QTL Mapping For Plant Architecture Traits And CRISPR/Cas9-mediated Generation Of Plant Architecture And Sex-related Gene Mutants In Asparagus Officinalis L.

Garden asparagus（Asparagus officinalis L.）,known as the“king of vegetables”,is a typical dioecious perennial cash crop due to the unique texture,flavor and high nutrition of its edible spears.The sex and ideal plant architecture of asparagus are highly correlated with its yield and quality.To understand the complex genetic component of plant architecture and the genetic basis of the sex determination could provide significant role in asparagus breeding for ideotype and sex controlling.Sequencing and assembly of asparagus genome has been completed by our international cooperation team in 2017.A total of 27,656 coding genes were predicted with functions of most genes remaining unclear.In recent years,CRISPR/cas9 genome editing technology has become an important tool for gene function analysis and molecular breeding in many crops,but it has not been employed in asparagus.In this study,QTL analysis and identification of candidate genes for plant architecture traits consisting plant height,stem diameter and the number of stems and other traits were carried out;meanwhile,an efficient CRISPR/Cas9 system for gene editing in Asparagus officinalis L.was established;the loss-of-function mutants of candidate gene related to asparagus plant architecture were obtained for analysis of gene biological function,and sex-related mutants based on asparagus sex determining genes SOFF and asp TDF1 editing were created through the system.Main results of this study were as follows:（1）The phenotypic data of plant height,stem diameter and the number of stems were obtained from a female plant of asparagus cultivar Tainan No.3（TN3）,a male plant of Asparagus dauricus（XA）and two F₁ and 100 F₂ progenies derived from interspecific hybridization between the two species in four growing seasons in two years.Analysis results of the phenotypic data showed that asparagus plant architecture was a complex quantitative trait,and significant positive correlations were found among plant height,stem diameter and the number of stems,especially between plant height and stem diameter（correlation coefficient=0.793,P<0.01）.Using the F₂ population of 100 individual plants,a high-density genetic linkage map of asparagus containing 10 linkage groups and 1218 markers,with the total and average map distance,4277.34 c M and 3.51 c M,respectively,was constructed by QTL Ici Mapping 4.2 software with the SNP developed by dd RAD-seq and STS and CAPS markers of related functional genes.QTL analysis was conducted using ICIM-ADD analysis method for plant height,stem diameter and the number of stems.A total of 17 QTLs related to plant architecture with a phenotypic variation explanation rate from 6.83%to 32.87%and a LOD value from3.67 to 10.93 were detected in the four different growth environmental conditions.The seven consistent major QTLs region was located between 40,304,599 and46,821,499 bp on chromosome 5,with a physical distance of about 6.52Mb.Ao SPL14a,a homologous gene of IPA1 of rice ideal plant architecture was identified from the major QTLs region with a total of 102 predicted genes.Five missense SNPs were discovered in exon 1 and exon 3 of Ao SPL14a between parents with significant differences from plant architecture,which might cause the dwarf phenotype of XA.The relative expression level of Ao SPL14a was increased in asparagus stems and cladodes,but it was significantly decreased in stems of XA.Therefore,Ao SPL14a,encoding a squamosa promoter-binding-like protein 14,is putatively acting as a candidate gene for plant architecture control in asparagus.（2）Due to the conserved characteristics of U6 gene in Arabidopsis thaliana,two U6 promoters Asp U6P3 and Asp U6P6 were cloned to drive sg RNA expression in asparagus.Based on Asp U6P3 and Asp U6P6 to drive sg RNA expression,four vectors of co-expression of sg RNA and Cas9 carrying hygromycin or kanamycin resistance genes were constructed.Using the candidate gene Ao SPL14a related to asparagus plant height and stem diameter as the target gene,four CRISPR/Cas9expression vectors with kanamycin resistance were constructed.The expression vectors were transformed respectively into the embryonic calli of asparagus line‘JXT4’mediated by Agrobacterium tumefaciens to obtain resistant calli.The sequencing results from resistant calli showed that the editing rates of the two vectors of 3KI1 and 3KI2 carrying Asp U6P3 promoter were 70.00%and 78.13%,respectively.It showed that asparagus endogenous U6 promoter Asp U6P3 had high transcriptional activity and could drive the expression of downstream sg RNAs.The positive rates of transgenic plants from the two vectors of 3KI1 and 3KI2 were46.67%and 44.00%,and the gene editing efficiency of regenerated plants was64.29%and 81.82%,respectively.In total,18 independent T₀ generation plants with gene editing of Ao SPL14a were obtained,including one homozygous mutation,one biallelic mutation,three heterozygous mutations and 13 chimeric mutations.Among them,10 mutants were predicted to be loss of function including a distinctive"triallel"SNP chimeric mutation.The results of genotyping showed that there were 25 mutation types including 71.28%base deletion as the most common mutation type,25.39%insertion mutation with mainly one base insertion and 3.33%compound mutation.Phenotype identification showed that the plant height and stem diameter decreased significantly in two 18-month-old and two 6-month-old mutants.Compared with the wild type,plant height was shortened in the homozygous and double-allelic mutants of 2-month-old seedlings.The directional mutation of Ao SPL14a gene mediated by CRISPR/cas9 system changed asparagus plant architecture,which further confirmed that Ao SPL14a was an important functional gene regulating plant architecture and stem diameter of asparagus.（3）Previous studies showed that most of the non-recombining sex determination region of the Y chromosome was hemizygous distinctively with the two most important genes,SOFF and asp TDF1,responsible for sex determination in Asparagus officinalis L.In order to further verify gene editing efficiency of the constructed CRISPR/Cas9 system in asparagus and create sex mutants,SOFF and asp TDF1 as target were edited with the system.Four CRISPR/Cas9 expression vectors of SOFF and asp TDF1 for single gene knockout were constructed and transformed into the embryogenic calli of asparagus XY male plant‘X7’,respectively.The results of genotyping of positive transgenic plants showed that one sg RNA each for SOFF and asp TDF1 led to the editing of target genes,and the gene editing efficiencies of regenerated plants were 36.36%and 60.00%,respectively.In total,4 independent T₀ generation plants with ten mutation types including one homozygous mutation and three chimeric mutations were obtained based on SOFF gene editing.A total of three independent T₀ generation plants with four mutation types were obtained by editing asp TDF1 gene including 3 chimeric mutant seedlings.The hemizygous,non-recombinining genes on the Y chromosome of plant were edited successfully based on CRISPR/Cas9 system for the first time.In conclusion,QTLs related to plant height,stem diameter and the number of stems were detected and a candidate gene,Ao SPL14a,was identified from a major QTLs region.Moreover,CRISPR/Cas9 system was successfully applied to knock out the endogenous gene Ao SPL14a in Asparagus officinalis L.It was confirmed that Ao SPL14a gene was an important functional gene to regulate plant architecture in asparagus by CRISPR/Cas9-mediated targeted mutagenesis of Ao SPL14a genes.Sex-related mutants were also obtained by CRISPR/Cas9-mediated targeted mutagenesis of sex determining genes SOFF and asp TDF1 in Asparagus officinalis L.The results laid a foundation of gene reservoir and material basis for revealing the genetic basis and molecular mechanism of plant architecture control and sex determination in asparagus,which provide new technical support for effective improvement on sex controlling and ideotype breeding of asparagus.