Estudios citogenéticos en citotipos apomícticos y sexuales de paspalum notatum y caracterización molecular de secuencias asociadas a la aposporía
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Date
2012
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FCA-UNR
Abstract
Paspalum notatum Flügge es una gramínea rizomatosa perenne ampliamente difundida en
las regiones tropicales y subtropicales de Sudamérica. Las razas tetraploides naturales de esta
especie se reproducen casi exclusivamente por apomixis de tipo apospórica. La aposporía (un
componente de la apomixis apospórica) en P. notatum es controlada por un locus único (denominado ACL) que presenta segregación distorsionada y supresión de la recombinación. La presencia de inversiones u otras alteraciones cromosómicas fueron propuestas para explicar el tipo
de herencia observado para la aposporía. La determinación de la estructura cromosómica que
contiene al ACL puede aportar conocimiento sobre la transmisión del carácter a la progenie y
contribuir a la identificación de secuencias codificantes asociadas con su expresión. Por otro
lado, varios marcadores moleculares completamente ligados a la aposporía y transcriptos de
ARNm diferencialmente expresados en inflorescencias de plantas apomícticas y sexuales fueron identificados en P. notatum y otras especies de gramíneas. Sin embargo, hasta el momento
solo unos pocos de ellos han sido estudiados en detalle a nivel genético y funcional.
El objetivo general de este trabajo de Tesis fue profundizar los estudios tendientes a determinar las características citogenéticas y moleculares del locus responsable de la aposporía en
P. notatum y caracterizar genes cuya expresión fue asociadas a la apomixis en gramíneas así
como secuencias genómicas localizadas específicamente en el locus de la aposporía.
En el Capítulo I se describen los estudios citogenéticos realizados en meiocitos de plantas
apomícticas y sexuales de P. notatum. Como material vegetal fueron incluidas cinco accesiones
apomícticas naturales, tres individuos sexuales obtenidos experimentalmente y 16 híbridos F1
clasificados por el modo de reproducción. Las observaciones citogenéticas revelaron que tanto
las plantas apomícticas como las sexuales presentaron algunas anormalidades meióticas como,
cromosomas rezagados, puentes de cromatina y micronúcleos. Sin embargo, un análisis cuantitativo de éstas reveló diferencias significativas entre las accesiones apomícticas y sexuales.
Asimismo, el análisis de los híbridos confirmó las diferencias observadas en los progenitores con
diferente modo de reproducción. Un estudio de FISH reveló que el ACL se encuentra en hemicigosis, muy próximo a regiones ricas en heterocromatina. Los resultados presentados en este
Capítulo demostraron que las plantas apomícticas presentan un mayor número de anormalidades meióticas en la anafase I que las sexuales y que estas anormalidades se transmiten a la
descendencia asociadas con el modo de reproducción. Asimismo fue posible determinar que el
locus responsable de la aposporía se transmite solo a uno de los productos de la meiosis I. Esta
estructura cromosómica podría explicar la distorsión de la segregación y la supresión de la recombinación asociada a la transmisión de la apomixis en la especie.
En el Capítulo II se describe la caracterización molecular de los genes SERK y EXS en P.
