Fine Mapping And Functional Analysis Of BnLLA10,a Major QTL For Lobed Leaf In Rapeseed

Leaf shape is an important agronomic trait,in Brassica germplasm,there are a wide range of natural variations ranging from entire leaves to lobed leaves(also be called"huaye").The functional significance of lobed leaves has been identified with advantages for gas exchange,canopy architecture and high-density planting over other leaves.However,a few studies on the molecular mechanism of lobed leaf formation in Brassica napus were reported.Previously,a lobed leaf line Tongling huaye(HY)and a serrated leave line Zhongshuang 9(Z9)were combined to develop segregated populations.In the present study,we used map-based cloning to identify the lobed leaf genes,and conducted a preliminary study on its regulation mechanism.The results were described as follows:1.The lobed-leaved Tongling huaye(HY)and the serrated-leaved Zhongshuang 9(Z9)were used as parents to construct segregated populations.The F1 plants from the reciprocal crosses exhibited intermediate leaf morphology.In each of the random F2 and BC1F2 subpopulations,the segregation ratio for the serrated,intermediate and lobed leaf types was approximately 1:2:1.These results indicated that the lobed-leaf trait is controlled by a nuclear locus with major effect,designated BnLLA10,without cytoplasmic genetic effects and the lobed leaves are incompletely dominant compared to serrated leaves.2.In the F2 segregated population,the BnLLA10 locus was initially located into A10 chromosome by using the BSA method.After a recombinant screening using more plants from the F2,F3,BC1F2,BC2F2 and BC3F2 populations,the BnLLA10 locus was further narrowed down to a 4 1.0-kb region.Five genes,designated BnaA10g26310D through BnaA10g26350D,were identified in the mapping region.Among the mapped genes,BnaA10g26320D(hereafter BnaA10.RCO)and BnaA10g26330D(hereafter BnaA10.LMI1)are LMI1-like paralogues that encode class I Homeodomain leucine-zipper(HD-Zip)transcription factors.Those genes were reported in determining leaf shape in cardamine and cotton,so tandemly duplicated homeobox genes BnaA10.RCO and BnaA10.LMI1 are the most likely candidate genes in BnLLA10.3.A comparison of genomic sequences from gene clones of both BnaA10.RCO and BnaA10.LMI1 revealed that an amino acid change between the HY and Z9 alleles led to a non-conservative change in the BnaA10.RCO coding sequence.In the 5’-and 3’-flanking regions of BnaA10.RCO,abundant polymorphisms were identified between Z9 and HY alleles.Similarly,the Comparative sequencing of BnaA10.LMI1 showed that no sequence variation was detected in the open reading frame of BnaA10.LMI1 among two parents,while a larger insertion of 2624bp and three SNPs in the BnaA10.LMI1 promoter sequence are unique to the two serrated-leaved inbred lines Z9 and J7005 compared to HY,and three SNPs in the 3’-flanking sequence.Thus,gene expression regulatory regions of BnaA10.RCO and BnaA10.LMI1 are main allelic variation respectively among the two parents.4.According to qRT-PCR results,BnaRCO and BnaLMI1 are dramatically up-regulated in HY compared with Z9-NIL in the shoot apex and leaves of seedlings,with BnaRCO showing the more pronounced increase than BnaLMI1.The expression profiles of BnaRCO and BnaLMI1 in different tissues of seedlings indicated their high expression in early leaves,implying their important function during early leaf development.The subcellular localization of BnaA10.RCO was mainly localized to nuclei.5.We developed transgenic B.napus lines that overexpressed of the allele BnaA10.RCOHY from HY and the allele BnaA10.RCOZ9 from Z9 under J9707genetic background respectively.The transgenic plants lines produced more serration along leaf margin,and even some plants with deep lobes like HY.In BnaA10.RCOZ9 OE lines under HY genetic backgrounds,most transgenic plants produced severely lobed-leaf with only few blade tissues left,resulting a higher blade complexity.But some transgenic lines from the transformation of BnaA10.RCOZ9 OE construct into HY background showed a cosuppression-like phenotype:a smooth leaf.By using CRISPR/Cas9 technology to knock out BnaA10.RCO in HY with lobed leaves,the transgenic plants were changed into serrated leaves.Those results indicated that BnaA10.RCO plays an important role in leaf lobe formation,while knocking out BnaC04.RCO did not change leaves shape.The knockout mutations of BnaA10.LMI1 gene were also induced using the CRISPR/Cas9 system in both HY(the lobed-leaf parent)and J9707(serrated leaf)genetic backgrounds.BnaA10.LMI1 null mutations in the HY background were sufficient to produce unlobed leaves,whereas null mutations in the J9707 background showed no obvious changes in leaf shape compared with the control.Collectively,our results indicate that BnaA10.LMI1 positively regulates the development of leaf lobes in B.napus.6.N-1-naphthylphtalamic acid(NPA)changed the lobed leaves of HY into entire leaves,with no serrations at all,which indicated that blocking auxin polar transport can change leaf shape in rapeseed.The auxin activity marker DR5 was similarly expressed in the juvenile leaves of HY and J9707,and no difference in free IAA content was observed between HY and Z9-NIL.Thus,these data indicate a possibility that BnLLA10 act in parallel to P/N1-mediated polar auxin transport to regulate leaf complexity in rapeseed.7.In Brassica rapa and Brassica juncea,the lobed leaves lines and the serrated leaves lines were crossed to study the genetic mechanism of lobed leaf formation.Though QTL-Seq,the lobed leaf genes were co-localized to the homologous segment of the A10 chromosome in the F2 segregated population,and all of the mapping segment contained the LMI1-like genes.Gene editing results verified the lobe formation is regulated by BjA10.RCO and has nothing to do with BjLMI1.The above results indicated that the lobed leaf formation are regulated by the conserved LMI1-like genes in different Brassica species,and the function of these genes are differentiated in Brassica species.