Volume 9, Issue 12, Pages 1559-1569 (December 2016) Deletion of a Stay-Green Gene Associates with Adaptive Selection in Brassica napus  Lunwen Qian, Kai Voss-Fels, Yixin Cui, Habib U. Jan, Birgit Samans, Christian Obermeier, Wei Qian, Rod J. Snowdon  Molecular Plant  Volume 9, Issue 12, Pages 1559-1569 (December 2016) DOI: 10.1016/j.molp.2016.10.017 Copyright © 2016 The Author Terms and Conditions

Figure 1 Detailed SNP Associations to Leaf CCI in the BnNYE1-A01 Gene Region of B. napus Chromosome A01 in 203 Chinese Semi-Winter Rapeseed Accessions. (A) The blue horizontal line indicates a threshold of genome-wide significance at a p value of 1.0 × 10−4. The green dots indicate the SNPs with the most significant associations to leaf CCI in the greenhouse in this haplotype region. The position of BnNYE1-A01 (BnaA01g22870D) is marked. The two SNPs labeled in red are located within this gene. The heatmap spans the SNP markers in LD with the most strongly associated SNPs. (B) Three haplogroups with frequency greater than 0.01 were identified in the BnNYE1-A01 haplotype region. The boxplots show that BnCCI-Hap2 has a higher leaf CCI than the other two haplogroups. The symbols indicate significant differences in the haplogroups compared with Hap2: *p ≤ 0.05, **p ≤ 0.01. Red triangles, not significant (p > 0.05). Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 2 Chlorophyll Content Phenotypes for SNP Haplogroups (A) The SNPs Bn-A01-p17905950 and Bn-A01-p17906322, located in intron 1 and exon 3 of BnNYE1-A01 (BnaA01g22870D) within a 0.63-kb haplotype block, exhibit significant associations with leaf CCI. (B) Boxplots showing the leaf CCI values for the three haplogroups with a frequency greater than 0.01 within the BnNYE1-A01 gene haplotype region. The symbols indicate significant differences in the haplogroups compared with BnNYE1-A01-Hap2 *p ≤ 0.05, **p ≤ 0.01. Black triangles, genotypes carrying BnNYE1-A01-Hap2. Red triangles, not significant (p > 0.05). Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 3 Phenotypic Values for Leaf CCI Related to Haplogroups. (A) Comparative analysis of CCI-Hap2 and BnNYE1-A01-Hap2 related to the leaf CCI. The haplogroups exhibited similar leaf CCI phenotype variations. (B) Comparative analysis of 16, 7, and 11 accessions, respectively, from BnNYE1-A01-Hap1, BnNYE1-A01-Hap2, and BnNYE1-A01-Hap3 corresponding to the leaf CCI in the greenhouse in 2014. The symbols indicate significant differences in the haplogroups compared with BnNYE1-A01-Hap2: *p ≤ 0.05 and **p ≤ 0.01. Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 4 Assay of Bn-A01-p17905950 and Bn-A01-p17906322 Showing Multiple Clusters in 203 Chinese Semi-winter Rapeseed Accessions Using Default Parameters. The red cluster represents AA homozygotes, the green AB heterozygotes, and the dark golden BB homozygotes, while the dark cluster group represents the same seven genotypes where these two SNPs were not called (NC). Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 5 A-subgenome Genetic Relationships Among the Diversity Panel. (A) Unweighted pair group matrix algorithm (UPGMA) genetic distance tree describing the genetic relationships among three natural ecotypes of rapeseed, the synthetic B. napus and B. rapa accessions, using 3000 SNPs from the A-subgenome with minor allele frequency (MAF) of ≥0.05. (B) Genetic distances based on the 3000 A-subgenome SNPs, revealing the close genetic relationship among the eight Asian B. rapa, eight resynthesized B. napus, and 60 Asian semi-winter rapeseed accessions. Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 6 Haplogroup Analysis of the BnNYE1-A01 Gene Region in the Three Rapeseed Ecotypes and B. rapa Accessions. Three haplogroups with a frequency greater than 0.01 were estimated in different population groups. Haplogroup BnNYE1-A01-Hap2 was only found in the semi-winter rapeseed and Asian groups, whereas BnNYE1-A01-Hap1 and BnNYE1-A01-Hap3 were found in all four material groups. Each circle represents a haplogroup, and the size of the circle is proportional to the number of accessions within the haplogroup: the largest group contains 403 accessions, the second largest 348, and the smallest 10. Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 7 Presence–Absence Analysis of the BnNYE1-A01 (BnaA01g22870D) Gene Region in Eight Synthetic B. napus Accessions, Compared with the Natural B. napus Accession PBY018, by Genomic Resequencing. Presence–absence is determined according to the normalized number of mapped sequence reads per 1000 bp. In five synthetic B. napus accessions (PBY034, PBY037, PBY038, PBY040, and PBY047) the normalized number of mapped reads was less than −1, and the relative coverage (%) less than 50, indicating deletion of BnNYE1-A01. Resynthesized accession PBY048 and the natural B. napus PBY018 show >0.5 coverage across the gene region, indicating presence of BnNYE1-A01. Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 8 Comparative Analysis of the Expression of 39 Differentially Expressed Genes (DEGs) from the Light-Harvesting Chlorophyll a/b Protein Complexes (LHC) in Siliques in the BnNYE1-A01 (Presence) and bnnye1-A01 (Deletion) Groups *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions

Figure 9 Association Mapping of Leaf Chlorophyll Content Index (CCI) and Plant Height (PH) across the 15 122 568- to 15 236 220-bp Haplotype Region of Chromosome A01 in 203 Chinese Semi-winter Rapeseed Accessions. (A) Green and red plots indicating significant associations between leaf CCI and PH in the greenhouse (ss_GH and bs_GH) and field (Germany, 2013 and 2014) in this haplotype region. The positions of B. napus orthologs of the five Arabidopsis genes are marked. (B) The heatmap spans the SNP markers in LD with the most strongly associated SNPs in 203 Chinese semi-winter rapeseed accessions. Molecular Plant 2016 9, 1559-1569DOI: (10.1016/j.molp.2016.10.017) Copyright © 2016 The Author Terms and Conditions