Functional Identification Of The ERECTA Family Genes And Creation Of Germplasm With Compact Architecture In Brassica Napus L.

Improving rapeseed yield and cultivating varieties suitable for mechanical harvesting are both important goals in rapeseed breeding,which can be simultaneously achieved by optimizing the plant architecture of current varieties to adapt them to the high-density planting manner.ERECTA(ER)encode a receptor-like protein kinase in Arabidopsis thaliana.And its mutant(er)is warmly welcome by researchers due to its compact and upright plant architecture.ER has two homologous genes,ERECTA LIKE1(ERL1)and ERECTA LIKE2(ERL2).Functional analysis of this gene family(ERfs)shows that they play an important role in plant growth and development;however,whether their paralogues can be used in Brassica napus remains to be determined.In this research,to answer this question preliminarily,the mutants associated with ERfs gene were created via the CRISPR/Cas9 system in Brassica napus,Moreover,we have successfully obtained a new germplasm resource that shows compact architecture without significant reduction in yield per plant,suggesting the potential of utilizing of ERfs in plant architecture improvement of Brassica napus.The main results obtained are as follows:1.Sequence alignment showed that there are two ER homologous copies,two ERL1 homologous copies,and one ERL2 homologous copy,respectively in the genome of Xiaoyun.Sequence of ERfs in Brassica napus have the same and consistent distribution of domains and conserved motifs,and the similar expression pattern as those of Arabidopsis,suggesting that ERfs may have conserved functions in the two species.2.To edit the three gene family members of BnaER,BnaERL1,and BnaERL2 in Brassica napus,three dual-target CRISPR/Cas9 vectors were constructed and introduced into the Xiaoyun genome.A total of 40,1,2,and 14 positive plants,were harvested for the constructs of BnaER-A9/C8,BnaERL1-A6/C3,BnaERL2-A10,and BnaEH(3 vectors mixed infection),respectively.From them,the homozygous mutants,such as Bnaer.a9/Bnaer.c8,Bnaerl1.a6/Bnaerl1.c3 and Bnaerl2.a10 were obtained for each gene finally.3.In rapid breeding environment,compared to the wild type,the Bnaer.a9/Bnaer.c8 mutant showed obvious altered phenotypes,like the compact inflorescences,dwarf plant,and shortened siliques,the increased number of branches and siliques per plant.In contrast,no significant change was observed for the number of ovules,seeds per silique,thousand-seed weight as well as oil content.Compared with the wild type,the Bnaerl1.a6/Bnaerl1.c3 mutant showed significantly reduced silique length,while the other phenotypes were not significantly modified.For Bnaerl2.a10 mutant there is no significant trait change was found.In the field environment,compared to the wild type,the phenotype of Bnaer.a9/Bnaer.c8 mutant is consistent with that under the rapid breeding conditions,except that thousand-seed weight is significantly reduced.Therefore,the Bnaer.a9/Bnaer.c8 mutant seems to have a potential in architecture improvement in rapeseed.4.Cytological observation found that the stem cortex of the Bnaer.a9/Bnaer.c8 mutant had reduced cell layers and irregular cell arrangement,while the number of cells in the silique wall were reduced,and the cells became longer with irregular shapes.So,it seems that the changed plant height and silique length may be caused by the abnormal growth and proliferation of cells in Bnaer.a9/Bnaer.c8 mutant.Analysis of endogenous hormones during silique development between Bnaer.a9/Bnaer.c8 mutants and Xiaoyun showed that there were significant differences in the contents of IAA and ABA during early silique development,suggesting that hormonal pathways likely play an important role in shaping the silique phenotype in Bnaer.a9/Bnaer.c8 mutants.5.More combinations of the BnaER,BnaERL1 and BnaERL2 mutants were created.Finally,we have successfully obtained the mutants,including Bnaer.a9/Bnaer.c8,Bnaerl1.a6/Bnaerl1.c3,Bnaerl2.a10,Bnaer.a9/Bnaer.c8 Bnaerl1.a6/Bnaerl1.c3 and Bnaer.a9/Bnaer.c8 Bnaerl1.c3 Bnaerl2.a10.The new mutants would construct a solid foundation towards further understanding the function of ERfs genes in Brassica napus.In conclusion,we created mutants and mutant combinations for each ERfs gene and preliminarily analyzed their biological functions of ERfs genes in Brassica napus.Our results showed that the Bnaer.a9/Bnaer.c8 mutant with advantages of compact plant architecture has a good potential in rapeseed breeding.All these mutants would be useful for the systematic study of plant architecture genetic control and plant architecture breeding in the future.