Fine Mapping And Cloning Of Key QTLs Related To In Vivo Haploid Induction And Super High Oil Haploid Inducer Development

Haploid breeding technology based on Stock6 derived inducers had developed to be one of the most important methods in maize breeding.However,both genetics bases and mechanism underlie had not been fully understood.This research focused on the fine mapping of haploid induction related QTLs,cloning and functional verification of ZmPLAl,development of super high oil inducers and the mechanism of heterofertilization during maize haploid induction.Summary of the research:1)Based on the fine mapping result of qhirl,Bacterial Artificial Chromosome library was constructed,and positive clone cover the mapping region was screened and sequenced.Based on the annotation information of the genes in the region,a patatin like phospholipase A(ZmPLAl)was predicted as the candidate for haploid induction.CRISPR/Cas9 was used for editing the candidate in the receptor materials,haploid plants was found in the selfed progenies and crossed progenies of the ZmPLAl edited lines,which verified the gene ZmPLAl responsible for haploid induction.2)Comparison between sequence of Zmplal in inducer and non-inducer demonstrated that 11 single nucleotide substitutions in total and a 4-bp insertion exist in the fourth exon of inducer,which leading to frame shift and premature of transcription termination.4-bp insertion was the reason for the weak/loss of function of ZMPLA1 and triggered haploid induction.RNAseq showed that the expression of Zmpla1 was up-regulated which may be the result of feedback regulation system.3)According to the mapping result of Prigge et al.,(2012),fine mapping of qhir8 was conducted.In the F2 population constructed by CAUHOI and UH400,the effect of qhir8 was confirmed,34 recombinants screened from 3989 F2 individuals were used for fine mapping.With the progeny test strategy,the mapping region was finally narrowed down to 207Kb,which paved the way for gene cloning.4)New high oil inducers was developed toward the goal of engineering application.An inducer line CAU2 with high HIR and the high oil plasm BHO was used as two parents.Molelar markerassisted selection of qhirl was used in F2 generation.From F3 generation,each individual was determined with haploid induction rate,individuals with high HIR was further determined with kernel oil content.High HIR and high KOC was selected for the next generation.After 6 round selection,stable lines with KOC>10%and HIR>10%was acquired and could be used for engineering haploid production and determination with automatic NMR haploid selection system.5)Heterofertilization during the haploid induction was studied in this research.With dual pollination method and delayed pollination with different time intervals,the frequency of heterofertilization was in positive correlation with HIR,inducers with high HIR could lead to more heterofertilization.In another hand,heterofertilization was affected by the time of sperm from different parents arriving the embryo sac,ie.,when pollen from different parents arriving the embryo sac simultaneously could lead to the highest frequency of heterofertilization.