Plant Disease • 2019 • 103:1068-1074 • https://doi.org/10.1094/PDIS-05-18-0831-RE Research Introgression of a Novel Ug99-Effective Stem Rust Resistance Gene into Wheat and Development of Dasypyrum villosum Chromosome-Specific Markers via Genotyping-by-Sequencing (GBS) Kaori Ando,1 Vandhana Krishnan,1 Sheri Rynearson,1 Matthew N. Rouse,2 Tatiana Danilova,3 Bernd Friebe,3 Deven See,4 and Michael O. Pumphrey1,† 1 Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164 2 United States Department of Agriculture–Agricultural Research Service (USDA-ARS), Cereal Disease Laboratory and Depart- ment of Plant Pathology, University of Minnesota, St. Paul, MN 55108 3 Wheat Genetics Resource Center, Kansas State University, Manhattan, KS 66506 4 USDA-ARS, Western Regional Small Grains Genotyping Laboratory and Department of Plant Pathology, Washington State University, Pullman, WA 99164 Abstract Dasypyrum villosum is a wild relative of common wheat (Triticum aes- line MS2V(2D) (20² +1¢ 2V[2D]). This is the first report of stem rust re- tivum L.) with resistance to Puccinia graminis f. tritici, the causal agent sistance on 2V, which was temporarily designated as SrTA10276-2V. of stem rust, including the highly virulent race TTKSK (Ug99). In order To facilitate the use of this gene in wheat improvement, a complete to transfer resistance, T. durum–D. villosum amphiploids were initially set of previously developed wheat–D. villosum disomic addition lines developed and used as a bridge to create wheat–D. villosum introgres- was subjected to genotyping-by-sequencing analysis to develop D. vil- sion lines. Conserved ortholog set (COS) markers were used to identify losum chromosome-specific markers. On average, 350 markers per D. villosum chromosome introgression lines, which were then sub- chromosome were identified. These markers can be used to develop di- jected to seedling P. graminis f. tritici resistance screening with race agnostic markers for D. villosum-derived genes of interest in wheat TTKSK. A COS marker-verified line carrying chromosome 2V with improvement. TTKSK resistance was further characterized by combined genomic in situ and fluorescent in situ analyses to confirm a monosomic substitution Keywords: cereals and grains, field crops, fungi, techniques. Dasypyrum villosum (L.) P. Candargy (2n = 2x = 14, VV) is an out- powdery mildew, eyespot, loose smuts, take-all disease, leaf spot, crossing tertiary gene pool relative of wheat (Triticum aestivum L.). wheat curl mite, Wheat spindle streak mosaic virus,leafrust,andstem The species originated around the northeastern Mediterranean and rust (De Pace et al. 2011; Gra˛dzielewska 2006). A collection of D. vil- Caucasus region (De Pace et al. 2011) and has been utilized for wheat losum accessions (n = 95) was previously screened, which revealed improvement due to abiotic stress tolerance, including salt and near-immunity of this species to wheat stem rust, caused by Puccinia drought, as well as resistance to several wheat diseases, including graminis f. tritici Erikss. & Hennings (Pumphrey et al. 2008). Stem rust has reemerged as a threat to world wheat production since the emergence of a new virulent race, TTKSK (also known Current address of K. Ando: USDA-ARS, Crop Improvement and Protection as Ug99), and evolution of related races (Singh et al. 2011, 2015). Research Unit, Salinas, CA 93905. Stem rust has been effectively controlled by using resistance genes throughout the world in recent decades; however, the Ug99 race Current address of V. Krishnan: Stanford Center for Genomics and Person- group defeated many effective wheat stem rust resistance (Sr) genes alized Medicine, Department of Genetics, Stanford University, School of and continues to evolve (Jin et al. 2008, 2009; Newcomb et al. 2016). Medicine, Palo Alto, CA, 94305. The Ug99 race group is virulent to the majority of genes of common † wheat origin. There are at least 41 Sr genes effective against the Ug99 Corresponding author: M. O. Pumphrey; E-mail: [email protected] race group to date (Hiebert et al. 2016; Rahmatov et al. 2016; Singh Funding: This project was supported by the College of Agriculture and Life et al. 2015). Thirteen genes are derived from common wheat back- Sciences, Cornell University as part of the Durable Rust Resistance in Wheat grounds and the remaining are derived from other species belonging project, which was funded by the Bill and Melinda Gates Foundation and the to the primary, secondary, and tertiary gene pools. One of the resis- United Kingdom Department for International Development. tance genes, Sr52, was identified on D. villosum chromosome 6VL via a Robertsonian translocation (T6AS·6V#3L) conferring resis- e-Xtra “ ” *The logo stands for electronic extra and indicates that one supple- tance to TTKSK (Qi et al. 2011). Sr52 is the only Sr gene derived mentary table, one supplementary file, and two supplementary figures are published online. from D. villosum. It is important to broaden the genetic basis of stem rust resistance genes by acquiring