Specific expression of apomixis-linked alleles revealed by comparative transcriptomic analysis of sexual and apomictic Paspalum simplex Morong flowers.

Apomixis is defined as clonal reproduction by seed. A comparative transcriptomic analysis was undertaken between apomictic and sexual genotypes of Paspalum simplex Morong to identify apomixis-related polymorphisms at the level of mRNA. cDNA-AFLP (amplified fragment length polymorphism) profiling of apomictic and sexual flowers at several stages of development yielded 202 amplicons that showed several kinds of expression specificities. Among these, the large majority consisted of amplicons that were present only in specific stages of development of the apomictic flowers. Ten percent of polymorphic amplicons were present with almost identical intensity in all stages of the apomictic flowers and never in the sexual flowers. Reverse transcription-PCR (RT-PCR) and Southern analyses of these amplicons showed that they belong to constitutively expressed alleles that are specifically present on the apomixis-controlling locus of P. simplex. The most frequent biological functions inferred from the sequence homology of the apomixis-linked alleles were related to signal transduction and nucleic acid/protein-binding activities. Most of these apomixis-linked alleles showed nonsense and frameshift mutations, revealing their probable pseudogene nature. None of the amplicons that were present only in specific stages of development of the apomictic flowers co-segregated with apomixis, indicating they did not originate from additional apomictic alleles but more probably from differential regulation of the same allele in apomictic and sexual flowers. The molecular functions inferred from sequence analysis of these latter amplicons were related to seed storage protein and regulatory genes of various types. The results are discussed regarding the possible role in apomictic reproduction of the differentially expressed genes in relation to their specificity of expression and inferred molecular functions.

[1]  Thomas Thiel,et al.  Molecular signatures of apomictic and sexual ovules in the Boechera holboellii complex. , 2009, The Plant journal : for cell and molecular biology.

[2]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[3]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[4]  J. Casacuberta,et al.  The Frequent Transcriptional Readthrough of the Tobacco Tnt1 Retrotransposon and Its Possible Implications for the Control of Resistance Genes , 2009, Journal of Molecular Evolution.

[5]  B. Larkins,et al.  The Development of Endosperm in Grasses1 , 2009, Plant Physiology.

[6]  R. Visser,et al.  A new and versatile method for the successful conversion of AFLP markers into simple single locus markers. , 2003, Nucleic acids research.

[7]  P. Vos,et al.  AFLP: a new technique for DNA fingerprinting. , 1995, Nucleic acids research.

[8]  H. El-Shemy,et al.  Antisense phenotypes reveal a functional expression of OsARF1, an auxin response factor, in transgenic rice. , 2009, Current issues in molecular biology.

[9]  S. Pessino,et al.  Non-radioactive mRNA fingerprinting to visualise gene expression in mature ovaries of Brachiaria hybrids derived from B. brizantha, an apomictic tropical forage , 1997 .

[10]  T. Adachi,et al.  Developmental expression of ASG- 1 during gametogenesis in apomictic guinea grass (Panicum maximum). , 2005, Journal of plant physiology.

[11]  H. Rolletschek,et al.  Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage. , 2007, The Plant journal : for cell and molecular biology.

[12]  B. Larkins,et al.  The opaque-2 mutation of maize differentially reduces zein gene transcription. , 1989, The Plant cell.

[13]  Sarah Hake,et al.  The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA , 2007, Nature Genetics.

[14]  I. Donnison,et al.  Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice , 2006, Theoretical and Applied Genetics.

[15]  S. Pessino,et al.  Gene expression analysis at the onset of aposporous apomixis in Paspalum notatum , 2008, Plant Molecular Biology.

[16]  A. Koltunow,et al.  Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency , 2001, Planta.

[17]  Richard B. Flavell,et al.  Plant molecular biology , 1978, Nature.

[18]  R. Sekhon,et al.  A Mutator Transposon Insertion Is Associated With Ectopic Expression of a Tandemly Repeated Multicopy Myb Gene pericarp color1 of Maize , 2008, Genetics.

[19]  M. H. Urbani,et al.  Cytogeography and reproduction of the Paspalum simplex polyploid complex , 2002, Plant Systematics and Evolution.

[20]  A. Koltunow,et al.  A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium , 2000, Planta.

[21]  M. Jamilena,et al.  Plant sex chromosomes: molecular structure and function , 2008, Cytogenetic and Genome Research.

[22]  J. Yazaki,et al.  Analysis of expressed sequence tags in apomictic guineagrass (Panicum maximum). , 2009, Journal of plant physiology.

[23]  A. Schnittger,et al.  A positive signal from the fertilization of the egg cell sets off endosperm proliferation in angiosperm embryogenesis , 2006, Nature Genetics.

[24]  D. Grimanelli,et al.  Seed development and inheritance studies in apomictic maize-Tripsacum hybrids reveal barriers for the transfer of apomixis into sexual crops. , 2009, The International journal of developmental biology.

[25]  G. Hagen,et al.  The Roles of Auxin Response Factor Domains in Auxin-Responsive Transcription Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.008417. , 2003, The Plant Cell Online.