notatum. El gen SERK (somatic embryogenesis receptor like kinase) fue asociado a la apomixis
en Poa pratensis. A partir del apilamiento de secuencias conocidas fue posible aislar dos secuencias genómicas de P. notatum con alta similitud a genes SERK: PnSERK1 (1750 pb) y
PnSERK2 (2050pb). Al menos 3 copias de PnSERK están presentes en el genoma de P. notatum. Estudios de expresión por PCR en tiempo real (qRT-PCR) en diferentes estadios del desarrollo reproductivo del genotipo Q4188 (sexual) y Q4117 (apomíctico) mostraron que PnSERK2
se expresa a niveles superiores de PnSERK1 y que muestra diferencias significativas en los
estadios de premeiosis, postmeiosis y antesis entre ambos genotipos. Experimentos de hibridación in situ de tejidos, mostraron una expresión contrastante entre el genotipo sexual y el apomíctico. Los resultados obtenidos indican que PnSERK2 presenta una expresión diferencial entre
plantas apomícticas y sexuales y podría formar parte de la cascada de genes asociados a la
expresión de la apomixis en P. notatum. Se analizó también el gen EXS. Una secuencia codificante para el dominio EXS fue identificada en un clon de BAC conteniendo marcadores moleculares completamente ligados a la aposporía en P. simplex. Mediante experimentos similares a
los descriptos anteriormente se aisló un fragmento de 2298 pb con alta similitud al gen EXS de
arroz y maíz de los genotipos Q4188 y Q4117. El análisis del número de copias reveló que existen entre 2 y 4 secuencias homólogas en el genoma de P. notatum. Estudios de expresión por
qRT-PCR no mostraron diferencias significativas entre los genotipos sexuales y apomícticos en
distintos estadios del desarrollo reproductivo. Sin embargo, estudios de hibridación in situ de
tejido mostraron expresión de EXS en tejido ovárico del genotipo sexual mientras que solo se
observó expresión en células que rodean a los sacos embrionarios en el genotipo apomíctico. El
análisis de mapeo in silico, indicó que PnEXS se localiza en regiones que ya han sido reportadas como asociadas a la aposporía. Estos resultados indican que PnEXS podría estar diferencialmente regulado en plantas apomícticas y sexuales.
En el Capítulo III se presentan los estudios realizados en la caracterización de secuencias
específicas del ACL en P. notatum y el mapeo de transcriptos de ARNm asociados con el modo
de reproducción. Varias de las secuencias correspondientes al ACL mostraron similitud con
elementos repetitivos, proteínas hipotéticas, proteínas ribosomales y dominios codificantes. El
mapeo de seis transcriptos expresados diferencialmente entre plantas apomícticas y sexuales
mostró que N54 resultó ligado en repulsión al ACL.
Los resultados obtenidos en esta Tesis permitieron detectar la presencia de un rearreglo
cromosomal que se trasmite a la progenie asociado a la aposporía. Además, se logró localizar el
ACL en un único cromosoma de P. notatum y determinar su condición hemicigota. Paralelamente, se pudo demostrar que PnSERK2 se encuentra funcionalmente asociado a la aposporía y
que PnEXS estaría regulado diferencialmente en plantas apomícticas y sexuales. El análisis de
marcadores 100% ligados al carácter puso en evidencia la presencia de secuencias codificantes
que podrían estar asociadas a la aposporía.
Paspalum notatum Flügge is a perennial rhizomatous grass species distributed in tropical and subtropical regions of South America. Natural tetraploid cytotypes reproduce by obligated aposporous apomixis. Apospory (a component of apomictic reproduction) in P. notatum is controlled by a single locus (ACL) with distorted segregation ratio and suppression of recombination. The presence of an inversion or other type of chromosomal alteration was proposed to explain the observed segregation fashion of the trait. Disclosing the chromosomal structure of the ACL can contribute to the understanding of the transmission of apospory and the identification of sequences associated with its expression. On the other hand, several molecular markers fully linked to the ACL as well as mRNA transcripts differentially expressed in inflorescences of apomictic and sexual plants were identified in P. notatum and other species of grasses. However, only a few of them have been studied in detail at the genetic and functional levels so far. The objective of this work was to determine