Construction Of A Libary Of CRISPR/Cas9 Mutants Associated With Silique Development And Seed Size In Brassica Napus L.

Brassica napus L.is the most important rapeseed type in China,with its seed yield highly related to silique development and seed size.In recent years,a number of QTL locus affecting silique developmental traits and seed size in B.napus have been identified by map-based cloning or genome-wide association analysis,but the number of cloned functional genes remains very small.The CRISPR/Cas9 gene-editing system is a powerful tool that can efficiently edit multiple homologous genes in polyploid crops.Thus,compared with some approaches reported previously,the CRISPR/Cas9-based mutant library is a high-throughput screening platform with obvious advantages.In this study,we screened out candidate genes regulating silique development and seed size by combining the data from transcriptome-wide association study(TWAS)and tissue-specific expression profiles throughout the reproductive period of B.napus.A sgRNA library targeting these candidate genes were ligated to the pRGEB32-Bn U6C01 vector to construct a robust library.By expressing these library vectors in the short-season B.napus inbred accession ‘Xiaoyun’,we obtained a comprehensive library of edited mutants of the target genes.The main results obtained are as follows:1.A total of 1905 candidate genes related to seed size and silique development regulation were screened based on TWAS data during silique development,transcriptome data during the whole reproductive period of ZS11 and other plantrelated functional gene studies.6124 sgRNAs were specifically designed mainly for 1739 candidate genes which have no more than four paralogues.The sgRNA library were synthesized and ligated to pRGEB32-Bn U6C01 to form a mixed plasmid library.Sequence analysis showed that 6120 sgRNAs were detected in the plasmid library,with a coverage rate of 99.93% and the skewness of 4.53,suggesting that the plasmid library has good coverage and homogeneity.2.A total of 621 T0 plants were obtained through genetic transformation,of which 567 were positive plants.Using specific sgRNA detection primers,we detected a total of 249 sgRNAs in 425 T0 plants.Of these,312 plants(73.24%)contained one sgRNA,20.42% of the individual plants contained two sgRNAs,while the plants carrying 3 and 4 sgRNAs shared a ratio of 1.64% and 0.70%,respectively.Only 4.00% of the plants failed to contain any sgRNA.Among the 312 plants containing one single sgRNA,163 different sgRNAs were detected,and 91.80% of these sgRNAs were contained by one to three independent T0 individuals,indicating that the mutant library was established with excellent uniformity.3.Of the 408 T0 plants analyzed,151 plants(37.00%)were edited in 84 target genes.Of the 249 sgRNAs detected,51.41% of the sgRNAs displayed the edition effect.The CRISPR/Cas9 system produced heterozygous or chimeric editing types such as single-base insertion and multiple-base deletion in all the T0 individuals.Homozygous mutant plants were isolated from 10 T1 families numbered as A03,A21,B29,B49,A68,A73,A79,A49,A29 and B27.The potential phenotypic variations of these mutants are to be investigated.In summary,we used the CRISPR/Cas9 technology to create a preliminary library of gene-edited mutants in B.napus,which has laid a solid foundation for screening candidate genes related to silique development and seed size in the future;moreover,this study has accumulated rich experiences for the rapid identification of other functional genes in a similar manner in B.napus.