Creating Mutants With Disorder Meiotic Aynapsis In Brassica Napus L.by CRISPR/Cas9

Meiosis is a key step in generational alternation of sexual reproductive organisms.In this process,homologous chromosomes complete a series of important events,such as recognition,pairing,synapse,fragment exchange between non-sister chromatids and separation.Synaptonemal complexes（SC）are formed between paired homologous chromosomes where exchanges were produced.AtZYP1 is a transverse filament protein that forms SC in model plant Arabidopsis thaliana.During meiosis of zyp1 mutant,SC could not form and the number of homologous chromosome exchanges decreased significantly,but the number of homeologous chromosomes exchanges increased.Brassica napus L.is an allotetraploid with a complex genome and few studies had been done on the genetic regulation of meiosis due to the lack of related mutants.In this study,we used CRISPR/Cas9 technology to edit four homologous genes of ZYP1 in B.napus to study its function in meiosis.Using these mutants as intermediate materials may also promote the exchange of chromosomes between rapeseed and its related species,thus introducing excellent genes into rapeseed.The main results are as follows:（1）By homologous alignment we identified two,two and four homologous genes of Arabidopsis AtZYP1 in genome of B.rapa,B.oleracea and B.napus,respectively.Based on these gene sequences,a double-target CRISPR/Cas9 vector targeting four copies of BnZYP1 in B.napus was constructed.Agrobacterium-mediated transformation was then carried out in Westar.A total of 143 independent transformants were obtained in the T₀generation,62 of which were Cas9-positive.Sequencing analysis showed that at least one copy of all the individual plants was successfully edited with a mutation rate of 100%.（2）The mutation types of T₀ plants varied greatly among different targets and different copies.The average probability of substitution for target 1 was 55.9%,insertion was 10%,missing was 8.8%,while target 2 was 34.6%,40.6%and 10%,respectively.The mutation rates of T₀ generation for BnaA07g20670D,BnaA07g34050D,BnaC06g20530D and BnaC06g38690D were 100%,88.7%,74.2%and 80.6%;The average probability of substitution for BnaA07g20670D was 50.4%,insertion was 19.5%%,missing was7.1%;BnaA07g34050D was 54.4%,25.2%and 7.8%,respectively;BnaC06g20530D was35.5%,46.8%and 17.7%,respectively;BnaC06g38690D was 21.2%,27.3%and 12.1%,respectively.No homozygous mutant with four copies was obtained in the T₀ generation.（3）The mutation of 32 T₁ plants from 4 T₀ generations was identified.The results showed that the mutation of T₀ generations could be inherited stably.At the same time,Cas9 protein could works in T₁ generations and produce new mutations,with a probability of 2.34%.Of the 32 T₁ plants,19 had mutations in four copies at amino acid level,and 2 of them had homozygous mutations in all four copies.（4）The pollen stainability of 7 plants（63.9%-82.7%）in the T₀ generation was significantly lower than that of wild type plants,and 4 plants had shrunken stamens,which showed male abortion and no self-crossing or hybrid seeds were obtained.Two mutant plants of T₁ generation also showed shrinkage of stamens,no normal pollen production,and three other mutant plants of T₁ generation had about only 25%pollen stainability.The pollen mother cells of these mutants showed frequent chromosome laggards in the metaphase I,anaphase I,metaphase II and anaphase II of meiosis.