Target Gene Cloning And Molecular Mechanism Elucidating For Major QTLs Of Pod Shattering Resistance In Brassica Napus L.

Oilseed rape(Brassica napus L.)is a leading oil crop in China.Currently,a prominent problem confronting rapeseed mechanical harvest is the serious yield loss resulting from pod shattering,which usually accounts for 20%yield loss during improper harvesting period.Genetic improvement of rapeseed pod shattering resistance is an important way to solve this problem.Therefore,it is of great scientific signifcance and industry value to discover favorable alleles for pod shattering resistance and understand the underlying molecular mechanism.Previously,we have identified two major QTL qSRI.A9.1 and qSRI.A9.2 for pod shattering resistance on chr A09,which can explain 11.26-12.07%and 27.58-38.11%phenotypic variation respectively.In this study,we successfully cloned the target genes for qSRI.A9.1 and qSRI.A9.2 and unraveled the underlying molecular mechanism of pod shattering resistance.One Arabidopsis SHP1 homologue located near the peak of qSRI.A9.1 interval was chosen as the candidate gene and named as BnSHP1.A9.Overexpression of BnSHP1.A9 in R1 which is resistant to pod shattering promoted pod shattering significantly.A 4.8 kb copia retrotransposon insertion was identified 252 bp upstream of ATG in BnSHP1.A9R2.BnSHP1.A9R1 was highly expressed in flower and siliques at different time point after flowering.In contrast,BnSHP1.A9R2 appears silenced in the corresponding organs and the repression of BnSHP1.A9R2 expresson is correlated with upstream copia retrotransposon insertion in two DH lines.Bisulfate sequencing revealed a high DNA methylation level in the copia retrotransposon sequence which may be responsible for the silence of BnSHP1.A9R2 and contribute to the positive effect for pod shattering resistance at qSRI.A9.1 in R2.We further identified five 24 nt siRNAs that might originate from the copia retrotransposon by small RNAs sequencing,suggesting that RdDM pathway may involve in the DNA methylation of copia retrotransposon and play a vital role in pod shattering resistance conferred by BnSHP1.A9R2.qSRI.A9.2 was defined at a 183 kb interval corresponding to the Darmor-bzh reference genome which contains 32 annotated genes.One Arabidopsis NST2 homologue was chosen as a candidate gene for qSRI.A9.2 and named as BnNST2.A9.Parental sequencing of BnNST2.A9 showed that the coding sequence of BnNST2.A9R2 is 969 bp in length with two exons.BnNST2.A9R1 harbors a "C" deletion and 629 bp Solo-LTR insertion sequence in exon 2.The "C" deletion causes a frameshift mutation.Transgenic overexpression,genetic complement and targeted mutagenesis by CRISPR-Cas9 confirmed a positive role of BnNST2.A9R2 in pod shattering.BnNST2.A9R1 is a null allele conferring pod shattering resistance and is the target gene of qSRI.A9.2.BnNST2.A9R2 encodes a typical NAC transcription factor with transcriptional activation activity and nuclear localization.High expression level of BnNST2.A9R2 are found in vascular bundles and other cell types with secondary wall deposition across the plant.Both overexpression and genetic complement of BnNST2.A9R2 led to remarkable increment of cell density in endocarp b layer and secondary wall deposition,but didn’t cause any changes in valve margin.RNA sequencing and qRT-PCR showed that many genes involved in secondary wall biosynthesis were upregulated in BnNST2.A9R2 OE lines.MYB46 homologue genes which are the key regulators of secondary wall formation were significantly induced by BnNST2.A9R2.EMSA and dual luciferases assay revealed that BnNST2.A9R2 could bind to a conserved SNBE1 cis-element in the promoter of six MYB46 homologues and activated the transcription of five MYB46 homologues.Overexpression of three MYB46 homologues in R1 respectively have also induced an enhanced cell density of endocarp b layer in silique valves,suggesting that MYB46 genes are direct targets of BnNST2.A9R2.In conclusion,two target genes of the major QTLs on A09 for pod shattering resistance in B.napus were cloned and the molecular mechanism of the resistant genes was revealed.A BnSHP1.A9BnNST2.A9R2-MYB46 module functions in secondary wall biosynthesis of silique valves and pod shattering in rapeseed was established,providing evidences to link previously seperate transcription regulation network of secondary wall formation and silique dehiscence together as well as new insights into molecular mechanism of pod shattering resistance.