the cytogenetic characteristic of apomictic and sexual tetraploid P. notatum and to characterize genes whose expression was associated with apomixis in grasses, as well as, a group of genomic sequences mapping at ACL in the species. Chapter I describes the studies carried out in meiocytes of apomictic and sexual plants of P. notatum. Five natural apomictic accessions, three completely sexual experimentallyobtained tetraploid individuals and 16 F1 hybrids classified by the mode of reproduction were used as plant material. The cytogenetic observations revealed that both sexual and apomictic plants showed meiotic abnormalities as, lagging chromosomes, chromatin bridges and micronuclei. However, a quantitative analysis of these configurations at anaphase I revealed significant differences between apomictic and sexual individuals. The analysis of hybrids confirmed the differences between accessions with different reproductive mode. FISH analysis showed that the ACL is hemizygous and it is placed close to a region rich in heterochromatin. These results indicated that apomictic plants showed a greater number of meiotic abnormalities in anaphase I than sexual ones and that abnormalities were transmitted to the offspring associated with the mode of reproduction. It was also possible to determine that the ACL is transmitted only to one of the products of meiosis I. This chromosomal structure could explain the segregation distortion and the suppression of recombination associated with transmission of apospory in the species. Chapter II describes the molecular characterization of genes SERK and EXS in P. notatum. The SERK (somatic embryogenesis receptor like kinase) gene was associated with apomixis in Poa pratensis. Two sequence PnSERK1 (1750 BP) and PnSERK2 (2050pb) were isolated. At least 3 copies of PnSERK were present in the P. notatum. Expression analysis by real time PCR (qRT-PCR) at different stages of the reproductive development of Q4188 (sexual) and Q4117 (apomictic) genotypes showed that PnSERK2 is expressed at higher levels than PnSERK1 and showed significant differences between genotypes at premeiosis, postmeiosis and anthesis. In situ hybridization with PnSERK2, showed a contrasting expression between the apomictic and sexual plants. Results presented in this work indicate that PnSERK2 participate in the cascade of genes associated with the expression of apomixis in P. notatum. On the other hand, a coding sequence showing an EXS domain was identified in a BAC clone containing molecular makers completely linked apospory in P. simplex. A fragment of 2298 pb highly similar to the EXS domain of rice and maize was isolated from genotypes Q4188 and Q4117. Copy number analysis revealed that there are between 2 and 4 homologous sequences in the P. notatum genome. Expression analysis by qRT-PCR at different stages of the reproductive development showed no significant differences among genotypes. However, in situ tissue hybridization showed expression of EXS in ovaries of the sexual genotype, while EXS was expressed only in the surrounding cells of the embryo sac in the apomictic genotype. In silico mapping, indicated that PnEXS is located in regions that have associated previously with apospory. These results indicate that EXS could be under a different expression control in sexual and apomictic plants. Chapter III presents studies focused on the characterization of specific sequences mapping at the ACL in P. notatum and the localization of mRNA transcripts associated with the mode of reproduction. Several sequences of the ACL showed similarity with repetitive elements, hypothetical proteins, ribosomal proteins and coding domains. Mapping assays of six transcripts differentially expressed between apomictic and sexual plants showed that clone N54 was linked in repulsion to the ACL. This can be a good candidate for further analysis. The results presented in this Thesis showed the presence of a chromosomal rearrangement associated with apospory that it is transmitted to the progeny. In addition, it was determined that ACL of P. notatum is placed in a hemizygous region. Analysis of PnSERK2 and PnEXS indicated that both genes are associated with apomixis. Moreover, analysis of markers 100% linked to the character revealed the presence of coding sequence in this region.
