Functional Studies Of MiR319 And Genetic Analysis Of Multi Inflorescence In Brassica Napus L.

Brassica napus L.,one of the important oil crops in China.The healthy development of rapeseed was severely restricted by low level of mechanization and it is the main direction of current breeding workers to cultivate high-yield and high-quality rapeseed varieties that are suitable for mechanized operation.Plant architecture is the main limiting factor for realizing mechanization and little is appreciated about the role of miRNAs in rapeseed plant architecture regulation.In this research,the targeted gene of miR319 precursor,newly discovered by Chen Li in Brassica napus,were identified and the transgenic materials of miR319 and its targeted genes were created to explore the regulation between miR319 and rapeseed plant architecture.Meanwhile,multi-inflorescence(MI)fulfills the requirement of mechanized harvesting and yield increase in rapeseed,but its genetic mechanism remains poorly understood.A multi-inflorescence mutant(MI)and a single-inflorescence material(L780)were used to explore the genetic pattern of multi-inflorescence trait,cause of character formation and clone multi-inflorescence gene in Brassica napus.The main results were as follows:The main results of miR319 regulating rapeseed plant architecture are as follows:1.The targeted genes predicted by degradome sequencing were verified by 5’ RACE experiment.The results indicated that miR319 could target Bna C03g38850D(TCP4),and the same result was obtained in a rapid transient assay in Nicotiana benthamiana.Expression pattern analysis showed that miR319 a was highly expressed in root,stem and shoot apex,while its targeted gene was highly expressed in leave,cotyledon and bud.2.The leaves of miR319 a overexpression lines were serrated in Arabidopsis.Compared with wild type,the expression level of miR319 a in transgenic plants were significantly increased,while the expression of its predicted target genes(At TCP3,At TCP4 and At TCP10)were significantly decreased.Overexpression of Bna C03g38850D(TCP4)in Arabidopsis could lead to reduced plant size and lower plant height after bolting.3.Overexpression of miR319 a led to curled leaves in Brassica napus.Abnormal apical development of transgenic plants before bolting were observed,which led to changes in rapeseed plant architecture,including reduced plant height,abnormal stems and reduced pod numbers.q RT-PCR analysis showed that miR319 a was significantly up-regulated in transgenic plants,and its target gene Bna C03g38850D(TCP4)was down-regulated.Meanwhile,the expression of SAM development-related genes,including CLV1,WUS,were significantly changed.4.Using CRISPR/Cas9 platform to knock out miR319 a precursor and its targeted genes Bna A08g01260D(TCP3)and 6 copies of TCP4 in Brassica napus.High-throughput Tracking of Mutations(Hi-TOM)sequencing technology was used to detect the editing efficiency of CRISPR/Cas9 knockout lines in T0 generation.The editing types of miR319 a included insertion,deletion and replacement,and the editing types of Bna A08g01260D(TCP3)included insertion and deletion.At present,homozygous editing plants have not been obtained.The main results obtained from fine-mapping of multi-inflorescence trait are as follows:1.The abnormal apical meristem resulted in the formation of multi-inflorescences character: compared with wild-type L780 parent,the MI mutant exhibited parallel main inflorescences,and division nodes on main stem were clearly visible.At the vegetative development stage,the seedlings of MI mutant exhibited more rosette leaves than L780.At the flowering stage,MI mutant exhibited lower plant height than that in L780,and significant higher number of main inflorescence pods at maturity.Combined with the result of stereomicroscope observation and q RT-PCR analysis of apical meristem,it can be implied that the natural variation of SAM in MI is caused by CLV-WUS feedback loop.2.Genetic pattern analysis of multi-inflorescence character: The phenotypic identification results of F1,RF1 and segregation population indicated that one pair of dominant genes were different between the two tested parental materials and no cytoplasmic effect was detected.The results of phenotypic investigation of BC populations in Lanzhou and Wuhan showed that the modified characters were generally affected by environment factors.3.The Brassica 60 K SNP array was employed and a total of 972 SNPs were detected between the two mixed pools,and these SNPs were distributed over 19 chromosomes.Combined with the results of Excel analysis and SSR screening,the candidate interval is locked in the range of 4-8 Mb on the A05 chromosome.4.A linkage map of the multi-inflorescence gene BnaMI was preliminarily constructed: The BC1 population was scaned by AFLP technique combined with BSA method and 7 pairs of 2,048 pairs of primer combinations linked to the MI genes were identified.Based on the sequencing information of AFLP markers,SSR primers were designed with reference to the Darmor-bzh genome.L04-2 and L06-14,the closest flanking markers were mapped at 4.4 c M and 9.3 c M distant from the BnaMI gene,respectively,the physical distance is 2.5 Mb.5.Fine mapping and candidate genes analysis of BnaMI: BnaMI was fine-mapped to a 55 kb genomic region combining with genotype and phenotype of 5,768 BCF1 individuals using a traditional mapping approach.Through comparative sequencing and expression analyses,combined with the annotation in Arabidopsis,five genes(Bna A05g08870 D,Bna A05g08900 D,Bna A05g08910 D,ORF12 and ORF13)were speculated as candidate genes controlling multi?inflorescence in Brassica napus.6.Cloning and transgenic verification of candidate genes: Five candidate genes were cloned and the genetic complementation experiment revealed that Bna A05g08900 D and Bna A05g08910 D were not target genes for the multi-inflorescence trait.Finally,Bna A05g08870 D,ORF12 and ORF13 were identified as candidate genes for the multi-inflorescence trait.