plants from the BC2F2:3 generation. The dominant marker Xucw108 was used for foreground selection, and heterozygous plants were identified through progeny testing. For RPG recovery, both genotypic and phenotypic selection was used. Introgression of the high GPC gene into the recipient background without yield loss was completed in 5 years, starting from 2009-10 (F1) and completed in 2013-14 (BC2F5). A conventional selection program would take the same time to reach BC2F5 but ensuring the transfer of GPC would not not be possible. Ten selected single plants from the BC2F3:4 generation had comparable yields of the parents with 26% higher GPC than the recurrent parent HUW 234. Eight selected plants had comparable yields and 34% higher GPC than HUW 468. Multi-row progenies (BC2F4 and BC2F5) of each selected plant were evaluated in yield traits with the donor and recipient parents as controls during 2012-13 and 2013-14. Two lines based on each recurrent parent were identified with significantly higher GPC with no yield penalty. The study reinforced the belief that MAS in combination with phenotypic selection could be a useful strategy to develop high GPC genotypes without sacrificing grain yield. These lines will be submitted to national trial where MAS derived lines require only two years of testing compared to four years for conventionally bred lines.

Bread wheat is the most important cereal crop in Turkey. Rusts (caused by Puccinia spp.) are the most significant diseases affecting wheat yield and quality on the Central Anatolian Plateau. The purpose of this study was to identify the reactions of 198 Turkish, white seeded, winter wheat genotypes developed by the Central Research Institute for Field Crops (CRIFC) and entered in preliminary yield trials. Adult plant and seedling tests were conducted for stripe rust whereas only seedling tests were conducted for leaf rust and stem rust. Evaluations were carried out at CRIFC, İkizce and Yenimahalle, in the 2014 season. For adult plant stripe rust assessments the materials were inoculated with a local Pst population (virulent on differentials carrying Yr2, Yr6, Yr7, Yr8, Yr9, Yr25, Yr27, YrSd, YrSu, and YrA). Stripe rust development on each entry was scored using the modified Cobb scale when the susceptible check Little Club had reached 80S in June 2014. Coefficients of infection were calculated and values below 20 were considered to be resistant. Seedlings were inoculated with local Pgt (avirulent on differentials with Sr24, Sr26, Sr27 and Sr31), Pt (avirulent on differentials with Lr9, Lr19, Lr24 and Lr28) and the Pst population. Reactions were scored for each entry at 14 days post-inoculation on standard 0-4 (LR and SR) or 0-9 (YR) scales. At the seedling stage, 56 (28%), 43 (22%), and 31 (31%) genotypes were resistant to SR, LR and YR, respectively. Eighty three (42%) lines were resistant to YR at the adult stage.