Karnal bunt caused by Tilletia indica is a quarantine disease of wheat causing huge economic losses due to the ban on the import of bunted grains. This study was designed to characterize pathogenicity, aggressiveness and genetic diversity of 68 Tilletia indica isolates collected from different geographic regions of Pakistan. Forty-six isolates were tested for their pathogenicity on eight wheat varieties, out of which three were non-aggressive. The coefficient of infection (CI) ranged from 15.73% (PB-25) to 10% (PB-68, PB-60, and PB-43). The isolates collected from central Punjab showed higher infestation compared to other isolates. Among the wheat varieties used for the aggressiveness study, WL-711 showed susceptible reaction with 10.88% CI, while NIFA-Barsat, HD-29, Janbaz, Bakhtawar-92, Tatara, and AARI 2011 showed resistance to the highly resistant response. These isolates were amplified using 31 random amplified polymorphic DNA (RAPD) markers and 32 inter-simple sequence repeat (ISSR) markers for diversity analysis. The principal component analysis (PCA) and analysis of molecular variance (AMOVA) showed greater divergence among isolates collected from Punjab and Khyber Pakhtunkhwa (KPK), with a moderate level of admixture. The isolates from Faisalabad (Punjab) were more aggressive compared to isolates from KPK and were clearly separated based on PCA, indicating the significant genetic distance in the populations. Our findings will assist breeders and pathologists in better understanding the pathogenic variability in Tilletia indica and in subsequent disease management.

Grain protein content (GPC) is extremely important for common wheat (Triticum aestivum L.) end-use quality. Identifying genetic loci for GPC is helpful in creating new varieties with good processing quality. Linmai2 and Zhong892 are two elite wheat varieties with differences in GPC. In the present study, a recombinant inbred line (RIL) population derived from Linmai2 and Zhong892 was used to identify GPC loci. Linkage mapping was conducted by combining the wheat 90K single nucleotide polymorphisms (SNPs) chip data and GPC from four environments. Five QTLs on chromosomes 1B, 2B, 4A, 4B and 6B were identified, and designated as QGPC.lfnu-1BL, QGPC.lfnu-2BS, QGPC.lfnu-4AS, QGPC.lfnu-4B and QGPC.lfnu-6BS, respectively, explaining 5.21–11.38% of the phenotypic variances. The favorable alleles of QGPC.lfnu-1BL, QGPC.lfnu-4AS, QGPC.lfnu-4B and QGPC.lfnu-6BS originated from Zhong892, while the favorable allele of QGPC.lfnu-2BS came from Linmai2. Among these, QGPC.lfnu-1BL, QGPC.lfnu-2BS, QGPC.lfnu-4AS, and QGPC.lfnu-4B are potentially novel loci, whereas QGPC.lfnu-6BS likely represents the re-detection of Gpc-B1. Additionally, competitive allele-specific PCR (KASP) markers suitable for breeding were developed, and genetic effects were validated in a natural population. Four KASP markers, KASP-GPC-1BL, KASP-GPC-2BS, KASP-GPC-4B and KASP-GPC-6BS, were significantly associated with GPC in a diverse panel of 140 wheat varieties. A total of twelve potential genes involved in nitrogen assimilation, amino acid biosynthesis and transport, and NPF transporter and protein turnover were proposed as candidate genes within the intervals of five QTLs. These findings identified stable QTLs, available KASP markers and promising candidate genes for marker assisted selection to accelerate wheat GPC improvement.