Unraveling Parental Origin And Evolution Of Populus Tomentosa Carr. And Creation Of High-throughput Mutants

Poplar is naturally distributed in the northern hemisphere.Extensive morphological variation and cross infiltration between species have produced many hybrids,which makes the genetic background of poplars extremely complicated.Highly complex genomes are becoming a bottleneck in poplar breeding and genomic research.Populus tomentosa is an important native tree species in our country and is considered a hybrid of the poplar.However,due to the lack of conclusive genomic evidence,its origin,evolution and genetic structure are still controversial in this field.To study the origin and genomic evolution of P.tomentosa,we selected the plantlet GM15 regenerated from P.tomentosa LM50 through anther induction approach as genome sequencing samples,and employed the Pac Bio + Hi-C + Illumina strategy to perform sequencing,de novo assembly and annotation.As result,we obtained a high-quality chromosomal-level genome.Subsequently,we conducted series analyses of parental origin and evolutionary events of genome,chromosomal structural variations,genome-wide allele-indels.Eventually,we revealed the parental origin and genomic evolution events of P.tomentosa.Furthermore,to find more solid data to support above result on parental origin of P.tomentosa,we also performed analyses of structure and evolution of chloroplast and mitochondria,and identified mother of P.tomentosa.On the basis of the above information,using sg RNA library transformation,we explored a new approach to creation mutants with high throughput in P.tomentosa.The main research results are as follows:(1)Plant regeneration induced by anthers of P.tomentosa.Using the elite clone P.tomentosa LM50,we establish anther regeneration system(callus induction medium was H medium + 1.0 mg / L NAA + 1.0 mg / L BA;differentiation induction medium was MS medium + 0.5mg / L NAA + 0.05 mg /L BA;rooting medium was 1/2 MS + 0.3 mg / L IBA),and obtained 27 anther regenerating plants,which were ideal candidates for P.tomentosa genome sequencing.(2)The first complete genome sequence of P.tomentosa with high quality.Using the plantlet(GM15)induced by anther of P.tomentosa,we combined a Pac Bio + Hi-C + Illumina strategy to sequence and assemble a 740.2 Mb genome,which comprises 38 chromosomes,with even 0.96 Mb contig N50 and17.13 Mb scaffold N50 and the biggest ones of 5.47 Mb and 46.68 Mb respectively,and 33.6% GC content.Integrating Hi-C analysis,phylogenetic trees and the genome and transcript data of several poplar species,we successfully portioned it into two sub-genomes(2×19 chromosomes): A(P.alba var.Pyramidalis)and D(P.adenopoda).Finally,59,124 high-quality genes were identified using P.tomentosa transcriptome data and protein database annotations,with the average coding sequence 1.31 Kb length and 430 amino acids each protein.(3)The parental origin and genome evolution of P.tomentosa.To study the parental origin of P.tomentosa and address dates of divergence and duplication events in poplars,using published genomes of Salix species,we performed comprehensive analyses through homologous gene clustering,collinearity comparison,genetic distance calculation,and phylogenetic tree construction,and found that Populus and Salix experienced a common whole genome duplication(WGD)event(Ks≈0.25),the divergence event between section Lecue and section Tacamahaca(P.trichocarpa)occurred at approximately 13.4 Mya.P.adenopoda was the first to separate from the Leuce family as an independent clade approximately 9.3Mya.Subsequently,the aspen tribe and white poplars tribe underwent a divergence event(approximately8.4 Mya)and respective speciation events(approximately 7.6–5.6 Mya,and 4.8–3.9 Mya).P.alba var.pyramidalis,gave rise to an independent variant of P.alba approximately 4.8 Mya.Approximately 3.9Mya,P.tomentosa,a new white poplar hybrid,was created by hybridization between P.adenopoda and P.alba var.pyramidalis.In addition,based on the fact that P.alba var.pyramidalis is a male clone,and phylogenetic analysis of chloroplast genome data of section Lecue,it was speculated that P.alba var.pyramidalis and P.adenopoda played male and female roles respectively during the formation of P.tomentosa.(4)Chromosome structure and allel-indel characteristics of P.tomentosa.There were abundant chromosome structural variations between two sub-genomics of P.tomentosa,total of 15,480 chromosome variant structures including 6,654 INS(insertion),6,231 DEL(deletion),INV 1,602(inversion),694 TRANS(translocation),and 299 CNV(copy number variation)were found.Among them,INS and DEL accounted for 83%,while the other three types accounted for 27%.In addition,a total of188,575 indels alleles were detected,which were randomly distributed on 15,052 alleles on 38 chromosomes of P.tomentosa.(5)Organelle genomic feature and maternal origin of P.tomentosa.Comprehensive organelle genomic analysis indicated that the poplar chloroplast genome sequence is highly conserved,and the number and distribution of repeat types are similar and conserved.The positions of the IR and SC boundaries are slightly different among the poplar species.There are 7 relatively large insertions in the chloroplast genome sequence of the poplar species,and the coding region is more conserved than the noncoding region.Subsequently,further phylogenetic analysis of six poplar chloroplast genomes revealed that P.tomentosa and P.adenopoda were most closely related.Following the genetic dogma of maternal inheritance of organelle,we concluded that P.adenopoda is the maternal parent of P.tomentosa,such conclusion further supported the inference in the result(3).(6)Creation of high-throughput mutant of P.tomentosa.Based on the poplar MYB gene family and related genes in the anthocyanin synthesis pathway,we designed and synthesized approximately2,500 sg RNAs,further constructed a CRISPR/Cas9-sg RNAs library,and conducted genetic transformation in poplars.Finally,286 transformed lines were selected from the medium with antibiotics.94 plants of them were positive by PCR identification,accounting for 32.87% of the total.Further analysis showed that 54 were single-target lines and 40 were multi-target lines.Subsequently,the target gene fragments were cloned and sequenced,the results indicated that a total of 22 target site sequences were mutated,and the mutation rate was 23.40%.This is the first attempt to create high-throughput mutants using sg RNA library transformation in P.tomentosa.It lays a foundation for high-throughput creation of mutants,mining and identification of functional genes,and molecular design and breeding.