A long-standing goal in plant breeding has been the ability to confer apomixis to agriculturally relevant species, which would require a deeper comprehension of the molecular basis of apomictic ...regulatory mechanisms. Eragrostis curvula (Schrad.) Nees is a perennial grass that includes both sexual and apomictic cytotypes. The availability of a reference transcriptome for this species would constitute a very important tool toward the identification of genes controlling key steps of the apomictic pathway. Here, we used Roche/454 sequencing technologies to generate reads from inflorescences of E. curvula apomictic and sexual genotypes that were de novo assembled into a reference transcriptome. Near 90% of the 49568 assembled isotigs showed sequence similarity to sequences deposited in the public databases. A gene ontology analysis categorized 27448 isotigs into at least one of the three main GO categories. We identified 11475 SSRs, and several of them were assayed in E curvula germoplasm using SSR-based primers, providing a valuable set of molecular markers that could allow direct allele selection. The differential contribution to each library of the spliced forms of several transcripts revealed the existence of several isotigs produced via alternative splicing of single genes. The reference transcriptome presented and validated in this work will be useful for the identification of a wide range of gene(s) related to agronomic traits of E. curvula, including those controlling key steps of the apomictic pathway in this species, allowing the extrapolation of the findings to other plant species.
Las enfermedades virales constituyen uno de los principales factores limitantes del cultivo de;
Lilium spp. Los virus más comunmente encontrados en Lilium spp. en diferentes países son Lily;
...symptomless virus (LSV), Lily mottle virus (LMoV) y Cucumber mosaic virus (CMV). El objetivo;
de esta tesis fue evaluar la presencia de LSV, LMoV y CMV en Lilium en Argentina, y en el caso;
de que estos virus estuvieran presentes se planteó estimar su incidencia en diferentes híbridos;
cultivados y localidades de producción, así como evaluar diferentes alternativas para obtener;
plantas de sanidad controlada. Se trabajó con híbridos de Lilium longiflorum (Ll), Asiáticos (As),;
Orientales (Or), Longiflorum × Asiáticos (LA), Longiflorum × Orientales (LO) y Orientales ×;
Trompeta (OT). Se llevó a cabo el reconocimiento de la sintomatología asociada a la infección viral;
y se identificaron áfidos posibles vectores de virus en parcelas de producción de Lilium. Se;
evaluaron plantas sintomáticas y asintomáticas mediante DAS-ELISA y RT-PCR. Para esto se;
utilizaron anticuerpos policlonales específicos contra las proteínas de la cápside de LSV, LMoV y;
CMV, así como primers degenerados y específicos, respectivamente. Se pudo detectar la presencia;
de los tres virus. Las secuencias del gen de la proteína de la cápside (CP) y de aminoácidos de la CP;
de LSV, LMoV y CMV, aislados desde plantas cultivadas en Argentina, presentaron una elevada;
similitud con las de otras cepas de los mismos virus registradas en el GenBank. Asimismo, se;
detectaron infecciones virales en bulbos provenientes de Holanda, lo cual indica una posible vía de;
entrada de los virus. La incidencia de virosis fue evaluada por medio de DAS-ELISA en plantas de;
bulbos provenientes de siete localidades de Argentina entre las latitudes 26° 56′ S y 43° 03′ S, y;
longitudes 65° 21′ O y 71° 29′ O. Los virus detectados en órden decreciente fueron LSV (42,1%),;
LMoV (35,6%) y CMV (20,0%) y se encontraron en infecciones simples o mixtas. La incidencia de;
virus varió entre los híbridos desde 36% (O ‘Montecristo’) a 94,7% (Ll ‘Avita’) en 2006, y desde;
38,9% (OT ‘Yelloween’) a 82,1% (LO ‘Triumphator’) en 2007, con una incidencia total de 64,1% y;
70,7% en 2006 y 2007, respectivamente. También se encontró una variación en la incidencia entre;
localidades. La mayor incidencia de virus (89,6 y 87,6% en 2006 y 2007, respectivamente) se;
observó en Bahía Blanca, y la menor incidencia fue detectada en Trevellin (47,4%) en 2006 y en;
Malargüe (48,6%) en 2007. La alta incidencia de virus en el cultivo de Lilium puede estar asociada a;
la utilización de bulbos infectados para la propagación y a la falta de medidas de control de;
vectores. Para ajustar un sistema de obtención de bulbos de sanidad controlada se utilizó material;
infectado con virus de híbridos cultivados en Argentina. Los bulbillos fueron producidos por;
scaling ex vitro o por multiplicación in vitro con o sin tratamiento de termoterapia y/o;
quimioterapia. Posteriormente, se extrajeron y cultivaron los ápices meristemáticos. La obtención;
de plantas sanas fue dependiente de los híbridos, de los virus presentes y de los tratamientos;
aplicados. La aplicación de termoterapia ex vitro en escamas de bulbos durante la diferenciación de;
bulbillos (scaling) y la obtención de meristemas desde dichos bulbillos se propone como una;
innovación para un protocolo exitoso de obtención de bulbos de Lilium spp. de sanidad controlada.
