Identification of candidate genes associated with phenotypic
plasticity through GWAS
Within the LD interval upstream and downstream of the significant SNPs
detected by GWAS, the B. napus homologs for seven candidate genes
known to participate in the lipid metabolism of Arabidopsis seeds
(http://aralip.plantbiology.msu.edu/) were detected: PMT6(BnaA05G0437100ZS), DAO1 (BnaA09G0636200ZS), MYB106(BnaA01G0418000ZS), HAD (BnaC02G0039300ZS), MIPS3(BnaC02G0039400ZS), DGD1 (BnaC05G0484900ZS) and PSD1(BnaC09G0366500ZS).
To investigate the variation in the promoter regions of these genes, the
methodology described by Li was followed(Li et al., 2021). In detail,
sequences surrounding the genes (PMT6 and DGD1 ) implicated
in SOC phenotypic plasticity were retrieved from the B. napuspan-genome information resource (BnPIR; http://cbi.hzau.edu.cn/bnapus/)
for the inbred lines ZS11, Zheyou7, Gangan, Shengli, No2127, Westar,
Quinta and Tapidor. The sequences of these two genes were also extracted
from this website. The colinear sequences among these eight inbred lines
were used as queries for a BLAST search against each other to identify
structural variations. Transposon elements (TEs) present in the
sequences were identified with a TE Library constructed by Extensive
de-novo TE Annotator (EDTA) (Ou et al., 2019). In addition, sequence
variation within the genes (DAO1, MYB106 and HAD ) was
analyzed with polyphen-2 (http://genetics.bwh.harvard.edu/pph2/)
and provean (http://provean.jcvi.org/), noting putative loss-of-function
alleles for each gene as predicted by at least one of the software
platforms (Supplementary Table S16).
To model the genetic effect dynamics along the environmental gradient,
SNPs significantly associated with slope were tested for association
with SOC within each environment using the mixed-model method in GAPIT.
These separate genetic effects were then regressed on the environmental
gradients to generate the fitted lines as the genetic effect continua,
defined as varied genetic effects with different environmental inputs
(Fig. 5D-F).
We used RNA sequencing data from 289 lines (subset of 505 lines) of
developing seeds at 20 days after flowering in WH2016 (Tang et al.,
2021). The expression levels of the plasticity genes were determined by
the abundance of transcripts and compared between two alleles with a
Wilcoxon test.