INVESTIGADORES
PESSINO Silvina Claudia
congresos y reuniones científicas
Título:
Molecular characterization of a PPIase gene linked to apomixis in Paspalum notatum
Autor/es:
SPOTO, NICOLÁS; STEIN, JULIANA; SIENA, LORENA; PESSINO, SILVINA; ORTIZ, JUAN PABLO AMELIO
Lugar:
Bahía Blanca
Reunión:
Workshop; V Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis; 2016
Institución organizadora:
CERZOS-CONICET
Resumen:
Paspalum notatum is an apomictic forage grass widely distributed in tropical and subtropical regions of South America. Apomixis is a natural form of asexual reproduction by seeds, which generates progenies genetically identical to the mother plant. This type of reproduction is considered as a deregulation of the sexual developmental pathway, produced by genetic and/or epigenetic factor/s. Apomixis in tetraploid P. notatum is controlled by a single locus (ACL) that showed a distorted segregation ratio and restriction in recombination. Molecular analysis have shown that the ACL includes protein coding and non-coding sequences, repetitive elements, and cytosine methylation. Comparative mapping studies showed that markers located at the long arms of chromosome 2 and 12 of rice, mapped completely linked to apomixis in the species. One of these markers (RFLP C932) (LOC_Os02g52290.1 of rice) codifies for a peptidyl-prolyl cis/trans isomerase protein of the FKBP family. This type of proteins are involved in protein folding and has been associated with cell division and cell elongation in Arabidopsis. The objective of this work was to characterize the orthologous sequences of locus LOC_Os02g52290.1 in P. notatum genome and to determine its relation with the apomictic trait. Tetraploid genotypes Q4188 (sexual), Q4117 (apomictic) and one F1 family of 21 individuals derived from them were used. The reproduction mode of each F1 plant was determined by cytoembryological observation of cleared ovules at anthesis, and by assaying the SCAR marker PNSA2, which generates a band of 180 bp completely linked to the trait. Individuals that showed only meiotic embryo sacs in their ovules (Polygonum type: egg cell, two polar nuclei and antipodals) were classified as sexual, while individuals showing at least one aposporic embryo sac (egg cell, two polar nuclei and absence of antipodals) were classified as apomicts. For the identification of orthologous PPIase sequence of P. notatum, the complete CDS of rice was retrieved from the Gramene web page (www.gramene.org). A BLAST search was carried out against a 454/Roche transcriptome database of the flower development of apomictic and sexual P. notatum genotypes in order to identify similar sequences. Afterward, specific PCR primers for each transcript (isotig) were designed for PCR amplification from genomic DNA. Genetic linkage of amplicons with the trait was performed by analyzing both parental genotypes, a bulk of 5 sexual and 5 apomictic F1 progenies and then all individual samples. The classification of the F1 individuals by both citoembryological observation and molecular analyses showed 14 sexual and 7 apomictic progenies. BLAST analysis on the 454 database using the rice PPIase as a query detected eight highly similar sequences. In the sexual genotype, five isotigs (alleles/splice variant) of 1,471-3,113 bp corresponding to the same isogroup (gene= isogrup00104) were identified. Among them, the isotig01380 (S= 333; E-value= 6e-91) was the most similar to the query in the phylogenetic analysis. The predicted protein (112 aa) was highly similar to the FKBP-type peptidyl-prolyl cis-trans isomerase of Sorghum bicolor (XP_002454586.1). In the apomictic genotype, three different sequences (isogroups = genes) were detected. The first one was a short transcript of 631 nt (isotig35750; S= 325; E-value= 2e-88) which encoded for a protein similar to the rice gene. The second one (isotig32671; S= 82; E-value= 4e-15), seems a truncated form of the PPIase gene, and the third one (isotig33823; S= 240; E-value= 8e-63) corresponded to a chimeric transcript containing a FKBP-domain and a DNAJ domain. The specific amplifications of all isotigs determined that only the short version of the rice gene (isotig35750) co-segregates with the ACL. This sequence showed a specific band presents in the apomictic parent and all apomictic progenies that was absent in sexual plants. Moreover, the same band was detected in several other natural apomictic accessions. The band of interest is being cloned and sequenced to confirm its identity. Specific fragments of sexual and apomictic isotigs will be amplified for expression analysis by qRT-PCR during sexual and apomictic reproductive development. Moreover, the complete genomic sequence will be isolated for characterizing the gene structure in both genotypes and analyze the epigenetic landscape associated with both sequences.