Email: rudra.bhattarai@research.uwa.edu.au

Metribuzin is a broad-spectrum herbicide that can also kill our crops from the same field while controlling the weeds, therefore metribuzin resistance identification in wheat is essential for successful controlling the weeds. One of the best approaches to identifying causal genes or their linked markers for metribuzin resistance in wheat is through the development and characterisation of near isogeneic lines (NILs).

Those causal genes or their linked markers are useful for marker-assisted breeding and selection for metribuzin resistance in wheat. Targeting a major metribuzin resistance Qsns.uwa.4A.2 genomic region, putative NILs were developed using the principle of heterogeneous inbreed family (HIF) method with the assistance of fast generation cycling system (FGCS), in a cross-population of Chuan Mai 25 (Resistance) and Ritchie (Susceptible).

NILs were confirmed through genotype-phenotype association analysis at F8 generation, with 200g ai per ha dose of metribuzin, the recommended dose to control weeds in Australian wheat fields. Pairs of contrasting NILs (NIL_3 and NIL_17) were studied using mRNA sequencing and protein sequencing (NIL_3, NIL_17 and NIL_10) to identify metribuzin resistance candidate genes and candidate proteins. Obtained candidate genes through mRNA sequencing and protein sequencing were validated using qPCR analysis. NILs targeting a major metribuzin resistance locus explaining 69% of the phenotypic variance were developed.

Seven pairs of NILs targeting Qsns.uwa.4A.2 were confirmed through genotype-phenotype association analysis. NILs (NIL_3 and NIL_17) were also validated using qPCR-based gene expression analysis revealed that nitrate excretion transporter and aspartyl protease were the major candidate genes. Novel molecular variants (SNPs at 714355066bp on 4A wheat chromosome) were identified within the gene nitrate excretion transporter (TraesCS4A03G1098900), from which a novel major allele for metribuzin resistance has been identified.

Additionally, other novel molecular variants (SNPs at 739694493bp on 4A wheat chromosome) were identified within the gene aspartyl protease (TraesCS4A03G1181200) from which a novel major allele for metribuzin has been identified within the same QTL region. Proteomic analysis using an isobaric tag (label-based) for relative and absolute quantification (iTRAQ) method identified candidate proteins WD repeats, AB hydrolase_1 and PsbP associated with metribuzin resistance.

The reason for detecting novel candidate genes through mRNA sequencing, but not detecting the same candidate genes/protein through proteomics analysis from the same QTL genomic region could be due to heterozygous novel SNPs obtained through mRNA sequence analysis from a locus linked to the candidate genes where such SNPs combination may not find through using the information of WheatRefSeq2.1 database through Proteomic analysis.

As compared to the allelic combination and their effects within genes TraesCS4A03G1098900 and TraesCS4A03G1181200, combinations of allelic variation could be different with the candidate genes TraesCS4A03G1099000 and TraesCS4A03G1181300 between contrasting isolines, respectively, though they are similar in functions. In this study, identified candidate genes from two different NIL pairs may have multiple novel alleles from a locus responsible for detecting common but some different levels of candidate gene expression between NIL pairs which can be utilised in marker-assisted wheat breeding for metribuzin resistance.