Fine mapping of a leaf flattening gene Bralcm through BSR-Seq in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Leaf flattening influences plant photosynthesis, thereby affecting product yield and quality. Here, we obtained a stably inherited leaf crinkled mutant (lcm), derived from the Chinese cabbage doubled haploid (DH) ‘FT’ line using EMS mutagenesis combined with isolated microspore culture. The crinkled phenotype was controlled by a single recessive nuclear gene, namely Bralcm, which was preliminarily mapped to chromosome A01 by bulked segregant analysis RNA-seq, and further between markers SSRS-1 and IndelD-20 using 1,575 recessive homozygous individuals in F2 population by a map-based cloning method. The target region physical distance was 126.69 kb, containing 23 genes; the marker SSRMG-4 co-segregated with the crinkled trait. Further, we found SSRMG-4 to be located on BraA01g007510.3C, a homolog of AHA2, which encodes H+-ATPase2, an essential enzyme in plant growth and development. Sequence analysis indicated a C to T transition in exon 7 of BraA01g007510.3C, resulting in a Thr (ACT) to Ile (ATT) amino acid change. Genotyping revealed that the leaf crinkled phenotype fully co-segregated with this SNP within the recombinants. qRT-PCR demonstrated that BraA01g007510.3C expression in lcm mutant leaves was dramatically higher than that in wild-type ‘FT’. Thus, BraA01g007510.3C is a strong candidate gene for Bralcm, and AHA2 is possibly associated with leaf flattening in Chinese cabbage.