new resistance genes from other The author(s) declare no conflict of interest. gene pools, not only to control the disease but also to prolong the du- rability of the genes by pyramiding (McDonald and Linde 2002). Accepted for publication 11 February 2019. Alien gene transfer for disease resistance control in wheat is not a new concept. In order to transfer resistance genes from D. villosum to wheat, addition, substitution, and translocation lines were devel- This article is in the public domain and not copyrightable. It may be freely ˛ reprinted with customary crediting of the source. The American Phytopath- oped (Gradzielewska 2006). Creating these lines required spe- ological Society, 2019. cialized techniques, including embryo rescue and chromosome 1068 Plant Disease / Vol. 103 No. 6 doubling techniques. To minimize the size of the transferred chromosome (TA7677) through DA7V#3 (TA7683) and a set of Robertsonian segments to reduce the potential for linkage to undesirable alleles on translocations (cRobTs) involving 12 of the 14 D. villosum chromosomes the same segments, inducing homologous recombination of chromo- were obtained from the Kansas State University Wheat Genetic Re- somes harboring resistance genes and developing chromosome-specific source Center (Table 1). Tetraploid T. durum ‘Rusty’ (PI 639869) single-nucleotide polymorphism (SNP) markers spanning target chro- (Klindworth et al. 2006), susceptible to all P. graminis f. tritici races mosomes will help track alien resistance in a wheat background. Also, tested in North America and races of exotic origin, was used as the it provides markers for targeted chromosome engineering. female in crosses with D. villosum to make amphiploids. A popula- D. villosum is highly heterogeneous due to open pollination; there- tion of F2:3 families of amphiploids Rusty/TA10276B//Rusty// fore, it is challenging to develop adequate markers (De Pace et al. 2011; TA2126B (Table 2) were created for allelism testing. CS was used Qi et al. 2011). However, with the advances in molecular techniques, for subsequent crosses with amphiploids to produce wheat–D. vil- additional chromosome-specific markers were developed for genotyp- losum single-chromosome introgression lines. Initially, the amphi- ing (Cao et al. 2009; He et al. 2013; Liu et al. 2011; Qi et al. 2011; ploids were used as female plants and CS was used as the pollen Zhang et al. 2006; R. Zhang et al. 2017; X. Zhang et al. 2017). Con- donor to produce F1 seed. These F1 plants were backcrossed to CS served ortholog set (COS) markers are developed based on genes as female to create BC1F1 progeny. The BC1F1 progeny were further which are highly conserved and low copy across a species and are suit- backcrossed to CS or self-pollinated until representative single D. vil- able for comparisons to closely related species (Molnar´ et al. 2013; losum introgression lines were isolated. D. villosum accession Pont et al. 2013; Quraishi et al. 2009). In total, 695 COS markers were TA10260 was used for DNA extraction to produce a D. villosum- developed based on rice-wheat orthologs and these markers are avail- specific genomic in situ hybridization (GISH) probe; and TA5617, able for cereal genome research (Quraishi et al. 2009). a 6AS·6VL translocation line, was used as a GISH-positive control. Genotyping-by-sequencing (GBS) is a recent high-throughput Embryo rescue and colchicine treatment. Immature embryos of method of genotyping species with no reference genomes (Poland Rusty/D. villosum hybridizations were removed from the spikes and Rife 2012; Poland et al. 2012). GBS reduces the genome infor- 14 days after pollination. Immature embryos were sterilized with mation by using restriction enzymes targeting intron and exon 20% commercial bleach solution (containing 6% NaOCl) for regions to capture the polymorphic regions. This approach is espe- 20 min followed by rinsing with sterilized water three times in a lam- cially suitable for species such as D. villosum with limited sequence inar hood. Immature embryos were isolated under a dissecting scope. information, because it can provide de novo sequence and marker in- The embryos were placed on wheat embryo rescue (ER) me- formation simultaneously. dium containing Murashige and Skoog (MS) basal salt mixture In this study, we created T. durum–D. villosum amphiploids from four D. villosum accessions. These amphiploids were screened with TTKSK and the resistant lines were advanced to develop wheat– Table 2. Parental and Rusty-Dasypyrum villosum amphiploid infection types in response to Puccinia graminis race TTKSK at seedling stage D. villosum single-chromosome introgression lines. A wheat–D. villosum monosomic substitution line containing D. villosum chro- Infection typea mosome 2V conferred TTKSK resistance. To facilitate the transfer D. villosum accessions Origin Parent A B of this new stem rust resistance gene to wheat, GBS was used to ge- notype a complete set of Chinese Spring (CS)–D. villosum addition TA2126 Croatia 0; 13- 0; lines to anchor D.