Insertion of a large retrotransposon TNT fragment in the promoter region of <i>Brgl1</i> results in a gray leaf phenotype in pakchoi

In plants, pentatricopeptide repeat (PPR) proteins perform post-transcriptional modifications on RNA and regulate photosynthesis. In this study, a natural gray leaf mutant, <italic>M579</italic>, was identified during the self-breeding process of the pakchoi (<italic>Brassica campestris</italic> L. ssp<italic>. chinensis</italic>) double haploid (DH) line '579'. The chlorophyll content and net photosynthetic rate of <italic>M579</italic> were lower than those of '579'. Mutant <italic>M579</italic> exhibited defective chloroplast development, characterized by a reduced number of grana stacks. Genetic analysis indicated that the gray leaf phenotype was controlled by a single pair of recessive nuclear genes, subsequently named <italic>Brgl1.</italic> Using bulked segregant analysis sequencing, <italic>Brgl1</italic> was initially mapped to chromosome A06. Further fine mapping using 1,877 recessive homozygous individuals from the F₂ population, together with a map-based cloning approach, localized <italic>Brgl1</italic> between markers SIN-33 and SIN-34. The target region spanned a physical distance of 8.069 kb and contained four genes. Further cloning revealed a 4,788 bp insertion in the promoter region of <italic>BraA06g036440.3C</italic> within the mapped interval, and this inserted fragment was identified as the retrotransposon TNT 1-94, suggesting that <italic>BraA06g036440.3C</italic> was the candidate gene for <italic>Brgl1</italic>. <italic>BraA06g036440.3C</italic> belonged to the P subfamily of the PPR gene family, encoding a hydrophilic, non-secretory protein with seven tandemly repeated PPR motifs. GUS activity analysis showed that <italic>Brgl1</italic> was primarily expressed in the leaves. The relative expression level of <italic>Brgl1</italic> was reduced in <italic>M579</italic> leaves. This study provides significant information regarding the molecular mechanisms underlying leaf color mutations in pakchoi.