A Systematic Dissection Of The Regulatory Mechanisms Underlying The Natural Variation Of Silique Length In Rapeseed

Silique length is an important trait tightly related to seed yield in rapeseed(Brassica napus L.).In rapeseed germplasm,the silique length shows extensive variation,but its regulatory genes and mechanisms are largely unknown.To answer this question,a systematic study at the genetic,physiological,cytological,and molecular levels(including transcriptome sequencing and candidate gene functional validation)was conducted using a core association population and the lines with extreme silique length.The main results are as follow:First,the developmental dynamics of the 23 extreme lines(12 long-and 11 short-silique)were successively measured from the initiation stage of silique development and fix stage of its length.The results showed that the long-and short-silique lines grew fast at 12 to 15 and 9 to 12 days after flowering,respectively;and the silique length difference between two types of lines was due to both growth rate and duration time.Based on this,several indexes(silique phytohormone content,leaf photosynthetic rate,and chlorophyll content,etc.)related to silique development were measured,of these auxin,ethylene and photosynthetic rate showed a significant difference.Second,the 23 extreme lines were subjected to microscopic observation at the initiation stage of silique development and fix stage of its length.The results showed that:(1)On the day of flowering,the average ovary length of long-and short-silique lines was 7.2 and 6.0 mm respectively,the former is only 1.2 times of the latter;and after four weeks after flowering,the average silique length of the long-and short-siliques lines was 78.4 and 38.2 mm respectively,showing 2.1 times difference.(2)From the flowering date to four weeks after flowering,in the long axial section of the silique wall,the average cell number the long-and short-silique lines increased 3.56 times and 2.38 times,respectively,and the cell length increased by an average of 3.04 times and 2.67 times,respectively.(3)The difference in the silique length of the long-and short-silique lines was mainly due to the difference in the number of cells(the former is 1.87 times of the latter),followed by the size of the cell(1.10 times in length).Third,a genome-wide association study was performed using 331 core accessions planted at ten different environments,which identified 13 significantly associated loci on A01,A04,A07,A09,and C03 chromosomes,explaining 6.2-19.2% of the phenotype variance.Of the two associated loci repeatedly detected on the A09 chromosome,qSL.A9-3 was found in almost all the investigated environments and explained the largest phenotypic variance,which was therefore chosen as the target for gene cloning.Based on the published reference genome physical maps,it was found that three associated loci had the same positions as the published silique length QTLs,and the remaining 10 should represent the new loci.Fourth,the two silique wall(at 12-DAF)pools from long-and short-silique lines were used to do transcriptomic analysis,a total of 3248 differentially expressed genes(DEGs)were identified,of which 1116 DEGs were up-regulated,and the other 2132 were down-regulated(long vs short).KEGG analysis showed that these DEGs were significantly enriched in 22 pathways,of which 11 reached extremely significant levels,including RNA,protein,misc,transport,development,hormone metabolism,cell wall,secondary metabolism,DNA,and biodegradation of xenobiotics.Fifth,by integrating GWAS and RNA-seq/function prediction,a total of 24 candidate genes were identified within genomic intervals of seven associated loci,which involving cell wall,protein,redox,bHLH,cell division,WRKY domain,cytochrome P450,F-box,RING/U-box,NAC,hormone(ETH and Aux/IAA,BR)metabolism,photosynthesis,Calvin cycle,RNA helicase,and MYB domain.A total of 17 candidate genes were located within the genomic interval of the target associated loci qSL.A9-3,of which two(BnaA9.ARF18 and BnaA9.CYP78A9)were known silique length/seed weight genes.Using 48 representative rapeseed lines chosen from our association panel,nine and five haplotypes were identified for BnaA9.ARF18 and BnaA9.CYP78A9 respectively,through gene cloning and sequencing.Of which two haplotypes with high frequency were related to silique length/seed weight,especially the expression abundance of BnaA9.CYP78A9 is highly correlated with the two traits.Based on this,two SNP markers were successfully developed at the coding and promoter region of BnaA9.ARF1 and BnaA9.CYP78A9,respectively,which were genotyped in our association panel and highly associated with silique length/seed weight.For the other 17 candidate genes,preliminary functional verification was first performed in Arabidopsis using Crispr-Cas9 based gene-editing technology.The results showed that the silique length of the successfully edited lines for BRH1(homologous to BnaA09g39200D)was significantly decreased.In summary,this study provides a relatively comprehensive and systematic explanation for the regulatory mechanisms underlying the natural variation of silique length in rapeseed from the genetic,physiological,cellular,and molecular levels.This study also identifies two key silique length genes and their haplotypes and preliminarily verifies a new functional gene in rapeseed.All of these results provide useful information for the genetic improvement of silique length in rapeseed.