Construction Of Mutant Library In Fragaria Pentaphylla And Study On Gene Function Of Mg-chelatase I Subunit (CHLI)

Low light condition is the main problem of strawberry production in winter protected cultivation in north China.Breeding or screening strawberry varieties suitable for low light cultivation is an important way to solve this problem.At present,however,most of our main strawberry varieties are introduced from Japan or Europe or America,the local varieties are few,which resulted in the degradation of the strawberry varieties in China,Moreover,the traditional hybridization breeding is time-consuming and challenging.There are abundant wild strawberry resources in China,the fragaria pentaphylla was one of these wild strawberry resources,which has strong cold and disease resistance and specific fruit aroma,and has high research and application value.Magnesium chelatase（Mg Ch）catalyzes the insertion of magnesium into protoporphyrin IX,a vital step in chlorophyll（Chl）biogenesis.The enzyme consists of three subunits,Mg Ch I subunit（CHLI）,Mg Ch D subunit（CHLD）,and Mg Ch H subunit（CHLH）.The CHLI subunit is an ATPase that mediates catalysis.Therefore,we performed ENU mutagenesis on the seeds of two lines of wild diploid strawberry F.pentaphylla,and the mutant library of M1 generation was created.We identified and characterized a yellow-green mutant in this mutant library and noted as p240.Notably,this mutant exhibited a light-green-leaf phenotype and higher photosynthesis ability compared with WT when under low light conditions.We identified a mutation in the 186th amino acid of the CHLI subunit responsible for this phenotype,and the gene function of CHLI was studied further.The main results obtained as follows:1.A mutant library of strawberry F.pentaphylla was createdWe identified the chromosome ploidy of two lines’Pink’and’White’of F.pentaphylla which were collected from Lueyang,Hanzhong,Shaanxi Province,and the chromoploidy identification by flow cytometry showed that both two lines were diploid.Through ENU mutagenesis,The M1 mutant libraries of F.pentaphylla were constructed and totally 200mutants were obtained,including 93‘White’lines and 107‘Pink’lines.There were similar phenotype of these two mutant lines,and these phenotype of the mutants mainly focused on leaf type,leaf color,plant type and floral organ.Among all these mutants,we observed a yellow-leaf mutant p240,which showed an obvious response to light intensity.Whether under natural sunlight or strong indoor light,the leaves are yellow-green,and turn from yellow-green to light-green under low light,and the photosynthetic capacity of the wild type was nearly twice as high as that of the WT under low light.Therefore,we studied it further.2.The candidate gene resulting in p240 yellow-green-leaf phenotype was identifiedWe conducted RNA-Seq analysis of leaves of p240 and WT,it was found that the differential expression genes of p240 and WT were significantly enriched in chlorophyll metabolism and carbon metabolism pathways.The genes of these pathways were aligned to the SNPs identified by RNA-Seq,found the genes with base mutation in p240 but no base mutation in WT consistent with reference genome,and finally,magnesium chelatase I subunit（CHLI）and phosphofructokinase 4（PFK4）were determined to be the possible genes causing yellow-leaf in p240.Among them,the mutation site of CHLI was glutamic acid at position 186 to lysine,and that of PFK4 was alanine at position 450 to serine.By knocking down and knocking out these two genes,the stable transformation of woodland strawberry Ruegen’and‘Hawaii 4’and Arabidopsis thaliana‘Col-0’showed that PFK4 was not associated with the phenotype of p240.Heterozygous gene editing lines and RNAi lines of CHLI showed the same phenotype as p240,while homozygous editing lines showed an albino phenotype.Therefore,CHLI was identified as a possible candidate gene for p240.Further complementation of CHLI in p240 suggested that CHLI was responsible for the phenotype of p240 and named Fp CHLI.3.Analysis of CHLI gene function in strawberryMagnesium chelatase catalyzes the insertion of magnesium into protoporphyrin IX,and is a vital step in chlorophyll biogenesis.The enzyme consists of three subunits,CHLI,CHLD,and CHLH.The CHLI subunit is an ATPase that mediates catalysis.Further analysis indicated that mutation in Glu186 of CHLI does not affect its subcellular localization nor the interaction between CHLI and CHLD.However,intramolecular interactions were impaired,leading to reduced ATPase and magnesium chelatase activity.These findings demonstrate that Glu¹⁸⁶ has a key role in enzyme function,affecting leaf coloration via the formation of hexameric ring itself.Metabolite analysis of chli mutants under high light conditions revealed significant changes in both nitrogen and carbon metabolism.Gene-editing heterozygous chli mutants accumulated more chlorophyll under low light conditions(50μmol m^-2 s^-1)than high light conditions,and its photosynthetic capacity was nearly 2 times higher than that of WT under low light.The results indicated that the phenotype of the hybrid gene-editing lines was consistent with that of p240 under different light condition.CHLI and its 186^th amino acid are expected to be the potential candidate genes and effective sites for molecular breeding in strawberry in the future.In addition,inoculation of CHLI-RNAi lines with xanthomonas fragariae YL19 showed that it enhanced the susceptibility of diploid woodland strawberry to x.fragariae,suggesting that Fve CHLI may positively regulate strawberry resistance to x.fragariae.In summary,we established a mutant library of fragaria pentaphylla and screened a yellow-leaf mutant p240 from the library.The candidate gene causing p240 phenotype was identified as CHLI by RNA-Seq SNPs analysis combined with phenotypic analysis of transgenic plants,and further the gene function of CHLI was analyzed.The results not only enriched the germplasm of wild strawberry,but also provided a theoretical basis for the study of CHLI gene function in horticultural crops and the breeding of strawberry germplasm resistant to stress.