Leaf trichome formation is a very important agronomic trait as it confers resistance to biotic and abiotic stresses, but the causal genes involved in this process in Brassica juncea remain largely unexplored. In this study, we first characterized the haplotypes of BjB02.GL1 among different inbred lines with leaf trichomes or glabrous leaves. A comparative analysis of the number and density of leaf trichomes between the two mustard inbred lines was then performed. BSA analysis of leaves with trichomes and glabrous pools from the F2 segregating population mapped the candidate genes on Chr.A06 and Chr.B02. Two candidate genes, BjA06.GL1 and BjB02.GL1, were subsequently cloned. After sequence alignment of the BjGL1 genes, both single-nucleotide polymorphisms (SNPs) and indel were identified in the BjA06.GL1 and BjB02.GL1 genes. And quantitative real-time polymerase chain reaction (qRT-PCR) analysis further confirmed that both the BjA06.GL1 and BjB02.GL1 genes were more highly expressed in leaves with trichomes than in glabrous leaves. As the leaf size increased, the leaf trichome density decreased. Gene editing of both BjA06.GL1 and BjB02.GL1 changed the leaf trichome to a glabrous leaf phenotype in mustard. In addition, plants with leaf trichomes presented greater resistance to aphids. Taken together, our results revealed that both BjA06.GL1 and BjB02.GL1 positively regulate leaf trichome formation and help increase aphid resistance in mustard. This study provides valuable resources and helps to elucidate the molecular mechanism of leaf trichome formation in B. juncea.

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