Gene Mapping And Allelism Test Of Maize Viviparous Mutant

Cloning and functional analysis of maize kernel mutants not only help us understand the development mechanism of maize kernels,but also provide great application value for maize breeding.In our laboratory,nine viviparous mutants were found during the seed reproduction process.After successive generations of self-crossing,three mutants with obvious phenotypeand stable inheritance were screened for further experiments.In this study,we identified and cloned the viviparous gene of the viviparous mutant vp-like4.The F2 genetic mapping population was constructed by crossing vp-like4 and the inbred line Mo17.Using BSR-Seq method,the target gene was mapped on chromosome 5 between 173.8 and 175.6 Mb.Genomic sequence information analysis and allelic test showed that vp-like4 is an allele of the gene Viviparous15(Vp15),which encodes a small subunit of molybdenide synthase and participates in the process of carotenoid cleavage to ABA.Alignment of the genomic sequences revealed a deletion of 60-bp at the second exon and 3,-untranslated region of Vp15 gene,resulting in a frameshift mutation.Two vp15 mutants reported previously(vp15-umu1)and(vp15-DR1126),both mutated from a Mutator transponson inserting in the second exon of Vp15 gene that were different from vp-like4.q RT-PCR showed that there was a significant difference in the expression level of Vp15 gene between vp-like4 mutant and normal grain.In summary,the mutant vp-like4 is a new allelic mutant of the Vp15 gene.Similarly,another viviparous mutant was mapped to the interval of 160.4-165.6 Mb on chromosome 3 taking the same stretagy.Genome sequence analysis and allelic test results indicated that vp-like8 is an allele of the gene Viviparous1(Vp1),which encodes a seed-specific transcription factor and enhances seed response to ABA.Alignment of the genomic sequence revealed a deletion of the 343 bp in the second intron of vp-like8 and the insertion of the 222 bp repeat in the third intron.It was reported that there was a deletion of 343 bp in the second intron for the vp1 mutant,the deletion of sequence is mutated in different ways.q RT-PCR showed that there was a significant difference in the expression of Vp1 gene between vp-like8 mutant and normal grain.In summary,the mutant vp-like8 is a new allelic mutant of the Vp1 gene.Notably,a new viviparous mutant vp-like5 was fine mapped.Different F2 mapping population were constructed by crossing vp-like5 with inbred line Zheng58 and Mo17 respectively.The target gene was located on chromosome 4 between 235.5 and 241.0 Mb by BSR-Seq method.And it was flanked by SSR marker bnlg589 and umc1109 using map-based cloning method.The physical distance between the two markers was 1.8 Mb,further expansion of the population and development of new molecular markers were needed to determine the candidate gene and conduct the next validation test.Based on the above results,among the viviparous mutants found in the laboratory,two mutants were identified to be allelic to Vp15 and Vp1,respectively.And a new viviparous mutant vp-like5 was also found.The identification of these mutants provided invaluable experimental materials for the study of the mechanism of vivipary in maize.