Material (Recipients, Donors, and Markers)
Recipient parents. Eleven (11) elite Indian bread wheat cultivars that were used in the present study as recipients included the following: (i) six cultivars (HD2967, PBW550 (Yr15), DBW17, PBW621, UP2338, and UP2382) cultivated in north western plain zone, (ii) two cultivars (HUW234 and HUW468) grown in north eastern plain zone, (iii) two cultivars (MACS 2496 and NI5439) used in peninsular zone and the (iv) a solitary cultivar (Lok1) is cultivated in central zone (Fig. S1). All recipient cultivars are cultivated under timely or late sown (HUW234) and irrigated conditions; only cv. NI5439 of peninsular zone is recommended for cultivation under rainfed and timely sown conditions.
Donors with desirable genes. The donors used in the present study included the following: (i) PBW343 (Gpc-B1/Yr36+Lr24), which is an improved version of an erstwhile popular cultivar PBW343, which later became susceptible to yellow rust, and was also resurrected through pyramiding of leaf and stripe rust resistance genes (Kumar et al. 2011; Sharma et al. 2021). (ii) Glupro (Gpc-B1/Yr36), an exotic genotype earlier developed by Jorge Dubcovsky and his team at the University of California (Davis), USA (Khan et al. 2000; Uauy et al. 2006).
Markers for foreground and background selections. For foreground selection following markers were used: (i) Gpc-B1 gene: Xuhw89, a marker closely linked (0.1 cM) to Gpc-B1 (Distelfeld et al. 2006), and Xucw108, a gene-based co-dominant functional marker (Uauy et al. 2006). (ii) Lr24: SCAR marker XSCS73719 (Prabhu et al. 2004). (iii) Yr15: gwm413 (Murphy et al. 2009); (iv) Yr36 (linked to Gpc-B1) gene-based markers: Xucw130 (Fu et el. 2009). For background selection, polymorphic SSR markers ranging in number from 102 to 126 were used.
Scheme for MABB
The breeding scheme followed for MABB is presented in Fig. 1. Eleven (11) crosses were made involving 11 recipient and the two donor genotypes [the donor genotype PBW343 (Gpc-B1/Yr36+Lr24) was used for eight recipients and Glupro (Gpc-B1/Yr36) was used for the remaining three recipients]. In BC1F1 to BC3F2 populations, foreground selection was exercised in order to recover plants heterozygous/homozygous for the desired allele from the donor parent. The positive plants in BC1F2/BC2F2/BC3F2 were selfed to obtain BC1F3/BC2F3/BC3F3 progenies, which were advanced up to BC1-3F4-7 generations before conducting field trial of MABB-derived progenies. Background selection accompanied the foreground selection up to BC1F3/BC2F3/BC3F3 generations; in some cases, background selection was performed after the recovery of the homozygous and stable progenies in order to estimate the percent recurrent parent genome recovery. Phenotypic selection was carried out whenever necessary in order to derive agronomically superior progenies.
DNA isolation, PCR, and marker analysis. DNA isolation was carried out using leaf samples of 30–40 days old plants by the CTAB method following Saghai-Maroof et al. (1984) with some modifications. DNA quality was checked on 1% agarose gels and quantification was carried out using Pico2000. Polymerase chain reaction (PCR) was carried out in 12-20 μL reaction volume containing 25–50 ng of genomic DNA, 1-Unit Taq polymerase (Sigma-Aldrich, USA/Bangalore Genie, India), 0.02 mM of each of four dNTPs (Sigma-Aldrich, USA/Bangalore Genie, India), 0.2 μM of forward and reverse primers (synthesized by IDT, USA), and 1X PCR buffer (10 mM Tris–HCl pH 8.4). PCR was performed using Applied Biosystem’s Thermal Cycler using the following PCR profile: 94 °C for 4-5 min, followed by 35 cycles of 94 °C for 30-60s, 57-60 °C (depending on the primer sequences) for 30s, and 72 °C for 1 min, with a final extension at 72 °C for 7-8 min. The amplified products were resolved on 2-2.5% agarose gel and visualized following ethidium bromide staining or on 6% PAGE and visualized following silver staining.
Grain quality tests: Percent grain protein content (GPC) was determined using Near-Infrared Transmittance in 1241 Grain Analyzer (FOSS, Sweden). The contents of Fe (ppm) and Zn (ppm) in the grains were estimated using Energy-Dispersive X-ray fluorescence (EDXRF spectrometer X-Supreme8000) (Paltridge et al. 2012). Sedimentation volume was worked out using micro-SDS sedimentation volume (MSDS-SV) test following Dick and Quick (1983).
Micro-baking for loaf volume: For estimation of loaf volume, the formulation included flour (100 parts), water (65%), salt (2 parts), fresh yeast (2.5 parts), which were mixed in the 10-g mixograph. The resulting dough which was molded, rested for 20 min at 28°C, remolded, proofed for 45 min at 28°C and 90% relative humidity, and baked at 200°C for 17 min (Gras and Bekes 1996). Loaf volume was measured by the mustard seed displacement method. Baking tests were done in duplicate.
