Screening And Genetic Research Of Four Novel Male Gametophytic Mutants And A Male Sterile Mutant In Arabidopsis Thaliana

The male gametophyte plays a critical role in sexual reproduction of angiosperms. Currently, the genes and pathways involved in male gametophytic development and function in flowering plants remain largely unknown. A large-scale mutant screen of Ds transposon insertion lines was employed to identify four mutants (KD360, KD361, KD362, and KD375) of Arabidopsis thaliana with defect in male gametophyte development and function. Genetic and morphologic analyses indicated that KD362 mutant was a completely male gametophytic lethal, but KD360, KD361 and KD375 mutants were partly male gametophytic lethal. In addition, KD360, KD362 and KD375 mutants affect only the male gametophyte, while KD361 mutation affects both the female and male gametophyte. We analyzed male gametophyte development of four mutants and found that the KD360, KD361 and KD375 mutants degenerated before the PMI, while the KD362 microspores arrested during the binucleate stage to trinucleate stage. Complete cosegregation of the phenotype with the antibiotic resistance marker and consistent segregation ratios suggested that Ds was tightly linked to or responsible for the reduced genetic transmission. The sequences flanking the Ds element were isolated with TAIL-PCR. The flanking sequences obtained were run against BLASTN to identify the genomic location of the Ds element. In KD360, Ds inserts into the exon of At1g44224, which encodes an ECA1 gametogenesis related family protein. In KD361, Ds inserts in the upstream of At4g00380, which encodes XH/XS domain-containing protein or XS zinc finger domain-containing protein. In KD362, Ds inserts not only in the first exon of At1g44224 but also between At2g01910 and At2g01913. In KD375, Ds inserts in the upstream of Atlg26815, which is similar to F-box family protein. Analyses the additional insertion lines of these genes suggested that At1g44224 and At4g00380 may be responsible for the reduced genetic transmission and pollen abortion. Pro::GUS transgenic Arabidopsis showed that At1g44224 and At4g00380 driven GUS activity during pollen maturation and also in germinating pollen.Besides four male gametophytic mutants, we obtained a male sterile mutant fortuitously. In ms360 mutant, after released from the tetrad, the microspores'exine and tapetum are abnormal. ms360 is a recessive male sterile mutant controlled by single gene. Using map-based cloning technique, we mapped MS360 gene close to the molecular marker nga162 and nga172 of Chromosome 3.