Screening of Candidate Leaf Morphology Genes by Integration of QTL Mapping and RNA Sequencing Technologies in Oilseed Rape (<i>Brassica napus</i> L.)

<div><p>Leaf size and shape play important roles in agronomic traits, such as yield, quality and stress responses. Wide variations in leaf morphological traits exist in cultivated varieties of many plant species. By now, the genetics of leaf shape and size have not been characterized in <i>Brassica napus</i>. In this study, a population of 172 recombinant inbred lines (RILs) was used for quantitative trait locus (QTL) analysis of leaf morphology traits. Furthermore, fresh young leaves of extreme lines with more leaf lobes (referred to as ‘A’) and extreme lines with fewer lobes (referred to as ‘B’) selected from the RIL population and leaves of dissected lines (referred to as ‘P’) were used for transcriptional analysis. A total of 31 QTLs for the leaf morphological traits tested in this study were identified on 12 chromosomes, explaining 5.32–39.34% of the phenotypic variation. There were 8, 6, 2, 5, 8, and 2 QTLs for PL (petiole length), PN (lobe number), LW (lamina width), LL (Lamina length), LL/LTL (the lamina size ratio) and LTL (leaf total length), respectively. In addition, 74, 1,166 and 1,272 differentially expressed genes (DEGs) were identified in ‘A vs B’, ‘A vs P’ and ‘B vs P’ comparisons, respectively. The Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to predict the functions of these DEGs. Gene regulators of leaf shape and size, such as <i>ASYMMETRIC LEAVES 2</i>, <i>gibberellin 20-oxidase 3</i>, genes encoding gibberellin-regulated family protein, genes encoding growth-regulating factor and KNOTTED1-like homeobox were also detected in DEGs. After integrating the QTL mapping and RNA sequencing data, 33 genes, including a gene encoding auxin-responsive GH3 family protein and a gene encoding sphere organelles protein-related gene, were selected as candidates that may control leaf shape. Our findings should be valuable for studies of the genetic control of leaf morphological trait regulation in <i>B</i>. <i>napus</i>.</p></div

Expression analysis of the orthologous genes that influence leaf morphology in <i>Arabidopsis</i>.

<p>A: Extreme lines with more lobes; B: extreme lines with fewer lobes; P: dissected lines.</p

Locations of significant QTLs for leaf morphological traits on the high-density SNP map.

<p>For simplicity, only the markers in the QTL confidence intervals, along with the two terminal markers at each end of the QTL-containing chromosomes, are shown. Full map data are provided in Liu et al., 2013. LTL: leaf total length (cm); LW: lamina width (cm); LL: lamina length (cm); PL: petiole length (cm); PN: lobe number; LL/LTL: the ratio of lamina width: leaf total length.</p

Screening of Candidate Leaf Morphology Genes by Integration of QTL Mapping and RNA Sequencing Technologies in Oilseed Rape (<i>Brassica napus</i> L.) - Fig 1

<p>Graphical representation of leaf morphological traits measured in this study (a), extreme lines (b). LL: lamina length; LW: lamina width; PL: petiole length; LTL: leaf total length and L: lobe(s). A: Extreme lines with more lobes; B: extreme lines with fewer lobes; P: dissected lines.</p

Significant QTLs associated with leaf morphological traits in the RIL population.

<p>LTL: leaf total length (cm); LW: lamina width (cm); LL: lamina length (cm); PL: petiole length (cm); PN: lobe number; LL/LTL: the ratio of lamina width: leaf total length. a, peak SNP location of the QTL; b, an additive value >0 indicates that additive effects came from GH06, or came from P174 otherwise; c, thresholds values; d, QTL size (cM); e, phenotypic variation.</p

Expression analysis of candidate genes for the regulation of leaf development identified using QTL and RNA-Seq analyses.

<p>A: Extreme lines with more lobes; B: extreme lines with fewer lobes; P: dissected lines.</p

Summary of read numbers from the RNA-Seq data for the three samples.

<p>A: Extreme lines with more lobes; B: extreme lines with fewer lobes; P: dissected lines.</p

qRT-PCR validation of the expression patterns of 25 randomly selected genes identified in transcriptome sequencing.

<p>A: Extreme lines with more lobes; B: extreme lines with fewer lobes; P: dissected lines.</p

Genome-Wide Association Mapping of Seed Coat Color in Brassica napus

Seed coat color is an extremely important breeding characteristic of Brassica napus. To elucidate the factors affecting the genetic architecture of seed coat color, a genome-wide association study (GWAS) of seed coat color was conducted with a diversity panel comprising 520 B. napus cultivars and inbred lines. In total, 22 single-nucleotide polymorphisms (SNPs) distributed on 7 chromosomes were found to be associated with seed coat color. The most significant SNPs were found in 2014 near Bn-scaff_15763_1-p233999, only 43.42 kb away from BnaC06g17050D, which is orthologous to Arabidopsis thaliana TRANSPARENT TESTA 12 (<i>TT12</i>), an important gene involved in the transportation of proanthocyanidin precursors into the vacuole. Two of eight repeatedly detected SNPs can be identified and digested by restriction enzymes. Candidate gene mining revealed that the relevant regions of significant SNP loci on the A09 and C08 chromosomes are highly homologous. Moreover, a comparison of the GWAS results to those of previous quantitative trait locus (QTL) studies showed that 11 SNPs were located in the confidence intervals of the QTLs identified in previous studies based on linkage analyses or association mapping. Our results provide insights into the genetic basis of seed coat color in B. napus, and the beneficial allele, SNP information, and candidate genes should be useful for selecting yellow seeds in B. napus breeding

Frequency distributions of leaf morphological traits in RIL lines.

<p>LTL: leaf total length (cm); LW: lamina width (cm); LL: lamina length (cm); PL: petiole length (cm); PN: lobe number; LL/LTL: the ratio of lamina width: leaf total length.</p