[26]  R. Van der Hoeven,et al.  Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. , 1996, The Plant journal : for cell and molecular biology.

[27]  Manjit Singh,et al.  Isolation of candidate genes for apomictic development in buffelgrass (Pennisetum ciliare) , 2007, Plant Molecular Biology.

[28]  Monica Alandete-Saez,et al.  Auxin-Dependent Patterning and Gamete Specification in the Arabidopsis Female Gametophyte , 2009, Science.

[29]  G. Hagen,et al.  Auxin Response Factors , 2001, Journal of Plant Growth Regulation.

[30]  Gang Wang,et al.  Expressional profiling study revealed unique expressional patterns and dramatic expressional divergence of maize α-zein super gene family , 2009, Plant Molecular Biology.

[31]  G. Barcaccia,et al.  SERK and APOSTART. Candidate Genes for Apomixis in Poa pratensis1[w] , 2005, Plant Physiology.

[32]  A. Depicker,et al.  Down-regulation of endogenes mediated by a transitive silencing signal. , 2006, RNA.

[33]  A. Evsikov,et al.  Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. , 2004, Developmental cell.

[34]  J. Reed,et al.  AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana , 2005, Development.

[35]  B. M. Johri.  Embryology of Angiosperms , 1984, Springer Berlin Heidelberg.

[36]  P A Ward,et al.  Molecular signatures of sepsis: multiorgan gene expression profiles of systemic inflammation. , 2001, The American journal of pathology.

[37]  U. Grossniklaus,et al.  Apomixis technology development—virgin births in farmers' fields? , 2004, Nature Biotechnology.

[38]  K. Yamato,et al.  Gene organization of the liverwort Y chromosome reveals distinct sex chromosome evolution in a haploid system , 2007, Proceedings of the National Academy of Sciences.

[39]  Ben Hui Liu,et al.  Statistical Genomics: Linkage, Mapping, and QTL Analysis , 1997 .

[40]  D. Rigden,et al.  Identification of differentially expressed cDNA sequences in ovaries of sexual and apomictic plants of Brachiaria brizantha , 2003, Plant Molecular Biology.

[41]  S. Arcioni,et al.  Feulgen-DNA densitometry of embryo sacs permits discrimination between sexual and apomictic plants in Paspalum simplex , 1999, Euphytica.

[42]  C. A. Dunn,et al.  Impact of transposable elements on the evolution of mammalian gene regulation , 2005, Cytogenetic and Genome Research.

[43]  H. Yoshida,et al.  Refunctionalization of the ancient rice blast disease resistance gene Pit by the recruitment of a retrotransposon as a promoter. , 2009, The Plant journal : for cell and molecular biology.

[44]  S. Arcioni,et al.  An AFLP marker tightly linked to apomixis reveals hemizygosity in a portion of the apomixis-controlling locus in Paspalum simplex. , 2002, Genome.

[45]  Emmanuelle Lerat,et al.  Influence of the transposable element neighborhood on human gene expression in normal and tumor tissues. , 2007, Gene.

[46]  S. Clough,et al.  Tissue-Specific Gene Silencing Mediated by a Naturally Occurring Chalcone Synthase Gene Cluster in Glycine max , 2004, The Plant Cell Online.

[47]  D. Schubert,et al.  Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects , 2004, The Plant Cell Online.

[48]  A. Levy,et al.  Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat , 2003, Nature Genetics.

[49]  J. Carman.  Asynchronous expression of duplicate genes in angiosperms may cause apomixis, bispory, tetraspory, and polyembryony , 1997 .

[50]  D. Grimanelli,et al.  Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum. , 2003, Genetics.

[51]  M. Hudson,et al.  Endogenous, Tissue-Specific Short Interfering RNAs Silence the Chalcone Synthase Gene Family in Glycine max Seed Coats[W][OA] , 2009, The Plant Cell Online.

[52]  O. Calderini,et al.  Comparative mapping reveals partial conservation of synteny at the apomixis locus in Paspalum spp. , 2003, Molecular Genetics and Genomics.

[53]  G. Barcaccia,et al.  Isolation of candidate genes for apomixis in Poa pratensis L. , 2004, Plant Molecular Biology.

[54]  G. Cervigni,et al.  Gene expression in diplosporous and sexual Eragrostis curvula genotypes with differing ploidy levels , 2008, Plant Molecular Biology.

[55]  R. Wing,et al.  Comparative gene expression in sexual and apomictic ovaries of Pennisetum ciliare (L.) Link. , 1996, Plant Molecular Biology.

[56]  T. Adachi,et al.  Isolation and Characterization of a Gene Expressed during Early Embryo Sac Development in Apomictic Guinea Grass (Panicum maximum) , 1999 .

[57]  Nature Genetics , 1991, Nature.

[58]  S. Arcioni,et al.  The chromosome segment related to apomixis in Paspalum simplex is homoeologous to the telomeric region of the long arm of rice chromosome 12 , 2001, Molecular Breeding.

[59]  G. Servant,et al.  Remodeling Yeast Gene Transcription by Activating the Ty1 Long Terminal Repeat Retrotransposon under Severe Adenine Deficiency , 2008, Molecular and Cellular Biology.