Leafy head is an important agronomic trait that determines the yield and quality of Chinese cabbage. The molecular mechanism underlying heading in Chinese cabbage has been the focus of research, and wide leaves are a prerequisite for leafy head formation. In our study, two allelic leafy heading-deficient mutants (lhd1 and lhd2) with narrow leaf phenotypes were screened in an ethyl methanesulfonate mutagenized population from a heading Chinese cabbage double haploid line “FT”. Genetic analysis revealed that the mutant trait was controlled by a recessive nuclear gene, which was found to be BraA10g000480.3C by MutMap and Kompetitive allele-specific PCR analyses. As BraA10g000480.3C was the ortholog of ANGUSTIFOLI in Arabidopsis, which has been found to regulate leaf width by controlling cortical microtubule arrangement and pavement cell shape, we named it BrAN. BrAN in mutant lhd1 carried an SNP (G to A) on intron 2 that co-segregated with the mutant phenotype, and disrupted the exon-intron splice junction generating intron retention and a putative truncated protein. BrAN in mutant lhd2 carried an SNP (G to A) on exon 4 leading to a premature stop codon. The ectopic overexpression of BrAN restored normal leaf phenotype due to abnormal cortical microtubule arrangement and pavement cell shape in the Arabidopsis an-t1 mutant. However, transformation of Bran did not rescue the an-t1 phenotype. These results indicate that BrAN contributes to leafy head formation of Chinese cabbage.
Leaf flattening plays a vital role in the establishment of plant architecture, which is closely related to plant photosynthesis and, thus, influences the product yield and quality of Chinese cabbage. In this study, we used the doubled haploid line ‘FT’ of Chinese cabbage as the wild type for ethyl methanesulfonate (EMS) mutagenesis and obtained a mutant cwm with stably inherited compact and wrinkled leaves. Genetic analysis revealed that the mutated trait was controlled by a single recessive nuclear gene, Brcwm. Brcwm was preliminarily mapped to chromosome A07 based on bulked segregant RNA sequencing (BSR-seq) and fine-mapped to a 205.66 kb region containing 39 genes between Indel12 and Indel21 using SSR and Indel analysis. According to the whole-genome re-sequencing results, we found that there was only one nonsynonymous single nucleotide polymorphism (SNP) (C to T) within the target interval on exon 4 of BraA07g021970.3C, which resulted in a proline to serine amino acid substitution. The mutated trait co-segregated with the SNP. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that BraA07g021970.3C expression was dramatically higher in ‘FT’ leaves than that in cwm leaves. BraA07g021970.3C is homologous to AT3G55000 encoding a protein related to cortical microtubule organization. A similar phenotype of dwarfism and wrinkled leaves was observed in the recessive homozygous mutant cwm-f1 of AT3G55000, and its T3 transgenic lines were restored to the Arabidopsis wild-type phenotype through ectopic overexpression of BraA07g021970.3C. These results verified that BraA07g021970.3C was the target gene essential for leaf flattening in Chinese cabbage.
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