Map Based Cloning Of The Barley Genic Male Sterile Gene Msg26

Barley is a diploid autogamous cereal ranking the fourth largest cereal crops worldwide and is primarily used for beer and feed stock.In China,most of the barley supply is imported because of an insufficient production.Improving the yield and quality of barley is of great importance for the food security in China.Utilization of crop heterosis can be applied to improve agronomically important.Plant male sterile genes are ideal tools for exploring crop heterosis.The cloning of barley male sterile genes provides tools for producing hybrid seeds,and facilitates our understanding that governs genic male sterility(GMS)in Triticeae.The barley GMS gene msg26 is mapped on the 4HL chromosome.Using the BSR-seq and chromosome walking,msg26 was mapped to a 0.02 cM interval that anchors to a low-quality assembly physical region in barley.The main results of this study are as follows:1.Phenotype of barley genic male sterile mutant: Compared with wild-type anthers,the anthers of the spontaneous mutant GSHO745 were pale yellow in color and lacked pollen grains in the mature stage.In addition,the F1 hybrid of GSHO745 and Morex produced viable pollen grains,as indicated by a pink color by the Alexander stain,which indicated that msg26 confers plant sporophytic male sterility.We examined meiosis among the male-fertile(MF)and male-sterile(MS)segregants from GSHO745.Compared with the MF groups,the pollen mother cells(PMCs)produced comparable tetrads.However,some of the early uninucleate microspores in the MS groups were poorly colored by the carbo-fuchsin stain and continued to degenerate in the middle uninucleate stage.By the late uninucleate stage,only highly condensed cellular debris were observed in anthers,which led to a non-pollen type male sterility.2.Fine mapping of the barley genic male sterile gene msg26: In this study,two F2 populations(PopM and Pop124)were mainly used to map msg26.Using 7,490 PopM plants,msg26 was mapped to the SP1M21 ~ SP1M4 interval,which is about 0.06 cM and anchors to a 446-kb region in the Morex reference sequence,but it contains a region that conflicts with the genetic map.The genetic analysis showed that an inversion(SP1M32 ~ SP1M37,ca.80 kb)occurred in the msg26 region of GSHO745.To overcome the inversion interference,we performed genetic mapping of msg26 in the wild barley population.The results showed that the inversion still existed in the wild barley population.Luckly,chromosome recombination occurred in the key recombinant(#124-372)of Pop124.Altogether,msg26 was further mapped to the SP1M14 ~ SP1M4 interval,which is less than 0.06 cM.3.Physical mapping and candidate gene identification of the barley genic male sterile gene msg26: Based on the collinearity of the msg26 target region among barley-wheat and barley-rice-maize,we found that the msg26 target region anchors to a low-quality assembly physical region on the 4HL chromosome of barley.After genetic map correction,msg26 was delimited in SPM14-SP1M49 interval,which is about 0.02 cM and associated with four candidate genes.Among the four candidate genes,only the male-sterile allele of HORVU4Hr1G074840 in GSHO745 was associated with a unique frameshift InDel that resulted in a variant protein as CYP704B: p.G436Qfs*70.Next,the HORVU4Hr1G074840 orthologues in wheat were specifically expressed in young spikes.In addition,HORVU4Hr1G074840 is located in a collinear region of the rice CYP704B2 and the maize CYP704B1 that were both involved in male reproductivity.Altogether,HORVU4Hr1G074840 appeared to be the only candidate for the MSG26 gene in barley.4.MSG26 is spatiotemporally expressed in anthers: HORVU4Hr1G074840 was specifically expressed in anthers during meiosis(S1 stage),and the expression level was high.in situ hybridization showed that HORVU4Hr1G074840 mRNA accumulated only in tapetum.Subcellular localization showed that the GFP6:HvCYP704B fusion protein appeared to be associated with the cytoplasm and endoplasmic reticulum.