José Guillermo Seijo

Molecular confirmation of species status for the allopolyploid cotton species, Gossypium ekmanianum Wittmack

Understanding the relationship between domesticated crop species and their wild relatives is paramount to germplasm maintenance and the utilization of wild relatives in breeding programs. Recently, Gossypium ekmanianum was resurrected as an independent species based on morphological analysis of specimens obtained from the Dominican Republic, where the original type specimen was collected. The molecular data presented here support the recognition of G. ekmanianum Wittmack as a distinct species that is phylogenetically close to G. hirsutum L. Analyses of chloroplast DNA data reveal species-specific, indel polymorphisms that unambiguously distinguish G. ekmanianum samples from other polyploid congeners. Furthermore, analysis of accessions that originated from the Dominican Republic demonstrate the cryptic inclusion of this sister taxon within the US National Plant Germplasm System, a germplasm collection maintained for diversity preservation and future breeding resources. The data presented here indicate that “wild” G. hirsutum accessions may include the closely related G. ekmanianum, and provide a method to easily distinguish the two.

Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE ( SERK ) genes in sexual and apomictic Paspalum notatum

The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatumSERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli. Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue.

Characterization of retrotransposon sequences expressed in inflorescences of apomictic and sexual Paspalum notatum plants.

Apomixis, an asexual mode of reproduction through seeds, holds much promise for agricultural advances. However, the molecular mechanisms underlying this trait are still poorly understood. We previously isolated several transcripts representing novel sequences differentially expressed in reproductive tissues of sexual and apomictic plants. Here, we report the characterization of two of these unknown RNA transcripts (experimental codes N17 and N22). Since original fragments showed no significant homologies to sequences at databases, preliminary genomic PCR experiments were carried out to discard possible contaminations. RACE extension on flanking regions provided longer sequences for the candidates and additional related transcripts, which revealed similarity to LTR retrotransposons carrying short transduplicated segments of protein-coding genes. Interestingly, some transduplicated segments corresponded to genes previously associated with apomictic development. Gene copy number estimations revealed a moderate representation of the elements in the genome, with significantly increased numbers in a sexual genotype with respect to an apomictic one. Genetic mapping of N17 showed that a copy of this particular element was located onto Paspalum notatum linkage group F3c, at a central non-recombinant region resembling a centromere. Expression analysis showed an increased activity of N17 and N22 sense strands in ovules of the sexual genotypes. A retrotransposon-specific differential display analysis aimed at detecting related sequences allowed the identification of a complex family, with the majority of its members represented in the sexual genotype. Our results suggest that these elements could be participating in regulatory pathways related to apomixis and sexuality.

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum

Key messagencRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes.AbstractApomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30–60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

Erratum to: Genome sizes in diploid and allopolyploid Arachis L. species (section Arachis)

Fil: Samoluk, Sergio Sebastian. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Nordeste. Instituto de Botanica del Nordeste (i); Argentina