RESUMEN
MAIN CONCLUSION: Although grass pea is an environmentally successful robust legume with major traits of interest for food and nutrition security, the genetic potential of this orphan crop has long been neglected. Grass pea (Lathyrus sativus L.) is a Neolithic plant that has survived millennia of cultivation and has spread over three continents. It is a robust legume crop that is considered one of the most resilient to climate changes and to be survival food during drought-triggered famines. The hardy penetrating root system allows the cultivation of grass pea in various soil types, including marginal ones. As an efficient nitrogen fixer, it meets its own nitrogen requirements and positively benefits subsequent crops. However, already in ancient India and Greece, overconsumption of the seeds and a crippling neurological disorder, later coined neurolathyrism, had been linked. Overemphasis of their suspected toxic properties has led to disregard the plant's exceptionally positive agronomic properties and dietary advantages. In normal socio-economic and environmental situations, in which grass pea is part of a balanced diet, neurolathyrism is virtually non-existent. The etiology of neurolathyrism has been oversimplified and the deficiency in methionine in the diet has been overlooked. In view of the global climate change, this very adaptable and nutritious orphan crop deserves more attention. Grass pea can become a wonder crop if the double stigma on its reputation as a toxic plant and as food of the poor can be disregarded. Additionally, recent research has exposed the potential of grass pea as a health-promoting nutraceutical. Development of varieties with an improved balance in essential amino acids and diet may be relevant to enhance the nutritional value without jeopardizing the multiple stress tolerance of this promising crop.
Asunto(s)
Productos Agrícolas , Lathyrus , Producción de Cultivos , Productos Agrícolas/crecimiento & desarrollo , Abastecimiento de Alimentos , Lathyrus/crecimiento & desarrollo , Valor Nutritivo , Plantas Medicinales/crecimiento & desarrolloRESUMEN
Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC-NBS-LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety 'Altar' that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus-induced gene silencing in tetraploid wheat lines. In contrast to most described NBS-LRR proteins, the N-terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS-LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10-based resistance is highly unusual both in its dependence on two, only distantly, related CC-NBS-LRR proteins, as well as in the pattern of diversifying selection in the N-terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.
Asunto(s)
Inmunidad Innata , Enfermedades de las Plantas/genética , Triticum/genética , Alelos , Secuencia de Aminoácidos , ADN de Plantas/genética , Silenciador del Gen , Genes de Plantas , Variación Genética , Datos de Secuencia Molecular , Poliploidía , Selección Genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Triticum/metabolismoRESUMEN
Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discover ies was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.
Asunto(s)
Regulación hacia Abajo , Silenciador del Gen , Poaceae/genética , ARN/genética , ARN/metabolismo , ADN Complementario/genética , Técnicas de Silenciamiento del Gen , Genes de Plantas/genética , Vectores Genéticos , Hordeum/genética , Secuencias Invertidas Repetidas , Virus del Mosaico/metabolismo , Oxidorreductasas/genética , Fenotipo , Plásmidos/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Triticum/genéticaRESUMEN
Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or loss-of-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids. This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. We have introduced into bread wheat double-stranded RNA-expressing constructs containing fragments of genes encoding Phytoene Desaturase (PDS) or the signal transducer of ethylene, Ethylene Insensitive 2 (EIN2). Transformed plants showed phenotypic changes that were stably inherited over at least two generations. These changes were very similar to mutant phenotypes of the two genes in diploid model plants. Quantitative real-time polymerase chain reaction revealed a good correlation between decreasing mRNA levels and increasingly severe phenotypes. RNAi silencing had the same quantitative effect on all three homoeologous genes. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Wheat seedlings with low mRNA levels for EIN2 were ethylene insensitive. Thus, EIN2 is a positive regulator of the ethylene-signaling pathway in wheat, very similar to its homologs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Our data show that RNAi results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.
Asunto(s)
Etilenos/metabolismo , Genómica/métodos , Oxidorreductasas/genética , Interferencia de ARN , Triticum/genética , Carotenoides/metabolismo , Etiquetas de Secuencia Expresada , Genoma de Planta , Fotoblanqueo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiología , Triticum/metabolismoRESUMEN
Production of transgenic plants is now routine for many of our crop species. Methods for the detailed molecular analysis of transgenic plants are available, but often the exact location of the transgene within the crop genome is poorly understood. As a starting point to understanding more about the site of transgene insertion, transgenes can be physically located using fluorescence in situ hybridization (FISH). This technique allows transgenes to be located to specific chromosome regions following the hybridization of a fluorescent labelled probe to a chromosome spread. The technique is sensitive enough to detect single transgene copies and can reveal information about the complexity of a transgene insertion site as well as identifying plants homozygous for the transgene. A FISH method is described that has been used successfully to detect single-transgene copies in mitotic metaphase chromosome preparations of wheat and barley.
Asunto(s)
Cromosomas de las Plantas/genética , Hordeum/genética , Plantas Modificadas Genéticamente/genética , Triticum/genética , Mapeo Cromosómico/métodos , Hibridación Fluorescente in Situ/métodos , Mitosis , Plantas Modificadas Genéticamente/citología , Plásmidos/genéticaRESUMEN
The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.
Asunto(s)
Mapeo Cromosómico , Hordeum/genética , Transformación Genética , Transgenes/genética , Bases de Datos Genéticas , Ligamiento Genético , Hibridación Fluorescente in Situ , Repeticiones de Minisatélite/genética , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
More than 50 leaf rust resistance (Lr) genes against the fungal pathogen Puccinia triticina have been identified in the wheat gene pool, and a large number of them have been extensively used in breeding. Of the 50 Lr genes, all are known only from their phenotype and/or map position except for Lr21, which was cloned recently. For many years, the problems of molecular work in the large (1.6 x 10(10) bp), highly repetitive (80%), and hexaploid bread wheat (Triticum aestivum L.) genome have hampered map-based cloning. Here, we report the isolation of the Lr gene Lr10 from hexaploid wheat by using a combination of subgenome map-based cloning and haplotype studies in the genus Triticum. Lr10 is a single-copy gene on chromosome 1AS. It encodes a CC-NBS-LRR type of protein with an N-terminal domain, which is under diversifying selection. When overexpressed in transgenic wheat plants, Lr10 confers enhanced resistance to leaf rust. Lr10 has similarities to RPM1 in Arabidopsis thaliana and to resistance gene analogs in rice and barley, but is not closely related to other wheat Lr genes based on Southern analysis. We conclude that map-based cloning of genes of agronomic importance in hexaploid wheat is now feasible, opening perspectives for molecular bread wheat improvement trough transgenic strategies and diagnostic allele detection.