Paspalum notatum Flügge is a perennial rhizomatous grass species distributed in tropical and subtropical regions of South America. Natural tetraploid cytotypes reproduce by obligated aposporous apomixis. Apospory (a component of apomictic reproduction) in P. notatum is controlled by a single locus (ACL) with distorted segregation ratio and suppression of recombination. The presence of an inversion or other type of chromosomal alteration was proposed to explain the observed segregation fashion of the trait. Disclosing the chromosomal structure of the ACL can contribute to the understanding of the transmission of apospory and the identification of sequences associated with its expression. On the other hand, several molecular markers fully linked to the ACL as well as mRNA transcripts differentially expressed in inflorescences of apomictic and sexual plants were identified in P. notatum and other species of grasses. However, only a few of them have been studied in detail at the genetic and functional levels so far. The objective of this work was to determine the cytogenetic characteristic of apomictic and sexual tetraploid P. notatum and to characterize genes whose expression was associated with apomixis in grasses, as well as, a group of genomic sequences mapping at ACL in the species. Chapter I describes the studies carried out in meiocytes of apomictic and sexual plants of P. notatum. Five natural apomictic accessions, three completely sexual experimentallyobtained tetraploid individuals and 16 F1 hybrids classified by the mode of reproduction were used as plant material. The cytogenetic observations revealed that both sexual and apomictic plants showed meiotic abnormalities as, lagging chromosomes, chromatin bridges and micronuclei. However, a quantitative analysis of these configurations at anaphase I revealed significant differences between apomictic and sexual individuals. The analysis of hybrids confirmed the differences between accessions with different reproductive mode. FISH analysis showed that the ACL is hemizygous and it is placed close to a region rich in heterochromatin. These results indicated that apomictic plants showed a greater number of meiotic abnormalities in anaphase I than sexual ones and that abnormalities were transmitted to the offspring associated with the mode of reproduction. It was also possible to determine that the ACL is transmitted only to one of the products of meiosis I. This chromosomal structure could explain the segregation distortion and the suppression of recombination associated with transmission of apospory in the species. Chapter II describes the molecular characterization of genes SERK and EXS in P. notatum. The SERK (somatic embryogenesis receptor like kinase) gene was associated with apomixis in Poa pratensis. Two sequence PnSERK1 (1750 BP) and PnSERK2 (2050pb) were isolated. At least 3 copies of PnSERK were present in the P. notatum. Expression analysis by real time PCR (qRT-PCR) at different stages of the reproductive development of Q4188 (sexual) and Q4117 (apomictic) genotypes showed that PnSERK2 is expressed at higher levels than PnSERK1 and showed significant differences between genotypes at premeiosis, postmeiosis and anthesis. In situ hybridization with PnSERK2, showed a contrasting expression between the apomictic and sexual plants. Results presented in this work indicate that PnSERK2 participate in the cascade of genes associated with the expression of apomixis in P. notatum. On the other hand, a coding sequence showing an EXS domain was identified in a BAC clone containing molecular makers completely linked apospory in P. simplex. A fragment of 2298 pb highly similar to the EXS domain of rice and maize was isolated from genotypes Q4188 and Q4117. Copy number analysis revealed that there are between 2 and 4 homologous sequences in the P. notatum genome. Expression analysis by qRT-PCR at different stages of the reproductive development showed no significant differences among genotypes. However, in situ tissue hybridization showed expression of EXS in ovaries of the sexual genotype, while EXS was expressed only in the surrounding cells of the embryo sac in the apomictic genotype. In silico mapping, indicated that PnEXS is located in regions that have associated previously with apospory. These results indicate that EXS could be under a different expression control in sexual and apomictic plants. Chapter III presents studies focused on the characterization of specific sequences mapping at the ACL in P. notatum and the localization of mRNA transcripts associated with the mode of reproduction. Several sequences of the ACL showed similarity with repetitive elements, hypothetical proteins, ribosomal proteins and coding domains. Mapping assays of six transcripts differentially expressed between apomictic and sexual plants showed that clone N54 was linked in repulsion to the ACL. This can be a good candidate for further analysis. The results presented in this Thesis showed the presence of a chromosomal rearrangement associated with apospory that it is transmitted to the progeny. In addition, it was determined that ACL of P. notatum is placed in a hemizygous region. Analysis of PnSERK2 and PnEXS indicated that both genes are associated with apomixis. Moreover, analysis of markers 100% linked to the character revealed the presence of coding sequence in this region.
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Keywords
Gramíneas, Paspalum notatum, Apomixis, Aposporía, Reproducción asexual