Generation of Asynaptic Mutants in Potato by Disrupting StDMC1 Gene Using RNA Interference Approach

Fixing the genomic composition and multiplication through true potato seed (TPS) is an important challenge in autotetraploid potato. Disrupted meiotic cDNA (DMC1) is a meiotic gene that plays a central role in DNA recombination through crossing over in meiosis. Using the Arabidopsis DMC1 (AtDMC1) gene sequence, we retrieved Solanum tuberosum DMC1(StDMC1) from the diploid potato genome, and subsequently, sense and antisense regions of the StDMC1 gene were amplified in potato cv. Kufri Jyoti. The sense and antisense fragments were confirmed by Sanger-sequencing and cloned in the pRI101 vector. Agrobacterium-mediated transformation of the RNAi construct resulted in 44% transformation efficiency, and a total of 137 mutant lines were obtained. These mutant lines were further validated through pollen viability testing, and selected lines were used for gene expression analysis. The acetocarmine-based pollen staining showed reduced pollen viability ranging from 14 to 21% in four DMC1 mutant lines (DMC4-37, DMC4-41, DMC6-20, and DMC6-21), as compared to the Kufri Jyoti control plants, which on average exhibited 78% pollen viability. The phenotypic data was supported by the reduced expression of the StDMC1 gene in these four mutant lines compared to the control Kufri Jyoti. The results confirmed the generation of StDMC1 knockdown lines. This is the first report of StDMC1 mutant line generation in tetraploid potatoes and will be a step forward in generating non-recombinant mutants through sexual reproduction in potatoes.

[1]  V. Bhardwaj,et al.  Biofortification Strategies to Improve Iron Concentrations in Potato Tubers: Lessons and Future Opportunities , 2021, Potato Research.

[2]  E. V. Perrino,et al.  Crop wild relatives: know how past and present to improve future research, conservation and utilization strategies, especially in Italy: a review , 2020, Genetic Resources and Crop Evolution.

[3]  Anil Kumar,et al.  A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’ , 2020, Physiology and Molecular Biology of Plants.

[4]  O. Ortiz,et al.  The Potato Crop: Its Agricultural, Nutritional and Social Contribution to Humankind , 2019 .

[5]  A. Pendle,et al.  Dmc1 is a candidate for temperature tolerance during wheat meiosis , 2019, Theoretical and Applied Genetics.

[6]  I. Szarejko,et al.  The dmc1 Mutant Allows an Insight Into the DNA Double-Strand Break Repair During Meiosis in Barley (Hordeum vulgare L.) , 2019, Front. Plant Sci..

[7]  D. K. Bishop,et al.  A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability , 2019, bioRxiv.

[8]  I. Colas,et al.  desynaptic5 carries a spontaneous semi-dominant mutation affecting Disrupted Meiotic cDNA 1 in barley , 2019, Journal of experimental botany.

[9]  J. Bradshaw.  Improving the Nutritional Value of Potatoes by Conventional Breeding and Genetic Modification , 2019, Quality Breeding in Field Crops.

[10]  E. Greene,et al.  The biochemistry of early meiotic recombination intermediates , 2018, Cell cycle.

[11]  M. Kumar,et al.  Breeding Potato for Quality Improvement , 2018, Potato - From Incas to All Over the World.

[12]  N. N. Sharma,et al.  RNAi-based transgene conferred extreme resistance to the geminivirus causing apical leaf curl disease in potato , 2018, Plant Biotechnology Reports.

[13]  G. Jander,et al.  RNAi-Mediated Simultaneous Resistance Against Three RNA Viruses in Potato , 2017, Molecular Biotechnology.

[14]  R. Visser,et al.  Silencing of six susceptibility genes results in potato late blight resistance , 2016, Transgenic Research.

[15]  N. Kandemir,et al.  Apomixis: new horizons in plant breeding , 2015 .

[16]  R. Visser,et al.  Development and analysis of a 20K SNP array for potato (Solanum tuberosum): an insight into the breeding history , 2015, Theoretical and Applied Genetics.

[17]  S. Sharma,et al.  Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease , 2015, Functional & Integrative Genomics.

[18]  E. Guchi.  Disease Management Practice on Potato (Solanum tuberosum L.) in Ethiopia , 2015 .

[19]  Rodney Rothstein,et al.  Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae , 2014, Genetics.

[20]  J. Keurentjes,et al.  Hybrid recreation by reverse breeding in Arabidopsis thaliana , 2014, Nature Protocols.

[21]  I. Colas,et al.  The Synaptonemal Complex Protein ZYP1 Is Required for Imposition of Meiotic Crossovers in Barley[W][OPEN] , 2014, Plant Cell.

[22]  M. Brown,et al.  DNA strand exchange and RecA homologs in meiosis. , 2014, Cold Spring Harbor perspectives in biology.

[23]  Ruslan Kalendar,et al.  FastPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis. , 2014, Methods in molecular biology.

[24]  F. Degroote,et al.  Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role , 2013, PLoS genetics.

[25]  Doris Chen,et al.  The Recombinases DMC1 and RAD51 Are Functionally and Spatially Separated during Meiosis in Arabidopsis[W][OA] , 2012, Plant Cell.

[26]  Baoming Tian,et al.  Dissecting 2 Meiotic Mutations (dmc1 and asy1) in Artificial Autopolyploid Arabidopsis thaliana , 2011 .

[27]  A. Gholizadeh,et al.  Genome wide cloning of maize meiotic recombinase Dmc1 and its functional structure through molecular phylogeny. , 2011, Genetics and molecular research : GMR.

[28]  Upendra Kumar Devisetty,et al.  The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis , 2010, BMC Research Notes.

[29]  Jiming Jiang,et al.  Agrobacterium-Mediated Transient Gene Expression and Silencing: A Rapid Tool for Functional Gene Assay in Potato , 2009, PloS one.

[30]  T. Shibata,et al.  Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins , 2008, Nucleic acids research.

[31]  Tai Wang,et al.  OsDMC1 is required for homologous pairing in Oryza sativa , 2007, Plant Molecular Biology.

[32]  Masatoshi Nei,et al.  Origins and evolution of the recA/RAD51 gene family: Evidence for ancient gene duplication and endosymbiotic gene transfer , 2006, Proceedings of the National Academy of Sciences.

[33]  A. Shinohara,et al.  Roles of RecA homologues Rad51 and Dmc1 during meiotic recombination , 2004, Cytogenetic and Genome Research.

[34]  J. Sainis,et al.  Cloning and characterization of the DMC1 genes in Oryza sativa , 2004 .

[35]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[36]  K. Chong,et al.  Isolation and characterization of OsDMC1, the rice homologue of the yeast DMC1 gene essential for meiosis , 2001, Sexual Plant Reproduction.

[37]  C. Bergounioux,et al.  Isolation and characterisation of the RAD51 and DMC1 homologs from Arabidopsis thaliana , 1998, Molecular and General Genetics MGG.

[38]  S. Tabata,et al.  Structural analysis of a recA-like gene in the genome of Arabidopsis thaliana. , 1995, DNA research : an international journal for rapid publication of reports on genes and genomes.

[39]  N. Miyajima,et al.  Characterization of cDNAs induced in meiotic prophase in lily microsporocytes. , 1994, DNA research : an international journal for rapid publication of reports on genes and genomes.

[40]  N. Simmonds.  Potato Breeding , 1970, Nature.