Map-based Cloning And Application Of The Male Sterility Gene MS7 In Zea Mays L.

The application of the hybrid breeding systems efficiently increases the agricultural yield because hybrid plants possess superior traits.Manual or mechanical detasseling of the female lines remains the predominant method in commercial hybrid seed production in maize.However,it is not only time-consuming,labor-intensive and expensive,but also reduces the yield of hybrid seed.There has been little industry use of chemical sterilants because of the risk of incomplete pollen sterility,a reduction on female fertility and its detrimental effect to the environment.The utilization of the natural male-sterile female lines is ideal for large-scale hybrid seed production.The three-line hybrid breeding system which employs the cytoplasmic male sterility(CMS)line is widely used to produce the hybrid seeds.Nuclear-encoded male sterility(NMS)or Genetic male sterility(GMS)lines have such advantages: the genetic diversity,without special restoration gene and disease resistant compared with cytoplasmic male sterility lines.In this study,the map-based cloning and functional characterization of the maize nuclear-encoded Zea mays Male sterility 7(ZmMs7)gene had been carried out,and the results were as following:1.Compared with the wild-type male fertile sibling,ms7-6007 and ms7gl1 displayed complete male sterility with no exerted anthers,but normal vegetative growth and female fertility.Microscopic comparison analysis between ms7-6007 and wild type was conducted.Compared with the normal phenotype in the wild type,the ms7-6007 mutant tapetal cells were swollen and lightly stained,along with the microspore aborted,leaving debris in the shrinkage locule,indicating the abnormal PCD of tapetal cells in the ms7-6007 mutant.2.The ms7 gene was defined to the interval of 180-kb length on maize chromosome 7.Six predicted genes are located in this region,including GRMZM5G890224,which encodes a putative PHD-finger transcription factor orthologous to rice PTC1 and Arabidopsis MS1.3.To analyze the expression pattern of ZmMs7,Semi-quantitative RT-PCR and quantitative real-time PCR were performed.The obtained results revealed that ZmMs7 was specifically expressed in post-meiotic anthers from the quartet to late-vacuolate microspore stages,with the highest expression level in the early-vacuolate microspore stage.Compared with the expression of ZmMs7 in Ms7/ms7-6007 male-fertile sibling,the ms7-6007 male-sterile mutant showed the lower ZmMs7 expression level.Together,these results proved that ZmMs7 is an anther-specific expression gene and may play a specific role during the development of anther and microspore.4.To confirm the above prediction,the functional complementation experiment was performed.The transgenic plants carrying the vector pZmMs7pro::ZmMs7 were then crossed with the ms7-6007 mutant.The transgenic plants rescued the male-sterile defect of ms7-6007 and recovered the fertility phenotype,demonstrating that the male-sterile phenotype of the ms7-6007 mutant results from the ZmMs7 gene mutation.5.The Multi-Control Sterility(MCS)transgenic maintainer lines are developed based on the ms7-6007 mutant transformed with MCS constructs containing the(i)ZmMs7 gene,(ii)α-amylase gene ZmAA and/or(iii)DNA adenine methylase gene Dam,(iv)red fluorescence protein gene DsRed2 or mCherry,and(v)herbicide-resistant gene Bar.Self-pollination of the MCS transgenic maintainer line produces transgenic red fluorescent seeds(transgenic maintainer seeds)and non-transgenic normal color seeds(male-sterile mutant seeds)at a 1:1 ratio.Moreover,our present data indicated that the transgene transmission rate through pollen of transgenic plants harboring two pollen-disrupted genes is lower than that containing one pollen-disrupted gene,at least in the bachground of Zheng58 inbred line.The MCS system has great potential to enhance the efficiency of maize male-sterile line propagation and commercial hybrid seed production.