Fine Mapping of a Locus Underlying the Ectopic Blade-Like Outgrowths on Leaf and Screening Its Candidate Genes in Rapeseed (Brassica napus L.)

Abstract

Leaf is an important organ for higher plants, and the shape of it is one of the crucial traits of crops. In this study, we investigated a unique aberrant leaf morphology trait in a mutational rapeseed material, which displayed ectopic blade-like outgrowths on the adaxial side of leaf. The abnormal line 132000B-3 was crossed with the normal line 827-3. Based on the F_{2 : 3} family, we constructed two DNA pools (normal pool and abnormal pool) by the bulked segregant analysis (BSA) method and performed whole genome re-sequencing (WGR), obtaining the single-nucleotide polymorphism (SNP) and insertion/deletion (InDel) data. The SNP-index method was used to calculate the Δ(SNP/InDel-index), and then an association region was identified on chromosome A10 with a length of 5.5 Mbp, harboring 1048 genes totally. Subsequently, the fine mapping was conducted by using the penta-primer amplification refractory mutation system (PARMS), and the associated region was narrowed down to a 35.1-kbp segment, containing only seven genes. These seven genes were then analyzed according to their annotations and finally, BnA10g0422620 and BnA10g0422610, orthologs of LATE MERISTEM IDENTITY1 (LMI1) gene from Arabidopsis and REDUCED COMPLEXITY (RCO) gene from its relative Cardamine hirsuta, respectively, were identified as the candidate genes responding to this blade-like outgrowth trait in rapeseed. This study provides a novel perspective into the leaf formation in Brassica plants.

Keywords: Brassica napus; LATE MERISTEM IDENTITY1; REDUCED COMPLEXITY; aberrant leaf; fine mapping; whole genome re-sequencing.

FIGURE 1

The schematic diagram for constructions of F_2:3 family and the two DNA pools.

FIGURE 2

The ectopic blade-like outgrowth phenotype. (a) A normal leaf from rapeseed line 827-3; (b) the abnormal leaf from 132000B-3. One ectopic blade-like outgrowth on the adaxial side of leaf developed margins and veins, but no obvious petioles.

FIGURE 3

The associated region on chromosome A10 identified by SNPs and InDels. (A) SNP-associated region; (B) InDel-associated region; (C) overlapped associated region. The peak regions above the threshold values are defined as an associated region. The x-axis represents chromosomal position. The y-axis shows the Δ(SNP-index), Δ(InDel-index), or Δ(ALL-index), respectively. Blue line: the threshold value line for the 95% confidence intervals (P-value = 5%).

FIGURE 4

Fine mapping of the candidate gene by PARMS. (A) A partial physical map of PARMS markers on chromosome A10 of rapeseed; (B) the locus was further located between Bn2132 and Bn2136; (C) genotyping in recombinant plants 187–192, 179–199, 197–018, 198–069, and 197–082.