Study Of Haploid Induction Mechanism Of The Wheat MATRILINEAL Gene And Development Of A Double Fluorescencebased Haploid Identification System

Wheat is not only one of the major crops in the world,but also has largest cultivating area.The tremendous food demand puts forward higher demand for wheat breeding.A solution to overcome this problem is to use efficient breeding technology.Double haploid technology(DH)can greatly reduce the breeding periods and provide efficient technical support for selecting varieties to meet the needs in a short time.One of the key steps in this technology is haploid induction(HI).The recent cloning of the underlying gene MATRILINEAL(MTL)/Zm PLA1/NLD in Stock 6 which encodes a pollen-specific phospholipase A.Chromosome fragmentation was detected in pollen sperm cells,and the gradual elimination of chromosomes was observed after pollination in maize HI lines,but when and how such DNA damage takes place remains unknown.At the same time,the lack of effective haploid identification system in wheat restricts the application of double haploid breeding.In this study,wheat cultivar Fielder was used a receptor for transgenic study.Ta MTL editing plants were obtained by using CRISPR/Cas9.Anthers and pollen of wheat MTL mutants were studied.Our work identified the mechanism of haploid induction after Ta MTL mutation.Meanwhile,haploid grains could be screened efficiently by establishing a double fluorescent labeling system.The main results are as follows:1.Ta MTL was specifically expressed in the trinuclear pollen grain stage(S12 stage).In situ hybridization showed that Ta MTL was mainly expressed in the male germ unit of pollen grains.According to the expression pattern of Ta MTL,it is speculated that Ta MTL is mainly involved in the growth and development of anthers,especially in the process of pollen mitosis.2.Transcriptome analysis showed that significant changes occurred in genes related to fatty acid metabolism and calcium signal transduction in mutant anthers,consistent with changes in fatty acid content detected in the mutant anthers.Scanning electron microscopy and transmission electron microscopy showed there were a large number of fatty acid-like metabolites on the surface of pollen grains.A large number of ROS related genes were found to have changed,which may affect ROS homeostasis.3.The production of ROS and the decrease of antioxidant enzyme activity were detected in the threenucleus pollen grain stage and later stages of the mutant.After anther staining and section observation,it was found that ROS was mainly concentrated in pollen grains.TUNEL assay detected strong signals of DNA damage.DNA damage was quantified by comet assay.The dynamic model of ROS accumulation and DNA damage in anthers was established.The analysis of the growth rate in different stages showed that the increase of reactive oxygen species preceded the generation of DNA damage which may lead to the production of haploid.4.Cassettes with red fluorescent protein(RFP)and green fluorescent protein(GFP)genes driven by endosperm-specific and embryo-specific promoters respectively were transferred into the induction line.Green color was specifically expressed in endosperm aleurone layer and red in embryo.In the grains of self-crossed or crossed lines,different fluorescence signals can be observed using different excitation light sources.Haploid grains were identified with green colors in the embryo,with no color in endosperm which should show a red color in diploid grains.The efficiency of our fluorescent haploid identification system had a positive detection rate at 70%.In summary,for the mechanism of wheat phospholipase gene Ta MTL inducing haploid production,a hypothesis is put forward.Ta MTL mutation leads to early termination of translation,loss of protein function,disorder of lipid metabolism in pollen cells at the trinuclear pollen stage,accumulation of palmitic acid and oleic acid,intermediate products of fatty acid synthesis in pollen,and then disorder of mitochondrial electron transport chain,resulting in excessive reactive oxygen species,The results showed that the chromosomes of sperm cells in pollen were damaged during the second mitosis and gradually eliminated after fertilization,resulting in haploid after pollination.At the same time,an efficient haploid screening system based on double fluorescent protein was established for the first time in wheat,which provided a technical guarantee for accelerating the process of wheat breeding.