SDS-PAGE (polyacrylamide gel electrophoresis): The proteins of flour samples were extracted and fractionated in 10% polyacrylamide gels using the method of Laemmli (1970) as modified by Payne et al. (1980).
The details of field experiments conducted for evaluation of MABB-derived progenies in the background of 11 recipient cultivars are summarized in Table S1. The number of progenies in the different recipient genetic backgrounds varied from 1 to 24. The experiments were conducted in randomized block designs each with 2-3 replications over 1-3 years and 1-5 locations. The plot size differed in different experiments with the largest plot size comprising 12 rows of 6m each and smallest plot size comprising 4 rows of 3m each (Table S1). Standard cultural practices were followed to raise the crop at different locations and years. In each plot, data were recorded on 1000-grain weight (g), grain yield (t/ha or q/ha), and GPC (%).
Screening for Rusts
Glass-house screening against three rusts. MAS-derived progenies in the backgrounds of cvs. Lok1 and HD2967 were screened for resistance to three rusts. Altogether, 16 pathotypes of the three rust pathogens occurring in different wheat-growing regions of India were used for this purpose. Screening for rust resistance of the MAS-derived progenies along with their recipient parents was carried out as described in Gautam et al. (2020). The seedlings were assayed for the infection types (ITs) against each pathotype at 15 days post-inoculation following Stakman et al. (1962). IT0 indicated resistant; ITs 1, 12, 2−, 2, and 22+ indicated moderately resistant; ITs 23 and 3 indicated moderately susceptible; and ITs 33+ and 3+ indicated susceptible. ITs 12, 22+, 23, and 33+ were based on the following criteria: 12 = small uredia with distinct chlorotic or necrotic areas; 22+ = uredia size similar to IT 2 but with more sporulation; 23 = same uredia size as for IT 3 but chlorosis is more pronounced; and 33+ = large uredia, coalescing with minor chlorosis or necrosis. Plus (+) and minus (-) signs indicate variation within a given infection type.
Field screening against leaf rust and stripe rust: Screening of MAS-derived progenies in the backgrounds of three recipient cvs. PBW550 (Yr15), DBW17, and PBW621 was done in field under artificial rust epidemics created by spraying the experimental material with the mixture of uredinospores of prevalent races of stripe rust (78S84, 49S119 196, and 110S119) and leaf rust (077-5, 104-2 + unknown races collected from farmer's field). Details of races and their virulence formulae are available elsewhere (Sharma et al. 2021).
For screening of the progenies for rust resistance under field conditions, the seeds of the progenies and the parents were planted in a non-replicated augmented block design in paired rows of 1 m. The distance within the paired rows was 22 cm and between two paired rows was 30cm. The planting of the seed material was done in the first fortnight of November each year. PBW621, PBW550, DBW17, PBW343, Agra local (for stripe rust and leaf rust) andC306 (for brown rust), each susceptible to a mixture of prevalent pathotypes having virulence for genes under consideration were planted as infector rows (at every 7th paired row) and in spreader rows (perpendicular to the 1 m paired rows) surrounding the plot for establishing sufficient inoculum. To ensure uniform disease distribution, rust infected pots were placed in fields between the experimental materials, and the spores that appeared naturally in the spreader rows were used to inoculate the infector rows.
For creating stripe rust epidemics, repeated spray inoculations with uredinospores of Puccinia striiformis were carried out. The inoculation was carried out in evening with an ultralow volume sprayer on alternate days beginning from the end of December to the end of January till stripe rust appeared on the susceptible checks/parents. For this purpose, the infected leaves of susceptible host (which was pre-inoculated to multiply the pathogen) were collected and immersed in water for extracting uredinospores. The inoculum was prepared by suspending rust uredinospores (@5.6 g/ha, which equates to 1000 spores per plant (Imtiaz et al. 2003) in 10 l of water using 5-7 drops of Tween-20.
The response to rusts was recorded at the reproductive stage using disease severity (DS) and infection response (IR) as the two measures according to the modified Cobb scale (Peterson et al. 1948). DS was measured as an estimation of percentage coverage (0, 5, 10, 187 20, 40, 60, 80, and 100) of rust pustules (uredinia) seen on the flag leaf. IR was scored as a reaction of the host to rust infection and was categorised as 0 =immune; R = resistant, MR = moderately resistant; MS = moderately susceptible and S = susceptible. Data were recorded three times at equal intervals (starting mid-January) when the flag leaves of the susceptible check cultivars showed a disease score of 80S (DS: 80; IR: S). Out of these three scores of a test line, the highest score toward susceptibility was used for the subsequent analysis.
Means and ANOVA: The means were calculated using Excel. The analysis of variance (ANOVA) was carried out using SPSS Statistics for Windows, version 16.0 (SPSS Inc., Chicago, Ill., USA).
Estimation of the recovery of recipient genome (RPG): The recovery of RPG was estimated using the following formula: RPG= [(X+1/2Y)/N] x 100. Where, X is the number of homozygous marker loci for recurrent parent allele, Y is the number of heterozygous marker loci for the parental alleles and N is the total number of parental polymorphic markers used for screening.