Gene Localization And Molecular Mechanism Of The Nuclear Male Sterile Line Pby-1 In Capsicum Annuum L.

Pepper（Capsicum annuum L.） is an important vegetable crop in China and the world.It belongs to cross pollination crop and has obvious heterosis in yield,quality,disease resistance（adverse）and other aspects.For a long time,male sterility mechanism has been a research hotspot in pepper breeding.Using male sterile lines in pepper is one of the main ways to realize heterosis and carry out pepper genetic breeding.Pby-1 is a mononuclear recessive gene male sterility mutant material found in the field by our group,and the molecular mechanism of its sterility is still unclear.In this study,we used the male sterility mutant material pby-1 and wild-type material PBY-1 as the research objects,and carried out the study on the localization of male sterility gene and molecular mechanism by using cytology,physiology,BSA-Seq,proteomics,q PCR and VIGS.The main findings are as follows:（1）The pby-1 plants showed normal growth,bud development and flowering habit,except that the floral organ structure was different from that of the wild-type material,with lighter degeneration of floral organs,slightly deflated anthers and very little or even no pollen in the anthers.The pollen of the male sterile line pby-1 plants was found to be non-viable by the in vitro pollen germination test.The results observed using semi-thin sections and transmission electron microscopy showed that the cell structure of pby-1 was damaged during pollen mother cell formation,meiosis and late mononucleosis;in late mononucleosis,no obvious germ cell nuclei were seen;premature death of the chorion layer（beginning of meiosis）may be the cause of male sterility.（2）In the three periods of meiosis,tetrad and binucleation,the protective enzyme activities of sterile and fertile lines were measured and found that the SOD activity of sterile line pby-1 was significantly lower than that of fertile line PBY-1,the POD activity was only slightly higher than that of PBY-1 in binucleation,and the CAT activity and MDA content of sterile lines were higher than those of PBY-1.The endogenous hormone contents were measured and found that the GA₃content of the sterile lines was lower than PBY-1 during meiosis,while the ZR,JA and ABA contents were higher than PBY-1;the GA₃,IAA and ZR contents of the sterile lines were higher than PBY-1 and the JA and ABA contents were lower than PBY-1 during tetrad;the GA₃,IAA,ZR,JA and ABA contents of the sterile lines were higher than PBY-1 during dicotyledonous stage.Changes in protective enzyme activity and abnormalities in endogenous hormone content as well as changes in hormone ratios during bud development may be responsible for the sterile line pby-1abortion by specifically blocking the transport of plastid exosomal sugars and other carbohydrates that affect pepper pollen development,anther endogenous substances,and pollen viability.（3）The results of the investigation on the genetic law of fertility of hybrid F₁,inbred F₂ and backcross BC₁ populations constructed by PBY-1 and pby-1 showed that the F₁ generation was fertile,and the segregation ratio of sterile plants to fertile plants in F₂ generation was 1:2.93,and that of sterile plants to fertile plants in BC₁ generation was 1:1.03.It was found that the sterile base was due to the single recessive nuclear sterility genes.Combined with the F2 fertility phenotype,a male sterile BSA pool of 30+30 in the parents and offspring was constructed.With Zunla as the reference genome,the sterility genes were localized by BSA-Seq technique,and finally12 associated regions were identified,all located on chromosome Chr07,totaling 70.02 Mb and containing 343 genes.The enriched candidate genes were hypothesized to be related to genes related to carbohydrate metabolism,lipid transport metabolism,and phytohormone signaling in the F₂ segregating population of pepper.（4）By proteome sequencing of pepper anthers,a total of 8516 proteins and 1646differentially expressed proteins（DAPs）were identified in three periods:meiosis,tetrad and binucleation,and 213/149,99/80 and 453/651 DAPs were identified respectively,which were up-regulated or down-regulated.These DAPs were mainly enriched in monoterpene biosynthesis,starch and sucrose metabolic pathways,and it is hypothesized that differences in accumulation and metabolism of amino acids,monoterpenoids and sugars are closely associated with pollen abortion in sterile pepper lines.The subcellular compartments of DAPs were localized,and most of the DAPs obtained from pepper anther protein sequencing were localized in chloroplasts,indicating that chloroplast proteins play a key role in anther development.（5）By combining genomic locus interval and proteomic analysis,13 candidate genes were screened,not only in the male sterility QTL region but also in the DAPs associated with male sterility from proteomic analysis.Thirteen candidate genes were verified by q PCR,and six candidate genes were screened:Capana07g000676,Capana07g000979,Capana07g000993,Capana07g001228,Capana07g001241 and Capana07g001294.Through bioinformatics analysis and literature search of the six candidate genes screened,it was found that Capana07g000676 and Capana07g001241 genes were highly expressed in anther tissue,which might be closely related to male sterility;VIGS technology was further used to study the function of these two genes.The VIGS-2 strain with Capana07g000676 gene silenced and the VIGS-8 strain with Capana07g001241 gene silenced showed sterile phenotype in flower organs and fruits.It is speculated that candidate genes Capana07g000676 and Capana07g001241 are male sterile genes in pepper.