Viral diseases are one of the main limiting factors in lily production. The viruses most commonly;
found in lilies worldwide are Lily symptomless virus (LSV), Lily mottle virus (LMoV) and;
Cucumber mosaic virus (CMV). The aim of this thesis was to evaluate the presence of LSV, LMoV;
and CMV in Lilium in Argentina, and when present, to estimate their incidence in different hybrids;
and localities, and also to evaluate different alternatives to produce virus-free plants. Hybrids;
evaluated belong to six groups: Lilium longiflorum (Ll), Asiatics (As), Orientals (Or), Longiflorum;
× Asiatics (LA), Longiflorum × Orientals (LO) and Orientals × Trumpet (OT). Different symptoms;
associated to viral infection were recognized and aphid species with potential capacity to transmit;
viruses were identified in Lilium production plots. Symptomatic and asymptomatic plants were;
evaluated by DAS-ELISA and RT-PCR using polyclonal antiserum against the capsid protein of;
LSV, LMoV and CMV, and with degenerate and specific primers, respectively. The gene;
nucleotide and amino acid sequences of the coat protein of LSV, LMoV and CMV were highly;
similar to those from other isolates of the same viruses registered in the GenBank. Infections were;
detected in bulbs imported from the Netherlands, which indicates a possible way of entry of viruses;
into our country. Virus incidence was evaluated by DAS-ELISA in bulbs coming from seven;
Argentinean localities. The areas surveyed were between latitude 26° 56′ S and 43° 03′ S, and;
longitude 65° 21′ W and 71° 29′ W. Virus infection in decreasing order were LSV (42.1%), LMoV;
(35.6%), and CMV (20.0%) and single or mixed infections were detected. Virus infection varied;
among hybrids from 36.0% (Oriental ‘Montecristo’) to 94.7% (Lilium longiflorum ‘Avita’) in 2006,;
and from 38.9% (OT ‘Yelloween’) to 82.1% (LO ‘Triumphator’) in 2007, with an overall infection;
incidence of 64.1 and 70.7% in 2006 and 2007, respectively. A variation in virus infection;
incidence among localities was also detected. The highest virus infection incidence (89.6 and 87.6%;
in 2006 and 2007, respectively) was observed in Bahía Blanca (38° 44′ S, 62° 16′ W). The lowest;
virus infection incidences, were 47.4% in Trevellin (43° 03′ S, 71° 29′ W) in 2006, and 48.6% in;
Malargüe (35° 28′ S, 69° 35′ W) in 2007. The high occurrence of viruses infecting lily crops in;
Argentina could be due to the use of infected bulbs during propagation and also to the absence of;
preventive virus vector control measures. The starting material to set a system for obtaining virusfree;
plants were infected hybrids grown in Argentina. For bulblet production, scales or scale;
sections were cultured ex vitro or in vitro, with or without thermotherapy and /or chemotherapy.;
Then, meristem-tips were cultured in vitro. Different percentages of virus-free plants were obtained;
depending on the hybrid, the treatment and the viruses present in infected tissues. The application of;
thermotherapy to bulb scales during the differentiation of bulblets (scaling) and the extraction of;
meristem-tips from these bulblets is an innovative proposal for a successful protocol for obtaining;
virus- free Lilium spp plants.
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