QTL Analysis For Culturability Traits And Cloning Of BOC1,Decreasing Callus Browning In Rice(Oryza Sativa L.)

Callus of indica rice is easy to brown with lower differentiation rate,which seriously affects the genetic transformation.It is of vital importance to identify genes that reducing callus browning and increasing genetic transformation efficiency in common wild rice(O.rufipogon Griff.),owing to the wild rice as an ancestral species of cultivated rice,containing many missing alleles in Asian cultivated rice(O.sativa L.).The QTLs for rice mature embryo culturability were analyzed using an Oryza rufipogon introgression line population in the background of indica cultivar 93-11.At the same time,a novel gene decreasing callus browning was isolated,cloned through positional cloning and researched on mechanism using the introgression line YIL25 in the background of indica cultivar Teqing.(1)A total of 25 quantitative trait loci related to tissue culturability were detected that included QTLs for callus browning index after one-and two-round subculture and callus regeneration rate using 127 O.rufipogon introgression lines in the background of indica cultivar,93-11.Among them,QTLs detected at the RM335 locus on chromosome 4 were found to control callus browning index after one-and two-round subculture and accounted for 11 and 15%of the phenotypic variation and QTLs detected at the RM341 locus on chromosome 2 were found to control regeneration rate with 10%contributions to the observed variation,at once,the alleles from Yuanjiang wild rice reduced callus browning and improved callus regeneration.(2)We screened YIL25 reducing callus browning using O.rufipogon introgression lines.The callus browning rate and index between YIL25,Yuanjiang common wild rice and F1 were lower than Teqing.Morphological analyses revealed that embryonic cells of YIL25 were more compact than that of Teqing,while Teqing had a large propotion of cells showing aging characteristics.(3)We developed segregating populations from the backcross between YIL25 and Teqing and BROWNING of CALLUS1(BOC1)controlling callus browning was delimited within an 18.6-kb region on the short arm of chromosome 3.Fortunately,there was just one candidate gene,which was LOC＿Os03g12820.We sequenced the coding region of BOC1 and found no sequence variations between YIL25 and Teqing and then compared～1.5-kb region of the promoter in the BOC1 sequences between YIL25 and Teqing,and identified 3 SNPs,one 337-bp insertion/deletion(indel)and one 1-bp indel variations.We generated a construct(pCPL)and transformed into the indica cultivar Teqing.Compared with the Teqing,independent transgenic plants were generated which callus-browning index significantly reduced.In contrast,an RNAi construct was introduced into the YIL25 and displayed the phenotype similar to Teqing.However,overexpression construct was introduced into Teqing and didn’t reduce callus browning.These transgenic complementary experiments verified that LOC＿Os03g12820 in the O.rufipogon genome was a key gene(BOC1)whose appropriate expression reduced callus browning.To identify the transformation frequency of YIL25,we detected independent transformation events in YIL25 and Teqing respectively.The results showed that the number of emerging Hyg-resistant calli per callus co-cultivated with Agrobacterium in YIL25 was much higher than that in Teqing,which demonstrated that YIL25 was a favourable genetic transformation material.Meanwhile we constructed pCPL,which co-cultivated with Agrobacterium in Teqing and the transformation effiency was significantly improved than that of pCAMBIA1300 vector,indicating that BOC1 increased genetic transformation while reduced callus browning.(4)The subcellular localization of BOC1 protein showed that the fluorescent signal was localized to nucleolus,indicating that BOC1 was a nuclear protein.The expression profiles of BOC1 were analyzed in different periods using qRT-PCR and the result showed that BOC1 was highest in the subculture 21 day of YIL25.RNA in situ hybridization revealed that BOC1 was predominantly expressed in the sites of compact and undead cells.(5)The BOC1 gene encodes a plant-specific SRO protein(homologous to RCD1),regulating oxidative stress and programmed cell death(PCD).Accumulation of H2O2 in Teqing aggravated oxidative stress and the content of MDA was significantly higher in Teqing which increased the degree of membrane lipid peroxidation.YIL25 generated more GSH under the catalysis of GR enzyme activity,ensuring the cells in a reducing environment.It is speculated that the excessive accumulation of ROS is the possible cause of the callus browning.Simultaneously,we discovered that the contents of SA,ACC and ethylene were significantly higher in Teqing than that of YIL25.RNA-seq analysis showed that the mechanism of BOC1 regulating callus browning might involve SA response,ET synthesis,oxidative stress and ROS metabolism,which elucidated that the BOC1 gene might act as a node for SA,ET and H2O2 regulating ROS homeostasis and inhibitting PCD.