Generation And Preliminary Function Analysis Of The Mutants Related To Seed Germination In Brassica Napus L.

Brassica napus L.is an important oil crop.Timely dormancy and germination of seeds are important to adapt to the biotic and abiotic stress.Plant hormones are one of the main physiological factors determining seed germination and dormancy.In this experiment,some loci related to seed germination were obtained through genome-wide association analysis.After the analysis of candidate genes in these intervals,some hormone related genes were found,such as Bna A10G0029000WE（Bna YUC3）.CRISPR/Cas9 technology was used to create related gene mutants and overexpression materials were also created.The main results are as follows:（1）Combining the resequencing data of 505 natural populations and the phenotypic data of seed germination rates,we identified 22 significant correlation loci.The upstream and downstream 10 kb of the significant locus were selected as the candidate interval.Annotate the genes in the candidate interval,we found that many hormones related genes are included in the candidate interval.Five genes associated with hormones such as Bna A10G0029000WE（Bna YUC3）were selected for verifying functions.Analysis of the important domains which were candidate genes contained,results showed that these domains were conserved in candidate proteins.（2）Tissue expression pattern analysis showed that candidate gene Bna YUC3 and Bna IAA12 were expressed in all tissues which shows constitutive expression;Bna GA3ox1was highly expressed in roots,stems and leaves;Bna TIFY5B was mainly expressed in seeds;Bna GA2ox1 was mainly expressed in stems.Hormone treated RNA-seq data showed that GA induced Bna YUC3.C05 and Bna GA2ox1.C06 expression.The expression of Bna TIFY5B.A05 and Bna TIFY5B.C04a was up-regulated in ABA treatment.The expression of Bna GA2ox1.C02 was down regulated in GA treatment.Bna YUC3.C05 and Bna GA3ox1.A06 was down regulated in ABA treatment.According to the expression pattern,these candidate genes play an important role in plant growth and development in response to hormones.（3）Furthermore,we use CRISPR/Cas9 and overexpression technology to create relevant mutant materials and overexpression materials.The results of CRISPR/Cas9positive plant editing showed that the editing efficiency of Bna YUC3,Bna GA3ox1,Bna IAA12 and Bna TIFY5B in T₀ generation was 0%～62.50%;The q RT-PCR results showed that the expression level of Bna YUC3.C05 OE,Bna IAA12.C05OE,Bna TIFY5B.C03 OE and Bna GA2ox1.C02 OE transgenic lines were significantly higher than that of wild-type plants.（4）The results of seed germination of T₁ generation overexpression lines showed that the seed germination of different overexpressed lines was different compared with that of the wild-type Westar which all showed a suppressive role in seed germination.Bna YUC3.C05 OE lines inhibited seed germination significantly at 24 h.The phenotype of Bna YUC3.C05 overexpression strain was preliminarily investigated,The results showed that the flowering period of Bna YUC3.C05 overexpression strain was significantly different from that of wild type.