QTL mapping for seedling and adult plant resistance to stripe and leaf rust in two winter wheat populations

The two recombinant inbred lines (RIL) populations developed by crossing Almaly × Avocet S (206 RILs) and Almaly × Anza (162 RILs) were used to detect the novel genomic regions associated with adult plant resistance (APR) and seedling or all-stage resistance (ASR) to yellow rust (YR) and leaf rust (LR). Both the populations were evaluated for YR APR in two environments (2018 and 2019) and LR APR in three environments (2018, 2019, and 2020) in the Anza population and two environments (2018 and 2019) in the Avocet population; both the populations were phenotyped for one environment during 2020 for LR and YR ASR and genotyped using high throughput DArTseq technology. A set of 51 QTLs including 22 for YR APR, nine for LR APR, nine for YR ASR, and 11 for LR ASR were identified. Also, a set of 13 stable QTLs including nine QTLs (QYR-APR-2A.1, QYR-APR-2A.2, QYR-APR-4D.2, QYR-APR-1B, QYR-APR-2B.1, QYR-APR-2B.2, QYR-APR-3D, QYR-APR-4D.1, and QYR-APR-4D.2) for YR APR and four QTLs (QLR-APR-4A, QLR-APR-2B, QLR-APR-3B, and QLR-APR-5A.2) for LR APR were identified. In silico analysis revealed that the key putative candidate genes such as Cytochrome P450, Protein kinase-like domain superfamily, Zinc-binding ribosomal protein, SANT/Myb domain, WRKY transcription factor, Nucleotide-sugar transporter, and NAC domain superfamily were in the QTL regions and involved in the regulation of host response towards the pathogen infection. The stable QTLs identified in this study are useful for developing rust-resistant varieties through marker-assisted selection (MAS).

Methods
Phenotypic dataset
Seedling resistance in greenhouse
The P. striiformis races were differentiated in 2020  using a set of 12 wheat lines developed in the Avocet wheat background and on nine supplemental wheat differentials using a method developed by Johnson et al. 1972. Determination of the type of plant reaction was carried out twice within 14–20 days after infection according to the Gassner and Straib accounting scale (Gassner and Straib, 1932). At the same time, reactions of 0, 1, and 2 points were assigned to the resistant type R (Resistant), and 3 and 4 points were assigned to the susceptible type S (Susceptible). The P. triticina races were also differentiated during 2020 using 20 near-isogenic lines (NILs) developed in Thatcher background each carrying one of the LR-resistant genes. (Kolmer et al., 2014; Schachtel et al., 2012; Kolmer and Ordonez, 2007). The virulence of the phenotypes was determined on these 20 differential lines and encoded with 0 and 1 for avirulence and virulence, respectively (Kolmer and Ordonez, 2007; Long and Kolmer, 1989). The Virulence Analysis Tools (Schachtel et al., 2012) was used for the nomenclature of P. triticina races. The type of response to leaf rust was determined twice within 14-20 days after infection according to the scale of Mains and Jackson (1926). Reactions of 0, 1, and 2 points were assigned to the resistant type R (Resistant), and 3 and 4 points were assigned to the susceptible type S (Susceptible).

The seedlings of the RIL population from Almaly × Avocet S cross along with the parents were inoculated with two races of P. striiformis i.e., 108E187 (Pst_1) and 110E191 (Pst_2) and two races of P. triticina i.e., MLTTH and TLTTR to determine the race-specific resistance. Similarly, the RIL population from Almaly × Anza cross along with parents were inoculated with two races of P. striiformis i.e., 108E187 (Pst_1) and 101E191 (Pst_3) and four races of P. triticina i.e., THTTQ, TCTTR, TCPTQ, and THTTR. The plants were infected with spores at 3-leaf stage and humid chamber was created for 24 hours. The seedling infection type of RIL was scored using the same approach as for rases differentiation. 

Phenotyping for Adult Plant Resistance in Field 
The field phenotyping for YR and LR APR was done during 2018 and 2019 for both the populations and an additional year during 2020 for LR APR for the Anza population at Kazakh Research Institute of Agriculture and Crop Production (KazNIIZiR), Almalybak. Pathogen racial mixtures from the local population were used to inoculate the mapping populations. The method of Roelfs et al. (1992) was followed for spore sampling, storage, and propagation. The pathogen was propagated in a greenhouse on the susceptible wheat variety, Morocco. The experimental wheat material was inoculated with a mixture of spores and talc in the ratio of 1:100 by spraying with an aqueous suspension of spores with 0.001% Tween-80 at a stem elongation stages (Z21-32). After infection, the plots were wrapped with plastic cover for 16-18 hours to create high humidity. After the manifestation of diseases on susceptible control varieties, an assessment (2–3 times) of rust resistance was carried out. Leaf and yellow rust resistance of wheat accessions was evaluated using the modified Cobb scale (Peterson et al., 1948; McIntosh et al., 1995).   The scoring was based both on disease severity (proportion of leaf area infected) and on the plant response to infection (reaction type). Plant responses were recorded as resistant (R), moderately resistant (MR), moderately susceptible (MS), and susceptible (S) reactions.

Genotypic dataset
The genomic DNA was extracted from parents and each RIL from both populations following the modified CTAB (cetyltrimethylammonium bromide) method (Dreisigacker et al., 2012). The DArTseq technology was used for genotyping of both the RILs in Genetic Analysis and Service for Agriculture (SAGA) lab based in Mexico (Edet et al., 2018). Briefly, the sequencing of mapping populations was carried out at 192-plexing on Illumina HiSeq2500 with 1 × 77-bp reads. Allele calls for SNPs were generated through proprietary analytical pipeline developed by DArT P/L (Sansaloni et al., 2011). Further, genetic locations of the SNPs were identified by using 100K consensus map given by SAGA (Sansaloni et al. unpublished).  

The markers were filtered and removed the monomorphic markers, markers with >30% missing data, high heterozygosity percentage (>30%), low allele frequency (<5%) using MS Excel. The BIN functionality in IciMapping 4.2 QTL software was used to remove redundant markers. A filtered set of 1293 and 1127 high-quality SNPs were finally used for QTL analysis in Anza and Avocet populations.